The proof that Covid-19 was a bioweapon created by the US

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Unread post by FOIA » 2024-2-1 22:39

The proof that Covid-19 was a bioweapon created by the US released by a court as a result of a FOIA request:

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The proof that Covid-19 was a bioweapon created by the US Text content

United States Department of the Interior
U. S. GEOLOGICAL SURVEY 12201 Sunrise Valley Drive Reston, Virginia 20192-0002
In Reply Refer To: December 5, 2023 U.S. Geological Survey
Attention: Judy Cearley
Post Office Box 66783
Albuquerque, New Mexico 87193
Ms. Emily Anne Kopp U.S. Right to Know Washington, DC 20012 emily@usrtk.org
Re: U.S. Geological Survey (USGS) Freedom of Information Act (FOIA) Tracking # DOI-USGS-2023-000257 – Response
Dear Ms. Kopp:
This letter is our response to your FOIA request submitted on October 14, 2022, in which you requested the following information:
Records maintained by U.S. Geological Survey by Tonie Rocke (trocke@usgs.gov), a research epidemiologist at the National Wildlife Health Center.
1. We request communications between Dr. Rocke and email addresses from the following domains: @ecohealthalliance.org, @wh.iov.cn.
2. We request communications that include the key term “Wuhan Institute of Virology.” We also request communications that include the terms “PREEMPT” and “DEFUSE,” but only those communications where all of the letters in each of these terms is capitalized.
The time period for this request is March 24, 2017 to the present. Please narrow the search results to exclude any published papers, organizational newsletters or other widely available published materials.
We are releasing, in part, one Portable Document Format (PDF) file, consisting of 1,412 pages. We reasonably foresee that disclosure would harm an interest protected by one or more of the nine exemptions to the FOIA’s general rule of disclosure and disclosure would be prohibited by law; therefore, portions of these materials are being withheld under the following FOIA exemptions:
Ms. Emily Anne Kopp 2

Exemption 3
Exemption 3 allows the withholding of information protected by a nondisclosure provision in a federal statute other than the FOIA. Specifically, 41 U.S.C. § 4702 (b)-(c) prohibits the release of contractor proposals unless they have been set forth or incorporated by reference in a final contract. Here, the proposals of the unsuccessful submitters are not releasable under FOIA because they were not set forth or incorporated by reference into the final contract. Therefore, we are withholding the proposal in full under Exemption 3. Approximately 175 pages contain redactions pursuant to Exemption 3.
Exemption 4
Exemption 4 protects “trade secrets and commercial or financial information obtained from a person [that is] privileged or confidential.” The withheld information is commercial or financial information. The company that supplied this information (the submitter) is considered a person, because the term “person,” under the FOIA, includes a wide range of entities including “corporations.” Additionally, the submitter does not customarily release this information to the public, so the information is confidential for the purposes of Exemption 4. Approximately 183 pages contain redactions pursuant to Exemption 4.
Exemption 5
Exemption 5 allows an agency to withhold “inter-agency or intra-agency memorandums or letters which would not be available by law to a party ... in litigation with the agency.” 5 U.S.C. § 552(b)(5). Exemption 5 therefore incorporates the privileges that protect materials from discovery in litigation, including the deliberative process, attorney work-product, attorney-client, and commercial information privileges. Two pages contain redactions under Exemption 5 because they qualify to be withheld under the following privilege:
Deliberative Process Privilege
The deliberative process privilege protects the decision-making process of government agencies and encourages the frank exchange of ideas on legal or policy matters by ensuring agencies are not forced to operate in a fish bowl. A number of policy purposes have been attributed to the deliberative process privilege, such us: (1) assuring that subordinates will feel free to provide the decisionmaker with their uninhibited opinions and recommendations; (2) protecting against premature disclosure of proposed policies; and (3) protecting against confusing the issues and misleading the public.
The deliberative process privilege protects materials that are both predecisional and deliberative. The privilege covers records that reflect the give-and-take of the consultative process and may include recommendations, draft documents, proposals, suggestions, and other subjective documents which reflect the personal opinions of the writer rather than the policy of the agency.
The materials that have been withheld under the deliberative process privilege of Exemption 5 are both predecisional and deliberative. They do not contain or represent formal or informal

agency policies or decisions. They are the result of frank and open discussions among agency Ms. Emily Anne Kopp 3
officials and their consultants. Their contents have been held confidential by all parties and public dissemination of the information would have a chilling effect on the agency’s deliberative processes; expose the agency’s decision-making process in such a way as to discourage candid discussion within the agency, and thereby undermine its ability to perform its mandated functions.
The deliberative process privilege does not apply to records created 25 years or more before the date on which the records were requested.
Exemption 6
Exemption 6 allows an agency to withhold “personnel and medical files and similar files the disclosure of which would constitute a clearly unwarranted invasion of personal privacy.” 5 U.S.C. § 552(b)(6).
The phrase “similar files” covers any agency records containing information about a particular individual that can be identified as applying to that individual. To determine whether releasing records containing information about a particular individual would constitute a clearly unwarranted invasion of personal privacy, we are required to balance the privacy interest that would be affected by disclosure against any public interest in the information.
Under the FOIA, the only relevant public interest to consider under the exemption is the extent to which the information sought would shed light on agency’s performance of its statutory duties or otherwise let citizens ‘know what their government is up to.’ The burden is on the requester to establish that disclosure would serve the public interest. When the privacy interest at stake and the public interest in disclosure have been determined, the two competing interests must be weighed against one another to determine which is the greater result of disclosure: the harm to personal privacy or the benefit to the public. The purposes for which the request for information is made do not impact this balancing test, as a release of information requested under the FOIA constitutes a release to the general public.
Approximately 281 pages contain redactions pursuant to Exemption 6. The information that has been withheld under Exemption 6 consists of personal home phone numbers, personal mobile phone numbers, non-public phone numbers of EcoHealth Alliance personnel, personal email addresses, names of unsuccessful bidders, user access information, and other personal matters. We have determined that the individuals to whom this information pertains have a substantial privacy interest in withholding it. Additionally, you have not provided information that explains a relevant public interest under the FOIA in the disclosure of this personal information and we have determined that the disclosure of this information would shed little or no light on the performance of the agency’s statutory duties. Because the harm to personal privacy is greater than whatever public interest may be served by disclosure, release of the information would constitute a clearly unwarranted invasion of the privacy of these individuals, and we are withholding the information under Exemption 6.

Please note that some pages contain redactions pursuant to more than one exemption. Judy Cearley, Government Information Specialist, is responsible for this partial denial. Jonathan Fleshner, with the U.S. Department of the Interior Office of the Solicitor, was consulted on this Ms. Emily Anne Kopp 4
response. The Defense Advanced Research Projects Agency, a component of the U.S. Department of Defense, was also consulted regarding its equities in the responsive records.
On November 1, 2022, we approved your request for a fee waiver. Therefore, there is no billable fee for the processing of this request.
You may appeal this response to the Department of the Interior’s FOIA/Privacy Act Appeals Officer. If you choose to appeal, the FOIA/Privacy Act Appeals Officer must receive your FOIA appeal no later than 90 workdays from the date of this letter. Appeals arriving or delivered after 5 p.m. Eastern Time, Monday through Friday, will be deemed received on the next workday.
Your appeal must be made in writing. You may submit your appeal and accompanying materials to the FOIA/Privacy Act Appeals Officer by mail, courier service, fax, or email. All communications concerning your appeal should be clearly marked with the words: "FREEDOM OF INFORMATION APPEAL." You must include an explanation of why you believe the USGS’s response is in error. You must also include with your appeal copies of all correspondence between you and USGS concerning your FOIA request, including your original FOIA request and USGS's response. Failure to include with your appeal all correspondence between you and USGS will result in the Department's rejection of your appeal, unless the FOIA/Privacy Act Appeals Officer determines (in the FOIA/Privacy Act Appeals Officer’s sole discretion) that good cause exists to accept the defective appeal.
Please include your name and daytime telephone number (or the name and telephone number of an appropriate contact), email address and fax number (if available) in case the FOIA/Privacy Act Appeals Officer needs additional information or clarification of your appeal.
DOI FOIA/Privacy Act Appeals Office Contact Information
Department of the Interior
Office of the Solicitor
1849 C Street, Northwest
MS-6556 MIB
Washington, DC 20240
Attn: FOIA/Privacy Act Appeals Office Telephone: (202) 208-5339
Fax: (202) 208-6677
Email: FOIA.Appeals@sol.doi.gov
The 2007 FOIA amendments created the Office of Government Information Services (OGIS) to

offer mediation services to resolve disputes between FOIA requesters and Federal agencies as a non-exclusive alternative to litigation. Using OGIS services does not affect your right to pursue litigation. You may contact OGIS in any of the following ways:
Office of Government Information Services National Archives and Records Administration 8601 Adelphi Road – OGIS
Ms. Emily Anne Kopp 5
College Park, Maryland 20740-6001 Telephone: (202) 741-5770
Fax: (202) 741-5769
Toll-free: 1-877-684-6448
E-mail: ogis@nara.gov
Web: https://www.archives.gov/ogis
Please note that using OGIS services does not affect the timing of filing an appeal with the Department’s FOIA & Privacy Act Appeals Officer. Contact information for the Department’s FOIA Public Liaison, who you may also seek dispute resolution services from, is available at https://www.doi.gov/foia/foiacenters.
This completes our response to your request. If you have any questions about our response to your request, please contact me by phone at (650) 329-4035 or by email at foia@usgs.gov.
Sincerely,
Judy Cearley
U.S. Geological Survey Government Information Specialist
Enclosure:
2021-006245 - Combined Records_Redacted.pdf (1,412 pages)

10/8/21, 11:00 AM
Mail - Richgels, Katherine L - Outlook
Re: Form 9-3090 - EcoHealth Alliance
Meicher, Lisa K <lmeicher@usgs.gov>
Tue 5/29/2018 1:47 PM
To: Richgels, Katherine L <krichgels@usgs.gov>
Wow! That was quick. I will get the remaining signatures too.
Lisa K. Meicher
Budget Analyst
USGS National Wildlife Health Center 6006 Schroeder Rd
Madison, WI 53711
608-270-2410
fax 608-270-2415 lmeicher@usgs.gov
On Tue, May 29, 2018 at 1:42 PM, Richgels, Katherine <krichgels@usgs.gov> wrote:
Ok, great. We need this form to then go to Jonathan and on to Leon (I believe). Can you usher it through the process for me?
Thanks, Katie
On Tue, May 29, 2018 at 1:40 PM, Ethics Office, GS-O <ethicsoffice@usgs.gov> wrote: Hello,
Please find the signed form attached.
Please let me know if you need anything else.
Thanks, Liz
USGS Ethics Office ~~~~~~~~~~~~
U.S. Geological Survey
https://outlook.office365.com/mail/id/AAMkADhiNzQ4MTAyLWU2OWYtNDZiMi04YWQ4LTkwMjYxMjIwZDU3MwBGAAAAAADUZu9K9h4rQJeL%2FM4BbYGwBwAbOFsLP%2BOFSZSmnCBcAmWVAA... 1/2

10/8/21, 11:00 AM Mail - Richgels, Katherine L - Outlook
12210 Sunrise Valley Drive, MS 603
Reston, VA 20192
EthicsOffice@usgs.gov
USGS Ethics Office website: www2.usgs.gov/quality_integrity/ethics
On Tue, May 29, 2018 at 2:20 PM, Meicher, Lisa <lmeicher@usgs.gov> wrote: Please review and sign the attached Form 9-3090.
Thanks! Lisa
Lisa K. Meicher
Budget Analyst
USGS National Wildlife Health Center 6006 Schroeder Rd
Madison, WI 53711
608-270-2410
fax 608-270-2415 lmeicher@usgs.gov
--
Katherine L. D. Richgels, Ph.D.
Branch Chief, Applied Wildlife Health Research Responsible Official, Federal Select Agent Program USGS National Wildlife Health Center
6006 Schroeder Rd
Madison, WI 53711
(608) 270 - 2450 (office)
(608) 381 - 2492 (cell)
(608) 270 - 2415 (fax)
krichgels@usgs.gov
www.nwhc.usgs.gov
https://outlook.office365.com/mail/id/AAMkADhiNzQ4MTAyLWU2OWYtNDZiMi04YWQ4LTkwMjYxMjIwZDU3MwBGAAAAAADUZu9K9h4rQJeL%2FM4BbYGwBwAbOFsLP%2BOFSZSmnCBcAmWVAA... 2/2













10/8/21, 10:59 AM
Mail - Richgels, Katherine L - Outlook
Form 9-3090 - EcoHealth Alliance
Meicher, Lisa K <lmeicher@usgs.gov>
Tue 5/29/2018 1:20 PM
To: Ethics Office, GS-O <ethicsoffice@usgs.gov> Cc: Richgels, Katherine L <krichgels@usgs.gov>
Please review and sign the attached Form 9-3090.
Thanks! Lisa
Lisa K. Meicher
Budget Analyst
USGS National Wildlife Health Center 6006 Schroeder Rd
Madison, WI 53711
608-270-2410
fax 608-270-2415 lmeicher@usgs.gov
https://outlook.office365.com/mail/id/AAMkADhiNzQ4MTAyLWU2OWYtNDZiMi04YWQ4LTkwMjYxMjIwZDU3MwBGAAAAAADUZu9K9h4rQJeL%2FM4BbYGwBwAbOFsLP%2BOFSZSmnCBcAmWVAA... 1/1













10/5/21, 4:41 PM Mail - Rocke, Tonie E - Outlook
..also want to add my thanks for your help geng this together Tonie!
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/3
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10/5/21, 4:41 PM
Mail - Rocke, Tonie E - Outlook
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4474
www.ecohealthalliance.org @PeterDaszak @EcoHealthNYC
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
From: Luke Hamel [mailto:hamel@ecohealthalliance.org]
Sent: Wednesday, March 28, 2018 1:00 PM
To: Rocke, Tonie
Cc: Peter Daszak; Alison Andre; Rachel Abbott; Richgels, Katherine Subject: DEFUSE documents as submitted
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 2/3

10/5/21, 4:41 PM
Mail - Rocke, Tonie E - Outlook
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct) (b) (6) (mobile)
www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics and promote conservation.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 3/3
(b) (6)
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.yadretsey dettimbus sa ,lasoporp
ESUFED ruo fo snoisrev lanif eht era yehT .uoy ot selif eseht dnes ot em deksa dah reteP
vog.sgsu@ekcort
1542-072-806
11735 IW ,nosidaM
.dR redeorhcS 6006
retneC htlaeH efildliW lanoitaN SGSU
ekcoR .E einoT
--
vog.sgsu@ekcort
1542-072-806
11735 IW ,nosidaM
.dR redeorhcS 6006
retneC htlaeH efildliW lanoitaN SGSU
ekcoR .E einoT
--
,einoT iH

10/8/21, 10:49 AM Mail - Richgels, Katherine L - Outlook
Fwd: [EXTERNAL] DEFUSE documents as submitted
Richgels, Katherine L <krichgels@usgs.gov>
Wed 3/28/2018 1:10 PM
To: Sleeman, Jonathan M <jsleeman@usgs.gov>; Center Director, GS-MWA-NWHC <nwhc_director@usgs.gov> 4 attachments (7 MB)
PREEMPT Volume 1 no ESS HR001118S0017 EcoHealthAlliance DEFUSE.pdf; Executive Slide HR001118S0017 EcoHealthAlliance DEFUSE.pptx; NWHC budget packet HR001118S0017 EcoHealthAlliance DEFUSE.xlsx; NWHC budget Justification HR001118S0017 EcoHealthAlliance DEFUSE.pdf;
FYI Tonie has submitted the PREEMPT grant. Katie
Sent from my Verizon, Samsung Galaxy smartphone
-------- Original message --------
From: "Rocke, Tonie" <trocke@usgs.gov>
Date: 3/28/18 2:07 PM (GMT-05:00)
To: "Richgels, Katherine" <krichgels@usgs.gov>
Subject: Fwd: [EXTERNAL] DEFUSE documents as submitted
---------- Forwarded message ----------
From: Luke Hamel <hamel@ecohealthalliance.org>
Date: Wed, Mar 28, 2018 at 11:59 AM
Subject: [EXTERNAL] DEFUSE documents as submitted
To: "Rocke, Tonie" <trocke@usgs.gov>
Cc: "Dr. Peter Daszak" <daszak@ecohealthalliance.org>, Alison Andre <andre@ecohealthalliance.org>, Rachel Abbott <rabbott@usgs.gov>, "Richgels, Katherine" <krichgels@usgs.gov>
Hi Tonie,
https://outlook.office365.com/mail/id/AAMkADhiNzQ4MTAyLWU2OWYtNDZiMi04YWQ4LTkwMjYxMjIwZDU3MwBGAAAAAADUZu9K9h4rQJeL%2FM4BbYGwBwAbOFsLP%2BOFSZSmnCBcAmWVAA... 1/2

10/8/21, 10:49 AM Mail - Richgels, Katherine L - Outlook
Peter had asked me to send these files to you. They are the final versions of our DEFUSE proposal, as submitted yesterday.
Attached files include:
Technical and Management Proposal (Vol. I) Executive Summary Slide
NWHC budget packet
NWHC budget justification
Best,
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(b) (6) (direct) (b) (6) (mobile) www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics and promote conservation.
--
Tonie E. Rocke
USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
608-270-2451
trocke@usgs.gov
https://outlook.office365.com/mail/id/AAMkADhiNzQ4MTAyLWU2OWYtNDZiMi04YWQ4LTkwMjYxMjIwZDU3MwBGAAAAAADUZu9K9h4rQJeL%2FM4BbYGwBwAbOFsLP%2BOFSZSmnCBcAmWVAA... 2/2

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c)) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

(b) (3) (B): 41 U.S.C. § 4702 (b)-(c), (b) (4), (b) (6)

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10/5/21, 2:40 PM Mail - Rocke, Tonie E - Outlook
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Project Overview
• PARC developed a unique spray technology for large area and high throughput aerosol delivery of highly viscous and concentrated fluids. These fluids can include a range of solutions, e.g., bioactive formulations. This technology has a potential application in large area inoculation of animals/humans with bioengineered formulations for pre-emptive reduction of disease transfer.
• PARC has expertise in fluid/aerosol delivery, leveraging the unique spray method that can aerosolize fluids independent of viscosity or bioactive concentration. This technique enables partners in the biological space to deliver bioactive formulations to animal models with improved chance of efficacy/bioavailability. Potential technical challenges to overcome will be systems integration with rapid development/preparation of pre-emptive agents (potentially with on- demand concentration and composition) and in testing the biological response with animal models.
• PARC can have significant involvement in Technical Area 2 of a PRE-EMPT project: development of a scalable aerosol delivery method for wide-scale inoculation of animal models.
Teaming Overview and Objectives
• PARC has worked with both commercial and university partners for applications of this technology.
• PARC has expertise in fluid delivery, droplet generation, and device and systems integration drawing on our long history with developing printing systems (ink-on-paper). PARC will leverage both previous and on-going work and our related IP portfolio on fluid delivery using platform technologies (spray, transdermal delivery) to meet the PRE-EMPT program objectives.
• PARC has the institutional assets to develop and fabricate new systems for spraying, as well as the background to help improve spray formulation for uptake in mucosal and other targeted membranes.
• PARC is well-positioned to advance its unique spray technology for the PRE-EMPT program, given its demonstrated scalability and wide applicability across different fluids (ranging from low to very high viscosity and independent of bioactive concentration/loading). PARC is looking for collaborators who will investigate disease transmission across animal species and develop the necessary pre-emptive biologicals to prevent such transmission. These engineered biologicals can then be delivered to animal models using the spray technology with maximum chance for efficacy and bioavailability.
Contact Information
Dr. Jerome Unidad; email: Jerome.Unidad@parc.com; telephone: 650-812-4209
3333 Coyote Hill Road Palo Alto, CA 94304 USA +1 650 812 4000 engage@parc.com www.parc.com

10/5/21, 2:41 PM Mail - Rocke, Tonie E - Outlook
Dear All,
I’ve aached a first rough dra of the DARPA abstract. Apologies for the delay. Unfortunately, edits to my Science paper came through on Friday and took many hours to do, so this delayed me. I’m right now in Geneva in my hotel at 3 am finishing these off before flying back to NYC from a WHO meeng.
Some important points:
1)
2)
3)
4) 5)
6)
Zhengli, Linfa, Ralph – Billy and I spoke with Tonie Rocke on Friday. Tonie is at the Naonal Wildlife Health Center, Madison USA, and has worked on wildlife vaccines: plague in prairie dogs, rabies in Jamaican fruit bats, white nose syndrome in US bats. We needed someone with experse in delivery of molecules/vaccines to wildlife because DARPA specifically lay that out. As you’ll see, Tonie is perfect for our project and will be able to do work at USGS NWHC and with Zhengli in China to help with TA2
Zhengli and Linfa – Aer I spoke with you both, I had a great conversaon with Ralph Baric. He proposed to work on recombinant chimeric spike proteins as a second line of aack. I think that is a perfect fit because 1) it’s his experse and he has published on it, 2) it will act as an alternave to the blue-sky and risky immune boosng work that Linfa/Peng have proposed. I hope you agree!
Ralph, Zhengli, Linfa, Tonie – as you can see, I have mangled the language/technical details for most of your secons. Pardon my lack of knowledge, and please dra a couple of paragraphs each to make your secons look correct. Thanks to Peng for giving me some text anyway – very useful, but please check what I’ve done with it.
All – please add some names and details on the team part so we get clarity in this on what staff you will need to do the work.
Please don’t worry about keeping this to the 8 page limit. Just add text here and there, references, and edit to make what I’ve wrien correct, and more excing. I will work on this on Saturday, Sunday and Monday to bring it down to 8 pages of very crisp, super-excing text. I also want as many of your good ideas in here, so that I can use this dra to build on for the full proposal.
Finally – please edit rapidly using tracked changes in word. If you don’t want to mess up endnote, please just insert references as comment boxes and we’ll pull them off the web.
Aleksei and Anna: please read the dra and work on some dra image designs that sum up the project flow. I’ll call you Thursday aernoon to discuss so you can finish them off.
Luke – please have a go at a first dra of the execuve summary slide. I’ll pick up from what you’ve done once you send it to me.
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10/5/21, 2:41 PM Mail - Rocke, Tonie E - Outlook
Thanks again to all of you for agreeing to collaborate on this proposal. From what I know of the compeon, what DARPA wants, and what we’re offering, I think we have an extremely strong team, so I’m looking forward to geng the full proposal together and winning this contract!
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
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DARPA – PREEMPT – HR001118S0017
Abstract Submission Requirements:
**8 pages with 12 point font or higher (smaller font may be used for figures, tables
and charts)
**Page limit includes all figures, tables, charts and the Executive Summary Slide **Copies of all documents submitted must be clearly labeled with the following:
-DARPA BAA number
-Proposer Organization
-Proposal title/Proposal short title
-Submission letter is optional and does not count towards page limit
A. Cover Sheet (does not count towards page limit):
Include the administrative and technical points of contact (name, address, phone, fax, email, lead organization). Also include the BAA number, title of the proposed project, primary subcontractors, estimated cost, duration of project, and the label “ABSTRACT.”
B. Executive Summary Slide:
Provide a one slide summary in PowerPoint that effectively and succinctly conveys the main objective, key innovations, expected impact, and other unique aspects of the proposed project. Use the slide template provided at http://www.fbo.gov.
**See slide template at bottom of document.
Clearly describe what is being proposed and what difference it will make (qualitatively and quantitatively), including brief answers to the following questions:
1. What is the proposed work attempting to accomplish or do?
We aim to defuse the potential for emergence of novel bat-origin high-zoonotic risk SARS-related coronaviruses in Southeast Asia. We envisage a scenario whereby the US warfighter is called on to intervene in a security hotspot in SE Asia for a period of 3-6 months. As planners begin choosing sites for the mission, they will use an app we will design to assess the background risk of a site harboring dangerous zoonotic viruses. If
PROJECT DEFUSE
C. Goals and Impact:

there is no alternative to a high-risk site, a tactical forward team will deploy automated delivery technology we will develop in caves that harbor bats carrying these viruses. These devices will release immune boosting molecules and chimeric polyvalent spike protein immune priming inocula to lower viral shedding from bats at the site for a few weeks or months, allowing our warfighters to execute the operation at lowered risk for spillover.
2. How is it done today? And what are the limitations?
Currently, there is no available technology to reduce the risk of exposure to novel coronaviruses from bats, other than avoid the regions where bats harbor these viruses. This includes large areas of southeast Asia where SARS-related CoVs are endemic in bats, which roost in caves during the day, but forage over wide areas at night, shedding virus in their feces and urine. The limitations of this lack of capacity are significant – we have shown evidence of recent spillover of SARS-related CoVs into people in southern China, and have identified viruses in this region that are capable of producing SARS-like illness in humanized mice, with no available vaccines or countermeasures. These viruses are a clear-and-present danger to our military personnel, and to global health security.
3. What is innovative in your approach and how does it compare to current practice and state-of-the-art (SOA)?
**Note: DARPA wants to know, “how the proposed project is revolutionary and how it significantly rises above the current state of the art
Our group has shown that bats harbor the highest proportion of potential zoonoses of any mammal group, and that they are able to live with high viral loads due to unique damping of their immune systems, likely as an evolutionary adaptation to flight. We will use this to design strategies to upregulate their immune response in their cave roosts, down-regulate viral replication, and reduce the risk of viral shedding and spillover (immune boosting strategy). At the same time, we will inoculate bats with novel chimeric polyvalent recombinant spike proteins to enhance their immune response against replication of specific, high-risk viruses (immune priming strategy). We will use our innovative modeling to design apps that identify the likelihood of any region harboring high-risk bat viruses. We will design novel, automated approaches to deliver both types of inoculum remotely into caves to reduce exposure risk during decontamination.
4. What are the key technical challenges in your approach and how do you plan to overcome these?
Decide which of following parts to talk about:

Modeling bat suitability
Inventory of caves
Sampling/testing
Reverse engineering, binding assays, mouse expts Modeling viral risk of evolution and spillover Modeling inoculation/defusing strategy
Immune modulation
Immune Booster recombinant production Gain-of-function issue.
Inoculum delivery
Mesocosm expts
Cave expts
5. Who will care and what will the impact be if you are successful?
This will have direct relevance to the warfighter. The potential for deployment to the region in which bat hosts of SARS-related CoVs exist is high – countries include security hotspots (Myanmar, Bangladesh, Pakistan, Lao, Korea). The ability to decontaminate and defuse these viruses will be useful in preventing potentially devastating illness. Furthermore, these technologies, if successful, can be adapted to hosts of other bat- origin CoVs (MERS, SADS), and potentially other zoonotic bat-origin viruses (Hendra, Nipah, EBOV). Finally, our approach is directly applicable to public health measures in the region to reduce the risk of spillover into the general population, as well as for food security by reducing the risk of viruses like SADS-CoV spilling over from bats into intensive pig farms, and devastating and industry, leading to potential civil unrest.
6. How much will it cost and how long will it take?
Will insert this later after calculating and brainstorming.
D. Technical Plan:
Outline and address all technical challenges inherent in the approach and possible solutions for overcoming potential problems. This section should provide appropriate specific milestones (quantitative, if possible) at intermediate stages of the project to demonstrate progress and a brief plan for accomplishment of the milestones. **Note: “The technical plan should demonstrate a deep understanding of the
technical challenges and present a credible (even if risky) plan to achieve
the program goal”
Key Terms/Aspects to Emphasize in Abstract
Commented [PD1]: Check on the duration of PREEMPT
46 months

● IACUC/IRB
○ DARPA wants to know who has experience w/ ACURO IACUC work.
■ EHA has multiple ACURO IACUC proposals (either approved or undergoing approval)
■ IRB also in place, just has to be modified
Rationale for the SE Asian SARS-related CoV – Rhinolophus bat target system, and immune priming/boosting: 1) Our group has shown that bats harbor a higher proportion of potentially zoonotic viruses than any other mammalian group (1), so that proof-of- concept for blocking viral spillover from this host group may lead to a bigger impact on global health security; 2) The Rhinolophus bats that harbor SARS like-CoVs are insectivorous and roost in dense colonies at a fixed, known location, yet disperse each night over wide distances from these sites. Defusing the risk of viral shedding in the roost will also defuse the risk of viral shedding over the population range. This would be difficult for rodent or primate reservoirs; 3) Bats are mammalian hosts, therefore immune modulating drugs trialed out in people may also work on bats. This would be less likely for an insect vector; 4) Members of our collaborative group has worked together on bats and their viruses for over 15 years, with a total of >100 yrs experience focused on bat-origin zoonoses among the key personnel. We have published much of the seminal work on the bat origins of SARS, Nipah, Hendra, and MERS viruses, and have opened new boundaries in studies of bat host-viral relationships ecologically, immunologically and virologically; 5) The South and Southeast Asian region where these bats occur is a security hotspot, with active political and ethnic conflicts, and displaced populations in Bangladesh, Pakistan, Myanmar, Thailand, Indonesia, Philippines and other countries. This is a likely potential site for US warfighter deployment; 6) We have worked for over 10 years on the SARS-related CoV – Rhinolophus bat system in China, demonstrating the origin of SARS-CoV within this host, the presence of SARSr-CoVs with remarkable sequence identity in the spike protein to SARS-CoV, their isolation and characterization of their ability to bind with human cells. We have demonstrated that chimeric SARS-CoV backbone with spike protein from SARSr-CoVs from our cave sites in Yunnan Province can infect a humanized mouse model and cause SARS-like illness, and that clinical signs are not reduced with SARS monoclonal therapy or vaccination. Finally, we have demonstrated that people living up to 6 kilometers from our cave site have evidence of SARSr-CoV antibodies (3% seroprevalence in 200+ cohort), suggesting active spillover, and marking these viruses as a clear-and-present danger of a new SARS-like pandemic; 7) SARSr-CoVs are transmitted among bats via fecal-oral route, making
Commented [PD2]: I know this is too long. I’ll edit later this weekend, but want to keep this text for the full proposal
Overview

sampling relatively easy (collection of fresh fecal pellets) and molecule or vaccine approaches feasible; 8) Proof-of-concept in this system may be rapidly scalable to other bat-coronavirus systems, e.g. MERS-CoV, SADS-CoV, and to other cave bat origin viruses.
Other important bat-origin zoonotic viruses (e.g. filoviruses, henipaviruses) have very rare spillover events - usually to a single index case, which makes validated prevention of spillover challenging. These viruses also show little strain diversity which makes modeling which evolutionary lines will be more high-risk, a challenge. SARSr-CoVs are diverse, with recombinants regularly identified in the field and lab. Furthermore, we have identified a single cave in Yunnan that harbors every gene from the SARS-CoV in a diversity of SARSr-CoVs within the bat population, making it an ideal evolutionary soup to target for intervention.
Finally, we believe that alternative approaches to transmission blocking, e.g. CRISPER-Cas are likely to be far less effective in bats because most bats are long-lived relative to their small size, with long inter-generational periods (2-5 years). Gene drives would likely take many decades to run through a population, so that proof-of-concept of transmission blocking in the DARPA time scale wouldn’t be possible. Furthermore, many bat species’ populations mix readily or migrate which would disperse the impact of gene drives, whereas targeting a small number of caves in a region for molecule or vaccine delivery would cover a very large dispersal area.
TA1: Develop and validate integrated, multiscale models that quantify the likelihood a human-capable virus will emerge from an animal reservoir residing in a “hot spot” geographic region
The DEFUSE modeling and analytics team will develop models to evaluate the likelihood of bat caves harboring high-risk SARSr-CoVs, evaluate the probability of specific SARS- related CoV spillover, and identify the most effective strategy for inoculation of immune boosting molecules and chimeric spike protein immune priming inocula.
We will collect specific data to inform our model building, validate assumptions and refine predictions. At the start of Yr 1, we will conduct a full inventory of host and virus distribution within our field sites, two caves in Yunnan Province, China. This builds on 8 years of surveillance in these caves and includes a cave in which we have identified all the genetic components of SARS-CoV distributed across a bat population. Two other caves will act as controls/comparison sites, in that we have not yet identified the high- risk SARSr-CoVs in that cave. We will assess: the population density, distribution and segregation of individual bats; changes in these daily, weekly and by season; viral prevalence and intensity in individuals; distribution of low- and high-risk SARSr-CoV strains, and how readily these are transmitted among bat species, age classes, genders; and using mark-recapture to assess metapopulation structure. To assess geographic

distribution of bat hosts, we have access to biological inventory data on all bat caves in Southern China, as well as information on species distributions across SE Asia from the literature and museum records. We will use radio- and satellite telemetry to identify the home range of each species of bat in the caves, to assess how widely the viral ‘plume’ could contaminate
We will build environmental niche models using the data above, and environmental and ecological correlates, and traits of cave species communities (eg. phylogenetic and functional diversity), to predict the species composition of bat caves across Southern China, South and SE Asia. We will validate these with data from the current project and data from PREDICT sampling in Thailand, Indonesia, Malaysia and other SE Asian countries. We will then use our unique database of bat host-viral relationships updated from our recent Nature paper (1) to assess the likelihood of low- or high-risk SARSr-CoVs being present in a cave at any site across the region. At the end of Yr 1, we will use these analyses to produce a prototype app for the warfighter that identifies the likelihood of bats harboring dangerous viral pathogens based on these analyses. The ‘high-risk bats near me’ app will be updated as new host-viral surveillance data comes on line from our project and others, to ground-truth and fine- tune its predictive capacity. Specifically, our telemetry data on bat movement will be used to assess how often bats from high-risk caves migrate to other colonies and potentially spread their high-risk strains.
The Wuhan Institute of Virology team will conduct viral testing on samples from all bat species in the caves as part of this inventory. Fecal, oral, blood and urogenital samples will be collected from bats using standard capture techniques as we have done for the last decade. In addition, tarps will be laid down in caves to assess the feasibility of surveys using pooled fresh fecal and urine samples. Assays will be designed to correlate viral load in an individual with viral shedding in a fecal sample. Once this is complete, surveys will continue largely on fecal samples so as not to disturb bat colonies and undermine longitudinal sampling capacity. Samples will be tested by PCR and spike proteins of all SARS-related CoVs sequenced. Analyses of phylogeny, recombination events, and further characterization of high-risk viruses (those with spike proteins close to SARS-CoV) will be carried out (REF). Isolation will be attempted on a subset of samples with novel SARSr-CoVs. will reverse engineer spike proteins in his lab to conduct binding assays to human ACE2 (the SARS-CoV receptor). Proteins that bind will then be inserted into SARS-CoV backbones, and inoculated into humanized mice to assess their capacity to cause SARS-like disease, and their ability to be blocked by monoclonal therapies, or vaccines against SARS-CoV (REF).
The modeling team will use these data to build models of 1) risk of viral
surrounding regions, and therefore how wide the risk zone is for the
warfighter positioned close to bat caves.
Commented [PD3]: Could add “ We will continue monitoring the human population proximal to these caves to assess the rates of viral spillover, and ground- truth which specific CoVs are able to infect people
Prof. Ralph Baric, UNC,
Commented [PD4]: Ralph, Zhengli. If we win this contract, I do not propose that all of this work will necessarily be conducted by Ralph, but I do want to stress the US side of this proposal so that DARPA are comfortable with our team. Once we get the funds, we can then allocate who does what exact work, and I believe that a lot of these assays can be done in Wuhan as well...

evolution and spillover, and 2) strategies to maximize inoculation strategy.
Data on the diversity of bat spike proteins, prevalence of recombinant CoVs, ability to bind and infect human cells, degree of clinical signs in mouse models, will be used to estimate evolutionary rates, rates of recombination, and capacity to generate novel strains capable of human infection. Using dynamic metapopulation models, we will estimate the flow of genes within each bat cave, based on the known host and viral assemblages. This will inform how rapidly new CoV strains with distinct phenotypic characteristics evolve. Because of our unique collaboration among world-class modelers, and coronavirologists, we will be able to test model predictions of viral capacity for spillover by conducting spike protein-based binding and cell culture experiments. The BSL-2 nature of work on SARSr-CoVs makes our system highly cost- effective relative to other bat-virus systems (e.g. Ebola, Marburg, Hendra, Nipah), which require BSL-4 level facilities for cell culture.
We will use modeling approaches, the data above, and other biological and ecological data to estimate how rapidly high-risk SARSr-CoVs will re-colonize a bat population following immune boosting or priming. We will obtain model estimates of the frequency of inoculation required for both approaches, what proportion of a population needs to be reached to have effective viral dampening, and whether specific seasons, or locations within a cave would be more effective to treat. We will then model the efficacy of different delivery methods (spray, swab, cave mouth automated delivery, deliver to specific sections of a cave).
TA2: Develop scalable approaches that target and suppress the animal virus in its reservoir(s) and/or vector(s), to reduce the likelihood of virus transmission into humans.
Our goal is to use two approaches to defuse the potential for SARS-related CoVs to emerge in people: 1) Immune Boosting: using the unique immunological features of bats that our group has discovered, we will inoculate live bats in cave mesocosms with immune modulators to up-regulate their naïve immunity to suppress viral replication and shedding; 2) Immune Priming: building on preliminary development of polyvalent chimeric recombinant molecules targeting diverse spike proteins from bat SARS-related CoVs, we will produce, and trial inoculation of live bats to suppress the replication and shedding of a broad range of dangerous SARS-related CoVs. Both lines of work will begin in Yr 1 and run parallel throughout the project.
Prof. Linfa Wang (Duke-NUS) will lead the work on immune boosting work, building on his pioneering work on bat immunity (2). This work provides evidence that that the long-term coexistence of bats and their viruses has led to an equilibrium between viral replication and host immunity, whereby bats have specifically down-

regulated their innate immune system as part of the fitness cost of flight (the only true flying mammals) (2). The nature of the weakened but not entirely lost functionality of bat innate immunity factors like STING, a central DNA-interferon (IFN) sensing molecule, may have profound impact for bats to maintain the balanced state of “effective response”, but not “over response” against viruses (3). A similar finding was also observed in bat IFNA studies, which is less abundant but was constitutively expressed without stimulation (4). Given native levels of SARSr-CoVs in individual bats with damped immunity, we propose to suppress bat SARSr-CoV by boosting bat innate immunity through the IFN pathway, and breaking the natural host-virus equilibrium. One of the potential problems with this approach is that it can lead to severe inflammation. However, this is unlikely to occur in bats, because they also have a naturally dampened inflammation response (5).
Previous work has shown that aerosol spraying or intranasal inoculation of IFN or other small molecules has led to reduce viral loads in humans, ferrets and mouse models (12-14). We will therefore initially trial inoculation of live bats with synthetic double-stranded RNA (Poly I:C) and assay for reduced viral loads (DETAILS, CITATION). We will generate universal bat interferon and apply to bats in the lab. Interferon has been used extensively clinically if no viral-specific drugs are available, e.g. against filoviruses (11). Secondly, bat replication of SARSr-CoV is sensitive to interferon treatments, as has been shown in our previous work (12). We will attempt to boost bat IFN by blocking bat-specific IFN negative regulator. Bat IFNA is naturally constitutively expressed but cannot be induced to a high level (4). This is unique to bats. We think there should be a negative regulatory factor in the bat interferon production pathway. We propose using CRISPRi to find out that negative regulator and then screen for chemicals targeting at this gene. We will attempt to boost bat IFN by activating dampened bat-specific IFN production pathways which include DNA-STING-dependent and ssRNA-TLR7 dependent pathways. These changes have been proved to bat-specific, suggesting that they are important in viruses/bats coexistence, and supported by our own work showing that a mutant bat STING restores antiviral functionality (3). By identifying small molecules to directly activate downstream of STING, we have chance to activate bat interferon and then help bats to clear viruses. Similar strategy applies to ssRNA-TLR7 dependent pathways. We will also attempt to boost bat IFN by activating functional bat IFN production pathways. We will investigate if there are other IFN production pathways in bats. We then boost bat immune responses by ligands specifically to these pathways, e.g. polyIC to TLR3-IFN pathway or 5’ppp-dsRNA to RIG-I- IFN pathway. A similar strategy has been tested successful in mouse model for SARS- CoV, IAV or HBV (6, 7). We believe treating wild bats with IFN-modulating small molecules by spraying is superior to other invasive strategies that might be considered

by DARPA, including genome editing (CRISPR or RNAi), vaccination or DIP bats, in terms of its deployability and scalability. Finally, we will inoculate bats with fragments of non- bat Coronavirus (DETAILS).
Prof. Ralph Baric (UNC) will lead the immune priming work, building on his track record in reverse-engineering and manipulating SARS-CoV, MERS-CoV and other virus spike proteins over the last two decades . He will develop recombinant chimeric spike- proteins (8) based on SARSr-CoVs we have already identified, and those we will discover and characterize during project DEFUSE.
please!
While there are clear advantages to working with fixed populations of cave-
dwelling bats, molecule or vaccine delivery is technically challenging. Dr. Tonie Rocke, who developed, trialed, field-tested and rolled out the prairie dog plague vaccine (9), and is currently working on vaccines to bat rabies (10, 11) and white-nose syndrome, will manage a series of experiments in the lab and field to perfect a delivery system for both arms of TA2.
We will conduct initial experiments on a lab colony of wild-caught Rhinolophus sinicus bats at Wuhan Institute of Zoology. We (Prof. Wang) have previous experience conducting infection experiments on this bat genus ...(details and citation if possible). First, we will use our recently proven technology to design LIPS assays to the specific high zoonotic-risk SARSr-CoVs (12). We will conduct serological analysis on bats captured for infection experiments, to assess prior exposure to specific strains. These LIPS assays will be made available for use in people to assess exposure of the general population around bat caves in China, and for potential use by the warfighter to assess exposure to SARSr-CoVs during combat missions.
Finally, work on a delivery method will be overseen by Dr. Tonie Rocke at the National Wildlife Health Center who has proven capacity to develop and take animal vaccines through to licensure (9). Using her captive Jamaican fruitbat colony (10, 11), Dr. Rocke will trial out the following strategies for delivery of the molecules, inocula proposed above: 1) aerosolization; 2) transdermally applied nanoparticles; 3) sticky edible spray that bats will groom from each other; 4) automated spray triggered by timers and movement detectors at critical cave entry points.. (Details and ideas please Tonie!). These approaches will then be trialed out on live bats in our three cave sites in Yunnan Province. Fieldwork will be conducted under the auspices of Dr. Rocke, EHA field staff, and Dr. Yunzhi Zhang (Yunnan CDC, Consultant with EcoHealth Alliance). Sections of bat caves will be cordoned off and different application methods trialed out. A small number of bats will be captured and assayed for viral load after treatment, but so as not to disturb the colony, most viral load work will be conducted on fresh fecal pellets
RALPH – clearly I didn’t really understand the
details of your approach. Can you add a couple of paragraphs here and some citations

collected daily on the cave floor. EHA has unique access to these sites in Yunnan Province, with our field teams conducting surveillance there for around 10 years, under the guidance of Drs. Shi and Zhang. In year 1 of project DEFUSE, we will seek permission for these experimental inoculations in cave sites in Yunnan from the Provincial Forestry Department. We do not envisage problems getting permission, as we have worked with the Forestry Department collaboratively for the last few years, we have the support of the Yunnan CDC, and we are releasing molecules that are not dangerous to people or wildlife.
E. Capabilities:
A brief summary of expertise of the team, including subcontractors and key personnel. A principal investigator for the project must be identified, and a description of the team’s organization. Include a description of the team’s organization including roles and responsibilities. Describe the organizational experience in this area, existing intellectual property required to complete the project, and any specialized facilities to be used as part of the project. List Government furnished materials or data assumed to be available.
**Note: While only the proposal requires an organization chart, it may be helpful to include in the abstract if we have the space.
• This organization chart would include (as applicable): (1) the programmatic relationship of team members; (2) the unique capabilities of team members; (3) the task responsibilities of team members; (4) the teaming strategy among the team members; (5) key personnel with the amount of effort to be expended by each person during each year.
The lead institution for Project DEFUSE is EcoHealth Alliance, New York, an international research non-profit focused on emerging zoonotic diseases. The project will be led by PI Dr. Peter Daszak, who has 20+ years’ experience managing lab, field and modeling research projects on emerging zoonoses, including as EHA institutional lead, Head of Modeling and Analytics, and member of the Executive Committee for the $130 million USAID EPT/PREDICT. Dr. Daszak will oversee and coordinate all project activities, as well as lead the modeling and analytic work for TA1. Dr. Billy Karesh, who has 40+ years’ experience managing wildlife disease and zoonotic disease projects, will manage partnership activities and relationships and outreach. Dr. Jon Epstein, who has 15 years’ experience working with bats and emerging zoonoses will coordinate work on bat immune priming and boosting trials. Dr. Kevin Olival and Dr. Noam Ross will manage and conduct the modeling and analytical approaches for this project.

Team:
Lead Organization: EcoHealth Alliance, New York
PI: Peter Daszak Ph.D., President & Chief Scientist, EcoHealth Alliance, 3 months/year Key Personnel:
Billy Karesh DVM, Executive VP for Policy & Health, 1 month/year
Kevin J. Olival Ph.D, VP for Scientific Research, 1 month/year
Jonathan H. Epstein DVM Ph.D., VP for Science & Outreach, 0.5 months/year
Carlos Zambrana-Torrelio Ph.D., Assoc. VP for Conservation & Health, 1 month/year Noam Ross Ph.D., Senior Research Scientist, 2 months/year
Evan Eskew, Research Scientist, 2 months/year
Hongying Li, Program Coordinator, China Programs, 3 months/year
TBD Postdoctoral Researcher modeling and analysis, 12 months/year
TBD Research Assistant, 12 months/year
TBD Program Assistant, 12 months/year
Guangjian Zhu Ph.D., Consultant Field Lead, China Programs, 6 months/year
Yunzhi Zhang Ph.D., Consultant, Yunnan CDC, China, 2 months/year
Subcontract #1: University of North Carolina Medical School Organizational Lead: Prof. Ralph Baric Ph.D., 2 months/year XXX
TBD Research Assistant, 12 months/year
Subcontract #2: USGS National Wildlife Health Center
Organizational Lead: Tonie Rocke Ph.D., 2 months/year, no salary requested TBD Research Technician, 9 months/year
Subcontract #3: Duke NUS, Singapore
Organizational Lead: Prof. Linfa Wang Ph.D., 2 months/year XXX
TBD Research Assistant, 12 months/year
XXX
Subcontract #4: Wuhan Institute of Virology, China Organizational Lead: Prof Zhengli Shi Ph.D., 2 months/year Peng Zhou Ph.D., 2 months/year
TBD Research Assistant, 12 months/year

F. If desired, include a brief bibliography
resumes of key performers.
Commented [PD5]: I’m planning to use my resume and Ralph’s. Linfa/Zhengli, I realize your resumes are also very impressive, but I am trying to downplay the non-US focus of this proposal so that DARPA doesn’t see this as a negative.
Links to relevant papers, reports, or
Do not include more than two resumes as part of the abstract.
**Resumes count against the abstract page limit.
Dr. Peter Daszak is President and Chief Scientist of EcoHealth Alliance, a US-based organization that conducts research and outreach programs on emerging zoonotic diseases. He has published over 300 scientific papers, including the first global map of EID hotspots, strategies to estimate unknown viral diversity in wildlife, predictive models of virus-host relationships, and evidence of the bat origin of SARS-CoV and other emerging viruses. Dr Daszak is Chair of the National Academy of Sciences, Engineering and Medicine’s Forum on Microbial Threats and is a member of the Executive Committee and the EHA institutional lead for USAID-EPT-PREDICT. He serves on the NRC Advisory Committee to the USGCRP, the DHS CEEZAD External Advisory Board, and the WHO R&D Blueprint Pathogen Prioritization expert group, and has advised the Director for Medical Preparedness Policy on the White House National Security Staff on global health issues. Dr Daszak won the 2000 CSIRO medal for collaborative research.
Prof. Ralph Baric is a UNC Lineberger Comprehensive Cancer Center member and Professor in the UNC-Chapel Hill Department of Epidemiology. His work focuses on coronaviruses as models to study the genetics of RNA virus transcription, replication, persistence, and cross species transmission. His work crosses the boundaries of microbiology, virology, immunology and epidemiology, looking especially at the population genetics of viruses to find the molecular building blocks for more effective vaccines.
**General Notes:
• DARPA will evaluate proposals using the following criteria, listed in
descending order of importance:
1) 5.1.1. Overall Scientific and Technical Merit
The proposed technical approach is innovative, feasible, achievable, and complete.
Task descriptions and associated technical elements provided are complete and in a logical sequence with all proposed deliverables clearly defined such that a final outcome that achieves the goal can be expected as a result of award. The proposal identifies

major technical risks and planned mitigation efforts are clearly defined and feasible. The proposed PREEMPT Risk Mitigation Plan effectively provides the following: an assessment of potential risks; proposed guidelines to ensure maximal biosafety and biosecurity; a risk management plan for responsible communications; and a plan to address how input from the Government and community stakeholders will be considered regarding communication and publication of potentially sensitive dual-use information.
2) 5.1.2. Potential Contribution and Relevance to the DARPA Mission
The potential contributions of the proposed effort are relevant to the national technology base. Specifically, DARPA’s mission is to make pivotal early technology investments that create or prevent strategic surprise for U.S. National Security. The proposer clearly demonstrates its capability to transition the technology to the research, industrial, and/or operational military communities in such a way as to enhance U.S. defense. In
addition, the evaluation will take into consideration the extent to which the proposed intellectual property (IP) rights will potentially impact the Government’s ability to transition the technology.
3) 5.1.3. Cost Realism
The proposed costs are realistic for the technical and management approach and accurately reflect the technical goals and objectives of the solicitation. The proposed costs are consistent with the proposer's Statement of Work and reflect a sufficient understanding of the costs and level of effort needed to successfully accomplish the proposed technical approach. The costs for the prime proposer and proposed subawardees are substantiated by the details provided in the proposal (e.g., the type and number of labor hours proposed per task, the types and quantities of materials, equipment and fabrication costs, travel and any other applicable costs and the basis for the estimates).
It is expected that the effort will leverage all available relevant prior research in order to obtain the maximum benefit from the available funding. For efforts with a likelihood of commercial application, appropriate direct cost sharing may be a positive factor in the evaluation.
DARPA recognizes that undue emphasis on cost may motivate proposers to
offer low-risk ideas with minimum uncertainty and to staff the effort with junior
personnel in order to be in a more competitive posture. DARPA discourages such cost
strategies.
Commented [EA6]: Please note

Citations
2. 3.
4. 5.
6. 7.
1.
K. J. Olival et al., Host and viral traits predict zoonotic spillover from mammals. Nature 546, 646-650 (2017).
G. Zhang et al., Comparative analysis of bat genomes provides insight into the evolution of flight and immunity. Science 339, 456-460 (2013).
J. Xie et al., Dampened STING-Dependent Interferon Activation in Bats. Cell host & microbe, (2018).
P. Zhou et al., Contraction of the type I IFN locus and unusual constitutive expression of IFN-αin bats. Proceedings of the National Academy of Sciences of the United States of America, 201518240-201518246 (2016).
M. Ahn, J. Cui, A. T. Irving, L.-F. Wang, Unique Loss of the PYHIN Gene Family in Bats Amongst Mammals: Implications for Inflammasome Sensing. Scientific Reports 6, (2016).
J. Zhao et al., Intranasal Treatment with Poly(I.C) Protects Aged Mice from Lethal Respiratory Virus Infections. Journal of Virology 86, 11416-11424 (2012).
J. Wu et al., Poly(I:C) Treatment Leads to Interferon-Dependent Clearance of Hepatitis B Virus in a Hydrodynamic Injection Mouse Model. Journal of Virology 88, 10421-10431 (2014).
Attachment 1: Executive Summary Slide template

8.
9. 10.
11. 12.
X. F. Deng et al., A Chimeric Virus-Mouse Model System for Evaluating the Function and Inhibition of Papain-Like Proteases of Emerging Coronaviruses. Journal of Virology 88, 11825-11833 (2014).
T. E. Rocke et al., Sylvatic Plague Vaccine Partially Protects Prairie Dogs (Cynomys spp.) in Field Trials. Ecohealth 14, 438-450 (2017).
B. Stading et al., Protection of bats (Eptesicus fuscus) against rabies following topical or oronasal exposure to a recombinant raccoon poxvirus vaccine. Plos Neglect. Trop. Dis. 11, (2017).
B. R. Stading et al., Infectivity of attenuated poxvirus vaccine vectors and immunogenicity of a raccoonpox vectored rabies vaccine in the Brazilian Free-tailed bat (Tadarida brasiliensis). Vaccine 34, 5352-5358 (2016).
P. Zhou et al., Fatal Swine Acute Diarrhea Syndrome caused by an HKU2- related Coronavirus of Bat Origin. Nature In press, (2018
).

10/5/21, 2:43 PM Mail - Rocke, Tonie E - Outlook
See my brief notes/edits in the aached.
I am working on a large grant here in SG and won’t be able to spend too much me unl next week. LF
Linfa (Lin-Fa) WANG, PhD FTSE
Professor & Director
Programme in Emerging Infecous Disease Duke-NUS Medical School,
8 College Road, Singapore 169857
Tel: +65 6516 8397
From: Peter Daszak [mailto:daszak@ecohealthalliance.org]
Sent: Thursday, 8 February, 2018 10:51 AM
To: Ralph Baric (rbaric@email.unc.edu); Wang Linfa; Zhengli Shi (zlshi@wh.iov.cn); William B. Karesh; Rocke, Tonie
Cc: Luke Hamel; Jonathon Musser; Anna Willoughby; Kevin Olival, PhD; Jon Epstein; Noam Ross; Aleksei Chmura; Anna Willoughby; Hongying Li
Subject: First (rough) draft of the DARPA abstract - Project DEFUSE
Importance: High
Dear All,
I’ve aached a first rough dra of the DARPA abstract. Apologies for the delay. Unfortunately, edits to my Science paper came through on Friday and took many hours to do, so this delayed me. I’m right now in Geneva in my hotel at 3 am finishing these off before flying back to NYC from a WHO meeng.
Some important points:
1)
2)
Zhengli, Linfa, Ralph – Billy and I spoke with Tonie Rocke on Friday. Tonie is at the Naonal Wildlife Health Center, Madison USA, and has worked on wildlife vaccines: plague in prairie dogs, rabies in Jamaican fruit bats, white nose syndrome in US bats. We needed someone with experse in delivery of molecules/vaccines to wildlife because DARPA specifically lay that out. As you’ll see, Tonie is perfect for our project and will be able to do work at USGS NWHC and with Zhengli in China to help with TA2
Zhengli and Linfa – Aer I spoke with you both, I had a great conversaon with Ralph Baric. He proposed to work on recombinant chimeric spike proteins as a second line of aack. I think that is a perfect fit because 1) it’s his experse and he has published on it, 2) it will act as an alternave to the blue-sky and risky immune boosng work that Linfa/Peng have proposed. I hope you agree!
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/3
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10/5/21, 2:43 PM
Mail - Rocke, Tonie E - Outlook
3)
4) 5)
6)
Ralph, Zhengli, Linfa, Tonie – as you can see, I have mangled the language/technical details for most of your secons. Pardon my lack of knowledge, and please dra a couple of paragraphs each to make your secons look correct. Thanks to Peng for giving me some text anyway – very useful, but please check what I’ve done with it.
All – please add some names and details on the team part so we get clarity in this on what staff you will need to do the work.
Please don’t worry about keeping this to the 8 page limit. Just add text here and there, references, and edit to make what I’ve wrien correct, and more excing. I will work on this on Saturday, Sunday and Monday to bring it down to 8 pages of very crisp, super-excing text. I also want as many of your good ideas in here, so that I can use this dra to build on for the full proposal.
Finally – please edit rapidly using tracked changes in word. If you don’t want to mess up endnote, please just insert references as comment boxes and we’ll pull them off the web.
Aleksei and Anna: please read the dra and work on some dra image designs that sum up the project flow. I’ll call you Thursday aernoon to discuss so you can finish them off.
Luke – please have a go at a first dra of the execuve summary slide. I’ll pick up from what you’ve done once you send it to me.
Thanks again to all of you for agreeing to collaborate on this proposal. From what I know of the compeon, what DARPA wants, and what we’re offering, I think we have an extremely strong team, so I’m looking forward to geng the full proposal together and winning this contract!
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
Important: This email is confidential and may be privileged. If you are not the
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10/5/21, 2:43 PM Mail - Rocke, Tonie E - Outlook
intended recipient, please delete it and notify us immediately; you should not copy or use it for any purpose, nor disclose its contents to any other person. Thank you.
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DARPA – PREEMPT – HR001118S0017
Abstract Submission Requirements:
**8 pages with 12 point font or higher (smaller font may be used for figures, tables
and charts)
**Page limit includes all figures, tables, charts and the Executive Summary Slide **Copies of all documents submitted must be clearly labeled with the following:
-DARPA BAA number
-Proposer Organization
-Proposal title/Proposal short title
-Submission letter is optional and does not count towards page limit
A. Cover Sheet (does not count towards page limit):
Include the administrative and technical points of contact (name, address, phone, fax, email, lead organization). Also include the BAA number, title of the proposed project, primary subcontractors, estimated cost, duration of project, and the label “ABSTRACT.”
B. Executive Summary Slide:
Provide a one slide summary in PowerPoint that effectively and succinctly conveys the main objective, key innovations, expected impact, and other unique aspects of the proposed project. Use the slide template provided at http://www.fbo.gov.
**See slide template at bottom of document.
Clearly describe what is being proposed and what difference it will make (qualitatively and quantitatively), including brief answers to the following questions:
1. What is the proposed work attempting to accomplish or do?
We aim to defuse the potential for emergence of novel bat-origin high-zoonotic risk SARS-related coronaviruses in Southeast Asia. We envisage a scenario whereby the US warfighter is called on to intervene in a security hotspot in SE Asia for a period of 3-6 months. As planners begin choosing sites for the mission, they will use an app we will design to assess the background risk of a site harboring dangerous zoonotic viruses. If
PROJECT DEFUSE
C. Goals and Impact:

there is no alternative to a high-risk site, a tactical forward team will deploy automated delivery technology we will develop in caves that harbor bats carrying these viruses. These devices will release immune boosting molecules and chimeric polyvalent spike protein immune priming inocula to lower viral shedding from bats at the site for a few weeks or months, allowing our warfighters to execute the operation at lowered risk for spillover.
2. How is it done today? And what are the limitations?
, there is no available technology to reduce the risk of exposure to novel
coronaviruses from bats, other than avoid the regions where bats harbor these viruses. This includes large areas of southeast Asia where SARS-related CoVs are endemic in bats, which roost in caves during the day, but forage over wide areas at night, shedding virus in their feces and urine. The limitations of this lack of capacity are significant – we have shown evidence of recent spillover of SARS-related CoVs into people in southern China, and have identified viruses in this region that are capable of producing SARS-like illness in humanized mice, with no available vaccines or countermeasures. These viruses are a clear-and-present danger to our military personnel, and to global health security.
3. What is innovative in your approach and how does it compare to current practice and state-of-the-art (SOA)?
**Note: DARPA wants to know, “how the proposed project is revolutionary and how it significantly rises above the current state of the art
Our group has shown that bats harbor the highest proportion of potential zoonoses of any mammal group, and that they are able to live with the host without causing diseases due to unique damping of certain pathways in their immune systems, likely as an evolutionary adaptation to flight. We will use this new finding to design strategies to upregulate their immune response in their cave roosts, down-regulate viral replication, and reduce the risk of viral shedding and spillover (broad immune boosting strategy). At the same time, we will inoculate bats with novel chimeric polyvalent recombinant spike proteins to enhance their immune response against replication of specific, high-risk viruses (targeted immune priming strategy). We will use our innovative modeling to design apps that identify the likelihood of any region harboring high-risk bat viruses. We will design novel, automated approaches to deliver both types of inoculum remotely into caves to reduce exposure risk during decontamination.
4. What are the key technical challenges in your approach and how do you plan to overcome these?
Decide which of following parts to talk about:
Commented [L1]: My understanding is that the project will have two parts: A) better risk assessment and modeling and B) risk defusing.
Do we need to say anything about A here?!
Deleted: high viral loads Deleted: their
Commented [L2]: This will become important late: while we are specifically targeting SARS-realted CoVS, this strategy will be applicable to ALL bat-borne viruses in future
Currently

Modeling bat
suitability
Commented [L3]: I have highlighted the ones which are most challenging and novel for this proposal
Formatted: Highlight
Formatted: Highlight Formatted: Highlight
Formatted: Highlight Formatted: Highlight
Modeling inoculation/defusing strategy
Immune modulation
Inoculum delivery
Cave expts
46 months
Commented [PD4]: Check on the duration of PREEMPT
Inventory of caves
Sampling/testing
Reverse engineering, binding assays, mouse expts Modeling viral risk of evolution and spillover
Immune Booster recombinant production Gain-of-function issue.
Mesocosm expts
5. Who will care and what will the impact be if you are successful?
This will have direct relevance to the warfighter. The potential for deployment to the region in which bat hosts of SARS-related CoVs exist is high – countries include security hotspots (Myanmar, Bangladesh, Pakistan, Lao, Korea). The ability to decontaminate and defuse these viruses will be useful in preventing potentially devastating illness. Furthermore, these technologies, if successful, can be adapted to hosts of other bat- origin CoVs (MERS, SADS), and potentially other zoonotic bat-origin viruses (Hendra, Nipah, EBOV). Finally, our approach is directly applicable to public health measures in the region to reduce the risk of spillover into the general population, as well as for food security by reducing the risk of viruses like SADS-CoV spilling over from bats into intensive pig farms, and devastating and industry, leading to potential civil unrest.
6. How much will it cost and how long will it take?
Will insert this later after calculating and brainstorming.
D. Technical Plan:
Outline and address all technical challenges inherent in the approach and possible solutions for overcoming potential problems. This section should provide appropriate specific milestones (quantitative, if possible) at intermediate stages of the project to demonstrate progress and a brief plan for accomplishment of the milestones. **Note: “The technical plan should demonstrate a deep understanding of the
technical challenges and present a credible (even if risky) plan to achieve
the program goal”
Key Terms/Aspects to Emphasize in Abstract

● IACUC/IRB
○ DARPA wants to know who has experience w/ ACURO IACUC work.
■ EHA has multiple ACURO IACUC proposals (either approved or undergoing approval)
■ IRB also in place, just has to be modified
Rationale for the SE Asian SARS-related CoV – Rhinolophus bat target system, and immune priming/boosting: 1) Our group has shown that bats harbor a higher proportion of potentially zoonotic viruses than any other mammalian group (1), so that proof-of- concept for blocking viral spillover from this host group may lead to a bigger impact on global health security; 2) The Rhinolophus bats that harbor SARS like-CoVs are insectivorous and roost in dense colonies at a fixed, known location, yet disperse each night over wide distances from these sites. Defusing the risk of viral shedding in the roost will also defuse the risk of viral shedding over the population range. This would be difficult for rodent or primate reservoirs; 3) Bats are mammalian hosts, therefore immune modulating drugs trialed out in people may also work on bats. This would be less likely for an insect vector; 4) Members of our collaborative group has worked together on bats and their viruses for over 15 years, with a total of >100 yrs experience focused on bat-origin zoonoses among the key personnel. We have published much of the seminal work on the bat origins of SARS, Nipah, Hendra, and MERS viruses, and have opened new boundaries in studies of bat host-viral relationships ecologically, immunologically and virologically; 5) The South and Southeast Asian region where these bats occur is a security hotspot, with active political and ethnic conflicts, and displaced populations in Bangladesh, Pakistan, Myanmar, Thailand, Indonesia, Philippines and other countries. This is a likely potential site for US warfighter deployment; 6) We have worked for over 10 years on the SARS-related CoV – Rhinolophus bat system in China, demonstrating the origin of SARS-CoV within this host, the presence of SARSr-CoVs with remarkable sequence identity in the spike protein to SARS-CoV, their isolation and characterization of their ability to bind with human cells. We have demonstrated that chimeric SARS-CoV backbone with spike protein from SARSr-CoVs from our cave sites in Yunnan Province can infect a humanized mouse model and cause SARS-like illness, and that clinical signs are not reduced with SARS monoclonal therapy or vaccination. Finally, we have demonstrated that people living up to 6 kilometers from our cave site have evidence of SARSr-CoV antibodies (3% seroprevalence in 200+ cohort), suggesting active spillover, and marking these viruses as a clear-and-present danger of a new SARS-like pandemic; 7) SARSr-CoVs are transmitted among bats via fecal-oral route, making
Commented [PD5]: I know this is too long. I’ll edit later this weekend, but want to keep this text for the full proposal
Overview

Commented [L6]: We need to provide background info about bat immunity and the track record of this group in the field
Commented [L7]: Peng: I am working on an important grant here in Singapore. Can you add a few points here? Thanks
sampling relatively easy (collection of fresh fecal pellets) and molecule or vaccine approaches feasible; 8) Proof-of-concept in this system may be rapidly scalable to other bat-coronavirus systems, e.g. MERS-CoV, SADS-CoV, and to other cave bat origin viruses.
Other important bat-origin zoonotic viruses (e.g. filoviruses, henipaviruses) have very rare spillover events - usually to a single index case, which makes validated prevention of spillover challenging. These viruses also show little strain diversity which makes modeling which evolutionary lines will be more high-risk, a challenge. SARSr-CoVs are diverse, with recombinants regularly identified in the field and lab. Furthermore, we have identified a single cave in Yunnan that harbors every gene from the SARS-CoV in a diversity of SARSr-CoVs within the bat population, making it an ideal evolutionary soup to target for intervention.
Finally, we believe that alternative approaches to transmission blocking, e.g. CRISPER-Cas are likely to be far less effective in bats because most bats are long-lived relative to their small size, with long inter-generational periods (2-5 years). Gene drives would likely take many decades to run through a population, so that proof-of-concept of transmission blocking in the DARPA time scale wouldn’t be possible. Furthermore, many bat species’ populations mix readily or migrate which would disperse the impact of gene drives, whereas targeting a small number of caves in a region for molecule or vaccine delivery would cover a very large dispersal area.
TA1: Develop and validate integrated, multiscale models that quantify the likelihood a human-capable virus will emerge from an animal reservoir residing in a “hot spot” geographic region
The DEFUSE modeling and analytics team will develop models to evaluate the likelihood of bat caves harboring high-risk SARSr-CoVs, evaluate the probability of specific SARS- related CoV spillover, and identify the most effective strategy for inoculation of immune boosting molecules and chimeric spike protein immune priming inocula.
We will collect specific data to inform our model building, validate assumptions and refine predictions. At the start of Yr 1, we will conduct a full inventory of host and virus distribution within our field sites, two caves in Yunnan Province, China. This builds on 8 years of surveillance in these caves and includes a cave in which we have identified all the genetic components of SARS-CoV distributed across a bat population. Two other caves will act as controls/comparison sites, in that we have not yet identified the high- risk SARSr-CoVs in that cave. We will assess: the population density, distribution and segregation of individual bats; changes in these daily, weekly and by season; viral prevalence and intensity in individuals; distribution of low- and high-risk SARSr-CoV strains, and how readily these are transmitted among bat species, age classes, genders; and using mark-recapture to assess metapopulation structure. To assess geographic

distribution of bat hosts, we have access to biological inventory data on all bat caves in Southern China, as well as information on species distributions across SE Asia from the literature and museum records. We will use radio- and satellite telemetry to identify the home range of each species of bat in the caves, to assess how widely the viral ‘plume’ could contaminate
We will build environmental niche models using the data above, and environmental and ecological correlates, and traits of cave species communities (eg. phylogenetic and functional diversity), to predict the species composition of bat caves across Southern China, South and SE Asia. We will validate these with data from the current project and data from PREDICT sampling in Thailand, Indonesia, Malaysia and other SE Asian countries. We will then use our unique database of bat host-viral relationships updated from our recent Nature paper (1) to assess the likelihood of low- or high-risk SARSr-CoVs being present in a cave at any site across the region. At the end of Yr 1, we will use these analyses to produce a prototype app for the warfighter that identifies the likelihood of bats harboring dangerous viral pathogens based on these analyses. The ‘high-risk bats near me’ app will be updated as new host-viral surveillance data comes on line from our project and others, to ground-truth and fine- tune its predictive capacity. Specifically, our telemetry data on bat movement will be used to assess how often bats from high-risk caves migrate to other colonies and potentially spread their high-risk strains.
The Wuhan Institute of Virology team will conduct viral testing on samples from all bat species in the caves as part of this inventory. Fecal, oral, blood and urogenital samples will be collected from bats using standard capture techniques as we have done for the last decade. In addition, tarps will be laid down in caves to assess the feasibility of surveys using pooled fresh fecal and urine samples. Assays will be designed to correlate viral load in an individual with viral shedding in a fecal sample. Once this is complete, surveys will continue largely on fecal samples so as not to disturb bat colonies and undermine longitudinal sampling capacity. Samples will be tested by PCR and spike proteins of all SARS-related CoVs sequenced. Analyses of phylogeny, recombination events, and further characterization of high-risk viruses (those with spike proteins close to SARS-CoV) will be carried out (REF). Isolation will be attempted on a subset of samples with novel SARSr-CoVs. will reverse engineer spike proteins in his lab to conduct binding assays to human ACE2 (the SARS-CoV receptor). Proteins that bind will then be inserted into SARS-CoV backbones, and inoculated into humanized mice to assess their capacity to cause SARS-like disease, and their ability to be blocked by monoclonal therapies, or vaccines against SARS-CoV (REF).
The modeling team will use these data to build models of 1) risk of viral
surrounding regions, and therefore how wide the risk zone is for the
warfighter positioned close to bat caves.
Commented [PD8]: Could add “ We will continue monitoring the human population proximal to these caves to assess the rates of viral spillover, and ground- truth which specific CoVs are able to infect people
Prof. Ralph Baric, UNC,
Commented [PD9]: Ralph, Zhengli. If we win this contract, I do not propose that all of this work will necessarily be conducted by Ralph, but I do want to stress the US side of this proposal so that DARPA are comfortable with our team. Once we get the funds, we can then allocate who does what exact work, and I believe that a lot of these assays can be done in Wuhan as well...

evolution and spillover, and 2) strategies to maximize inoculation strategy.
Data on the diversity of bat spike proteins, prevalence of recombinant CoVs, ability to bind and infect human cells, degree of clinical signs in mouse models, will be used to estimate evolutionary rates, rates of recombination, and capacity to generate novel strains capable of human infection. Using dynamic metapopulation models, we will estimate the flow of genes within each bat cave, based on the known host and viral assemblages. This will inform how rapidly new CoV strains with distinct phenotypic characteristics evolve. Because of our unique collaboration among world-class modelers, and coronavirologists, we will be able to test model predictions of viral capacity for spillover by conducting spike protein-based binding and cell culture experiments. The BSL-2 nature of work on SARSr-CoVs makes our system highly cost- effective relative to other bat-virus systems (e.g. Ebola, Marburg, Hendra, Nipah), which require BSL-4 level facilities for cell culture.
We will use modeling approaches, the data above, and other biological and ecological data to estimate how rapidly high-risk SARSr-CoVs will re-colonize a bat population following immune boosting or priming. We will obtain model estimates of the frequency of inoculation required for both approaches, what proportion of a population needs to be reached to have effective viral dampening, and whether specific seasons, or locations within a cave would be more effective to treat. We will then model the efficacy of different delivery methods (spray, swab, cave mouth automated delivery, deliver to specific sections of a cave).
TA2: Develop scalable approaches that target and suppress the animal virus in its reservoir(s) and/or vector(s), to reduce the likelihood of virus transmission into humans.
Our goal is to use two approaches to defuse the potential for SARS-related CoVs to emerge in people: 1) Immune Boosting: using the unique immunological features of bats that our group has discovered, we will inoculate live bats in cave mesocosms with immune modulators to up-regulate their naïve immunity to suppress viral replication and shedding; 2) Immune Priming: building on preliminary development of polyvalent chimeric recombinant molecules targeting diverse spike proteins from bat SARS-related CoVs, we will produce, and trial inoculation of live bats to suppress the replication and shedding of a broad range of dangerous SARS-related CoVs. Both lines of work will begin in Yr 1 and run parallel throughout the project.
Prof. Linfa Wang (Duke-NUS) will lead the work on immune boosting work, building on his pioneering work on bat immunity (2). This work provides evidence that that the long-term coexistence of bats and their viruses has led to an equilibrium between viral replication and host immunity, whereby bats have specifically down-

regulated their innate immune system as part of the fitness cost of flight (the only true flying mammals) (2). The nature of the weakened but not entirely lost functionality of bat innate immunity factors like STING, a central DNA-interferon (IFN) sensing molecule, may have profound impact for bats to maintain the balanced state of “effective response”, but not “over response” against viruses (3). A similar finding was also observed in bat IFNA studies, which is less abundant but was constitutively expressed without stimulation (4). Given native levels of SARSr-CoVs in individual bats with damped immunity, we propose to suppress bat SARSr-CoV by boosting bat innate immunity through the IFN pathway, and breaking the natural host-virus equilibrium. One of the potential problems with this approach is that it can lead to severe inflammation. However, this is unlikely to occur in bats, because they also have a naturally dampened inflammation response (5).
Previous work has shown that aerosol spraying or intranasal inoculation of IFN or other small molecules has led to reduce viral loads in humans, ferrets and mouse models (12-14). We will therefore initially trial inoculation of live bats with synthetic double-stranded RNA (Poly I:C) and assay for reduced viral loads (DETAILS, CITATION). We will generate universal bat interferon and apply to bats in the lab. Interferon has been used extensively clinically if no viral-specific drugs are available, e.g. against filoviruses (11). Secondly, bat replication of SARSr-CoV is sensitive to interferon treatments, as has been shown in our previous work (12). We will attempt to boost bat IFN by blocking bat-specific IFN negative regulator. Bat IFNA is naturally constitutively expressed but cannot be induced to a high level (4). This is unique to bats. We think there should be a negative regulatory factor in the bat interferon production pathway. We propose using CRISPRi to find out that negative regulator and then screen for chemicals targeting at this gene. We will attempt to boost bat IFN by activating dampened bat-specific IFN production pathways which include DNA-STING-dependent and ssRNA-TLR7 dependent pathways. These changes have been proved to bat-specific, suggesting that they are important in viruses/bats coexistence, and supported by our own work showing that a mutant bat STING restores antiviral functionality (3). By identifying small molecules to directly activate downstream of STING, we have chance to activate bat interferon and then help bats to clear viruses. Similar strategy applies to ssRNA-TLR7 dependent pathways. We will also attempt to boost bat IFN by activating functional bat IFN production pathways. We will investigate if there are other IFN production pathways in bats. We then boost bat immune responses by ligands specifically to these pathways, e.g. polyIC to TLR3-IFN pathway or 5’ppp-dsRNA to RIG-I- IFN pathway. A similar strategy has been tested successful in mouse model for SARS- CoV, IAV or HBV (6, 7). We believe treating wild bats with IFN-modulating small molecules by spraying is superior to other invasive strategies that might be considered

Deleted: We will conduct initial experiments on a lab colony of wild-caught
Deleted: on this bat
Deleted: ...(details and citation if possible).
by DARPA, including genome editing (CRISPR or RNAi), vaccination or DIP bats, in terms of its deployability and scalability. Finally, we will inoculate bats with fragments of non- bat Coronavirus (DETAILS).
Prof. Ralph Baric (UNC) will lead the immune priming work, building on his track record in reverse-engineering and manipulating SARS-CoV, MERS-CoV and other virus spike proteins over the last two decades . He will develop recombinant chimeric spike- proteins (8) based on SARSr-CoVs we have already identified, and those we will discover and characterize during project DEFUSE.
please!
While there are clear advantages to working with fixed populations of cave-
dwelling bats, molecule or vaccine delivery is technically challenging. Dr. Tonie Rocke, who developed, trialed, field-tested and rolled out the prairie dog plague vaccine (9), and is currently working on vaccines to bat rabies (10, 11) and white-nose syndrome, will manage a series of experiments in the lab and field to perfect a delivery system for both arms of TA2.
We have found that the immune dampening features are highly conserved in all bat species tested so far. Duke-NUS has established a breeding colony of cave nectar bats for experimental use (one of very few experimental bat breeding colonies in the world and the only one in SE Asia!). So our initial proof of concept test can be done in this experimental colony. We will then extend the test to a small group of wild-caught Rhinolophus sinicus bats at Wuhan Institute of Zoology. We (Prof. Wang) have previous experience conducting SARS-CoV infection experiments with bat species from the same genus in the BSL4 facility at the Australian Animal Health Laboratory in Australia (L.Wang, unpublished results). First, we will use our recently proven technology to design LIPS assays to the specific high zoonotic-risk SARSr-CoVs (12). We will conduct serological analysis on bats captured for infection experiments, to assess prior exposure to specific strains. These LIPS assays will be made available for use in people to assess exposure of the general population around bat caves in China, and for potential use by the warfighter to assess exposure to SARSr-CoVs during combat missions.
Finally, work on a delivery method will be overseen by Dr. Tonie Rocke at the National Wildlife Health Center who has proven capacity to develop and take animal vaccines through to licensure (9). Using her captive Jamaican fruitbat colony (10, 11), Dr. Rocke will trial out the following strategies for delivery of the molecules, inocula proposed above: 1) aerosolization; 2) transdermally applied nanoparticles; 3) sticky edible spray that bats will groom from each other; 4) automated spray triggered by timers and movement detectors at critical cave entry points.. (Details and ideas please Tonie!). These approaches will then be trialed out on live bats in our three cave sites in
RALPH – clearly I didn’t really understand the
details of your approach. Can you add a couple of paragraphs here and some citations

Yunnan Province. Fieldwork will be conducted under the auspices of Dr. Rocke, EHA field staff, and Dr. Yunzhi Zhang (Yunnan CDC, Consultant with EcoHealth Alliance). Sections of bat caves will be cordoned off and different application methods trialed out. A small number of bats will be captured and assayed for viral load after treatment, but so as not to disturb the colony, most viral load work will be conducted on fresh fecal pellets collected daily on the cave floor. EHA has unique access to these sites in Yunnan Province, with our field teams conducting surveillance there for around 10 years, under the guidance of Drs. Shi and Zhang. In year 1 of project DEFUSE, we will seek permission for these experimental inoculations in cave sites in Yunnan from the Provincial Forestry Department. We do not envisage problems getting permission, as we have worked with the Forestry Department collaboratively for the last few years, we have the support of the Yunnan CDC, and we are releasing molecules that are not dangerous to people or wildlife.
E. Capabilities:
A brief summary of expertise of the team, including subcontractors and key personnel. A principal investigator for the project must be identified, and a description of the team’s organization. Include a description of the team’s organization including roles and responsibilities. Describe the organizational experience in this area, existing intellectual property required to complete the project, and any specialized facilities to be used as part of the project. List Government furnished materials or data assumed to be available.
**Note: While only the proposal requires an organization chart, it may be helpful to include in the abstract if we have the space.
• This organization chart would include (as applicable): (1) the programmatic relationship of team members; (2) the unique capabilities of team members; (3) the task responsibilities of team members; (4) the teaming strategy among the team members; (5) key personnel with the amount of effort to be expended by each person during each year.
The lead institution for Project DEFUSE is EcoHealth Alliance, New York, an international research non-profit focused on emerging zoonotic diseases. The project will be led by PI Dr. Peter Daszak, who has 20+ years’ experience managing lab, field and modeling research projects on emerging zoonoses, including as EHA institutional lead, Head of Modeling and Analytics, and member of the Executive Committee for the $130 million USAID EPT/PREDICT. Dr. Daszak will oversee and coordinate all project activities, as well as lead the modeling and analytic work for TA1. Dr. Billy Karesh, who has 40+ years’

experience managing wildlife disease and zoonotic disease projects, will manage partnership activities and relationships and outreach. Dr. Jon Epstein, who has 15 years’ experience working with bats and emerging zoonoses will coordinate work on bat immune priming and boosting trials. Dr. Kevin Olival and Dr. Noam Ross will manage and conduct the modeling and analytical approaches for this project.
Team:
Lead Organization: EcoHealth Alliance, New York
PI: Peter Daszak Ph.D., President & Chief Scientist, EcoHealth Alliance, 3 months/year Key Personnel:
Billy Karesh DVM, Executive VP for Policy & Health, 1 month/year
Kevin J. Olival Ph.D, VP for Scientific Research, 1 month/year
Jonathan H. Epstein DVM Ph.D., VP for Science & Outreach, 0.5 months/year
Carlos Zambrana-Torrelio Ph.D., Assoc. VP for Conservation & Health, 1 month/year Noam Ross Ph.D., Senior Research Scientist, 2 months/year
Evan Eskew, Research Scientist, 2 months/year
Hongying Li, Program Coordinator, China Programs, 3 months/year
TBD Postdoctoral Researcher modeling and analysis, 12 months/year
TBD Research Assistant, 12 months/year
TBD Program Assistant, 12 months/year
Guangjian Zhu Ph.D., Consultant Field Lead, China Programs, 6 months/year
Yunzhi Zhang Ph.D., Consultant, Yunnan CDC, China, 2 months/year
Subcontract #1: University of North Carolina Medical School Organizational Lead: Prof. Ralph Baric Ph.D., 2 months/year XXX
TBD Research Assistant, 12 months/year
Subcontract #2: USGS National Wildlife Health Center
Organizational Lead: Tonie Rocke Ph.D., 2 months/year, no salary requested TBD Research Technician, 9 months/year
Subcontract #3: Duke NUS, Singapore
Organizational Lead: Prof. Linfa Wang Ph.D., 2 months/year XXX
TBD Research Assistant, 12 months/year
XXX

Subcontract #4: Wuhan Institute of Virology, China Organizational Lead: Prof Zhengli Shi Ph.D., 2 months/year Peng Zhou Ph.D., 2 months/year
TBD Research Assistant, 12 months/year
Links to relevant papers, reports, or
Do not include more than two resumes as part of the abstract.
**Resumes count against the abstract page limit.
Dr. Peter Daszak is President and Chief Scientist of EcoHealth Alliance, a US-based organization that conducts research and outreach programs on emerging zoonotic diseases. He has published over 300 scientific papers, including the first global map of EID hotspots, strategies to estimate unknown viral diversity in wildlife, predictive models of virus-host relationships, and evidence of the bat origin of SARS-CoV and other emerging viruses. Dr Daszak is Chair of the National Academy of Sciences, Engineering and Medicine’s Forum on Microbial Threats and is a member of the Executive Committee and the EHA institutional lead for USAID-EPT-PREDICT. He serves on the NRC Advisory Committee to the USGCRP, the DHS CEEZAD External Advisory Board, and the WHO R&D Blueprint Pathogen Prioritization expert group, and has advised the Director for Medical Preparedness Policy on the White House National Security Staff on global health issues. Dr Daszak won the 2000 CSIRO medal for collaborative research.
Prof. Ralph Baric is a UNC Lineberger Comprehensive Cancer Center member and Professor in the UNC-Chapel Hill Department of Epidemiology. His work focuses on coronaviruses as models to study the genetics of RNA virus transcription, replication, persistence, and cross species transmission. His work crosses the boundaries of microbiology, virology, immunology and epidemiology, looking especially at the population genetics of viruses to find the molecular building blocks for more effective vaccines.
**General Notes:
• DARPA will evaluate proposals using the following criteria, listed in
descending order of importance:
F. If desired, include a brief bibliography
resumes of key performers.
Commented [PD10]: I’m planning to use my resume and Ralph’s. Linfa/Zhengli, I realize your resumes are also very impressive, but I am trying to downplay the non-US focus of this proposal so that DARPA doesn’t see this as a negative.

1) 5.1.1. Overall Scientific and Technical Merit
The proposed technical approach is innovative, feasible, achievable, and complete.
Task descriptions and associated technical elements provided are complete and in a logical sequence with all proposed deliverables clearly defined such that a final outcome that achieves the goal can be expected as a result of award. The proposal identifies major technical risks and planned mitigation efforts are clearly defined and feasible. The proposed PREEMPT Risk Mitigation Plan effectively provides the following: an assessment of potential risks; proposed guidelines to ensure maximal biosafety and biosecurity; a risk management plan for responsible communications; and a plan to address how input from the Government and community stakeholders will be considered regarding communication and publication of potentially sensitive dual-use information.
2) 5.1.2. Potential Contribution and Relevance to the DARPA Mission
The potential contributions of the proposed effort are relevant to the national technology base. Specifically, DARPA’s mission is to make pivotal early technology investments that create or prevent strategic surprise for U.S. National Security. The proposer clearly demonstrates its capability to transition the technology to the research, industrial, and/or operational military communities in such a way as to enhance U.S. defense. In
addition, the evaluation will take into consideration the extent to which the proposed intellectual property (IP) rights will potentially impact the Government’s ability to transition the technology.
3) 5.1.3. Cost Realism
The proposed costs are realistic for the technical and management approach and accurately reflect the technical goals and objectives of the solicitation. The proposed costs are consistent with the proposer's Statement of Work and reflect a sufficient understanding of the costs and level of effort needed to successfully accomplish the proposed technical approach. The costs for the prime proposer and proposed subawardees are substantiated by the details provided in the proposal (e.g., the type and number of labor hours proposed per task, the types and quantities of materials, equipment and fabrication costs, travel and any other applicable costs and the basis for the estimates).
It is expected that the effort will leverage all available relevant prior research in order to obtain the maximum benefit from the available funding. For efforts with a likelihood of commercial application, appropriate direct cost sharing may be a positive factor in the evaluation. DARPA recognizes that undue emphasis on cost may motivate proposers to

offer low-risk ideas with minimum uncertainty and to staff the effort with junior
personnel in order to be in a more competitive posture. DARPA discourages such cost
strategies.
Commented [EA11]: Please note
Citations
2. 3.
4. 5.
1.
K. J. Olival et al., Host and viral traits predict zoonotic spillover from mammals. Nature 546, 646-650 (2017).
G. Zhang et al., Comparative analysis of bat genomes provides insight into the evolution of flight and immunity. Science 339, 456-460 (2013).
J. Xie et al., Dampened STING-Dependent Interferon Activation in Bats. Cell host & microbe, (2018).
P. Zhou et al., Contraction of the type I IFN locus and unusual constitutive expression of IFN-αin bats. Proceedings of the National Academy of Sciences of the United States of America, 201518240-201518246 (2016).
M. Ahn, J. Cui, A. T. Irving, L.-F. Wang, Unique Loss of the PYHIN Gene Family in Bats Amongst Mammals: Implications for Inflammasome Sensing. Scientific Reports 6, (2016).
Attachment 1: Executive Summary Slide template

6. 7.
8.
9. 10.
11. 12.
J. Zhao et al., Intranasal Treatment with Poly(I.C) Protects Aged Mice from Lethal Respiratory Virus Infections. Journal of Virology 86, 11416-11424 (2012).
J. Wu et al., Poly(I:C) Treatment Leads to Interferon-Dependent Clearance of Hepatitis B Virus in a Hydrodynamic Injection Mouse Model. Journal of Virology 88, 10421-10431 (2014).
X. F. Deng et al., A Chimeric Virus-Mouse Model System for Evaluating the Function and Inhibition of Papain-Like Proteases of Emerging Coronaviruses. Journal of Virology 88, 11825-11833 (2014).
T. E. Rocke et al., Sylvatic Plague Vaccine Partially Protects Prairie Dogs (Cynomys spp.) in Field Trials. Ecohealth 14, 438-450 (2017).
B. Stading et al., Protection of bats (Eptesicus fuscus) against rabies following topical or oronasal exposure to a recombinant raccoon poxvirus vaccine. Plos Neglect. Trop. Dis. 11, (2017).
B. R. Stading et al., Infectivity of attenuated poxvirus vaccine vectors and immunogenicity of a raccoonpox vectored rabies vaccine in the Brazilian Free-tailed bat (Tadarida brasiliensis). Vaccine 34, 5352-5358 (2016).
P. Zhou et al., Fatal Swine Acute Diarrhea Syndrome caused by an HKU2- related Coronavirus of Bat Origin. Nature In press, (2018
).

10/5/21, 2:43 PM Mail - Rocke, Tonie E - Outlook
I have built in my comments atop of Linfa’s comments. ralph
From: Wang Linfa [mailto:linfa.wang@duke-nus.edu.sg]
Sent: Thursday, February 8, 2018 7:25 AM
To: Peter Daszak <daszak@ecohealthalliance.org>; Baric, Ralph S <rbaric@email.unc.edu>; Zhengli Shi (zlshi@wh.iov.cn) <zlshi@wh.iov.cn>; William B. Karesh <karesh@ecohealthalliance.org>; Rocke, Tonie <trocke@usgs.gov>
Cc: Luke Hamel <hamel@ecohealthalliance.org>; Jonathon Musser <musser@ecohealthalliance.org>; Anna Willoughby <willoughby@ecohealthalliance.org>; Kevin Olival, PhD <olival@ecohealthalliance.org>; Jon Epstein <epstein@ecohealthalliance.org>; Noam Ross <ross@ecohealthalliance.org>; Aleksei Chmura <chmura@ecohealthalliance.org>; Anna Willoughby <willoughby@ecohealthalliance.org>; Hongying Li <li@ecohealthalliance.org>
Subject: RE: First (rough) dra of the DARPA abstract - Project DEFUSE
See my brief notes/edits in the aached.
I am working on a large grant here in SG and won’t be able to spend too much me unl next week. LF
Linfa (Lin-Fa) WANG, PhD FTSE
Professor & Director
Programme in Emerging Infecous Disease Duke-NUS Medical School,
8 College Road, Singapore 169857
Tel: +65 6516 8397
From: Peter Daszak [mailto:daszak@ecohealthalliance.org]
Sent: Thursday, 8 February, 2018 10:51 AM
To: Ralph Baric (rbaric@email.unc.edu); Wang Linfa; Zhengli Shi (zlshi@wh.iov.cn); William B. Karesh; Rocke, Tonie
Cc: Luke Hamel; Jonathon Musser; Anna Willoughby; Kevin Olival, PhD; Jon Epstein; Noam Ross; Aleksei Chmura; Anna Willoughby; Hongying Li
Subject: First (rough) draft of the DARPA abstract - Project DEFUSE
Importance: High
Dear All,
I’ve aached a first rough dra of the DARPA abstract. Apologies for the delay. Unfortunately, edits to my Science paper came through on Friday and took many hours to do, so this delayed me. I’m right now in Geneva in my
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10/5/21, 2:43 PM Mail - Rocke, Tonie E - Outlook
hotel at 3 am finishing these off before flying back to NYC from a WHO meeng. Some important points:
1)
2)
3)
4) 5)
6)
Zhengli, Linfa, Ralph – Billy and I spoke with Tonie Rocke on Friday. Tonie is at the Naonal Wildlife Health Center, Madison USA, and has worked on wildlife vaccines: plague in prairie dogs, rabies in Jamaican fruit bats, white nose syndrome in US bats. We needed someone with experse in delivery of molecules/vaccines to wildlife because DARPA specifically lay that out. As you’ll see, Tonie is perfect for our project and will be able to do work at USGS NWHC and with Zhengli in China to help with TA2
Zhengli and Linfa – Aer I spoke with you both, I had a great conversaon with Ralph Baric. He proposed to work on recombinant chimeric spike proteins as a second line of aack. I think that is a perfect fit because 1) it’s his experse and he has published on it, 2) it will act as an alternave to the blue-sky and risky immune boosng work that Linfa/Peng have proposed. I hope you agree!
Ralph, Zhengli, Linfa, Tonie – as you can see, I have mangled the language/technical details for most of your secons. Pardon my lack of knowledge, and please dra a couple of paragraphs each to make your secons look correct. Thanks to Peng for giving me some text anyway – very useful, but please check what I’ve done with it.
All – please add some names and details on the team part so we get clarity in this on what staff you will need to do the work.
Please don’t worry about keeping this to the 8 page limit. Just add text here and there, references, and edit to make what I’ve wrien correct, and more excing. I will work on this on Saturday, Sunday and Monday to bring it down to 8 pages of very crisp, super-excing text. I also want as many of your good ideas in here, so that I can use this dra to build on for the full proposal.
Finally – please edit rapidly using tracked changes in word. If you don’t want to mess up endnote, please just insert references as comment boxes and we’ll pull them off the web.
Aleksei and Anna: please read the dra and work on some dra image designs that sum up the project flow. I’ll call you Thursday aernoon to discuss so you can finish them off.
Luke – please have a go at a first dra of the execuve summary slide. I’ll pick up from what you’ve done once you send it to me.
Thanks again to all of you for agreeing to collaborate on this proposal. From what I know of the compeon, what DARPA wants, and what we’re offering, I think we have an extremely strong team, so I’m looking forward to geng the full proposal together and winning this contract!
Cheers, Peter
Peter Daszak
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10/5/21, 2:43 PM Mail - Rocke, Tonie E - Outlook
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DARPA – PREEMPT – HR001118S0017
Abstract Submission Requirements:
**8 pages with 12 point font or higher (smaller font may be used for figures, tables
and charts)
**Page limit includes all figures, tables, charts and the Executive Summary Slide **Copies of all documents submitted must be clearly labeled with the following:
-DARPA BAA number
-Proposer Organization
-Proposal title/Proposal short title
-Submission letter is optional and does not count towards page limit
A. Cover Sheet (does not count towards page limit):
Include the administrative and technical points of contact (name, address, phone, fax, email, lead organization). Also include the BAA number, title of the proposed project, primary subcontractors, estimated cost, duration of project, and the label “ABSTRACT.”
B. Executive Summary Slide:
Provide a one slide summary in PowerPoint that effectively and succinctly conveys the main objective, key innovations, expected impact, and other unique aspects of the proposed project. Use the slide template provided at http://www.fbo.gov.
**See slide template at bottom of document.
Clearly describe what is being proposed and what difference it will make (qualitatively and quantitatively), including brief answers to the following questions:
1. What is the proposed work attempting to accomplish or do?
We aim to defuse the potential for emergence of novel bat-origin high-zoonotic risk SARS-related coronaviruses in Southeast Asia. We envisage a scenario whereby the US warfighter is called on to intervene in a security hotspot in SE Asia for a period of 3-6 months. As planners begin choosing sites for the mission, they will use an app we will design to assess the background risk of a site harboring dangerous zoonotic viruses. If
PROJECT DEFUSE
C. Goals and Impact:

there is no alternative to a high-risk site, a tactical forward team will deploy automated delivery technology we will develop in caves that harbor bats carrying these viruses. These devices will release immune boosting molecules and chimeric polyvalent spike protein immune priming inocula to lower viral shedding from bats at the site for a few weeks or months, allowing our warfighters to execute the operation at lowered risk for spillover.
2. How is it done today? And what are the limitations?
, there is no available technology to reduce the risk of exposure to novel
coronaviruses from bats, other than avoid the regions where bats harbor these viruses. This includes large areas of southeast Asia where SARS-related CoVs are endemic in bats, which roost in caves during the day, but forage over wide areas at night, shedding virus in their feces and urine. The limitations of this lack of capacity are significant – we have shown evidence of recent spillover of SARS-related CoVs into people in southern China, and have identified viruses in this region that are capable of producing SARS-like illness in humanized mice, with no available vaccines or countermeasures. These viruses are a clear-and-present danger to our military personnel, and to global health security.
3. What is innovative in your approach and how does it compare to current practice and state-of-the-art (SOA)?
**Note: DARPA wants to know, “how the proposed project is revolutionary and how it significantly rises above the current state of the art
Our group has shown that bats harbor the highest proportion of potential zoonoses of any mammal group, and that they are able to live with the host without causing diseases due to unique damping of certain pathways in their immune systems, likely in part as an evolutionary adaptation to flight. We will use this new finding to design strategies like small molecule Rig like receptor (RLR) or Toll like receptor (TLR) agonists to upregulate their immune response in their cave roosts, down-regulate viral replication, and reduce the risk of viral shedding and spillover (broad immune boosting strategy). At the same time, we will inoculate bats with novel chimeric polyvalent recombinant spike proteins to enhance their immune response against replication of specific, high-risk viruses (targeted immune priming strategy). We will use our innovative modeling to design apps that identify the likelihood of any region harboring high-risk bat viruses. We will design novel, automated approaches to deliver both types of inoculum remotely into caves to reduce exposure risk during decontamination.
4. What are the key technical challenges in your approach and how do you plan to overcome these?
Commented [L1]: My understanding is that the project will have two parts: A) better risk assessment and modeling and B) risk defusing.
Do we need to say anything about A here?!
Deleted: high viral loads Deleted: their
Commented [L2]: This will become important late: while we are specifically targeting SARS-realted CoVS, this strategy will be applicable to ALL bat-borne viruses in future
Commented [BRS3]: I thought we were also going to use innate immune antagonists to boost baseline immunity, which should attenuate virus burden in animals?
Isn’t this supposed to be a two pronged approach that are complementary, e.g., in that innate immune agonists will also boost immunity to recombinant spike vaccines.
Currently

Decide which of following parts to talk about:
Sampling/testing
Reverse engineering, binding assays, mouse expts Modeling viral risk of evolution and spillover
Gain-of-function issue. Mesocosm expts
5. Who will care and what will the impact be if you are successful?
This will have direct relevance to the warfighter. The potential for deployment to the region in which bat hosts of SARS-related CoVs exist is high – countries include security hotspots (Myanmar, Bangladesh, Pakistan, Lao, Korea, Vietnam and Cambodia?). The ability to decontaminate and defuse these viruses will be useful in preventing potentially devastating illness. Furthermore, these technologies, if successful, can be adapted to hosts of other bat-origin CoVs (MERS, SARS and related prepandemic zoonotic strains), and potentially other zoonotic bat-origin viruses (Hendra, Nipah, EBOV). Finally, our approach is directly applicable to public health measures in the region to reduce the risk of spillover into the general population, as well as for food security by reducing the risk of viruses like SADS-CoV spilling over from bats into intensive pig farms, and devastating and industry, leading to potential civil unrest.
6. How much will it cost and how long will it take?
Will insert this later after calculating and brainstorming.
D. Technical Plan:
Outline and address all technical challenges inherent in the approach and possible solutions for overcoming potential problems. This section should provide appropriate specific milestones (quantitative, if possible) at intermediate stages of the project to demonstrate progress and a brief plan for accomplishment of the milestones. **Note: “The technical plan should demonstrate a deep understanding of the
technical challenges and present a credible (even if risky) plan to achieve
Modeling bat
suitability
Commented [L4]: I have highlighted the ones which are most challenging and novel for this proposal
Formatted: Highlight
Formatted: Highlight Formatted: Highlight
Formatted: Highlight Formatted: Highlight
Deleted: D
Commented [PD5]: Check on the duration of PREEMPT
Inventory of caves
Modeling inoculation/defusing strategy
Immune modulation
Immune Booster recombinant production
Inoculum delivery
Cave expts
46 months

the program goal”
Key Terms/Aspects to Emphasize in Abstract ● IACUC/IRB
○ DARPA wants to know who has experience w/ ACURO IACUC work.
■ EHA has multiple ACURO IACUC proposals (either approved or
undergoing approval)
■ IRB also in place, just has to be modified
Rationale for the SE Asian SARS-related CoV – Rhinolophus bat target system, and immune priming/boosting: 1) Our group has shown that bats harbor a higher proportion of potentially highly heterogeneous zoonotic viruses than any other mammalian group (1), so that proof-of-concept for blocking viral spillover from this host group may lead to a bigger impact on global health security; 2) The Rhinolophus bats that harbor SARS like-CoVs are insectivorous and roost in dense colonies at fixed, known locations, yet disperse each night over wide distances from these sites. Defusing the risk of viral shedding in the roost will also defuse the risk of viral shedding over the population range. This would be difficult for rodent or primate reservoirs; 3) Bats are mammalian hosts, therefore immune modulating drugs evaluated in people and rodents may also work on bats. This would be less likely for an insect vector; 4) Members of our collaborative group has worked together on bats and their viruses for over 15 years, with a total of >100 yrs experience focused on bat-origin zoonoses among the key personnel. We have published much of the seminal work on the bat origins of SARS, Nipah, Hendra, and MERS viruses, and have opened new boundaries in studies of bat host-viral relationships ecologically, immunologically and virologically; 5) The South and Southeast Asian region where these bats occur is a security hotspot, with active political and ethnic conflicts, and displaced populations in Bangladesh, Pakistan, Myanmar, Thailand, Indonesia, Philippines and other . This is a likely potential site for US warfighter deployment; 6) We have worked for over 10 years on the SARS-related CoV – Rhinolophus bat system in China, demonstrating the origin of SARS-CoV within this host, the presence of with remarkable sequence identity in the spike protein to SARS-CoV, their isolation and characterization of their ability to bind and replicate efficiently in primary human lung airway cells. We have demonstrated that chimeric
SARS-CoV backbone with spike protein from SARSr-CoVs from our cave sites in Yunnan Province can infect a humanized mouse model and cause SARS-like illness, and that clinical signs are not reduced with SARS monoclonal therapy or vaccination. Finally, we have demonstrated that people living up to 6 kilometers from our cave site have
Overview
Commented [PD6]: I know this is too long. I’ll edit later this weekend, but want to keep this text for the full proposal
Deleted: a
Deleted: trialed out
Commented [BRS7]: About 90,000 of the 550,000 deployed US military are in se asian, mostly japan and south korea.
Commented [BRS8]: What abbreviation mean
Deleted: bind Deleted: with
countries
SARSr-CoVs

evidence of SARSr-CoV antibodies (3% seroprevalence in 200+ cohort), suggesting active spillover, and marking these viruses as a clear-and-present danger of a new SARS-like pandemic; 7) SARSr-CoVs are transmitted among bats via fecal-oral route, making sampling relatively easy (collection of fresh fecal pellets) and molecule or vaccine approaches feasible; 8) Proof-of-concept in this system may be rapidly scalable to other bat-coronavirus systems, e.g. MERS-CoV, SADS-CoV, and to other cave bat origin viruses.
Other important bat-origin zoonotic viruses (e.g. filoviruses, henipaviruses) have very rare spillover events - usually to a single index case, which makes validated prevention of spillover challenging. These viruses also show little strain diversity which makes modeling which evolutionary lines will be more high-risk, a . SARSr-CoVs are diverse, with recombinants regularly identified in the field and lab. Furthermore, we have identified SARS-like strains in a single cave in Yunnan that harbor every gene found in the human SARS-CoV strains detected during the 2002-2003 epidemic. Within this bat population, an ideal evolutionary soup exists which can produce new human strains by high frequency RNA recombination and presents a perfect target for 21st generation
intervention strategies.
Finally, we believe that alternative approaches to transmission blocking, e.g.
CRISPER-Cas gene drives that are likely to be far less effective in bats because most bats are long-lived relative to their small size, long inter-generational periods (2-5 yrs) and low progeny (~1-2 pups). Gene drives would likely take many decades to run through a
population, so that proof-of-concept of transmission blocking in the DARPA time scale wouldn’t be possible. Furthermore, many bat species’ populations mix readily or migrate which would disperse the impact of gene drives, whereas targeting a small number of caves in a region for molecule or vaccine delivery would cover a very large dispersal area.
TA1: Develop and validate integrated, multiscale models that quantify the likelihood a human-capable virus will emerge from an animal reservoir residing in a “hot spot” geographic region
The DEFUSE modeling and analytics team will develop models to evaluate the likelihood of bat caves harboring high-risk SARSr-CoVs, evaluate the probability of specific SARS- related CoV spillover, and identify the most effective strategy for inoculation of immune boosting molecules and chimeric spike protein immune priming inocula.
We will collect specific data to inform our model building, validate assumptions and refine predictions. At the start of Yr 1, we will conduct a full inventory of host and virus distribution within our field sites, two caves in Yunnan Province, China. This builds on 8 years of surveillance in these caves and includes a cave in which we have identified all the genetic components of the 2002-2003 epidemic SARS-CoV distributed across a
Commented [BRS9]: These viruses can either be cultured and/or recovered using reverse genetic strategies.
Commented [BRS10]: Filoviruses pretty diverse, although not anywhere near as diverse as cov. Is this a sampling thing or not likely remains unclear?
Deleted: s
Deleted: from the
Deleted: in a diversity of SARSr-CoVs w Deleted: e
Deleted: making it
Deleted: to
Formatted: Superscript
Deleted: with
Commented [BRS11]: Is this correct? Deleted: 2-5 years
Commented [L12]: We need to provide background info about bat immunity and the track record of this group in the field
Commented [L13]: Peng: I am working on an important grant here in Singapore. Can you add a few points here? Thanks
challenge

bat population. Two other caves will act as controls/comparison sites, in that we have not yet identified the high-risk SARSr-CoVs in that cave. We will assess: the population density, distribution and segregation of individual bats; changes in these daily, weekly and by season; viral prevalence and intensity in individuals; distribution of low- and high-risk SARSr-CoV strains, and how readily these are transmitted among bat species, age classes, genders; and using mark-recapture to assess metapopulation structure. To assess geographic distribution of bat hosts, we have access to biological inventory data on all bat caves in Southern China, as well as information on species distributions across SE Asia from the literature and museum records. We will use radio- and satellite telemetry to identify the home range of each species of bat in the caves, to assess how widely the viral ‘plume’ could contaminate
We will build environmental niche models using the data above, and environmental and ecological correlates, and traits of cave species communities (eg. phylogenetic and functional diversity), to predict the species composition of bat caves across Southern China, South and SE Asia. We will validate these with data from the current project and data from PREDICT sampling in Thailand, Indonesia, Malaysia and other SE Asian countries. We will then use our unique database of bat host-viral relationships updated from our recent Nature paper (1) to assess the likelihood of low- or high-risk SARSr-CoVs being present in a cave at any site across the region. At the end of Yr 1, we will use these analyses to produce a prototype app for the warfighter that identifies the likelihood of bats harboring dangerous viral pathogens based on these analyses. The ‘high-risk bats near me’ app will be updated as new host-viral surveillance data comes on line from our project and others, to ground-truth and fine- tune its predictive capacity. Specifically, our telemetry data on bat movement will be used to assess how often bats from high-risk caves migrate to other colonies and potentially spread their high-risk strains.
The Wuhan Institute of Virology team will conduct viral testing on samples from all bat species in the caves as part of this inventory. Fecal, oral, blood and urogenital samples will be collected from bats using standard capture techniques as we have done for the last decade. In addition, tarps will be laid down in caves to assess the feasibility of surveys using pooled fresh fecal and urine samples. Assays will be designed to correlate viral load in an individual with viral shedding in a fecal sample. Once this is complete, surveys will continue largely on fecal samples so as not to disturb bat colonies and undermine longitudinal sampling capacity. Samples will be tested by PCR and spike proteins of all SARS-related CoVs sequenced. Analyses of phylogeny, recombination events, and further characterization of high-risk viruses (those with spike proteins close to SARS-CoV) will be carried out (REF). Isolation will be attempted on a subset of
Commented [BRS14]: Is surveillance in these other caves equally robust over the past 8 yrs?
Commented [PD15]: Could add “ We will continue monitoring the human population proximal to these caves to assess the rates of viral spillover, and ground- truth which specific CoVs are able to infect people
surrounding regions, and therefore how
wide the risk zone is for the warfighter positioned close to bat caves.

samples with novel SARSr-CoVs. will reverse engineer spike
proteins in his lab to conduct binding assays to human ACE2 (the SARS-CoV receptor).
Their group have also devised new strategies to culture SARS-like bat coronaviruses, allowing biological characterization of both high risk strains that can replicate in primary human cells and low risk strains that can only replicate in the presence of exogenous enhancers. Viral spike glycoproteins that bind receptor will then be inserted into SARS- CoV backbones, and inoculated into human cells and humanized mice to assess their capacity to cause SARS-like disease, and their ability to be blocked by monoclonal therapies, or vaccines against SARS-CoV ((PMC5798318, PMC5567817, PMC5380844, PMC5578707, PMC4801244, PMC4797993). The Baric group has also demonstrated that a nucleoside analogue inhibitor, GS-5734 (Gilead Inc), blocks epidemic, preepidemic and zoonotic SARS-CoV and SARS-like bat coronavirus replication in primary human airway cells and in mice (PMC5567817). Consequently, they will evaluate the ability of this drug to block replication of newly disovered pre-epidemic and zoonotic high risk strains. As the drug has been used to effectively treat Ebola virus infected patients (PMC4967715, PMC5583641) as well and has potent activity against Nipha and Hendra viruses (PMC5338263), an alternative intervention for military personnel is prophylactic treatment treatment prior to deployment into high risk settings.
The modeling team will use these data to build models of 1) risk of viral evolution and spillover, and 2) strategies to maximize inoculation strategy.
Data on the diversity of bat spike proteins, prevalence of recombinant CoVs, ability to bind and infect human cells, degree of clinical signs in mouse models, will be used to estimate evolutionary rates, rates of recombination, and capacity to generate novel strains capable of human infection. Using dynamic metapopulation models, we will estimate the flow of genes within each bat cave, based on the known host and viral assemblages. This will inform how rapidly new CoV strains with distinct phenotypic characteristics evolve. Because of our unique collaboration among world-class modelers, and coronavirologists, we will be able to test model predictions of viral capacity for spillover by conducting spike protein-based binding and cell culture experiments.
.
We will use modeling approaches, the data above, and other biological and
ecological data to estimate how rapidly high-risk SARSr-CoVs will re-colonize a bat population following immune boosting or priming. We will obtain model estimates of the frequency of inoculation required for both approaches, what proportion of a population needs to be reached to have effective viral dampening, and whether specific seasons, or locations within a cave would be more effective to treat. We will then model
Prof. Ralph Baric, UNC,
Commented [PD16]: Ralph, Zhengli. If we win this contract, I do not propose that all of this work will necessarily be conducted by Ralph, but I do want to stress the US side of this proposal so that DARPA are comfortable with our team. Once we get the funds, we can then allocate who does what exact work, and I believe that a lot of these assays can be done in Wuhan as well...
Deleted: P
Deleted: REF)
The BSL-2 nature of work on SARSr-CoVs makes our system highly cost-
effective relative to other bat-virus systems (e.g. Ebola, Marburg, Hendra, Nipah), which
require BSL-4 level facilities for cell culture
Commented [BRS17]: IN the US, these recombinant SARS CoV are studied under BSL3, not BSL2, especially important for those that are able to bind and replicate in primary human cells.
In china, might be growin these virus under bsl2. US reseachers will likely freak out.

the efficacy of different delivery methods (spray, swab, cave mouth automated delivery, deliver to specific sections of a cave).
TA2: Develop scalable approaches that target and suppress the animal virus in its reservoir(s) and/or vector(s), to reduce the likelihood of virus transmission into humans.
Our goal is to use two approaches to defuse the potential for SARS-related CoVs to emerge in people: 1) Immune Boosting: using the unique immunological features of bats that our group has discovered, we will inoculate live bats in cave mesocosms with immune to up-regulate their naïve immunity to suppress viral replication and shedding; 2) Immune Priming: building on preliminary development of polyvalent chimeric recombinant molecules targeting diverse spike proteins from bat SARS-related CoVs, we will produce, and trial inoculation of live bats to suppress the replication and shedding of a broad range of dangerous SARS-related CoVs. Both lines of work will begin in Yr 1 and run parallel throughout the project.
Prof. Linfa Wang (Duke-NUS) will lead the work on immune boosting work, building on his pioneering work on bat immunity (2). This work provides evidence that that the long-term coexistence of bats and their viruses has led to an equilibrium between viral replication and host immunity, whereby bats have specifically down- regulated their innate immune system as part of the fitness cost of flight (the only true flying mammals) (2). The nature of the weakened but not entirely lost functionality of bat innate immunity factors like STING, a central DNA-interferon (IFN) sensing molecule, may have profound impact for bats to maintain the balanced state of “effective response”, but not “over response” against viruses (3). A similar finding was also observed in bat IFNA studies, which is less abundant but was constitutively expressed without stimulation (4). Given native levels of SARSr-CoVs in individual bats with damped immunity, we propose to suppress bat SARSr-CoV by boosting bat innate immunity through the IFN pathway, and breaking the natural host-virus equilibrium. One of the potential problems with this approach is that it can lead to severe inflammation. However, this is unlikely to occur in bats, because they also have a naturally dampened inflammation response (5).
Previous work has shown that aerosol spraying or intranasal inoculation of IFN or other small molecules has led to reduce viral loads in humans, ferrets and mouse models (12-14). We will therefore initially trial inoculation of live bats with synthetic double-stranded RNA (Poly I:C) and assay for reduced viral loads (DETAILS, CITATION). We will generate universal bat interferon and apply to bats in the lab. Interferon has been used extensively clinically if no viral-specific drugs are available, e.g. against filoviruses (11). Secondly, bat replication of SARSr-CoV is sensitive to interferon
Commented [BRS18]: Like what
Commented [BRS19]: Transient low level Chronic inflammation sounds better
modulators

treatments, as has been shown in our previous work (12). We will attempt to boost bat IFN by blocking bat-specific IFN negative regulator. Bat IFNA is naturally constitutively expressed but cannot be induced to a high level (4).
We will attempt to boost bat IFN by activating dampened bat-specific IFN production pathways which include DNA-STING-dependent
and ssRNA-TLR7 dependent pathways. These changes have been proved to bat-specific, suggesting that they are important in viruses/bats coexistence, and supported by our own work showing that a mutant bat STING restores antiviral functionality (3). By identifying small molecules to directly activate downstream of STING, we have chance to activate bat interferon and then help bats to clear viruses. Similar strategy applies to ssRNA-TLR7 dependent pathways. We will also attempt to boost bat IFN by activating functional bat IFN production pathways. We will investigate if there are other IFN production pathways in bats. We then boost bat immune responses by ligands specifically to these pathways, e.g. polyIC to TLR3-IFN pathway or 5’ppp-dsRNA to RIG-I- IFN . A similar strategy has been tested successful in mouse model for SARS- CoV, IAV or HBV (6, 7). We believe treating wild bats with IFN-modulating small molecules by spraying is superior to other invasive strategies that might be considered by DARPA, including genome editing (CRISPR or RNAi), or DIP bats, in terms
of its deployability and scalability. Finally, we will inoculate bats with fragments of non- bat Coronavirus (DETAILS).
Prof. Ralph Baric (UNC) will lead the immune priming work, building on his track record in reverse-engineering and manipulating SARS-CoV, MERS-CoV and other virus spike proteins over the last two decades . He will develop recombinant chimeric spike- proteins (8) based on SARSr-CoVs we have already identified, and those we will discover and characterize during project DEFUSE.
please!
While there are clear advantages to working with fixed populations of cave-
dwelling bats, molecule or vaccine delivery is technically challenging. Dr. Tonie Rocke, who developed, trialed, field-tested and rolled out the prairie dog plague vaccine (9), and is currently working on vaccines to bat rabies (10, 11) and white-nose syndrome, will manage a series of experiments in the lab and field to perfect a delivery system for both arms of TA2.
We have found that the immune dampening features are highly conserved in all bat species tested so far. Duke-NUS has established a breeding colony of cave nectar bats for experimental use (one of very few experimental bat breeding colonies in the
there should be a negative regulatory factor in the bat interferon production pathway.
We propose using CRISPRi to find out that negative regulator and then screen for
This is unique to bats. We think
chemicals targeting at this gene.
Commented [BRS20]: This could easily take longer than 3 years. Poly ic, IFN or any type of TLR agonist might be more robust. Might want to test in captive bats infected with SARS or select SARS like viruses, like SHC014, which we could provide.
Commented [BRS21]: We have several papers showing importance of TLR3 and TLR4 signaling in control of SARS pathogenesis. PMC4447251, PMC5473747
Commented [BRS22]: Don’t attack the other arm of the program. And I disagree that its superior to vaccination, which potentially provides long-term immunity.
Formatted: Highlight
Commented [BRS23]: The structure of the SARS-CoV spike glycoprotein has been solved and the addition of two proline residues at positions V1060P and L1061P stabilize the prefusion state of the trimer, including key neutralizing epitopes in the receptor binding domain (PMC5584442). In parallel, the spike trimers or the receptor binding domain can be incorporated into alphavirus vectored or nanoparticle vaccines for delivery, either as aerosols, in baits, or as large droplet delivery vehicles (PMC4058772, PMC5423355, PMC2883479, PMC5578707, PMC3014161). Initially, we will test various delivery vehicles in controlled conditions in bats in a laboratory setting, taking the best candidate forward for testing in the field.
The Baric laboratory has built recombinant S pike glycoproteins harboring structurally defined domains from SARS epidemic strains, pre-epidemic strains like SCH014 and zoonotic strains like HKU3. It is anticipated that recombinant S glycoprotein based vaccines harboring immunogenic blocks across the group 2B coronaviruses will induce broad based immune responses that simultaneously reduce genetically heterogeneous virus burdens in bats, thereby reducing disease risk in these animals for multiple years (PMC3977350,
PMC2588415).
pathway
vaccination
RALPH – clearly I didn’t really understand the
details of your approach. Can you add a couple of paragraphs here and some citations

Deleted: We will conduct initial experiments on a lab colony of ...
Deleted: on this bat
Deleted: ...(details and citation if possible).
world and the only one in SE Asia!). So our initial proof of concept test can be done in this experimental colony. We will then extend the test to a small group of wild-caught Rhinolophus sinicus bats at Wuhan Institute of Zoology. We (Prof. Wang) have previous experience conducting SARS-CoV infection experiments with bat species from the same genus in the BSL4 facility at the Australian Animal Health Laboratory in Australia (L.Wang, unpublished results). First, we will use our recently proven technology to design LIPS assays to the specific high zoonotic-risk SARSr-CoVs (12). We will conduct serological analysis on bats captured for infection experiments, to assess prior exposure to specific strains. These LIPS assays will be made available for use in people to assess exposure of the general population around bat caves in China, and for potential use by the warfighter to assess exposure to SARSr-CoVs during combat missions.
Finally, work on a delivery method will be overseen by Dr. Tonie Rocke at the National Wildlife Health Center who has proven capacity to develop and take animal vaccines through to licensure (9). Using her captive Jamaican fruitbat colony (10, 11), Dr. Rocke will trial out the following strategies for delivery of the molecules, inocula proposed above: 1) aerosolization; 2) transdermally applied nanoparticles; 3) sticky edible spray that bats will groom from each other; 4) automated spray triggered by timers and movement detectors at critical cave entry points.. (Details and ideas please Tonie!). These approaches will then be trialed out on live bats in our three cave sites in Yunnan Province. Fieldwork will be conducted under the auspices of Dr. Rocke, EHA field staff, and Dr. Yunzhi Zhang (Yunnan CDC, Consultant with EcoHealth Alliance). Sections of bat caves will be cordoned off and different application methods trialed out. A small number of bats will be captured and assayed for viral load after treatment, but so as not to disturb the colony, most viral load work will be conducted on fresh fecal pellets collected daily on the cave floor. EHA has unique access to these sites in Yunnan Province, with our field teams conducting surveillance there for around 10 years, under the guidance of Drs. Shi and Zhang. In year 1 of project DEFUSE, we will seek permission for these experimental inoculations in cave sites in Yunnan from the Provincial Forestry Department. We do not envisage problems getting permission, as we have worked with the Forestry Department collaboratively for the last few years, we have the support of the Yunnan CDC, and we are releasing molecules that are not dangerous to people or wildlife.
E. Capabilities:
A brief summary of expertise of the team, including subcontractors and key personnel. A principal investigator for the project must be identified, and a description of the team’s organization. Include a description of the team’s organization including roles and

responsibilities. Describe the organizational experience in this area, existing intellectual property required to complete the project, and any specialized facilities to be used as part of the project. List Government furnished materials or data assumed to be available.
**Note: While only the proposal requires an organization chart, it may be helpful to include in the abstract if we have the space.
• This organization chart would include (as applicable): (1) the programmatic relationship of team members; (2) the unique capabilities of team members; (3) the task responsibilities of team members; (4) the teaming strategy among the team members; (5) key personnel with the amount of effort to be expended by each person during each year.
The lead institution for Project DEFUSE is EcoHealth Alliance, New York, an international research non-profit focused on emerging zoonotic diseases. The project will be led by PI Dr. Peter Daszak, who has 20+ years’ experience managing lab, field and modeling research projects on emerging zoonoses, including as EHA institutional lead, Head of Modeling and Analytics, and member of the Executive Committee for the $130 million USAID EPT/PREDICT. Dr. Daszak will oversee and coordinate all project activities, as well as lead the modeling and analytic work for TA1. Dr. Billy Karesh, who has 40+ years’ experience managing wildlife disease and zoonotic disease projects, will manage partnership activities and relationships and outreach. Dr. Jon Epstein, who has 15 years’ experience working with bats and emerging zoonoses will coordinate work on bat immune priming and boosting trials. Dr. Kevin Olival and Dr. Noam Ross will manage and conduct the modeling and analytical approaches for this project.
Team:
Lead Organization: EcoHealth Alliance, New York
PI: Peter Daszak Ph.D., President & Chief Scientist, EcoHealth Alliance, 3 months/year Key Personnel:
Billy Karesh DVM, Executive VP for Policy & Health, 1 month/year
Kevin J. Olival Ph.D, VP for Scientific Research, 1 month/year
Jonathan H. Epstein DVM Ph.D., VP for Science & Outreach, 0.5 months/year
Carlos Zambrana-Torrelio Ph.D., Assoc. VP for Conservation & Health, 1 month/year Noam Ross Ph.D., Senior Research Scientist, 2 months/year
Evan Eskew, Research Scientist, 2 months/year
Hongying Li, Program Coordinator, China Programs, 3 months/year
TBD Postdoctoral Researcher modeling and analysis, 12 months/year
TBD Research Assistant, 12 months/year

TBD Program Assistant, 12 months/year
Guangjian Zhu Ph.D., Consultant Field Lead, China Programs, 6 months/year Yunzhi Zhang Ph.D., Consultant, Yunnan CDC, China, 2 months/year
Subcontract #1: University of North Carolina Medical School Organizational Lead: Prof. Ralph Baric Ph.D., 2 months/year Dr. Tim Sheahan (6 months/yr)
Dr. Amy Sims (4 months/yr)
Sarah Leist, Postdoctoral fellow (4 months/yr) Boyd Yount, Research Analyst, 12 months/year
Trevor Scobey, Research Technician, 6 months/yr
Subcontract #2: USGS National Wildlife Health Center
Organizational Lead: Tonie Rocke Ph.D., 2 months/year, no salary requested TBD Research Technician, 9 months/year
Subcontract #3: Duke NUS, Singapore
Organizational Lead: Prof. Linfa Wang Ph.D., 2 months/year XXX
TBD Research Assistant, 12 months/year
XXX
Subcontract #4: Wuhan Institute of Virology, China Organizational Lead: Prof Zhengli Shi Ph.D., 2 months/year Peng Zhou Ph.D., 2 months/year
TBD Research Assistant, 12 months/year
Links to relevant papers, reports, or
Do not include more than two resumes as part of the abstract.
**Resumes count against the abstract page limit.
Dr. Peter Daszak is President and Chief Scientist of EcoHealth Alliance, a US-based organization that conducts research and outreach programs on emerging zoonotic diseases. He has published over 300 scientific papers, including the first global map of EID hotspots, strategies to estimate unknown viral diversity in wildlife, predictive models of virus-host relationships, and evidence of the bat origin of SARS-CoV and other emerging viruses. Dr Daszak is Chair of the National Academy of Sciences, Engineering
Deleted: XXX Deleted: TBD Deleted: ssistant
F. If desired, include a brief bibliography
resumes of key performers.
Commented [PD24]: I’m planning to use my resume and Ralph’s. Linfa/Zhengli, I realize your resumes are also very impressive, but I am trying to downplay the non-US focus of this proposal so that DARPA doesn’t see this as a negative.

and Medicine’s Forum on Microbial Threats and is a member of the Executive Committee and the EHA institutional lead for USAID-EPT-PREDICT. He serves on the NRC Advisory Committee to the USGCRP, the DHS CEEZAD External Advisory Board, and the WHO R&D Blueprint Pathogen Prioritization expert group, and has advised the Director for Medical Preparedness Policy on the White House National Security Staff on global health issues. Dr Daszak won the 2000 CSIRO medal for collaborative research.
Prof. Ralph Baric is a UNC Lineberger Comprehensive Cancer Center member and Professor in the UNC-Chapel Hill Department of Epidemiology and Department of Microbiology and Immunology . His work focuses on coronaviruses as models to study the genetics of RNA virus transcription, replication, persistence, and cross species transmission and pathogenesis. Dr. Baric and his group have developed a platform strategy to access the potential “preepidemic” risk associated with zoonotic virus cross species transmission potential and evaluation of countermeasure potential to control future outbreaks of disease ( , PMC4801244, PMC4797993). His work crosses the boundaries of microbiology, virology, immunology and epidemiology, looking especially at the population genetics of viruses to find the molecular building blocks for more effective vaccines.
**General Notes:
• DARPA will evaluate proposals using the following criteria, listed in
descending order of importance:
1) 5.1.1. Overall Scientific and Technical Merit
The proposed technical approach is innovative, feasible, achievable, and complete.
Task descriptions and associated technical elements provided are complete and in a logical sequence with all proposed deliverables clearly defined such that a final outcome that achieves the goal can be expected as a result of award. The proposal identifies major technical risks and planned mitigation efforts are clearly defined and feasible. The proposed PREEMPT Risk Mitigation Plan effectively provides the following: an assessment of potential risks; proposed guidelines to ensure maximal biosafety and biosecurity; a risk management plan for responsible communications; and a plan to address how input from the Government and community stakeholders will be considered regarding communication and publication of potentially sensitive dual-use information.
PMC5798318
,
PMC5567817
, PMC5380844,
PMC5578707
Formatted: Font: (Default) Arial, 11 pt, Font color: Accent 1

2) 5.1.2. Potential Contribution and Relevance to the DARPA Mission
The potential contributions of the proposed effort are relevant to the national technology base. Specifically, DARPA’s mission is to make pivotal early technology investments that create or prevent strategic surprise for U.S. National Security. The proposer clearly demonstrates its capability to transition the technology to the research, industrial, and/or operational military communities in such a way as to enhance U.S. defense. In addition, the evaluation will take into consideration the extent to which the proposed intellectual property (IP) rights will potentially impact the Government’s ability to transition the technology.
3) 5.1.3. Cost Realism
The proposed costs are realistic for the technical and management approach and accurately reflect the technical goals and objectives of the solicitation. The proposed costs are consistent with the proposer's Statement of Work and reflect a sufficient understanding of the costs and level of effort needed to successfully accomplish the proposed technical approach. The costs for the prime proposer and proposed subawardees are substantiated by the details provided in the proposal (e.g., the type and number of labor hours proposed per task, the types and quantities of materials, equipment and fabrication costs, travel and any other applicable costs and the basis for the estimates).
It is expected that the effort will leverage all available relevant prior research in order to obtain the maximum benefit from the available funding. For efforts with a likelihood of commercial application, appropriate direct cost sharing may be a positive factor in the evaluation.
DARPA recognizes that undue emphasis on cost may motivate proposers to
Deleted: ¶
offer low-risk ideas with minimum uncertainty and to staff the effort with junior
personnel in order to be in a more competitive posture. DARPA discourages such cost
strategies.
Commented [EA25]: Please note

Citations
2. 3.
4. 5.
6. 7.
1.
K. J. Olival et al., Host and viral traits predict zoonotic spillover from mammals. Nature 546, 646-650 (2017).
G. Zhang et al., Comparative analysis of bat genomes provides insight into the evolution of flight and immunity. Science 339, 456-460 (2013).
J. Xie et al., Dampened STING-Dependent Interferon Activation in Bats. Cell host & microbe, (2018).
P. Zhou et al., Contraction of the type I IFN locus and unusual constitutive expression of IFN-αin bats. Proceedings of the National Academy of Sciences of the United States of America, 201518240-201518246 (2016).
M. Ahn, J. Cui, A. T. Irving, L.-F. Wang, Unique Loss of the PYHIN Gene Family in Bats Amongst Mammals: Implications for Inflammasome Sensing. Scientific Reports 6, (2016).
J. Zhao et al., Intranasal Treatment with Poly(I.C) Protects Aged Mice from Lethal Respiratory Virus Infections. Journal of Virology 86, 11416-11424 (2012).
J. Wu et al., Poly(I:C) Treatment Leads to Interferon-Dependent Clearance of Hepatitis B Virus in a Hydrodynamic Injection Mouse Model. Journal of Virology 88, 10421-10431 (2014).
Attachment 1: Executive Summary Slide template

8.
9. 10.
11. 12.
X. F. Deng et al., A Chimeric Virus-Mouse Model System for Evaluating the Function and Inhibition of Papain-Like Proteases of Emerging Coronaviruses. Journal of Virology 88, 11825-11833 (2014).
T. E. Rocke et al., Sylvatic Plague Vaccine Partially Protects Prairie Dogs (Cynomys spp.) in Field Trials. Ecohealth 14, 438-450 (2017).
B. Stading et al., Protection of bats (Eptesicus fuscus) against rabies following topical or oronasal exposure to a recombinant raccoon poxvirus vaccine. Plos Neglect. Trop. Dis. 11, (2017).
B. R. Stading et al., Infectivity of attenuated poxvirus vaccine vectors and immunogenicity of a raccoonpox vectored rabies vaccine in the Brazilian Free-tailed bat (Tadarida brasiliensis). Vaccine 34, 5352-5358 (2016).
P. Zhou et al., Fatal Swine Acute Diarrhea Syndrome caused by an HKU2- related Coronavirus of Bat Origin. Nature In press, (2018
).

From: Sent: To: Cc:
Subject: Attachments:
Likewise, I added my comments to Ralph's document. I added some detail, but not too much, so let me know if you want more. Best -Tonie
On Thu, Feb 8, 2018 at 10:22 AM, Baric, Ralph S <rbaric@email.unc.edu> wrote: I have built in my comments atop of Linfa’s comments. ralph
From: Wang Linfa [mailto:linfa.wang@duke-nus.edu.sg]
Sent: Thursday, February 8, 2018 7:25 AM
To: Peter Daszak <daszak@ecohealthalliance.org>; Baric, Ralph S <rbaric@email.unc.edu>; Zhengli Shi (zlshi@wh.iov.cn) <zlshi@wh.iov.cn>; William B. Karesh <karesh@ecohealthalliance.org>; Rocke, Tonie <trocke@usgs.gov>
Cc: Luke Hamel <hamel@ecohealthalliance.org>; Jonathon Musser <musser@ecohealthalliance.org>; Anna Willoughby <willoughby@ecohealthalliance.org>; Kevin Olival, PhD <olival@ecohealthalliance.org>; Jon Epstein <epstein@ecohealthalliance.org>; Noam Ross <ross@ecohealthalliance.org>; Aleksei Chmura <chmura@ecohealthalliance.org>; Anna Willoughby <willoughby@ecohealthalliance.org>; Hongying Li <li@ecohealthalliance.org>
Subject: RE: First (rough) draft of the DARPA abstract - Project DEFUSE
See my brief notes/edits in the attached.
I am working on a large grant here in SG and won’t be able to spend too much time until next week.
LF
Linfa (Lin-Fa) WANG, PhD FTSE
Rocke, Tonie <trocke@usgs.gov>
Thursday, February 8, 2018 10:01 AM
Baric, Ralph S
Wang Linfa; Peter Daszak; Zhengli Shi (zlshi@wh.iov.cn); William B. Karesh; Luke Hamel; Jonathon Musser; Anna Willoughby; Kevin Olival, PhD; Jon Epstein; Noam Ross; Aleksei Chmura; Hongying Li
Re: First (rough) draft of the DARPA abstract - Project DEFUSE
DARPA (PREEMPT) Abstract EcoHealth Alliance DEFUSE 1st Draft-LW180208-rsb- ter.docx
1

Professor & Director
Programme in Emerging Infectious Disease Duke-NUS Medical School,
8 College Road, Singapore 169857
Tel: +65 6516 8397
From: Peter Daszak [mailto:daszak@ecohealthalliance.org]
Sent: Thursday, 8 February, 2018 10:51 AM
To: Ralph Baric (rbaric@email.unc.edu); Wang Linfa; Zhengli Shi (zlshi@wh.iov.cn); William B. Karesh; Rocke, Tonie Cc: Luke Hamel; Jonathon Musser; Anna Willoughby; Kevin Olival, PhD; Jon Epstein; Noam Ross; Aleksei Chmura; Anna Willoughby; Hongying Li
Subject: First (rough) draft of the DARPA abstract - Project DEFUSE
Importance: High
Dear All,
I’ve attached a first rough draft of the DARPA abstract. Apologies for the delay. Unfortunately, edits to my Science paper came through on Friday and took many hours to do, so this delayed me. I’m right now in Geneva in my hotel at 3 am finishing these off before flying back to NYC from a WHO meeting.
Some important points:
1) Zhengli, Linfa, Ralph – Billy and I spoke with Tonie Rocke on Friday. Tonie is at the National Wildlife Health Center, Madison USA, and has worked on wildlife vaccines: plague in prairie dogs, rabies in Jamaican fruit bats, white nose syndrome in US bats. We needed someone with expertise in delivery of molecules/vaccines to wildlife because DARPA specifically lay that out. As you’ll see, Tonie is perfect for our project and will be able to do work at USGS NWHC and with Zhengli in China to help with TA2
2) Zhengli and Linfa – After I spoke with you both, I had a great conversation with Ralph Baric. He proposed to work on recombinant chimeric spike proteins as a second line of attack. I think that is a perfect fit because 1) it’s his expertise and he has published on it, 2) it will act as an alternative to the blue-sky and risky immune boosting work that Linfa/Peng have proposed. I hope you agree!
2

3) Ralph, Zhengli, Linfa, Tonie – as you can see, I have mangled the language/technical details for most of your sections. Pardon my lack of knowledge, and please draft a couple of paragraphs each to make your sections look correct. Thanks to Peng for giving me some text anyway – very useful, but please check what I’ve done with it.
4) All – please add some names and details on the team part so we get clarity in this on what staff you will need to do the work.
5) Please don’t worry about keeping this to the 8 page limit. Just add text here and there, references, and edit to make what I’ve written correct, and more exciting. I will work on this on Saturday, Sunday and Monday to bring it down to 8 pages of very crisp, super-exciting text. I also want as many of your good ideas in here, so that I can use this draft to build on for the full proposal.
6) Finally – please edit rapidly using tracked changes in word. If you don’t want to mess up endnote, please just insert references as comment boxes and we’ll pull them off the web.
Aleksei and Anna: please read the draft and work on some draft image designs that sum up the project flow. I’ll call you Thursday afternoon to discuss so you can finish them off.
Luke – please have a go at a first draft of the executive summary slide. I’ll pick up from what you’ve done once you send it to me.
Thanks again to all of you for agreeing to collaborate on this proposal. From what I know of the competition, what DARPA wants, and what we’re offering, I think we have an extremely strong team, so I’m looking forward to getting the full proposal together and winning this contract!
Cheers,
Peter
3


DARPA – PREEMPT – HR001118S0017
Abstract Submission Requirements:
**8 pages with 12 point font or higher (smaller font may be used for figures, tables
and charts)
**Page limit includes all figures, tables, charts and the Executive Summary Slide **Copies of all documents submitted must be clearly labeled with the following:
-DARPA BAA number
-Proposer Organization
-Proposal title/Proposal short title
-Submission letter is optional and does not count towards page limit
A. Cover Sheet (does not count towards page limit):
Include the administrative and technical points of contact (name, address, phone, fax, email, lead organization). Also include the BAA number, title of the proposed project, primary subcontractors, estimated cost, duration of project, and the label “ABSTRACT.”
B. Executive Summary Slide:
Provide a one slide summary in PowerPoint that effectively and succinctly conveys the main objective, key innovations, expected impact, and other unique aspects of the proposed project. Use the slide template provided at http://www.fbo.gov.
**See slide template at bottom of document.
Clearly describe what is being proposed and what difference it will make (qualitatively and quantitatively), including brief answers to the following questions:
1. What is the proposed work attempting to accomplish or do?
We aim to defuse the potential for emergence of novel bat-origin high-zoonotic risk SARS-related coronaviruses in Southeast Asia. We envisage a scenario whereby the US warfighter is called on to intervene in a security hotspot in SE Asia for a period of 3-6 months. As planners begin choosing sites for the mission, they will use an app we will design to assess the background risk of a site harboring dangerous zoonotic viruses. If
PROJECT DEFUSE
C. Goals and Impact:

there is no alternative to a high-risk site, a tactical forward team will deploy automated delivery technology we will develop in caves that harbor bats carrying these viruses. These devices will release immune boosting molecules and chimeric polyvalent spike protein immune priming inocula to lower viral shedding from bats at the site for a few weeks or months, allowing our warfighters to execute the operation at lowered risk for spillover.
2. How is it done today? And what are the limitations?
Currently, there is no available technology to reduce the risk of exposure to novel coronaviruses from bats, other than avoid the regions where bats harbor these viruses. This includes large areas of southeast Asia where SARS-related CoVs are endemic in bats, which roost in caves during the day, but forage over wide areas at night, shedding virus in their feces and urine. The limitations of this lack of capacity are significant – we have shown evidence of recent spillover of SARS-related CoVs into people in southern China, and have identified viruses in this region that are capable of producing SARS-like illness in humanized mice, with no available vaccines or countermeasures. These viruses are a clear-and-present danger to our military personnel, and to global health security.
3. What is innovative in your approach and how does it compare to current practice and state-of-the-art (SOA)?
**Note: DARPA wants to know, “how the proposed project is revolutionary and how it significantly rises above the current state of the art
Our group has shown that bats harbor the highest proportion of potential zoonoses of any mammal group, and that they are able to live with the host without causing diseases due to unique damping of certain pathways in their immune systems, likely in part as an evolutionary adaptation to flight. We will use this new finding to design strategies, like small molecule Rig like receptor (RLR) or Toll like receptor (TLR) agonists, to upregulate their immune response in their cave roosts, down-regulate viral replication, and reduce the risk of viral shedding and spillover (broad immune boosting strategy). At the same time, we will inoculate bats with novel chimeric polyvalent recombinant spike proteins to enhance their immune response against replication of specific, high-risk viruses (targeted immune priming strategy). We will use our innovative modeling to design apps that identify the likelihood of any region harboring high-risk bat viruses. We will design novel, automated approaches to deliver both types of inoculum remotely into caves to reduce exposure risk during decontamination.
4. What are the key technical challenges in your approach and how do you plan to overcome these?

Decide which of following parts to talk about:
Sampling/testing
Reverse engineering, binding assays, mouse expts Modeling viral risk of evolution and spillover
Gain-of-function issue.
Inoculum delivery
Mesocosm expts
Cave expts
5. Who will care and what will the impact be if you are successful?
This will have direct relevance to the warfighter. The potential for deployment to the region in which bat hosts of SARS-related CoVs exist is high – countries include security hotspots (Myanmar, Bangladesh, Pakistan, Lao, Korea, Vietnam and Cambodia?). The ability to decontaminate and defuse these viruses will be useful in preventing potentially devastating illness. Furthermore, these technologies, if successful, can be adapted to hosts of other bat-origin CoVs (MERS, SARS and related prepandemic zoonotic strains), and potentially other zoonotic bat-origin viruses (Hendra, Nipah, EBOV). Finally, our approach is directly applicable to public health measures in the region to reduce the risk of spillover into the general population, as well as for food security by reducing the risk of viruses like SADS-CoV spilling over from bats into intensive pig farms and devastating the industry, leading to potential civil unrest.
6. How much will it cost and how long will it take?
Will insert this later after calculating and brainstorming. 46 months
D. Technical Plan:
Outline and address all technical challenges inherent in the approach and possible solutions for overcoming potential problems. This section should provide appropriate specific milestones (quantitative, if possible) at intermediate stages of the project to demonstrate progress and a brief plan for accomplishment of the milestones. **Note: “The technical plan should demonstrate a deep understanding of the
technical challenges and present a credible (even if risky) plan to achieve
Modeling bat suitability
Inventory of caves
Modeling inoculation/defusing strategy
Immune modulation
Immune Booster recombinant production

the program goal”
Key Terms/Aspects to Emphasize in Abstract ● IACUC/IRB
○ DARPA wants to know who has experience w/ ACURO IACUC work.
■ EHA has multiple ACURO IACUC proposals (either approved or
undergoing approval)
■ IRB also in place, just has to be modified
Overview
Rationale for the SE Asian SARS-related CoV – Rhinolophus bat target system, and immune priming/boosting: 1) Our group has shown that bats harbor a higher proportion of potentially highly heterogeneous zoonotic viruses than any other mammalian group (1), so that proof-of-concept for blocking viral spillover from this host group may lead to a bigger impact on global health security; 2) The Rhinolophus bats that harbor SARS like-CoVs are insectivorous and roost in dense colonies at fixed, known locations, yet disperse each night over wide distances from these sites. Defusing the risk of viral shedding in the roost will also defuse the risk of viral shedding over the population range. This would be difficult for rodent or primate reservoirs; 3) Bats are mammalian hosts, therefore immune modulating drugs evaluated in people and rodents may also work on bats. This would be less likely for an insect vector; 4) Members of our collaborative group has worked together on bats and their viruses for over 15 years, with a total of >100 yrs experience focused on bat-origin zoonoses among the key personnel. We have published much of the seminal work on the bat origins of SARS, Nipah, Hendra, and MERS viruses, and have opened new boundaries in studies of bat host-viral relationships ecologically, immunologically and virologically; 5) The South and Southeast Asian region where these bats occur is a security hotspot, with active political and ethnic conflicts, and displaced populations in Bangladesh, Pakistan, Myanmar, Thailand, Indonesia, Philippines and other countries. This is a likely potential site for US warfighter deployment; 6) We have worked for over 10 years on the SARS-related CoV – Rhinolophus bat system in China, demonstrating the origin of SARS-CoV within this host, the presence of SARSr-CoVs with remarkable sequence identity in the spike protein to SARS-CoV, their isolation and characterization of their ability to bind and replicate efficiently in primary human lung airway cells. We have demonstrated that chimeric SARS-CoV backbone with spike protein from SARSr-CoVs from our cave sites in Yunnan Province can infect a humanized mouse model and cause SARS-like illness, and that clinical signs are not reduced with SARS monoclonal therapy or vaccination. Finally, we have demonstrated that people living up to 6 kilometers from our cave site have

evidence of SARSr-CoV antibodies (3% seroprevalence in 200+ cohort), suggesting active spillover, and marking these viruses as a clear-and-present danger of a new SARS-like pandemic; 7) SARSr-CoVs are transmitted among bats via fecal-oral route, making sampling relatively easy (collection of fresh fecal pellets) and molecule or vaccine approaches feasible; 8) Proof-of-concept in this system may be rapidly scalable to other bat-coronavirus systems, e.g. MERS-CoV, SADS-CoV, and to other cave bat origin viruses.
Other important bat-origin zoonotic viruses (e.g. filoviruses, henipaviruses) have very rare spillover events - usually to a single index case- making validated prevention of spillover challenging. These viruses also show little strain diversity ,which also makes it more difficult to model which evolutionary lines are high-risk. Conversely, SARSr-CoVs are diverse, with recombinants regularly identified in the field and lab. Furthermore, we have identified SARS-like strains in a single cave in Yunnan that harbor every gene found in the human SARS-CoV strains detected during the 2002-2003 epidemic. Within this bat population, an ideal evolutionary soup exists that could produce new human strains by high frequency RNA recombination, and thus, it presents a perfect target for 21st generation intervention strategies.
Finally, we believe that alternative approaches to transmission blocking, e.g. CRISPER-Cas gene drives that are likely to be far less effective in bats because most bats are long-lived relative to their small size, have long inter-generational periods (2-5 yrs) and low progeny (~1-2 pups). Gene drives would likely take many decades to run through a population, so that proof-of-concept of transmission blocking in the DARPA time scale wouldn’t be possible. Furthermore, many bat species’ populations mix readily or migrate which would disperse the impact of gene drives, whereas targeting a small number of caves in a region for molecule or vaccine delivery would cover a very large dispersal area.
TA1: Develop and validate integrated, multiscale models that quantify the likelihood a human-capable virus will emerge from an animal reservoir residing in a “hot spot” geographic region
The DEFUSE modeling and analytics team will develop models to evaluate the likelihood of bat caves harboring high-risk SARSr-CoVs, evaluate the probability of specific SARS- related CoV spillover, and identify the most effective strategy for inoculation of immune boosting molecules and chimeric spike protein immune priming inocula.
We will collect specific data to inform our model building, validate assumptions and refine predictions. At the start of Yr 1, we will conduct a full inventory of host and virus distribution within our field sites, two caves in Yunnan Province, China. This builds on 8 years of surveillance in these caves and includes a cave in which we have identified all the genetic components of the 2002-2003 epidemic SARS-CoV distributed across a

bat population. Two other caves will act as controls/comparison sites, in that we have not yet identified the high-risk SARSr-CoVs in that cave. We will assess: the population density, distribution and segregation of individual bats; changes in these daily, weekly and by season; viral prevalence and intensity in individuals; distribution of low- and high-risk SARSr-CoV strains, and how readily these are transmitted among bat species, age classes, genders; and using mark-recapture to assess metapopulation structure. To assess geographic distribution of bat hosts, we have access to biological inventory data on all bat caves in Southern China, as well as information on species distributions across SE Asia from the literature and museum records. We will use radio- and satellite telemetry to identify the home range of each species of bat in the caves, to assess how widely the viral ‘plume’ could contaminate surrounding regions, and therefore how wide the risk zone is for the warfighter positioned close to bat caves.
We will build environmental niche models using the data above, environmental and ecological correlates, and traits of cave species communities (eg. phylogenetic and functional diversity), to predict the species composition of bat caves across Southern China, South and SE Asia. We will validate these with data from the current project and data from PREDICT sampling in Thailand, Indonesia, Malaysia and other SE Asian countries. We will then use our unique database of bat host-viral relationships updated from our recent Nature paper (1) to assess the likelihood of low- or high-risk SARSr- CoVs being present in a cave at any site across the region. At the end of Yr 1, we will use these analyses to produce a prototype app for the warfighter that identifies the likelihood of bats harboring dangerous viral pathogens based on these analyses. The ‘high-risk bats near me’ app will be updated as new host-viral surveillance data comes on line from our project and others, to ground-truth and fine-tune its predictive capacity. Specifically, our telemetry data on bat movement will be used to assess how often bats from high-risk caves migrate to other colonies and potentially spread their high-risk strains.
The Wuhan Institute of Virology team will conduct viral testing on samples from all bat species in the caves as part of this inventory. Fecal, oral, blood and urogenital samples will be collected from bats using standard capture techniques as we have done for the last decade. In addition, tarps will be laid down in caves to assess the feasibility of surveys using pooled fresh fecal and urine samples. Assays will be designed to correlate viral load in an individual with viral shedding in a fecal sample. Once this is complete, surveys will continue largely on fecal samples so as not to disturb bat colonies and undermine longitudinal sampling capacity. Samples will be tested by PCR and spike proteins of all SARS-related CoVs sequenced. Analyses of phylogeny, recombination events, and further characterization of high-risk viruses (those with spike proteins close to SARS-CoV) will be carried out (REF). Isolation will be attempted on a subset of

samples with novel SARSr-CoVs. Prof. Ralph Baric, UNC, will reverse engineer spike proteins in his lab to conduct binding assays to human ACE2 (the SARS-CoV receptor). Their group have also devised new strategies to culture SARS-like bat coronaviruses, allowing biological characterization of both high risk strains that can replicate in primary human cells and low risk strains that can only replicate in the presence of exogenous enhancers. Viral spike glycoproteins that bind receptor will then be inserted into SARS- CoV backbones, and inoculated into human cells and humanized mice to assess their capacity to cause SARS-like disease, and their ability to be blocked by monoclonal therapies, or vaccines against SARS-CoV ((PMC5798318, PMC5567817, PMC5380844, PMC5578707, PMC4801244, PMC4797993). The Baric group has also demonstrated that a nucleoside analogue inhibitor, GS-5734 (Gilead Inc), blocks epidemic, preepidemic and zoonotic SARS-CoV and SARS-like bat coronavirus replication in primary human airway cells and in mice (PMC5567817). Consequently, they will evaluate the ability of this drug to block replication of newly disovered pre-epidemic and zoonotic high risk strains. As the drug has been used to effectively treat Ebola virus infected patients (PMC4967715, PMC5583641) as well and has potent activity against Nipha and Hendra viruses (PMC5338263), an alternative intervention for military personnel is prophylactic treatment treatment prior to deployment into high risk settings.
The modeling team will use these data to build models of 1) risk of viral evolution and spillover, and 2) strategies to maximize inoculation strategy.
Data on the diversity of bat spike proteins, prevalence of recombinant CoVs, ability to bind and infect human cells, degree of clinical signs in mouse models, will be used to estimate evolutionary rates, rates of recombination, and capacity to generate novel strains capable of human infection. Using dynamic metapopulation models, we will estimate the flow of genes within each bat cave, based on the known host and viral assemblages. This will inform how rapidly new CoV strains with distinct phenotypic characteristics evolve. Because of our unique collaboration among world-class modelers and coronavirologists, we will be able to test model predictions of viral capacity for spillover by conducting spike protein-based binding and cell culture experiments. The BSL-2 nature of work on SARSr-CoVs makes our system highly cost-effective relative to other bat-virus systems (e.g. Ebola, Marburg, Hendra, Nipah), which require BSL-4 level facilities for cell culture.
We will use modeling approaches, the data above, and other biological and ecological data to estimate how rapidly high-risk SARSr-CoVs will re-colonize a bat population following immune boosting or priming. We will obtain model estimates of the frequency of inoculation required for both approaches, what proportion of a population needs to be reached to have effective viral dampening, and whether specific seasons, or locations within a cave would be more effective to treat. We will then model

the efficacy of different delivery methods (spray, swab, cave mouth automated delivery, deliver to specific sections of a cave).
TA2: Develop scalable approaches that target and suppress the animal virus in its reservoir(s) and/or vector(s), to reduce the likelihood of virus transmission into humans.
Our goal is to test? two approaches to defuse the potential for SARS-related CoVs to emerge in people: 1) Immune Boosting: using the unique immunological features of bats that our group has discovered, we will inoculate live bats in cave mesocosms with immune modulators designed to up-regulate their naïve immunity and assess their ability to suppress viral replication and shedding; 2) Immune Priming: building on preliminary development of polyvalent chimeric recombinant molecules targeting diverse spike proteins from bat SARS-related CoVs, we will conduct inoculation trials with live bats to assess suppression of replication and shedding of a broad range of dangerous SARS-related CoVs. Both lines of work will begin in Yr 1 and run parallel throughout the project.
Prof. Linfa Wang (Duke-NUS) will lead the work on immune boosting work, building on his pioneering work on bat immunity (2). This work provides evidence that that the long-term coexistence of bats and their viruses has led to an equilibrium between viral replication and host immunity, whereby bats have specifically down- regulated their innate immune system as part of the fitness cost of flight (the only true flying mammals) (2). The nature of the weakened but not entirely lost functionality of bat innate immunity factors like STING, a central DNA-interferon (IFN) sensing molecule, may have profound impact for bats to maintain the balanced state of “effective response”, but not “over response” against viruses (3). A similar finding was also observed in bat IFNA studies, which is less abundant but was constitutively expressed without stimulation (4). Given native levels of SARSr-CoVs in individual bats with damped immunity, we propose to suppress bat SARSr-CoV by boosting bat innate immunity through the IFN pathway, and breaking the natural host-virus equilibrium. One of the potential problems with this approach is that it can lead to severe inflammation. However, this is unlikely to occur in bats, because they also have a naturally dampened inflammation response (5).
Previous work has shown that aerosol spraying or intranasal inoculation of IFN or other small molecules has led to reduce viral loads in humans, ferrets and mouse models (12-14). We will therefore initially trial inoculation of live bats with synthetic double-stranded RNA (Poly I:C) and assay for reduced viral loads (DETAILS, CITATION). We will generate universal bat interferon and apply to bats in the lab. Interferon has been used extensively clinically if no viral-specific drugs are available, e.g. against

filoviruses (11). Secondly, bat replication of SARSr-CoV is sensitive to interferon treatments, as has been shown in our previous work (12). We will attempt to boost bat IFN by blocking bat-specific IFN negative regulator. Bat IFNA is naturally constitutively expressed but cannot be induced to a high level (4). This is unique to bats. We think there should be a negative regulatory factor in the bat interferon production pathway. We propose using CRISPRi to find out that negative regulator and then screen for chemicals targeting at this gene. We will attempt to boost bat IFN by activating dampened bat-specific IFN production pathways which include DNA-STING-dependent and ssRNA-TLR7 dependent pathways. These changes have been proved to bat-specific, suggesting that they are important in viruses/bats coexistence, and supported by our own work showing that a mutant bat STING restores antiviral functionality (3). By identifying small molecules to directly activate downstream of STING, we have chance to activate bat interferon and then help bats to clear viruses. Similar strategy applies to ssRNA-TLR7 dependent pathways. We will also attempt to boost bat IFN by activating functional bat IFN production pathways. We will investigate if there are other IFN production pathways in bats. We then boost bat immune responses by ligands specifically to these pathways, e.g. polyIC to TLR3-IFN pathway or 5’ppp-dsRNA to RIG-I- IFN pathway. A similar strategy has been tested successful in mouse model for SARS- CoV, IAV or HBV (6, 7). We believe treating wild bats with IFN-modulating small molecules by spraying is superior to other invasive strategies that might be considered by DARPA, including genome editing (CRISPR or RNAi), vaccination or DIP bats, in terms of its deployability and scalability. Finally, we will inoculate bats with fragments of non- bat Coronavirus (DETAILS).
Prof. Ralph Baric (UNC) will lead the immune priming work, building on his track record in reverse-engineering and manipulating SARS-CoV, MERS-CoV and other virus spike proteins over the last two decades . He will develop recombinant chimeric spike- proteins (8) based on SARSr-CoVs we have already identified, and those we will discover and characterize during project DEFUSE.
While there are clear advantages to working with fixed populations of cave- dwelling bats, molecule or vaccine delivery is technically challenging. Dr. Tonie Rocke, who developed, trialed, field-tested and rolled out the prairie dog plague vaccine (9), and is currently working on vaccines to bat rabies (10, 11) and white-nose syndrome, will manage a series of experiments in the lab and field to perfect a delivery system for both arms of TA2.
We have found that the immune dampening features are highly conserved in all bat species tested so far. Duke-NUS has established a breeding colony of cave nectar
RALPH – clearly I didn’t really understand the
details of your approach. Can you add a couple of paragraphs here and some citations
please!

bats for experimental use (one of very few experimental bat breeding colonies in the world and the only one in SE Asia!). So our initial proof of concept test can be done in this experimental colony. We will then extend the test to a small group of wild-caught Rhinolophus sinicus bats at Wuhan Institute of Zoology. We (Prof. Wang) have previous experience conducting SARS-CoV infection experiments with bat species from the same genus in the BSL4 facility at the Australian Animal Health Laboratory in Australia (L.Wang, unpublished results). First, we will use our recently proven technology to design LIPS assays to the specific high zoonotic-risk SARSr-CoVs (12). We will conduct serological analysis on bats captured for infection experiments, to assess prior exposure to specific strains. These LIPS assays will be made available for use in people to assess exposure of the general population around bat caves in China, and for potential use by the warfighter to assess exposure to SARSr-CoVs during combat missions.
Finally, work on a delivery method will be overseen by Dr. Tonie Rocke at the US Geological Survey, National Wildlife Health Center, who has proven capacity to develop and take animal vaccines through to licensure (9). Using locally acquired insectiverous bats like Tadarida brasiliensis or Eptesicus fuscus (10, 11) as proxies, Dr. Rocke will further develop and assess delivery vehices (mediums) and methods of delivery for the molecules, inocula proposed above, including: 1) transdermally applied nanoparticles; 2) sticky edible gels that bats will groom from themselves and each other; 3) aerosolization via spayers that could be used in cave settings; and 4) automated sprays triggered by timers and movement detectors at critical cave entry points. Simple gels have already been used to vaccinate big brown bats against rabies (11) in a laboratory setting, and hand delivery of these gels containing biomarkers (no vaccine) to vampire bats (Desmodus rotundus) in Peru and Mexico have shown they are readily consumed and transferred between bats. Methods to improve uptake (different gels, nanoparticles) and mechanize delivery methods (aerosolization) will be tested first in a laboratry setting, and secondly in local field settings using the biomarker, rhodamine B (which marks hair and whiskers upon consumption) to assess uptake by bats. The most optimal approaches will then be tested on live bats in our three cave sites in Yunnan Province with the most successful immunomodulators developed in TA1?. Fieldwork will be conducted under the auspices of Dr. Rocke, EHA field staff, and Dr. Yunzhi Zhang (Yunnan CDC, Consultant with EcoHealth Alliance). Sections of bat caves will be cordoned off and different application methods tested. A small number of bats will be captured and assayed for viral load after treatment, but so as not to disturb the colony, most viral load work will be conducted on fresh fecal pellets collected daily on the cave floor. EHA has unique access to these sites in Yunnan Province, with our field teams conducting surveillance there for around 10 years, under the guidance of Drs. Shi and Zhang. In year 1 of project DEFUSE, we will seek permission for these experimental

inoculations in cave sites in Yunnan from the Provincial Forestry Department. We do not envisage problems getting permission, as we have worked with the Forestry Department collaboratively for the last few years, we have the support of the Yunnan CDC, and we are releasing molecules that are not dangerous to people or wildlife.
E. Capabilities:
A brief summary of expertise of the team, including subcontractors and key personnel. A principal investigator for the project must be identified, and a description of the team’s organization. Include a description of the team’s organization including roles and responsibilities. Describe the organizational experience in this area, existing intellectual property required to complete the project, and any specialized facilities to be used as part of the project. List Government furnished materials or data assumed to be available.
**Note: While only the proposal requires an organization chart, it may be helpful to include in the abstract if we have the space.
• This organization chart would include (as applicable): (1) the programmatic relationship of team members; (2) the unique capabilities of team members; (3) the task responsibilities of team members; (4) the teaming strategy among the team members; (5) key personnel with the amount of effort to be expended by each person during each year.
The lead institution for Project DEFUSE is EcoHealth Alliance, New York, an international research non-profit focused on emerging zoonotic diseases. The project will be led by PI Dr. Peter Daszak, who has 20+ years’ experience managing lab, field and modeling research projects on emerging zoonoses, including as EHA institutional lead, Head of Modeling and Analytics, and member of the Executive Committee for the $130 million USAID EPT/PREDICT. Dr. Daszak will oversee and coordinate all project activities, as well as lead the modeling and analytic work for TA1. Dr. Billy Karesh, who has 40+ years’ experience managing wildlife disease and zoonotic disease projects, will manage partnership activities and relationships and outreach. Dr. Jon Epstein, who has 15 years’ experience working with bats and emerging zoonoses will coordinate work on bat immune priming and boosting trials. Dr. Kevin Olival and Dr. Noam Ross will manage and conduct the modeling and analytical approaches for this project.
Team:
Lead Organization: EcoHealth Alliance, New York
PI: Peter Daszak Ph.D., President & Chief Scientist, EcoHealth Alliance, 3 months/year

Key Personnel:
Billy Karesh DVM, Executive VP for Policy & Health, 1 month/year
Kevin J. Olival Ph.D, VP for Scientific Research, 1 month/year
Jonathan H. Epstein DVM Ph.D., VP for Science & Outreach, 0.5 months/year
Carlos Zambrana-Torrelio Ph.D., Assoc. VP for Conservation & Health, 1 month/year Noam Ross Ph.D., Senior Research Scientist, 2 months/year
Evan Eskew, Research Scientist, 2 months/year
Hongying Li, Program Coordinator, China Programs, 3 months/year
TBD Postdoctoral Researcher modeling and analysis, 12 months/year
TBD Research Assistant, 12 months/year
TBD Program Assistant, 12 months/year
Guangjian Zhu Ph.D., Consultant Field Lead, China Programs, 6 months/year
Yunzhi Zhang Ph.D., Consultant, Yunnan CDC, China, 2 months/year
Subcontract #1: University of North Carolina Medical School Organizational Lead: Prof. Ralph Baric Ph.D., 2 months/year Dr. Tim Sheahan (6 months/yr)
Dr. Amy Sims (4 months/yr)
Sarah Leist, Postdoctoral fellow (4 months/yr) Boyd Yount, Research Analyst, 12 months/year Trevor Scobey, Research Technician, 6 months/yr
Subcontract #2: USGS National Wildlife Health Center
Organizational Lead: Tonie Rocke Ph.D., 2 months/year, no salary requested TBD Research Technician, 9 months/year
Subcontract #3: Duke NUS, Singapore
Organizational Lead: Prof. Linfa Wang Ph.D., 2 months/year XXX
TBD Research Assistant, 12 months/year
XXX
Subcontract #4: Wuhan Institute of Virology, China Organizational Lead: Prof Zhengli Shi Ph.D., 2 months/year Peng Zhou Ph.D., 2 months/year
TBD Research Assistant, 12 months/year
F. If desired, include a brief bibliography

Links to relevant papers, reports, or resumes of key performers.
Do not include more than two resumes as part of the abstract.
**Resumes count against the abstract page limit.
Dr. Peter Daszak is President and Chief Scientist of EcoHealth Alliance, a US-based organization that conducts research and outreach programs on emerging zoonotic diseases. He has published over 300 scientific papers, including the first global map of EID hotspots, strategies to estimate unknown viral diversity in wildlife, predictive models of virus-host relationships, and evidence of the bat origin of SARS-CoV and other emerging viruses. Dr Daszak is Chair of the National Academy of Sciences, Engineering and Medicine’s Forum on Microbial Threats and is a member of the Executive Committee and the EHA institutional lead for USAID-EPT-PREDICT. He serves on the NRC Advisory Committee to the USGCRP, the DHS CEEZAD External Advisory Board, and the WHO R&D Blueprint Pathogen Prioritization expert group, and has advised the Director for Medical Preparedness Policy on the White House National Security Staff on global health issues. Dr Daszak won the 2000 CSIRO medal for collaborative research.
Prof. Ralph Baric is a UNC Lineberger Comprehensive Cancer Center member and Professor in the UNC-Chapel Hill Department of Epidemiology and Department of Microbiology and Immunology . His work focuses on coronaviruses as models to study the genetics of RNA virus transcription, replication, persistence, and cross species transmission and pathogenesis. Dr. Baric and his group have developed a platform strategy to access the potential “preepidemic” risk associated with zoonotic virus cross species transmission potential and evaluation of countermeasure potential to control future outbreaks of disease (PMC5798318, PMC5567817, PMC5380844, PMC5578707, PMC4801244, PMC4797993). His work crosses the boundaries of microbiology, virology, immunology and epidemiology, looking especially at the population genetics of viruses to find the molecular building blocks for more effective vaccines.
**General Notes:
• DARPA will evaluate proposals using the following criteria, listed in
descending order of importance:
1) 5.1.1. Overall Scientific and Technical Merit
The proposed technical approach is innovative, feasible, achievable, and complete.

Task descriptions and associated technical elements provided are complete and in a logical sequence with all proposed deliverables clearly defined such that a final outcome that achieves the goal can be expected as a result of award. The proposal identifies major technical risks and planned mitigation efforts are clearly defined and feasible. The proposed PREEMPT Risk Mitigation Plan effectively provides the following: an assessment of potential risks; proposed guidelines to ensure maximal biosafety and biosecurity; a risk management plan for responsible communications; and a plan to address how input from the Government and community stakeholders will be considered regarding communication and publication of potentially sensitive dual-use information.
2) 5.1.2. Potential Contribution and Relevance to the DARPA Mission
The potential contributions of the proposed effort are relevant to the national technology base. Specifically, DARPA’s mission is to make pivotal early technology investments that create or prevent strategic surprise for U.S. National Security. The proposer clearly demonstrates its capability to transition the technology to the research, industrial, and/or operational military communities in such a way as to enhance U.S. defense. In addition, the evaluation will take into consideration the extent to which the proposed intellectual property (IP) rights will potentially impact the Government’s ability to transition the technology.
3) 5.1.3. Cost Realism
The proposed costs are realistic for the technical and management approach and accurately reflect the technical goals and objectives of the solicitation. The proposed costs are consistent with the proposer's Statement of Work and reflect a sufficient understanding of the costs and level of effort needed to successfully accomplish the proposed technical approach. The costs for the prime proposer and proposed subawardees are substantiated by the details provided in the proposal (e.g., the type and number of labor hours proposed per task, the types and quantities of materials, equipment and fabrication costs, travel and any other applicable costs and the basis for the estimates).
It is expected that the effort will leverage all available relevant prior research in order to obtain the maximum benefit from the available funding. For efforts with a likelihood of commercial application, appropriate direct cost sharing may be a positive factor in the evaluation. DARPA recognizes that undue emphasis on cost may motivate proposers to offer low-risk ideas with minimum uncertainty and to staff the effort with junior personnel in order to be in a more competitive posture. DARPA discourages such cost strategies.

Citations
2. 3.
4. 5.
6. 7.
1.
Attachment 1: Executive Summary Slide template
K. J. Olival et al., Host and viral traits predict zoonotic spillover from mammals. Nature 546, 646-650 (2017).
G. Zhang et al., Comparative analysis of bat genomes provides insight into the evolution of flight and immunity. Science 339, 456-460 (2013).
J. Xie et al., Dampened STING-Dependent Interferon Activation in Bats. Cell host & microbe, (2018).
P. Zhou et al., Contraction of the type I IFN locus and unusual constitutive expression of IFN-αin bats. Proceedings of the National Academy of Sciences of the United States of America, 201518240-201518246 (2016).
M. Ahn, J. Cui, A. T. Irving, L.-F. Wang, Unique Loss of the PYHIN Gene Family in Bats Amongst Mammals: Implications for Inflammasome Sensing. Scientific Reports 6, (2016).
J. Zhao et al., Intranasal Treatment with Poly(I.C) Protects Aged Mice from Lethal Respiratory Virus Infections. Journal of Virology 86, 11416-11424 (2012).
J. Wu et al., Poly(I:C) Treatment Leads to Interferon-Dependent Clearance of Hepatitis B Virus in a Hydrodynamic Injection Mouse Model. Journal of Virology 88, 10421-10431 (2014).

8. X. F. Deng et al., A Chimeric Virus-Mouse Model System for Evaluating the Function and Inhibition of Papain-Like Proteases of Emerging Coronaviruses.
Journal of Virology 88, 11825-11833 (2014).
9. T. E. Rocke et al., Sylvatic Plague Vaccine Partially Protects Prairie Dogs
(Cynomys spp.) in Field Trials. Ecohealth 14, 438-450 (2017).
10. B. Stading et al., Protection of bats (Eptesicus fuscus) against rabies following topical or oronasal exposure to a recombinant raccoon poxvirus vaccine. Plos
Neglect. Trop. Dis. 11, (2017).
11. B. R. Stading et al., Infectivity of attenuated poxvirus vaccine vectors and
immunogenicity of a raccoonpox vectored rabies vaccine in the Brazilian Free-tailed bat (Tadarida brasiliensis). Vaccine 34, 5352-5358 (2016).
12. P. Zhou et al., Fatal Swine Acute Diarrhea Syndrome caused by an HKU2- related Coronavirus of Bat Origin. Nature In press, (2018
).

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10/5/21, 2:45 PM Mail - Rocke, Tonie E - Outlook
From: Peter Daszak [mailto:daszak@ecohealthalliance.org]
Sent: Thursday, 8 February, 2018 10:51 AM
To: Ralph Baric (rbaric@email.unc.edu); Wang Linfa; Zhengli Shi (zlshi@wh.iov.cn); William B. Karesh; Rocke, Tonie
Cc: Luke Hamel; Jonathon Musser; Anna Willoughby; Kevin Olival, PhD; Jon Epstein; Noam Ross; Aleksei Chmura; Anna Willoughby; Hongying Li
Subject: First (rough) draft of the DARPA abstract - Project DEFUSE
Importance: High
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EcoHealth Alliance
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+1.212.380.4471 (direct) +1.212.380.4465 (fax) @noamross (twitter) www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics
and promote conservation.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 4/4
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DARPA – PREEMPT – HR001118S0017
Abstract Submission Requirements:
**8 pages with 12 point font or higher (smaller font may be used for figures, tables
and charts)
**Page limit includes all figures, tables, charts and the Executive Summary Slide **Copies of all documents submitted must be clearly labeled with the following:
-DARPA BAA number
-Proposer Organization
-Proposal title/Proposal short title
-Submission letter is optional and does not count towards page limit
A. Cover Sheet (does not count towards page limit):
Include the administrative and technical points of contact (name, address, phone, fax, email, lead organization). Also include the BAA number, title of the proposed project, primary subcontractors, estimated cost, duration of project, and the label “ABSTRACT.”
B. Executive Summary Slide:
Provide a one slide summary in PowerPoint that effectively and succinctly conveys the main objective, key innovations, expected impact, and other unique aspects of the proposed project. Use the slide template provided at http://www.fbo.gov.
**See slide template at bottom of document.
Clearly describe what is being proposed and what difference it will make (qualitatively and quantitatively), including brief answers to the following questions:
1. What is the proposed work attempting to accomplish or do?
We aim to defuse the potential for emergence of novel bat-origin high-zoonotic risk SARS-related coronaviruses in Southeast Asia. We envisage a scenario whereby the US warfighter is called on to intervene in a security hotspot in SE Asia for a period of 3-6 months. As planners begin choosing sites for the mission, they will use an app we will design to assess the background risk of a site harboring dangerous zoonotic viruses. If
PROJECT DEFUSE
C. Goals and Impact:

there is no alternative to a high-risk site, a tactical forward team will deploy automated delivery technology we will develop in caves that harbor bats carrying these viruses. These devices will release immune boosting molecules and chimeric polyvalent spike protein immune priming inocula to lower viral shedding from bats at the site for a few weeks or months, allowing our warfighters to execute the operation at lowered risk for spillover.
2. How is it done today? And what are the limitations?
Currently, there is no available technology to reduce the risk of exposure to novel coronaviruses from bats, other than avoid the regions where bats harbor these viruses. This includes large areas of southeast Asia where SARS-related CoVs are endemic in bats, which roost in caves during the day, but forage over wide areas at night, shedding virus in their feces and urine. The limitations of this lack of capacity are significant – we have shown evidence of recent spillover of SARS-related CoVs into people in southern China, and have identified viruses in this region that are capable of producing SARS-like illness in humanized mice, with no available vaccines or countermeasures. These viruses are a clear-and-present danger to our military personnel, and to global health security.
3. What is innovative in your approach and how does it compare to current practice and state-of-the-art (SOA)?
**Note: DARPA wants to know, “how the proposed project is revolutionary and how it significantly rises above the current state of the art
Our group has shown that bats harbor the highest proportion of potential zoonoses of any mammal group, and that they are able to live with high viral loads due to unique damping of their immune systems, likely as an evolutionary adaptation to flight. We will use this to design strategies to upregulate their immune response in their cave roosts, down-regulate viral replication, and reduce the risk of viral shedding and spillover (immune boosting strategy). At the same time, we will inoculate bats with novel chimeric polyvalent recombinant spike proteins to enhance their immune response against replication of specific, high-risk viruses (immune priming strategy). We will use our innovative modeling to design apps that identify the likelihood of any region harboring high-risk bat viruses. We will design novel, automated approaches to deliver both types of inoculum remotely into caves to reduce exposure risk during decontamination.
4. What are the key technical challenges in your approach and how do you plan to overcome these?
Decide which of following parts to talk about:

Deleted: 46 months
Modeling bat suitability
Inventory of caves
Sampling/testing
Reverse engineering, binding assays, mouse expts Modeling viral risk of evolution and spillover Modeling inoculation/defusing strategy
Immune modulation
Immune Booster recombinant production Gain-of-function issue.
Inoculum delivery
Mesocosm expts
Cave expts
Model validation
5. Who will care and what will the impact be if you are successful?
This will have direct relevance to the warfighter. The potential for deployment to the region in which bat hosts of SARS-related CoVs exist is high – countries include security hotspots (Myanmar, Bangladesh, Pakistan, Lao, Korea). The ability to decontaminate and defuse these viruses will be useful in preventing potentially devastating illness. Furthermore, these technologies, if successful, can be adapted to hosts of other bat- origin CoVs (MERS, SADS), and potentially other zoonotic bat-origin viruses (Hendra, Nipah, EBOV). In the region directly surrounding our study site, these bat hosts currently roost in unoccupied military bases that may be used by troops at a future time. Finally, our approach is directly applicable to public health measures in the region to reduce the risk of spillover into the general population, as well as for food security by reducing the risk of viruses like SADS-CoV spilling over from bats into intensive pig farms, and devastating and industry, leading to potential civil unrest.
6. How much will it cost and how long will it take?
Will insert this later after calculating and brainstorming. 42 months.
D. Technical Plan:
Outline and address all technical challenges inherent in the approach and possible solutions for overcoming potential problems. This section should provide appropriate specific milestones (quantitative, if possible) at intermediate stages of the project to demonstrate progress and a brief plan for accomplishment of the milestones. **Note: “The technical plan should demonstrate a deep understanding of the

technical challenges and present a credible (even if risky) plan to achieve
the program goal”
Key Terms/Aspects to Emphasize in Abstract ● IACUC/IRB
○ DARPA wants to know who has experience w/ ACURO IACUC work.
■ EHA has multiple ACURO IACUC proposals (either approved or
undergoing approval)
■ IRB also in place, just has to be modified
Rationale for the SE Asian SARS-related CoV – Rhinolophus bat target system, and immune priming/boosting: 1) Our group has shown that bats harbor a higher proportion of potentially zoonotic viruses than any other mammalian group (1), so that proof-of- concept for blocking viral spillover from this host group may lead to a bigger impact on global health security; 2) The Rhinolophus bats that harbor SARS like-CoVs are insectivorous and roost in dense colonies at a fixed, known location, yet disperse each night over wide distances from these sites. Defusing the risk of viral shedding in the roost will also defuse the risk of viral shedding over the population range. This would be difficult for rodent or primate reservoirs; 3) Bats are mammalian hosts, therefore immune modulating drugs trialed out in people may also work on bats. This would be less likely for an insect vector; 4) Members of our collaborative group has worked together on bats and their viruses for over 15 years, with a total of >100 yrs experience focused on bat-origin zoonoses among the key personnel. We have published much of the seminal work on the bat origins of SARS, Nipah, Hendra, and MERS viruses, and have opened new boundaries in studies of bat host-viral relationships ecologically, immunologically and virologically; 5) The South and Southeast Asian region where these bats occur is a security hotspot, with active political and ethnic conflicts, and displaced populations in Bangladesh, Pakistan, Myanmar, Thailand, Indonesia, Philippines and other countries. This is a likely potential site for US warfighter deployment; 6) We have worked for over 10 years on the SARS-related CoV – Rhinolophus bat system in China, demonstrating the origin of SARS-CoV within this host, the presence of SARSr-CoVs with remarkable sequence identity in the spike protein to SARS-CoV, their isolation and characterization of their ability to bind with human cells. We have demonstrated that chimeric SARS-CoV backbone with spike protein from SARSr-CoVs from our cave sites in Yunnan Province can infect a humanized mouse model and cause SARS-like illness, and that clinical signs are not reduced with SARS monoclonal therapy or vaccination. Finally, we have demonstrated that people living up to 6 kilometers from our cave site have
Commented [PD3]: I know this is too long. I’ll edit later this weekend, but want to keep this text for the full proposal
Overview

evidence of SARSr-CoV antibodies (3% seroprevalence in 200+ cohort), suggesting active spillover, and marking these viruses as a clear-and-present danger of a new SARS-like pandemic. This also gives us the unique ability to validate our models on a significant number of actual spillover events, not only experimental infections; 7) SARSr-CoVs are transmitted among bats via fecal-oral route, making sampling relatively easy (collection of fresh fecal pellets) and molecule or vaccine approaches feasible; 8) Proof-of-concept in this system may be rapidly scalable to other bat-coronavirus systems, e.g. MERS-CoV, SADS-CoV, and to other cave bat origin viruses.
Other important bat-origin zoonotic viruses (e.g. filoviruses, henipaviruses) have very rare spillover events - usually to a single index case, which makes validated prevention of spillover challenging. These viruses also show little strain diversity which makes modeling which evolutionary lines will be more high-risk, a challenge. SARSr-CoVs are diverse, with recombinants regularly identified in the field and lab. Furthermore, we have identified a single cave in Yunnan that harbors every gene from the SARS-CoV in a diversity of SARSr-CoVs within the bat population, making it an ideal evolutionary soup to target for intervention.
Finally, we believe that alternative approaches to transmission blocking, e.g. CRISPER-Cas are likely to be far less effective in bats because most bats are long-lived relative to their small size, with long inter-generational periods (2-5 years). Gene drives would likely take many decades to run through a population, so that proof-of-concept of transmission blocking in the DARPA time scale wouldn’t be possible. Furthermore, many bat species’ populations mix readily or migrate which would disperse the impact of gene drives, whereas targeting a small number of caves in a region for molecule or vaccine delivery would cover a very large dispersal area.
TA1: Develop and validate integrated, multiscale models that quantify the likelihood a human-capable virus will emerge from an animal reservoir residing in a “hot spot” geographic region
The DEFUSE modeling and analytics team will develop models to evaluate the likelihood of bat caves harboring high-risk SARSr-CoVs, evaluate the probability of specific SARS- related CoV spillover, and identify the most effective strategy for inoculation of immune boosting molecules and chimeric spike protein immune priming inocula.
We will collect specific data to inform our model building, validate assumptions and refine predictions. At the start of Yr 1, we will conduct a full inventory of host and virus distribution within our field sites, two caves in Yunnan Province, China. This builds on 8 years of surveillance in these caves and the surrounding region and includes a cave in which we have identified all the genetic components of SARS-CoV distributed across a bat population. Two other caves will act as controls/comparison sites, in that we have

not yet identified the high-risk SARSr-CoVs in those caves. We will assess: the population density, distribution and segregation of individual bats; changes in these daily, weekly and by season; viral prevalence and intensity in individuals; distribution of low- and high-risk SARSr-CoV strains, and how readily these are transmitted among bat species, age classes, genders; and using mark-recapture to assess metapopulation structure. To assess geographic distribution of bat hosts, we have access to biological inventory data on all bat caves in Southern China, as well as information on species distributions across SE Asia from the literature and museum records. We will use radio- and satellite telemetry to identify the home range of each species of bat in the caves, to assess how widely the viral ‘plume’ could contaminate
We will build environmental niche models using the data above, and environmental and ecological correlates, and traits of cave species communities (eg. phylogenetic and functional diversity), to predict the species composition of bat caves across Southern China, South and SE Asia. We will validate these with data from the current project and data from PREDICT sampling in Thailand, Indonesia, Malaysia and other SE Asian countries. We will then use our unique database of bat host-viral relationships updated from our recent Nature paper (1) to assess the likelihood of low- or high-risk SARSr-CoVs being present in a cave at any site across the region. At the end of Yr 1, we will use these analyses to produce a prototype app for the warfighter that identifies the likelihood of bats harboring dangerous viral pathogens based on these analyses. The ‘high-risk bats near me’ app will be updated as new host-viral surveillance data comes on line from our project and others, to ground-truth and fine- tune its predictive capacity. Specifically, our telemetry data on bat movement will be used to assess how often bats from high-risk caves migrate to other colonies and potentially spread their high-risk strains.
The Wuhan Institute of Virology team will conduct viral testing on samples from all bat species in the caves as part of this inventory. Fecal, oral, blood and urogenital samples will be collected from bats using standard capture techniques as we have done for the last decade. In addition, tarps will be laid down in caves to assess the feasibility of surveys using pooled fresh fecal and urine samples. Assays will be designed to correlate viral load in an individual with viral shedding in a fecal sample. Once this is complete, surveys will continue largely on fecal samples so as not to disturb bat colonies and undermine longitudinal sampling capacity. Samples will be tested by PCR and spike proteins of all SARS-related CoVs sequenced. Analyses of phylogeny, recombination events, and further characterization of high-risk viruses (those with spike proteins close to SARS-CoV) will be carried out (REF). Isolation will be attempted on a subset of samples with novel SARSr-CoVs. will reverse engineer spike
Deleted: that
surrounding regions, and therefore how
wide the risk zone is for the warfighter positioned close to bat caves.
Commented [PD4]: Could add “ We will continue monitoring the human population proximal to these caves to assess the rates of viral spillover, and ground- truth which specific CoVs are able to infect people
Commented [PD5]: Ralph, Zhengli. If we win this contract, I do not propose that all of this work will necessarily be conducted by Ralph, but I do want to stress the US side of this proposal so that DARPA are comfortable with our team. Once we get the funds, we can then allocate who does what exact work, and I believe that a lot of these assays can be done in Wuhan as well...
Prof. Ralph Baric, UNC,

Formatted: Indent: First line: 0.5"
Deleted: Data on the diversity of bat spike proteins, prevalence of recombinant CoVs, ability to bind and infect human cells, degree of clinical signs in mouse models, will be used to estimate evolutionary rates, rates of recombination, and capacity to generate novel strains capable of human infection. Using dynamic metapopulation models, we will estimate the flow of genes within each bat cave, based on the known host and viral assemblages. This will inform how rapidly new CoV strains with distinct phenotypic characteristics evolve.
Deleted:
Deleted: , the data above, Deleted: and
Deleted: to estimate how rapidly high-risk SARSr-CoVs will re-colonize a bat population following immune boosting or priming.
Deleted: We will
Deleted: obtain model estimates of the
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Deleted: of inoculation required for both approaches
Deleted: , what proportion of a population needs to be reached to have effective viral dampening, and whether specific
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Deleted: within a cave would be more effective to treat.
We will then model the efficacy of
Deleted: delivery, deliver to specific sections of a cave Deleted: .
proteins in his lab to conduct binding assays to human ACE2 (the SARS-CoV receptor). Proteins that bind will then be inserted into SARS-CoV backbones, and inoculated into humanized mice to assess their capacity to cause SARS-like disease, and their ability to be blocked by monoclonal therapies, or vaccines against SARS-CoV (REF).
Using both samples from our previous work and new sampling of the human population in the region surrounding our sites, we will determine which viral strains in addition to SARS-CoV have successfully jumped into humans.
The modeling team will use these data to build models of 1) risk of viral evolution and spillover, and 2) strategies to maximize inoculation strategy.
First, based on binding and infection assays in mouse models, we will develop genotype- to-phenotype models to predict viral ability to infect host cells based on genetic traits. Secondly, data on diversity of bat spike proteins, prevalence of recombinant CoVs, and flow of genes within each bat cave via bat movement and migration, will be used to estimate evolutionary rates, rates of recombination, and capacity to generate novel strains capable of human infection. Finally, ecological data, including viral host species and species home ranges will be used to estimate the likelihood of spillover into human populations.
Because of our unique collaboration among world-class modelers, and coronavirologists, we will be able to test model predictions of viral capacity for spillover by conducting spike protein-based binding and cell culture experiments. The BSL-2 nature of work on SARSr-CoVs makes our system highly cost-effective relative to other bat-virus systems (e.g. Ebola, Marburg, Hendra, Nipah), which require BSL-4 level facilities for cell culture. In addition, the high frequency of SARSr-CoV spillover events into the human population in this region gives us the allows us to validate models to a degree not possible in systems where spillover events are extremely rare.
We will use stochastic simulation modeling approaches to characterize the dynamics of viral circulation in these bat populations using the data above and other biological and ecological data. Using this model, we will estimate the frequency,
efficacy, and population coverage required for our intervention approaches to effectively suppress the viral population. We will determine the seasons, locations within a cave, and different delivery methods (spray, swab, cave mouth automated) that will be most effective. Finally we will determine the time frame the treatment will be effective until re-colonization or evolution will cause a return of a high-risk SARSr-CoV to the population.
TA2: Develop scalable approaches that target and suppress the animal virus in its reservoir(s) and/or vector(s), to reduce the likelihood of virus transmission into humans.

Our goal is to use two approaches to defuse the potential for SARS-related CoVs to emerge in people: 1) Immune Boosting: using the unique immunological features of bats that our group has discovered, we will inoculate live bats in cave mesocosms with immune modulators to up-regulate their naïve immunity to suppress viral replication and shedding; 2) Immune Priming: building on preliminary development of polyvalent chimeric recombinant molecules targeting diverse spike proteins from bat SARS-related CoVs, we will produce, and trial inoculation of live bats to suppress the replication and shedding of a broad range of dangerous SARS-related CoVs. Both lines of work will begin in Yr 1 and run parallel throughout the project.
Prof. Linfa Wang (Duke-NUS) will lead the work on immune boosting work, building on his pioneering work on bat immunity (2). This work provides evidence that that the long-term coexistence of bats and their viruses has led to an equilibrium between viral replication and host immunity, whereby bats have specifically down- regulated their innate immune system as part of the fitness cost of flight (the only true flying mammals) (2). The nature of the weakened but not entirely lost functionality of bat innate immunity factors like STING, a central DNA-interferon (IFN) sensing molecule, may have profound impact for bats to maintain the balanced state of “effective response”, but not “over response” against viruses (3). A similar finding was also observed in bat IFNA studies, which is less abundant but was constitutively expressed without stimulation (4). Given native levels of SARSr-CoVs in individual bats with damped immunity, we propose to suppress bat SARSr-CoV by boosting bat innate immunity through the IFN pathway, and breaking the natural host-virus equilibrium. One of the potential problems with this approach is that it can lead to severe inflammation. However, this is unlikely to occur in bats, because they also have a naturally dampened inflammation response (5).
Previous work has shown that aerosol spraying or intranasal inoculation of IFN or other small molecules has led to reduce viral loads in humans, ferrets and mouse models (12-14). We will therefore initially trial inoculation of live bats with synthetic double-stranded RNA (Poly I:C) and assay for reduced viral loads (DETAILS, CITATION). We will generate universal bat interferon and apply to bats in the lab. Interferon has been used extensively clinically if no viral-specific drugs are available, e.g. against filoviruses (11). Secondly, bat replication of SARSr-CoV is sensitive to interferon treatments, as has been shown in our previous work (12). We will attempt to boost bat IFN by blocking bat-specific IFN negative regulator. Bat IFNA is naturally constitutively expressed but cannot be induced to a high level (4). This is unique to bats. We think there should be a negative regulatory factor in the bat interferon production pathway. We propose using CRISPRi to find out that negative regulator and then screen for chemicals targeting at this gene. We will attempt to boost bat IFN by activating

dampened bat-specific IFN production pathways which include DNA-STING-dependent and ssRNA-TLR7 dependent pathways. These changes have been proved to bat-specific, suggesting that they are important in viruses/bats coexistence, and supported by our own work showing that a mutant bat STING restores antiviral functionality (3). By identifying small molecules to directly activate downstream of STING, we have chance to activate bat interferon and then help bats to clear viruses. Similar strategy applies to ssRNA-TLR7 dependent pathways. We will also attempt to boost bat IFN by activating functional bat IFN production pathways. We will investigate if there are other IFN production pathways in bats. We then boost bat immune responses by ligands specifically to these pathways, e.g. polyIC to TLR3-IFN pathway or 5’ppp-dsRNA to RIG-I- IFN pathway. A similar strategy has been tested successful in mouse model for SARS- CoV, IAV or HBV (6, 7). We believe treating wild bats with IFN-modulating small molecules by spraying is superior to other invasive strategies that might be considered by DARPA, including genome editing (CRISPR or RNAi), vaccination or DIP bats, in terms of its deployability and scalability. Finally, we will inoculate bats with fragments of non- bat Coronavirus (DETAILS).
Prof. Ralph Baric (UNC) will lead the immune priming work, building on his track record in reverse-engineering and manipulating SARS-CoV, MERS-CoV and other virus spike proteins over the last two decades . He will develop recombinant chimeric spike- proteins (8) based on SARSr-CoVs we have already identified, and those we will discover and characterize during project DEFUSE.
please!
While there are clear advantages to working with fixed populations of cave-
dwelling bats, molecule or vaccine delivery is technically challenging. Dr. Tonie Rocke, who developed, trialed, field-tested and rolled out the prairie dog plague vaccine (9), and is currently working on vaccines to bat rabies (10, 11) and white-nose syndrome, will manage a series of experiments in the lab and field to perfect a delivery system for both arms of TA2.
We will conduct initial experiments on a lab colony of wild-caught Rhinolophus sinicus bats at Wuhan Institute of Zoology. We (Prof. Wang) have previous experience conducting infection experiments on this bat genus ...(details and citation if possible). First, we will use our recently proven technology to design LIPS assays to the specific high zoonotic-risk SARSr-CoVs (12). We will conduct serological analysis on bats captured for infection experiments, to assess prior exposure to specific strains. These LIPS assays will be made available for use in people to assess exposure of the general population around bat caves in China, and for potential use by the warfighter to assess exposure to SARSr-CoVs during combat missions.
RALPH – clearly I didn’t really understand the
details of your approach. Can you add a couple of paragraphs here and some citations

Finally, work on a delivery method will be overseen by Dr. Tonie Rocke at the National Wildlife Health Center who has proven capacity to develop and take animal vaccines through to licensure (9). Using her captive Jamaican fruitbat colony (10, 11), Dr. Rocke will trial out the following strategies for delivery of the molecules, inocula proposed above: 1) aerosolization; 2) transdermally applied nanoparticles; 3) sticky edible spray that bats will groom from each other; 4) automated spray triggered by timers and movement detectors at critical cave entry points.. (Details and ideas please Tonie!). These approaches will then be trialed out on live bats in our three cave sites in Yunnan Province. Fieldwork will be conducted under the auspices of Dr. Rocke, EHA field staff, and Dr. Yunzhi Zhang (Yunnan CDC, Consultant with EcoHealth Alliance). Sections of bat caves will be cordoned off and different application methods trialed out. A small number of bats will be captured and assayed for viral load after treatment, but so as not to disturb the colony, most viral load work will be conducted on fresh fecal pellets collected daily on the cave floor. EHA has unique access to these sites in Yunnan Province, with our field teams conducting surveillance there for around 10 years, under the guidance of Drs. Shi and Zhang. In year 1 of project DEFUSE, we will seek permission for these experimental inoculations in cave sites in Yunnan from the Provincial Forestry Department. We do not envisage problems getting permission, as we have worked with the Forestry Department collaboratively for the last few years, we have the support of the Yunnan CDC, and we are releasing molecules that are not dangerous to people or wildlife.
E. Capabilities:
A brief summary of expertise of the team, including subcontractors and key personnel. A principal investigator for the project must be identified, and a description of the team’s organization. Include a description of the team’s organization including roles and responsibilities. Describe the organizational experience in this area, existing intellectual property required to complete the project, and any specialized facilities to be used as part of the project. List Government furnished materials or data assumed to be available.
**Note: While only the proposal requires an organization chart, it may be helpful to include in the abstract if we have the space.
• This organization chart would include (as applicable): (1) the programmatic relationship of team members; (2) the unique capabilities of team members; (3) the task responsibilities of team members; (4) the teaming strategy among the team members; (5) key personnel with the amount of effort to be expended by each person during each year.

The lead institution for Project DEFUSE is EcoHealth Alliance, New York, an international research non-profit focused on emerging zoonotic diseases. The project will be led by PI Dr. Peter Daszak, who has 20+ years’ experience managing lab, field and modeling research projects on emerging zoonoses, including as EHA institutional lead, Head of Modeling and Analytics, and member of the Executive Committee for the $130 million USAID EPT/PREDICT. Dr. Daszak will oversee and coordinate all project activities, as well as lead the modeling and analytic work for TA1. Dr. Billy Karesh, who has 40+ years’ experience managing wildlife disease and zoonotic disease projects, will manage partnership activities and relationships and outreach. Dr. Jon Epstein, who has 15 years’ experience working with bats and emerging zoonoses will coordinate work on bat immune priming and boosting trials. Dr. Kevin Olival and Dr. Noam Ross will manage and conduct the modeling and analytical approaches for this project.
Team:
Lead Organization: EcoHealth Alliance, New York
PI: Peter Daszak Ph.D., President & Chief Scientist, EcoHealth Alliance, 3 months/year Key Personnel:
Billy Karesh DVM, Executive VP for Policy & Health, 1 month/year
Kevin J. Olival Ph.D, VP for Scientific Research, 1 month/year
Jonathan H. Epstein DVM Ph.D., VP for Science & Outreach, 0.5 months/year
Carlos Zambrana-Torrelio Ph.D., Assoc. VP for Conservation & Health, 1 month/year Noam Ross Ph.D., Senior Research Scientist, 2 months/year
Evan Eskew, Research Scientist, 2 months/year
Hongying Li, Program Coordinator, China Programs, 3 months/year
TBD Postdoctoral Researcher modeling and analysis, 12 months/year
TBD Research Assistant, 12 months/year
TBD Program Assistant, 12 months/year
Guangjian Zhu Ph.D., Consultant Field Lead, China Programs, 6 months/year
Yunzhi Zhang Ph.D., Consultant, Yunnan CDC, China, 2 months/year
Subcontract #1: University of North Carolina Medical School Organizational Lead: Prof. Ralph Baric Ph.D., 2 months/year XXX
TBD Research Assistant, 12 months/year
Subcontract #2: USGS National Wildlife Health Center
Organizational Lead: Tonie Rocke Ph.D., 2 months/year, no salary requested

TBD Research Technician, 9 months/year
Subcontract #3: Duke NUS, Singapore
Organizational Lead: Prof. Linfa Wang Ph.D., 2 months/year XXX
TBD Research Assistant, 12 months/year
XXX
Subcontract #4: Wuhan Institute of Virology, China Organizational Lead: Prof Zhengli Shi Ph.D., 2 months/year Peng Zhou Ph.D., 2 months/year
TBD Research Assistant, 12 months/year
Links to relevant papers, reports, or
Do not include more than two resumes as part of the abstract.
**Resumes count against the abstract page limit.
Dr. Peter Daszak is President and Chief Scientist of EcoHealth Alliance, a US-based organization that conducts research and outreach programs on emerging zoonotic diseases. He has published over 300 scientific papers, including the first global map of EID hotspots, strategies to estimate unknown viral diversity in wildlife, predictive models of virus-host relationships, and evidence of the bat origin of SARS-CoV and other emerging viruses. Dr Daszak is Chair of the National Academy of Sciences, Engineering and Medicine’s Forum on Microbial Threats and is a member of the Executive Committee and the EHA institutional lead for USAID-EPT-PREDICT. He serves on the NRC Advisory Committee to the USGCRP, the DHS CEEZAD External Advisory Board, and the WHO R&D Blueprint Pathogen Prioritization expert group, and has advised the Director for Medical Preparedness Policy on the White House National Security Staff on global health issues. Dr Daszak won the 2000 CSIRO medal for collaborative research.
Prof. Ralph Baric is a UNC Lineberger Comprehensive Cancer Center member and Professor in the UNC-Chapel Hill Department of Epidemiology. His work focuses on coronaviruses as models to study the genetics of RNA virus transcription, replication, persistence, and cross species transmission. His work crosses the boundaries of microbiology, virology, immunology and epidemiology, looking especially at the population genetics of viruses to find the molecular building blocks for more effective vaccines.
F. If desired, include a brief bibliography
resumes of key performers.
Commented [PD6]: I’m planning to use my resume and Ralph’s. Linfa/Zhengli, I realize your resumes are also very impressive, but I am trying to downplay the non-US focus of this proposal so that DARPA doesn’t see this as a negative.

**General Notes:
• DARPA will evaluate proposals using the following criteria, listed in
descending order of importance:
1) 5.1.1. Overall Scientific and Technical Merit
The proposed technical approach is innovative, feasible, achievable, and complete.
Task descriptions and associated technical elements provided are complete and in a logical sequence with all proposed deliverables clearly defined such that a final outcome that achieves the goal can be expected as a result of award. The proposal identifies major technical risks and planned mitigation efforts are clearly defined and feasible. The proposed PREEMPT Risk Mitigation Plan effectively provides the following: an assessment of potential risks; proposed guidelines to ensure maximal biosafety and biosecurity; a risk management plan for responsible communications; and a plan to address how input from the Government and community stakeholders will be considered regarding communication and publication of potentially sensitive dual-use information.
2) 5.1.2. Potential Contribution and Relevance to the DARPA Mission
The potential contributions of the proposed effort are relevant to the national technology base. Specifically, DARPA’s mission is to make pivotal early technology investments that create or prevent strategic surprise for U.S. National Security. The proposer clearly demonstrates its capability to transition the technology to the research, industrial, and/or operational military communities in such a way as to enhance U.S. defense. In
addition, the evaluation will take into consideration the extent to which the proposed intellectual property (IP) rights will potentially impact the Government’s ability to transition the technology.
3) 5.1.3. Cost Realism
The proposed costs are realistic for the technical and management approach and accurately reflect the technical goals and objectives of the solicitation. The proposed costs are consistent with the proposer's Statement of Work and reflect a sufficient understanding of the costs and level of effort needed to successfully accomplish the proposed technical approach. The costs for the prime proposer and proposed

subawardees are substantiated by the details provided in the proposal (e.g., the type and number of labor hours proposed per task, the types and quantities of materials, equipment and fabrication costs, travel and any other applicable costs and the basis for the estimates).
It is expected that the effort will leverage all available relevant prior research in order to obtain the maximum benefit from the available funding. For efforts with a likelihood of commercial application, appropriate direct cost sharing may be a positive factor in the evaluation.
DARPA recognizes that undue emphasis on cost may motivate proposers to
offer low-risk ideas with minimum uncertainty and to staff the effort with junior
personnel in order to be in a more competitive posture. DARPA discourages such cost
strategies.
Commented [EA7]: Please note
Citations
2.
1. K. J. Olival et al., Host and viral traits predict zoonotic spillover from mammals. Nature 546, 646-650 (2017).
G. Zhang et al., Comparative analysis of bat genomes provides insight into the evolution of flight and immunity. Science 339, 456-460 (2013).
Attachment 1: Executive Summary Slide template

3. 4.
5. 6.
7. 8.
9. 10.
11. 12.
J. Xie et al., Dampened STING-Dependent Interferon Activation in Bats. Cell host & microbe, (2018).
P. Zhou et al., Contraction of the type I IFN locus and unusual constitutive expression of IFN-αin bats. Proceedings of the National Academy of Sciences of the United States of America, 201518240-201518246 (2016).
M. Ahn, J. Cui, A. T. Irving, L.-F. Wang, Unique Loss of the PYHIN Gene Family in Bats Amongst Mammals: Implications for Inflammasome Sensing. Scientific Reports 6, (2016).
J. Zhao et al., Intranasal Treatment with Poly(I.C) Protects Aged Mice from Lethal Respiratory Virus Infections. Journal of Virology 86, 11416-11424 (2012).
J. Wu et al., Poly(I:C) Treatment Leads to Interferon-Dependent Clearance of Hepatitis B Virus in a Hydrodynamic Injection Mouse Model. Journal of Virology 88, 10421-10431 (2014).
X. F. Deng et al., A Chimeric Virus-Mouse Model System for Evaluating the Function and Inhibition of Papain-Like Proteases of Emerging Coronaviruses. Journal of Virology 88, 11825-11833 (2014).
T. E. Rocke et al., Sylvatic Plague Vaccine Partially Protects Prairie Dogs (Cynomys spp.) in Field Trials. Ecohealth 14, 438-450 (2017).
B. Stading et al., Protection of bats (Eptesicus fuscus) against rabies following topical or oronasal exposure to a recombinant raccoon poxvirus vaccine. Plos Neglect. Trop. Dis. 11, (2017).
B. R. Stading et al., Infectivity of attenuated poxvirus vaccine vectors and immunogenicity of a raccoonpox vectored rabies vaccine in the Brazilian Free-tailed bat (Tadarida brasiliensis). Vaccine 34, 5352-5358 (2016).
P. Zhou et al., Fatal Swine Acute Diarrhea Syndrome caused by an HKU2- related Coronavirus of Bat Origin. Nature In press, (2018
).

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From: Peter Daszak [mailto:daszak@ecohealthalliance.org]
Sent: Thursday, 8 February, 2018 10:51 AM
To: Ralph Baric (rbaric@email.unc.edu); Wang Linfa; Zhengli Shi (zlshi@wh.iov.cn); William B. Karesh; Rocke, Tonie
Cc: Luke Hamel; Jonathon Musser; Anna Willoughby; Kevin Olival, PhD; Jon Epstein; Noam Ross; Aleksei Chmura; Anna Willoughby; Hongying Li
Subject: First (rough) draft of the DARPA abstract - Project DEFUSE
Importance: High
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EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics
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DARPA – PREEMPT – HR001118S0017
Abstract Submission Requirements:
**8 pages with 12 point font or higher (smaller font may be used for figures, tables
and charts)
**Page limit includes all figures, tables, charts and the Executive Summary Slide **Copies of all documents submitted must be clearly labeled with the following:
-DARPA BAA number
-Proposer Organization
-Proposal title/Proposal short title
-Submission letter is optional and does not count towards page limit
A. Cover Sheet (does not count towards page limit):
Include the administrative and technical points of contact (name, address, phone, fax, email, lead organization). Also include the BAA number, title of the proposed project, primary subcontractors, estimated cost, duration of project, and the label “ABSTRACT.”
B. Executive Summary Slide:
Provide a one slide summary in PowerPoint that effectively and succinctly conveys the main objective, key innovations, expected impact, and other unique aspects of the proposed project. Use the slide template provided at http://www.fbo.gov.
**See slide template at bottom of document.
Clearly describe what is being proposed and what difference it will make (qualitatively and quantitatively), including brief answers to the following questions:
1. What is the proposed work attempting to accomplish or do?
We aim to defuse the potential for emergence of novel bat-origin high-zoonotic risk SARS-related coronaviruses in Southeast Asia. We envisage a scenario whereby the US warfighter is called on to intervene in a security hotspot in SE Asia for a period of 3-6 months. As planners begin choosing sites for the mission, they will use an app we will design to assess the background risk of a site harboring dangerous zoonotic viruses. If
PROJECT DEFUSE
C. Goals and Impact:

there is no alternative to a high-risk site, a tactical forward team will deploy automated delivery technology we will develop in caves that harbor bats carrying these viruses. These devices will release immune boosting molecules and chimeric polyvalent spike protein immune priming inocula to lower viral shedding from bats at the site for a few weeks or months, allowing our warfighters to execute the operation at lowered risk for spillover.
2. How is it done today? And what are the limitations?
, there is no available technology to reduce the risk of exposure to novel
coronaviruses from bats, other than avoid the regions where bats harbor these viruses. This includes large areas of southeast Asia where SARS-related CoVs are endemic in bats, which roost in caves during the day, but forage over wide areas at night, shedding virus in their feces and urine. The limitations of this lack of capacity are significant – we have shown evidence of recent spillover of SARS-related CoVs into people in southern China, and have identified viruses in this region that are capable of producing SARS-like illness in humanized mice, with no available vaccines or countermeasures. These viruses are a clear-and-present danger to our military personnel, and to global health security.
3. What is innovative in your approach and how does it compare to current practice and state-of-the-art (SOA)?
**Note: DARPA wants to know, “how the proposed project is revolutionary and how it significantly rises above the current state of the art
Our group has shown that bats harbor the highest proportion of potential zoonoses of any mammal group, and that they are able to live with the host without causing diseases due to unique damping of certain pathways in their immune systems, likely in part as an evolutionary adaptation to flight. We will use this new finding to design strategies like small molecule Rig like receptor (RLR) or Toll like receptor (TLR) agonists to upregulate their immune response in their cave roosts, down-regulate viral replication, and reduce the risk of viral shedding and spillover (broad immune boosting strategy). At the same time, we will inoculate bats with novel chimeric polyvalent recombinant spike proteins to enhance their immune response against replication of specific, high-risk viruses (targeted immune priming strategy). We will use our innovative modeling to design apps that identify the likelihood of any region harboring high-risk bat viruses. We will design novel, automated approaches to deliver both types of inoculum remotely into caves to reduce exposure risk during decontamination.
4. What are the key technical challenges in your approach and how do you plan to overcome these?
Commented [L1]: My understanding is that the project will have two parts: A) better risk assessment and modeling and B) risk defusing.
Do we need to say anything about A here?!
Deleted: high viral loads Deleted: their
Commented [L2]: This will become important late: while we are specifically targeting SARS-realted CoVS, this strategy will be applicable to ALL bat-borne viruses in future
Commented [BRS3]: I thought we were also going to use innate immune antagonists to boost baseline immunity, which should attenuate virus burden in animals?
Isn’t this supposed to be a two pronged approach that are complementary, e.g., in that innate immune agonists will also boost immunity to recombinant spike vaccines.
Currently

Decide which of following parts to talk about:
Sampling/testing
Reverse engineering, binding assays, mouse expts Modeling viral risk of evolution and spillover
Gain-of-function issue. Mesocosm expts
5. Who will care and what will the impact be if you are successful?
This will have direct relevance to the warfighter. The potential for deployment to the region in which bat hosts of SARS-related CoVs exist is high – countries include security hotspots (Myanmar, Bangladesh, Pakistan, Lao, Korea, Vietnam and Cambodia?). The ability to decontaminate and defuse these viruses will be useful in preventing potentially devastating illness. Furthermore, these technologies, if successful, can be adapted to hosts of other bat-origin CoVs (MERS, SARS and related prepandemic zoonotic strains), and potentially other zoonotic bat-origin viruses (Hendra, Nipah, EBOV). Finally, our approach is directly applicable to public health measures in the region to reduce the risk of spillover into the general population, as well as for food security by reducing the risk of viruses like Severe Acute Diarrheal Syndrome CoV spilling over from bats into intensive pig farms, and devastating and industry, leading to potential civil unrest.
6. How much will it cost and how long will it take?
Will insert this later after calculating and brainstorming.
D. Technical Plan:
Outline and address all technical challenges inherent in the approach and possible solutions for overcoming potential problems. This section should provide appropriate specific milestones (quantitative, if possible) at intermediate stages of the project to demonstrate progress and a brief plan for accomplishment of the milestones. **Note: “The technical plan should demonstrate a deep understanding of the
Modeling bat
suitability
Commented [L4]: I have highlighted the ones which are most challenging and novel for this proposal
Formatted: Highlight
Formatted: Highlight Formatted: Highlight
Formatted: Highlight Formatted: Highlight
Deleted: D
Deleted: -
Commented [PD5]: Check on the duration of PREEMPT
Inventory of caves
Modeling inoculation/defusing strategy
Immune modulation
Immune Booster recombinant production
Inoculum delivery
Cave expts
46 months

technical challenges and present a credible (even if risky) plan to achieve
the program goal”
Key Terms/Aspects to Emphasize in Abstract ● IACUC/IRB
○ DARPA wants to know who has experience w/ ACURO IACUC work.
■ EHA has multiple ACURO IACUC proposals (either approved or
undergoing approval)
■ IRB also in place, just has to be modified
○ EHA has more than 50 years experience with IACUC (Jon, Billy, Linfa, & Ralph combined, including free-ranging and captive bat species) proposals and currently we have 3 DoD funded projects approved or undergoing ACURO review.
Rationale for the SE Asian SARS-related CoV – Rhinolophus bat target system, and immune priming/boosting: 1) Our group has shown that bats harbor a higher proportion of potentially highly heterogeneous zoonotic viruses than any other mammalian group (1), so that proof-of-concept for blocking viral spillover from this host group may lead to a bigger impact on global health security; 2) The Rhinolophus bats that harbor SARS like-CoVs are insectivorous, common, have a broad geographic range throughout Asia;roost in dense colonies at fixed, known locations, yet disperse each night over wide
distances from these sites. Defusing the risk of viral shedding in the roost will also defuse the risk of viral shedding over the population range. This would be difficult for rodent or primate reservoirs; 3) Bats are mammalian hosts, therefore immune modulating drugs evaluated in people and rodents may also work on bats. This would be less likely for an insect vector; 4) Members of our collaborative group have worked together on bats and their viruses for over 15 years, with a total of >100 yrs experience focused on bat-origin zoonoses among the key personnel. We have published much of the seminal work on the bat origins of SARS, Nipah, Hendra, and MERS viruses, and have opened new boundaries in studies of bat host-viral relationships ecologically, immunologically and virologically; 5) The South and Southeast Asian region where these bats occur is a security hotspot, with active political and ethnic conflicts, and displaced populations in Bangladesh, Pakistan, Myanmar, Thailand, Indonesia, Philippines and other . This is a likely potential site for US warfighter deployment; 6) We have worked for 15 years on the SARS-related CoV – Rhinolophus bat system in China,
demonstrating the origin of SARS-CoV within this host, the presence of remarkable sequence identity in the spike protein to SARS-CoV, their isolation and
Formatted
Commented [PD6]: I know this is too long. I’ll edit later this weekend, but want to keep this text for the full proposal
Deleted: and Deleted: a
Deleted: trialed out Deleted: s
Commented [BRS7]: About 90,000 of the 550,000 deployed US military are in se asian, mostly japan and south korea.
Deleted: over
Deleted: 0
Commented [BRS8]: What abbreviation mean
Overview
countries
SARSr-CoVs
with

characterization of their ability to bind and replicate efficiently in primary human lung airway cells. We have demonstrated that chimeric SARS-CoV backbone with spike protein from SARSr-CoVs from our cave sites in Yunnan Province can infect a humanized mouse model and cause SARS-like illness, and that clinical signs are not reduced with SARS monoclonal therapy or vaccination. Finally, we have demonstrated that people living up to 6 kilometers from our cave site have evidence of SARSr-CoV antibodies (3% seroprevalence in 200+ cohort), suggesting active spillover, and marking these viruses as a clear-and-present danger of a new SARS-like pandemic; 7) SARSr-CoVs are transmitted among bats via fecal-oral route, making sampling relatively easy (collection of fresh fecal pellets) and molecule or vaccine approaches feasible; 8) Proof-of-concept in this system may be rapidly scalable to other bat-coronavirus systems, e.g. MERS-CoV, SADS-CoV, and to other cave bat origin viruses.
O
makes modeling which evolutionary lines will be more high-risk, a
are diverse, with recombinants regularly identified in the field and lab. Furthermore, we have identified SARS-like strains in a single cave in Yunnan that harbor every gene found in the human SARS-CoV strains detected during the 2002-2003 epidemic. Within this bat population, an ideal evolutionary soup exists which can produce new human strains by high frequency RNA recombination and presents a perfect target for 21st generation intervention strategies.
Finally, we believe that alternative approaches to transmission blocking, e.g. CRISPER-Cas gene drives that are likely to be far less effective in bats because most bats are long-lived relative to their small size, long inter-generational periods (2-5 yrs) and low progeny (~ per year). Gene drives would likely take many decades to run through a population, so that proof-of-concept of transmission blocking in the DARPA time scale wouldn’t be possible. Furthermore, many bat species’ populations mix readily or migrate which would disperse the impact of gene drives, whereas targeting a small number of caves in a region for molecule or vaccine delivery would cover a very large dispersal area.
TA1: Develop and validate integrated, multiscale models that quantify the likelihood a human-capable virus will emerge from an animal reservoir residing in a “hot spot” geographic region
The DEFUSE modeling and analytics team will develop models to evaluate the likelihood of bat caves harboring high-risk SARSr-CoVs, evaluate the probability of specific SARS- related CoV spillover, and identify the most effective strategy for inoculation of immune
Deleted: bind Deleted: with
Commented [BRS9]: These viruses can either be cultured and/or recovered using reverse genetic strategies.
Commented [J10]: However, we may want to highlight that our approach of immune modulation may also reduce the viral load of filoviruses and henipaviruses in cave bat populations. Our work has found Ebola Reston in cave bats in the Philippines (Mineopterus spp.) and henipaviruses and filoviruses have been identified in insectivorous bats in China.
Commented [BRS11]: Filoviruses pretty diverse, although not anywhere near as diverse as cov. Is this a sampling thing or not likely remains unclear?
Deleted: s
Deleted: from the
Deleted: in a diversity of SARSr-CoVs w Deleted: e
Deleted: making it
Deleted: to
Formatted: Superscript
Deleted: with
Commented [BRS12]: Is this correct? Commented [J13]: Yes, correct Deleted: 2-5 years
Commented [L14]: We need to provide background info about bat immunity and the track record of this group in the field
Commented [L15]: Peng: I am working on an important grant here in Singapore. Can you add a few points here? Thanks
ther important bat-origin zoonotic viruses (e.g. filoviruses, henipaviruses) have
very rare spillover events - usually to a single index case, which makes validated
prevention of spillover challenging.
These viruses also show little strain diversity which
challenge
. SARSr-CoVs
1-2 pups

boosting molecules and chimeric spike protein immune priming inocula.
We will collect specific data to inform our model building, validate assumptions
and refine predictions. At the start of Yr 1, we will conduct a full inventory of host and virus distribution within our field sites, two caves in Yunnan Province, China. This builds on 8 years of surveillance in these caves and includes a cave in which we have identified all the genetic components of the 2002-2003 epidemic SARS-CoV distributed across a bat population. Two other caves will act as controls/comparison sites, in that we have not yet identified the high-risk SARSr-CoVs in these caves. We will assess: the population
density, distribution and segregation of individual bats; changes in these daily, weekly and by season; viral prevalence and intensity in individuals; distribution and seasonal shedding of low- and high-risk SARSr-CoV strains, and how readily these are transmitted among bat species, age classes, genders; and using mark-recapture to assess metapopulation structure. To assess geographic distribution of bat hosts, we have access to biological inventory data on all bat caves in Southern China, as well as information on species distributions across SE Asia from the literature and museum records. We will use radio- GPStelemetry to identify the home range of each species of bat in the caves, to identify additional roost sites; to assess how widely the viral ‘plume’ could contaminate
We will build ecological niche models using the data above, and environmental and ecological correlates, and traits of cave species communities (eg. phylogenetic and functional diversity), to predict the species composition of bat caves across Southern China, South and SE Asia. We will validate these with data from the current project and data from PREDICT sampling in Thailand, Indonesia, Malaysia and other SE Asian countries. We will then use our unique database of bat host-viral relationships updated from our recent Nature paper (1) to assess the likelihood of low- or high-risk SARS-CoVs being present in a cave at any site across the region. At the end of Yr 1, we will use these analyses to produce a prototype app for the warfighter that identifies the likelihood of bats harboring dangerous viral pathogens based on these analyses. The ‘high-risk bats near me’ app will be updated as new host-viral surveillance data comes on line from our project and others, to ground-truth and fine-tune its predictive capacity. Specifically, our telemetry data on bat movement will be used to assess how often and how far bats from high-risk caves migrate to other colonies and potentially spread their high-risk strains.
The Wuhan Institute of Virology team will conduct viral testing on samples from all bat species in the caves as part of this inventory. Fecal, oral, blood and urogenital samples will be collected from bats using standard capture techniques as we have done for the last decade. In addition, tarps will be laid down in caves to collect fresh fecal and
Commented [BRS16]: Is surveillance in these other caves equally robust over the past 8 yrs?
Deleted: at
Deleted: and satellite
Commented [PD17]: Could add “ We will continue monitoring the human population proximal to these caves to assess the rates of viral spillover, and ground- truth which specific CoVs are able to infect people
Deleted: environmental
Deleted: r
surrounding regions, and therefore how wide the risk zone is for the
warfighter positioned close to bat caves.
Deleted: assess the feasibility Deleted: of surveys using pooled

urine samples. Assays will be designed to correlate viral load in an individual with viral shedding in a fecal sample. Once this is complete, surveys will continue largely on fecal samples so as not to disturb bat colonies and undermine longitudinal sampling capacity. Samples will be tested by PCR and spike proteins of all SARS-related CoVs sequenced. Analyses of phylogeny, recombination events, and further characterization of high-risk viruses (those with spike proteins close to SARS-CoV) will be carried out (REF). Isolation will be attempted on a subset of samples with novel SARSr-CoVs. Prof. Ralph Baric, UNC, will reverse engineer spike proteins in his lab to conduct binding assays to human ACE2 (the SARS-CoV receptor). Their group have also devised new strategies to culture SARS- like bat coronaviruses, allowing biological characterization of both high risk strains that can replicate in primary human cells and low risk strains that can only replicate in the presence of exogenous enhancers. Viral spike glycoproteins that bind receptor will then be inserted into SARS-CoV backbones, and inoculated into human cells and humanized mice to assess their capacity to cause SARS-like disease, and their ability to be blocked by monoclonal therapies, or vaccines against SARS-CoV ((PMC5798318, PMC5567817, PMC5380844, PMC5578707, PMC4801244, PMC4797993). The Baric group has also demonstrated that a nucleoside analogue inhibitor, GS-5734 (Gilead Inc), blocks epidemic, preepidemic and zoonotic SARS-CoV and SARS-like bat coronavirus replication in primary human airway cells and in mice (PMC5567817). Consequently, they will evaluate the ability of this drug to block replication of newly discovered pre-epidemic and zoonotic high risk strains. As the drug has been used to effectively treat Ebola virus infected patients (PMC4967715, PMC5583641) as well and has potent activity against Nipah and Hendra viruses (PMC5338263), an alternative intervention for military personnel is prophylactic treatment treatment prior to deployment into high risk settings.
The modeling team will use these data to build models of 1) risk of viral evolution and spillover, and 2) strategies to maximize inoculation strategy.
Data on the diversity of bat spike proteins, prevalence of recombinant CoVs, ability to bind and infect human cells, degree of clinical signs in mouse models, will be used to estimate evolutionary rates, rates of recombination, and capacity to generate novel strains capable of human infection. Using dynamic metapopulation models, we will estimate the flow of genes within each bat cave, based on the known host and viral assemblages. This will inform how rapidly new CoV strains with distinct phenotypic characteristics evolve. Because of our unique collaboration among world-class modelers, and virologists with coronavirus expertise, we will be able to test model predictions of viral capacity for spillover by conducting spike protein-based binding and cell culture experiments.
The BSL-3 nature of work on SARSr-CoVs makes our system
Commented [PD18]: Ralph, Zhengli. If we win this contract, I do not propose that all of this work will necessarily be conducted by Ralph, but I do want to stress the US side of this proposal so that DARPA are comfortable with our team. Once we get the funds, we can then allocate who does what exact work, and I believe that a lot of these assays can be done in Wuhan as well...
Commented [J19]: Can we culture any bat coronaviruses? It might be good to broaden this so we can include novel beta CoVs that we may discover which look like they may be transmissible to people
Deleted: P Deleted: REF)
Deleted: h
Deleted: corona Deleted: 2
highly cost-effective relative to other bat-virus systems (e.g. Ebola, Marburg, Hendra,

Nipah), which require BSL-4 level facilities for cell culture
Commented [BRS20]: IN the US, these recombinant SARS CoV are studied under BSL3, not BSL2, especially important for those that are able to bind and replicate in primary human cells.
In china, might be growin these virus under bsl2. US reseachers will likely freak out.
Deleted: , Deleted: ,
Deleted: re
modulators
Commented [BRS21]: Like what
The nature of the weakened but not entirely lost functionality of
bat innate immunity factors like STING, a central DNA-interferon (IFN) sensing molecule,
may have profound impact for bats to maintain the balanced state of “effective
response”, but not “over response” against viruses
Commented [J22]: Linfa: Do we know if these findings from Pteropus also apply to Rhinolophus? If not, we should be careful with the assertions we make...
We can make the argument that these two families of bats are more closely related than to other
Commented [BRS23]: Transient low level Chronic inflammation sounds better
.
We will use modeling approaches informed by field and experimental data including the data above and other biological and ecological data, to estimate how
rapidly high-risk SARSr-CoVs will re-colonize a bat population following immune boosting or priming. We will obtain model estimates of the frequency of inoculation required for both approaches, what proportion of a population needs to be reached to have effective viral dampening, and whether specific seasons, or locations within a cave would be most effective to treat. We will then model the efficacy of different delivery methods (spray, swab, cave mouth automated delivery, deliver to specific sections of a cave).
TA2: Develop scalable approaches that target and suppress the animal virus in its reservoir(s) and/or vector(s), to reduce the likelihood of virus transmission into humans.
Our goal is to use two approaches to defuse the potential for SARS-related CoVs to emerge in people: 1) Immune Boosting: using the unique immunological features of bats that our group has discovered, we will inoculate live bats in cave mesocosms with immune to up-regulate their naïve immunity to suppress viral replication and shedding; 2) Immune Priming: building on preliminary development of polyvalent chimeric recombinant molecules targeting diverse spike proteins from bat SARS-related CoVs, we will produce, and trial inoculation of live bats to suppress the replication and shedding of a broad range of dangerous SARS-related CoVs. Both lines of work will begin in Yr 1 and run parallel throughout the project.
Prof. Linfa Wang (Duke-NUS) will lead the work on immune boosting work, building on his pioneering work on bat immunity (2). This work provides evidence that that the long-term coexistence of bats and their viruses has led to an equilibrium between viral replication and host immunity, whereby bats have specifically down- regulated their innate immune system as part of the fitness cost of flight (the only true flying mammals) (2).
(3). A similar finding was also observed in bat IFNA studies, which is less abundant but was constitutively expressed
without stimulation (4). Given native levels of SARSr-CoVs in individual bats with damped immunity, we propose to suppress bat SARSr-CoV by boosting bat innate immunity through the IFN pathway, and breaking the natural host-virus equilibrium. One of the potential problems with this approach is that it can lead to severe

inflammation. However, this is unlikely to occur in bats, because they also have a naturally dampened inflammation response (5).
Previous work has shown that aerosol spraying or intranasal inoculation of IFN or other small molecules has led to reduce viral loads in humans, ferrets and mouse models (12-14). We will therefore initially trial inoculation of live bats with synthetic double-stranded RNA (Poly I:C) and assay for reduced viral loads (DETAILS, CITATION). We will generate universal bat interferon and apply to bats in the lab. Interferon has been used extensively clinically if no viral-specific drugs are available, e.g. against filoviruses (11). Secondly, bat replication of SARSr-CoV is sensitive to interferon treatments, as has been shown in our previous work (12). We will attempt to boost bat IFN by blocking bat-specific IFN negative regulator. Bat IFNA is naturally constitutively expressed but cannot be induced to a high level (4).
We will attempt to boost bat IFN by activating dampened bat-specific IFN production pathways which include DNA-STING-dependent
and ssRNA-TLR7 dependent pathways. These changes have been proved to be bat- specific, suggesting that they are important in viruses/bats coexistence, and supported by our own work showing that a mutant bat STING restores antiviral functionality (3). By identifying small molecules to directly activate downstream of STING, we have chance to activate bat interferon and then help bats to clear viruses. Similar strategy applies to ssRNA-TLR7 dependent pathways. We will also attempt to boost bat IFN by activating functional bat IFN production pathways. We will investigate if there are other IFN
We then boost bat immune responses by ligands specifically to these pathways, e.g. polyIC to TLR3-IFN pathway or 5’ppp-dsRNA to RIG-I-
IFN . A similar strategy has been tested successful in mouse model for SARS-
CoV, IAV or HBV (6, 7). We believe treating wild bats with IFN-modulating small molecules by spraying is superior to other invasive strategies that might be considered by DARPA, including genome editing (CRISPR or RNAi), or DIP bats, in terms of its deployability and scalability. Finally, we will inoculate bats with fragments of non- bat Coronavirus (DETAILS).
Prof. Ralph Baric (UNC) will lead the immune priming work, building on his track record in reverse-engineering and manipulating SARS-CoV, MERS-CoV and other virus spike proteins over the last two decades . He will develop recombinant chimeric spike- proteins (8) based on SARSr-CoVs we have already identified, and those we will discover and characterize during project DEFUSE.
please!
Commented [BRS24]: This could easily take longer than 3 years. Poly ic, IFN or any type of TLR agonist might be more robust. Might want to test in captive bats infected with SARS or select SARS like viruses, like SHC014, which we could provide.
Commented [J25]: If we’re proposing experimental work with bats, we should spcify that we’ll use SARS-CoV & SADS-CoV host species (Rhinolophus) which can be readily obtained by our Chinese colleagues at WIV
Commented [BRS26]: We have several papers showing importance of TLR3 and TLR4 signaling in control of SARS pathogenesis. PMC4447251, PMC5473747
Commented [BRS27]: Don’t attack the other arm of the program. And I disagree that its superior to vaccination, which potentially provides long-term immunity.
Commented [J28]: Agree with Ralph – and this mechanism of delivery would probably be the same for vaccination attempts(intranasal or oral via grooming droplets from fur).
Formatted: Highlight
Commented [BRS29]: The structure of the SARS-CoV spike glycoprotein has been solved and the addition of two proline residues at positions V1060P and L1061P stabilize the prefusion state of the trimer, including key neutralizing epitopes in the receptor binding domain (PMC5584442). In parallel, the spike trimers or the receptor binding domain can be incorporated into alphavirus vectored or nanoparticle vaccines for delivery, either as aerosols, in baits, or as large droplet delivery vehicles (PMC4058772, PMC5423355, PMC2883479, PMC5578707, PMC3014161). Initially, we will test various delivery vehicles in controlled conditions in bats in a laboratory setting, taking the best candidate forward for testing in the field.
The Baric laboratory has built recombinant S pike glycoproteins harboring structurally defined domains from SARS epidemic strains, pre-epidemic strains like SCH014 and zoonotic strains like HKU3. It is anticipated that recombinant S glycoprotein based vaccines harboring immunogenic blocks across the group 2B coronaviruses will induce broad based immune responses that simultaneously reduce genetically heterogeneous virus burdens in bats, thereby reducing disease risk (and transmission risk to people) in these animals for multiple years (PMC3977350, PMC2588415).
This is unique to bats. We think
there should be a negative regulatory factor in the bat interferon production pathway.
We propose using CRISPRi to find out that negative regulator and then screen for
chemicals targeting at this gene.
production pathways in bats.
pathway
vaccination
RALPH – clearly I didn’t really understand the
details of your approach. Can you add a couple of paragraphs here and some citations

While there are clear advantages to working with fixed populations of cave- dwelling bats, molecule or vaccine delivery is technically challenging. Dr. Tonie Rocke, who developed, trialed, field-tested and rolled out the prairie dog plague vaccine (9), and is currently working on vaccines to bat rabies (10, 11) and white-nose syndrome, will manage a series of experiments in the lab and field to perfect a delivery system for both arms of TA2.
We have found that the immune dampening features are highly conserved in all . Duke-NUS has established a breeding colony of cave nectar bats for experimental use (one of very few experimental bat breeding colonies in the world and the only one in SE Asia!). So our initial proof of concept test can be done in
this experimental colony. We will then extend the test to a small group of wild-caught Rhinolophus sinicus bats at Wuhan Institute of Zoology. We (Prof. Wang) have previous experience conducting SARS-CoV infection experiments with bat species from the same genus in the BSL4 facility at the Australian Animal Health Laboratory in Australia (L.Wang, unpublished results). First, we will use our recently proven technology to design LIPS assays to the specific high zoonotic-risk SARSr-CoVs (12). We will conduct serological analysis on bats captured for infection experiments, to assess prior exposure to specific strains. These LIPS assays will be made available for use in people to assess exposure of the general population around bat caves in China, and for potential use by the warfighter to assess exposure to SARSr-CoVs during combat missions.
Finally, work on a delivery method for immunological countermeasures will be overseen by Dr. Tonie Rocke at the National Wildlife Health Center who has proven capacity to develop and tak gh to licensure (9). Using her captive Jamaican fruitbat colony (10, 11), Dr. Rocke will trial out the following strategies for delivery of the molecules, inocula proposed above: 1) aerosolization; 2) transdermally applied nanoparticles; 3) sticky edible spray that bats will groom from each other; 4) automated spray triggered by timers and movement detectors at critical cave entry points.. (Details and ideas please Tonie!). These approaches will then be tested on wild bats in our three cave sites in Yunnan Province. Fieldwork will be conducted under the auspices of Dr. Rocke, EHA field staff, and Dr. Yunzhi Zhang (Yunnan CDC, Consultant with EcoHealth Alliance).
. A small number of bats will be captured and assayed for viral load and immune function after treatment, but so as not to disturb the colony, most viral load work will be conducted on fresh fecal pellets collected daily on the cave
floor. EHA has unique access to these sites in Yunnan Province, with our field teams conducting surveillance there for around 10 years, under the guidance of Drs. Shi and Zhang. In year 1 of project DEFUSE, we will seek permission for these experimental inoculations in cave sites in Yunnan from the Provincial Forestry Department. We do
bat species tested so far
Commented [J30]: Eonycterus and Pteropus are evolutionarily related to Rhinolophus – we may want to have some language asserting our confidence that what we know about bat immunity so far will apply to SARS CoV reservoir species.
Deleted: We will conduct initial experiments on a lab colony of ...
Deleted: on this bat
Deleted: ...(details and citation if possible).
Commented [J31]: We should be clear as to whether we’re deploying a vaccine or an immune-modulator that promotes innate immunity. When we mention Tonie’s experience with vaccine deployment it looks like that what we’re planning.
Deleted: trialed out Deleted: live
Commented [J32]: This probably won’t work as bats may move throughout the cave – mixing application techniques. It would be more practical to use a different technique on each cave.
e animal vaccines throu
Sections of bat caves will be cordoned off and different
application methods trialed out

not envisage problems getting permission, as we have worked with the Forestry Department collaboratively for the last few years, we have the support of the Yunnan CDC, and we are releasing molecules that are not dangerous to people or wildlife.
E. Capabilities:
A brief summary of expertise of the team, including subcontractors and key personnel. A principal investigator for the project must be identified, and a description of the team’s organization. Include a description of the team’s organization including roles and responsibilities. Describe the organizational experience in this area, existing intellectual property required to complete the project, and any specialized facilities to be used as part of the project. List Government furnished materials or data assumed to be available.
**Note: While only the proposal requires an organization chart, it may be helpful to include in the abstract if we have the space.
• This organization chart would include (as applicable): (1) the programmatic relationship of team members; (2) the unique capabilities of team members; (3) the task responsibilities of team members; (4) the teaming strategy among the team members; (5) key personnel with the amount of effort to be expended by each person during each year.
The lead institution for Project DEFUSE is EcoHealth Alliance, New York, an international research non-profit focused on emerging zoonotic diseases. The project will be led by PI Dr. Peter Daszak, who has 20+ years’ experience managing lab, field and modeling research projects on emerging zoonoses, including as EHA institutional lead, Head of Modeling and Analytics, and member of the Executive Committee for the $130 million USAID EPT/PREDICT. Dr. Daszak will oversee and coordinate all project activities, as well as lead the modeling and analytic work for TA1. Dr. Billy Karesh, who has 40+ years’ experience managing wildlife disease and zoonotic disease projects, will manage partnership activities and relationships and outreach. Dr. Jon Epstein, who has 15 years’ experience working with bats and emerging zoonoses, including SARSr-CoVs and MERS- CoV, will coordinate work on bat immune priming and boosting trials. Dr. Kevin Olival and Dr. Noam Ross will manage and conduct the modeling and analytical approaches for this project. The EHA team has extensive experience working with the other team members on previous and current research including Dr. Wang (15+ years); Dr. Shi (15+ years); Dr. Baric (5+ years) and
Team:
Commented [J33]: [via CCM-NWHC partnership]
Dr. Rocke (15+ years)

Deleted: XXX Deleted: TBD Deleted: ssistant
Lead Organization: EcoHealth Alliance, New York
PI: Peter Daszak Ph.D., President & Chief Scientist, EcoHealth Alliance, 3 months/year Key Personnel:
Billy Karesh DVM, Executive VP for Policy & Health, 1 month/year
Kevin J. Olival Ph.D, VP for Scientific Research, 1 month/year
Jonathan H. Epstein DVM Ph.D., VP for Science & Outreach, 0.5 months/year
Carlos Zambrana-Torrelio Ph.D., Assoc. VP for Conservation & Health, 1 month/year Noam Ross Ph.D., Senior Research Scientist, 2 months/year
Evan Eskew, Research Scientist, 2 months/year
Hongying Li, Program Coordinator, China Programs, 3 months/year
TBD Postdoctoral Researcher modeling and analysis, 12 months/year
TBD Research Assistant, 12 months/year
TBD Program Assistant, 12 months/year
Guangjian Zhu Ph.D., Consultant Field Lead, China Programs, 6 months/year
Yunzhi Zhang Ph.D., Consultant, Yunnan CDC, China, 2 months/year
Subcontract #1: University of North Carolina Medical School Organizational Lead: Prof. Ralph Baric Ph.D., 2 months/year Dr. Tim Sheahan (6 months/yr)
Dr. Amy Sims (4 months/yr)
Sarah Leist, Postdoctoral fellow (4 months/yr) Boyd Yount, Research Analyst, 12 months/year
Trevor Scobey, Research Technician, 6 months/yr
Subcontract #2: USGS National Wildlife Health Center
Organizational Lead: Tonie Rocke Ph.D., 2 months/year, no salary requested TBD Research Technician, 9 months/year
Subcontract #3: Duke NUS, Singapore
Organizational Lead: Prof. Linfa Wang Ph.D., 2 months/year XXX
TBD Research Assistant, 12 months/year
XXX
Subcontract #4: Wuhan Institute of Virology, China Organizational Lead: Prof Zhengli Shi Ph.D., 2 months/year Peng Zhou Ph.D., 2 months/year
TBD Research Assistant, 12 months/year

F. If desired, include a brief bibliography
resumes of key performers.
Commented [PD34]: I’m planning to use my resume and Ralph’s. Linfa/Zhengli, I realize your resumes are also very impressive, but I am trying to downplay the non-US focus of this proposal so that DARPA doesn’t see this as a negative.
PMC5798318
,
PMC5567817
, PMC5380844,
PMC5578707
Formatted: Font: (Default) Arial, 11 pt, Font color: Accent 1
Links to relevant papers, reports, or
Do not include more than two resumes as part of the abstract.
**Resumes count against the abstract page limit.
Dr. Peter Daszak is President and Chief Scientist of EcoHealth Alliance, a US-based organization that conducts research and outreach programs on emerging zoonotic diseases. He has published over 300 scientific papers, including the first global map of EID hotspots, strategies to estimate unknown viral diversity in wildlife, predictive models of virus-host relationships, and evidence of the bat origin of SARS-CoV and other emerging viruses. Dr Daszak is Chair of the National Academy of Sciences, Engineering and Medicine’s Forum on Microbial Threats and is a member of the Executive Committee and the EHA institutional lead for USAID-EPT-PREDICT. He serves on the NRC Advisory Committee to the USGCRP, the DHS CEEZAD External Advisory Board, and the WHO R&D Blueprint Pathogen Prioritization expert group, and has advised the Director for Medical Preparedness Policy on the White House National Security Staff on global health issues. Dr Daszak won the 2000 CSIRO medal for collaborative research.
Prof. Ralph Baric is a UNC Lineberger Comprehensive Cancer Center member and Professor in the UNC-Chapel Hill Department of Epidemiology and Department of Microbiology and Immunology . His work focuses on coronaviruses as models to study the genetics of RNA virus transcription, replication, persistence, and cross species transmission and pathogenesis. Dr. Baric and his group have developed a platform strategy to access the potential “preepidemic” risk associated with zoonotic virus cross species transmission potential and evaluation of countermeasure potential to control future outbreaks of disease ( , PMC4801244, PMC4797993). His work crosses the boundaries of microbiology, virology, immunology and epidemiology, looking especially at the population genetics of viruses to find the molecular building blocks for more effective vaccines.
**General Notes:
• DARPA will evaluate proposals using the following criteria, listed in
descending order of importance:

Deleted: ¶
1) 5.1.1. Overall Scientific and Technical Merit
The proposed technical approach is innovative, feasible, achievable, and complete.
Task descriptions and associated technical elements provided are complete and in a logical sequence with all proposed deliverables clearly defined such that a final outcome that achieves the goal can be expected as a result of award. The proposal identifies major technical risks and planned mitigation efforts are clearly defined and feasible. The proposed PREEMPT Risk Mitigation Plan effectively provides the following: an assessment of potential risks; proposed guidelines to ensure maximal biosafety and biosecurity; a risk management plan for responsible communications; and a plan to address how input from the Government and community stakeholders will be considered regarding communication and publication of potentially sensitive dual-use information.
2) 5.1.2. Potential Contribution and Relevance to the DARPA Mission
The potential contributions of the proposed effort are relevant to the national technology base. Specifically, DARPA’s mission is to make pivotal early technology investments that create or prevent strategic surprise for U.S. National Security. The proposer clearly demonstrates its capability to transition the technology to the research, industrial, and/or operational military communities in such a way as to enhance U.S. defense. In addition, the evaluation will take into consideration the extent to which the proposed intellectual property (IP) rights will potentially impact the Government’s ability to transition the technology.
3) 5.1.3. Cost Realism
The proposed costs are realistic for the technical and management approach and accurately reflect the technical goals and objectives of the solicitation. The proposed costs are consistent with the proposer's Statement of Work and reflect a sufficient understanding of the costs and level of effort needed to successfully accomplish the proposed technical approach. The costs for the prime proposer and proposed subawardees are substantiated by the details provided in the proposal (e.g., the type and number of labor hours proposed per task, the types and quantities of materials, equipment and fabrication costs, travel and any other applicable costs and the basis for the estimates).
It is expected that the effort will leverage all available relevant prior research in order to obtain the maximum benefit from the available funding. For efforts with a likelihood of commercial application, appropriate direct cost sharing may be a positive factor in the evaluation.
DARPA recognizes that undue emphasis on cost may motivate proposers to
offer low-risk ideas with minimum uncertainty and to staff the effort with junior

personnel in order to be in a more competitive posture. DARPA discourages such cost
strategies.
Commented [EA35]: Please note
Citations
Attachment 1: Executive Summary Slide template
1. K. J. Olival et al., Host and viral traits predict zoonotic spillover from
mammals. Nature 546, 646-650 (2017).
2. G. Zhang et al., Comparative analysis of bat genomes provides insight into the
evolution of flight and immunity. Science 339, 456-460 (2013).
3. J. Xie et al., Dampened STING-Dependent Interferon Activation in Bats. Cell
host & microbe, (2018).
4. P. Zhou et al., Contraction of the type I IFN locus and unusual constitutive
expression of IFN-αin bats. Proceedings of the National Academy of Sciences of
the United States of America, 201518240-201518246 (2016).
5. M. Ahn, J. Cui, A. T. Irving, L.-F. Wang, Unique Loss of the PYHIN Gene Family
in Bats Amongst Mammals: Implications for Inflammasome Sensing. Scientific
Reports 6, (2016).
6. J. Zhao et al., Intranasal Treatment with Poly(I.C) Protects Aged Mice from
Lethal Respiratory Virus Infections. Journal of Virology 86, 11416-11424
(2012).

Commented [J36]: Is this reference correct? I couldn’t find it online.
7. J. Wu et al., Poly(I:C) Treatment Leads to Interferon-Dependent Clearance of
Hepatitis B Virus in a Hydrodynamic Injection Mouse Model. Journal of
Virology 88, 10421-10431 (2014).
8. X. F. Deng et al., A Chimeric Virus-Mouse Model System for Evaluating the
Function and Inhibition of Papain-Like Proteases of Emerging Coronaviruses.
Journal of Virology 88, 11825-11833 (2014).
9. T. E. Rocke et al., Sylvatic Plague Vaccine Partially Protects Prairie Dogs
(Cynomys spp.) in Field Trials. Ecohealth 14, 438-450 (2017).
10. B. Stading et al., Protection of bats (Eptesicus fuscus) against rabies following
topical or oronasal exposure to a recombinant raccoon poxvirus vaccine. Plos
Neglect. Trop. Dis. 11, (2017).
11. B. R. Stading et al., Infectivity of attenuated poxvirus vaccine vectors and
immunogenicity of a raccoonpox vectored rabies vaccine in the Brazilian
Free-tailed bat (Tadarida brasiliensis). Vaccine 34, 5352-5358 (2016).
12. P. Zhou et al., Fatal Swine Acute Diarrhea Syndrome caused by an HKU2-
related Coronavirus of Bat Origin. Nature In press, (2018
).

From:
Sent:
To:
Subject: Attachments:
Hi Katie: As I mentioned to you by phone, I have been asked to collaborate on a proposal to DARPA with EcoHealth Alliance. I am forwarding you and Jonathan the first draft I received to keep you in the loop so you know what is going on. There is alot here I need to correct (i.e. we don't have a captive Jamaican fruit bat colony but we are thinking of setting up a vampire bat colony at UW) and I will be working on editing the draft proposal.
Also, I have been asked to collaborate on a second proposal by another group (BU emerging infectious disease unit) for the same RFA that involves morbillivirus and rabies in vampire bats in Latin America, although I haven't seen a draft of that proposal yet.
At this point, I plan to participate in both proposals as there is no restriction in that regard, but let me know soon if you have any questions/concerns. Due date for abstracts is approaching very rapidly - Feb 13. No guarantee of funding of course, but just the fact that we are being asked to collaborate on these proposals should be taken as evidence of the relevance of the NWHC research branch, which seems to be in question lately. -Tonie
---------- Forwarded message ----------
From: Peter Daszak <daszak@ecohealthalliance.org>
Date: Wed, Feb 7, 2018 at 8:51 PM
Subject: First (rough) draft of the DARPA abstract - Project DEFUSE
To: "Ralph Baric (rbaric@email.unc.edu)" <rbaric@email.unc.edu>, Wang Linfa <linfa.wang@duke-nus.edu.sg>, "Zhengli Shi (zlshi@wh.iov.cn)" <zlshi@wh.iov.cn>, "William B. Karesh" <karesh@ecohealthalliance.org>, "Rocke, Tonie" <trocke@usgs.gov>
Cc: Luke Hamel <hamel@ecohealthalliance.org>, Jonathon Musser <musser@ecohealthalliance.org>, Anna Willoughby <willoughby@ecohealthalliance.org>, "Kevin Olival, PhD" <olival@ecohealthalliance.org>, Jon Epstein <epstein@ecohealthalliance.org>, Noam Ross <ross@ecohealthalliance.org>, Aleksei Chmura <chmura@ecohealthalliance.org>, Hongying Li <li@ecohealthalliance.org>
Dear All,
I’ve attached a first rough draft of the DARPA abstract. Apologies for the delay. Unfortunately, edits to my Science paper came through on Friday and took many hours to do, so this delayed me. I’m right now in Geneva in my hotel at 3 am finishing these off before flying back to NYC from a WHO meeting.
Rocke, Tonie <trocke@usgs.gov>
Thursday, February 8, 2018 6:28 AM
Richgels, Katherine; Jonathan M Sleeman
Fwd: First (rough) draft of the DARPA abstract - Project DEFUSE DARPA (PREEMPT) Abstract EcoHealth Alliance DEFUSE 1st Draft.docx
Some important points:
1

1) Zhengli, Linfa, Ralph – Billy and I spoke with Tonie Rocke on Friday. Tonie is at the National Wildlife Health Center, Madison USA, and has worked on wildlife vaccines: plague in prairie dogs, rabies in Jamaican fruit bats, white nose syndrome in US bats. We needed someone with expertise in delivery of molecules/vaccines to wildlife because DARPA specifically lay that out. As you’ll see, Tonie is perfect for our project and will be able to do work at USGS NWHC and with Zhengli in China to help with TA2
2) Zhengli and Linfa – After I spoke with you both, I had a great conversation with Ralph Baric. He proposed to work on recombinant chimeric spike proteins as a second line of attack. I think that is a perfect fit because 1) it’s his expertise and he has published on it, 2) it will act as an alternative to the blue-sky and risky immune boosting work that Linfa/Peng have proposed. I hope you agree!
3) Ralph, Zhengli, Linfa, Tonie – as you can see, I have mangled the language/technical details for most of your sections. Pardon my lack of knowledge, and please draft a couple of paragraphs each to make your sections look correct. Thanks to Peng for giving me some text anyway – very useful, but please check what I’ve done with it.
4) All – please add some names and details on the team part so we get clarity in this on what staff you will need to do the work.
5) Please don’t worry about keeping this to the 8 page limit. Just add text here and there, references, and edit to make what I’ve written correct, and more exciting. I will work on this on Saturday, Sunday and Monday to bring it down to 8 pages of very crisp, super-exciting text. I also want as many of your good ideas in here, so that I can use this draft to build on for the full proposal.
6) Finally – please edit rapidly using tracked changes in word. If you don’t want to mess up endnote, please just insert references as comment boxes and we’ll pull them off the web.
Aleksei and Anna: please read the draft and work on some draft image designs that sum up the project flow. I’ll call you Thursday afternoon to discuss so you can finish them off.
Luke – please have a go at a first draft of the executive summary slide. I’ll pick up from what you’ve done once you send it to me.
2

Thanks again to all of you for agreeing to collaborate on this proposal. From what I know of the competition, what DARPA wants, and what we’re offering, I think we have an extremely strong team, so I’m looking forward to getting the full proposal together and winning this contract!
Cheers,
Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge research into the critical connections between human and wildlife health and delicate ecosystems. With this science we develop solutions that prevent pandemics and promote conservation.
--
Tonie E. Rocke
3

USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
608-270-2451
trocke@usgs.gov
4

DARPA – PREEMPT – HR001118S0017
Abstract Submission Requirements:
**8 pages with 12 point font or higher (smaller font may be used for figures, tables
and charts)
**Page limit includes all figures, tables, charts and the Executive Summary Slide **Copies of all documents submitted must be clearly labeled with the following:
-DARPA BAA number
-Proposer Organization
-Proposal title/Proposal short title
-Submission letter is optional and does not count towards page limit
A. Cover Sheet (does not count towards page limit):
Include the administrative and technical points of contact (name, address, phone, fax, email, lead organization). Also include the BAA number, title of the proposed project, primary subcontractors, estimated cost, duration of project, and the label “ABSTRACT.”
B. Executive Summary Slide:
Provide a one slide summary in PowerPoint that effectively and succinctly conveys the main objective, key innovations, expected impact, and other unique aspects of the proposed project. Use the slide template provided at http://www.fbo.gov.
**See slide template at bottom of document.
Clearly describe what is being proposed and what difference it will make (qualitatively and quantitatively), including brief answers to the following questions:
1. What is the proposed work attempting to accomplish or do?
We aim to defuse the potential for emergence of novel bat-origin high-zoonotic risk SARS-related coronaviruses in Southeast Asia. We envisage a scenario whereby the US warfighter is called on to intervene in a security hotspot in SE Asia for a period of 3-6 months. As planners begin choosing sites for the mission, they will use an app we will design to assess the background risk of a site harboring dangerous zoonotic viruses. If
PROJECT DEFUSE
C. Goals and Impact:

there is no alternative to a high-risk site, a tactical forward team will deploy automated delivery technology we will develop in caves that harbor bats carrying these viruses. These devices will release immune boosting molecules and chimeric polyvalent spike protein immune priming inocula to lower viral shedding from bats at the site for a few weeks or months, allowing our warfighters to execute the operation at lowered risk for spillover.
2. How is it done today? And what are the limitations?
Currently, there is no available technology to reduce the risk of exposure to novel coronaviruses from bats, other than avoid the regions where bats harbor these viruses. This includes large areas of southeast Asia where SARS-related CoVs are endemic in bats, which roost in caves during the day, but forage over wide areas at night, shedding virus in their feces and urine. The limitations of this lack of capacity are significant – we have shown evidence of recent spillover of SARS-related CoVs into people in southern China, and have identified viruses in this region that are capable of producing SARS-like illness in humanized mice, with no available vaccines or countermeasures. These viruses are a clear-and-present danger to our military personnel, and to global health security.
3. What is innovative in your approach and how does it compare to current practice and state-of-the-art (SOA)?
**Note: DARPA wants to know, “how the proposed project is revolutionary and how it significantly rises above the current state of the art
Our group has shown that bats harbor the highest proportion of potential zoonoses of any mammal group, and that they are able to live with high viral loads due to unique damping of their immune systems, likely as an evolutionary adaptation to flight. We will use this to design strategies to upregulate their immune response in their cave roosts, down-regulate viral replication, and reduce the risk of viral shedding and spillover (immune boosting strategy). At the same time, we will inoculate bats with novel chimeric polyvalent recombinant spike proteins to enhance their immune response against replication of specific, high-risk viruses (immune priming strategy). We will use our innovative modeling to design apps that identify the likelihood of any region harboring high-risk bat viruses. We will design novel, automated approaches to deliver both types of inoculum remotely into caves to reduce exposure risk during decontamination.
4. What are the key technical challenges in your approach and how do you plan to overcome these?
Decide which of following parts to talk about:

Modeling bat suitability
Inventory of caves
Sampling/testing
Reverse engineering, binding assays, mouse expts Modeling viral risk of evolution and spillover Modeling inoculation/defusing strategy
Immune modulation
Immune Booster recombinant production Gain-of-function issue.
Inoculum delivery
Mesocosm expts
Cave expts
5. Who will care and what will the impact be if you are successful?
This will have direct relevance to the warfighter. The potential for deployment to the region in which bat hosts of SARS-related CoVs exist is high – countries include security hotspots (Myanmar, Bangladesh, Pakistan, Lao, Korea). The ability to decontaminate and defuse these viruses will be useful in preventing potentially devastating illness. Furthermore, these technologies, if successful, can be adapted to hosts of other bat- origin CoVs (MERS, SADS), and potentially other zoonotic bat-origin viruses (Hendra, Nipah, EBOV). Finally, our approach is directly applicable to public health measures in the region to reduce the risk of spillover into the general population, as well as for food security by reducing the risk of viruses like SADS-CoV spilling over from bats into intensive pig farms, and devastating and industry, leading to potential civil unrest.
6. How much will it cost and how long will it take?
Will insert this later after calculating and brainstorming.
D. Technical Plan:
Outline and address all technical challenges inherent in the approach and possible solutions for overcoming potential problems. This section should provide appropriate specific milestones (quantitative, if possible) at intermediate stages of the project to demonstrate progress and a brief plan for accomplishment of the milestones. **Note: “The technical plan should demonstrate a deep understanding of the
technical challenges and present a credible (even if risky) plan to achieve
the program goal”
Key Terms/Aspects to Emphasize in Abstract
Commented [PD1]: Check on the duration of PREEMPT
46 months

● IACUC/IRB
○ DARPA wants to know who has experience w/ ACURO IACUC work.
■ EHA has multiple ACURO IACUC proposals (either approved or undergoing approval)
■ IRB also in place, just has to be modified
Rationale for the SE Asian SARS-related CoV – Rhinolophus bat target system, and immune priming/boosting: 1) Our group has shown that bats harbor a higher proportion of potentially zoonotic viruses than any other mammalian group (1), so that proof-of- concept for blocking viral spillover from this host group may lead to a bigger impact on global health security; 2) The Rhinolophus bats that harbor SARS like-CoVs are insectivorous and roost in dense colonies at a fixed, known location, yet disperse each night over wide distances from these sites. Defusing the risk of viral shedding in the roost will also defuse the risk of viral shedding over the population range. This would be difficult for rodent or primate reservoirs; 3) Bats are mammalian hosts, therefore immune modulating drugs trialed out in people may also work on bats. This would be less likely for an insect vector; 4) Members of our collaborative group has worked together on bats and their viruses for over 15 years, with a total of >100 yrs experience focused on bat-origin zoonoses among the key personnel. We have published much of the seminal work on the bat origins of SARS, Nipah, Hendra, and MERS viruses, and have opened new boundaries in studies of bat host-viral relationships ecologically, immunologically and virologically; 5) The South and Southeast Asian region where these bats occur is a security hotspot, with active political and ethnic conflicts, and displaced populations in Bangladesh, Pakistan, Myanmar, Thailand, Indonesia, Philippines and other countries. This is a likely potential site for US warfighter deployment; 6) We have worked for over 10 years on the SARS-related CoV – Rhinolophus bat system in China, demonstrating the origin of SARS-CoV within this host, the presence of SARSr-CoVs with remarkable sequence identity in the spike protein to SARS-CoV, their isolation and characterization of their ability to bind with human cells. We have demonstrated that chimeric SARS-CoV backbone with spike protein from SARSr-CoVs from our cave sites in Yunnan Province can infect a humanized mouse model and cause SARS-like illness, and that clinical signs are not reduced with SARS monoclonal therapy or vaccination. Finally, we have demonstrated that people living up to 6 kilometers from our cave site have evidence of SARSr-CoV antibodies (3% seroprevalence in 200+ cohort), suggesting active spillover, and marking these viruses as a clear-and-present danger of a new SARS-like pandemic; 7) SARSr-CoVs are transmitted among bats via fecal-oral route, making
Commented [PD2]: I know this is too long. I’ll edit later this weekend, but want to keep this text for the full proposal
Overview

sampling relatively easy (collection of fresh fecal pellets) and molecule or vaccine approaches feasible; 8) Proof-of-concept in this system may be rapidly scalable to other bat-coronavirus systems, e.g. MERS-CoV, SADS-CoV, and to other cave bat origin viruses.
Other important bat-origin zoonotic viruses (e.g. filoviruses, henipaviruses) have very rare spillover events - usually to a single index case, which makes validated prevention of spillover challenging. These viruses also show little strain diversity which makes modeling which evolutionary lines will be more high-risk, a challenge. SARSr-CoVs are diverse, with recombinants regularly identified in the field and lab. Furthermore, we have identified a single cave in Yunnan that harbors every gene from the SARS-CoV in a diversity of SARSr-CoVs within the bat population, making it an ideal evolutionary soup to target for intervention.
Finally, we believe that alternative approaches to transmission blocking, e.g. CRISPER-Cas are likely to be far less effective in bats because most bats are long-lived relative to their small size, with long inter-generational periods (2-5 years). Gene drives would likely take many decades to run through a population, so that proof-of-concept of transmission blocking in the DARPA time scale wouldn’t be possible. Furthermore, many bat species’ populations mix readily or migrate which would disperse the impact of gene drives, whereas targeting a small number of caves in a region for molecule or vaccine delivery would cover a very large dispersal area.
TA1: Develop and validate integrated, multiscale models that quantify the likelihood a human-capable virus will emerge from an animal reservoir residing in a “hot spot” geographic region
The DEFUSE modeling and analytics team will develop models to evaluate the likelihood of bat caves harboring high-risk SARSr-CoVs, evaluate the probability of specific SARS- related CoV spillover, and identify the most effective strategy for inoculation of immune boosting molecules and chimeric spike protein immune priming inocula.
We will collect specific data to inform our model building, validate assumptions and refine predictions. At the start of Yr 1, we will conduct a full inventory of host and virus distribution within our field sites, two caves in Yunnan Province, China. This builds on 8 years of surveillance in these caves and includes a cave in which we have identified all the genetic components of SARS-CoV distributed across a bat population. Two other caves will act as controls/comparison sites, in that we have not yet identified the high- risk SARSr-CoVs in that cave. We will assess: the population density, distribution and segregation of individual bats; changes in these daily, weekly and by season; viral prevalence and intensity in individuals; distribution of low- and high-risk SARSr-CoV strains, and how readily these are transmitted among bat species, age classes, genders; and using mark-recapture to assess metapopulation structure. To assess geographic

distribution of bat hosts, we have access to biological inventory data on all bat caves in Southern China, as well as information on species distributions across SE Asia from the literature and museum records. We will use radio- and satellite telemetry to identify the home range of each species of bat in the caves, to assess how widely the viral ‘plume’ could contaminate
We will build environmental niche models using the data above, and environmental and ecological correlates, and traits of cave species communities (eg. phylogenetic and functional diversity), to predict the species composition of bat caves across Southern China, South and SE Asia. We will validate these with data from the current project and data from PREDICT sampling in Thailand, Indonesia, Malaysia and other SE Asian countries. We will then use our unique database of bat host-viral relationships updated from our recent Nature paper (1) to assess the likelihood of low- or high-risk SARSr-CoVs being present in a cave at any site across the region. At the end of Yr 1, we will use these analyses to produce a prototype app for the warfighter that identifies the likelihood of bats harboring dangerous viral pathogens based on these analyses. The ‘high-risk bats near me’ app will be updated as new host-viral surveillance data comes on line from our project and others, to ground-truth and fine- tune its predictive capacity. Specifically, our telemetry data on bat movement will be used to assess how often bats from high-risk caves migrate to other colonies and potentially spread their high-risk strains.
The Wuhan Institute of Virology team will conduct viral testing on samples from all bat species in the caves as part of this inventory. Fecal, oral, blood and urogenital samples will be collected from bats using standard capture techniques as we have done for the last decade. In addition, tarps will be laid down in caves to assess the feasibility of surveys using pooled fresh fecal and urine samples. Assays will be designed to correlate viral load in an individual with viral shedding in a fecal sample. Once this is complete, surveys will continue largely on fecal samples so as not to disturb bat colonies and undermine longitudinal sampling capacity. Samples will be tested by PCR and spike proteins of all SARS-related CoVs sequenced. Analyses of phylogeny, recombination events, and further characterization of high-risk viruses (those with spike proteins close to SARS-CoV) will be carried out (REF). Isolation will be attempted on a subset of samples with novel SARSr-CoVs. will reverse engineer spike proteins in his lab to conduct binding assays to human ACE2 (the SARS-CoV receptor). Proteins that bind will then be inserted into SARS-CoV backbones, and inoculated into humanized mice to assess their capacity to cause SARS-like disease, and their ability to be blocked by monoclonal therapies, or vaccines against SARS-CoV (REF).
The modeling team will use these data to build models of 1) risk of viral
surrounding regions, and therefore how wide the risk zone is for the
warfighter positioned close to bat caves.
Commented [PD3]: Could add “ We will continue monitoring the human population proximal to these caves to assess the rates of viral spillover, and ground- truth which specific CoVs are able to infect people
Prof. Ralph Baric, UNC,
Commented [PD4]: Ralph, Zhengli. If we win this contract, I do not propose that all of this work will necessarily be conducted by Ralph, but I do want to stress the US side of this proposal so that DARPA are comfortable with our team. Once we get the funds, we can then allocate who does what exact work, and I believe that a lot of these assays can be done in Wuhan as well...

evolution and spillover, and 2) strategies to maximize inoculation strategy.
Data on the diversity of bat spike proteins, prevalence of recombinant CoVs, ability to bind and infect human cells, degree of clinical signs in mouse models, will be used to estimate evolutionary rates, rates of recombination, and capacity to generate novel strains capable of human infection. Using dynamic metapopulation models, we will estimate the flow of genes within each bat cave, based on the known host and viral assemblages. This will inform how rapidly new CoV strains with distinct phenotypic characteristics evolve. Because of our unique collaboration among world-class modelers, and coronavirologists, we will be able to test model predictions of viral capacity for spillover by conducting spike protein-based binding and cell culture experiments. The BSL-2 nature of work on SARSr-CoVs makes our system highly cost- effective relative to other bat-virus systems (e.g. Ebola, Marburg, Hendra, Nipah), which require BSL-4 level facilities for cell culture.
We will use modeling approaches, the data above, and other biological and ecological data to estimate how rapidly high-risk SARSr-CoVs will re-colonize a bat population following immune boosting or priming. We will obtain model estimates of the frequency of inoculation required for both approaches, what proportion of a population needs to be reached to have effective viral dampening, and whether specific seasons, or locations within a cave would be more effective to treat. We will then model the efficacy of different delivery methods (spray, swab, cave mouth automated delivery, deliver to specific sections of a cave).
TA2: Develop scalable approaches that target and suppress the animal virus in its reservoir(s) and/or vector(s), to reduce the likelihood of virus transmission into humans.
Our goal is to use two approaches to defuse the potential for SARS-related CoVs to emerge in people: 1) Immune Boosting: using the unique immunological features of bats that our group has discovered, we will inoculate live bats in cave mesocosms with immune modulators to up-regulate their naïve immunity to suppress viral replication and shedding; 2) Immune Priming: building on preliminary development of polyvalent chimeric recombinant molecules targeting diverse spike proteins from bat SARS-related CoVs, we will produce, and trial inoculation of live bats to suppress the replication and shedding of a broad range of dangerous SARS-related CoVs. Both lines of work will begin in Yr 1 and run parallel throughout the project.
Prof. Linfa Wang (Duke-NUS) will lead the work on immune boosting work, building on his pioneering work on bat immunity (2). This work provides evidence that that the long-term coexistence of bats and their viruses has led to an equilibrium between viral replication and host immunity, whereby bats have specifically down-

regulated their innate immune system as part of the fitness cost of flight (the only true flying mammals) (2). The nature of the weakened but not entirely lost functionality of bat innate immunity factors like STING, a central DNA-interferon (IFN) sensing molecule, may have profound impact for bats to maintain the balanced state of “effective response”, but not “over response” against viruses (3). A similar finding was also observed in bat IFNA studies, which is less abundant but was constitutively expressed without stimulation (4). Given native levels of SARSr-CoVs in individual bats with damped immunity, we propose to suppress bat SARSr-CoV by boosting bat innate immunity through the IFN pathway, and breaking the natural host-virus equilibrium. One of the potential problems with this approach is that it can lead to severe inflammation. However, this is unlikely to occur in bats, because they also have a naturally dampened inflammation response (5).
Previous work has shown that aerosol spraying or intranasal inoculation of IFN or other small molecules has led to reduce viral loads in humans, ferrets and mouse models (12-14). We will therefore initially trial inoculation of live bats with synthetic double-stranded RNA (Poly I:C) and assay for reduced viral loads (DETAILS, CITATION). We will generate universal bat interferon and apply to bats in the lab. Interferon has been used extensively clinically if no viral-specific drugs are available, e.g. against filoviruses (11). Secondly, bat replication of SARSr-CoV is sensitive to interferon treatments, as has been shown in our previous work (12). We will attempt to boost bat IFN by blocking bat-specific IFN negative regulator. Bat IFNA is naturally constitutively expressed but cannot be induced to a high level (4). This is unique to bats. We think there should be a negative regulatory factor in the bat interferon production pathway. We propose using CRISPRi to find out that negative regulator and then screen for chemicals targeting at this gene. We will attempt to boost bat IFN by activating dampened bat-specific IFN production pathways which include DNA-STING-dependent and ssRNA-TLR7 dependent pathways. These changes have been proved to bat-specific, suggesting that they are important in viruses/bats coexistence, and supported by our own work showing that a mutant bat STING restores antiviral functionality (3). By identifying small molecules to directly activate downstream of STING, we have chance to activate bat interferon and then help bats to clear viruses. Similar strategy applies to ssRNA-TLR7 dependent pathways. We will also attempt to boost bat IFN by activating functional bat IFN production pathways. We will investigate if there are other IFN production pathways in bats. We then boost bat immune responses by ligands specifically to these pathways, e.g. polyIC to TLR3-IFN pathway or 5’ppp-dsRNA to RIG-I- IFN pathway. A similar strategy has been tested successful in mouse model for SARS- CoV, IAV or HBV (6, 7). We believe treating wild bats with IFN-modulating small molecules by spraying is superior to other invasive strategies that might be considered

by DARPA, including genome editing (CRISPR or RNAi), vaccination or DIP bats, in terms of its deployability and scalability. Finally, we will inoculate bats with fragments of non- bat Coronavirus (DETAILS).
Prof. Ralph Baric (UNC) will lead the immune priming work, building on his track record in reverse-engineering and manipulating SARS-CoV, MERS-CoV and other virus spike proteins over the last two decades . He will develop recombinant chimeric spike- proteins (8) based on SARSr-CoVs we have already identified, and those we will discover and characterize during project DEFUSE.
please!
While there are clear advantages to working with fixed populations of cave-
dwelling bats, molecule or vaccine delivery is technically challenging. Dr. Tonie Rocke, who developed, trialed, field-tested and rolled out the prairie dog plague vaccine (9), and is currently working on vaccines to bat rabies (10, 11) and white-nose syndrome, will manage a series of experiments in the lab and field to perfect a delivery system for both arms of TA2.
We will conduct initial experiments on a lab colony of wild-caught Rhinolophus sinicus bats at Wuhan Institute of Zoology. We (Prof. Wang) have previous experience conducting infection experiments on this bat genus ...(details and citation if possible). First, we will use our recently proven technology to design LIPS assays to the specific high zoonotic-risk SARSr-CoVs (12). We will conduct serological analysis on bats captured for infection experiments, to assess prior exposure to specific strains. These LIPS assays will be made available for use in people to assess exposure of the general population around bat caves in China, and for potential use by the warfighter to assess exposure to SARSr-CoVs during combat missions.
Finally, work on a delivery method will be overseen by Dr. Tonie Rocke at the National Wildlife Health Center who has proven capacity to develop and take animal vaccines through to licensure (9). Using her captive Jamaican fruitbat colony (10, 11), Dr. Rocke will trial out the following strategies for delivery of the molecules, inocula proposed above: 1) aerosolization; 2) transdermally applied nanoparticles; 3) sticky edible spray that bats will groom from each other; 4) automated spray triggered by timers and movement detectors at critical cave entry points.. (Details and ideas please Tonie!). These approaches will then be trialed out on live bats in our three cave sites in Yunnan Province. Fieldwork will be conducted under the auspices of Dr. Rocke, EHA field staff, and Dr. Yunzhi Zhang (Yunnan CDC, Consultant with EcoHealth Alliance). Sections of bat caves will be cordoned off and different application methods trialed out. A small number of bats will be captured and assayed for viral load after treatment, but so as not to disturb the colony, most viral load work will be conducted on fresh fecal pellets
RALPH – clearly I didn’t really understand the
details of your approach. Can you add a couple of paragraphs here and some citations

collected daily on the cave floor. EHA has unique access to these sites in Yunnan Province, with our field teams conducting surveillance there for around 10 years, under the guidance of Drs. Shi and Zhang. In year 1 of project DEFUSE, we will seek permission for these experimental inoculations in cave sites in Yunnan from the Provincial Forestry Department. We do not envisage problems getting permission, as we have worked with the Forestry Department collaboratively for the last few years, we have the support of the Yunnan CDC, and we are releasing molecules that are not dangerous to people or wildlife.
E. Capabilities:
A brief summary of expertise of the team, including subcontractors and key personnel. A principal investigator for the project must be identified, and a description of the team’s organization. Include a description of the team’s organization including roles and responsibilities. Describe the organizational experience in this area, existing intellectual property required to complete the project, and any specialized facilities to be used as part of the project. List Government furnished materials or data assumed to be available.
**Note: While only the proposal requires an organization chart, it may be helpful to include in the abstract if we have the space.
• This organization chart would include (as applicable): (1) the programmatic relationship of team members; (2) the unique capabilities of team members; (3) the task responsibilities of team members; (4) the teaming strategy among the team members; (5) key personnel with the amount of effort to be expended by each person during each year.
The lead institution for Project DEFUSE is EcoHealth Alliance, New York, an international research non-profit focused on emerging zoonotic diseases. The project will be led by PI Dr. Peter Daszak, who has 20+ years’ experience managing lab, field and modeling research projects on emerging zoonoses, including as EHA institutional lead, Head of Modeling and Analytics, and member of the Executive Committee for the $130 million USAID EPT/PREDICT. Dr. Daszak will oversee and coordinate all project activities, as well as lead the modeling and analytic work for TA1. Dr. Billy Karesh, who has 40+ years’ experience managing wildlife disease and zoonotic disease projects, will manage partnership activities and relationships and outreach. Dr. Jon Epstein, who has 15 years’ experience working with bats and emerging zoonoses will coordinate work on bat immune priming and boosting trials. Dr. Kevin Olival and Dr. Noam Ross will manage and conduct the modeling and analytical approaches for this project.

Team:
Lead Organization: EcoHealth Alliance, New York
PI: Peter Daszak Ph.D., President & Chief Scientist, EcoHealth Alliance, 3 months/year Key Personnel:
Billy Karesh DVM, Executive VP for Policy & Health, 1 month/year
Kevin J. Olival Ph.D, VP for Scientific Research, 1 month/year
Jonathan H. Epstein DVM Ph.D., VP for Science & Outreach, 0.5 months/year
Carlos Zambrana-Torrelio Ph.D., Assoc. VP for Conservation & Health, 1 month/year Noam Ross Ph.D., Senior Research Scientist, 2 months/year
Evan Eskew, Research Scientist, 2 months/year
Hongying Li, Program Coordinator, China Programs, 3 months/year
TBD Postdoctoral Researcher modeling and analysis, 12 months/year
TBD Research Assistant, 12 months/year
TBD Program Assistant, 12 months/year
Guangjian Zhu Ph.D., Consultant Field Lead, China Programs, 6 months/year
Yunzhi Zhang Ph.D., Consultant, Yunnan CDC, China, 2 months/year
Subcontract #1: University of North Carolina Medical School Organizational Lead: Prof. Ralph Baric Ph.D., 2 months/year XXX
TBD Research Assistant, 12 months/year
Subcontract #2: USGS National Wildlife Health Center
Organizational Lead: Tonie Rocke Ph.D., 2 months/year, no salary requested TBD Research Technician, 9 months/year
Subcontract #3: Duke NUS, Singapore
Organizational Lead: Prof. Linfa Wang Ph.D., 2 months/year XXX
TBD Research Assistant, 12 months/year
XXX
Subcontract #4: Wuhan Institute of Virology, China Organizational Lead: Prof Zhengli Shi Ph.D., 2 months/year Peng Zhou Ph.D., 2 months/year
TBD Research Assistant, 12 months/year

Links to relevant papers, reports, or
Do not include more than two resumes as part of the abstract.
**Resumes count against the abstract page limit.
Dr. Peter Daszak is President and Chief Scientist of EcoHealth Alliance, a US-based organization that conducts research and outreach programs on emerging zoonotic diseases. He has published over 300 scientific papers, including the first global map of EID hotspots, strategies to estimate unknown viral diversity in wildlife, predictive models of virus-host relationships, and evidence of the bat origin of SARS-CoV and other emerging viruses. Dr Daszak is Chair of the National Academy of Sciences, Engineering and Medicine’s Forum on Microbial Threats and is a member of the Executive Committee and the EHA institutional lead for USAID-EPT-PREDICT. He serves on the NRC Advisory Committee to the USGCRP, the DHS CEEZAD External Advisory Board, and the WHO R&D Blueprint Pathogen Prioritization expert group, and has advised the Director for Medical Preparedness Policy on the White House National Security Staff on global health issues. Dr Daszak won the 2000 CSIRO medal for collaborative research.
Prof. Ralph Baric is a UNC Lineberger Comprehensive Cancer Center member and Professor in the UNC-Chapel Hill Department of Epidemiology. His work focuses on coronaviruses as models to study the genetics of RNA virus transcription, replication, persistence, and cross species transmission. His work crosses the boundaries of microbiology, virology, immunology and epidemiology, looking especially at the population genetics of viruses to find the molecular building blocks for more effective vaccines.
**General Notes:
• DARPA will evaluate proposals using the following criteria, listed in
descending order of importance:
1) 5.1.1. Overall Scientific and Technical Merit
The proposed technical approach is innovative, feasible, achievable, and complete.
Task descriptions and associated technical elements provided are complete and in a logical sequence with all proposed deliverables clearly defined such that a final outcome that achieves the goal can be expected as a result of award. The proposal identifies
Commented [PD5]: I’m planning to use my resume and Ralph’s. Linfa/Zhengli, I realize your resumes are also very impressive, but I am trying to downplay the non-US focus of this proposal so that DARPA doesn’t see this as a negative.
F. If desired, include a brief bibliography
resumes of key performers.

major technical risks and planned mitigation efforts are clearly defined and feasible. The proposed PREEMPT Risk Mitigation Plan effectively provides the following: an assessment of potential risks; proposed guidelines to ensure maximal biosafety and biosecurity; a risk management plan for responsible communications; and a plan to address how input from the Government and community stakeholders will be considered regarding communication and publication of potentially sensitive dual-use information.
2) 5.1.2. Potential Contribution and Relevance to the DARPA Mission
The potential contributions of the proposed effort are relevant to the national technology base. Specifically, DARPA’s mission is to make pivotal early technology investments that create or prevent strategic surprise for U.S. National Security. The proposer clearly demonstrates its capability to transition the technology to the research, industrial, and/or operational military communities in such a way as to enhance U.S. defense. In
addition, the evaluation will take into consideration the extent to which the proposed intellectual property (IP) rights will potentially impact the Government’s ability to transition the technology.
3) 5.1.3. Cost Realism
The proposed costs are realistic for the technical and management approach and accurately reflect the technical goals and objectives of the solicitation. The proposed costs are consistent with the proposer's Statement of Work and reflect a sufficient understanding of the costs and level of effort needed to successfully accomplish the proposed technical approach. The costs for the prime proposer and proposed subawardees are substantiated by the details provided in the proposal (e.g., the type and number of labor hours proposed per task, the types and quantities of materials, equipment and fabrication costs, travel and any other applicable costs and the basis for the estimates).
It is expected that the effort will leverage all available relevant prior research in order to obtain the maximum benefit from the available funding. For efforts with a likelihood of commercial application, appropriate direct cost sharing may be a positive factor in the evaluation.
DARPA recognizes that undue emphasis on cost may motivate proposers to
offer low-risk ideas with minimum uncertainty and to staff the effort with junior
personnel in order to be in a more competitive posture. DARPA discourages such cost
strategies.
Commented [EA6]: Please note

Citations
2. 3.
4. 5.
6. 7.
1.
K. J. Olival et al., Host and viral traits predict zoonotic spillover from mammals. Nature 546, 646-650 (2017).
G. Zhang et al., Comparative analysis of bat genomes provides insight into the evolution of flight and immunity. Science 339, 456-460 (2013).
J. Xie et al., Dampened STING-Dependent Interferon Activation in Bats. Cell host & microbe, (2018).
P. Zhou et al., Contraction of the type I IFN locus and unusual constitutive expression of IFN-αin bats. Proceedings of the National Academy of Sciences of the United States of America, 201518240-201518246 (2016).
M. Ahn, J. Cui, A. T. Irving, L.-F. Wang, Unique Loss of the PYHIN Gene Family in Bats Amongst Mammals: Implications for Inflammasome Sensing. Scientific Reports 6, (2016).
J. Zhao et al., Intranasal Treatment with Poly(I.C) Protects Aged Mice from Lethal Respiratory Virus Infections. Journal of Virology 86, 11416-11424 (2012).
J. Wu et al., Poly(I:C) Treatment Leads to Interferon-Dependent Clearance of Hepatitis B Virus in a Hydrodynamic Injection Mouse Model. Journal of Virology 88, 10421-10431 (2014).
Attachment 1: Executive Summary Slide template

8.
9. 10.
11. 12.
X. F. Deng et al., A Chimeric Virus-Mouse Model System for Evaluating the Function and Inhibition of Papain-Like Proteases of Emerging Coronaviruses. Journal of Virology 88, 11825-11833 (2014).
T. E. Rocke et al., Sylvatic Plague Vaccine Partially Protects Prairie Dogs (Cynomys spp.) in Field Trials. Ecohealth 14, 438-450 (2017).
B. Stading et al., Protection of bats (Eptesicus fuscus) against rabies following topical or oronasal exposure to a recombinant raccoon poxvirus vaccine. Plos Neglect. Trop. Dis. 11, (2017).
B. R. Stading et al., Infectivity of attenuated poxvirus vaccine vectors and immunogenicity of a raccoonpox vectored rabies vaccine in the Brazilian Free-tailed bat (Tadarida brasiliensis). Vaccine 34, 5352-5358 (2016).
P. Zhou et al., Fatal Swine Acute Diarrhea Syndrome caused by an HKU2- related Coronavirus of Bat Origin. Nature In press, (2018
).

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yldipar yrev gnihcaorppa si stcartsba rof etad euD .snrecnoc/snoitseuq
yna evah uoy fi noos wonk em tel tub ,drager taht ni noitcirtser
on si ereht sa slasoporp htob ni etapicitrap ot nalp I ,tniop siht tA
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lanoitaN eht ta si einoT .yadirF no ekcoR einoT htiw ekops I dna ylliB – hplaR ,afniL ,ilgnehZ )1
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.krow eht od ot deen lliw uoy ffats
tahw no siht ni ytiralc teg ew os trap maet eht no sliated dna seman emos dda esaelp – llA )4

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Hongying Li, MPH
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460 West 34th Street – 17th floor New York, NY 10001
1.917.573.2178 (U.S. mobile) 86.130.4112.0837 (China mobile) Hongying Li (Skype)
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EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics
and promote conservation.
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ESUFED tcejorP - tcartsba APRAD eht fo tfard )hguor( tsriF :eR

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From: Peter Daszak [mailto:daszak@ecohealthalliance.org]
Sent: Thursday, 8 February, 2018 10:51 AM
To: Ralph Baric (rbaric@email.unc.edu); Wang Linfa; Zhengli Shi (zlshi@wh.iov.cn); William B. Karesh; Rocke, Tonie
Cc: Luke Hamel; Jonathon Musser; Anna Willoughby; Kevin Olival, PhD; Jon Epstein; Noam Ross; Aleksei Chmura; Anna Willoughby; Hongying Li
Subject: First (rough) draft of the DARPA abstract - Project DEFUSE
Importance: High
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.krow eht od ot deen lliw uoy ffats tahw
no siht ni ytiralc teg ew os trap maet eht no sliated dna seman emos dda esaelp – llA )4
.ti htiw enod evʼI tahw kcehc esaelp tub ,lufesu yrev – yawyna txet
emos em gnivig rof gneP ot sknahT .tcerroc kool snoitces ruoy ekam ot hcae shpargarap
fo elpuoc a tfard esaelp dna ,egdelwonk fo kcal ym nodraP .snoitces ruoy fo tsom rof sliated
lacinhcet/egaugnal eht delgnam evah I ,ees nac uoy sa – einoT ,afniL ,ilgnehZ ,hplaR )3
!eerga uoy epoh I .desoporp evah
gneP/afniL taht krow gnitsoob enummi yksir dna yks-eulb eht ot evitanretla na sa tca lliw ti
)2 ,ti no dehsilbup sah eh dna esitrepxe sih sʼti )1 esuaceb tif tcefrep a si taht kniht I .kcatta
fo enil dnoces a sa snietorp ekips ciremihc tnanibmocer no krow ot desoporp eH .ciraB
hplaR htiw noitasrevnoc taerg a dah I ,htob uoy htiw ekops I retfA – afniL dna ilgnehZ )2
od ot elba eb lliw dna tcejorp ruo rof tcefrep si einoT ,ees llʼuoy sA .tuo taht yal yllacificeps
2AT htiw pleh ot anihC ni ilgnehZ htiw dna CHWN SGSU ta krow
APRAD esuaceb efildliw ot seniccav/selucelom fo yreviled ni esitrepxe htiw enoemos
ni eugalp :seniccav efildliw no dekrow sah dna ,ASU nosidaM ,retneC htlaeH efildliW lanoitaN
dedeen eW .stab SU ni emordnys eson etihw ,stab tiurf naciamaJ ni seibar ,sgod eiriarp
eht ta si einoT .yadirF no ekcoR einoT htiw ekops I dna ylliB – hplaR ,afniL ,ilgnehZ )1
:stniop tnatropmi emoS
ot sruoh ynam koot dna yadirF no hguorht emac repap ecneicS ym ot stide ,yletanutrofnU
ffo eseht gnihsinif ma 3 ta letoh ym ni aveneG ni won thgir mʼI .em deyaled siht os ,od
.gniteem OHW a morf CYN ot kcab gniylf erofeb
.yaled eht rof seigolopA .tcartsba APRAD eht fo tfard hguor tsrif a dehcatta evʼI
,llA raeD

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ecnaillA htlaeHocE
tnediserP
kazsaD reteP
reteP
,sreehC
!tcartnoc
ylemertxe na evah ew kniht I ,gnireffo erʼew tahw dna ,stnaw APRAD tahw ,noititepmoc eht
siht gninniw dna rehtegot lasoporp lluf eht gnitteg ot drawrof gnikool mʼI os ,maet gnorts
fo wonk I tahw morF .lasoporp siht no etaroballoc ot gnieerga rof uoy fo lla ot niaga sknahT
.em ot ti dnes uoy ecno enod evʼuoy
tahw morf pu kcip llʼI .edils yrammus evitucexe eht fo tfard tsrif a ta og a evah esaelp – ekuL
.ffo meht hsinif nac uoy os ssucsid ot noonretfa yadsruhT uoy llac llʼI .wolf tcejorp eht
pu mus taht sngised egami tfard emos no krow dna tfard eht daer esaelp :annA dna ieskelA
.bew eht ffo meht llup llʼew dna sexob tnemmoc sa secnerefer tresni tsuj esaelp ,etondne
pu ssem ot tnaw tʼnod uoy fI .drow ni segnahc dekcart gnisu yldipar tide esaelp – yllaniF )6
.lasoporp lluf eht rof
no dliub ot tfard siht esu nac I taht os ,ereh ni saedi doog ruoy fo ynam sa tnaw osla I .txet
siht no krow lliw I .gniticxe erom dna ,tcerroc nettirw evʼI tahw ekam ot tide dna ,secnerefer
gniticxe-repus ,psirc yrev fo segap 8 ot nwod ti gnirb ot yadnoM dna yadnuS ,yadrutaS no
,ereht dna ereh txet dda tsuJ .timil egap 8 eht ot siht gnipeek tuoba yrrow tʼnod esaelP )5

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Vice President for Science and Outreach
@epsteinjon
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10/5/21, 2:49 PM Mail - Rocke, Tonie E - Outlook
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics
and promote conservation.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 6/6

DARPA – PREEMPT – HR001118S0017
Abstract Submission Requirements:
**8 pages with 12 point font or higher (smaller font may be used for figures, tables
and charts)
**Page limit includes all figures, tables, charts and the Executive Summary Slide **Copies of all documents submitted must be clearly labeled with the following:
-DARPA BAA number
-Proposer Organization
-Proposal title/Proposal short title
-Submission letter is optional and does not count towards page limit
A. Cover Sheet (does not count towards page limit):
Include the administrative and technical points of contact (name, address, phone, fax, email, lead organization). Also include the BAA number, title of the proposed project, primary subcontractors, estimated cost, duration of project, and the label “ABSTRACT.”
B. Executive Summary Slide:
Provide a one slide summary in PowerPoint that effectively and succinctly conveys the main objective, key innovations, expected impact, and other unique aspects of the proposed project. Use the slide template provided at http://www.fbo.gov.
**See slide template at bottom of document.
Clearly describe what is being proposed and what difference it will make (qualitatively and quantitatively), including brief answers to the following questions:
1. What is the proposed work attempting to accomplish or do?
We aim to defuse the potential for emergence of novel bat-origin high-zoonotic risk SARS-related coronaviruses in Southeast Asia. We envisage a scenario whereby the US warfighter is called on to intervene in a security hotspot in SE Asia for a period of 3-6 months. As planners begin choosing sites for the mission, they will use an app we will design to assess the background risk of a site harboring dangerous zoonotic viruses. If
PROJECT DEFUSE
C. Goals and Impact:

there is no alternative to a high-risk site, a tactical forward team will deploy automated delivery technology we will develop in caves that harbor bats carrying these viruses. These devices will release immune boosting molecules and
at the site for a few weeks or months, allowing our warfighters to execute the operation at lowered risk for
spillover.
2. How is it done today? And what are the limitations?
Currently, there is no available technology to reduce the risk of exposure to novel coronaviruses from bats, other than avoid the regions where bats harbor these viruses. This includes large areas of southeast Asia where SARS-related CoVs are endemic in bats, which roost in caves during the day, but forage over wide areas at night, shedding virus in their feces and urine. The limitations of this lack of capacity are significant – we have shown evidence of recent spillover of SARS-related CoVs into people in southern China, and have identified viruses in this region that are capable of producing SARS-like illness in humanized mice, with no available vaccines or countermeasures. These viruses are a clear-and-present danger to our military personnel, and to global health security.
3. What is innovative in your approach and how does it compare to current practice and state-of-the-art (SOA)?
**Note: DARPA wants to know, “how the proposed project is revolutionary and how it significantly rises above the current state of the art
Our group has shown that bats harbor the highest proportion of potential zoonoses of any mammal group, and that they are able to coexist and spillover viruses due to unique features of their immune systems, likely as an evolutionary adaptation to flight. We will use this to design strategies to upregulate their immune response in their cave roosts, down-regulate viral replication, and reduce the risk of viral shedding and spillover (immune boosting strategy). At the same time, we will inoculate bats with novel chimeric polyvalent recombinant spike proteins to enhance their immune response against replication of specific, high-risk viruses ( ). We will use our innovative modeling to design apps that identify the likelihood of any region harboring high-risk bat viruses. We will design novel, automated approaches to deliver both types of inoculum remotely into caves to reduce exposure risk during decontamination.
4. What are the key technical challenges in your approach and how do you plan to overcome these?
Decide which of following parts to talk about:
chimeric polyvalent spike
protein immune priming inocula to lower viral shedding from bats
Commented [p1]: so far all evidences point out that bat WON”T generate high Ab titer, whether under naturally or experimental infection. This is very unique compared to other mammals. We hypothesis this is because of a dampened bat antibody response. Thus I suggest a pre-exposure using immune boosting to bats, and a post-exposure using spike to HUMAN!
Deleted: live with high viral loads Deleted: damping
Commented [p2]: same comment as above
Commented [p3]: sampling and testing might be challenging as we haven’t done a thorough analysis in a certain region. We probably need to sequencing full genome, which is costly as well. I guess we mention the issue that need more money but not revolutionary technique...
immune priming strategy

Modeling bat suitability
Reverse engineering, binding assays, mouse expts
Formatted: Highlight
Formatted: Highlight Formatted: Highlight Formatted: Highlight
Formatted: Highlight
Formatted: Highlight Formatted: Highlight Formatted: Highlight
Modeling viral risk of evolution and spillover
Modeling inoculation/defusing strategy
Immune Booster recombinant production
Inoculum delivery
Mesocosm expts
Cave expts
46 months
Commented [PD4]: Check on the duration of PREEMPT
Inventory of caves Sampling/testing
Immune modulation Gain-of-function issue.
5. Who will care and what will the impact be if you are successful?
This will have direct relevance to the warfighter. The potential for deployment to the region in which bat hosts of SARS-related CoVs exist is high – countries include security hotspots (Myanmar, Bangladesh, Pakistan, Lao, Korea). The ability to decontaminate and defuse these viruses will be useful in preventing potentially devastating illness. Furthermore, these technologies, if successful, can be adapted to hosts of other bat- origin CoVs (MERS, SADS), and potentially other zoonotic bat-origin viruses (Hendra, Nipah, EBOV). Finally, our approach is directly applicable to public health measures in the region to reduce the risk of spillover into the general population, as well as for food security by reducing the risk of viruses like SADS-CoV spilling over from bats into intensive pig farms, and devastating and industry, leading to potential civil unrest.
6. How much will it cost and how long will it take?
Will insert this later after calculating and brainstorming.
D. Technical Plan:
Outline and address all technical challenges inherent in the approach and possible solutions for overcoming potential problems. This section should provide appropriate specific milestones (quantitative, if possible) at intermediate stages of the project to demonstrate progress and a brief plan for accomplishment of the milestones. **Note: “The technical plan should demonstrate a deep understanding of the
technical challenges and present a credible (even if risky) plan to achieve
the program goal”
Key Terms/Aspects to Emphasize in Abstract

● IACUC/IRB
○ DARPA wants to know who has experience w/ ACURO IACUC work.
■ EHA has multiple ACURO IACUC proposals (either approved or undergoing approval)
■ IRB also in place, just has to be modified
Rationale for the SE Asian SARS-related CoV – Rhinolophus bat target system, and immune priming/boosting: 1) Our group has shown that bats harbor a higher proportion of potentially zoonotic viruses than any other mammalian group (1), so that proof-of- concept for blocking viral spillover from this host group may lead to a bigger impact on global health security; 2) The Rhinolophus bats that harbor SARS like-CoVs are insectivorous and roost in dense colonies at a fixed, known location, yet disperse each night over wide distances from these sites. Defusing the risk of viral shedding in the roost will also defuse the risk of viral shedding over the population range. This would be difficult for rodent or primate reservoirs; 3) Bats are mammalian hosts, therefore immune modulating drugs trialed out in people may also work on bats. This would be less likely for an insect vector; 4) Members of our collaborative group has worked together on bats and their viruses for over 15 years, with a total of >100 yrs experience focused on bat-origin zoonoses among the key personnel. We have published much of the seminal work on the bat origins of SARS, Nipah, Hendra, and MERS viruses, and have opened new boundaries in studies of bat host-viral relationships ecologically, immunologically and virologically; 5) The South and Southeast Asian region where these bats occur is a security hotspot, with active political and ethnic conflicts, and displaced populations in Bangladesh, Pakistan, Myanmar, Thailand, Indonesia, Philippines and other countries. This is a likely potential site for US warfighter deployment; 6) We have worked for over 10 years on the SARS-related CoV – Rhinolophus bat system in China, demonstrating the origin of SARS-CoV within this host, the presence of SARSr-CoVs with remarkable sequence identity in the spike protein to SARS-CoV, their isolation and characterization of their ability to bind with human cells. We have demonstrated that chimeric SARS-CoV backbone with spike protein from SARSr-CoVs from our cave sites in Yunnan Province can infect a humanized mouse model and cause SARS-like illness, and that clinical signs are not reduced with SARS monoclonal therapy or vaccination. Finally, we have demonstrated that people living up to 6 kilometers from our cave site have evidence of SARSr-CoV antibodies (3% seroprevalence in 200+ cohort), suggesting active spillover, and marking these viruses as a clear-and-present danger of a new SARS-like pandemic; 7) SARSr-CoVs are transmitted among bats via fecal-oral route, making
Commented [PD5]: I know this is too long. I’ll edit later this weekend, but want to keep this text for the full proposal
Overview

sampling relatively easy (collection of fresh fecal pellets) and molecule or vaccine approaches feasible; 8) Proof-of-concept in this system may be rapidly scalable to other bat-coronavirus systems, e.g. MERS-CoV, SADS-CoV, and to other cave bat origin viruses.
Other important bat-origin zoonotic viruses (e.g. ) have
very rare spillover events - usually to a single index case, which makes validated prevention of spillover challenging. These viruses also show little strain diversity which makes modeling which evolutionary lines will be more high-risk, a challenge. SARSr-CoVs are diverse, with identified in the field and lab. Furthermore, we have identified a single cave in Yunnan that harbors every gene from the SARS-CoV in a diversity of SARSr-CoVs within the bat population, making it an ideal evolutionary soup to target for intervention.
Finally, we believe that alternative approaches to transmission blocking, e.g. CRISPER-Cas are likely to be far less effective in bats because most bats are long-lived relative to their small size, with long inter-generational periods (2-5 years). Gene drives would likely take many decades to run through a population, so that proof-of-concept of transmission blocking in the DARPA time scale wouldn’t be possible (same to rodents and primates). Furthermore, many bat species’ populations mix readily or migrate which would disperse the impact of gene drives, whereas targeting a small number of caves in a region for molecule or vaccine delivery would cover a very large dispersal area.
TA1: Develop and validate integrated, multiscale models that quantify the likelihood a human-capable virus will emerge from an animal reservoir residing in a “hot spot” geographic region
The DEFUSE modeling and analytics team will develop models to evaluate the likelihood of bat caves harboring high-risk SARSr-CoVs, evaluate the probability of specific SARS- related CoV spillover, and identify the most effective strategy for inoculation of immune boosting molecules and chimeric spike protein immune priming inocula.
We will collect specific data to inform our model building, validate assumptions and refine predictions. At the start of Yr 1, we will conduct a full inventory of host and virus distribution within our field sites, two caves in Yunnan Province, China. This builds on 8 years of surveillance in these caves and includes a cave in which we have identified all the genetic components of SARS-CoV distributed across a bat . Two other caves will act as controls/comparison sites, in that we have not yet identified the high-risk SARSr-CoVs in that cave. We will assess: the population density, distribution and segregation of individual bats; changes in these daily, weekly and by season; viral prevalence and intensity in individuals; distribution of low- and high-risk SARSr-CoV strains, and how readily these are transmitted among bat species, age classes, genders; and using mark-recapture to assess metapopulation structure. To assess geographic
recombinants regularly
filoviruses, henipaviruses
Commented [p6]: this contradict to previous comments that our model can apply to EBOV bats
Commented [p7]: caution that too frequent recombination can also make modeling difficult
population
Commented [D8]: Ge et al., Nature, 2013; Yang et al., Journal of Virology; Hu et al., PLoS pathogens, 2017

distribution of bat hosts, we have access to biological inventory data on all bat caves in Southern China, as well as information on species distributions across SE Asia from the literature and museum records. We will use radio- and satellite telemetry to identify the home range of each species of bat in the caves, to assess how widely the viral ‘plume’ could contaminate
We will build environmental niche models using the data above, and environmental and ecological correlates, and traits of cave species communities (eg. phylogenetic and functional diversity), to predict the species composition of bat caves across Southern China, South and SE Asia. We will validate these with data from the current project and data from PREDICT sampling in Thailand, Indonesia, Malaysia and other SE Asian countries. We will then use our unique database of bat host-viral relationships updated from our recent Nature paper (1) to assess the likelihood of low- or high-risk SARSr-CoVs being present in a cave at any site across the region. At the end of Yr 1, we will use these analyses to produce a prototype app for the warfighter that identifies the likelihood of bats harboring dangerous viral pathogens based on these analyses. The ‘high-risk bats near me’ app will be updated as new host-viral surveillance data comes on line from our project and others, to ground-truth and fine- tune its predictive capacity. Specifically, our telemetry data on bat movement will be used to assess how often bats from high-risk caves migrate to other colonies and potentially spread their high-risk strains.
The Wuhan Institute of Virology team will conduct viral testing on samples from all bat species in the caves as part of this inventory. Fecal, oral, blood and urogenital samples will be collected from bats using standard capture techniques as we have done for the last decade. In addition, tarps will be laid down in caves to assess the feasibility of surveys using pooled fresh fecal and urine samples. Assays will be designed to correlate viral load in an individual with viral shedding in a fecal sample. Once this is complete, surveys will continue largely on fecal samples so as not to disturb bat colonies and undermine longitudinal sampling capacity. Samples will be tested by PCR and spike proteins of all SARS-related CoVs sequenced. Analyses of phylogeny, recombination events, and further characterization of high-risk viruses (those with spike proteins close to SARS-CoV) will be carried out (REF). Isolation will be attempted on a subset of samples with novel SARSr-CoVs.
proteins in his lab to conduct binding assays to human ACE2 (the SARS-CoV receptor). Proteins that bind will then be inserted into SARS-CoV backbones, and inoculated into humanized mice to assess their capacity to cause SARS-like disease, and their ability to be blocked by monoclonal therapies, or vaccines against SARS-CoV (REF).
The modeling team will use these data to build models of 1) risk of viral
surrounding regions, and therefore how wide the risk zone is for the
warfighter positioned close to bat caves.
Commented [PD9]: Could add “ We will continue monitoring the human population proximal to these caves to assess the rates of viral spillover, and ground- truth which specific CoVs are able to infect people
Commented [D10]: Ge et al., Nature, 2013; Yang et al., Journal of Virology; Hu et al., PLoS pathogens, 2017
Commented [D11]: Bat serum samples will be tested for antibody (particularly neutralization antibody) against the SARSr-CoV to evaluate the prevalence and lifetime of antibody in bats. Human samples will be collected from people living around cave and tested for SARSr-CoV infection (Wang et al., Virologica Sinica, 2018).
Commented [PD12]: Ralph, Zhengli. If we win this contract, I do not propose that all of this work will necessarily be conducted by Ralph, but I do want to stress the US side of this proposal so that DARPA are comfortable with our team. Once we get the funds, we can then allocate who does what exact work, and I believe that a lot of these assays can be done in Wuhan as well...
Prof. Ralph Baric, UNC,
will reverse engineer spike

evolution and spillover, and 2) strategies to maximize inoculation strategy.
Data on the diversity of bat spike proteins, prevalence of recombinant CoVs, ability to bind and infect human cells, degree of clinical signs in mouse models, will be used to estimate evolutionary rates, rates of recombination, and capacity to generate novel strains capable of human infection. Using dynamic metapopulation models, we will estimate the flow of genes within each bat cave, based on the known host and viral assemblages. This will inform how rapidly new CoV strains with distinct phenotypic characteristics evolve. Because of our unique collaboration among world-class modelers, and coronavirologists, we will be able to test model predictions of viral capacity for spillover by conducting spike protein-based binding and cell culture experiments. The BSL-2 nature of work on SARSr-CoVs makes our system highly cost- effective relative to other bat-virus systems (e.g. Ebola, Marburg, Hendra, Nipah), which require BSL-4 level facilities for cell culture.
We will use modeling approaches, the data above, and other biological and ecological data to estimate how rapidly high-risk SARSr-CoVs will re-colonize a bat population following immune boosting or priming. We will obtain model estimates of the frequency of inoculation required for both approaches, what proportion of a population needs to be reached to have effective viral dampening, and whether specific seasons, or locations within a cave would be more effective to treat. We will then model the efficacy of different delivery methods (spray, swab, cave mouth automated delivery, deliver to specific sections of a cave).
TA2: Develop scalable approaches that target and suppress the animal virus in its reservoir(s) and/or vector(s), to reduce the likelihood of virus transmission into humans.
Our goal is to use two approaches to defuse the potential for SARS-related CoVs to emerge in people: 1) Immune Boosting: using the unique immunological features of bats that our group has discovered, we will inoculate live bats in cave mesocosms with immune modulators to up-regulate their naïve immunity to suppress viral replication and shedding; 2) Immune Priming: building on preliminary development of polyvalent chimeric recombinant molecules targeting diverse spike proteins from bat SARS-related CoVs, we will produce, and trial inoculation of live bats to suppress the replication and shedding of a broad range of dangerous SARS-related CoVs. Both lines of work will begin in Yr 1 and run parallel throughout the project.
Prof. Linfa Wang (Duke-NUS) will lead the work on immune boosting work, building on his pioneering work on bat immunity (2). This work provides evidence that that the long-term coexistence of bats and their viruses has led to an equilibrium between viral replication and host immunity, whereby bats have specifically down-

regulated their innate immune system as part of the fitness cost of flight (the only true flying mammals) (2). The nature of the weakened but not entirely lost functionality of bat innate immunity factors like STING, a central DNA-interferon (IFN) sensing molecule, may have profound impact for bats to maintain the balanced state of “effective response”, but not “over response” against viruses (3). A similar finding was also observed in bat IFNA studies, which is less abundant but was constitutively expressed without stimulation (4). Given native levels of SARSr-CoVs in individual bats with damped immunity, we propose to suppress bat SARSr-CoV by boosting bat innate immunity through the IFN pathway, and breaking the natural host-virus equilibrium. One of the potential problems with this approach is that it can lead to severe inflammation. However, this is unlikely to occur in bats, because they also have a naturally dampened inflammation response (5).
work has shown that aerosol spraying or intranasal inoculation of IFN or
other small molecules has led to reduce viral loads in humans, ferrets and mouse models (12-14). Thus, bat interferon would be our first choice. Firstly, we will generate universal bat interferon and apply to bats in the lab. Interferon has been used extensively clinically if no viral-specific drugs are available, e.g. against filoviruses (11). Replication of SARSr-CoV is sensitive to interferon treatments, as has been shown in our previous work (12). Secondly, we will also attempt to boost bat IFN by blocking bat- specific IFN negative regulator. Bat IFNA is naturally constitutively expressed but cannot be induced to a high level (4). This is unique to bats. We think there should be a negative regulatory factor in the bat interferon production pathway. We propose using CRISPRi to find out that negative regulator and then screen for chemicals targeting at this gene. Thirdly, We will attempt to boost bat IFN by activating dampened bat-specific IFN production pathways which include DNA-STING-dependent and ssRNA-TLR7 dependent pathways. These changes have been proved to bat-specific, suggesting that they are important in viruses/bats coexistence, and supported by our own work showing that a mutant bat STING restores antiviral functionality (3). By identifying small molecules to directly activate downstream of STING, we have chance to activate bat interferon and then help bats to clear viruses. Similar strategy applies to ssRNA-TLR7 dependent pathways. Fourthly, We will also attempt to boost bat IFN by activating functional bat IFN production pathways, e.g. polyIC to TLR3-IFN pathway or 5’ppp- dsRNA to RIG-I-IFN pathway and assay for reduced viral loads. A similar strategy has been tested successful in mouse model for SARS-CoV, IAV or HBV (6, 7). Lastly, we believe treating wild bats with IFN-modulating small molecules by spraying is superior to other invasive strategies that might be considered by DARPA, including genome editing (CRISPR or RNAi), vaccination or DIP bats, in terms of its deployability and scalability. Finally, we will inoculate bats with fragments of (DETAILS).
Commented [p13]: Sorry Peter I rearranged this part in a logical order: interferon first, then modulate unique bat interferon pathways, then modulate usual interferon pathways. I tried not to put polyI:C first, as which is not bat unique.
Moved (insertion) [1]
Deleted: Secondly, bat r Moved (insertion) [2] Deleted: W
Deleted: gene.
Moved (insertion) [3]
Deleted: We will therefore initially trial inoculation of live bats with synthetic double-stranded RNA (Poly I:C) and assay for reduced viral loads (DETAILS, CITATION). We will generate universal bat interferon and apply to bats in the lab. Interferon has been used extensively clinically if no viral-specific drugs are available, e.g. against filoviruses (11). Secondly, bat replication of SARSr-CoV is sensitive to interferon treatments, as has been shown in our previous work (12). We will attempt to boost bat IFN by blocking bat-specific IFN negative regulator. Bat IFNA is naturally constitutively expressed but cannot be induced to a high level (4). This is unique to bats. We think there should be a negative regulatory factor in the bat interferon production pathway. We propose using CRISPRi to find out that negative regulator and then screen for chemicals targeting at this gene. We will attempt to boost bat IFN by activating dampened bat-specific IFN production pathways which include DNA-STING-dependent and ssRNA-TLR7 dependent pathways. These changes have been proved to bat-specific, suggesting that they are important in viruses/bats coexistence, and supported by our own work showing that a mutant bat STING restores antiviral functionality (3). By identifying small molecules to directly activate downstream of STING, we have chance to activate bat interferon and then help bats to clear viruses. Similar strategy applies to ssRNA-TLR7 dependent pathways. We will also attempt to boost bat IFN by activating functional bat IFN production
Moved up [1]: generate universal bat interferon and apply to bats in the lab. Interferon has been used extensively clinically if no viral-specific drugs are available,
Moved up [2]: We will attempt to boost bat IFN by blocking bat-specific IFN negative regulator. Bat IFNA is naturally constitutively expressed but cannot be induced to
Moved up [3]: We will attempt to boost bat IFN by activating dampened bat-specific IFN production pathways which include DNA-STING-dependent and ssRNA-TLR7
Deleted: W
Commented [p14]: same comment as above, I think it should be a backup post-exposure to human but not to bats
Commented [石15]: why with non-bat coronavirus?
Previous
... [1]
non-bat Coronavirus

Prof. Ralph Baric (UNC) will lead the immune priming work, building on his track record in reverse-engineering and manipulating SARS-CoV, MERS-CoV and other virus spike proteins over the last two decades . He will develop recombinant chimeric spike- proteins (8) based on SARSr-CoVs we have already identified, and those we will discover and characterize during project DEFUSE.
please!
While there are clear advantages to working with fixed populations of cave-
dwelling bats, molecule or vaccine delivery is technically challenging. Dr. Tonie Rocke, who developed, trialed, field-tested and rolled out the prairie dog plague vaccine (9), and is currently working on vaccines to bat rabies (10, 11) and white-nose syndrome, will manage a series of experiments in the lab and field to perfect a delivery system for both arms of TA2.
We will conduct initial experiments on a lab colony of wild-caught Rhinolophus sinicus bats at Wuhan Institute of Zoology. We (Prof. Wang) have previous experience conducting infection experiments on this bat genus ...(details and citation if possible). First, we will use our recently proven technology to design LIPS assays to the specific high zoonotic-risk SARSr-CoVs (12). We will conduct serological analysis on bats captured for infection experiments, to assess prior exposure to specific strains. These LIPS assays will be made available for use in people to assess exposure of the general population around bat caves in China, and for potential use by the warfighter to assess exposure to SARSr-CoVs during combat missions.
Finally, work on a delivery method will be overseen by Dr. Tonie Rocke at the National Wildlife Health Center who has proven capacity to develop and take animal vaccines through to licensure (9). Using her captive Jamaican fruitbat colony (10, 11), Dr. Rocke will trial out the following strategies for delivery of the molecules, inocula proposed above: 1) aerosolization; 2) transdermally applied nanoparticles; 3) sticky edible spray that bats will groom from each other; 4) automated spray triggered by timers and movement detectors at critical cave entry points.. (Details and ideas please Tonie!). These approaches will then be trialed out on live bats in our three cave sites in Yunnan Province. Fieldwork will be conducted under the auspices of Dr. Rocke, EHA field staff, and Dr. Yunzhi Zhang (Yunnan CDC, Consultant with EcoHealth Alliance). Sections of bat caves will be cordoned off and different application methods trialed out. A small number of bats will be captured and assayed for viral load after treatment, but so as not to disturb the colony, most viral load work will be conducted on fresh fecal pellets collected daily on the cave floor. EHA has unique access to these sites in Yunnan Province, with our field teams conducting surveillance there for around 10 years, under the guidance of Drs. Shi and Zhang. In year 1 of project DEFUSE, we will seek permission
RALPH – clearly I didn’t really understand the
details of your approach. Can you add a couple of paragraphs here and some citations

for these experimental inoculations in cave sites in Yunnan from the Provincial Forestry Department. We do not envisage problems getting permission, as we have worked with the Forestry Department collaboratively for the last few years, we have the support of the Yunnan CDC, and we are releasing molecules that are not dangerous to people or wildlife.
E. Capabilities:
A brief summary of expertise of the team, including subcontractors and key personnel. A principal investigator for the project must be identified, and a description of the team’s organization. Include a description of the team’s organization including roles and responsibilities. Describe the organizational experience in this area, existing intellectual property required to complete the project, and any specialized facilities to be used as part of the project. List Government furnished materials or data assumed to be available.
**Note: While only the proposal requires an organization chart, it may be helpful to include in the abstract if we have the space.
• This organization chart would include (as applicable): (1) the programmatic relationship of team members; (2) the unique capabilities of team members; (3) the task responsibilities of team members; (4) the teaming strategy among the team members; (5) key personnel with the amount of effort to be expended by each person during each year.
The lead institution for Project DEFUSE is EcoHealth Alliance, New York, an international research non-profit focused on emerging zoonotic diseases. The project will be led by PI Dr. Peter Daszak, who has 20+ years’ experience managing lab, field and modeling research projects on emerging zoonoses, including as EHA institutional lead, Head of Modeling and Analytics, and member of the Executive Committee for the $130 million USAID EPT/PREDICT. Dr. Daszak will oversee and coordinate all project activities, as well as lead the modeling and analytic work for TA1. Dr. Billy Karesh, who has 40+ years’ experience managing wildlife disease and zoonotic disease projects, will manage partnership activities and relationships and outreach. Dr. Jon Epstein, who has 15 years’ experience working with bats and emerging zoonoses will coordinate work on bat immune priming and boosting trials. Dr. Kevin Olival and Dr. Noam Ross will manage and conduct the modeling and analytical approaches for this project.
Team:
Lead Organization: EcoHealth Alliance, New York

PI: Peter Daszak Ph.D., President & Chief Scientist, EcoHealth Alliance, 3 months/year Key Personnel:
Billy Karesh DVM, Executive VP for Policy & Health, 1 month/year
Kevin J. Olival Ph.D, VP for Scientific Research, 1 month/year
Jonathan H. Epstein DVM Ph.D., VP for Science & Outreach, 0.5 months/year
Carlos Zambrana-Torrelio Ph.D., Assoc. VP for Conservation & Health, 1 month/year Noam Ross Ph.D., Senior Research Scientist, 2 months/year
Evan Eskew, Research Scientist, 2 months/year
Hongying Li, Program Coordinator, China Programs, 3 months/year
TBD Postdoctoral Researcher modeling and analysis, 12 months/year
TBD Research Assistant, 12 months/year
TBD Program Assistant, 12 months/year
Guangjian Zhu Ph.D., Consultant Field Lead, China Programs, 6 months/year
Yunzhi Zhang Ph.D., Consultant, Yunnan CDC, China, 2 months/year
Subcontract #1: University of North Carolina Medical School Organizational Lead: Prof. Ralph Baric Ph.D., 2 months/year XXX
TBD Research Assistant, 12 months/year
Subcontract #2: USGS National Wildlife Health Center
Organizational Lead: Tonie Rocke Ph.D., 2 months/year, no salary requested TBD Research Technician, 9 months/year
Subcontract #3: Duke NUS, Singapore
Organizational Lead: Prof. Linfa Wang Ph.D., 2 months/year XXX
TBD Research Assistant, 12 months/year
XXX
Subcontract #4: Wuhan Institute of Virology, China Organizational Lead: Prof Zhengli Shi Ph.D., 2 months/year Peng Zhou Ph.D., 2 months/year
TBD Research Assistant, 12 months/year
Links to relevant papers, reports, or
Do not include more than two resumes as part of the abstract.
F. If desired, include a brief bibliography
Commented [PD16]: I’m planning to use my resume and Ralph’s. Linfa/Zhengli, I realize your resumes are also very impressive, but I am trying to downplay the non-US focus of this proposal so that DARPA doesn’t see this as a negative.
resumes of key performers.

**Resumes count against the abstract page limit.
Dr. Peter Daszak is President and Chief Scientist of EcoHealth Alliance, a US-based organization that conducts research and outreach programs on emerging zoonotic diseases. He has published over 300 scientific papers, including the first global map of EID hotspots, strategies to estimate unknown viral diversity in wildlife, predictive models of virus-host relationships, and evidence of the bat origin of SARS-CoV and other emerging viruses. Dr Daszak is Chair of the National Academy of Sciences, Engineering and Medicine’s Forum on Microbial Threats and is a member of the Executive Committee and the EHA institutional lead for USAID-EPT-PREDICT. He serves on the NRC Advisory Committee to the USGCRP, the DHS CEEZAD External Advisory Board, and the WHO R&D Blueprint Pathogen Prioritization expert group, and has advised the Director for Medical Preparedness Policy on the White House National Security Staff on global health issues. Dr Daszak won the 2000 CSIRO medal for collaborative research.
Prof. Ralph Baric is a UNC Lineberger Comprehensive Cancer Center member and Professor in the UNC-Chapel Hill Department of Epidemiology. His work focuses on coronaviruses as models to study the genetics of RNA virus transcription, replication, persistence, and cross species transmission. His work crosses the boundaries of microbiology, virology, immunology and epidemiology, looking especially at the population genetics of viruses to find the molecular building blocks for more effective vaccines.
**General Notes:
• DARPA will evaluate proposals using the following criteria, listed in
descending order of importance:
1) 5.1.1. Overall Scientific and Technical Merit
The proposed technical approach is innovative, feasible, achievable, and complete.
Task descriptions and associated technical elements provided are complete and in a logical sequence with all proposed deliverables clearly defined such that a final outcome that achieves the goal can be expected as a result of award. The proposal identifies major technical risks and planned mitigation efforts are clearly defined and feasible. The proposed PREEMPT Risk Mitigation Plan effectively provides the following: an assessment of potential risks; proposed guidelines to ensure maximal biosafety and

biosecurity; a risk management plan for responsible communications; and a plan to address how input from the Government and community stakeholders will be considered regarding communication and publication of potentially sensitive dual-use information.
2) 5.1.2. Potential Contribution and Relevance to the DARPA Mission
The potential contributions of the proposed effort are relevant to the national technology base. Specifically, DARPA’s mission is to make pivotal early technology investments that create or prevent strategic surprise for U.S. National Security. The proposer clearly demonstrates its capability to transition the technology to the research, industrial, and/or operational military communities in such a way as to enhance U.S. defense. In
addition, the evaluation will take into consideration the extent to which the proposed intellectual property (IP) rights will potentially impact the Government’s ability to transition the technology.
3) 5.1.3. Cost Realism
The proposed costs are realistic for the technical and management approach and accurately reflect the technical goals and objectives of the solicitation. The proposed costs are consistent with the proposer's Statement of Work and reflect a sufficient understanding of the costs and level of effort needed to successfully accomplish the proposed technical approach. The costs for the prime proposer and proposed subawardees are substantiated by the details provided in the proposal (e.g., the type and number of labor hours proposed per task, the types and quantities of materials, equipment and fabrication costs, travel and any other applicable costs and the basis for the estimates).
It is expected that the effort will leverage all available relevant prior research in order to obtain the maximum benefit from the available funding. For efforts with a likelihood of commercial application, appropriate direct cost sharing may be a positive factor in the evaluation.
DARPA recognizes that undue emphasis on cost may motivate proposers to
offer low-risk ideas with minimum uncertainty and to staff the effort with junior
personnel in order to be in a more competitive posture. DARPA discourages such cost
strategies.
Commented [EA17]: Please note

Citations
2. 3.
4. 5.
6. 7.
1.
K. J. Olival et al., Host and viral traits predict zoonotic spillover from mammals. Nature 546, 646-650 (2017).
G. Zhang et al., Comparative analysis of bat genomes provides insight into the evolution of flight and immunity. Science 339, 456-460 (2013).
J. Xie et al., Dampened STING-Dependent Interferon Activation in Bats. Cell host & microbe, (2018).
P. Zhou et al., Contraction of the type I IFN locus and unusual constitutive expression of IFN-αin bats. Proceedings of the National Academy of Sciences of the United States of America, 201518240-201518246 (2016).
M. Ahn, J. Cui, A. T. Irving, L.-F. Wang, Unique Loss of the PYHIN Gene Family in Bats Amongst Mammals: Implications for Inflammasome Sensing. Scientific Reports 6, (2016).
J. Zhao et al., Intranasal Treatment with Poly(I.C) Protects Aged Mice from Lethal Respiratory Virus Infections. Journal of Virology 86, 11416-11424 (2012).
J. Wu et al., Poly(I:C) Treatment Leads to Interferon-Dependent Clearance of Hepatitis B Virus in a Hydrodynamic Injection Mouse Model. Journal of Virology 88, 10421-10431 (2014).
Attachment 1: Executive Summary Slide template

8.
9. 10.
11. 12.
X. F. Deng et al., A Chimeric Virus-Mouse Model System for Evaluating the Function and Inhibition of Papain-Like Proteases of Emerging Coronaviruses. Journal of Virology 88, 11825-11833 (2014).
T. E. Rocke et al., Sylvatic Plague Vaccine Partially Protects Prairie Dogs (Cynomys spp.) in Field Trials. Ecohealth 14, 438-450 (2017).
B. Stading et al., Protection of bats (Eptesicus fuscus) against rabies following topical or oronasal exposure to a recombinant raccoon poxvirus vaccine. Plos Neglect. Trop. Dis. 11, (2017).
B. R. Stading et al., Infectivity of attenuated poxvirus vaccine vectors and immunogenicity of a raccoonpox vectored rabies vaccine in the Brazilian Free-tailed bat (Tadarida brasiliensis). Vaccine 34, 5352-5358 (2016).
P. Zhou et al., Fatal Swine Acute Diarrhea Syndrome caused by an HKU2- related Coronavirus of Bat Origin. Nature In press, (2018
).

Page 8: [1] Deleted peterzhou 2/8/18 4:40:00 PM

10/5/21, 2:50 PM Mail - Rocke, Tonie E - Outlook
Luke, aached is the DARPA abstract.
First thing tomorrow, can you go through the references, and create links to the papers on NCBI. Just turn each reference citaon: “(1)” for example, into a live link to the paper on NCBI. We only have 2 spare lines, so no room to turn each of these into a PMC numbered ref, as Ralph did for his, but please make sure the citaon in parentheses is blue, so it’s clear it’s a live link on the final pdf.
Ralph, Lina, Peng, Zhengli, Tonie – please give this a quick read to make sure I’ve not said anything completely wrong. I’ve had to reduce the text a lot to hit the page limit, but I sll think it’s a great proposal.
I’ll finish off the exec summary slide and <500 wd abstract now.
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
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DARPA – PREEMPT – HR001118S0017- PROJECT DEFUSE
C. Goals and Impact:
1. What is the proposed work attempting to accomplish or do?
We will defuse the potential for emergence of novel bat-origin high-zoonotic risk SARS-related coronaviruses. We envisage a scenario whereby the US warfighter is deployed to a security hotspot in SE Asia. As planners choose sites for the mission, they will use an app we will design based on machine-learning models of the ecological and evolutionary potential of bat viruses to spillover. This will allow rapid assessment of the background risk of a site harboring dangerous zoonotic viruses. If there is no alternative site, a tactical forward team will deploy automated delivery technology we will develop in caves that harbor bats carrying these viruses. These devices will release broadscale immune boosting molecules and chimeric polyvalent spike protein targeted immune priming inocula to upregulate the naturally damped innate immune response of bats, and lower viral shedding from bats at the site for a few weeks or months, allowing our warfighters to execute the operation at lowered risk for spillover.
2. How is it done today? And what are the limitations?
There is no available current technology to reduce the risk of exposure to novel coronaviruses from bats. Models of bat host capacity to harbor viruses, of ecological and environmental drivers of their emergence, and of the evolutionary potential of different strains to spillover are rudimentary. No vaccines, or therapeutics exist for SARSr-CoVs, and exposure mitigation strategis are non-existent. SARSr-CoVs are endemic in Asian, African (1), and European bats (2) that roost in caves but forage widely at night, shedding virus in their feces and urine. The limitations of this lack of capacity are significant – we have shown evidence of recent spillover of SARSr-CoVs into people in China, and have isolated strains capable of producing SARS-like illness in humanized mice that doesn’t respond to antibody treatment or vaccination. These viruses are a clear-and-present danger to our military, and to global health security.
3. What is innovative in your approach and how does it compare to current
practice and state-of-the-art (SOA)?
Our group leads the world in predictive models of viral emergence. We will build on our hotspots, machine-learning, ecological niche and genotype-phenotype modeling by incorporating unique datasets to validate and refine hotspot risk maps of viral emergene in SE Asia and beyond. Our group has shown that bats coexist with lethal viruses by damping innate immunity pathways, likely as an evolutionary adaptation to flight. We will use this insight to design strategies, like small molecule Rig like receptor (RLR) or Toll like receptor (TLR) agonists, to upregulate bat immunity in their cave roosts, down- regulate viral replication, and reduce the risk of viral shedding and spillover (broadscale immune boosting strategy). We will complement this by inoculating bats with novel chimeric polyvalent recombinant spike proteins to enhance their immune response against specific, high-risk viruses (targeted immune priming strategy), especially when their immune response is boosted as above. We will design novel methods to deliver

these inocula remotely to reduce exposure risk during decontamination.
4. What are the key technical challenges in your approach and how do you plan to overcome these?
Modeling: Previous models have suffered from a lack of data to validate them. We have access to unique datasets that will allow us to validate our approach, including biodiversity surveys of bat caves across S. China, 10+ years of bat viral testing data in China, and 10 other countries (from NIAID and USAID EPT PREDICT work). Uniquely, we will validate our models of viral evolution/spillover risk using serology (based on LIPS assays) in local populations, who have high (~3%) serology to bat SARSr-CoVs. Identifying Immune boosting and priming inocula: Some of our approaches are novel and challenging (e.g. using CRISPRi to find the negative regulator for bat interferon production), and others are unproven in bats (e.g. Poly IC). We will begin all immune boosting and priming experiments at the beginning of the project, running them simultaneously and competitively, so that we field trial only the most efficient, cost- effective and scalable approaches.
5. Who will care and what will the impact be if you are successful?
This will have direct relevance to the warfighter. Potential deployment to regions where SARSr-CoVs exist is high – countries include security hotspots in Asia (e.g. Myanmar, Bangladesh, Pakistan, Korea, Vietnam), Africa and Eastern Europe. The ability to decontaminate and defuse these viruses may prevent potentially devastating illness. These technologies could be adapted to hosts of other bat-origin CoVs (e.g. MERS-CoV, SADS-CoV), and potentially other zoonotic bat-origin viruses (Hendra, Nipah, EBOV), with benefits to livestock production, food security and global public health.
6. How much will it cost and how long will it take?
D. Technical Plan:
Overview
The SARSr-CoV-bat system, and immune modulation focus: Our group’s 15 yrs work on the SARSr-CoV – Rhinolophus bat system in China has identified and isolated SARSr-CoVs with remarkable sequence identity in the spike protein to SARS-CoV (e.g. SCH014 & WIV-1). We have shown they bind and replicate efficiently in primary human lung airway cells and that chimeras with SARSr-CoV spike proteins in a SARS-CoV backbone cause SARS-like illness in humanized mice, with clinical signs that are not reduced by SARS monoclonal therapy or vaccination. We have identified a single cave site in Yunnan Province where bat SARSr-CoVs contain all the genetic components of epidemic SARS- CoV (7-9). We have now shown that people living up to 6 kilometers from this cave have SARSr-CoV antibodies (3% seroprevalence in 200+ cohort), suggesting active spillover,
Aleksei/Luke to fill out
SPACE SPACE SPACE

and marking these viruses as a clear-and-present danger of a new SARS-like pandemic. Our work on bat immunology suggests that bats’ unique flying ability has led to downregulated innate immune genes, and their ability to coexist with viruses such as SARSr-CoVs, henipa- and filoviruses that are lethal in many other mammals (3). We have identified bat-specific constitutively expressed bat interferon, a dampened STING- interferon production pathway (4, 5), and have identified a series of other innate immunity factors that are dampened in bats (6).
Our bat-CoV system has significant advantages for experiment and intervention. Firstly, these viruses are fecal-orally transmitted within bat populations, so sampling can be achieved from fresh fecal pellet collection. They are BSL-3, not -4, agents, so that experimental manipulation and infection is simpler. They have frequent spillover events, making it possible to validate predictive models of spillover by sampling people. They are diverse, with frequent recombination and different strains exhibiting differential host cell binding and spillover potential. Finally, we have identified SARSr-CoV strains in a single cave in Yunnan that harbor all of the epidemic SARS-CoV genes. This specific bat population harbors an ideal evolutionary soup that could produce new human strains by high frequency RNA recombination, and thus, it presents a perfect target for next generation, technology-forward intervention strategies.
TA1: Develop and validate integrated, multiscale models that quantify the likelihood a human-capable virus will emerge from an animal reservoir residing in a “hot spot” geographic region
The DEFUSE modeling and analytics team led by Drs Daszak, Ross, Olival, EHA, will build ecological niche models of environmental and ecological correlates, and traits of cave bat communities to predict species composition of bat caves across Southern China, South and SE Asia. We will then use a series of datasets we have built to produce host- virus risk models for the region. These include our unique database of bat host-viral relationships (7); biological inventory data on all bat caves in Southern China; and modeled species distribution data for all bats. We will parameterize the model with data from three cave sites in Yunnan, China (one with high-risk SARSr-CoVs, two other control/comparison sites), including: radio- and GPS-telemetry to identify home range and additional roost sites for each bat species; inventory of bat population density, distribution and segregation and their daily, weekly and seasonal changes; viral prevalence and individual viral load; shedding of low- and high-risk SARSr-CoV strains among bat species, age classes, genders; and telemetry and mark-recapture data to assess metapopulation structure and inter-cave connectivity. We will test and validate model predictions of a cave’s viral spillover potential with data from prior PREDICT sampling in 7 other Asian countries. At the end of Yr 1, we will produce a prototype app for the warfighter that identifies the likelihood of bats harboring dangerous viral pathogens in a region. The ‘high-risk bats near me’ app will be updated real-time with surveillance data (e.g. field-deployable iphone and android compatible echolocation data) from our project and others, to ground-truth and fine-tune its predictive capacity.
The Wuhan Institute of Virology team will test bat fecal, oral, blood and urogenital samples for SARSr-CoVs. We will correlate viral load data from these samples

with fresh fecal pellets from individuals and from tarps laid on cave floors. We will rapidly move to fecal pellet assays to reduce roost disturbance. SARSr-CoV spike proteins will be sequenced, analyzed phylogenetically, for recombination events, and high-risk viruses (spike proteins close to SARS-CoV) characterized and isolated. The UNC team will reverse engineer spike proteins to conduct binding assays to human ACE2 (the SARS-CoV receptor). They will culture SARS-like bat coronaviruses to distinguish high risk strains that can replicate in primary human cells and low risk strains that require exogenous enhancers. Viral spike glycoproteins that bind receptors will be inserted into SARS-CoV backbones, inoculated into human cells and humanized mice to assess capacity to cause SARS-like disease, and to be blocked by monoclonal therapies, the nucleoside analogue inhibitor GS-5734 (8) or vaccines against SARS-CoV (8-13).
The EHA modeling team will use these data to build models of risk of viral evolution and spillover. These genotype-to-phenotype machine-learning models will predict viral ability to infect host cells based on genetic traits and results of receptor binding and mouse infection assays. Using data on diversity of spike proteins, recombinant CoVs, and flow of genes within each bat cave via bat movement and migration, we will estimate evolutionary rates, rates of recombination, and capacity to generate novel strains capable of human infection. Finally, virus-host relationship and bat home range data will be used to estimate spillover potential - extending models well beyond our field sites. We will then validate model predictions of viral spillover risk by 1) conducting spike protein-based binding and cell culture experiments, and 2) identifying spillover strains in people near our bat cave sites. Our preliminary work on this shows ~3% serology to SARSr-CoVs, using a specific ELISA (14). We will design LIPS assays to the specific high- and low- zoonotic-risk SARSr-CoVs identified in this project as we have done previously (15). We will use banked and newly collected human sera from these populations to test for presence of antibodies to the high- and low-risk SARSr- CoVs identified by our modeling. We will then model optimal strategies to maximize inoculation efficacy for TA2, using machine-learning stochastic simulation modeling informed by field and experimental data to characterize viral circulation dynamics in bats. We will estimate frequency and population coverage required for our intervention approaches to suppress viral spillover. We will determine the seasons, locations within a cave, and delivery methods (spray, swab, or automated cave mouth or drone) that will be most effective. Finally we will determine the time period treatment will be effective for, until re-colonization or evolution leads to return of a high-risk SARSr-CoV.
TA2: Develop scalable approaches that target and suppress the animal virus in its reservoir(s) and/or vector(s), to reduce the likelihood of virus transmission into humans.
We will evaluate two approaches to defuse SARS-related CoV spillover potential: 1) Broadscale Immune Boosting: using the unique immune damping in bats that our group has discovered, we will inoculate live bats with immune modulators like bat interferon designed to up-regulate their naïve immunity and then assess their ability to suppress viral replication and shedding; 2) Targeted Immune Priming: building on preliminary development of polyvalent chimeric recombinant SARSr-CoV spike proteins, we will

conduct inoculation trials with live bats to assess suppression of replication and shedding of a broad range of dangerous SARS-related CoVs.
Both lines of work will begin in Yr 1 and run parallel. Prof. Linfa Wang (Duke- NUS) will lead the immune boosting work, building on his pioneering work on bat immunity (3) which shows that the long-term coexistence of bats and their viruses has led to equilibrium between viral replication and host immunity. This is likely due to down-regulation of their innate immune system as a fitness cost of flight (3). The weakened functionality of bat innate immunity factors like STING, a central DNA- interferon (IFN) sensing molecule, may allow bats to maintain an effective, but not over- response to viruses (4). A similar finding was observed for bat IFNA, which is less abundant but constitutively expressed without stimulation (5). Given high native SARSr- CoV load in bats, we aim to boost bat innate immunity through the IFN pathway, break the host-virus equilibrium to suppress bat SARSr-CoV replication and shedding.
We will trial the following concurrently and competitively for efficiency, cost and scalability: i) Universal bat interferon. Aerosol spraying or intranasal inoculation of IFN or other small molecules reduces viral loads in humans, ferrets and mouse models (16, 17). Interferon has been used clinically when antiviral drugs are unavailable, e.g. against filoviruses (18). Replication of SARSr-CoV is sensitive to interferon treatments, as shown in our previous work (16); ii) Boosting bat IFN by blocking bat-specific IFN negative regulators. Uniquely, bat IFNA is naturally constitutively expressed but cannot be induced to a high level (5), indicating a negative regulatory factor in the bat interferon production pathway. We will use CRISPRi to identify the negative regulator and then screen for compounds targeting this gene; iii) Activating dampened bat-specific IFN production pathways which include DNA-STING-dependent and ssRNA-TLR7-dependent pathways. Our work showing that mutant bat STING restores antiviral functionality suggests these pathways are important in bat-viral coexistence (4). By identifying small molecules to directly activate downstream of STING, we will activate bat interferon and promote viral clearance. A similar strategy will be applied to ssRNA-TLR7-dependent pathways; iv) Activating functional bat IFN production pathways, e.g. polyIC to TLR3-IFN pathway or 5’ppp-dsRNA to RIG-I-IFN pathway. A similar strategy has been demonstrated in a mouse model for SARS-CoV, IAV and HBV (17, 19); v) Inoculating crude coronavirus fragments to upregulate innate immune responses to specific CoVs – a partial step towards the targeted immune priming work below.
Prof. Ralph Baric (UNC) will lead the immune priming work. He will develop recombinant chimeric spike-proteins (20) from our known SARSr-CoVs, and those we characterize during project DEFUSE. The structure of the SARS-CoV spike glycoprotein has been solved and the addition of two proline residues at positions V1060P and L1061P stabilize the prefusion state of the trimer, including key neutralizing epitopes in the receptor binding domain (21). In parallel, the spike trimers or the receptor binding domain can be incorporated into alphavirus vectored or nanoparticle vaccines for delivery, either as aerosols, in baits, or as large droplet delivery vehicles (11, 22-25). We will test these in controlled lab conditions, taking the best candidate forward for testing in the field. We have built recombinant spike glycoproteins harboring structurally defined domains from SARS epidemic strains, pre-epidemic strains like SCH014 and

zoonotic strains like HKU3. It is anticipated that recombinant S glycoprotein based vaccines harboring immunogenic blocks across the group 2B coronaviruses will induce broadscale immune responses that simultaneously reduce genetically heterogeneous virus burdens in bats, potentially reducing disease risk (and transmission risk to people) in these animals for longer periods (26, 27).
The immune dampening features are highly conserved in all bat species tested so far. Duke-NUS has established the only experimental breeding colony of cave bats (Eonycteris spelaea) in SE Asia. This genus is evolutionarily closely related to Rhinolophus spp. (the hosts of SARSr-CoVs), so we have confidence that results will be transferable. Our initial proof-of-concept tests will be in this experimental colony, extended to a small group of wild-caught Rhinolophus sinicus bats at Wuhan Institute of Zoology. We (Prof. Wang) have previous experience conducting SARS-CoV infection experiments with Rhinolophus sp. bats in the BSL-4 facility at CSIRO, AAHL (L.Wang, unpublished results).
Finally, work on a delivery method for our immune boosting and priming molecules will be overseen by Dr. Tonie Rocke at the USGS, National Wildlife Health Center who has previously developed animal vaccines through to licensure (28). Using locally acquired insectivorous bats (29, 30) we will assess delivery vehicles and methods including: 1) transdermally applied nanoparticles; 2) series of sticky edible gels that bats will groom from themselves and each other; 3) aerosolization via sprayers that could be used in cave settings; 4) automated sprays triggered by timers and movement detectors at critical cave entry points; 5) sprays delivered by remote controlled drone. We have already used simple gels to vaccinate bats against rabies in the lab (29), and hand delivered these containing biomarkers to vampire bats in Peru and Mexico to show they are readily consumed and transferred among bats. In our bat colony, we will trial delivery vehicles using the biomarker rhodamine B (which marks hair and whiskers upon consumption) to assess uptake. The most optimal approaches will then be tested on wild bats in our three cave sites in Yunnan Province with the most successful immunomodulators from TA2. Fieldwork will be conducted under the auspices of Dr. Yunzhi Zhang (Yunnan CDC, Consultant at EcoHealth Alliance). A small number of bats will be captured and assayed for viral load and immune function after treatment, but so as not to disturb the colony, most viral load work will be conducted on fresh fecal pellets collected daily on the cave floor. EHA has had unique access to these sites for around 10 years, under the guidance of Drs. Shi and Zhang. In year 1 of project DEFUSE, we will seek permission for experimental inoculations from the Provincial Forestry Department. We expect to be successful, as we have worked with the Forestry Department collaboratively for 10 years, with support of the Yunnan CDC, and we are releasing molecules that are not dangerous to people or wildlife. EHA has a proven track record of rapidly obtaining IACUC and DoD ACURO approval for bat research.
E. Capabilities:
The lead institution for Project DEFUSE is EcoHealth Alliance, New York, an international research organization focused on emerging zoonotic diseases. The PI, Dr. Peter Daszak, has 25+ years’ experience managing lab, field and modeling research projects on emerging zoonoses. Dr. Daszak will commit 3 months annually to oversee and

coordinate all project activities, and lead modeling and analytic work for TA1. Dr. Billy Karesh has 40+ years’ experience leading zoonotic and wildlife disease projects, and will commit 1 month annually to manage partnership activities and outreach. Dr. Jon Epstein, with 15 years’ experience working emerging bat zoonoses will coordinate animal trials. Dr. Kevin Olival and Dr. Noam Ross will manage and conduct the modeling and analytical approaches for this project. Support staff include field surveillance teams, modeling analysts, and consultants based in Yunnan Province, China, to oversee field trials. The EHA team has worked extensively with all other collaborators: Prof. Wang (15+ years); Dr. Shi (15+ years); Prof. Baric (5+ years) and Dr. Rocke (15+ years).
Subcontracts: #1 to Prof. Ralph Baric, UNC, to oversee reverse engineering of SARSr-CoVs, BSL-3 humanized mouse experimental infections, design and testing of immune priming inocula based on recombinant spike proteins. Assisted by senior personnel Dr. Tim Sheahan, Dr. Amy Sims, and support staff; #2 to Prof. Linfa Wang, Duke NUS, to oversee the immune boosting approach, captive bat experiments, and analyze immunological and virological responses to immune boosting inocula; #3 to Dr. Zhengli Shi, Wuhan Institute of Virology, to conduct PCR testing, viral discovery and isolation from bat samples collected in China, spike protein binding assays, and some humanized mouse work, as well as experimental inoculation of Rhinolophus bats. Her team will include Dr. Peng Zhou and support staff; #4 to Dr. Tonie Rocke, USGS National Wildlife Health Center, to refine delivery mechanisms for both immune boosting and immune priming treatments. With a research technician, Dr. Rocke will use a captive colony of bats at NWHC for initial trials, and oversee cave experiments in China.
F. Links to published papers, resume of two key performers
Dr. Peter Daszak is President and Chief Scientist of EcoHealth Alliance, a US-based research organization focused on emerging zoonotic diseases. His >300 scientific papers include the first global map of EID hotspots (31, 32), estimates of unknown viral diversity (33), predictive models of virus-host relationships (7), and evidence of the bat origin of SARS-CoV (34, 35) and other emerging viruses (36-39). He is Chair of the NASEM Forum on Microbial Threats, and is a member of the Executive Committee and the EHA institutional lead for the $130 million USAID-EPT-PREDICT. He serves on the NRC Advisory Committee to the USGCRP, the DHS CEEZAD External Advisory Board, the WHO R&D Blueprint Pathogen Prioritization expert group, and has advised the Director for Medical Preparedness Policy on the White House National Security Staff on global health issues. Dr Daszak won the 2000 CSIRO medal for collaborative research.
Prof. Ralph Baric is a UNC Lineberger Comprehensive Cancer Center member and Professor in the UNC-Chapel Hill Dept. of Epidemiology and Dept. of Microbiology & Immunology. His work focuses on coronaviruses as models to study the genetics of RNA virus transcription, replication, persistence, cross species transmission and pathogenesis. His group has developed a platform strategy to access the potential “pre- epidemic” risk associated with zoonotic virus cross species transmission potential and evaluation of countermeasure potential to control future outbreaks of disease (8-13).

Citations
1. P. L. Quan et al., Identification of a Severe Acute Respiratory Syndrome Coronavirus-Like Virus in a Leaf-Nosed Bat in Nigeria. Mbio 1, (2010).
2. J. F. Drexler et al., Genomic Characterization of Severe Acute Respiratory Syndrome-Related Coronavirus in European Bats and Classification of Coronaviruses Based on Partial RNA-Dependent RNA Polymerase Gene Sequences. Journal of Virology 84, 11336-11349 (2010).
3. G. Zhang et al., Comparative analysis of bat genomes provides insight into the evolution of flight and immunity. Science 339, 456-460 (2013).
4. J. Xie et al., Dampened STING-Dependent Interferon Activation in Bats. Cell host & microbe, (2018).
5. P. Zhou et al., Contraction of the type I IFN locus and unusual constitutive expression of IFN-αin bats. Proceedings of the National Academy of Sciences of the United States of America, 201518240-201518246 (2016).
6. M. Ahn, J. Cui, A. T. Irving, L.-F. Wang, Unique Loss of the PYHIN Gene Family in Bats Amongst Mammals: Implications for Inflammasome Sensing. Scientific Reports 6, (2016).
7. K. J. Olival et al., Host and viral traits predict zoonotic spillover from mammals. Nature 546, 646-650 (2017).
8. T. P. Sheahan et al., Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses. Sci Transl Med 9, (2017).
9. V. D. Menachery et al., MERS-CoV and H5N1 influenza virus antagonize antigen presentation by altering the epigenetic landscape. Proc Natl Acad Sci U S A 115, E1012-E1021 (2018).
10. S. J. Anthony et al., Further Evidence for Bats as the Evolutionary Source of Middle East Respiratory Syndrome Coronavirus. MBio 8, (2017).
11. A. S. Cockrell et al., A mouse model for MERS coronavirus-induced acute respiratory distress syndrome. Nat Microbiol 2, 16226 (2016).
12. V. D. Menachery et al., SARS-like WIV1-CoV poised for human emergence. Proc Natl Acad Sci U S A 113, 3048-3053 (2016).
13. V. D. Menachery et al., A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence. Nat Med 21, 1508-1513 (2015).
14. N. Wang et al., Serological evidence of bat SARS-related coronavirus infection in humans, China. Virologica Sinica In press, (2018).
15. P. Zhou et al., Fatal Swine Acute Diarrhea Syndrome caused by an HKU2-related Coronavirus of Bat Origin. Nature In press, (2018).
16. B. M. Farr, J. M. Gwaltney, Jr., K. F. Adams, F. G. Hayden, Intranasal interferon- alpha 2 for prevention of natural rhinovirus colds. Antimicrob Agents Chemother 26, 31-34 (1984).
17. D. Kugel et al., Intranasal Administration of Alpha Interferon Reduces Seasonal Influenza A Virus Morbidity in Ferrets. Journal of Virology 83, 3843-3851 (2009).

18. L. M. Smith et al., Interferon-beta Therapy Prolongs Survival in Rhesus Macaque Models of Ebola and Marburg Hemorrhagic Fever. Journal of Infectious Diseases 208, 310-318 (2013).
19. J. Zhao et al., Intranasal Treatment with Poly(I.C) Protects Aged Mice from Lethal Respiratory Virus Infections. Journal of Virology 86, 11416-11424 (2012).
20. X. F. Deng et al., A Chimeric Virus-Mouse Model System for Evaluating the Function and Inhibition of Papain-Like Proteases of Emerging Coronaviruses. Journal of Virology 88, 11825-11833 (2014).
21. J. Pallesen et al., Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen. Proc Natl Acad Sci U S A 114, E7348-E7357 (2017).
22. C. M. Coleman et al., Purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice. Vaccine 32, 3169-3174 (2014).
23. C. M. Coleman et al., MERS-CoV spike nanoparticles protect mice from MERS- CoV infection. Vaccine 35, 1586-1589 (2017).
24. L. Du et al., A 219-mer CHO-expressing receptor-binding domain of SARS-CoV S protein induces potent immune responses and protective immunity. Viral immunology 23, 211-219 (2010).
25. T. Sheahan et al., Successful vaccination strategies that protect aged mice from lethal challenge from influenza virus and heterologous severe acute respiratory syndrome coronavirus. J Virol 85, 217-230 (2011).
26. S. Agnihothram et al., A mouse model for Betacoronavirus subgroup 2c using a bat coronavirus strain HKU5 variant. MBio 5, e00047-00014 (2014).
27. M. M. Becker et al., Synthetic recombinant bat SARS-like coronavirus is infectious in cultured cells and in mice. Proc Natl Acad Sci U S A 105, 19944- 19949 (2008).
28. T. E. Rocke et al., Sylvatic Plague Vaccine Partially Protects Prairie Dogs (Cynomys spp.) in Field Trials. Ecohealth 14, 438-450 (2017).
29. B. Stading et al., Protection of bats (Eptesicus fuscus) against rabies following topical or oronasal exposure to a recombinant raccoon poxvirus vaccine. Plos Neglect. Trop. Dis. 11, (2017).
30. B. R. Stading et al., Infectivity of attenuated poxvirus vaccine vectors and immunogenicity of a raccoonpox vectored rabies vaccine in the Brazilian Free- tailed bat (Tadarida brasiliensis). Vaccine 34, 5352-5358 (2016).
31. K. E. Jones et al., Global trends in emerging infectious diseases. Nature 451, 990- 993 (2008).
32. T. Allen et al., Global hotspots and correlates of emerging zoonotic diseases. Nat Commun 8, 1124 (2017).
33. S. J. Anthony et al., A Strategy to Estimate Unknown Viral Diversity in Mammals. mBio 4, (2013).
34. X. Y. Ge et al., Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature 503, 535-538 (2013).
35. W. Li et al., Bats are natural reservoirs of SARS-like coronaviruses. Science 310, 676-679 (2005).

36. A. N. Alagaili et al., Middle East respiratory syndrome coronavirus infection in dromedary camels in saudi arabia. MBio 5, (2014).
37. N. Homaira et al., Nipah virus outbreak with person-to-person transmission in a district of Bangladesh, 2007. Epidemiology and Infection 138, 1630-1636 (2010).
38. M. S. Islam et al., Nipah Virus Transmission from Bats to Humans Associated with Drinking Traditional Liquor Made from Date Palm Sap, Bangladesh, 2011–2014. Emerging Infectious Disease journal 22, 664 (2016).
39. K. J. Olival et al., Ebola virus antibodies in fruit bats, bangladesh. Emerg Infect Dis 19, 270-273 (2013).

10/5/21, 2:51 PM
Mail - Rocke, Tonie E - Outlook


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Tue 2/13/2018 12:03 AM

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RE: Final draft DARPA 500 wd summary
RE: Final draft DARPA 500 wd
summary
This message was sent with High importance.
Peter Daszak <daszak@ecohealthalliance.org>
Tue 2/13/2018 12:03 AM
org> Cc: William B. Karesh <karesh@ecohealthalliance.org>; Noam Ross <ross@ecohealthalliance.org>; Ralph Baric
Peter Daszak
To: Luke Hamel <hamel@ecohealthalliance.org>;
PREEMPT_Abstract_Sum...
20 KB

It’s 497 words now.
It’s 497 words now.
Cheers, Peter
Peter Daszak
President
Cheers, Peter
Peter Daszak
President
EcoHealth AllianceEcoHealth Alliance 460 West 34th Street – 17th Floor
460 West 34th Street – 17th Floor New York, NY 10001
New York, NY 10001
www.ecohealthalliance.org www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical

<daszak@ecohealthalliance.
Jonathon Musser <musser@ecohealthalliance.org>
(rbaric@email.unc.edu) <rbaric@email.unc.edu>; wang To: Luke Hamel <hamel@ecohealthalliance.org>; Jonathon Musser <muss
linfa <linfa.wang@duke-nus.edu.sg>;
Cc: William B. Karesh <karesh@ecohealthalliance.org>; Noam Ross <ross@
(peng.zhou@wh.iov.cn) <peng.zhou@wh.iov.cn>;
Zhengli Shi (zlshi@wh.iov.cn) <zlshi@wh.iov.cn>; Alison
Andre <andre@ecohealthalliance.org>; Aleksei Chmura
<chmura@ecohealthalliance.org>; Anna Willoughby
<willoughby@ecohealthalliance.org>; Rocke, Tonie E
<trocke@usgs.gov>
EcoHealth Alliance leads cung-edge research connecons between human and wildlife health and delicate
into the crical connecons between human and ecosystems. With this science we develop soluons that
wildlife health and delicate ecosystems. With this prevent pandemics and promote conservaon.
science we develop soluons that prevent pandemics and promote conservaon.
From: Peter Daszak
Sent: Tuesday, February 13, 2018 12:23 AM
From: Peter Daszak
To: Luke Hamel (hamel@ecohealthalliance.org); Jonathon
Sent: Tuesday, February 13, 2018 12:23 AM
To: Luke Hamel (hamel@ecohealthalliance.org); Cc: William B Karesh; Noam Ross; Ralph Baric
Musser
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/1
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鹏周

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e
a

Technical Approach: Our goal is to defuse the potential for spillover of novel bat-origin high-zoonotic risk SARS-related coronaviruses in Southeast Asia. In TA1 we will develop host-pathogen ecological niche models to predict the species composition of bat caves across Southeast Asia. We will parameterize this with a full inventory of host and virus distribution at our field sites, three caves in Yunnan Province, China and a series of unique datasets on bat host-viral relationships. By the end of Y1, we will use these to create a prototype app for the warfighter that identifies the likelihood of bats harboring dangerous viral pathogens at any site across Asia. We will intensively sample bats at our field sites to sequence SARSr-CoV spike proteins, reverse engineer them to conduct binding assays, and insert them into SARS-CoV backbones to infect humanized mice to assess capacity to cause SARS-like disease. Our modeling team will use these data to build machine-learning genotype-phenotype models of viral evolution and spillover risk. We will uniquely validate these with human serology data through LIPS assays designed to assess which spike proteins allow spillover into people.
In TA2, we will evaluate two approaches to reduce SARSr-CoV shedding in cave bats: (1) Broadscale Immune Boosting, in which we will inoculate bats with immune modulators to upregulate their innate immune response and downregulate viral replication; (2) Targeted Immune Priming, in which we will inoculate bats with novel chimeric polyvalent recombinant spike proteins to enhance innate immunity against specific, high-risk viruses. We will trial inoculum delivery methods on captive bats including automated aerosolization, transdermal nanoparticle application and edible, adhesive gels. We will use stochastic simulation modeling informed by field and experimental data to characterize viral dynamics in our cave sites, to maximize timing, inoculation protocol, delivery method and efficacy of viral suppression. The most effective delivery method and immune modulating treatments will be trialed in our experimental cave sites in Yunnan Province, with reduction in viral shedding as proof-of- concept.
Management Approach: Members of our collaborative group have worked together on bats and their viruses for over 15 years. The lead organization, EcoHealth Alliance, will oversee all modeling, lab, and fieldwork. EHA staff will develop models to evaluate the probability of specific SARS-related CoV spillover, and identify the most effective strategy for delivery of both immune boosting and immune targeting inocula. Specific work will be subcontracted to the following organizations:
1) Prof. Ralph Baric, UNC, will lead the immune priming work, building on his track record in reverse-engineering and manipulating SARS-CoV, MERS-CoV and other virus spike proteins over the last two decades.
2) Prof. Linfa Wang, Duke-NUS, will lead work on immune boosting, building from his groups’ pioneering work on bat immunity.
3) Dr. Zhengli Shi, Wuhan Institute of Virology will conduct viral testing on all collected samples, binding assays and some humanized mouse work.
4) Dr. Tonie Rocke, USGS National Wildlife Health Center will develop a delivery method for immunological countermeasures, following from her work on vaccine delivery in wildlife, including bats.

10/5/21, 2:52 PM Mail - Rocke, Tonie E - Outlook
Hi Luke and Jonathon.
Here’s the edited, final summary slide.
Please insert the re-drawn figure, and the budget numbers from Aleksei tomorrow before you submit
NB –there are other things to check on the Abstract, including budget numbers, meline, the references, and then please do a final check on the compliance with DARPA instrucons for all!
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
From: Peter Daszak
Sent: Tuesday, February 13, 2018 12:23 AM
To: Luke Hamel (hamel@ecohealthalliance.org); Jonathon Musser
Cc: William B. Karesh; Noam Ross; Ralph Baric (rbaric@email.unc.edu); wang linfa; 周鹏 (peng.zhou@wh.iov.cn); Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura (chmura@ecohealthalliance.org); 'Anna Willoughby (willoughby@ecohealthalliance.org)'; Rocke, Tonie
Subject: Final draft DARPA abstract
Importance: High
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/2
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10/5/21, 2:52 PM Mail - Rocke, Tonie E - Outlook
Luke, aached is the DARPA abstract.
First thing tomorrow, can you go through the references, and create links to the papers on NCBI. Just turn each reference citaon: “(1)” for example, into a live link to the paper on NCBI. We only have 2 spare lines, so no room to turn each of these into a PMC numbered ref, as Ralph did for his, but please make sure the citaon in parentheses is blue, so it’s clear it’s a live link on the final pdf.
Ralph, Lina, Peng, Zhengli, Tonie – please give this a quick read to make sure I’ve not said anything completely wrong. I’ve had to reduce the text a lot to hit the page limit, but I sll think it’s a great proposal.
I’ll finish off the exec summary slide and <500 wd abstract now.
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 2/2

Executive Summary: Proposal Title EcoHealth Alliance; Dr. Peter Daszak
CONCEPT
Provide graphic.
APPROACH
Describe new ideas.
IMPACT
Describe need and problem being addressed. Describe goal.
CONTEXT
Describe existing approaches; compare to state of the art.
Phase I
Phase II
Total
Proposed
$-
$-
$-
xHuman Use/ x Animal Use
HR001118S0017 PREEMPT
1

Executive Summary: DEFUSE EcoHealth Alliance; Dr. Peter Daszak
CONCEPT
Pathogen Prediction: APPROACH
• Host-Pathogen Ecology: Develop host-pathogen ecological niche models based on unique bat and viral data, to
estimate likelihood of spillover of SARS-related CoVs into human populations. Doing so will enhance predictive ability
of models beyond sampling sites in China to cover all Asia.
• Mobile Application: Create ‘Reservoirs Near Me’ mobile application, to assess background risk of disease spillover for
any site across Southeast Asia.
• Binding and Humanized mouse asssays: Utilize team’s unique collaboration between world-class modelers and
virologists with CoV expertise to conduct spike protein-based binding and humanized mice experiments.
• Use results to test machine-learning genotype-to-phenotype model predictions of viral spillover risk.
• Genotype-Phenotype Models: Develop models to estimate evolutionary rates, rates of recombination, and capacity to generate novel strains capable of human infection. Inputs to include: Diversity of bat spike proteins, prevalence of recombinant CoVs, and flow of genes within each bat cave via bat movement and migration.
• Validation with Human Sera: Analyze new and existing human serum samples to validate model outputs. Given frequent SARSr-CoV spillover events into local human populations, this can be done to a degree not possible in systems where spillover events are rare.
Intervention Development: (2 parallel approaches)
• (1) Broadscale Immune Boosting Strategy: Inoculate bats with immune modulators to upregulate innate immune response and downregulate viral replication, transiently reducing risk of viral shedding and spillover.
• (2) Targeted Immune Priming Strategy: Inoculate bats with novel chimeric polyvalent recombinant spike proteins to enhance innate immune response against specific, high-risk viruses.
• Viral Dynamics: Develop stochastic simulation models to estimate the frequency, efficacy, and population coverage required for intervention approaches to effectively suppress the viral population.
• Field Deployment and Testing: Uilize team’s expertise in wildlife vaccine delivery to assess and deploy effective molecule delivery methods, including: automated aerosolization technology that inoculates bats as they leave cave roost; remote controlled drone technology; transdermal nanoparticle application; and application of edible, adhesive gels that bats ingest when grooming fur of self and others.
CONTEXT
• No technology currently exists to reduce the risk of exposure to novel bat Coronaviruses.
• Our team has conducted pioneering research on modeling disease emergence, understanding Coronavirus virology, bat immunity, and wildlife vaccine delivery. Our previous work provides proof-of-concept for: (1) predictive ‘hotspot’ modeling; (2) upregulating bat immune response through the STING IFN pathway, (2) developing recombinant chimeric spike-proteins from SARS and SARSr-CoVs and (3) delivering immunological countermeasures to wildlife (including multiple bat species).
• The DEFUSE approach is broadly effective, scalable, economical and achievable in the allotted time frame. It also poses little environmental risk, and presents no threat to local livestock or human populations.
• While CRISPR-Cas9 gene drives are being considered for many disease research applications, the technique is unlikely to be effective in suppressing viral transmission in bat hosts. Bats are relatively long- lived, highly mobile, and have long inter-generational periods (2-5 years) with low progeny (1-2 pups). Furthermore, gene drive technology could have far-reaching, negative ecological consequences and its effectiveness cannot be evaluated within the defined Period of Performance.
IMPACT
• Recent and ongoing security concerns within South and SE Asia make the region a likely deployment site for US warfighters. Troops deployed to the region face increased disease risk from SARS and related bat viruses, as bats shed these pathogens through urine and feces while foraging over large areas at night.
• Our work in Yunnan Province, China has shown that (1) SARSr-CoVs are capable of producing SARS-like illness in humanized mice that are not affected by monoclonal or vaccine treatment, and (2) that spillover into local human populations is frequent. With no available vaccine or alternative method to counter these SARS-related viruses, US defense forces and national security are placed at risk.
• Our goal is to “DEFUSE” the potential for emergence of novel bat-origin high zoonotic risk SARSr-CoVs in Southeast Asia. In doing so, we will not only safeguard the US warfighter, but also reduce SARSr-CoV exposure for local communities and their livestock, improving food security Global Health Security.
• If successful, our strategy can be adapted to hosts of other bat-origin CoVs (MERS-CoV in the Middle East and other SARS-related pre-pandemic zoonotic strains in Africa, e.g. Nigeria), and potentially other zoonotic bat-origin viruses (Hendra, Nipah, Ebola viruses).
Phase I
Phase II
Total
Proposed
$-
$-
$-
xHuman Use/ x Animal Use
HR001118S0017 PREEMPT 2

10/5/21, 2:53 PM Mail - Rocke, Tonie E - Outlook
Hi Peer,
It actually reads well now and I hope the selecon commiee will “buy in” our brave ideas!
Nothing to add a or change, other than an English queson: we used “dampened immunity of bats” in most places, but you use the expression of “damping innate immunity pathways” on page 1. Is there a difference between “damping” and “dampening”?
Thanks
LF
Linfa (Lin-Fa) Wang, PhD FTSE
Professor & Director
Programme in Emerging Infecous Diseases Duke-NUS Medical School
8 College Road, Singapore 169875
Tel: +65 65168397
From: Peter Daszak [mailto:daszak@ecohealthalliance.org]
Sent: Tuesday, 13 February 2018 1:23 PM
To: Luke Hamel <hamel@ecohealthalliance.org>; Jonathon Musser <musser@ecohealthalliance.org>
Cc: William B. Karesh <karesh@ecohealthalliance.org>; Noam Ross <ross@ecohealthalliance.org>; Ralph Baric (rbaric@email.unc.edu) <rbaric@email.unc.edu>; Wang Linfa <linfa.wang@duke-nus.edu.sg>; 周鹏 (peng.zhou@wh.iov.cn) <peng.zhou@wh.iov.cn>; Zhengli Shi (zlshi@wh.iov.cn) <zlshi@wh.iov.cn>; Alison Andre <andre@ecohealthalliance.org>; Aleksei Chmura <chmura@ecohealthalliance.org>; Anna Willoughby <willoughby@ecohealthalliance.org>; Rocke, Tonie <trocke@usgs.gov>
Subject: Final dra DARPA abstract
Importance: High
Luke, aached is the DARPA abstract.
First thing tomorrow, can you go through the references, and create links to the papers on NCBI. Just turn each reference citaon: “(1)” for example, into a live link to the paper on NCBI. We only have 2 spare lines, so no room to turn each of these into a PMC numbered ref, as Ralph did for his, but please make sure the citaon in parentheses is blue, so it’s clear it’s a live link on the final pdf.
Ralph, Lina, Peng, Zhengli, Tonie – please give this a quick read to make sure I’ve not said anything completely wrong. I’ve had to reduce the text a lot to hit the page limit, but I sll think it’s a great proposal.
I’ll finish off the exec summary slide and <500 wd abstract now.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/2
>vog.sgsu@ekcort<
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arumhCieskelA;>gro.ecnaillahtlaehoce@erdna<erdnAnosilA;>nc.voi.hw@ihslz<)nc.voi.hw@ihslz(
ihSilgnehZ;>nc.voi.hw@uohz.gnep<)nc.voi.hw@uohz.gnep(鹏周;>ude.cnu.liame@cirabr<)ude.cnu.liame@cirabr(
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tcartsba APRAD tfard laniF :ER

10/5/21, 2:53 PM
Mail - Rocke, Tonie E - Outlook
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
Important: This email is confidential and may be privileged. If you are not the intended recipient, please delete it and notify us immediately; you should not copy or use it for any purpose, nor disclose its contents to any other person. Thank you.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 2/2

10/5/21, 2:53 PM Mail - Rocke, Tonie E - Outlook
Dear Peter,
Another English quesons (sorry, I seem to have all these English quesons today!): you have used “inoculate” to cover all of the proposed approaches in this proposal. If we decide to “spray” chemicals for bats to breath in, can we sll consider this as “inoculate”?! Or shall we be more general and used “apply” instead, by stang that “we will apply immune modulators and vaccine to bats”?!
Thanks
Linfa (Lin-Fa) Wang, PhD FTSE
Professor & Director
Programme in Emerging Infecous Diseases Duke-NUS Medical School
8 College Road, Singapore 169875
Tel: +65 65168397
From: Peter Daszak [mailto:daszak@ecohealthalliance.org]
Sent: Tuesday, 13 February 2018 2:04 PM
To: Luke Hamel <hamel@ecohealthalliance.org>; Jonathon Musser <musser@ecohealthalliance.org>
Cc: William B. Karesh <karesh@ecohealthalliance.org>; Noam Ross <ross@ecohealthalliance.org>; Ralph Baric (rbaric@email.unc.edu) <rbaric@email.unc.edu>; Wang Linfa <linfa.wang@duke-nus.edu.sg>; 周鹏 (peng.zhou@wh.iov.cn) <peng.zhou@wh.iov.cn>; Zhengli Shi (zlshi@wh.iov.cn) <zlshi@wh.iov.cn>; Alison Andre <andre@ecohealthalliance.org>; Aleksei Chmura <chmura@ecohealthalliance.org>; Anna Willoughby <willoughby@ecohealthalliance.org>; Rocke, Tonie <trocke@usgs.gov>
Subject: RE: Final dra DARPA 500 wd summary
Importance: High
It’s 497 words now.
Cheers, Peter
Peter Daszak
President
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/3
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10/5/21, 2:53 PM
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
From: Peter Daszak
Sent: Tuesday, February 13, 2018 12:23 AM
To: Luke Hamel (hamel@ecohealthalliance.org); Jonathon Musser
Cc: William B. Karesh; Noam Ross; Ralph Baric (rbaric@email.unc.edu); wang linfa; 周鹏 (peng.zhou@wh.iov.cn); Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura (chmura@ecohealthalliance.org); 'Anna Willoughby (willoughby@ecohealthalliance.org)'; Rocke, Tonie
Subject: Final draft DARPA abstract
Importance: High
Luke, aached is the DARPA abstract.
First thing tomorrow, can you go through the references, and create links to the papers on NCBI. Just turn each reference citaon: “(1)” for example, into a live link to the paper on NCBI. We only have 2 spare lines, so no room to turn each of these into a PMC numbered ref, as Ralph did for his, but please make sure the citaon in parentheses is blue, so it’s clear it’s a live link on the final pdf.
Ralph, Lina, Peng, Zhengli, Tonie – please give this a quick read to make sure I’ve not said anything completely wrong. I’ve had to reduce the text a lot to hit the page limit, but I sll think it’s a great proposal.
I’ll finish off the exec summary slide and <500 wd abstract now.
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
Mail - Rocke, Tonie E - Outlook
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 2/3

10/5/21, 2:53 PM Mail - Rocke, Tonie E - Outlook
Important: This email is confidential and may be privileged. If you are not the intended recipient, please delete it and notify us immediately; you should not copy or use it for any purpose, nor disclose its contents to any other person. Thank you.
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10/5/21, 2:53 PM Mail - Rocke, Tonie E - Outlook
Hi, Peter ,
It was really great proposal ! Just a couple of minor issues :
1, at 3, you used "spike protein", for the immune part. But you used "CoV fragments" for immunization in the actual ta2 part. Please be consistent. Also be consistent if we use CoV fragments to immunize bats or just "inoculate " (bat or human ).
2, last paragraph : Duke-Nus but not Duke Nus. Prof. Zhengli Shi, rather than Dr. ?
All good for other parts! Best wishes ,
Peng
Luke, aached is the DARPA abstract.
First thing tomorrow, can you go through the references, and create links to the papers on NCBI. Just turn each reference citaon: “(1)” for example, into a live link to the paper on NCBI. We only have 2 spare lines, so no room to turn each of these into a PMC numbered ref, as Ralph did for his, but please make sure the citaon in parentheses is blue, so it’s clear it’s a live link on the final pdf.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/2
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10/5/21, 2:53 PM Mail - Rocke, Tonie E - Outlook
Ralph, Lina, Peng, Zhengli, Tonie – please give this a quick read to make sure I’ve not said anything completely wrong. I’ve had to reduce the text a lot to hit the page limit, but I sll think it’s a great proposal.
I’ll finish off the exec summary slide and <500 wd abstract now.
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 2/2

From: Sent: To: Cc:
Subject: Attachments:
Just minor edits. Looks good. -Tonie
On Mon, Feb 12, 2018 at 11:23 PM, Peter Daszak <daszak@ecohealthalliance.org> wrote:
Luke, attached is the DARPA abstract.
First thing tomorrow, can you go through the references, and create links to the papers on NCBI. Just turn each reference citation: “(1)” for example, into a live link to the paper on NCBI. We only have 2 spare lines, so no room to turn each of these into a PMC numbered ref, as Ralph did for his, but please make sure the citation in parentheses is blue, so it’s clear it’s a live link on the final pdf.
Ralph, Lina, Peng, Zhengli, Tonie – please give this a quick read to make sure I’ve not said anything completely wrong. I’ve had to reduce the text a lot to hit the page limit, but I still think it’s a great proposal.
I’ll finish off the exec summary slide and <500 wd abstract now.
Cheers,
Peter
Rocke, Tonie <trocke@usgs.gov>
Monday, February 12, 2018 11:48 PM
Peter Daszak
Luke Hamel; Jonathon Musser; William B. Karesh; Noam Ross; Ralph Baric (rbaric@email.unc.edu); wang linfa; 周鹏 (peng.zhou@wh.iov.cn); Zhengli Shi
(zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura; Anna Willoughby Re: Final draft DARPA abstract
DARPA PREEMPT DEFUSE abstract 3_TRedits.docx
1

Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge research into the critical connections between human and wildlife health and delicate ecosystems. With this science we develop solutions that prevent pandemics and promote conservation.
--
Tonie E. Rocke
USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
608-270-2451
trocke@usgs.gov
2

DARPA – PREEMPT – HR001118S0017- PROJECT DEFUSE
C. Goals and Impact:
1. What is the proposed work attempting to accomplish or do?
We will defuse the potential for emergence of novel bat-origin high-zoonotic risk SARS-related coronaviruses. We envisage a scenario whereby the US warfighter is deployed to a security hotspot in SE Asia. As planners choose sites for the mission, they will use an app we will design based on machine-learning models of the ecological and evolutionary potential of bat viruses to spillover. This will allow rapid assessment of the background risk of a site harboring dangerous zoonotic viruses. If there is no alternative site, a tactical forward team will deploy automated delivery technology we will develop in caves that harbor bats carrying these viruses. These devices will release broadscale immune boosting molecules and chimeric polyvalent spike protein targeted immune priming inocula to upregulate the naturally damped innate immune response of bats, and lower viral shedding from bats at the site for a few weeks or months, allowing our warfighters to execute the operation at lowered risk for spillover.
2. How is it done today? And what are the limitations?
There is no available current technology to reduce the risk of exposure to novel coronaviruses from bats. Models of bat host capacity to harbor viruses, of ecological and environmental drivers of their emergence, and of the evolutionary potential of different strains to spillover are rudimentary. No vaccines or therapeutics exist for SARSr-CoVs, and exposure mitigation strategies are non-existent. SARSr-CoVs are endemic in Asian, African (1), and European bats (2) that roost in caves but forage widely at night, shedding virus in their feces and urine. The limitations of this lack of capacity are significant – we have shown evidence of recent spillover of SARSr-CoVs into people in China and have isolated strains capable of producing SARS-like illness in humanized mice that don’t respond to antibody treatment or vaccination. These viruses are a clear-and- present danger to our military and to global health security.
3. What is innovative in your approach and how does it compare to current
practice and state-of-the-art (SOA)?
Our group leads the world in predictive models of viral emergence. We will build on our hotspots, machine-learning, ecological niche and genotype-phenotype modeling by incorporating unique datasets to validate and refine hotspot risk maps of viral emergene in SE Asia and beyond. Our group has shown that bats coexist with lethal viruses by damping innate immunity pathways, likely as an evolutionary adaptation to flight. We will use this insight to design strategies, like small molecule Rig like receptor (RLR) or Toll like receptor (TLR) agonists, to upregulate bat immunity in their cave roosts, down- regulate viral replication, and reduce the risk of viral shedding and spillover (broadscale immune boosting strategy). We will complement this by inoculating bats with novel chimeric polyvalent recombinant spike proteins to enhance their immune response against specific, high-risk viruses (targeted immune priming strategy), especially when their immune response is boosted as above. We will design novel methods to deliver

these inocula remotely to reduce exposure risk during decontamination.
4. What are the key technical challenges in your approach and how do you plan to overcome these?
Modeling: Previous models have suffered from a lack of data to validate them. We have access to unique datasets that will allow us to validate our approach, including biodiversity surveys of bat caves across S. China, 10+ years of bat viral testing data in China, and 10 other countries (from NIAID and USAID EPT PREDICT work). Uniquely, we will validate our models of viral evolution/spillover risk using serology (based on LIPS assays) in local populations that have high (~3%) seroprevelance to bat SARSr-CoVs. Identifying Immune boosting and priming inocula: Some of our approaches are novel and challenging (e.g. using CRISPRi to find the negative regulator for bat interferon production), and others are unproven in bats (e.g. Poly IC). We will begin all immune boosting and priming experiments at the beginning of the project, running them simultaneously and competitively, so that we field trial only the most efficient, cost- effective and scalable approaches.
5. Who will care and what will the impact be if you are successful?
This will have direct relevance to the warfighter. Potential deployment to regions where SARSr-CoVs exist is high – countries include security hotspots in Asia (e.g. Myanmar, Bangladesh, Pakistan, Korea, Vietnam), Africa and Eastern Europe. The ability to decontaminate and defuse these viruses may prevent potentially devastating illness. These technologies could be adapted to hosts of other bat-origin CoVs (e.g. MERS-CoV, SADS-CoV) and potentially other zoonotic bat-origin viruses (Hendra, Nipah, EBOV), with benefits to livestock production, food security and global public health.
6. How much will it cost and how long will it take?
D. Technical Plan:
Overview
The SARSr-CoV-bat system, and immune modulation focus: Our group’s 15 yrs work on the SARSr-CoV – Rhinolophus bat system in China has identified and isolated SARSr-CoVs with remarkable sequence identity in the spike protein to SARS-CoV (e.g. SCH014 & WIV-1). We have shown they bind and replicate efficiently in primary human lung airway cells and that chimeras with SARSr-CoV spike proteins in a SARS-CoV backbone cause SARS-like illness in humanized mice, with clinical signs that are not reduced by SARS monoclonal therapy or vaccination. We have identified a single cave site in Yunnan Province where bat SARSr-CoVs contain all the genetic components of epidemic SARS- CoV (7-9). We have now shown that people living up to 6 kilometers from this cave have SARSr-CoV antibodies (3% seroprevalence in 200+ cohort), suggesting active spillover,
Aleksei/Luke to fill out
SPACE SPACE SPACE

and marking these viruses as a clear-and-present danger of a new SARS-like pandemic. Our work on bat immunology suggests that bats’ unique flying ability has led to downregulated innate immune genes, and their ability to coexist with viruses such as SARSr-CoVs, henipa- and filoviruses that are lethal in many other mammals (3). We have identified bat-specific constitutively expressed bat interferon, a dampened STING- interferon production pathway (4, 5), and have identified a series of other innate immunity factors that are dampened in bats (6).
Our bat-CoV system has significant advantages for experimentation and intervention. Firstly, these viruses are fecal-orally transmitted within bat populations, so sampling can be achieved from fresh fecal pellet collection. They are BSL-3, not -4, agents, so that experimental manipulation and infection is simpler. They have frequent spillover events, making it possible to validate predictive models of spillover by sampling people. They are diverse, with frequent recombination and different strains exhibiting differential host cell binding and spillover potential. Finally, we have identified SARSr- CoV strains in a single cave in Yunnan that harbor all of the epidemic SARS-CoV genes. This specific bat population harbors an ideal evolutionary soup that could produce new human strains by high frequency RNA recombination, and thus, it presents a perfect target for next generation, technology-forward intervention strategies.
TA1: Develop and validate integrated, multiscale models that quantify the likelihood a human-capable virus will emerge from an animal reservoir residing in a “hot spot” geographic region
The DEFUSE modeling and analytics team, led by Drs Daszak, Ross, Olival, EHA, will build ecological niche models of environmental and ecological correlates and traits of cave bat communities to predict species composition of bat caves across Southern China, South and SE Asia. We will then use a series of datasets we have built to produce host- virus risk models for the region. These include our unique database of bat host-viral relationships (7); biological inventory data on all bat caves in Southern China; and modeled species distribution data for all bats. We will parameterize the model with data from three cave sites in Yunnan, China (one with high-risk SARSr-CoVs, two other control/comparison sites), including: radio- and GPS-telemetry to identify home range and additional roost sites for each bat species; inventory of bat population density, distribution and segregation and their daily, weekly and seasonal changes; viral prevalence and individual viral load; shedding of low- and high-risk SARSr-CoV strains among bat species, age classes, genders; and telemetry and mark-recapture data to assess metapopulation structure and inter-cave connectivity. We will test and validate model predictions of a cave’s viral spillover potential with data from prior PREDICT sampling in 7 other Asian countries. At the end of Yr 1, we will produce a prototype app for the warfighter that identifies the likelihood of bats harboring dangerous viral pathogens in a region. The ‘high-risk bats near me’ app will be updated real-time with surveillance data (e.g. field-deployable iphone and android compatible echolocation data) from our project and others, to ground-truth and fine-tune its predictive capacity.
The Wuhan Institute of Virology team will test bat fecal, oral, blood and urogenital samples for SARSr-CoVs. We will correlate viral load data from these samples

with fresh fecal pellets from individuals and from tarps laid on cave floors. We will rapidly move to fecal pellet assays to reduce roost disturbance. SARSr-CoV spike proteins will be sequenced, analyzed phylogenetically for recombination events, and high-risk viruses (spike proteins close to SARS-CoV) characterized and isolated. The UNC team will reverse-engineer spike proteins to conduct binding assays to human ACE2 (the SARS-CoV receptor). They will culture SARS-like bat coronaviruses to distinguish high risk strains that can replicate in primary human cells and low risk strains that require exogenous enhancers. Viral spike glycoproteins that bind receptors will be inserted into SARS-CoV backbones, inoculated into human cells and humanized mice to assess capacity to cause SARS-like disease, and to be blocked by monoclonal therapies, the nucleoside analogue inhibitor GS-5734 (8) or vaccines against SARS-CoV (8-13).
The EHA modeling team will use these data to build models of risk of viral evolution and spillover. These genotype-to-phenotype machine-learning models will predict viral ability to infect host cells based on genetic traits and results of receptor binding and mouse infection assays. Using data on diversity of spike proteins, recombinant CoVs, and flow of genes within each bat cave via bat movement and migration, we will estimate evolutionary rates, rates of recombination, and capacity to generate novel strains capable of human infection. Finally, virus-host relationship and bat home range data will be used to estimate spillover potential - extending models well beyond our field sites. We will then validate model predictions of viral spillover risk by 1) conducting spike protein-based binding and cell culture experiments, and 2) identifying spillover strains in people near our bat cave sites. Our preliminary work on this shows ~3% seroprevalence to SARSr-CoVs, using a specific ELISA (14). We will design LIPS assays to the specific high- and low- zoonotic-risk SARSr-CoVs identified in this project as we have done previously (15). We will use banked and newly collected human sera from these populations to test for presence of antibodies to the high- and low-risk SARSr-CoVs identified by our modeling. We will then model optimal strategies to maximize inoculation efficacy for TA2, using machine-learning stochastic simulation modeling informed by field and experimental data to characterize viral circulation dynamics in bats. We will estimate frequency and population coverage required for our intervention approaches to suppress viral spillover. We will determine the seasons, locations within a cave, and delivery methods (spray, swab, or automated cave mouth or drone) that will be most effective. Finally we will determine the time period treatment will be effective for, until re-colonization or evolution leads to return of a high-risk SARSr-CoV.
TA2: Develop scalable approaches that target and suppress the animal virus in its reservoir(s) and/or vector(s), to reduce the likelihood of virus transmission into humans.
We will evaluate two approaches to defuse SARS-related CoV spillover potential: 1) Broadscale Immune Boosting: using the unique immune damping in bats that our group has discovered, we will inoculate live bats with immune modulators like bat interferon designed to up-regulate their naïve immunity and then assess their ability to suppress viral replication and shedding; 2) Targeted Immune Priming: building on preliminary

development of polyvalent chimeric recombinant SARSr-CoV spike proteins, we will conduct inoculation trials with live bats to assess suppression of replication and shedding of a broad range of dangerous SARS-related CoVs.
Both lines of work will begin in Yr 1 and run parallel. Prof. Linfa Wang (Duke- NUS) will lead the immune boosting work, building on his pioneering work on bat immunity (3) which shows that the long-term coexistence of bats and their viruses has led to equilibrium between viral replication and host immunity. This is likely due to down-regulation of their innate immune system as a fitness cost of flight (3). The weakened functionality of bat innate immunity factors like STING, a central DNA- interferon (IFN) sensing molecule, may allow bats to maintain an effective, but not over- response to viruses (4). A similar finding was observed for bat IFNA, which is less abundant but constitutively expressed without stimulation (5). Given high native SARSr- CoV load in bats, we aim to boost bat innate immunity through the IFN pathway, break the host-virus equilibrium to suppress bat SARSr-CoV replication and shedding.
We will trial the following, concurrently and competitively, for efficiency, cost and scalability: i) Universal bat interferon. Aerosol spraying or intranasal inoculation of IFN or other small molecules reduces viral loads in humans, ferrets and mouse models (16, 17). Interferon has been used clinically when antiviral drugs are unavailable, e.g. against filoviruses (18). Replication of SARSr-CoV is sensitive to interferon treatments, as shown in our previous work (16); ii) Boosting bat IFN by blocking bat-specific IFN negative regulators. Uniquely, bat IFNA is naturally constitutively expressed but cannot be induced to a high level (5), indicating a negative regulatory factor in the bat interferon production pathway. We will use CRISPRi to identify the negative regulator and then screen for compounds targeting this gene; iii) Activating dampened bat- specific IFN production pathways which include DNA-STING-dependent and ssRNA-TLR7- dependent pathways. Our work showing that mutant bat STING restores antiviral functionality suggests these pathways are important in bat-viral coexistence (4). By identifying small molecules to directly activate downstream of STING, we will activate bat interferon and promote viral clearance. A similar strategy will be applied to ssRNA- TLR7-dependent pathways; iv) Activating functional bat IFN production pathways, e.g. polyIC to TLR3-IFN pathway or 5’ppp-dsRNA to RIG-I-IFN pathway. A similar strategy has been demonstrated in a mouse model for SARS-CoV, IAV and HBV (17, 19); v) Inoculating crude coronavirus fragments to upregulate innate immune responses to specific CoVs – a partial step towards the targeted immune priming work below.
Prof. Ralph Baric (UNC) will lead the immune priming work. He will develop recombinant chimeric spike-proteins (20) from our known SARSr-CoVs, and those we characterize during project DEFUSE. The structure of the SARS-CoV spike glycoprotein has been solved and the addition of two proline residues at positions V1060P and L1061P stabilize the prefusion state of the trimer, including key neutralizing epitopes in the receptor binding domain (21). In parallel, the spike trimers or the receptor binding domain can be incorporated into alphavirus vectored or nanoparticle vaccines for delivery, either as aerosols, in baits, or as large droplet delivery vehicles (11, 22-25). We will test these in controlled lab conditions, taking the best candidate forward for testing in the field. We have built recombinant spike glycoproteins harboring structurally

defined domains from SARS epidemic strains, pre-epidemic strains like SCH014 and zoonotic strains like HKU3. It is anticipated that recombinant S glycoprotein based vaccines harboring immunogenic blocks across the group 2B coronaviruses will induce broadscale immune responses that simultaneously reduce genetically heterogeneous virus burdens in bats, potentially reducing disease risk (and transmission risk to people) in these animals for longer periods (26, 27).
The immune dampening features are highly conserved in all bat species tested so far. Duke-NUS has established the only experimental breeding colony of cave bats (Eonycteris spelaea) in SE Asia. This genus is evolutionarily closely related to Rhinolophus spp. (the hosts of SARSr-CoVs), so we have confidence that results will be transferable. Our initial proof-of-concept tests will be in this experimental colony, extended to a small group of wild-caught Rhinolophus sinicus bats at Wuhan Institute of Zoology. We (Prof. Wang) have previous experience conducting SARS-CoV infection experiments with Rhinolophus sp. bats in the BSL-4 facility at CSIRO, AAHL (L.Wang, unpublished results).
Finally, work on a delivery method for our immune boosting and priming molecules will be overseen by Dr. Tonie Rocke at the USGS, National Wildlife Health Center who has previously developed animal vaccines through to licensure (28). Using locally acquired insectivorous bats (29, 30) we will assess delivery vehicles and methods including: 1) transdermally applied nanoparticles; 2) series of sticky edible gels that bats will groom from themselves and each other; 3) aerosolization via sprayers that could be used in cave settings; 4) automated sprays triggered by timers and movement detectors at critical cave entry points; 5) sprays delivered by remote controlled drone. We have already used simple gels to vaccinate bats against rabies in the lab (29), and hand delivered these containing biomarkers to vampire bats in Peru and Mexico to show they are readily consumed and transferred among bats. In our bat colony, we will trial delivery vehicles using the biomarker rhodamine B (which marks hair and whiskers upon consumption) to assess uptake. The most optimal approaches will then be tested on wild bats in our three cave sites in Yunnan Province with the most successful immunomodulators from TA2. Fieldwork will be conducted under the auspices of Dr. Yunzhi Zhang (Yunnan CDC, Consultant at EcoHealth Alliance). A small number of bats will be captured and assayed for viral load and immune function after treatment, but so as not to disturb the colony, most viral load work will be conducted on fresh fecal pellets collected daily on the cave floor. EHA has had unique access to these sites for around 10 years, under the guidance of Drs. Shi and Zhang. In year 1 of project DEFUSE, we will seek permission for experimental inoculations from the Provincial Forestry Department. We expect to be successful, as we have worked with the Forestry Department collaboratively for 10 years, with support of the Yunnan CDC, and we are releasing molecules that are not dangerous to people or wildlife. EHA has a proven track record of rapidly obtaining IACUC and DoD ACURO approval for bat research.
E. Capabilities:
The lead institution for Project DEFUSE is EcoHealth Alliance, New York, an international research organization focused on emerging zoonotic diseases. The PI, Dr. Peter Daszak, has 25+ years’ experience managing lab, field and modeling research projects on

emerging zoonoses. Dr. Daszak will commit 3 months annually to oversee and coordinate all project activities, and lead modeling and analytic work for TA1. Dr. Billy Karesh has 40+ years’ experience leading zoonotic and wildlife disease projects, and will commit 1 month annually to manage partnership activities and outreach. Dr. Jon Epstein, with 15 years’ experience working emerging bat zoonoses will coordinate animal trials. Dr. Kevin Olival and Dr. Noam Ross will manage and conduct the modeling and analytical approaches for this project. Support staff include field surveillance teams, modeling analysts, and consultants based in Yunnan Province, China, to oversee field trials. The EHA team has worked extensively with all other collaborators: Prof. Wang (15+ years); Dr. Shi (15+ years); Prof. Baric (5+ years) and Dr. Rocke (15+ years).
Subcontracts: #1 to Prof. Ralph Baric, UNC, to oversee reverse engineering of SARSr-CoVs, BSL-3 humanized mouse experimental infections, design and testing of immune priming inocula based on recombinant spike proteins. Assisted by senior personnel Dr. Tim Sheahan, Dr. Amy Sims, and support staff; #2 to Prof. Linfa Wang, Duke NUS, to oversee the immune boosting approach, captive bat experiments, and analyze immunological and virological responses to immune boosting inocula; #3 to Dr. Zhengli Shi, Wuhan Institute of Virology, to conduct PCR testing, viral discovery and isolation from bat samples collected in China, spike protein binding assays, and some humanized mouse work, as well as experimental inoculation of Rhinolophus bats. Her team will include Dr. Peng Zhou and support staff; #4 to Dr. Tonie Rocke, USGS National Wildlife Health Center, to refine delivery mechanisms for both immune boosting and immune priming treatments. With a research technician, Dr. Rocke will use a captive colony of bats at NWHC for initial trials, and oversee cave experiments in China.
F. Links to published papers, resume of two key performers
Dr. Peter Daszak is President and Chief Scientist of EcoHealth Alliance, a US-based research organization focused on emerging zoonotic diseases. His >300 scientific papers include the first global map of EID hotspots (31, 32), estimates of unknown viral diversity (33), predictive models of virus-host relationships (7), and evidence of the bat origin of SARS-CoV (34, 35) and other emerging viruses (36-39). He is Chair of the NASEM Forum on Microbial Threats, and is a member of the Executive Committee and the EHA institutional lead for the $130 million USAID-EPT-PREDICT. He serves on the NRC Advisory Committee to the USGCRP, the DHS CEEZAD External Advisory Board, the WHO R&D Blueprint Pathogen Prioritization expert group, and has advised the Director for Medical Preparedness Policy on the White House National Security Staff on global health issues. Dr Daszak won the 2000 CSIRO medal for collaborative research.
Prof. Ralph Baric is a UNC Lineberger Comprehensive Cancer Center member and Professor in the UNC-Chapel Hill Dept. of Epidemiology and Dept. of Microbiology & Immunology. His work focuses on coronaviruses as models to study the genetics of RNA virus transcription, replication, persistence, cross species transmission and pathogenesis. His group has developed a platform strategy to access the potential “pre- epidemic” risk associated with zoonotic virus cross species transmission potential and evaluation of countermeasure potential to control future outbreaks of disease (8-13).

Citations
1. P. L. Quan et al., Identification of a Severe Acute Respiratory Syndrome Coronavirus-Like Virus in a Leaf-Nosed Bat in Nigeria. Mbio 1, (2010).
2. J. F. Drexler et al., Genomic Characterization of Severe Acute Respiratory Syndrome-Related Coronavirus in European Bats and Classification of Coronaviruses Based on Partial RNA-Dependent RNA Polymerase Gene Sequences. Journal of Virology 84, 11336-11349 (2010).
3. G. Zhang et al., Comparative analysis of bat genomes provides insight into the evolution of flight and immunity. Science 339, 456-460 (2013).
4. J. Xie et al., Dampened STING-Dependent Interferon Activation in Bats. Cell host & microbe, (2018).
5. P. Zhou et al., Contraction of the type I IFN locus and unusual constitutive expression of IFN-αin bats. Proceedings of the National Academy of Sciences of the United States of America, 201518240-201518246 (2016).
6. M. Ahn, J. Cui, A. T. Irving, L.-F. Wang, Unique Loss of the PYHIN Gene Family in Bats Amongst Mammals: Implications for Inflammasome Sensing. Scientific Reports 6, (2016).
7. K. J. Olival et al., Host and viral traits predict zoonotic spillover from mammals. Nature 546, 646-650 (2017).
8. T. P. Sheahan et al., Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses. Sci Transl Med 9, (2017).
9. V. D. Menachery et al., MERS-CoV and H5N1 influenza virus antagonize antigen presentation by altering the epigenetic landscape. Proc Natl Acad Sci U S A 115, E1012-E1021 (2018).
10. S. J. Anthony et al., Further Evidence for Bats as the Evolutionary Source of Middle East Respiratory Syndrome Coronavirus. MBio 8, (2017).
11. A. S. Cockrell et al., A mouse model for MERS coronavirus-induced acute respiratory distress syndrome. Nat Microbiol 2, 16226 (2016).
12. V. D. Menachery et al., SARS-like WIV1-CoV poised for human emergence. Proc Natl Acad Sci U S A 113, 3048-3053 (2016).
13. V. D. Menachery et al., A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence. Nat Med 21, 1508-1513 (2015).
14. N. Wang et al., Serological evidence of bat SARS-related coronavirus infection in humans, China. Virologica Sinica In press, (2018).
15. P. Zhou et al., Fatal Swine Acute Diarrhea Syndrome caused by an HKU2-related Coronavirus of Bat Origin. Nature In press, (2018).
16. B. M. Farr, J. M. Gwaltney, Jr., K. F. Adams, F. G. Hayden, Intranasal interferon- alpha 2 for prevention of natural rhinovirus colds. Antimicrob Agents Chemother 26, 31-34 (1984).

17. D. Kugel et al., Intranasal Administration of Alpha Interferon Reduces Seasonal Influenza A Virus Morbidity in Ferrets. Journal of Virology 83, 3843-3851 (2009).
18. L. M. Smith et al., Interferon-beta Therapy Prolongs Survival in Rhesus Macaque Models of Ebola and Marburg Hemorrhagic Fever. Journal of Infectious Diseases
208, 310-318 (2013).
19. J. Zhao et al., Intranasal Treatment with Poly(I.C) Protects Aged Mice from Lethal
Respiratory Virus Infections. Journal of Virology 86, 11416-11424 (2012).
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Function and Inhibition of Papain-Like Proteases of Emerging Coronaviruses.
Journal of Virology 88, 11825-11833 (2014).
21. J. Pallesen et al., Immunogenicity and structures of a rationally designed
prefusion MERS-CoV spike antigen. Proc Natl Acad Sci U S A 114, E7348-E7357
(2017).
22. C. M. Coleman et al., Purified coronavirus spike protein nanoparticles induce
coronavirus neutralizing antibodies in mice. Vaccine 32, 3169-3174 (2014).
23. C. M. Coleman et al., MERS-CoV spike nanoparticles protect mice from MERS-
CoV infection. Vaccine 35, 1586-1589 (2017).
24. L. Du et al., A 219-mer CHO-expressing receptor-binding domain of SARS-CoV S
protein induces potent immune responses and protective immunity. Viral
immunology 23, 211-219 (2010).
25. T. Sheahan et al., Successful vaccination strategies that protect aged mice from
lethal challenge from influenza virus and heterologous severe acute respiratory
syndrome coronavirus. J Virol 85, 217-230 (2011).
26. S. Agnihothram et al., A mouse model for Betacoronavirus subgroup 2c using a
bat coronavirus strain HKU5 variant. MBio 5, e00047-00014 (2014).
27. M. M. Becker et al., Synthetic recombinant bat SARS-like coronavirus is
infectious in cultured cells and in mice. Proc Natl Acad Sci U S A 105, 19944-
19949 (2008).
28. T. E. Rocke et al., Sylvatic Plague Vaccine Partially Protects Prairie Dogs (Cynomys
spp.) in Field Trials. Ecohealth 14, 438-450 (2017).
29. B. Stading et al., Protection of bats (Eptesicus fuscus) against rabies following
topical or oronasal exposure to a recombinant raccoon poxvirus vaccine. Plos
Neglect. Trop. Dis. 11, (2017).
30. B. R. Stading et al., Infectivity of attenuated poxvirus vaccine vectors and
immunogenicity of a raccoonpox vectored rabies vaccine in the Brazilian Free-
tailed bat (Tadarida brasiliensis). Vaccine 34, 5352-5358 (2016).
31. K. E. Jones et al., Global trends in emerging infectious diseases. Nature 451, 990-
993 (2008).
32. T. Allen et al., Global hotspots and correlates of emerging zoonotic diseases. Nat
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33. S. J. Anthony et al., A Strategy to Estimate Unknown Viral Diversity in Mammals.
mBio 4, (2013).
34. X. Y. Ge et al., Isolation and characterization of a bat SARS-like coronavirus that
uses the ACE2 receptor. Nature 503, 535-538 (2013).

35. W. Li et al., Bats are natural reservoirs of SARS-like coronaviruses. Science 310, 676-679 (2005).
36. A. N. Alagaili et al., Middle East respiratory syndrome coronavirus infection in dromedary camels in saudi arabia. MBio 5, (2014).
37. N. Homaira et al., Nipah virus outbreak with person-to-person transmission in a district of Bangladesh, 2007. Epidemiology and Infection 138, 1630-1636 (2010).
38. M. S. Islam et al., Nipah Virus Transmission from Bats to Humans Associated with Drinking Traditional Liquor Made from Date Palm Sap, Bangladesh, 2011–2014. Emerging Infectious Disease journal 22, 664 (2016).
39. K. J. Olival et al., Ebola virus antibodies in fruit bats, bangladesh. Emerg Infect Dis 19, 270-273 (2013).

10/5/21, 2:54 PM Mail - Rocke, Tonie E - Outlook
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Mail - Rocke, Tonie E - Outlook
www.ecohealthalliance.org
Dr. Noam Ross
Senior Research Scientist
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
+1.212.380.4471 (direct) +1.212.380.4465 (fax) @noamross (twitter) www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics
and promote conservation.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 2/2
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DARPA – PREEMPT – HR001118S0017- PROJECT DEFUSE
C. Goals and Impact:
1. What is the proposed work attempting to accomplish or do?
We will defuse the potential for emergence of novel bat-origin high-zoonotic risk SARS-related coronaviruses. We envisage a scenario whereby the US warfighter is deployed to a security hotspot in SE Asia. As planners choose sites for the mission, they will use an app we will design based on machine-learning models of the ecological and evolutionary potential of bat viruses to spillover. This will allow rapid assessment of the background risk of a site harboring dangerous zoonotic viruses. If there is no alternative site, a tactical forward team will deploy automated delivery technology we will develop in caves that harbor bats carrying these viruses. These devices will release broadscale immune boosting molecules and chimeric polyvalent spike protein targeted immune priming inocula to upregulate the naturally damped innate immune response of bats, and lower viral shedding from bats at the site for a few weeks or months, allowing our warfighters to execute the operation at lowered risk for spillover.
2. How is it done today? And what are the limitations?
There is no available current technology to reduce the risk of exposure to novel coronaviruses from bats. Models of bat host capacity to harbor viruses, of ecological and environmental drivers of their emergence, and of the evolutionary potential of different strains to spillover are rudimentary. No vaccines or therapeutics exist for SARSr-CoVs, and exposure mitigation strategies are non-existent. SARSr-CoVs are endemic in Asian, African (1), and European bats (2) that roost in caves but forage widely at night, shedding virus in their feces and urine. The limitations of this lack of capacity are significant – we have shown evidence of recent spillover of SARSr-CoVs into people in China and have isolated strains capable of producing SARS-like illness in humanized mice that don’t respond to antibody treatment or vaccination. These viruses are a clear-and- present danger to our military and to global health security.
3. What is innovative in your approach and how does it compare to current
practice and state-of-the-art (SOA)?
Our group leads the world in predictive models of viral emergence. We will build on our machine-learning models of spillover hotspots, host-pathogen ecological niches and genotype-phenotype mapping by incorporating unique datasets to validate and refine hotspot risk maps of viral emergence in SE Asia and beyond. Our group has shown that bats coexist with lethal viruses by damping innate immunity pathways, likely as an evolutionary adaptation to flight. We will use this insight to design strategies, like small molecule Rig like receptor (RLR) or Toll like receptor (TLR) agonists, to upregulate bat immunity in their cave roosts, down-regulate viral replication, and reduce the risk of viral shedding and spillover (broadscale immune boosting strategy). We will complement this by inoculating bats with novel chimeric polyvalent recombinant spike proteins to enhance their immune response against specific, high-risk viruses (targeted immune priming strategy), especially when their immune response is boosted as above.

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We will design novel methods to deliver these inocula remotely to reduce exposure risk during decontamination.
4. What are the key technical challenges in your approach and how do you plan to overcome these?
Modeling: Previous models have suffered from a lack of data to validate them. We have access to unique datasets that will allow us to validate our approach, including biodiversity surveys of bat caves across S. China, 10+ years of bat viral testing data in China, and 10 other countries (from NIAID and USAID EPT PREDICT work). Uniquely, we will validate our models of viral evolution/spillover risk using serology (based on LIPS assays) in local populations that have high (~3%) seroprevelance to bat SARSr-CoVs. Identifying Immune boosting and priming inocula: Some of our approaches are novel and challenging (e.g. using CRISPRi to find the negative regulator for bat interferon production), and others are unproven in bats (e.g. Poly IC). We will begin all immune boosting and priming experiments at the beginning of the project, running them simultaneously and competitively, so that we field trial only the most efficient, cost- effective and scalable approaches.
5. Who will care and what will the impact be if you are successful?
This will have direct relevance to the warfighter. Potential deployment to regions where SARSr-CoVs exist is high – countries include security hotspots in Asia (e.g. Myanmar, Bangladesh, Pakistan, Korea, Vietnam), Africa and Eastern Europe. The ability to decontaminate and defuse these viruses may prevent potentially devastating illness. These technologies could be adapted to hosts of other bat-origin CoVs (e.g. MERS-CoV, SADS-CoV) and potentially other zoonotic bat-origin viruses (Hendra, Nipah, EBOV), with benefits to livestock production, food security and global public health.
6. How much will it cost and how long will it take?
Aleksei/Luke to fill out
SPACE SPACE SPACE
D. Technical Plan:
Overview
The SARSr-CoV-bat system, and immune modulation focus: Our group’s 15 yrs work on the SARSr-CoV – Rhinolophus bat system in China has identified and isolated SARSr-CoVs with remarkable sequence identity in the spike protein to SARS-CoV (e.g. SCH014 & WIV-1). We have shown they bind and replicate efficiently in primary human lung airway cells and that chimeras with SARSr-CoV spike proteins in a SARS-CoV backbone cause SARS-like illness in humanized mice, with clinical signs that are not reduced by SARS monoclonal therapy or vaccination. We have identified a single cave site in Yunnan Province where bat SARSr-CoVs contain all the genetic components of epidemic SARS- CoV (7-9). We have now shown that people living up to 6 kilometers from this cave have

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SARSr-CoV antibodies (3% seroprevalence in 200+ cohort), suggesting active spillover, and marking these viruses as a clear-and-present danger of a new SARS-like pandemic. Our work on bat immunology suggests that bats’ unique flying ability has led to downregulated innate immune genes, and their ability to coexist with viruses such as SARSr-CoVs, henipa- and filoviruses that are lethal in many other mammals (3). We have identified bat-specific constitutively expressed bat interferon, a dampened STING- interferon production pathway (4, 5), and have identified a series of other innate immunity factors that are dampened in bats (6).
Our bat-CoV system has significant advantages for experimentation and intervention. Firstly, these viruses are fecal-orally transmitted within bat populations, so sampling can be achieved from fresh fecal pellet collection. They are BSL-3, not -4, agents, so that experimental manipulation and infection is simpler. They have frequent spillover events, making it possible to validate predictive models of spillover by sampling people. They are diverse, with frequent recombination and different strains exhibiting differential host cell binding and spillover potential. Finally, we have identified SARSr- CoV strains in a single cave in Yunnan that harbor all of the epidemic SARS-CoV genes. This specific bat population harbors an ideal evolutionary soup that could produce new human strains by high frequency RNA recombination, and thus, it presents a perfect target for next generation, technology-forward intervention strategies.
TA1: Develop and validate integrated, multiscale models that quantify the likelihood a human-capable virus will emerge from an animal reservoir residing in a “hot spot” geographic region
The DEFUSE modeling and analytics team, led by Drs Daszak, Ross, Olival, EHA, will build ecological niche models of environmental and ecological correlates and traits of cave bat communities to predict species composition of bat caves across Southern China, South and SE Asia. We will then use a series of datasets we have built to produce host- virus risk models for the region. These include our unique database of bat host-viral relationships (7); biological inventory data on all bat caves in Southern China; and modeled species distribution data for all bats. We will parameterize the model with data from three cave sites in Yunnan, China (one with high-risk SARSr-CoVs, two other control/comparison sites), including: radio- and GPS-telemetry to identify home range and additional roost sites for each bat species; inventory of bat population density, distribution and segregation and their daily, weekly and seasonal changes; viral prevalence and individual viral load; shedding of low- and high-risk SARSr-CoV strains among bat species, age classes, genders; and telemetry and mark-recapture data to assess metapopulation structure and inter-cave connectivity. We will test and validate model predictions of a cave’s viral spillover potential with data from prior PREDICT sampling in 7 other Asian countries. At the end of Yr 1, we will produce a prototype app for the warfighter that identifies the likelihood of bats harboring dangerous viral pathogens in a region. The ‘high-risk bats near me’ app will be updated real-time with surveillance data (e.g. field-deployable iphone and android compatible echolocation data) from our project and others, to ground-truth and fine-tune its predictive capacity.
The Wuhan Institute of Virology team will test bat fecal, oral, blood and

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urogenital samples for SARSr-CoVs. We will correlate viral load data from these samples with fresh fecal pellets from individuals and from tarps laid on cave floors. We will rapidly move to fecal pellet assays to reduce roost disturbance. SARSr-CoV spike proteins will be sequenced, analyzed phylogenetically for recombination events, and high-risk viruses (spike proteins close to SARS-CoV) characterized and isolated. The UNC team will reverse-engineer spike proteins to conduct binding assays to human ACE2 (the SARS-CoV receptor). They will culture SARS-like bat coronaviruses to distinguish high risk strains that can replicate in primary human cells and low risk strains that require exogenous enhancers. Viral spike glycoproteins that bind receptors will be inserted into SARS-CoV backbones, inoculated into human cells and humanized mice to assess capacity to cause SARS-like disease, and to be blocked by monoclonal therapies, the nucleoside analogue inhibitor GS-5734 (8) or vaccines against SARS-CoV (8-13).
The EHA modeling team will use these data to build models of risk of viral evolution and spillover. These genotype-to-phenotype machine-learning models will predict viral ability to infect host cells based on genetic traits and results of receptor binding and mouse infection assays. Using data on diversity of spike proteins, recombinant CoVs, and flow of genes within each bat cave via bat movement and migration, we will estimate evolutionary rates, rates of recombination, and capacity to generate novel strains capable of human infection. Finally, virus-host relationship and bat home range data will be used to estimate spillover potential - extending models well beyond our field sites. We will then validate model predictions of viral spillover risk by 1) conducting spike protein-based binding and cell culture experiments, and 2) identifying spillover strains in people near our bat cave sites. Our preliminary work on this shows ~3% seroprevalence to SARSr-CoVs, using a specific ELISA (14). We will design LIPS assays to the specific high- and low- zoonotic-risk SARSr-CoVs identified in this project as we have done previously (15). We will use banked and newly collected human sera from these populations to test for presence of antibodies to the high- and low-risk SARSr-CoVs identified by our modeling. We will then model optimal strategies to maximize inoculation efficacy for TA2, using stochastic simulation modeling informed by field and experimental data to characterize viral circulation dynamics in bats. We will estimate frequency and population coverage required for our intervention approaches to suppress viral spillover. We will determine the seasons, locations within a cave, and delivery methods (spray, swab, or automated cave mouth or drone) that will be most effective. Finally we will determine the time period treatment will be effective for, until re-colonization or evolution leads to return of a high-risk SARSr-CoV.
TA2: Develop scalable approaches that target and suppress the animal virus in its reservoir(s) and/or vector(s), to reduce the likelihood of virus transmission into humans.
We will evaluate two approaches to defuse SARS-related CoV spillover potential: 1) Broadscale Immune Boosting: using the unique immune damping in bats that our group has discovered, we will inoculate live bats with immune modulators like bat interferon designed to up-regulate their naïve immunity and then assess their ability to suppress viral replication and shedding; 2) Targeted Immune Priming: building on preliminary

development of polyvalent chimeric recombinant SARSr-CoV spike proteins, we will conduct inoculation trials with live bats to assess suppression of replication and shedding of a broad range of dangerous SARS-related CoVs.
Both lines of work will begin in Yr 1 and run parallel. Prof. Linfa Wang (Duke- NUS) will lead the immune boosting work, building on his pioneering work on bat immunity (3) which shows that the long-term coexistence of bats and their viruses has led to equilibrium between viral replication and host immunity. This is likely due to down-regulation of their innate immune system as a fitness cost of flight (3). The weakened functionality of bat innate immunity factors like STING, a central DNA- interferon (IFN) sensing molecule, may allow bats to maintain an effective, but not over- response to viruses (4). A similar finding was observed for bat IFNA, which is less abundant but constitutively expressed without stimulation (5). Given high native SARSr- CoV load in bats, we aim to boost bat innate immunity through the IFN pathway, break the host-virus equilibrium to suppress bat SARSr-CoV replication and shedding.
We will trial the following, concurrently and competitively, for efficiency, cost and scalability: i) Universal bat interferon. Aerosol spraying or intranasal inoculation of IFN or other small molecules reduces viral loads in humans, ferrets and mouse models (16, 17). Interferon has been used clinically when antiviral drugs are unavailable, e.g. against filoviruses (18). Replication of SARSr-CoV is sensitive to interferon treatments, as shown in our previous work (16); ii) Boosting bat IFN by blocking bat-specific IFN negative regulators. Uniquely, bat IFNA is naturally constitutively expressed but cannot be induced to a high level (5), indicating a negative regulatory factor in the bat interferon production pathway. We will use CRISPRi to identify the negative regulator and then screen for compounds targeting this gene; iii) Activating dampened bat- specific IFN production pathways which include DNA-STING-dependent and ssRNA-TLR7- dependent pathways. Our work showing that mutant bat STING restores antiviral functionality suggests these pathways are important in bat-viral coexistence (4). By identifying small molecules to directly activate downstream of STING, we will activate bat interferon and promote viral clearance. A similar strategy will be applied to ssRNA- TLR7-dependent pathways; iv) Activating functional bat IFN production pathways, e.g. polyIC to TLR3-IFN pathway or 5’ppp-dsRNA to RIG-I-IFN pathway. A similar strategy has been demonstrated in a mouse model for SARS-CoV, IAV and HBV (17, 19); v) Inoculating crude coronavirus fragments to upregulate innate immune responses to specific CoVs – a partial step towards the targeted immune priming work below.
Prof. Ralph Baric (UNC) will lead the immune priming work. He will develop recombinant chimeric spike-proteins (20) from our known SARSr-CoVs, and those we characterize during project DEFUSE. The structure of the SARS-CoV spike glycoprotein has been solved and the addition of two proline residues at positions V1060P and L1061P stabilize the prefusion state of the trimer, including key neutralizing epitopes in the receptor binding domain (21). In parallel, the spike trimers or the receptor binding domain can be incorporated into alphavirus vectored or nanoparticle vaccines for delivery, either as aerosols, in baits, or as large droplet delivery vehicles (11, 22-25). We will test these in controlled lab conditions, taking the best candidate forward for testing in the field. We have built recombinant spike glycoproteins harboring structurally

defined domains from SARS epidemic strains, pre-epidemic strains like SCH014 and zoonotic strains like HKU3. It is anticipated that recombinant S glycoprotein based vaccines harboring immunogenic blocks across the group 2B coronaviruses will induce broadscale immune responses that simultaneously reduce genetically heterogeneous virus burdens in bats, potentially reducing disease risk (and transmission risk to people) in these animals for longer periods (26, 27).
The immune dampening features are highly conserved in all bat species tested so far. Duke-NUS has established the only experimental breeding colony of cave bats (Eonycteris spelaea) in SE Asia. This genus is evolutionarily closely related to Rhinolophus spp. (the hosts of SARSr-CoVs), so we have confidence that results will be transferable. Our initial proof-of-concept tests will be in this experimental colony, extended to a small group of wild-caught Rhinolophus sinicus bats at Wuhan Institute of Zoology. We (Prof. Wang) have previous experience conducting SARS-CoV infection experiments with Rhinolophus sp. bats in the BSL-4 facility at CSIRO, AAHL (L.Wang, unpublished results).
Finally, work on a delivery method for our immune boosting and priming molecules will be overseen by Dr. Tonie Rocke at the USGS, National Wildlife Health Center who has previously developed animal vaccines through to licensure (28). Using locally acquired insectivorous bats (29, 30) we will assess delivery vehicles and methods including: 1) transdermally applied nanoparticles; 2) series of sticky edible gels that bats will groom from themselves and each other; 3) aerosolization via sprayers that could be used in cave settings; 4) automated sprays triggered by timers and movement detectors at critical cave entry points; 5) sprays delivered by remote controlled drone. We have already used simple gels to vaccinate bats against rabies in the lab (29), and hand delivered these containing biomarkers to vampire bats in Peru and Mexico to show they are readily consumed and transferred among bats. In our bat colony, we will trial delivery vehicles using the biomarker rhodamine B (which marks hair and whiskers upon consumption) to assess uptake. The most optimal approaches will then be tested on wild bats in our three cave sites in Yunnan Province with the most successful immunomodulators from TA2. Fieldwork will be conducted under the auspices of Dr. Yunzhi Zhang (Yunnan CDC, Consultant at EcoHealth Alliance). A small number of bats will be captured and assayed for viral load and immune function after treatment, but so as not to disturb the colony, most viral load work will be conducted on fresh fecal pellets collected daily on the cave floor. EHA has had unique access to these sites for around 10 years, under the guidance of Drs. Shi and Zhang. In year 1 of project DEFUSE, we will seek permission for experimental inoculations from the Provincial Forestry Department. We expect to be successful, as we have worked with the Forestry Department collaboratively for 10 years, with support of the Yunnan CDC, and we are releasing molecules that are not dangerous to people or wildlife. EHA has a proven track record of rapidly obtaining IACUC and DoD ACURO approval for bat research.
E. Capabilities:
The lead institution for Project DEFUSE is EcoHealth Alliance, New York, an international research organization focused on emerging zoonotic diseases. The PI, Dr. Peter Daszak, has 25+ years’ experience managing lab, field and modeling research projects on

emerging zoonoses. Dr. Daszak will commit 3 months annually to oversee and coordinate all project activities, and lead modeling and analytic work for TA1. Dr. Billy Karesh has 40+ years’ experience leading zoonotic and wildlife disease projects, and will commit 1 month annually to manage partnership activities and outreach. Dr. Jon Epstein, with 15 years’ experience working emerging bat zoonoses will coordinate animal trials. Dr. Kevin Olival and Dr. Noam Ross will manage and conduct the modeling and analytical approaches for this project. Support staff include field surveillance teams, modeling analysts, and consultants based in Yunnan Province, China, to oversee field trials. The EHA team has worked extensively with all other collaborators: Prof. Wang (15+ years); Dr. Shi (15+ years); Prof. Baric (5+ years) and Dr. Rocke (15+ years).
Subcontracts: #1 to Prof. Ralph Baric, UNC, to oversee reverse engineering of SARSr-CoVs, BSL-3 humanized mouse experimental infections, design and testing of immune priming inocula based on recombinant spike proteins. Assisted by senior personnel Dr. Tim Sheahan, Dr. Amy Sims, and support staff; #2 to Prof. Linfa Wang, Duke NUS, to oversee the immune boosting approach, captive bat experiments, and analyze immunological and virological responses to immune boosting inocula; #3 to Dr. Zhengli Shi, Wuhan Institute of Virology, to conduct PCR testing, viral discovery and isolation from bat samples collected in China, spike protein binding assays, and some humanized mouse work, as well as experimental inoculation of Rhinolophus bats. Her team will include Dr. Peng Zhou and support staff; #4 to Dr. Tonie Rocke, USGS National Wildlife Health Center, to refine delivery mechanisms for both immune boosting and immune priming treatments. With a research technician, Dr. Rocke will use a captive colony of bats at NWHC for initial trials, and oversee cave experiments in China.
F. Links to published papers, resume of two key performers
Dr. Peter Daszak is President and Chief Scientist of EcoHealth Alliance, a US-based research organization focused on emerging zoonotic diseases. His >300 scientific papers include the first global map of EID hotspots (31, 32), estimates of unknown viral diversity (33), predictive models of virus-host relationships (7), and evidence of the bat origin of SARS-CoV (34, 35) and other emerging viruses (36-39). He is Chair of the NASEM Forum on Microbial Threats, and is a member of the Executive Committee and the EHA institutional lead for the $130 million USAID-EPT-PREDICT. He serves on the NRC Advisory Committee to the USGCRP, the DHS CEEZAD External Advisory Board, the WHO R&D Blueprint Pathogen Prioritization expert group, and has advised the Director for Medical Preparedness Policy on the White House National Security Staff on global health issues. Dr Daszak won the 2000 CSIRO medal for collaborative research.
Prof. Ralph Baric is a UNC Lineberger Comprehensive Cancer Center member and Professor in the UNC-Chapel Hill Dept. of Epidemiology and Dept. of Microbiology & Immunology. His work focuses on coronaviruses as models to study the genetics of RNA virus transcription, replication, persistence, cross species transmission and pathogenesis. His group has developed a platform strategy to access the potential “pre- epidemic” risk associated with zoonotic virus cross species transmission potential and evaluation of countermeasure potential to control future outbreaks of disease (8-13).

Citations
1. P. L. Quan et al., Identification of a Severe Acute Respiratory Syndrome Coronavirus-Like Virus in a Leaf-Nosed Bat in Nigeria. Mbio 1, (2010).
2. J. F. Drexler et al., Genomic Characterization of Severe Acute Respiratory Syndrome-Related Coronavirus in European Bats and Classification of Coronaviruses Based on Partial RNA-Dependent RNA Polymerase Gene Sequences. Journal of Virology 84, 11336-11349 (2010).
3. G. Zhang et al., Comparative analysis of bat genomes provides insight into the evolution of flight and immunity. Science 339, 456-460 (2013).
4. J. Xie et al., Dampened STING-Dependent Interferon Activation in Bats. Cell host & microbe, (2018).
5. P. Zhou et al., Contraction of the type I IFN locus and unusual constitutive expression of IFN-αin bats. Proceedings of the National Academy of Sciences of the United States of America, 201518240-201518246 (2016).
6. M. Ahn, J. Cui, A. T. Irving, L.-F. Wang, Unique Loss of the PYHIN Gene Family in Bats Amongst Mammals: Implications for Inflammasome Sensing. Scientific Reports 6, (2016).
7. K. J. Olival et al., Host and viral traits predict zoonotic spillover from mammals. Nature 546, 646-650 (2017).
8. T. P. Sheahan et al., Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses. Sci Transl Med 9, (2017).
9. V. D. Menachery et al., MERS-CoV and H5N1 influenza virus antagonize antigen presentation by altering the epigenetic landscape. Proc Natl Acad Sci U S A 115, E1012-E1021 (2018).
10. S. J. Anthony et al., Further Evidence for Bats as the Evolutionary Source of Middle East Respiratory Syndrome Coronavirus. MBio 8, (2017).
11. A. S. Cockrell et al., A mouse model for MERS coronavirus-induced acute respiratory distress syndrome. Nat Microbiol 2, 16226 (2016).
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13. V. D. Menachery et al., A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence. Nat Med 21, 1508-1513 (2015).
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16. B. M. Farr, J. M. Gwaltney, Jr., K. F. Adams, F. G. Hayden, Intranasal interferon- alpha 2 for prevention of natural rhinovirus colds. Antimicrob Agents Chemother 26, 31-34 (1984).

17. D. Kugel et al., Intranasal Administration of Alpha Interferon Reduces Seasonal Influenza A Virus Morbidity in Ferrets. Journal of Virology 83, 3843-3851 (2009).
18. L. M. Smith et al., Interferon-beta Therapy Prolongs Survival in Rhesus Macaque Models of Ebola and Marburg Hemorrhagic Fever. Journal of Infectious Diseases
208, 310-318 (2013).
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Respiratory Virus Infections. Journal of Virology 86, 11416-11424 (2012).
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Function and Inhibition of Papain-Like Proteases of Emerging Coronaviruses.
Journal of Virology 88, 11825-11833 (2014).
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prefusion MERS-CoV spike antigen. Proc Natl Acad Sci U S A 114, E7348-E7357
(2017).
22. C. M. Coleman et al., Purified coronavirus spike protein nanoparticles induce
coronavirus neutralizing antibodies in mice. Vaccine 32, 3169-3174 (2014).
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protein induces potent immune responses and protective immunity. Viral
immunology 23, 211-219 (2010).
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lethal challenge from influenza virus and heterologous severe acute respiratory
syndrome coronavirus. J Virol 85, 217-230 (2011).
26. S. Agnihothram et al., A mouse model for Betacoronavirus subgroup 2c using a
bat coronavirus strain HKU5 variant. MBio 5, e00047-00014 (2014).
27. M. M. Becker et al., Synthetic recombinant bat SARS-like coronavirus is
infectious in cultured cells and in mice. Proc Natl Acad Sci U S A 105, 19944-
19949 (2008).
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spp.) in Field Trials. Ecohealth 14, 438-450 (2017).
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topical or oronasal exposure to a recombinant raccoon poxvirus vaccine. Plos
Neglect. Trop. Dis. 11, (2017).
30. B. R. Stading et al., Infectivity of attenuated poxvirus vaccine vectors and
immunogenicity of a raccoonpox vectored rabies vaccine in the Brazilian Free-
tailed bat (Tadarida brasiliensis). Vaccine 34, 5352-5358 (2016).
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mBio 4, (2013).
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uses the ACE2 receptor. Nature 503, 535-538 (2013).

35. W. Li et al., Bats are natural reservoirs of SARS-like coronaviruses. Science 310, 676-679 (2005).
36. A. N. Alagaili et al., Middle East respiratory syndrome coronavirus infection in dromedary camels in saudi arabia. MBio 5, (2014).
37. N. Homaira et al., Nipah virus outbreak with person-to-person transmission in a district of Bangladesh, 2007. Epidemiology and Infection 138, 1630-1636 (2010).
38. M. S. Islam et al., Nipah Virus Transmission from Bats to Humans Associated with Drinking Traditional Liquor Made from Date Palm Sap, Bangladesh, 2011–2014. Emerging Infectious Disease journal 22, 664 (2016).
39. K. J. Olival et al., Ebola virus antibodies in fruit bats, bangladesh. Emerg Infect Dis 19, 270-273 (2013).

10/5/21, 2:55 PM Mail - Rocke, Tonie E - Outlook
Good point Linfa – changed to ‘treatment’ or ‘applicaon’ throughout.
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
From: Wang Linfa [mailto:linfa.wang@duke-nus.edu.sg]
Sent: Tuesday, February 13, 2018 1:10 AM
To: Peter Daszak; Luke Hamel; Jonathon Musser
Cc: William B. Karesh; Noam Ross; Ralph Baric (rbaric@email.unc.edu); 周鹏 (peng.zhou@wh.iov.cn); Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura; Anna Willoughby; Rocke, Tonie
Subject: RE: Final draft DARPA abstract
Hi Peer,
It actually reads well now and I hope the selecon commiee will “buy in” our brave ideas!
Nothing to add a or change, other than an English queson: we used “dampened immunity of bats” in most places, but you use the expression of “damping innate immunity pathways” on page 1. Is there a difference between “damping” and “dampening”?
Thanks
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>vog.sgsu@ekcort<
EeinoT,ekcoR;>gro.ecnaillahtlaehoce@ybhguolliw<ybhguolliWannA;>gro.ecnaillahtlaehoce@arumhc<
arumhCieskelA;>gro.ecnaillahtlaehoce@erdna<erdnAnosilA;>nc.voi.hw@ihslz<)nc.voi.hw@ihslz(
ihSilgnehZ;>nc.voi.hw@uohz.gnep<)nc.voi.hw@uohz.gnep(鹏周;>ude.cnu.liame@cirabr<)ude.cnu.liame@cirabr(
ciraBhplaR;>gro.ecnaillahtlaehoce@ssor<ssoRmaoN;>gro.ecnaillahtlaehoce@hserak<hseraK.BmailliW:cC
>gro.ecnaillahtlaehoce@ressum<
ressuMnohtanoJ;>gro.ecnaillahtlaehoce@lemah<lemaHekuL;>gs.ude.sun-ekud@gnaw.afnil<afniLgnaW:oT
>gro.ecnaillahtlaehoce@kazsad<kMaA1z2s7a810D2/r31e/t2euPT
tcartsba APRAD tfard laniF :ER

10/5/21, 2:55 PM
Mail - Rocke, Tonie E - Outlook
LF
Linfa (Lin-Fa) Wang, PhD FTSE
Professor & Director
Programme in Emerging Infecous Diseases Duke-NUS Medical School
8 College Road, Singapore 169875
Tel: +65 65168397
From: Peter Daszak [mailto:daszak@ecohealthalliance.org]
Sent: Tuesday, 13 February 2018 1:23 PM
To: Luke Hamel <hamel@ecohealthalliance.org>; Jonathon Musser <musser@ecohealthalliance.org>
Cc: William B. Karesh <karesh@ecohealthalliance.org>; Noam Ross <ross@ecohealthalliance.org>; Ralph Baric (rbaric@email.unc.edu) <rbaric@email.unc.edu>; Wang Linfa <linfa.wang@duke-nus.edu.sg>; 周鹏 (peng.zhou@wh.iov.cn) <peng.zhou@wh.iov.cn>; Zhengli Shi (zlshi@wh.iov.cn) <zlshi@wh.iov.cn>; Alison Andre <andre@ecohealthalliance.org>; Aleksei Chmura <chmura@ecohealthalliance.org>; Anna Willoughby <willoughby@ecohealthalliance.org>; Rocke, Tonie <trocke@usgs.gov>
Subject: Final dra DARPA abstract
Importance: High
Luke, aached is the DARPA abstract.
First thing tomorrow, can you go through the references, and create links to the papers on NCBI. Just turn each reference citaon: “(1)” for example, into a live link to the paper on NCBI. We only have 2 spare lines, so no room to turn each of these into a PMC numbered ref, as Ralph did for his, but please make sure the citaon in parentheses is blue, so it’s clear it’s a live link on the final pdf.
Ralph, Lina, Peng, Zhengli, Tonie – please give this a quick read to make sure I’ve not said anything completely wrong. I’ve had to reduce the text a lot to hit the page limit, but I sll think it’s a great proposal.
I’ll finish off the exec summary slide and <500 wd abstract now.
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote
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10/5/21, 2:55 PM Mail - Rocke, Tonie E - Outlook
conservaon.
Important: This email is confidential and may be privileged. If you are not the intended recipient, please delete it and notify us immediately; you should not copy or use it for any purpose, nor disclose its contents to any other person. Thank you.
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10/5/21, 2:56 PM
Mail - Rocke, Tonie E - Outlook
Thanks Peng – made the changes now.
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
From: peng.zhou [mailto:peng.zhou@wh.iov.cn]
Sent: Tuesday, February 13, 2018 2:26 AM
To: Peter Daszak
Cc: Luke Hamel; Jonathon Musser; William B. Karesh; Noam Ross; Ralph Baric (rbaric@email.unc.edu); wang linfa; Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura; Anna Willoughby; Rocke, Tonie
Subject: Re: Final draft abstract
Hi, Peter ,
It was really great proposal ! Just a couple of minor issues :
1, at 3, you used "spike protein", for the immune part. But you used "CoV fragments" for immunization in the actual ta2 part. Please be consistent. Also be consistent if we use CoV fragments to immunize bats or just "inoculate " (bat or human ).
2, last paragraph : Duke-Nus but not Duke Nus. Prof. Zhengli Shi, rather than Dr. ?
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>vog.sgsu@ekcort<EeinoT,ekcoR;>gro.ecnaillahtlaehoce@ybhguolliw<
ybhguolliWannA;>gro.ecnaillahtlaehoce@arumhc<arumhCieskelA;>gro.ecnaillahtlaehoce@erdna<erdnAnosilA
;>nc.voi.hw@ihslz<)nc.voi.hw@ihslz(ihSilgnehZ;>gs.ude.sun-ekud@gnaw.afnil<afnilgnaw;>ude.cnu.liame@cirabr<
)ude.cnu.liame@cirabr(ciraBhplaR;>gro.ecnaillahtlaehoce@ssor<ssoRmaoN;>gro.ecnaillahtlaehoce@hserak<
hseraK.BmailliW;>gro.ecnaillahtlaehoce@ressum<ressuMnohtanoJ;>gro.ecnaillahtlaehoce@lemah<lemaHekuL:cC
>nc.voi.hw@uohz.gnep<uohz.gnep:oT
>gro.ecnaillahtlaehoce@kazsad<kMaA1z2s7a810D2/r31e/t2euPT
tcartsba tfard laniF :ER

10/5/21, 2:56 PM
Mail - Rocke, Tonie E - Outlook
All good for other parts! Best wishes ,
Peng
邮箱:peng.zhou@wh.iov.cn 签名由 网易邮箱大师 定制
在2018年02月13日 13:23,Peter Daszak 写道:
Luke, aached is the DARPA abstract.
周鹏
First thing tomorrow, can you go through the references, and create links to the papers on NCBI. Just turn each reference citaon: “(1)” for example, into a live link to the paper on NCBI. We only have 2 spare lines, so no room to turn each of these into a PMC numbered ref, as Ralph did for his, but please make sure the citaon in parentheses is blue, so it’s clear it’s a live link on the final pdf.
Ralph, Lina, Peng, Zhengli, Tonie – please give this a quick read to make sure I’ve not said anything completely wrong. I’ve had to reduce the text a lot to hit the page limit, but I sll think it’s a great proposal.
I’ll finish off the exec summary slide and <500 wd abstract now.
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
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10/5/21, 2:56 PM Mail - Rocke, Tonie E - Outlook
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10/5/21, 2:56 PM Mail - Rocke, Tonie E - Outlook
Great – thanks Tonie and Noam – all changes made now..
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
From: Rocke, Tonie [mailto:trocke@usgs.gov]
Sent: Tuesday, February 13, 2018 2:48 AM
To: Peter Daszak
Cc: Luke Hamel; Jonathon Musser; William B. Karesh; Noam Ross; Ralph Baric (rbaric@email.unc.edu); wang linfa; 周鹏 (peng.zhou@wh.iov.cn); Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura; Anna Willoughby Subject: Re: Final draft DARPA abstract
Just minor edits. Looks good. -Tonie
On Mon, Feb 12, 2018 at 11:23 PM, Peter Daszak <daszak@ecohealthalliance.org> wrote: Luke, attached is the DARPA abstract.
First thing tomorrow, can you go through the references, and create links to the papers on NCBI. Just turn each reference citation: “(1)” for example, into a live link to the paper on NCBI. We only have 2 spare lines, so no room to turn each of these into a PMC numbered ref, as Ralph did for his, but please make sure the citation in parentheses is blue, so it’s clear it’s a live link on the final pdf.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/2
>gro.ecnaillahtlaehoce@ybhguolliw<
ybhguolliWannA;>gro.ecnaillahtlaehoce@arumhc<arumhCieskelA;>gro.ecnaillahtlaehoce@erdna<
erdnAnosilA;>nc.voi.hw@ihslz<)nc.voi.hw@ihslz(ihSilgnehZ;>nc.voi.hw@uohz.gnep<
)nc.voi.hw@uohz.gnep(鹏周;>gs.ude.sun-ekud@gnaw.afnil<afnilgnaw;>ude.cnu.liame@cirabr<
)ude.cnu.liame@cirabr(ciraBhplaR;>gro.ecnaillahtlaehoce@ssor<ssoRmaoN;>gro.ecnaillahtlaehoce@hserak<
hseraK.BmailliW;>gro.ecnaillahtlaehoce@ressum<ressuMnohtanoJ;>gro.ecnaillahtlaehoce@lemah<lemaHekuL:cC
>vog.sgsu@ekcort<EeinoT,ekcoR:oT
>gro.ecnaillahtlaehoce@kazsad<kMaA1z2s7a810D2/r31e/t2euPT
tcartsba APRAD tfard laniF :ER

10/5/21, 2:56 PM Mail - Rocke, Tonie E - Outlook
Ralph, Lina, Peng, Zhengli, Tonie – please give this a quick read to make sure I’ve not said anything completely wrong. I’ve had to reduce the text a lot to hit the page limit, but I still think it’s a great proposal.
I’ll finish off the exec summary slide and <500 wd abstract now.
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge research into the critical connections between human and wildlife health and delicate ecosystems. With this science we develop solutions that prevent pandemics and promote conservation.
--
Tonie E. Rocke
USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
608-270-2451
trocke@usgs.gov
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10/5/21, 2:57 PM Mail - Rocke, Tonie E - Outlook
Here’s the Abstract version 4 for your files. Luke – please do the following ASAP today:
1.
2. 3. 4. 5.
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
From: Noam Ross [mailto:ross@ecohealthalliance.org]
Sent: Tuesday, February 13, 2018 6:22 AM
To: Peter Daszak; Luke Hamel
Cc: Jonathon Musser; William B. Karesh; Ralph Baric (rbaric@email.unc.edu); wang linfa; Rocke, Tonie; 周鹏 (peng.zhou@wh.iov.cn); Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura; Anna Willoughby Subject: Re: Final draft DARPA abstract
Added just a couple of small edits on modeling phrasing, done on top of Tonie's edits. On Tue, Feb 13, 2018 at 2:47 AM Rocke, Tonie <trocke@usgs.gov> wrote:
Fix the references – remove the endnote links, and use the format: “(1,2)”, but making these blue, and an embedded hyperlink to the NCBI paper
Insert the corrected figure
Make sure we have the right format throughout – including grant number etc. at the top Insert the meline and budget
Send it in!
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>gro.ecnaillahtlaehoce@ybhguolliw<ybhguolliWannA
;>gro.ecnaillahtlaehoce@arumhc<arumhCieskelA;>gro.ecnaillahtlaehoce@erdna<erdnAnosilA;>nc.voi.hw@ihslz<
)nc.voi.hw@ihslz(ihSilgnehZ;>nc.voi.hw@uohz.gnep<)nc.voi.hw@uohz.gnep(鹏周;>vog.sgsu@ekcort<
EeinoT,ekcoR;>gs.ude.sun-ekud@gnaw.afnil<afnilgnaw;>ude.cnu.liame@cirabr<)ude.cnu.liame@cirabr(
ciraBhplaR;>gro.ecnaillahtlaehoce@hserak<hseraK.BmailliW;>gro.ecnaillahtlaehoce@ressum<ressuMnohtanoJ:cC
>gro.ecnaillahtlaehoce@lemah<lemaHekuL;>gro.ecnaillahtlaehoce@ssor<ssoRmaoN:oT
>gro.ecnaillahtlaehoce@kazsad<kMaA1z2s7a810D2/r31e/t2euPT
tcartsba APRAD tfard laniF :ER

10/5/21, 2:57 PM Mail - Rocke, Tonie E - Outlook
Just minor edits. Looks good. -Tonie
On Mon, Feb 12, 2018 at 11:23 PM, Peter Daszak <daszak@ecohealthalliance.org> wrote: Luke, attached is the DARPA abstract.
First thing tomorrow, can you go through the references, and create links to the papers on NCBI. Just turn each reference citation: “(1)” for example, into a live link to the paper on NCBI. We only have 2 spare lines, so no room to turn each of these into a PMC numbered ref, as Ralph did for his, but please make sure the citation in parentheses is blue, so it’s clear it’s a live link on the final pdf.
Ralph, Lina, Peng, Zhengli, Tonie – please give this a quick read to make sure I’ve not said anything completely wrong. I’ve had to reduce the text a lot to hit the page limit, but I still think it’s a great proposal.
I’ll finish off the exec summary slide and <500 wd abstract now.
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473 www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge research into the critical connections between human and wildlife health and delicate ecosystems. With this science we develop solutions that prevent pandemics and promote conservation.
--
Tonie E. Rocke
USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
608-270-2451
trocke@usgs.gov
--
Dr. Noam Ross
Senior Research Scientist
EcoHealth Alliance
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10/5/21, 2:57 PM
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+1.212.380.4471 (direct) +1.212.380.4465 (fax) @noamross (twitter) www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics and promote conservation.
Mail - Rocke, Tonie E - Outlook
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DARPA – PREEMPT – HR001118S0017- PROJECT DEFUSE
C. Goals and Impact:
1. What is the proposed work attempting to accomplish or do?
We will defuse the potential for emergence of novel bat-origin high-zoonotic risk SARS-related coronaviruses. We envisage a scenario whereby the US warfighter is deployed to a security hotspot in SE Asia. As planners choose sites for the mission, they will use an app we will design based on machine-learning models of the ecological and evolutionary potential of bat viruses to spillover. This will allow rapid assessment of the background risk of a site harboring dangerous zoonotic viruses. If there is no alternative site, a tactical forward team will deploy automated delivery technology we will develop in caves that harbor bats carrying these viruses. These devices will release broadscale immune boosting molecules and chimeric polyvalent spike protein targeted immune priming treatments to upregulate the naturally damped innate immune response of bats, and lower viral shedding from bats at the site for a few weeks or months, allowing our warfighters to execute the operation at lowered risk for spillover.
2. How is it done today? And what are the limitations?
There is no available current technology to reduce the risk of exposure to novel coronaviruses from bats. Models of bat host capacity to harbor viruses, of ecological and environmental drivers of their emergence, and of the evolutionary potential of different strains to spillover are rudimentary. No vaccines or therapeutics exist for SARSr-CoVs, and exposure mitigation strategies are non-existent. SARSr-CoVs are endemic in Asian, African (1), and European bats (2) that roost in caves but forage widely at night, shedding virus in their feces and urine. The limitations of this lack of capacity are significant – we have shown evidence of recent spillover of SARSr-CoVs into people in China and have isolated strains capable of producing SARS-like illness in humanized mice that don’t respond to antibody treatment or vaccination. These viruses are a clear-and- present danger to our military and to global health security.
3. What is innovative in your approach and how does it compare to current
practice and state-of-the-art (SOA)?
Our group leads the world in predictive models of viral emergence. We will build on our machine-learning models of spillover hotspots, host-pathogen ecological niches and genotype-phenotype mapping by incorporating unique datasets to validate and refine hotspot risk maps of viral emergence in SE Asia and beyond. Our group has shown that bats coexist with lethal viruses by damping innate immunity pathways, likely as an evolutionary adaptation to flight. We will use this insight to design strategies, like small molecule Rig like receptor (RLR) or Toll like receptor (TLR) agonists, to upregulate bat immunity in their cave roosts, down-regulate viral replication, and reduce the risk of viral shedding and spillover (broadscale immune boosting strategy). We will complement this by treating bats with novel chimeric polyvalent recombinant spike proteins to enhance their immune response against specific, high-risk viruses (targeted immune priming strategy), especially when their immune response is boosted as above.

We will design novel methods to deliver these applications remotely to reduce exposure risk during decontamination.
4. What are the key technical challenges in your approach and how do you plan to overcome these?
Modeling: Previous models have suffered from a lack of data to validate them. We have access to unique datasets that will allow us to validate our approach, including biodiversity surveys of bat caves across S. China, 10+ years of bat viral testing data in China, and 10 other countries (from NIAID and USAID EPT PREDICT work). Uniquely, we will validate our models of viral evolution/spillover risk using serology (based on LIPS assays) in local populations that have high (~3%) seroprevelance to bat SARSr-CoVs. Identifying Immune boosting and priming treatments: Some of our approaches are novel and challenging (e.g. using CRISPRi to find the negative regulator for bat interferon production), and others are unproven in bats (e.g. Poly IC). We will begin all immune boosting and priming experiments at the beginning of the project, running them simultaneously and competitively, so that we field trial only the most efficient, cost- effective and scalable approaches.
5. Who will care and what will the impact be if you are successful?
This will have direct relevance to the warfighter. Potential deployment to regions where SARSr-CoVs exist is high – countries include security hotspots in Asia (e.g. Myanmar, Bangladesh, Pakistan, Korea, Vietnam), Africa and Eastern Europe. The ability to decontaminate and defuse these viruses may prevent potentially devastating illness. These technologies could be adapted to hosts of other bat-origin CoVs (e.g. MERS-CoV, SADS-CoV) and potentially other zoonotic bat-origin viruses (Hendra, Nipah, EBOV), with benefits to livestock production, food security and global public health.
6. How much will it cost and how long will it take?
D. Technical Plan:
Overview
The SARSr-CoV-bat system, and immune modulation focus: Our group’s 15 yrs work on the SARSr-CoV – Rhinolophus bat system in China has identified and isolated SARSr-CoVs with remarkable sequence identity in the spike protein to SARS-CoV (e.g. SCH014 & WIV-1). We have shown they bind and replicate efficiently in primary human lung airway cells and that chimeras with SARSr-CoV spike proteins in a SARS-CoV backbone cause SARS-like illness in humanized mice, with clinical signs that are not reduced by SARS monoclonal therapy or vaccination. We have identified a single cave site in Yunnan Province where bat SARSr-CoVs contain all the genetic components of epidemic SARS- CoV (7-9). We have now shown that people living up to 6 kilometers from this cave have
Aleksei/Luke to fill out
SPACE SPACE SPACE

SARSr-CoV antibodies (3% seroprevalence in 200+ cohort), suggesting active spillover, and marking these viruses as a clear-and-present danger of a new SARS-like pandemic. Our work on bat immunology suggests that bats’ unique flying ability has led to downregulated innate immune genes, and their ability to coexist with viruses such as SARSr-CoVs, henipa- and filoviruses that are lethal in many other mammals (3). We have identified bat-specific constitutively expressed bat interferon, a dampened STING- interferon production pathway (4, 5), and have identified a series of other innate immunity factors that are dampened in bats (6).
Our bat-CoV system has significant advantages for experimentation and intervention. Firstly, these viruses are fecal-orally transmitted within bat populations, so sampling can be achieved from fresh fecal pellet collection. They are BSL-3, not -4, agents, so that experimental manipulation and infection is simpler. They have frequent spillover events, making it possible to validate predictive models of spillover by sampling people. They are diverse, with frequent recombination and different strains exhibiting differential host cell binding and spillover potential. Finally, we have identified SARSr- CoV strains in a single cave in Yunnan that harbor all of the epidemic SARS-CoV genes. This specific bat population harbors an ideal evolutionary soup that could produce new human strains by high frequency RNA recombination, and thus, it presents a perfect target for next generation, technology-forward intervention strategies.
TA1: Develop and validate integrated, multiscale models that quantify the likelihood a human-capable virus will emerge from an animal reservoir residing in a “hot spot” geographic region
The DEFUSE modeling and analytics team, led by Drs Daszak, Ross, Olival, EHA, will build ecological niche models of environmental and ecological correlates and traits of cave bat communities to predict species composition of bat caves across Southern China, South and SE Asia. We will then use a series of datasets we have built to produce host- virus risk models for the region. These include our unique database of bat host-viral relationships (7); biological inventory data on all bat caves in Southern China; and modeled species distribution data for all bats. We will parameterize the model with data from three cave sites in Yunnan, China (one with high-risk SARSr-CoVs, two other control/comparison sites), including: radio- and GPS-telemetry to identify home range and additional roost sites for each bat species; inventory of bat population density, distribution and segregation and their daily, weekly and seasonal changes; viral prevalence and individual viral load; shedding of low- and high-risk SARSr-CoV strains among bat species, age classes, genders; and telemetry and mark-recapture data to assess metapopulation structure and inter-cave connectivity. We will test and validate model predictions of a cave’s viral spillover potential with data from prior PREDICT sampling in 7 other Asian countries. At the end of Yr 1, we will produce a prototype app for the warfighter that identifies the likelihood of bats harboring dangerous viral pathogens in a region. The ‘high-risk bats near me’ app will be updated real-time with surveillance data (e.g. field-deployable iphone and android compatible echolocation data) from our project and others, to ground-truth and fine-tune its predictive capacity.
The Wuhan Institute of Virology team will test bat fecal, oral, blood and

urogenital samples for SARSr-CoVs. We will correlate viral load data from these samples with fresh fecal pellets from individuals and from tarps laid on cave floors. We will rapidly move to fecal pellet assays to reduce roost disturbance. SARSr-CoV spike proteins will be sequenced, analyzed phylogenetically for recombination events, and high-risk viruses (spike proteins close to SARS-CoV) characterized and isolated. The UNC team will reverse-engineer spike proteins to conduct binding assays to human ACE2 (the SARS-CoV receptor). They will culture SARS-like bat coronaviruses to distinguish high risk strains that can replicate in primary human cells and low risk strains that require exogenous enhancers. Viral spike glycoproteins that bind receptors will be inserted into SARS-CoV backbones, inoculated into human cells and humanized mice to assess capacity to cause SARS-like disease, and to be blocked by monoclonal therapies, the nucleoside analogue inhibitor GS-5734 (8) or vaccines against SARS-CoV (8-13).
The EHA modeling team will use these data to build models of risk of viral evolution and spillover. These genotype-to-phenotype machine-learning models will predict viral ability to infect host cells based on genetic traits and results of receptor binding and mouse infection assays. Using data on diversity of spike proteins, recombinant CoVs, and flow of genes within each bat cave via bat movement and migration, we will estimate evolutionary rates, rates of recombination, and capacity to generate novel strains capable of human infection. Finally, virus-host relationship and bat home range data will be used to estimate spillover potential - extending models well beyond our field sites. We will then validate model predictions of viral spillover risk by 1) conducting spike protein-based binding and cell culture experiments, and 2) identifying spillover strains in people near our bat cave sites. Our preliminary work on this shows ~3% seroprevalence to SARSr-CoVs, using a specific ELISA (14). We will design LIPS assays to the specific high- and low- zoonotic-risk SARSr-CoVs identified in this project as we have done previously (15). We will use banked and newly collected human sera from these populations to test for presence of antibodies to the high- and low-risk SARSr-CoVs identified by our modeling. We will then model optimal strategies to maximize treatment efficacy for TA2, using stochastic simulation modeling informed by field and experimental data to characterize viral circulation dynamics in bats. We will estimate frequency and population coverage required for our intervention approaches to suppress viral spillover. We will determine the seasons, locations within a cave, and delivery methods (spray, swab, or automated cave mouth or drone) that will be most effective. Finally we will determine the time period treatment will be effective for, until re-colonization or evolution leads to return of a high-risk SARSr-CoV.
TA2: Develop scalable approaches that target and suppress the animal virus in its reservoir(s) and/or vector(s), to reduce the likelihood of virus transmission into humans.
We will evaluate two approaches to defuse SARS-related CoV spillover potential: 1) Broadscale Immune Boosting: using the unique immune damping in bats that our group has discovered, we will apply immune modulators like bat interferon to live bats, to up- regulate their naïve immunity and then assess their ability to suppress viral replication and shedding; 2) Targeted Immune Priming: building on preliminary development of

polyvalent chimeric recombinant SARSr-CoV spike proteins, we will conduct application trials with live bats to assess suppression of replication and shedding of a broad range of dangerous SARS-related CoVs.
Both lines of work will begin in Yr 1 and run parallel. Prof. Linfa Wang (Duke- NUS) will lead the immune boosting work, building on his pioneering work on bat immunity (3) which shows that the long-term coexistence of bats and their viruses has led to equilibrium between viral replication and host immunity. This is likely due to down-regulation of their innate immune system as a fitness cost of flight (3). The weakened functionality of bat innate immunity factors like STING, a central DNA- interferon (IFN) sensing molecule, may allow bats to maintain an effective, but not over- response to viruses (4). A similar finding was observed for bat IFNA, which is less abundant but constitutively expressed without stimulation (5). Given high native SARSr- CoV load in bats, we aim to boost bat innate immunity through the IFN pathway, break the host-virus equilibrium to suppress bat SARSr-CoV replication and shedding.
We will trial the following, concurrently and competitively, for efficiency, cost and scalability: i) Universal bat interferon. Aerosol spraying or intranasal application of IFN or other small molecules reduces viral loads in humans, ferrets and mouse models (16, 17). Interferon has been used clinically when antiviral drugs are unavailable, e.g. against filoviruses (18). Replication of SARSr-CoV is sensitive to interferon treatments, as shown in our previous work (16); ii) Boosting bat IFN by blocking bat-specific IFN negative regulators. Uniquely, bat IFNA is naturally constitutively expressed but cannot be induced to a high level (5), indicating a negative regulatory factor in the bat interferon production pathway. We will use CRISPRi to identify the negative regulator and then screen for compounds targeting this gene; iii) Activating dampened bat- specific IFN production pathways which include DNA-STING-dependent and ssRNA-TLR7- dependent pathways. Our work showing that mutant bat STING restores antiviral functionality suggests these pathways are important in bat-viral coexistence (4). By identifying small molecules to directly activate downstream of STING, we will activate bat interferon and promote viral clearance. A similar strategy will be applied to ssRNA- TLR7-dependent pathways; iv) Activating functional bat IFN production pathways, e.g. polyIC to TLR3-IFN pathway or 5’ppp-dsRNA to RIG-I-IFN pathway. A similar strategy has been demonstrated in a mouse model for SARS-CoV, IAV and HBV (17, 19); v) Inoculating crude coronavirus fragments to upregulate innate immune responses to specific CoVs – a partial step towards the targeted immune priming work below.
Prof. Ralph Baric (UNC) will lead the immune priming work. He will develop recombinant chimeric spike-proteins (20) from our known SARSr-CoVs, and those we characterize during project DEFUSE. The structure of the SARS-CoV spike glycoprotein has been solved and the addition of two proline residues at positions V1060P and L1061P stabilize the prefusion state of the trimer, including key neutralizing epitopes in the receptor binding domain (21). In parallel, the spike trimers or the receptor binding domain can be incorporated into alphavirus vectored or nanoparticle vaccines for delivery, either as aerosols, in baits, or as large droplet delivery vehicles (11, 22-25). We will test these in controlled lab conditions, taking the best candidate forward for testing in the field. We have built recombinant spike glycoproteins harboring structurally

defined domains from SARS epidemic strains, pre-epidemic strains like SCH014 and zoonotic strains like HKU3. It is anticipated that recombinant S glycoprotein based vaccines harboring immunogenic blocks across the group 2B coronaviruses will induce broadscale immune responses that simultaneously reduce genetically heterogeneous virus burdens in bats, potentially reducing disease risk (and transmission risk to people) in these animals for longer periods (26, 27).
The immune dampening features are highly conserved in all bat species tested so far. Duke-NUS has established the only experimental breeding colony of cave bats (Eonycteris spelaea) in SE Asia. This genus is evolutionarily closely related to Rhinolophus spp. (the hosts of SARSr-CoVs), so we have confidence that results will be transferable. Our initial proof-of-concept tests will be in this experimental colony, extended to a small group of wild-caught Rhinolophus sinicus bats at Wuhan Institute of Zoology. We (Prof. Wang) have previous experience conducting SARS-CoV infection experiments with Rhinolophus sp. bats in the BSL-4 facility at CSIRO, AAHL (L.Wang, unpublished results).
Finally, work on a delivery method for our immune boosting and priming molecules will be overseen by Dr. Tonie Rocke at the USGS, National Wildlife Health Center who has previously developed animal vaccines through to licensure (28). Using locally acquired insectivorous bats (29, 30) we will assess delivery vehicles and methods including: 1) transdermally applied nanoparticles; 2) series of sticky edible gels that bats will groom from themselves and each other; 3) aerosolization via sprayers that could be used in cave settings; 4) automated sprays triggered by timers and movement detectors at critical cave entry points; 5) sprays delivered by remote controlled drone. We have already used simple gels to vaccinate bats against rabies in the lab (29), and hand delivered these containing biomarkers to vampire bats in Peru and Mexico to show they are readily consumed and transferred among bats. In our bat colony, we will trial delivery vehicles using the biomarker rhodamine B (which marks hair and whiskers upon consumption) to assess uptake. The most optimal approaches will then be tested on wild bats in our three cave sites in Yunnan Province with the most successful immunomodulators from TA2. Fieldwork will be conducted under the auspices of Dr. Yunzhi Zhang (Yunnan CDC, Consultant at EcoHealth Alliance). A small number of bats will be captured and assayed for viral load and immune function after treatment, but so as not to disturb the colony, most viral load work will be conducted on fresh fecal pellets collected daily on the cave floor. EHA has had unique access to these sites for around 10 years, under the guidance of Drs. Shi and Zhang. In year 1 of project DEFUSE, we will seek permission for experimental trials from the Provincial Forestry Department. We expect to be successful, as we have worked with the Forestry Department collaboratively for 10 years, with support of the Yunnan CDC, and we are releasing molecules that are not dangerous to people or wildlife. EHA has a proven track record of rapidly obtaining IACUC and DoD ACURO approval for bat research.
E. Capabilities:
The lead institution for Project DEFUSE is EcoHealth Alliance, New York, an international research organization focused on emerging zoonotic diseases. The PI, Dr. Peter Daszak, has 25+ years’ experience managing lab, field and modeling research projects on

emerging zoonoses. Dr. Daszak will commit 3 months annually to oversee and coordinate all project activities, and lead modeling and analytic work for TA1. Dr. Billy Karesh has 40+ years’ experience leading zoonotic and wildlife disease projects, and will commit 1 month annually to manage partnership activities and outreach. Dr. Jon Epstein, with 15 years’ experience working emerging bat zoonoses will coordinate animal trials. Dr. Kevin Olival and Dr. Noam Ross will manage and conduct the modeling and analytical approaches for this project. Support staff include field surveillance teams, modeling analysts, and consultants based in Yunnan Province, China, to oversee field trials. The EHA team has worked extensively with all other collaborators: Prof. Wang (15+ years); Dr. Shi (15+ years); Prof. Baric (5+ years) and Dr. Rocke (15+ years).
Subcontracts: #1 to Prof. Ralph Baric, UNC, to oversee reverse engineering of SARSr-CoVs, BSL-3 humanized mouse experimental infections, design and testing of immune priming treatments based on recombinant spike proteins. Assisted by senior personnel Dr. Tim Sheahan, Dr. Amy Sims, and support staff; #2 to Prof. Linfa Wang, Duke NUS, to oversee the immune boosting approach, captive bat experiments, and analyze immunological and virological responses to immune boosting treatments; #3 to Dr. Zhengli Shi, Wuhan Institute of Virology, to conduct PCR testing, viral discovery and isolation from bat samples collected in China, spike protein binding assays, and some humanized mouse work, as well as experimental trials on Rhinolophus bats. Her team will include Dr. Peng Zhou and support staff; #4 to Dr. Tonie Rocke, USGS National Wildlife Health Center, to refine delivery mechanisms for both immune boosting and immune priming treatments. With a research technician, Dr. Rocke will use a captive colony of bats at NWHC for initial trials, and oversee cave experiments in China.
F. Links to published papers, resume of two key performers
Dr. Peter Daszak is President and Chief Scientist of EcoHealth Alliance, a US-based research organization focused on emerging zoonotic diseases. His >300 scientific papers include the first global map of EID hotspots (31, 32), estimates of unknown viral diversity (33), predictive models of virus-host relationships (7), and evidence of the bat origin of SARS-CoV (34, 35) and other emerging viruses (36-39). He is Chair of the NASEM Forum on Microbial Threats, and is a member of the Executive Committee and the EHA institutional lead for the $130 million USAID-EPT-PREDICT. He serves on the NRC Advisory Committee to the USGCRP, the DHS CEEZAD External Advisory Board, the WHO R&D Blueprint Pathogen Prioritization expert group, and has advised the Director for Medical Preparedness Policy on the White House National Security Staff on global health issues. Dr Daszak won the 2000 CSIRO medal for collaborative research.
Prof. Ralph Baric is a UNC Lineberger Comprehensive Cancer Center member and Professor in the UNC-Chapel Hill Dept. of Epidemiology and Dept. of Microbiology & Immunology. His work focuses on coronaviruses as models to study the genetics of RNA virus transcription, replication, persistence, cross species transmission and pathogenesis. His group has developed a platform strategy to access the potential “pre- epidemic” risk associated with zoonotic virus cross species transmission potential and evaluation of countermeasure potential to control future outbreaks of disease (8-13).

Citations
1. P. L. Quan et al., Identification of a Severe Acute Respiratory Syndrome Coronavirus-Like Virus in a Leaf-Nosed Bat in Nigeria. Mbio 1, (2010).
2. J. F. Drexler et al., Genomic Characterization of Severe Acute Respiratory Syndrome-Related Coronavirus in European Bats and Classification of Coronaviruses Based on Partial RNA-Dependent RNA Polymerase Gene Sequences. Journal of Virology 84, 11336-11349 (2010).
3. G. Zhang et al., Comparative analysis of bat genomes provides insight into the evolution of flight and immunity. Science 339, 456-460 (2013).
4. J. Xie et al., Dampened STING-Dependent Interferon Activation in Bats. Cell host & microbe, (2018).
5. P. Zhou et al., Contraction of the type I IFN locus and unusual constitutive expression of IFN-αin bats. Proceedings of the National Academy of Sciences of the United States of America, 201518240-201518246 (2016).
6. M. Ahn, J. Cui, A. T. Irving, L.-F. Wang, Unique Loss of the PYHIN Gene Family in Bats Amongst Mammals: Implications for Inflammasome Sensing. Scientific Reports 6, (2016).
7. K. J. Olival et al., Host and viral traits predict zoonotic spillover from mammals. Nature 546, 646-650 (2017).
8. T. P. Sheahan et al., Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses. Sci Transl Med 9, (2017).
9. V. D. Menachery et al., MERS-CoV and H5N1 influenza virus antagonize antigen presentation by altering the epigenetic landscape. Proc Natl Acad Sci U S A 115, E1012-E1021 (2018).
10. S. J. Anthony et al., Further Evidence for Bats as the Evolutionary Source of Middle East Respiratory Syndrome Coronavirus. MBio 8, (2017).
11. A. S. Cockrell et al., A mouse model for MERS coronavirus-induced acute respiratory distress syndrome. Nat Microbiol 2, 16226 (2016).
12. V. D. Menachery et al., SARS-like WIV1-CoV poised for human emergence. Proc Natl Acad Sci U S A 113, 3048-3053 (2016).
13. V. D. Menachery et al., A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence. Nat Med 21, 1508-1513 (2015).
14. N. Wang et al., Serological evidence of bat SARS-related coronavirus infection in humans, China. Virologica Sinica In press, (2018).
15. P. Zhou et al., Fatal Swine Acute Diarrhea Syndrome caused by an HKU2-related Coronavirus of Bat Origin. Nature In press, (2018).
16. B. M. Farr, J. M. Gwaltney, Jr., K. F. Adams, F. G. Hayden, Intranasal interferon- alpha 2 for prevention of natural rhinovirus colds. Antimicrob Agents Chemother 26, 31-34 (1984).

17. D. Kugel et al., Intranasal Administration of Alpha Interferon Reduces Seasonal Influenza A Virus Morbidity in Ferrets. Journal of Virology 83, 3843-3851 (2009).
18. L. M. Smith et al., Interferon-beta Therapy Prolongs Survival in Rhesus Macaque Models of Ebola and Marburg Hemorrhagic Fever. Journal of Infectious Diseases
208, 310-318 (2013).
19. J. Zhao et al., Intranasal Treatment with Poly(I.C) Protects Aged Mice from Lethal
Respiratory Virus Infections. Journal of Virology 86, 11416-11424 (2012).
20. X. F. Deng et al., A Chimeric Virus-Mouse Model System for Evaluating the
Function and Inhibition of Papain-Like Proteases of Emerging Coronaviruses.
Journal of Virology 88, 11825-11833 (2014).
21. J. Pallesen et al., Immunogenicity and structures of a rationally designed
prefusion MERS-CoV spike antigen. Proc Natl Acad Sci U S A 114, E7348-E7357
(2017).
22. C. M. Coleman et al., Purified coronavirus spike protein nanoparticles induce
coronavirus neutralizing antibodies in mice. Vaccine 32, 3169-3174 (2014).
23. C. M. Coleman et al., MERS-CoV spike nanoparticles protect mice from MERS-
CoV infection. Vaccine 35, 1586-1589 (2017).
24. L. Du et al., A 219-mer CHO-expressing receptor-binding domain of SARS-CoV S
protein induces potent immune responses and protective immunity. Viral
immunology 23, 211-219 (2010).
25. T. Sheahan et al., Successful vaccination strategies that protect aged mice from
lethal challenge from influenza virus and heterologous severe acute respiratory
syndrome coronavirus. J Virol 85, 217-230 (2011).
26. S. Agnihothram et al., A mouse model for Betacoronavirus subgroup 2c using a
bat coronavirus strain HKU5 variant. MBio 5, e00047-00014 (2014).
27. M. M. Becker et al., Synthetic recombinant bat SARS-like coronavirus is
infectious in cultured cells and in mice. Proc Natl Acad Sci U S A 105, 19944-
19949 (2008).
28. T. E. Rocke et al., Sylvatic Plague Vaccine Partially Protects Prairie Dogs (Cynomys
spp.) in Field Trials. Ecohealth 14, 438-450 (2017).
29. B. Stading et al., Protection of bats (Eptesicus fuscus) against rabies following
topical or oronasal exposure to a recombinant raccoon poxvirus vaccine. Plos
Neglect. Trop. Dis. 11, (2017).
30. B. R. Stading et al., Infectivity of attenuated poxvirus vaccine vectors and
immunogenicity of a raccoonpox vectored rabies vaccine in the Brazilian Free-
tailed bat (Tadarida brasiliensis). Vaccine 34, 5352-5358 (2016).
31. K. E. Jones et al., Global trends in emerging infectious diseases. Nature 451, 990-
993 (2008).
32. T. Allen et al., Global hotspots and correlates of emerging zoonotic diseases. Nat
Commun 8, 1124 (2017).
33. S. J. Anthony et al., A Strategy to Estimate Unknown Viral Diversity in Mammals.
mBio 4, (2013).
34. X. Y. Ge et al., Isolation and characterization of a bat SARS-like coronavirus that
uses the ACE2 receptor. Nature 503, 535-538 (2013).

35. W. Li et al., Bats are natural reservoirs of SARS-like coronaviruses. Science 310, 676-679 (2005).
36. A. N. Alagaili et al., Middle East respiratory syndrome coronavirus infection in dromedary camels in saudi arabia. MBio 5, (2014).
37. N. Homaira et al., Nipah virus outbreak with person-to-person transmission in a district of Bangladesh, 2007. Epidemiology and Infection 138, 1630-1636 (2010).
38. M. S. Islam et al., Nipah Virus Transmission from Bats to Humans Associated with Drinking Traditional Liquor Made from Date Palm Sap, Bangladesh, 2011–2014. Emerging Infectious Disease journal 22, 664 (2016).
39. K. J. Olival et al., Ebola virus antibodies in fruit bats, bangladesh. Emerg Infect Dis 19, 270-273 (2013).

10/5/21, 3:05 PM Mail - Rocke, Tonie E - Outlook
Thanks Peter.
Dear Luke,
It will be good if you can circulate the final/submied version to us all as well.
Fingers crossed.
LF
Linfa (Lin-Fa) WANG, PhD FTSE
Professor & Director
Programme in Emerging Infecous Disease Duke-NUS Medical School,
8 College Road, Singapore 169857
Tel: +65 6516 8397
From: Peter Daszak [mailto:daszak@ecohealthalliance.org]
Sent: Tuesday, 13 February, 2018 9:21 PM
To: Noam Ross; Luke Hamel
Cc: Jonathon Musser; William B. Karesh; Ralph Baric (rbaric@email.unc.edu); Wang Linfa; Rocke, Tonie; 周鹏 (peng.zhou@wh.iov.cn); Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura; Anna Willoughby Subject: RE: Final draft DARPA abstract
Importance: High
Here’s the Abstract version 4 for your files. Luke – please do the following ASAP today:
1. Fix the references – remove the endnote links, and use the format: “(1,2)”, but making these blue, and an embedded hyperlink to the NCBI paper
2. Insert the corrected figure
3. Make sure we have the right format throughout – including grant number etc. at the top
4. Insert the meline and budget
5. Send it in!
Cheers, Peter
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/3
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10/5/21, 3:05 PM
Mail - Rocke, Tonie E - Outlook
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
From: Noam Ross [mailto:ross@ecohealthalliance.org]
Sent: Tuesday, February 13, 2018 6:22 AM
To: Peter Daszak; Luke Hamel
Cc: Jonathon Musser; William B. Karesh; Ralph Baric (rbaric@email.unc.edu); wang linfa; Rocke, Tonie; 鹏 (peng.zhou@wh.iov.cn); Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura; Anna Willoughby Subject: Re: Final draft DARPA abstract
Added just a couple of small edits on modeling phrasing, done on top of Tonie's edits.
On Tue, Feb 13, 2018 at 2:47 AM Rocke, Tonie <trocke@usgs.gov> wrote: Just minor edits. Looks good. -Tonie
On Mon, Feb 12, 2018 at 11:23 PM, Peter Daszak <daszak@ecohealthalliance.org> wrote: Luke, attached is the DARPA abstract.
First thing tomorrow, can you go through the references, and create links to the papers on NCBI. Just turn each reference citation: “(1)” for example, into a live link to the paper on NCBI. We only have 2 spare lines, so no room to turn each of these into a PMC numbered ref, as Ralph did for his, but please make sure the citation in parentheses is blue, so it’s clear it’s a live link on the final pdf.
Ralph, Lina, Peng, Zhengli, Tonie – please give this a quick read to make sure I’ve not said anything completely wrong. I’ve had to reduce the text a lot to hit the page limit, but I still think it’s a great proposal.
I’ll finish off the exec summary slide and <500 wd abstract now.
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 2/3


10/5/21, 3:05 PM Mail - Rocke, Tonie E - Outlook
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473 www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge research into the critical connections between human and wildlife health and delicate ecosystems. With this science we develop solutions that prevent pandemics and promote conservation.
--
Tonie E. Rocke
USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
608-270-2451
trocke@usgs.gov
--
Dr. Noam Ross
Senior Research Scientist
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
+1.212.380.4471 (direct) +1.212.380.4465 (fax) @noamross (twitter) www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics and promote conservation.
Important: This email is confidential and may be privileged. If you are not the intended recipient, please delete it and notify us immediately; you should not copy or use it for any purpose, nor disclose its contents to any other person. Thank you.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 3/3

10/5/21, 3:06 PM
Mail - Rocke, Tonie E - Outlook
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct) (b) (6) (mobile)
www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics
and promote conservation.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/4
(b) (6)
!lla rof snoitcurtsni APRAD htiw ecnailpmoc eht no kcehc lanif a od esaelp neht dna ,secnerefer
eht ,enilemit ,srebmun tegdub gnidulcni ,tcartsbA eht no kcehc ot sgniht rehto era ereht– BN
timbus
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,reteP iH
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>vog.sgsu@ekcort<EeinoT,ekcoR;>gro.ecnaillahtlaehoce@ybhguolliw<ybhguolliWannA
)nc.voi.hw@ihslz(ihSilgnehZ;>nc.voi.hw@uohz.gnep<)nc.voi.hw@uohz.gnep(鹏周;>gs.ude.sun-ekud@gnaw.afnil<
ssoRmaoN;>gro.ecnaillahtlaehoce@hserak<hseraK.BmailliW;>gro.ecnaillahtlaehoce@ressum<ressuMnohtanoJ:cC
afnilgnaw;>ude.cnu.liame@cirabr<)ude.cnu.liame@cirabr(ciraBhplaR;>gro.ecnaillahtlaehoce@ssor<
>gro.ecnaillahtlaehoce@kazsad<kazsaDreteP:oT
>gro.ecnaillahtlaehoce@lemah<MlAe91m88a10H2/3e1/k2euuLT
edils yrammuS cexE APRAD laniF :eR

10/5/21, 3:06 PM Mail - Rocke, Tonie E - Outlook
From: Peter Daszak
Sent: Tuesday, February 13, 2018 12:23 AM
To: Luke Hamel (hamel@ecohealthalliance.org); Jonathon Musser
Cc: William B. Karesh; Noam Ross; Ralph Baric (rbaric@email.unc.edu); wang linfa; 周鹏 (peng.zhou@wh.iov.cn); Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura (chmura@ecohealthalliance.org); 'Anna Willoughby (willoughby@ecohealthalliance.org)'; Rocke, Tonie Subject: Final draft DARPA abstract
Importance: High
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 2/4
.noitavresnoc etomorp dna scimednap
tneverp taht snoitulos poleved ew ecneics siht htiW .smetsysoce etaciled dna htlaeh efildliw
dna namuh neewteb snoitcennoc lacitirc eht otni hcraeser egde-gnittuc sdael ecnaillA htlaeHocE
gro.ecnaillahtlaehoce.www
3744-083-212 1+ .leT
10001 YN ,kroY weN
roolF ht71 – teertS ht43 tseW 064
ecnaillA htlaeHocE
tnediserP
kazsaD reteP
reteP
,sreehC

10/5/21, 3:06 PM Mail - Rocke, Tonie E - Outlook
www.ecohealthalliance.org
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 3/4
3744-083-212 1+ .leT
10001 YN ,kroY weN
roolF ht71 – teertS ht43 tseW 064
ecnaillA htlaeHocE
tnediserP
kazsaD reteP
reteP
,sreehC
.won tcartsba dw 005< dna edils yrammus cexe eht ffo hsinif llʼI
.lasoporp taerg a sʼti
kniht llits I tub ,timil egap eht tih ot tol a txet eht ecuder ot dah evʼI .gnorw yletelpmoc gnihtyna
dias ton evʼI erus ekam ot daer kciuq a siht evig esaelp – einoT ,ilgnehZ ,gneP ,aniL ,hplaR
.fdp lanif
eht no knil evil a sʼti raelc sʼti os ,eulb si sesehtnerap ni noitatic eht erus ekam esaelp tub ,sih rof
did hplaR sa ,fer derebmun CMP a otni eseht fo hcae nrut ot moor on os ,senil eraps 2 evah ylno
eW .IBCN no repap eht ot knil evil a otni ,elpmaxe rof ”)1(“ :noitatic ecnerefer hcae nrut tsuJ
.IBCN no srepap eht ot sknil etaerc dna ,secnerefer eht hguorht og uoy nac ,worromot gniht tsriF
.tcartsba APRAD eht si dehcatta ,ekuL

10/5/21, 3:06 PM Mail - Rocke, Tonie E - Outlook
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 4/4
.noitavresnoc etomorp dna scimednap
tneverp taht snoitulos poleved ew ecneics siht htiW .smetsysoce etaciled dna htlaeh efildliw
dna namuh neewteb snoitcennoc lacitirc eht otni hcraeser egde-gnittuc sdael ecnaillA htlaeHocE

10/5/21, 3:08 PM
Mail - Rocke, Tonie E - Outlook
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct) (b) (6) (mobile)
www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics
and promote conservation.
Here’s the Abstract version 4 for your files. Luke – please do the following ASAP today:
1. Fix the references – remove the endnote links, and use the format: “(1,2)”, but making these blue, and an embedded hyperlink to the NCBI paper
2. Insert the corrected figure
3. Make sure we have the right format throughout – including grant number etc. at the top
4. Insert the meline and budget
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/4
(b) (6)
:etorw >gro.ecnaillahtlaehoce@kazsad< kazsaD reteP ,MA 128 ta 8102 ,31 beF ,euT nO
,tseB
.reteP ,yawa thgir smeti eseht no trats ll'I .yletulosbA
ybhguolliWannA;>gro.ecnaillahtlaehoce@arumhc<arumhCieskelA;>gro.ecnaillahtlaehoce@erdna<
>gro.ecnaillahtlaehoce@ybhguolliw<
erdnAnosilA;>nc.voi.hw@ihslz<)nc.voi.hw@ihslz(ihSilgnehZ;>nc.voi.hw@uohz.gnep<
)nc.voi.hw@uohz.gnep(鹏周;>vog.sgsu@ekcort<EeinoT,ekcoR;>gs.ude.sun-ekud@gnaw.afnil<
afnilgnaw;>ude.cnu.liame@cirabr<)ude.cnu.liame@cirabr(ciraBhplaR;>gro.ecnaillahtlaehoce@hserak<
hseraK.BmailliW;>gro.ecnaillahtlaehoce@ressum<ressuMnohtanoJ;>gro.ecnaillahtlaehoce@ssor<ssoRmaoN:cC
>gro.ecnaillahtlaehoce@kazsad<kazsaDreteP:oT
>gro.ecnaillahtlaehoce@lemah<MlAe32m88a10H2/3e1/k2euuLT
tcartsba APRAD tfard laniF :eR

10/5/21, 3:08 PM Mail - Rocke, Tonie E - Outlook
5. Send it in!
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473 www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
From: Noam Ross [mailto:ross@ecohealthalliance.org]
Sent: Tuesday, February 13, 2018 6:22 AM
To: Peter Daszak; Luke Hamel
Cc: Jonathon Musser; William B. Karesh; Ralph Baric (rbaric@email.unc.edu); wang linfa; Rocke, Tonie; 周鹏 (peng.zhou@wh.iov.cn); Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura; Anna Willoughby Subject: Re: Final draft DARPA abstract
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 2/4
:etorw >vog.sgsu@ekcort< einoT ,ekcoR MA 742 ta 8102 ,31 beF ,euT nO
.stide s'einoT fo pot no enod ,gnisarhp gniledom no stide llams fo elpuoc a tsuj deddA

10/5/21, 3:08 PM Mail - Rocke, Tonie E - Outlook
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 3/4
10001 YN ,kroY weN
roolF ht71 – teertS ht43 tseW 064
ecnaillA htlaeHocE
tnediserP
kazsaD reteP
reteP
,sreehC
.won tcartsba dw 005< dna edils yrammus cexe eht ffo hsinif llʼI
.lasoporp taerg a sʼti
kniht llits I tub ,timil egap eht tih ot tol a txet eht ecuder ot dah evʼI .gnorw yletelpmoc gnihtyna
dias ton evʼI erus ekam ot daer kciuq a siht evig esaelp – einoT ,ilgnehZ ,gneP ,aniL ,hplaR
.fdp lanif eht no knil evil
a sʼti raelc sʼti os ,eulb si sesehtnerap ni noitatic eht erus ekam esaelp tub ,sih rof did hplaR sa
,fer derebmun CMP a otni eseht fo hcae nrut ot moor on os ,senil eraps 2 evah ylno eW .IBCN
no repap eht ot knil evil a otni ,elpmaxe rof ”)1(“ :noitatic ecnerefer hcae nrut tsuJ .IBCN
no srepap eht ot sknil etaerc dna ,secnerefer eht hguorht og uoy nac ,worromot gniht tsriF
.tcartsba APRAD eht si dehcatta ,ekuL
:etorw >gro.ecnaillahtlaehoce@kazsad< kazsaD reteP ,MP 3211 ta 8102 ,21 beF ,noM nO
einoT- .doog skooL .stide ronim tsuJ

10/5/21, 3:08 PM
Mail - Rocke, Tonie E - Outlook
www.ecohealthalliance.org
Dr. Noam Ross
Senior Research Scientist
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
+1.212.380.4471 (direct) +1.212.380.4465 (fax) @noamross (twitter) www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics and promote conservation.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 4/4
--
vog.sgsu@ekcort
1542-072-806
11735 IW ,nosidaM
.dR redeorhcS 6006
retneC htlaeH efildliW lanoitaN SGSU
ekcoR .E einoT
--
.noitavresnoc etomorp dna scimednap tneverp
taht snoitulos poleved ew ecneics siht htiW .smetsysoce etaciled dna htlaeh efildliw dna
namuh neewteb snoitcennoc lacitirc eht otni hcraeser egde-gnittuc sdael ecnaillA htlaeHocE
3744-083-212 1+ .leT

10/5/21, 3:08 PM
Mail - Rocke, Tonie E - Outlook
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct) (b) (6) (mobile)
www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics
and promote conservation.
Thanks Peter.
Dear Luke,
It will be good if you can circulate the final/submied version to us all as well.
Fingers crossed.
LF
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/5
(b) (6)
:etorw >gs.ude.sun-ekud@gnaw.afnil< afniL gnaW ,MA 958 ta 8102 ,31 beF ,euT nO
,tseB
.etelpmoc s'ti ecno noisrev lanif eht dnuora dnes ot erus eb ll'I
,afniL iH
>gro.ecnaillahtlaehoce@ybhguolliw<
ybhguolliWannA;>gro.ecnaillahtlaehoce@arumhc<arumhCieskelA;>gro.ecnaillahtlaehoce@erdna<
erdnAnosilA;>nc.voi.hw@ihslz<)nc.voi.hw@ihslz(ihSilgnehZ;>nc.voi.hw@uohz.gnep<
)nc.voi.hw@uohz.gnep(鹏周;>vog.sgsu@ekcort<EeinoT,ekcoR;>ude.cnu.liame@cirabr<)ude.cnu.liame@cirabr(
ressuMnohtanoJ;>gro.ecnaillahtlaehoce@ssor<ssoRmaoN;>gro.ecnaillahtlaehoce@kazsad<kazsaDreteP:cC
ciraBhplaR;>gro.ecnaillahtlaehoce@hserak<hseraK.BmailliW;>gro.ecnaillahtlaehoce@ressum<
>gs.ude.sun-ekud@gnaw.afnil<afniLgnaW:oT
>gro.ecnaillahtlaehoce@lemah<MlAe62m88a10H2/3e1/k2 euuLT
tcartsba APRAD tfard laniF :eR

10/5/21, 3:08 PM
Mail - Rocke, Tonie E - Outlook
Linfa (Lin-Fa) WANG, PhD FTSE
Professor & Director
Programme in Emerging Infecous Disease Duke-NUS Medical School,
8 College Road, Singapore 169857
Tel: +65 6516 8397
From: Peter Daszak [mailto:daszak@ecohealthalliance.org]
Sent: Tuesday, 13 February, 2018 9:21 PM
To: Noam Ross; Luke Hamel
Cc: Jonathon Musser; William B. Karesh; Ralph Baric (rbaric@email.unc.edu); Wang Linfa; Rocke, Tonie; 周鹏 (peng.zhou@wh.iov.cn); Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura; Anna Willoughby Subject: RE: Final draft DARPA abstract
Importance: High
Here’s the Abstract version 4 for your files. Luke – please do the following ASAP today:
1. Fix the references – remove the endnote links, and use the format: “(1,2)”, but making these blue, and an embedded hyperlink to the NCBI paper
2. Insert the corrected figure
3. Make sure we have the right format throughout – including grant number etc. at the top
4. Insert the meline and budget
5. Send it in!
Cheers,
Peter
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 2/5

10/5/21, 3:08 PM
Mail - Rocke, Tonie E - Outlook
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473 www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
From: Noam Ross [mailto:ross@ecohealthalliance.org]
Sent: Tuesday, February 13, 2018 6:22 AM
To: Peter Daszak; Luke Hamel
Cc: Jonathon Musser; William B. Karesh; Ralph Baric (rbaric@email.unc.edu); wang linfa; Rocke, Tonie; 鹏 (peng.zhou@wh.iov.cn); Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura; Anna Willoughby Subject: Re: Final draft DARPA abstract
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 3/5
no repap eht ot knil evil a otni ,elpmaxe rof ”)1(“ :noitatic ecnerefer hcae nrut tsuJ .IBCN
no srepap eht ot sknil etaerc dna ,secnerefer eht hguorht og uoy nac ,worromot gniht tsriF
.tcartsba APRAD eht si dehcatta ,ekuL
:etorw >gro.ecnaillahtlaehoce@kazsad< kazsaD reteP ,MP 3211 ta 8102 ,21 beF ,noM nO
einoT- .doog skooL .stide ronim tsuJ
:etorw >vog.sgsu@ekcort< einoT ,ekcoR MA 742 ta 8102 ,31 beF ,euT nO
.stide s'einoT fo pot no enod ,gnisarhp gniledom no stide llams fo elpuoc a tsuj deddA


10/5/21, 3:08 PM Mail - Rocke, Tonie E - Outlook
www.ecohealthalliance.org
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 4/5
.noitavresnoc etomorp dna scimednap tneverp
taht snoitulos poleved ew ecneics siht htiW .smetsysoce etaciled dna htlaeh efildliw dna
namuh neewteb snoitcennoc lacitirc eht otni hcraeser egde-gnittuc sdael ecnaillA htlaeHocE
3744-083-212 1+ .leT
10001 YN ,kroY weN
roolF ht71 – teertS ht43 tseW 064
ecnaillA htlaeHocE
tnediserP
kazsaD reteP
reteP
,sreehC
.won tcartsba dw 005< dna edils yrammus cexe eht ffo hsinif llʼI
kniht llits I tub ,timil egap eht tih ot tol a txet eht ecuder ot dah evʼI .gnorw yletelpmoc gnihtyna
.lasoporp taerg a sʼti
dias ton evʼI erus ekam ot daer kciuq a siht evig esaelp – einoT ,ilgnehZ ,gneP ,aniL ,hplaR
a sʼti raelc sʼti os ,eulb si sesehtnerap ni noitatic eht erus ekam esaelp tub ,sih rof did hplaR sa
.fdp lanif eht no knil evil
,fer derebmun CMP a otni eseht fo hcae nrut ot moor on os ,senil eraps 2 evah ylno eW .IBCN

10/5/21, 3:08 PM
Mail - Rocke, Tonie E - Outlook
Dr. Noam Ross
Senior Research Scientist
EcoHealth Alliance
460 West 34th Street – 17th Floor
New York, NY 10001
+1.212.380.4471 (direct) +1.212.380.4465 (fax) @noamross (twitter) www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics and promote conservation.
Important: This email is confidential and may be privileged. If you are not the intended recipient, please delete it and notify us immediately; you should not copy or use it for any purpose, nor disclose its contents to any other person. Thank you.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 5/5
--
vog.sgsu@ekcort
1542-072-806
11735 IW ,nosidaM
.dR redeorhcS 6006
retneC htlaeH efildliW lanoitaN SGSU
ekcoR .E einoT
--

10/5/21, 3:09 PM
Mail - Rocke, Tonie E - Outlook
Thanks
Linfa (Lin-Fa) WANG, PhD FTSE
Professor & Director
Programme in Emerging Infecous Disease Duke-NUS Medical School,
8 College Road, Singapore 169857
Tel: +65 6516 8397
From: Luke Hamel [mailto:hamel@ecohealthalliance.org]
Sent: Tuesday, 13 February, 2018 10:26 PM
To: Wang Linfa
Cc: Peter Daszak; Noam Ross; Jonathon Musser; William B. Karesh; Ralph Baric (rbaric@email.unc.edu); Rocke, Tonie; 周鹏 (peng.zhou@wh.iov.cn); Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura; Anna Willoughby Subject: Re: Final draft DARPA abstract
Hi Linfa,
I'll be sure to send around the final version once it's complete. Best,
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct)
(mobile)
www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics and promote conservation.
On Tue, Feb 13, 2018 at 8:59 AM, Wang Linfa <linfa.wang@duke-nus.edu.sg> wrote: Thanks Peter.
(b) (6)
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/4
>gro.ecnaillahtlaehoce@ybhguolliw<
ybhguolliWannA;>gro.ecnaillahtlaehoce@arumhc<arumhCieskelA;>gro.ecnaillahtlaehoce@erdna<
erdnAnosilA;>nc.voi.hw@ihslz<)nc.voi.hw@ihslz(ihSilgnehZ;>nc.voi.hw@uohz.gnep<
)nc.voi.hw@uohz.gnep(鹏周;>vog.sgsu@ekcort<EeinoT,ekcoR;>ude.cnu.liame@cirabr<)ude.cnu.liame@cirabr(
ciraBhplaR;>gro.ecnaillahtlaehoce@hserak<hseraK.BmailliW;>gro.ecnaillahtlaehoce@ressum<
ressuMnohtanoJ;>gro.ecnaillahtlaehoce@ssor<ssoRmaoN;>gro.ecnaillahtlaehoce@kazsad<kazsaDreteP:cC
>gro.ecnaillahtlaehoce@lemah<lemaHekuL:oT
>gs.ude.sun-ekud@gnaw.afnilM<Aa8f28ni81L02/g31n/2aeuWT
tcartsba APRAD tfard laniF :ER
(b) (6)

10/5/21, 3:09 PM Mail - Rocke, Tonie E - Outlook
Dear Luke,
It will be good if you can circulate the final/submied version to us all as well.
Fingers crossed.
LF
Linfa (Lin-Fa) WANG, PhD FTSE
Professor & Director
Programme in Emerging Infecous Disease Duke-NUS Medical School,
8 College Road, Singapore 169857
Tel: +65 6516 8397
From: Peter Daszak [mailto:daszak@ecohealthalliance.org]
Sent: Tuesday, 13 February, 2018 9:21 PM
To: Noam Ross; Luke Hamel
Cc: Jonathon Musser; William B. Karesh; Ralph Baric (rbaric@email.unc.edu); Wang Linfa; Rocke, Tonie; 周鹏 (peng.zhou@wh.iov.cn); Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura; Anna Willoughby Subject: RE: Final draft DARPA abstract
Importance: High
Here’s the Abstract version 4 for your files. Luke – please do the following ASAP today:
1. Fix the references – remove the endnote links, and use the format: “(1,2)”, but making these blue, and an embedded hyperlink to the NCBI paper
2. Insert the corrected figure
3. Make sure we have the right format throughout – including grant number etc. at the top
4. Insert the meline and budget
5. Send it in!
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
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10/5/21, 3:09 PM Mail - Rocke, Tonie E - Outlook
Tel. +1 212-380-4473 www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
From: Noam Ross [mailto:ross@ecohealthalliance.org]
Sent: Tuesday, February 13, 2018 6:22 AM
To: Peter Daszak; Luke Hamel
Cc: Jonathon Musser; William B. Karesh; Ralph Baric (rbaric@email.unc.edu); wang linfa; Rocke, Tonie; 鹏 (peng.zhou@wh.iov.cn); Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura; Anna Willoughby Subject: Re: Final draft DARPA abstract
Added just a couple of small edits on modeling phrasing, done on top of Tonie's edits.
On Tue, Feb 13, 2018 at 2:47 AM Rocke, Tonie <trocke@usgs.gov> wrote: Just minor edits. Looks good. -Tonie
On Mon, Feb 12, 2018 at 11:23 PM, Peter Daszak <daszak@ecohealthalliance.org> wrote: Luke, attached is the DARPA abstract.
First thing tomorrow, can you go through the references, and create links to the papers on NCBI. Just turn each reference citation: “(1)” for example, into a live link to the paper on NCBI. We only have 2 spare lines, so no room to turn each of these into a PMC numbered ref, as Ralph did for his, but please make sure the citation in parentheses is blue, so it’s clear it’s a live link on the final pdf.
Ralph, Lina, Peng, Zhengli, Tonie – please give this a quick read to make sure I’ve not said anything completely wrong. I’ve had to reduce the text a lot to hit the page limit, but I still think it’s a great proposal.
I’ll finish off the exec summary slide and <500 wd abstract now.
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473 www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge research into the critical connections between human and wildlife health and delicate ecosystems. With this science we develop solutions that prevent pandemics
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10/5/21, 3:09 PM
Mail - Rocke, Tonie E - Outlook
and promote conservation.
--
Tonie E. Rocke
USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
608-270-2451
trocke@usgs.gov
--
Dr. Noam Ross
Senior Research Scientist
EcoHealth Alliance
460 West 34th Street – 17th Floor
New York, NY 10001
+1.212.380.4471 (direct) +1.212.380.4465 (fax) @noamross (twitter) www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics and promote conservation.
Important: This email is confidential and may be privileged. If you are not the intended recipient, please delete it and notify us immediately; you should not copy or use it for any purpose, nor disclose its contents to any other person. Thank you.
Important: This email is confidential and may be privileged. If you are not the intended recipient, please delete it and notify us immediately; you should not copy or use it for any purpose, nor disclose its contents to any other person. Thank you.
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10/5/21, 3:09 PM
Mail - Rocke, Tonie E - Outlook
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
From: Luke Hamel [mailto:hamel@ecohealthalliance.org]
Sent: Tuesday, February 13, 2018 9:19 AM
To: Peter Daszak
Cc: Jonathon Musser; William B. Karesh; Noam Ross; Ralph Baric (rbaric@email.unc.edu); wang linfa; 周鹏 (peng.zhou@wh.iov.cn); Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura; Anna Willoughby; Rocke, Tonie
Subject: Re: Final DARPA Exec Summary slide Hi Peter,
Jonathon and I will be sure to do a final check on everything. Also, I'm not seeing the final summary slide. Could you please reattach it?
Best,
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edils yrammuS cexE APRAD laniF :ER

10/5/21, 3:09 PM
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(b) (6) (direct) (b) (6) (mobile) www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics and promote conservation.
On Tue, Feb 13, 2018 at 1:03 AM, Peter Daszak <daszak@ecohealthalliance.org> wrote: Hi Luke and Jonathon.
Here’s the edited, final summary slide.
Please insert the re-drawn figure, and the budget numbers from Aleksei tomorrow before you submit
NB –there are other things to check on the Abstract, including budget numbers, timeline, the references, and then please do a final check on the compliance with DARPA instructions for all!
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473 www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge research into the critical connections between human and wildlife health and delicate ecosystems. With this science we develop solutions that prevent pandemics and promote conservation.
From: Peter Daszak
Sent: Tuesday, February 13, 2018 12:23 AM
To: Luke Hamel (hamel@ecohealthalliance.org); Jonathon Musser
Cc: William B. Karesh; Noam Ross; Ralph Baric (rbaric@email.unc.edu); wang linfa; 周鹏 (peng.zhou@wh.iov.cn);
Mail - Rocke, Tonie E - Outlook
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10/5/21, 3:09 PM Mail - Rocke, Tonie E - Outlook
Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura (chmura@ecohealthalliance.org); 'Anna Willoughby (willoughby@ecohealthalliance.org)'; Rocke, Tonie
Subject: Final draft DARPA abstract
Importance: High
Luke, attached is the DARPA abstract.
First thing tomorrow, can you go through the references, and create links to the papers on NCBI. Just turn each reference citation: “(1)” for example, into a live link to the paper on NCBI. We only have 2 spare lines, so no room to turn each of these into a PMC numbered ref, as Ralph did for his, but please make sure the citation in parentheses is blue, so it’s clear it’s a live link on the final pdf.
Ralph, Lina, Peng, Zhengli, Tonie – please give this a quick read to make sure I’ve not said anything completely wrong. I’ve had to reduce the text a lot to hit the page limit, but I still think it’s a great proposal.
I’ll finish off the exec summary slide and <500 wd abstract now.
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473 www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge research into the critical connections between human and wildlife health and delicate ecosystems. With this science we develop solutions that prevent pandemics and promote conservation.
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Executive Summary: Proposal Title EcoHealth Alliance; Dr. Peter Daszak
CONCEPT
Provide graphic.
APPROACH
Describe new ideas.
IMPACT
Describe need and problem being addressed. Describe goal.
CONTEXT
Describe existing approaches; compare to state of the art.
Phase I
Phase II
Total
Proposed
$-
$-
$-
xHuman Use/ x Animal Use
HR001118S0017 PREEMPT
1

Executive Summary: DEFUSE EcoHealth Alliance; Dr. Peter Daszak
CONCEPT
Pathogen Prediction: APPROACH
• Host-Pathogen Ecology: Develop host-pathogen ecological niche models based on unique bat and viral data, to
estimate likelihood of spillover of SARS-related CoVs into human populations. Doing so will enhance predictive ability
of models beyond sampling sites in China to cover all Asia.
• Mobile Application: Create ‘Reservoirs Near Me’ mobile application, to assess background risk of disease spillover for
any site across Southeast Asia.
• Binding and Humanized mouse asssays: Utilize team’s unique collaboration between world-class modelers and
virologists with CoV expertise to conduct spike protein-based binding and humanized mice experiments.
• Use results to test machine-learning genotype-to-phenotype model predictions of viral spillover risk.
• Genotype-Phenotype Models: Develop models to estimate evolutionary rates, rates of recombination, and capacity to generate novel strains capable of human infection. Inputs to include: Diversity of bat spike proteins, prevalence of recombinant CoVs, and flow of genes within each bat cave via bat movement and migration.
• Validation with Human Sera: Analyze new and existing human serum samples to validate model outputs. Given frequent SARSr-CoV spillover events into local human populations, this can be done to a degree not possible in systems where spillover events are rare.
Intervention Development: (2 parallel approaches)
• (1) Broadscale Immune Boosting Strategy: Inoculate bats with immune modulators to upregulate innate immune response and downregulate viral replication, transiently reducing risk of viral shedding and spillover.
• (2) Targeted Immune Priming Strategy: Inoculate bats with novel chimeric polyvalent recombinant spike proteins to enhance innate immune response against specific, high-risk viruses.
• Viral Dynamics: Develop stochastic simulation models to estimate the frequency, efficacy, and population coverage required for intervention approaches to effectively suppress the viral population.
• Field Deployment and Testing: Uilize team’s expertise in wildlife vaccine delivery to assess and deploy effective molecule delivery methods, including: automated aerosolization technology that inoculates bats as they leave cave roost; remote controlled drone technology; transdermal nanoparticle application; and application of edible, adhesive gels that bats ingest when grooming fur of self and others.
CONTEXT
• No technology currently exists to reduce the risk of exposure to novel bat Coronaviruses.
• Our team has conducted pioneering research on modeling disease emergence, understanding Coronavirus virology, bat immunity, and wildlife vaccine delivery. Our previous work provides proof-of-concept for: (1) predictive ‘hotspot’ modeling; (2) upregulating bat immune response through the STING IFN pathway, (2) developing recombinant chimeric spike-proteins from SARS and SARSr-CoVs and (3) delivering immunological countermeasures to wildlife (including multiple bat species).
• The DEFUSE approach is broadly effective, scalable, economical and achievable in the allotted time frame. It also poses little environmental risk, and presents no threat to local livestock or human populations.
• While CRISPR-Cas9 gene drives are being considered for many disease research applications, the technique is unlikely to be effective in suppressing viral transmission in bat hosts. Bats are relatively long- lived, highly mobile, and have long inter-generational periods (2-5 years) with low progeny (1-2 pups). Furthermore, gene drive technology could have far-reaching, negative ecological consequences and its effectiveness cannot be evaluated within the defined Period of Performance.
IMPACT
• Recent and ongoing security concerns within South and SE Asia make the region a likely deployment site for US warfighters. Troops deployed to the region face increased disease risk from SARS and related bat viruses, as bats shed these pathogens through urine and feces while foraging over large areas at night.
• Our work in Yunnan Province, China has shown that (1) SARSr-CoVs are capable of producing SARS-like illness in humanized mice that are not affected by monoclonal or vaccine treatment, and (2) that spillover into local human populations is frequent. With no available vaccine or alternative method to counter these SARS-related viruses, US defense forces and national security are placed at risk.
• Our goal is to “DEFUSE” the potential for emergence of novel bat-origin high zoonotic risk SARSr-CoVs in Southeast Asia. In doing so, we will not only safeguard the US warfighter, but also reduce SARSr-CoV exposure for local communities and their livestock, improving food security Global Health Security.
• If successful, our strategy can be adapted to hosts of other bat-origin CoVs (MERS-CoV in the Middle East and other SARS-related pre-pandemic zoonotic strains in Africa, e.g. Nigeria), and potentially other zoonotic bat-origin viruses (Hendra, Nipah, Ebola viruses).
Phase I
Phase II
Total
Proposed
$-
$-
$-
xHuman Use/ x Animal Use
HR001118S0017 PREEMPT 2

10/5/21, 3:10 PM Mail - Rocke, Tonie E - Outlook
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10/5/21, 3:10 PM Mail - Rocke, Tonie E - Outlook
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10/5/21, 3:10 PM Mail - Rocke, Tonie E - Outlook
Cheers, Peter
Peter Daszak
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10/5/21, 3:10 PM
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
From: Luke Hamel [mailto:hamel@ecohealthalliance.org]
Sent: Tuesday, February 13, 2018 9:19 AM
To: Peter Daszak
Cc: Jonathon Musser; William B. Karesh; Noam Ross; Ralph Baric (rbaric@email.unc.edu); wang linfa; 周鹏 (peng.zhou@wh.iov.cn); Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura; Anna Willoughby; Rocke, Tonie
Subject: Re: Final DARPA Exec Summary slide
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct)
Mail - Rocke, Tonie E - Outlook
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10/5/21, 3:10 PM Mail - Rocke, Tonie E - Outlook
From: Peter Daszak
Sent: Tuesday, February 13, 2018 12:23 AM
To: Luke Hamel (hamel@ecohealthalliance.org); Jonathon Musser
Cc: William B. Karesh; Noam Ross; Ralph Baric (rbaric@email.unc.edu); wang linfa; 周鹏 (peng.zhou@wh.iov.cn); Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura (chmura@ecohealthalliance.org); 'Anna Willoughby (willoughby@ecohealthalliance.org)'; Rocke, Tonie Subject: Final draft DARPA abstract
Importance: High
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gro.ecnaillahtlaehoce.www
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dna namuh neewteb snoitcennoc lacitirc eht otni hcraeser egde-gnittuc sdael ecnaillA htlaeHocE
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10/5/21, 3:11 PM Mail - Rocke, Tonie E - Outlook
Dear All,
Apologies – forgot to send you the final versions of the abstract and the execuve summary slide for your records...
Re. the total budget – please don’t view that as final – they’re planning to give out $40 million over the 3.5 years total to anything from 1 to 6 projects. Given that there were probably 3 or 4 other viable teams in the room at the proposer’s conference, I think that $14.8 million is probably the maximum they would give any project, and it’s more likely that they’ll fund three or four at 8 million and two or three smaller projects. They’ll hopefully give some clearer guidance as we move towards a full proposal.
The due date for proposals is March 27th, 4pm EST (NY me). That means we need to start honing in on our plans for the full proposal. I’m meeng with our team here on Thursday to start working on the next steps, and it would be good to have calls with all of you this week to start refining the ideas and building out the proposal. Luke will follow up with mes that work for phone calls.
Thanks again for your openness and clever ideas, and I look forward to working with you all to get the best possible full proposal submied on me!!!
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
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...spets txen dna ,tcartsba APRAD tfard laniF :ER

10/5/21, 3:11 PM Mail - Rocke, Tonie E - Outlook
From: Peter Daszak
Sent: Tuesday, February 13, 2018 8:15 AM
To: 'Wang Linfa'; Luke Hamel; Jonathon Musser
Cc: William B. Karesh; Noam Ross; Ralph Baric (rbaric@email.unc.edu); 周鹏 (peng.zhou@wh.iov.cn); Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura; Anna Willoughby; Rocke, Tonie
Subject: RE: Final draft DARPA abstract
Good point Linfa – changed to ‘treatment’ or ‘applicaon’ throughout.
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
From: Wang Linfa [mailto:linfa.wang@duke-nus.edu.sg]
Sent: Tuesday, February 13, 2018 1:10 AM
To: Peter Daszak; Luke Hamel; Jonathon Musser
Cc: William B. Karesh; Noam Ross; Ralph Baric (rbaric@email.unc.edu); 周鹏 (peng.zhou@wh.iov.cn); Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura; Anna Willoughby; Rocke, Tonie
Subject: RE: Final draft DARPA abstract
Hi Peer,
It actually reads well now and I hope the selecon commiee will “buy in” our brave ideas!
Nothing to add a or change, other than an English queson: we used “dampened immunity of bats” in most places, but you use the expression of “damping innate immunity pathways” on page 1. Is there a difference between “damping” and “dampening”?
Thanks
LF
Linfa (Lin-Fa) Wang, PhD FTSE
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10/5/21, 3:11 PM
Professor & Director
Programme in Emerging Infecous Diseases Duke-NUS Medical School
8 College Road, Singapore 169875
Tel: +65 65168397
From: Peter Daszak [mailto:daszak@ecohealthalliance.org]
Sent: Tuesday, 13 February 2018 1:23 PM
To: Luke Hamel <hamel@ecohealthalliance.org>; Jonathon Musser <musser@ecohealthalliance.org>
Cc: William B. Karesh <karesh@ecohealthalliance.org>; Noam Ross <ross@ecohealthalliance.org>; Ralph Baric (rbaric@email.unc.edu) <rbaric@email.unc.edu>; Wang Linfa <linfa.wang@duke-nus.edu.sg>; 周鹏 (peng.zhou@wh.iov.cn) <peng.zhou@wh.iov.cn>; Zhengli Shi (zlshi@wh.iov.cn) <zlshi@wh.iov.cn>; Alison Andre <andre@ecohealthalliance.org>; Aleksei Chmura <chmura@ecohealthalliance.org>; Anna Willoughby <willoughby@ecohealthalliance.org>; Rocke, Tonie <trocke@usgs.gov>
Subject: Final dra DARPA abstract
Importance: High
Luke, aached is the DARPA abstract.
First thing tomorrow, can you go through the references, and create links to the papers on NCBI. Just turn each reference citaon: “(1)” for example, into a live link to the paper on NCBI. We only have 2 spare lines, so no room to turn each of these into a PMC numbered ref, as Ralph did for his, but please make sure the citaon in parentheses is blue, so it’s clear it’s a live link on the final pdf.
Ralph, Lina, Peng, Zhengli, Tonie – please give this a quick read to make sure I’ve not said anything completely wrong. I’ve had to reduce the text a lot to hit the page limit, but I sll think it’s a great proposal.
I’ll finish off the exec summary slide and <500 wd abstract now.
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
Mail - Rocke, Tonie E - Outlook
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DARPA – PREEMPT – HR001118S0017- PROJECT DEFUSE
ABSTRACT - COVER PAGE
TITLE: PROJECT DEFUSE
FUNDER AND BAA: DARPA – PREEMPT (HR001118S0017)
TECHNICAL & LEAD POINT OF CONTACT:
ADMINISTRATIVE POINT OF CONTACT:
DR. PETER DASZAK
ECOHEALTH ALLIANCE
460 WEST 34TH STREET
17TH FLOOR
NEW YORK, NY 10001
Phone: +1.212.380.4474
Fax: +1.212.380.4465
(e) daszak@ecohealthalliance.org
MR. LUKE HAMEL
ECOHEALTH ALLIANCE
460 WEST 34TH STREET
17TH FLOOR
NEW YORK, NY 10001
Phone: +(b) (6)
Fax: +1.212.380.4465
(e) hamel@ecohealthalliance.org
LEAD ORGANIZATION: EcoHealth Alliance PRIMARY SUBCONTRACTORS:
Duke-National University Singapore Medical School
National Wildlife Health Center, United States Geological Survey University of North Carolina, School of Medicine
Wuhan Institute of Virology
ESTIMATED COST: $14,799,998.00 PROJECT DURATION: 3.5 Years
1

DARPA – PREEMPT – HR001118S0017- PROJECT DEFUSE
C. Goals and Impact:
1. What is the proposed work attempting to accomplish or do?
We will defuse the potential for emergence of novel bat-origin high-zoonotic risk SARS- related coronaviruses. We envisage a scenario whereby the US warfighter is deployed to a security hotspot in SE Asia. As planners choose sites for the mission, they will use an app we will design based on machine-learning models of the ecological and evolutionary potential of bat viruses to spillover. This will allow rapid assessment of the background risk of a site harboring dangerous zoonotic viruses. If there is no alternative site, a tactical forward team will deploy automated delivery technology we will develop in caves that harbor bats carrying these viruses. These devices will release broadscale immune boosting molecules and chimeric polyvalent spike protein targeted immune priming treatments to upregulate the naturally damped innate immune response of bats, and lower viral shedding from bats at the site for a few weeks or months, allowing
our warfighters to execute the operation at lowered risk for spillover.
2. How is it done today? And what are the limitations?
Other than PPE, there is no available current technology to reduce the risk of exposure to novel coronaviruses from bats. Models of bat host capacity to harbor viruses, of ecological and environmental drivers of their emergence, and of the evolutionary potential of different strains to spillover are rudimentary. No vaccines or therapeutics exist for SARSr-CoVs, and exposure mitigation strategies are non-existent. SARSr-CoVs are endemic in Asian, African (1), and European bats (2) that roost in caves but forage widely at night, shedding virus in their feces and urine. The limitations of this lack of capacity are significant – we have shown evidence of recent spillover of SARSr-CoVs into people in China and have isolated strains capable of producing SARS-like illness in humanized mice that don’t respond to antibody treatment or vaccination. These viruses are a clear-and-present danger to our military and to global health security.
3. What is innovative in your approach and how does it compare to current practice and state- of-the-art (SOA)?
Our group leads the world in predictive models of viral emergence. We will build on our machine-learning models of spillover hotspots, host-pathogen ecological niches and genotype- phenotype mapping by incorporating unique datasets to validate and refine hotspot risk maps of viral emergence in SE Asia and beyond. Our group has shown that bats coexist with lethal viruses by damping innate immunity pathways, likely as an evolutionary adaptation to flight. We will use this insight to design strategies, like small molecule Rig like receptor (RLR) or Toll like receptor (TLR) agonists, to upregulate bat immunity in their cave roosts, down-regulate viral replication, and reduce the risk of viral shedding and spillover (broadscale immune boosting strategy). We will complement this by treating bats with novel chimeric polyvalent recombinant spike proteins to enhance their immune response against specific, high-risk viruses (targeted immune priming strategy), especially when their immune response is boosted as above. We will design novel methods to deliver these applications remotely to reduce exposure risk during decontamination.
2

DARPA – PREEMPT – HR001118S0017- PROJECT DEFUSE
4. What are the key technical challenges in your approach and how do you plan to
overcome these?
Modeling: Previous models have suffered from a lack of data to validate them. We have access to unique datasets that will allow us to validate our approach, including biodiversity surveys of bat caves across S. China, 10+ years of bat viral testing data in China, and 10 other countries (from NIH-NIAID and USAID EPT PREDICT work). Uniquely, we will validate our models of viral evolution/spillover risk using human serology (based on LIPS assays) in local populations that have high (~3%) seroprevalence to bat SARSr-CoVs. Identifying Immune boosting and priming treatments: Some of our approaches are novel and challenging (e.g. using CRISPRi to find the negative regulator for bat interferon production), and others are unproven in bats (e.g. Poly IC). We will begin all immune boosting and priming experiments at the beginning of the project, running them simultaneously and competitively, so that we field trial only the most efficient, cost-effective and scalable approaches.
5. Who will care and what will the impact be if you are successful?
This will have direct relevance to the warfighter. Potential deployment to regions where SARSr- CoVs exist is high – countries include security hotspots in Asia (e.g. Myanmar, Bangladesh, Pakistan, Korea, Vietnam), Africa and Eastern Europe. The ability to decontaminate and defuse these viruses may prevent potentially devastating illness. These technologies could be adapted to hosts of other bat-origin CoVs (e.g. MERS-CoV, SADS-CoV) and potentially other zoonotic bat- origin viruses (Hendra, Nipah, EBOV), with benefits to livestock production, food security and global public health.
6. How much will it cost and how long will it take?
Proposed
D. Technical Plan:
Overview
Phase I (24 months)
$8,414,104
Phase II (18 months) $6,385,894
Total $14,799,998
The SARSr-CoV-bat system, and immune modulation focus: Our group’s 15 yrs work on the SARSr-CoV – Rhinolophus bat system in China has identified and isolated SARSr-CoVs with remarkable sequence identity in the spike protein to SARS-CoV (e.g. SCH014 & WIV-1). We have shown they bind and replicate efficiently in primary human lung airway cells and that chimeras with SARSr-CoV spike proteins in a SARS-CoV backbone cause SARS-like illness in humanized mice, with clinical signs that are not reduced by SARS monoclonal therapy or vaccination. We have identified a single cave site in Yunnan Province where bat SARSr-CoVs contain all the genetic components of epidemic SARS-CoV (7,8,9). We have now shown that people living up to 6 kilometers from this cave have SARSr-CoV antibodies (3% seroprevalence in 200+ cohort), suggesting active spillover, and marking these viruses as a clear-and-present danger of a new SARS-like pandemic. Our work on bat immunology suggests that bats’ unique flying ability has led to downregulated innate immune genes, and their ability to coexist with viruses such as
3

DARPA – PREEMPT – HR001118S0017- PROJECT DEFUSE
SARSr-CoVs, henipa- and filoviruses that are lethal in many other mammals (3). We have identified bat-specific constitutively expressed bat interferon, a dampened STING-interferon production pathway (4, 5), and have identified a series of other innate immunity factors that are dampened in bats (6).
Our bat-CoV system has significant advantages for experimentation and intervention. Firstly, these viruses are fecal-orally transmitted within bat populations, so sampling can be achieved from fresh fecal pellet collection. They are BSL-3, not -4, agents, so that experimental manipulation and infection is simpler. They have frequent spillover events, making it possible to validate predictive models of spillover by sampling people. They are diverse, with frequent recombination and different strains exhibiting differential host cell binding and spillover potential. Finally, we have identified SARSr-CoV strains in a single cave in Yunnan that harbor all of the epidemic SARS-CoV genes. This specific bat population harbors an ideal evolutionary soup that could produce new human strains by high frequency RNA recombination, and thus, it presents a perfect target for next generation, technology-forward intervention strategies.
TA1: Develop and validate integrated, multiscale models that quantify the likelihood a human-capable virus will emerge from an animal reservoir residing in a “hot spot” geographic region
The DEFUSE modeling and analytics team, led by Drs. Daszak, Ross, Olival, EHA, will build ecological niche models of environmental and ecological correlates and traits of cave bat communities to predict species composition of bat caves across Southern China, South and SE Asia. We will then use a series of datasets we have built to produce host-virus risk models for the region. These include our unique database of bat host-viral relationships (7); biological inventory data on all bat caves in Southern China; and modeled species distribution data for all bats. We will parameterize the model with data from three cave sites in Yunnan, China (one with high-risk SARSr-CoVs, two other control/comparison sites), including: radio- and GPS- telemetry to identify home range and additional roost sites for each bat species; inventory of bat population density, distribution and segregation and their daily, weekly and seasonal changes; viral prevalence and individual viral load; shedding of low- and high-risk SARSr-CoV strains among bat species, age classes, genders; and telemetry and mark-recapture data to assess metapopulation structure and inter-cave connectivity. We will test and validate model predictions of a cave’s viral spillover potential with data from prior PREDICT sampling in 7 other Asian countries. At the end of Yr 1, we will produce a prototype app for the warfighter that identifies the likelihood of bats harboring dangerous viral pathogens in a region. The ‘high-risk bats near me’ app will be updated real-time with surveillance data (e.g. field-deployable iPhone and android compatible echolocation data) from our project and others, to ground-truth and fine-tune its predictive capacity.
The Wuhan Institute of Virology team will test bat fecal, oral, blood and urogenital samples for SARSr-CoVs. We will correlate viral load data from these samples with fresh fecal pellets from individuals and from tarps laid on cave floors. We will rapidly move to fecal pellet assays to reduce roost disturbance. SARSr-CoV spike proteins will be sequenced, analyzed phylogenetically for recombination events, and high-risk viruses (spike proteins close to SARS- CoV) characterized and isolated. The UNC team will reverse-engineer spike proteins to conduct
4

DARPA – PREEMPT – HR001118S0017- PROJECT DEFUSE
binding assays to human ACE2 (the SARS-CoV receptor). They will culture SARS-like bat coronaviruses to distinguish high-risk strains that can replicate in primary human cells and low risk strains that require exogenous enhancers. Viral spike glycoproteins that bind receptors will be inserted into SARS-CoV backbones, inoculated into human cells and humanized mice to assess capacity to cause SARS-like disease, and to be blocked by monoclonal therapies, the nucleoside analogue inhibitor GS-5734(8) or vaccines against SARS-CoV (8,9,10,11,12,13).
The EHA modeling team will use these data to build models of risk of viral evolution and spillover. These genotype-to-phenotype machine-learning models will predict viral ability to infect host cells based on genetic traits and results of receptor binding and mouse infection assays. Using data on diversity of spike proteins, recombinant CoVs, and flow of genes within each bat cave via bat movement and migration, we will estimate evolutionary rates, rates of recombination, and capacity to generate novel strains capable of human infection. Finally, virus-host relationship and bat home range data will be used to estimate spillover potential - extending models well beyond our field sites. We will then validate model predictions of viral spillover risk by 1) conducting spike protein-based binding and cell culture experiments, and 2) identifying spillover strains in people near our bat cave sites. Our preliminary work on this shows ~3% seroprevalence to SARSr-CoVs, using a specific ELISA (14). We will design LIPS assays to the specific high- and low- zoonotic-risk SARSr-CoVs identified in this project as we have done previously (15). We will use banked and newly collected human sera from these populations to test for presence of antibodies to the high- and low-risk SARSr-CoVs identified by our modeling. We will then model optimal strategies to maximize treatment efficacy for TA2, using stochastic simulation modeling informed by field and experimental data to characterize viral circulation dynamics in bats. We will estimate frequency and population coverage required for our intervention approaches to suppress viral spillover. We will determine the seasons, locations within a cave, and delivery methods (spray, swab, or automated cave mouth or drone) that will be most effective. Finally we will determine the time period treatment will be effective for, until re-colonization or evolution leads to return of a high-risk SARSr-CoV.
TA2: Develop scalable approaches that target and suppress the animal virus in its reservoir(s) and/or vector(s), to reduce the likelihood of virus transmission into humans.
We will evaluate two approaches to defuse SARS-related CoV spillover potential: 1) Broadscale Immune Boosting: using the unique immune damping in bats that our group has discovered, we will apply immune modulators like bat interferon to live bats, to up-regulate their naïve immunity and then assess their ability to suppress viral replication and shedding; 2) Targeted Immune Priming: building on preliminary development of polyvalent chimeric recombinant SARSr-CoV spike proteins, we will conduct application trials with live bats to assess suppression of replication and shedding of a broad range of dangerous SARS-related CoVs.
Both lines of work will begin in Yr 1 and run parallel. Prof. Linfa Wang (Duke-NUS) will lead the immune boosting work, building on his pioneering work on bat immunity (3) which shows that the long-term coexistence of bats and their viruses has led to equilibrium between viral replication and host immunity. This is likely due to down-regulation of their innate immune system as a fitness cost of flight (3). The weakened functionality of bat innate immunity factors like STING, a central DNA-interferon (IFN) sensing molecule, may allow bats to maintain an
5

DARPA – PREEMPT – HR001118S0017- PROJECT DEFUSE
effective, but not over-response to viruses (4). A similar finding was observed for bat IFNA, which is less abundant but constitutively expressed without stimulation (5). Given high native SARSr-CoV load in bats, we aim to boost bat innate immunity through the IFN pathway, break the host-virus equilibrium to suppress bat SARSr-CoV replication and shedding.
We will trial the following, concurrently and competitively, for efficiency, cost and scalability: i) Universal bat interferon. Aerosol spraying or intranasal application of IFN or other small molecules reduces viral loads in humans, ferrets and mouse models (16, 17). Interferon has been used clinically when antiviral drugs are unavailable, e.g. against filoviruses (18). Replication of SARSr-CoV is sensitive to interferon treatments, as shown in our previous work (16); ii) Boosting bat IFN by blocking bat-specific IFN negative regulators. Uniquely, bat IFNA is naturally constitutively expressed but cannot be induced to a high level (5), indicating a negative regulatory factor in the bat interferon production pathway. We will use CRISPRi to identify the negative regulator and then screen for compounds targeting this gene; iii) Activating dampened bat-specific IFN production pathways which include DNA-STING- dependent and ssRNA-TLR7-dependent pathways. Our work showing that mutant bat STING restores antiviral functionality suggests these pathways are important in bat-viral coexistence (4). By identifying small molecules to directly activate downstream of STING, we will activate bat interferon and promote viral clearance. A similar strategy will be applied to ssRNA-TLR7- dependent pathways; iv) Activating functional bat IFN production pathways, e.g. polyIC to TLR3- IFN pathway or 5’ppp-dsRNA to RIG-I-IFN pathway. A similar strategy has been demonstrated in a mouse model for SARS-CoV, IAV and HBV (17, 19); v) Inoculating crude coronavirus fragments to upregulate innate immune responses to specific CoVs – a partial step towards the targeted immune priming work below.
Prof. Ralph Baric (UNC) will lead the immune priming work. He will develop recombinant chimeric spike-proteins (20) from our known SARSr-CoVs, and those we characterize during project DEFUSE. The structure of the SARS-CoV spike glycoprotein has been solved and the addition of two proline residues at positions V1060P and L1061P stabilize the prefusion state of the trimer, including key neutralizing epitopes in the receptor binding domain (21). In parallel, the spike trimers or the receptor binding domain can be incorporated into alphavirus vectored or nanoparticle vaccines for delivery, either as aerosols, in baits, or as large droplet delivery vehicles (11, 22,23,24,25). We will test these in controlled lab conditions, taking the best candidate forward for testing in the field. We have built recombinant spike glycoproteins harboring structurally defined domains from SARS epidemic strains, pre-epidemic strains like SCH014 and zoonotic strains like HKU3. It is anticipated that recombinant S glycoprotein based vaccines harboring immunogenic blocks across the group 2B coronaviruses will induce broad scale immune responses that simultaneously reduce genetically heterogeneous virus burdens in bats, potentially reducing disease risk (and transmission risk to people) in these animals for longer periods (26, 27).
The immune dampening features are highly conserved in all bat species tested so far. Duke-NUS has established the only experimental breeding colony of cave bats (Eonycteris spelaea) in SE Asia. This genus is evolutionarily related to Rhinolophus spp. (the hosts of SARSr- CoVs), so we have confidence that results will be transferable. Our initial proof-of-concept tests will be in this experimental colony, extended to a small group of wild-caught Rhinolophus
6

DARPA – PREEMPT – HR001118S0017- PROJECT DEFUSE
sinicus bats at Wuhan Institute of Zoology. We (Prof. Wang) have previous experience conducting SARS-CoV infection experiments with Rhinolophus sp. bats in the BSL-4 facility at CSIRO, AAHL (L.Wang, unpublished results).
Finally, work on a delivery method for our immune boosting and priming molecules will be overseen by Dr. Tonie Rocke at the USGS, National Wildlife Health Center who has previously developed animal vaccines through to licensure (28). Using locally acquired insectivorous bats (29, 30) we will assess delivery vehicles and methods including: 1) transdermally applied nanoparticles; 2) series of sticky edible gels that bats will groom from themselves and each other; 3) aerosolization via sprayers that could be used in cave settings; 4) automated sprays triggered by timers and movement detectors at critical cave entry points, and 5) sprays delivered by remote controlled drone. We have already used simple gels to vaccinate bats against rabies in the lab (29), and hand delivered these containing biomarkers to vampire bats in Peru and Mexico to show they are readily consumed and transferred among bats. In our bat colony, we will trial delivery vehicles using the biomarker rhodamine B (which marks hair and whiskers upon consumption) to assess uptake. The most optimal approaches will then be tested on wild bats in our three cave sites in Yunnan Province with the most successful immunomodulators from TA2. Fieldwork will be conducted under the auspices of Dr. Yunzhi Zhang (Yunnan CDC, Consultant at EcoHealth Alliance). A small number of bats will be captured and assayed for viral load and immune function after treatment, but so as not to disturb the colony, most viral load work will be conducted on fresh fecal pellets collected daily on the cave floor. EHA has had unique access to these sites for around 10 years, under the guidance of Drs. Shi and Zhang. In year 1 of project DEFUSE, we will seek permission for experimental trials from the Provincial Forestry Department. We expect to be successful, as we have worked with the Forestry Department collaboratively for 10 years, with support of the Yunnan CDC, and we are releasing molecules that are not dangerous to people or wildlife. EHA has a proven track record of rapidly obtaining IACUC and DoD ACURO approval for bat research.
E. Capabilities:
The lead institution for Project DEFUSE is EcoHealth Alliance, New York, an international research organization focused on emerging zoonotic diseases. The PI, Dr. Peter Daszak, has 25+ years’ experience managing lab, field and modeling research projects on emerging zoonoses. Dr. Daszak will commit 3 months annually to oversee and coordinate all project activities, and lead modeling and analytic work for TA1. Dr. Billy Karesh has 40+ years’ experience leading zoonotic and wildlife disease projects, and will commit 1 month annually to manage partnership activities and outreach. Dr. Jon Epstein, with 15 years’ experience working emerging bat zoonoses will coordinate animal trials. Dr. Kevin Olival and Dr. Noam Ross will manage and conduct the modeling and analytical approaches for this project. Support staff include field surveillance teams, modeling analysts, and consultants based in Yunnan Province, China, to oversee field trials. The EHA team has worked extensively with all other collaborators: Prof. Wang (15+ years); Dr. Shi (15+ years); Prof. Baric (5+ years) and Dr. Rocke (15+ years).
Subcontracts: #1 to Prof. Ralph Baric, UNC, to oversee reverse engineering of SARSr- CoVs, BSL-3 humanized mouse experimental infections, design and testing of immune priming treatments based on recombinant spike proteins. Assisted by senior personnel Dr. Tim
7

DARPA – PREEMPT – HR001118S0017- PROJECT DEFUSE
Sheahan, Dr. Amy Sims, and support staff; #2 to Prof. Linfa Wang, Duke NUS, to oversee the immune boosting approach, captive bat experiments, and analyze immunological and virological responses to immune boosting treatments; #3 to Dr. Zhengli Shi, Wuhan Institute of Virology, to conduct PCR testing, viral discovery and isolation from bat samples collected in China, spike protein binding assays, and some humanized mouse work, as well as experimental trials on Rhinolophus bats. Her team will include Dr. Peng Zhou and support staff; #4 to Dr. Tonie Rocke, USGS National Wildlife Health Center, to refine delivery mechanisms for both immune boosting and immune priming treatments. With a research technician, Dr. Rocke will use a captive colony of bats at NWHC for initial trials, and oversee cave experiments in China.
F. Links to published papers, resume of two key performers
Dr. Peter Daszak is President and Chief Scientist of EcoHealth Alliance, a US-based research organization focused on emerging zoonotic diseases. His >300 scientific papers include the first global map of EID hotspots (31, 32), estimates of unknown viral diversity (33), predictive models of virus-host relationships (7), and evidence of the bat origin of SARS-CoV (34, 35) and other emerging viruses (36,37,38,39). He is Chair of the NASEM Forum on Microbial Threats, and is a member of the Executive Committee and the EHA institutional lead for the $130 million USAID- EPT-PREDICT. He serves on the NRC Advisory Committee to the USGCRP, the DHS CEEZAD External Advisory Board, the WHO R&D Blueprint Pathogen Prioritization expert group, and has advised the Director for Medical Preparedness Policy on the White House National Security Staff on global health issues. Dr. Daszak won the 2000 CSIRO medal for collaborative research.
Prof. Ralph Baric is a UNC Lineberger Comprehensive Cancer Center member and Professor in the UNC-Chapel Hill Dept. of Epidemiology and Dept. of Microbiology & Immunology. His work focuses on coronaviruses as models to study the genetics of RNA virus transcription, replication, persistence, cross species transmission and pathogenesis. His group has developed a platform strategy to access the potential “pre-epidemic” risk associated with zoonotic virus cross species transmission potential and evaluation of countermeasure potential to control future outbreaks of disease (8,9,10,11,12,13).
8

Executive Summary: DEFUSE EcoHealth Alliance; Dr. Peter Daszak
Host-Pathogen Prediction Host-Pathogen Niche Modeling
Assays in humanized mice
Machine learning genotype-phenotype mapping
Modeling recombination and evolution
Validate models using human serology
Intervention Development Immune boosting
Immune priming
Captive experiments
Simulating host-viral dynamics
Field testing and deployment
CONCEPT
Host-Pathogen Prediction: APPROACH
• Host-Pathogen Ecology: Develop host-pathogen ecological niche models based on unique bat and viral data, to
estimate likelihood of spillover of SARS-related CoVs into human populations. Doing so will enhance predictive ability
of models beyond sampling sites in China to cover all Asia.
• Mobile Application: Create ‘Reservoirs Near Me’ mobile application, to assess background risk of disease spillover for
any site across Southeast Asia.
• Binding and Humanized mouse assays: Utilize team’s unique collaboration between world-class modelers and
virologists with CoV expertise to conduct spike protein-based binding and humanized mice experiments.
• Use results to test machine-learning genotype-to-phenotype model predictions of viral spillover risk.
• Genotype-Phenotype Models: Develop models to estimate evolutionary rates, rates of recombination, and capacity to generate novel strains capable of human infection. Inputs to include: Diversity of bat spike proteins, prevalence of recombinant CoVs, and flow of genes within each bat cave via bat movement and migration.
• Validation with Human Sera: Analyze new and existing human serum samples to validate model outputs. Given frequent SARSr-CoV spillover events into local human populations, this can be done to a degree not possible in systems where spillover events are rare.
Intervention Development: (2 parallel approaches)
• (1) Broadscale Immune Boosting Strategy: Inoculate bats with immune modulators to upregulate innate immune response and downregulate viral replication, transiently reducing risk of viral shedding and spillover.
• (2) Targeted Immune Priming Strategy: Inoculate bats with novel chimeric polyvalent recombinant spike proteins to enhance innate immune response against specific, high-risk viruses.
• Viral Dynamics: Develop stochastic simulation models to estimate the frequency, efficacy, and population coverage required for intervention approaches to effectively suppress the viral population.
• Field Deployment and Testing: Utilize team’s expertise in wildlife vaccine delivery to assess and deploy effective molecule delivery methods, including: automated aerosolization technology that inoculates bats as they leave cave roost; remote controlled drone technology; transdermal nanoparticle application; and application of edible, adhesive gels that bats ingest when grooming fur of self and others.
CONTEXT
• No technology currently exists to reduce the risk of exposure to novel bat Coronaviruses.
• Our team has conducted pioneering research on modeling disease emergence, understanding Coronavirus virology, bat immunity, and wildlife vaccine delivery. Our previous work provides proof-of-concept for: (1) predictive ‘hotspot’ modeling; (2) upregulating bat immune response through the STING IFN pathway, (2) developing recombinant chimeric spike-proteins from SARS and SARSr-CoVs and (3) delivering immunological countermeasures to wildlife (including multiple bat species).
• The DEFUSE approach is broadly effective, scalable, economical and achievable in the allotted time frame. It also poses little environmental risk, and presents no threat to local livestock or human populations.
• While CRISPR-Cas9 gene drives are being considered for many disease research applications, the technique is unlikely to be effective in suppressing viral transmission in bat hosts. Bats are relatively long- lived, highly mobile, and have long inter-generational periods (2-5 years) with low progeny (1-2 pups). Furthermore, gene drive technology could have far-reaching, negative ecological consequences and its effectiveness cannot be evaluated within the defined Period of Performance.
IMPACT
• Recent and ongoing security concerns within South and SE Asia make the region a likely deployment site for US warfighters. Troops deployed to the region face increased disease risk from SARS and related bat viruses, as bats shed these pathogens through urine and feces while foraging over large areas at night.
• Our work in Yunnan Province, China has shown that (1) SARSr-CoVs are capable of producing SARS-like illness in humanized mice that are not affected by monoclonal or vaccine treatment, and (2) that spillover into local human populations is frequent. With no available vaccine or alternative method to counter these SARS-related viruses, US defense forces and national security are placed at risk.
• Our goal is to “DEFUSE” the potential for emergence of novel bat-origin high zoonotic risk SARSr-CoVs in Southeast Asia. In doing so, we will not only safeguard the US warfighter, but also reduce SARSr-CoV exposure for local communities and their livestock, improving food security and Global Health Security.
• If successful, our strategy can be adapted to hosts of other bat-origin CoVs (MERS-CoV in the Middle East and other SARS-related pre-pandemic zoonotic strains in Africa, e.g. Nigeria), and potentially other zoonotic bat-origin viruses (Hendra, Nipah, Ebola viruses).
Phase I
Phase II
Total
Proposed
$8,414,104
$6,385,894
$14,799,998
xHuman Use/ x Animal Use
HR001118S0017 PREEMPT 1

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10/5/21, 3:13 PM Mail - Rocke, Tonie E - Outlook
From: Peter Daszak
Sent: Tuesday, February 13, 2018 8:15 AM
To: 'Wang Linfa'; Luke Hamel; Jonathon Musser
Cc: William B. Karesh; Noam Ross; Ralph Baric (rbaric@email.unc.edu); 周鹏 (peng.zhou@wh.iov.cn); Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura; Anna Willoughby; Rocke, Tonie
Subject: RE: Final draft DARPA abstract
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10/5/21, 3:13 PM Mail - Rocke, Tonie E - Outlook
From: Wang Linfa [mailto:linfa.wang@duke-nus.edu.sg]
Sent: Tuesday, February 13, 2018 1:10 AM
To: Peter Daszak; Luke Hamel; Jonathon Musser
Cc: William B. Karesh; Noam Ross; Ralph Baric (rbaric@email.unc.edu); 周鹏 (peng.zhou@wh.iov.cn); Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura; Anna Willoughby; Rocke, Tonie
Subject: RE: Final draft DARPA abstract
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!saedi evarb ruo ”ni yub“ lliw eettimmoc noitceles eht epoh I dna won llew sdaer yllautca tI
,reeP iH

10/5/21, 3:13 PM Mail - Rocke, Tonie E - Outlook
www.ecohealthalliance.org
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.lasoporp taerg a sʼti
dias ton evʼI erus ekam ot daer kciuq a siht evig esaelp – einoT ,ilgnehZ ,gneP ,aniL ,hplaR
.fdp
lanif eht no knil evil a sʼti raelc sʼti os ,eulb si sesehtnerap ni noitatic eht erus ekam esaelp tub ,sih
ylno eW .IBCN no repap eht ot knil evil a otni ,elpmaxe rof ”)1(“ :noitatic ecnerefer hcae nrut tsuJ
rof did hplaR sa ,fer derebmun CMP a otni eseht fo hcae nrut ot moor on os ,senil eraps 2 evah
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10/5/21, 3:13 PM Mail - Rocke, Tonie E - Outlook
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10/5/21, 3:14 PM
Mail - Rocke, Tonie E - Outlook
Thanks and fingers crossed!
Linfa (Lin-Fa) Wang, PhD FTSE
Professor & Director
Programme in Emerging Infecous Diseases Duke-NUS Medical School
8 College Road, Singapore 169875
Tel: +65 65168397
From: Peter Daszak <daszak@ecohealthalliance.org>
Date: Wednesday, 21 February 2018 at 5:47 AM
To: Wang Linfa <linfa.wang@duke-nus.edu.sg>, Luke Hamel <hamel@ecohealthalliance.org>, Jonathon Musser <musser@ecohealthalliance.org>
Cc: William Karesh <karesh@ecohealthalliance.org>, Noam Ross <ross@ecohealthalliance.org>, Ralph Baric <rbaric@email.unc.edu>, <peng.zhou@wh.iov.cn>, zlshi <zlshi@wh.iov.cn>, Alison Andre <andre@ecohealthalliance.org>, Aleksei Chmura <chmura@ecohealthalliance.org>, Anna Willoughby <willoughby@ecohealthalliance.org>, Tonie Rocke <trocke@usgs.gov>
Subject: RE: Final dra DARPA abstract, and next steps...
Dear All,
Apologies – forgot to send you the final versions of the abstract and the execuve summary slide for your records...
Re. the total budget – please don’t view that as final – they’re planning to give out $40 million over the 3.5 years total to anything from 1 to 6 projects. Given that there were probably 3 or 4 other viable teams in the room at the proposer’s conference, I think that $14.8 million is probably the maximum they would give any project, and it’s more likely that they’ll fund three or four at 8 million and two or three smaller projects. They’ll hopefully give some clearer guidance as we move towards a full proposal.
The due date for proposals is March 27th, 4pm EST (NY me). That means we need to start honing in on our plans for the full proposal. I’m meeng with our team here on Thursday to start working on the next steps, and it would be good to have calls with all of you this week to start refining the ideas and building out the proposal. Luke will follow up with mes that work for phone calls.
Thanks again for your openness and clever ideas, and I look forward to working with you all to get the best possible full proposal submied on me!!!
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
From: Peter Daszak
Sent: Tuesday, February 13, 2018 8:15 AM
To: 'Wang Linfa'; Luke Hamel; Jonathon Musser
Cc: William B. Karesh; Noam Ross; Ralph Baric (rbaric@email.unc.edu); 周鹏 (peng.zhou@wh.iov.cn); Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura; Anna Willoughby; Rocke, Tonie
Subject: RE: Final draft DARPA abstract
Good point Linfa – changed to ‘treatment’ or ‘applicaon’ throughout.
Cheers, Peter
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鹏周
>vog.sgsu@ekcort<EeinoT,ekcoR;>gro.ecnaillahtlaehoce@ybhguolliw<ybhguolliWannA;>gro.ecnaillahtlaehoce@arumhc<
arumhCieskelA;>gro.ecnaillahtlaehoce@erdna<erdnAnosilA;>nc.voi.hw@ihslz<)nc.voi.hw@ihslz(ihSilgnehZ;>nc.voi.hw@uohz.gnep<)nc.voi.hw@uohz.gnep(
鹏周;>ude.cnu.liame@cirabr<)ude.cnu.liame@cirabr(ciraBhplaR;>gro.ecnaillahtlaehoce@ssor<ssoRmaoN;>gro.ecnaillahtlaehoce@hserak<hseraK.BmailliW:cC
>gro.ecnaillahtlaehoce@ressum<ressuMnohtanoJ;>gro.ecnaillahtlaehoce@lemah<lemaHekuL;>gro.ecnaillahtlaehoce@kazsad<kazsaDreteP:oT
>gs.ude.sun-ekud@gnaw.afnil<MPa01f9ni81L02/g02n/2aeuWT
...spets txen dna ,tcartsba APRAD tfard laniF :eR

10/5/21, 3:14 PM
Mail - Rocke, Tonie E - Outlook
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
From: Wang Linfa [mailto:linfa.wang@duke-nus.edu.sg]
Sent: Tuesday, February 13, 2018 1:10 AM
To: Peter Daszak; Luke Hamel; Jonathon Musser
Cc: William B. Karesh; Noam Ross; Ralph Baric (rbaric@email.unc.edu); 周鹏 (peng.zhou@wh.iov.cn); Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura; Anna Willoughby; Rocke, Tonie
Subject: RE: Final draft DARPA abstract
Hi Peer,
It actually reads well now and I hope the selecon commiee will “buy in” our brave ideas!
Nothing to add a or change, other than an English queson: we used “dampened immunity of bats” in most places, but you use the expression of “damping innate immunity pathways” on page 1. Is there a difference between “damping” and “dampening”?
Thanks
LF
Linfa (Lin-Fa) Wang, PhD FTSE
Professor & Director
Programme in Emerging Infecous Diseases Duke-NUS Medical School
8 College Road, Singapore 169875
Tel: +65 65168397
From: Peter Daszak [mailto:daszak@ecohealthalliance.org]
Sent: Tuesday, 13 February 2018 1:23 PM
To: Luke Hamel <hamel@ecohealthalliance.org>; Jonathon Musser <musser@ecohealthalliance.org>
Cc: William B. Karesh <karesh@ecohealthalliance.org>; Noam Ross <ross@ecohealthalliance.org>; Ralph Baric (rbaric@email.unc.edu) <rbaric@email.unc.edu>; Wang Linfa <linfa.wang@duke-nus.edu.sg>; 周鹏 (peng.zhou@wh.iov.cn) <peng.zhou@wh.iov.cn>; Zhengli Shi (zlshi@wh.iov.cn) <zlshi@wh.iov.cn>; Alison Andre <andre@ecohealthalliance.org>; Aleksei Chmura <chmura@ecohealthalliance.org>; Anna Willoughby <willoughby@ecohealthalliance.org>; Rocke, Tonie <trocke@usgs.gov> Subject: Final dra DARPA abstract
Importance: High
Luke, aached is the DARPA abstract.
First thing tomorrow, can you go through the references, and create links to the papers on NCBI. Just turn each reference citaon: “(1)” for example, into a live link to the paper on NCBI. We only have 2 spare lines, so no room to turn each of these into a PMC numbered ref, as Ralph did for his, but please make sure the citaon in parentheses is blue, so it’s clear it’s a live link on the final pdf.
Ralph, Lina, Peng, Zhengli, Tonie – please give this a quick read to make sure I’ve not said anything completely wrong. I’ve had to reduce the text a lot to hit the page limit, but I sll think it’s a great proposal.
I’ll finish off the exec summary slide and <500 wd abstract now.
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4473
www.ecohealthalliance.org
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
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Important: This email is confidential and may be privileged. If you are not the intended recipient, please delete it and notify us immediately; you should not copy or use it for any purpose, nor disclose its contents to any other person. Thank you.
Important: This email is confidential and may be privileged. If you are not the intended recipient, please delete it and notify us immediately; you should not copy or use it for any purpose, nor disclose its contents to any other person. Thank you.
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10/5/21, 3:14 PM Mail - Rocke, Tonie E - Outlook
Thanks and fingers crossed!
By the way, there is a tiny mistake on the first line of page 7: "sinicus bats at Wuhan Institute of Zoology", should be Wuhan institute of virology. Hope this won't affect too much...
Cheers, Peng
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ylbaborp si noillim 8.41$ taht kniht I ,ecnerefnoc sʼresoporp eht ta moor eht ni smaet elbaiv rehto
4 ro 3 ylbaborp erew ereht taht neviG .stcejorp 6 ot 1 morf gnihtyna ot latot sraey 5.3 eht revo
noillim 04$ tuo evig ot gninnalp erʼyeht – lanif sa taht weiv tʼnod esaelp – tegdub latot eht .eR
...sdrocer ruoy rof
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"ssoR maoN" ,>gro.ecnaillahtlaehoce@hserak< "hseraK .B mailliW" :送抄
"lemaH ekuL" ,>gs.ude.sun-ekud@gnaw.afnil< "afniL gnaW" :人件收
>gro.ecnaillahtlaehoce@kazsad< "kazsaD reteP":人件发
)三期星( 417450 12-20-8102:间时送发
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>vog.sgsu@ekcort<
EeinoT,ekcoR;>gro.ecnaillahtlaehoce@ybhguolliw<ybhguolliWannA;>gro.ecnaillahtlaehoce@arumhc<
arumhCieskelA;>gro.ecnaillahtlaehoce@erdna<erdnAnosilA;>nc.voi.hw@ihslz<)nc.voi.hw@ihslz(
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>nc.voi.hw@uohzM.Ag92n1e81p02</12/2鹏de周W
...spets txen dna ,tcartsba APRAD tfard laniF :ER :eR

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.noitavresnoc etomorp dna scimednap
tneverp taht snoitulos poleved ew ecneics siht htiW .smetsysoce etaciled dna htlaeh efildliw
dna namuh neewteb snoitcennoc lacitirc eht otni hcraeser egde-gnittuc sdael ecnaillA htlaeHocE
gro.ecnaillahtlaehoce.www
3744-083-212 1+ .leT
10001 YN ,kroY weN
roolF ht71 – teertS ht43 tseW 064
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tnediserP
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teg ot lla uoy htiw gnikrow ot drawrof kool I dna ,saedi revelc dna ssennepo ruoy rof niaga sknahT
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trats ot keew siht uoy fo lla htiw sllac evah ot doog eb dluow ti dna ,spets txen eht no gnikrow
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.lasoporp lluf a sdrawot evom
8 ta ruof ro eerht dnuf llʼyeht taht ylekil erom sʼti dna ,tcejorp yna evig dluow yeht mumixam eht

10/5/21, 3:14 PM Mail - Rocke, Tonie E - Outlook
From: Peter Daszak
Sent: Tuesday, February 13, 2018 8:15 AM
To: 'Wang Linfa'; Luke Hamel; Jonathon Musser
Cc: William B. Karesh; Noam Ross; Ralph Baric (rbaric@email.unc.edu); 周鹏 (peng.zhou@wh.iov.cn); Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura; Anna Willoughby; Rocke, Tonie
Subject: RE: Final draft DARPA abstract
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 3/6
tneverp taht snoitulos poleved ew ecneics siht htiW .smetsysoce etaciled dna htlaeh efildliw
dna namuh neewteb snoitcennoc lacitirc eht otni hcraeser egde-gnittuc sdael ecnaillA htlaeHocE
gro.ecnaillahtlaehoce.www
3744-083-212 1+ .leT
10001 YN ,kroY weN
roolF ht71 – teertS ht43 tseW 064
ecnaillA htlaeHocE
tnediserP
kazsaD reteP
reteP
,sreehC
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10/5/21, 3:14 PM Mail - Rocke, Tonie E - Outlook
From: Wang Linfa [mailto:linfa.wang@duke-nus.edu.sg]
Sent: Tuesday, February 13, 2018 1:10 AM
To: Peter Daszak; Luke Hamel; Jonathon Musser
Cc: William B. Karesh; Noam Ross; Ralph Baric (rbaric@email.unc.edu); 周鹏 (peng.zhou@wh.iov.cn); Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura; Anna Willoughby; Rocke, Tonie
Subject: RE: Final draft DARPA abstract
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.noitavresnoc etomorp dna scimednap

10/5/21, 3:14 PM Mail - Rocke, Tonie E - Outlook
周鹏
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Important: This email is confidential and may be privileged. If you are not the intended recipient, please delete it and notify us immediately; you should not copy or use it for any purpose, nor disclose its contents to any other person. Thank you.
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RE: RE: Final draft DARPA abstract, and next steps...
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Rocke Lab
RGE Women'... 7 Invasive Sp... 47 Discover groups Manage groups
ecosystems. With this science we develop soluons that
2
www.ecohealthalliance.org
Peter Daszak <daszak@ecohealthalliance.
Wed 2/21/2018 11:01 AM
change that in the full proposal!
Cheers, Peter
Cheers,
Peter
EcoHealth Alliance
Peter Daszak
President
460 West 34th Street – 17th Floor
RE: RE: Final draft DARPA abstract,
and next steps...
This message was sent with High importance.
Peter Daszak <daszak@ecohealthalliance.org>
Wed 2/21/2018 11:01 AM
To: <peng.zhou@wh.iov.cn> 
Musser <musser@ecohealthalliance.org>; William B.
Karesh <karesh@ecohealthalliance.org>; Noam Ross To: <peng.zhou@wh.iov.cn>
<ross@ecohealthalliance.org>; Ralph Baric
Cc: Wang Linfa <linfa.wang@duke-nus.edu.sg>; Luke Hamel <hamel@eco
(rbaric@email.unc.edu) <rbaric@email.unc.edu>;
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Andre <andre@ecohealthalliance.org>; Aleksei Chmura
<chmura@ecohealthalliance.org>; Anna Willoughby <willoughby@ecohealthalliance.org>; Rocke, Tonie E <trocke@usgs.gov>
Ouch – great that you spoed that mistake and I’ll definitely change that in the full proposal!
Peter Daszak
President
Tel. +1 212-380-4473
EcoHealth Alliance
www.ecohealthalliance.org th th 460 West 34 Street – 17
Floor
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate
Tel. +1 212-380-4473
prevent pandemics and promote conservaon.
EcoHealth Alliance leads cung-edge research
From: 周鹏 [mailto:peng.zhou@wh.iov.cn]
into the crical connecons between human and
Sent: Wednesday, February 21, 2018 2:30 AM
To: Peter Daszak wildlife health and delicate ecosystems. With this
science we develop soluons that prevent
Cc: Wang Linfa; Luke Hamel; Jonathon Musser; William B. pandemics and promote conservaon.
Karesh; Noam Ross; Ralph Baric (rbaric@email.unc.edu); Zhengli Shi (zlshi@wh.iov.cn); Alison Andre; Aleksei Chmura;
From: 周鹏 [mailto:peng.zhou@wh.iov.cn] Anna Willoughby; Rocke, Tonie
Sent: Wednesday, February 21, 2018 2:30 AM https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/1
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鹏周
鹏周

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10/5/21, 3:15 PM
Mail - Rocke, Tonie E - Outlook
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct)
(mobile) www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics
and promote conservation.
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DARPA – PREEMPT PROPOSAL OUTLINE
NOTE: 36 pages max - 12 pt. font or higher (font can be smaller for tables, charts, figures)
VOLUME I – Technical and Management Proposal
Section I – Administrative
A) Cover Page (labeled “Proposal: Volume I”) B) Official Transmittal Letter
Section II – Detailed Proposal Information
A)
1) What is the proposed work attempting to accomplish or do?
We will defuse the potential for emergence of novel bat-origin high-zoonotic risk SARS- related coronaviruses. We envisage a scenario whereby the US warfighter is deployed to a security hotspot in SE Asia. As planners choose sites for the mission, they will use an app we will design based on machine-learning models of the ecological and evolutionary potential of bat viruses to spillover. This will allow rapid assessment of the background risk of a site harboring dangerous zoonotic viruses. If there is no alternative site, a tactical forward team will deploy automated delivery technology we will develop in caves that harbor bats carrying these viruses. These devices will release broadscale immune boosting molecules and chimeric polyvalent spike protein targeted immune priming treatments to upregulate the naturally damped innate immune response of bats, and lower viral shedding from bats at the site for a few weeks or months, allowing
our warfighters to execute the operation at lowered risk for spillover.
2) How is it done today, and what are the limitations?
Other than PPE, there is no available current technology to reduce the risk of exposure to novel coronaviruses from bats. Models of bat host capacity to harbor viruses, of ecological and environmental drivers of their emergence, and of the evolutionary potential of different strains to spillover are rudimentary. No vaccines or therapeutics exist for SARSr-CoVs, and exposure mitigation strategies are non-existent. SARSr-CoVs are endemic in Asian, African (1), and European bats (2) that roost in caves but forage widely at night, shedding virus in their feces and urine. The limitations of this lack of capacity are significant – we have shown evidence of recent spillover of SARSr-CoVs into people in China and have isolated strains capable of producing SARS-like illness in humanized mice that don’t respond to antibody treatment or vaccination. These viruses are a clear-and-present danger to our military and to global health security.
Executive Summary
(questions/answers below from abstract).
Commented [EA1]: Perhaps some of the more detailed information in this section could be moved to the ‘Goals and Impacts’ section

3) What is innovative in your approach?
Our group leads the world in predictive models of viral emergence. We will build on our machine-learning models of spillover hotspots, host-pathogen ecological niches and genotype- phenotype mapping by incorporating unique datasets to validate and refine hotspot risk maps of viral emergence in SE Asia and beyond. Our group has shown that bats coexist with lethal viruses by damping innate immunity pathways, likely as an evolutionary adaptation to flight. We will use this insight to design strategies, like small molecule Rig like receptor (RLR) or Toll like receptor (TLR) agonists, to upregulate bat immunity in their cave roosts, down-regulate viral replication, and reduce the risk of viral shedding and spillover (broadscale immune boosting strategy). We will complement this by treating bats with novel chimeric polyvalent recombinant spike proteins to enhance their immune response against specific, high-risk viruses (targeted immune priming strategy), especially when their immune response is boosted as above. We will design novel methods to deliver these applications remotely to reduce exposure risk during decontamination.
4) What are the key technical challenges in your approach and how do you plan to overcome these?
Modeling: Previous models have suffered from a lack of data to validate them. We have access to unique datasets that will allow us to validate our approach, including biodiversity surveys of bat caves across S. China, 10+ years of bat viral testing data in China, and 10 other countries (from NIH-NIAID and USAID EPT PREDICT work). Uniquely, we will validate our models of viral evolution/spillover risk using human serology (based on LIPS assays) in local populations that have high (~3%) seroprevalence to bat SARSr-CoVs. Identifying Immune boosting and priming treatments: Some of our approaches are novel and challenging (e.g. using CRISPRi to find the negative regulator for bat interferon production), and others are unproven in bats (e.g. Poly IC). We will begin all immune boosting and priming experiments at the beginning of the project, running them simultaneously and competitively, so that we field trial only the most efficient, cost-effective and scalable approaches.
5) Who or what will be affected and what will be the impact?
This will have direct relevance to the warfighter. Potential deployment to regions where SARSr- CoVs exist is high – countries include security hotspots in Asia (e.g. Myanmar, Bangladesh, Pakistan, Korea, Vietnam), Africa and Eastern Europe. The ability to decontaminate and defuse these viruses may prevent potentially devastating illness. These technologies could be adapted to hosts of other bat-origin CoVs (e.g. MERS-CoV, SADS-CoV) and potentially other zoonotic bat- origin viruses (Hendra, Nipah, EBOV), with benefits to livestock production, food security and global public health.

B) ExecutiveSummarySlide(mustuseprovidedtemplate) C)
• Clearly describe what the team is trying to achieve and the difference it will make (qualitatively and quantitatively) if successful.
• Describe the innovative aspects of the project in the context of existing capabilities and approaches, clearly delineating the uniqueness and benefits of this project in the context of the state of the art, alternative approaches, and other projects from the past and present.
• Describe how the proposed project is revolutionary and how it significantly rises above the current state of the art.
• Describe the deliverables associated with the proposed project and any plans to commercialize the technology, transition it to a customer, or further the work.
Overview
The SARSr-CoV-bat system, and immune modulation focus: Our group’s 15 yrs work on the SARSr-CoV – Rhinolophus bat system in China has identified and isolated SARSr-CoVs with remarkable sequence identity in the spike protein to SARS-CoV (e.g. SCH014 & WIV-1). We have shown they bind and replicate efficiently in primary human lung airway cells and that chimeras with SARSr-CoV spike proteins in a SARS-CoV backbone cause SARS-like illness in humanized mice, with clinical signs that are not reduced by SARS monoclonal therapy or vaccination. We have identified a single cave site in Yunnan Province where bat SARSr-CoVs contain all the genetic components of epidemic SARS-CoV (7,8,9). We have now shown that people living up to 6 kilometers from this cave have SARSr-CoV antibodies (3% seroprevalence in 200+ cohort), suggesting active spillover, and marking these viruses as a clear-and-present danger of a new SARS-like pandemic. Our work on bat immunology suggests that bats’ unique flying ability has led to downregulated innate immune genes, and their ability to coexist with viruses such as SARSr-CoVs, henipa- and filoviruses that are lethal in many other mammals (3). We have identified bat-specific constitutively expressed bat interferon, a dampened STING-interferon production pathway (4, 5), and have identified a series of other innate immunity factors that are dampened in bats (6).
Our bat-CoV system has significant advantages for experimentation and intervention. Firstly, these viruses are fecal-orally transmitted within bat populations, so sampling can be achieved from fresh fecal pellet collection. They are BSL-3, not -4, agents, so that experimental manipulation and infection is simpler. They have frequent spillover events, making it possible to validate predictive models of spillover by sampling people. They are diverse, with frequent recombination and different strains exhibiting differential host cell binding and spillover potential. Finally, we have identified SARSr-CoV strains in a single cave in Yunnan that harbor all of the epidemic SARS-CoV genes. This specific bat population harbors an ideal evolutionary soup that could produce new human strains by high frequency RNA recombination, and thus, it presents a perfect target for next generation, technology-forward intervention strategies.
Commented [EA2]: Detailed text from the ‘Executive Summary’ section may be better suited here
Goals and Impact

D) Technical Plan
• Outline and address technical challenges inherent in the approach and possible solutions for overcoming potential problems.
• Provide appropriate measurable milestones (quantitative if possible) and program metrics (see “metrics” attachment) at intermediate stages of the program to demonstrate progress, and a plan for achieving the milestones.
• Demonstrate a deep understanding of the technical challenges.
• Present a credible (even if risky) plan to achieve the program goal.
• Discuss mitigation of technical risk.
• Address TA1 and TA2 proposal content requirements (see “objectives” attachment)
TA1: Develop and validate integrated, multiscale models that quantify the likelihood a human-capable virus will emerge from an animal reservoir residing in a “hot spot” geographic region
The DEFUSE modeling and analytics team, led by Drs. Daszak, Ross, Olival, EHA, will build ecological niche models of environmental and ecological correlates and traits of cave bat communities to predict species composition of bat caves across Southern China, South and SE Asia. We will then use a series of datasets we have built to produce host-virus risk models for the region. These include our unique database of bat host-viral relationships (7); biological inventory data on all bat caves in Southern China; and modeled species distribution data for all bats. We will parameterize the model with data from three cave sites in Yunnan, China (one with high-risk SARSr-CoVs, two other control/comparison sites), including: radio- and GPS-telemetry to identify home range and additional roost sites for each bat species; inventory of bat population density, distribution and segregation and their daily, weekly and seasonal changes; viral prevalence and individual viral load; shedding of low- and high-risk SARSr-CoV strains among bat species, age classes, genders; and telemetry and mark-recapture data to assess metapopulation structure and inter-cave connectivity. We will test and validate model predictions of a cave’s viral spillover potential with data from prior PREDICT sampling in 7 other Asian countries. At the end of Yr 1, we will produce a prototype app for the warfighter that identifies the likelihood of bats harboring dangerous viral pathogens in a region. The ‘high-risk bats near me’ app will be updated real-time with surveillance data (e.g. field-deployable iPhone and android compatible echolocation data) from our project and others, to ground-truth and fine-tune its predictive capacity.
The Wuhan Institute of Virology team will test bat fecal, oral, blood and urogenital samples for SARSr-CoVs. We will correlate viral load data from these samples with fresh fecal pellets from individuals and from tarps laid on cave floors. We will rapidly move to fecal pellet assays to reduce roost disturbance. SARSr-CoV spike proteins will be sequenced, analyzed phylogenetically for recombination events, and high-risk viruses (spike proteins close to SARS- CoV) characterized and isolated. The UNC team will reverse-engineer spike proteins to conduct binding assay to human ACE2 (the SARS-CoV receptor). They will culture SARS-like bat coronaviruses to distinguish high-risk strains that can replicate in primary human cells and low risk strains that require exogenous enhancers. Viral spike glycoproteins that bind receptors will be inserted into SARS-CoV backbones, inoculated into human cells and humanized mice to assess capacity to cause SARS-like disease, and to be blocked by monoclonal therapies, the nucleoside analogue inhibitor GS-5734(8)or vaccines against SARS-CoV(8,9,10,11,12,13).
The EHA modeling team will use these data to build models of risk of viral evolution and

spillover. These genotype-to-phenotype machine-learning models will predict viral ability to infect host cells based on genetic traits and results of receptor binding and mouse infection assays. Using data on diversity of spike proteins, recombinant CoVs, and flow of genes within each bat cave via bat movement and migration, we will estimate evolutionary rates, rates of recombination, and capacity to generate novel strains capable of human infection. Finally, virus- host relationship and bat home range data will be used to estimate spillover potential-extending models well beyond our field sites. We will then validate model predictions of viral spillover risk by 1) conducting spike protein-based binding and cell culture experiments, and 2) identifying spillover strains in people near our bat cave sites. Our preliminary work on this shows ~3% seroprevalence to SARSr-CoVs, using a specific ELISA (14).We will design LIPS assays to the specific high-and low-zoonotic-risk SARSr-CoVs identified in this project as we have done previously(15).We will use banked and newly collected human sera from these populations to test for presence of antibodies to the high-and low-risk SARSr-CoVs identified by our modeling. We will then model optimal strategies to maximize treatment efficacy for TA2,using stochastic simulation modeling informed by field and experimental data to characterize viral circulation dynamics in bats. We will estimate frequency and population coverage required for our intervention approaches to suppress viral spillover. We will determine the seasons, locations within a cave, and delivery methods (spray, swab, or automated cave mouth or drone)that will be most effective. Finally we will determine the time period treatment will be effective for, until re-colonization or evolution leads to return of a high-risk SARSr-CoV.
TA2: Develop scalable approaches that target and suppress the animal virus in its reservoir(s)and/or vector(s), to reduce the likelihood of virus transmission into humans.
We will evaluate two approaches to defuse SARS-related CoV spillover potential: 1) Broadscale Immune Boosting: using the unique immune damping in bat that our group has discovered, we will apply immune modulators like bat interferon to live bats, to up-regulate their naïve immunity and then assess their ability to suppress viral replication and shedding;2) Targeted Immune Priming: building on preliminary development of polyvalent chimeric recombinant SARSr-CoV spike proteins, we will conduct application trials with live bats to assess suppression of replication and shedding of abroad range of dangerous SARS-related CoVs.
Both lines of work will begin in Yr 1and run parallel. Prof. Linfa Wang (Duke-NUS) will lead the immune boosting work, building on his pioneering work on bat immunity (3) which shows that the long-term coexistence of bats and their viruses has led to equilibrium between viral replication and host immunity. This is likely due to down-regulation of their innate immune system as a fitness cost of flight (3). The weakened functionality of bat innate immunity factors like STING, a central DNA-interferon (IFN) sensing molecule, may allow bats to maintain an effective, but not over-response to viruses (4). A similar finding was observed for bat IFNA, which is less abundant but constitutively expressed without stimulation (5). Given high native SARSr-CoV load in bats, we aim to boost bat innate immunity through the IFN pathway, break the host-virus equilibrium to suppress bat SARSr-CoV replication and shedding.
We will trial the following, concurrently and competitively, for efficiency, cost and scalability: i) Universal bat interferon. Aerosol spraying or intranasal application of IFN or other small molecules reduces viral loads in humans, ferrets and mouse models (16, 17). Interferon has been used clinically when antiviral drugs are unavailable, e.g. against filoviruses (18). Replication of SARSr-CoV is sensitive to interferon treatments, as shown in our previous work

(16); ii) Boosting bat IFN by blocking bat-specific IFN negative regulators. Uniquely, bat IFNA is naturally constitutively expressed but cannot be induced to a high level (5), indicating a negative regulatory factor in the bat interferon production pathway. We will use CRISPRi to identify the negative regulator and then screen for compounds targeting this gene; iii) Activating dampened bat-specific IFN production pathways which include DNA-STING-dependent and ssRNA-TLR7-dependent pathways. Our work showing that mutant bat STING restores antiviral functionality suggests these pathways are important in bat-viral coexistence (4). By identifying small molecules to directly activate downstream of STING, we will activate bat interferon and promote viral clearance. A similar strategy will be applied to ssRNA-TLR7-dependent pathways; iv) Activating functional bat IFN production pathways, e.g. polyIC to TLR3-IFN pathway or 5’ppp-dsRNA to RIG-I-IFN pathway. A similar strategy has been demonstrated in a mouse model for SARS-CoV, IAV and HBV (17, 19); v) Inoculating crude coronavirus fragments to upregulate innate immune responses to specific CoVs – a partial step towards the targeted immune priming work below.
Prof. Ralph Baric (UNC) will lead the immune priming work. He will develop recombinant chimeric spike-proteins (20) from our known SARSr-CoVs, and those we characterize during project DEFUSE. The structure of the SARS-CoV spike glycoprotein has been solved and the addition of two proline residues at positions V1060P and L1061P stabilize the prefusion state of the trimer, including key neutralizing epitopes in the receptor binding domain (21). In parallel, the spike trimers or the receptor binding domain can be incorporated into alphavirus vectored or nanoparticle vaccines for delivery, either as aerosols, in baits, or as large droplet delivery vehicles (11, 22,23,24,25). We will test these in controlled lab conditions, taking the best candidate forward for testing in the field. We have built recombinant spike glycoproteins harboring structurally defined domains from SARS epidemic strains, pre-epidemic strains like SCH014 and zoonotic strains like HKU3. It is anticipated that recombinant S glycoprotein based vaccines harboring immunogenic blocks across the group 2B coronaviruses will induce broad scale immune responses that simultaneously reduce genetically heterogeneous virus burdens in bats, potentially reducing disease risk (and transmission risk to people) in these animals for longer periods (26, 27).
The immune dampening features are highly conserved in all bat species tested so far. Duke- NUS has established the only experimental breeding colony of cave bats (Eonycteris spelaea) in SE Asia. This genus is evolutionarily related to Rhinolophus spp. (the hosts of SARSr-CoVs), so we have confidence that results will be transferable. Our initial proof-of-concept tests will be in this experimental colony, extended to a small group of wild-caught Rhinolophus sinicus bats at Wuhan Institute of Zoology. We (Prof. Wang) have previous experience conducting SARS-CoV infection experiments with Rhinolophus sp. bats in the BSL-4 facility at CSIRO, AAHL (L.Wang, unpublished results).
Finally, work on a delivery method for our immune boosting and priming molecules will be overseen by Dr. Tonie Rocke at the USGS, National Wildlife Health Center who has previously developed animal vaccines through to licensure (28). Using locally acquired insectivorous bats (29, 30) we will assess delivery vehicles and methods including: 1) transdermally applied nanoparticles; 2) series of sticky edible gels that bats will groom from themselves and each other; 3) aerosolization via sprayers that could be used in cave settings; 4) automated sprays triggered by timers and movement detectors at critical cave entry points, and 5) sprays delivered by remote controlled drone. We have already used simple gels to vaccinate bats against rabies in the lab (29), and hand delivered these containing biomarkers to vampire bats in Peru and Mexico to show they are readily consumed and transferred among bats. In our bat

colony, we will trial delivery vehicles using the biomarker rhodamine B (which marks hair and whiskers upon consumption) to assess uptake. The most optimal approaches will then be tested on wild bats in our three cave sites in Yunnan Province with the most successful immunomodulators from TA2. Fieldwork will be conducted under the auspices of Dr. Yunzhi Zhang (Yunnan CDC, Consultant at EcoHealth Alliance). A small number of bats will be captured and assayed for viral load and immune function after treatment, but so as not to disturb the colony, most viral load work will be conducted on fresh fecal pellets collected daily on the cave floor. EHA has had unique access to these sites for around 10 years, under the guidance of Drs. Shi and Zhang. In year 1 of project DEFUSE, we will seek permission for experimental trials from the Provincial Forestry Department. We expect to be successful, as we have worked with the Forestry Department collaboratively for 10 years, with support of the Yunnan CDC, and we are releasing molecules that are not dangerous to people or wildlife. EHA has a proven track record of rapidly obtaining IACUC and DoD ACURO approval for bat research.
E) ManagementPlan
• Provide a summary of expertise of the team, including any subcontractors, and key personnel who will be doing the work. Resumes count against the page count.
• Identify a principal investigator for the project.
• Provide a clear description of the team’s organization
• Include an organization chart with the following information, as applicable:
A) Programmatic relationship of team members
B) Unique capabilities of team members
C) Task responsibilities of team members
D) Teaming strategy among the team members
E) Key personnel with amount of effort to be expended by each during each year
• Provide a detailed plan for coordination including explicit guidelines for interaction among collaborators/subcontractors of the proposed effort.
• Include risk management approaches.
• Describe any formal teaming agreements that are required to execute this program.
The lead institution for Project DEFUSE is EcoHealth Alliance, New York, an international research organization focused on emerging zoonotic diseases. The PI, Dr. Peter Daszak, has 25+ years’ experience managing lab, field and modeling research projects on emerging zoonoses. Dr. Daszak will commit 3 months annually to oversee and coordinate all project activities, and lead modeling and analytic work for TA1. Dr. Billy Karesh has 40+ years’ experience leading zoonotic and wildlife disease projects, and will commit 1 month annually to manage partnership activities and outreach. Dr. Jon Epstein, with 15 years’ experience working

emerging bat zoonoses will coordinate animal trials. Dr. Kevin Olival and Dr. Noam Ross will manage and conduct the modeling and analytical approaches for this project. Support staff include field surveillance teams, modeling analysts, and consultants based in Yunnan Province, China, to oversee field trials. The EHA team has worked extensively with all other collaborators: Prof. Wang (15+ years); Dr. Shi (15+ years); Prof. Baric (5+ years) and Dr. Rocke (15+ years). Subcontracts: #1 to Prof. Ralph Baric, UNC, to oversee reverse engineering of SARSr-CoVs, BSL-3 humanized mouse experimental infections, design and testing of immune priming treatments based on recombinant spike proteins. Assisted by senior personnel Dr. Tim Sheahan, Dr. Amy Sims, and support staff; #2 to Prof. Linfa Wang, Duke NUS, to oversee the immune boosting approach, captive bat experiments, and analyze immunological and virological responses to immune boosting treatments; #3 to Dr. Zhengli Shi, Wuhan Institute of Virology, to conduct PCR testing, viral discovery and isolation from bat samples collected in China, spike protein binding assays, and some humanized mouse work, as well as experimental trials on Rhinolophus bats. Her team will include Dr. Peng Zhou and support staff; #4 to Dr. Tonie Rocke, USGS National Wildlife Health Center, to refine delivery mechanisms for both immune boosting and immune priming treatments. With a research technician, Dr. Rocke will use a captive colony of bats at NWHC for initial trials, and oversee cave experiments in China.
Dr. Peter Daszak is President and Chief Scientist of EcoHealth Alliance, a US-based research organization focused on emerging zoonotic diseases. His >300 scientific papers include the first global map of EID hotspots (31, 32), estimates of unknown viral diversity (33), predictive models of virus-host relationships (7), and evidence of the bat origin of SARS-CoV (34, 35) and other emerging viruses (36,37,38,39). He is Chair of the NASEM Forum on Microbial Threats, and is a member of the Executive Committee and the EHA institutional lead for the $130 million USAID- EPT-PREDICT. He serves on the NRC Advisory Committee to the USGCRP, the DHS CEEZAD External Advisory Board, the WHO R&D Blueprint Pathogen Prioritization expert group, and has advised the Director for Medical Preparedness Policy on the White House National Security Staff on global health issues. Dr. Daszak won the 2000 CSIRO medal for collaborative research.
Prof. Ralph Baric is a UNC Lineberger Comprehensive Cancer Center member and Professor in the UNC-Chapel Hill Dept. of Epidemiology and Dept. of Microbiology & Immunology. His work focuses on coronaviruses as models to study the genetics of RNA virus transcription, replication, persistence, cross species transmission and pathogenesis. His group has developed a platform strategy to access the potential “pre-epidemic” risk associated with zoonotic virus cross species transmission potential and evaluation of countermeasure potential to control future outbreaks of disease (8,9,10,11,12,13).
F) Capabilities
• Describe organizational experience in relevant subject area(s), existing intellectual property, specialized facilities, and any Government-furnished materials or information.
• Discuss any work in closely related research areas and previous accomplishments.

(The following information was taken from the ‘Goals and Impact’ section of the document).
The SARSr-CoV-bat system, and immune modulation focus: Our group’s 15 yrs work on the SARSr-CoV – Rhinolophus bat system in China has identified and isolated SARSr-CoVs with remarkable sequence identity in the spike protein to SARS-CoV (e.g. SCH014 & WIV-1). We have shown they bind and replicate efficiently in primary human lung airway cells and that chimeras with SARSr-CoV spike proteins in a SARS-CoV backbone cause SARS-like illness in humanized mice, with clinical signs that are not reduced by SARS monoclonal therapy or vaccination. We have identified a single cave site in Yunnan Province where bat SARSr-CoVs contain all the genetic components of epidemic SARS-CoV (7,8,9). We have now shown that people living up to 6 kilometers from this cave have SARSr-CoV antibodies (3% seroprevalence in 200+ cohort), suggesting active spillover, and marking these viruses as a clear-and-present danger of a new SARS-like pandemic. Our work on bat immunology suggests that bats’ unique flying ability has led to downregulated innate immune genes, and their ability to coexist with viruses such as SARSr-CoVs, henipa- and filoviruses that are lethal in many other mammals (3). We have identified bat-specific constitutively expressed bat interferon, a dampened STING-interferon production pathway (4, 5), and have identified a series of other innate immunity factors that are dampened in bats (6).
G) Statement of Work (SOW)
• Provide a detailed task breakdown, citing specific tasks and their connection to the interim milestones and program metrics.

NOTE: The SOW must not include proprietary information. • For each task/subtask, provide:
o A detailed description of the approach to be taken to accomplish each defined task/subtask.
o Identification of the primary organization responsible for task execution (prime contractor, subcontractor(s), consultant(s), by name).
o A measurable milestone, i.e., a deliverable, demonstration, or other event/activity that marks task completion. Include quantitative metrics.
o A definition of all deliverables (e.g., data, reports, software) to be provided to the Government in support of the proposed tasks/subtasks.
(TA1) Task 1: Collect ecological and environmental data from Yunnan Province cave sites. Description and execution:
Each phase of the program (Phase I base and Phase II option) should be separately defined in
the SOW and each task should be identified by TA (1 or 2).
Commented [EA3]: I’ve taken tasks from the ‘Technical Plan’ section above, but these tasks may need to be reorganized according to Phase (I or II).
Also, feel free to alter names of tasks, as well as task/subtask classifications
Phase I:

Preliminary Data: Organization leading task:
Progress Metrics: Deliverable(s):
(TA1) Task 2: Construct niche models to predict species composition of bat caves across South and Southeast Asia
Description and execution: Preliminary Data:
Organization leading task: Progress Metrics: Deliverable(s):
(TA1) Subtask 2.1: Construct host-pathogen risk models Description and execution:
Preliminary Data: Organization leading task:
Progress Metrics: Deliverable(s):
(TA1) Subtask 2.2: Develop prototype app for the warfighter Description and execution:
Preliminary Data: Organization leading task:
Progress Metrics: Deliverable(s):

(TA1) Task 3: Analyze bat samples for SARSr-CoVs Description and execution:
Preliminary Data: Organization leading task:
Progress Metrics: Deliverable(s):
(TA1) Subtask 3.1 Develop recombinant chimeric spike proteins from characterized SARSr-CoVs
Description and execution: Preliminary Data:
Organization leading task: Progress Metrics: Deliverable(s):
(TA1) Subtask 3.2 Conduct assays in humanized mice to identify high-risk SARSr-CoV strains
Description and execution: Preliminary Data:
Organization leading task: Progress Metrics: Deliverable(s):
Trial experimental approaches aimed towards ‘Broadscale Immune
Boosting’ using experimental bat colonies
Description and execution: Preliminary Data:
Organization leading task:
Commented [EA4]: This task may carry over into Phase II. Might want to break up between Phases I and II.
(TA2) Task 4:

Progress Metrics: Deliverable(s):
Trial experimental approaches aimed towards ‘Immune Targeting’
using experimental bat colonies
Description and execution: Preliminary Data:
Organization leading task: Progress Metrics: Deliverable(s):
(TA2) Task 6: Develop and assess delivery methods for immune boosting and priming molecules
Description and execution: Preliminary Data:
Organization leading task: Progress Metrics: Deliverable(s):
(TA1) Task 7: Construct genotype-to-phenotype machine learning models to predict risk of viral evolution and spillover
Description and execution:
Preliminary Data: Organization leading task:
Progress Metrics: Deliverable(s):
(TA2) Subtask 7.1: Validate model predictions of viral spillover risk Description and execution:
Preliminary Data:
Commented [EA5]: This task may carry over into Phase II. Might want to break up between Phases I and II.
(TA2) Task 5:

Organization leading task: Progress Metrics: Deliverable(s):
Phase II:
(TA1) Task 1: Design LIPS assays to specific high- and low- zoonotic risk SARSr-CoVs
Description and execution: Preliminary Data:
Organization leading task: Progress Metrics: Deliverable(s):
(TA1) Task 2: Construct models to maximize efficacy of intervention approaches Description and execution:
Preliminary Data: Organization leading task:
Progress Metrics: Deliverable(s):
(TA2) Task 3: Deploy most effective molecule delivery methods on bat colonies of Yunnan Province caves
Description and execution:
Preliminary Data: Organization leading task:
Progress Metrics:

Deliverable(s):
• (task name, duration, work breakdown structure element as applicable, performing organization), milestones, and the interrelationships among tasks.
NOTE: Task structure must be consistent with that in the SOW.
• Measurable milestones should be clearly articulated and defined in time relative to the start of the project.
• Indicate the types of partners (e.g., government, private industry, non-profit)
• Submit a timeline with incremental milestones toward successful engagement.
NOTE: begin transition activities during the early stages of the program (Phase I).
• Describe any potential DARPA roles.
J) PREEMPT Risk Mitigation Plan
• Provide the following:
o An assessment of potential risks to public health, agriculture, plants, animals, the
environment, and national security.
o Guidelines the proposer will follow to ensure maximal biosafety and biosecurity.
o A communication plan that addresses content, timing, and the extent of distribution of potentially sensitive dual-use information. The plan must also address how input from DARPA, other government, and community stakeholders will be taken into account in decisions regarding communication and publication of potentially sensitive dual-use information.
K) Ethical, Legal, Societal Implications (ELSI)
• Address potential ethical, legal, and societal implications of the proposed technology.
Section III – Additional Information (doesn’t count against 36 pg. limit)
H) Schedule and Milestones
Provide a detailed schedule showing tasks
I) PREEMPT
Transition Plan
Commented [EA6]: Description from the BAA:
PREEMPT Transition Plan
Proposers must include a PREEMPT Technology Transition Plan. Proposers must indicate the types of partners (e.g., government, private industry, non-profit) they plan to pursue and submit a timeline with incremental milestones toward successful engagement. Proposers should begin transition activities during the early stages of the program (Phase I). Awardees must include
DARPA in the development of transition relationships. If the transition plan includes a start-up company, a business development strategy must be included as well. The extent by which the proposed intellectual property (IP) rights will impede the Government’s ability to transition thetechnology will be considered in the proposal evaluation.

A) BriefBibliography(nopagelimitindicated–canbepublished/unpublished) B) Upto3relevantpapersattached(optional)

10/5/21, 3:21 PM
Mail - Rocke, Tonie E - Outlook
Brian Baker
Assistant Managing Editor, EcoHealth 460 West 34th Street, 17th Floor
New York, NY 10001
1.212.380.4498 (direct) brian.hartman.baker (Skype)
Website: www.ecohealth.net
Submissions and Log-in: https://mc.manuscriptcentral.com/ecohealth Author Instructions: http://www.ecohealth.net/submit.php
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10/5/21, 3:21 PM
Mail - Rocke, Tonie E - Outlook
Brian Baker
Assistant Managing Editor, EcoHealth 460 West 34th Street, 17th Floor
New York, NY 10001
1.212.380.4498 (direct) brian.hartman.baker (Skype)
Website: www.ecohealth.net
Submissions and Log-in: https://mc.manuscriptcentral.com/ecohealth Author Instructions: http://www.ecohealth.net/submit.php
Subject Title: PREEMPT call with Peter Hi Tonie,
My name is Luke Hamel and I am a Program Assistant at EcoHealth Alliance, helping to coordinate efforts for DARPA PREEMPT.
As the full project proposal is due March 27th, Peter was hoping to speak with you in order to discuss further details of the proposal and establish a timeline for moving forward.
If you are available, Peter would like to speak with you on either of the following dates:
Thu. 3/1 between 11:30 AM - 3:00 PM (ET) Fri. 3/2 between 1:00 PM - 5:00 PM (ET)
Could you please respond with a time that works for you, or provide an alternative date/time that is more convenient? Thank you very much.
Best,
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct)
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(b) (6)

10/5/21, 3:21 PM
Mail - Rocke, Tonie E - Outlook
(mobile) www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics and promote conservation.
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(b) (6)
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10/5/21, 3:22 PM Mail - Rocke, Tonie E - Outlook
Dear All,
Please see aached, a revised template for the PREEMPT Full Proposal. For the moment, please focus your aenon on Secon G (Statement of Work), as this is where technical informaon for all tasks and subtasks must be detailed. The other secons can remain as they are for now – I’ll edit these.
Please start draing your secons as indicated to expand the details of the prelim data, work plans etc. to fill out the tasks/subtasks for which your instuon has been highlighted. Duke-NUS and Wuhan (Peng and Zhengli) – I’m assuming you’re going to dra things together as possible, so I’ve put the names of both together. If this isn’t correct – please arrange among yourselves to decide who will lead which part.
Regarding the wring of each secon, keep in mind the following:
1. 2. 3. 4. 5.
6. 7.
8.
If you want to add a subtask, or change the tles, please feel free, but make a note (comment box) so we know that you changed it – no need to use ‘track changes’
Include as much detailed, technical informaon as necessary. Don't worry about the page limit. I can remove text as needed, later on.
For each task/subtask that you write, be sure to include preliminary data if applicable (e.g. no. of caves sampled, no. of samples collected, etc.)
Include any relevant figures, tables or charts – the more the beer, we can always delete/edit/shrink down later on
We think the ‘Descripon and execuon’ bullet is DARPA-speak for ‘Research Plan’ or ‘Plan of work’, i.e. where you lay out the strategy, the raonale, and the technical details of how you are going to achieve each goal.
Please put in some ideas for the bullets on ‘Progress metrics’ and ‘deliverables’. We’ll make sure we go back to these aer the first dras are collated, so that we write this in a uniform way that will appeal to DARPA.
Be sure to include any relevant references. Please use EndNote if possible; otherwise send list of references to me (Peter), cc’ing Luke Hamel. Note that some of the references are embedded as links to Pubmed webpages. I’ll be converng these back to Endnote later on, so ignore for now. We can insert as many references as we like because they’re not included in the page length.
Please send dras back to me by Wed. 3rd March (Eastern me – NYC me). Earlier if possible! As soon as they start coming in, I’ll be incorporang text, eding and adding to different secons so we have a good dra by the end of that week.
Addionally, please send me a list of all personnel you plan to include on your team, as soon as you are able. Along with names, please provide (1) Number of months to be commied to project, and (2) % effort for each member of your team (including yourself). This can be approximate right now and don’t worry about this affecng budget – it won’t - we’re going to keep that level as suggested previously...
Cheers,
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10/5/21, 3:22 PM
Mail - Rocke, Tonie E - Outlook
Peter
Peter Daszak President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4474 www.ecohealthalliance.org @PeterDaszak @EcoHealthNYC
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
-----Original Message-----
From: Peter Daszak
Sent: Tuesday, February 27, 2018 2:14 PM
To: Zhengli Shi (zlshi@wh.iov.cn); Ralph Baric (rbaric@email.unc.edu); 周鹏 (peng.zhou@wh.iov.cn); 'Wang Linfa'; Rocke, Tonie
Cc: Danielle Anderson (danielle.anderson@duke-nus.edu.sg); 'aaron.irving@duke-nus.edu.sg'; 'antonee_baric@med.unc.edu'; 'sims0018@email.unc.edu'; Luke Hamel (hamel@ecohealthalliance.org) Subject: For our DARPA PREEMPT conversaons this week: HR001118S0017-PREEMPT-PA-001 Proposal Abstract Status
Importance: High
Dear All,
Good news from DARPA - they like our abstract and we're officially invited for a full proposal. From the aached leer, it looks like they've got a lot of proposals asking for too much $$$, but there are some clear ways we can hedge against any possible cuts. We can talk further about this, and about fleshing out the technical details on our calls this week.
I'm working on scheduling a call with the DARPA team for Thursday of Friday this week - 15 mins to go through how these bullets in the leer above will affect our full proposal. It'll just be me and Luke, but we can think about key quesons to ask them..
Re. the full proposal. Luke has taken the abstract text and started populang the full proposal framework (aached), to give us an idea of what we need to write. It's not a huge effort, but it'll have to be technically sound, but sll tell the overall 'story' that DARPA want to hear - i.e. we can provide proof-of-concept of blocking spillover based on this novel and interesng approach.
Look forward to talking with all of you.
Cheers,
Peter
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10/5/21, 3:22 PM
Mail - Rocke, Tonie E - Outlook
Peter Daszak President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4474
www.ecohealthalliance.org
@PeterDaszak @EcoHealthNYC
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
-----Original Message-----
From: PREEMPT [mailto:PREEMPT@darpa.mil]
Sent: Tuesday, February 27, 2018 8:51 AM
To:
Cc:
Subject: HR001118S0017-PREEMPT-PA-001 Proposal Abstract Status
(b) (6)
Thank you for your interest in the Biological Technologies Office's PREvenng EMerging Pathogenic Threats (PREEMPT) program. Please find your proposal abstract status aached.
(b) (6)
(b) (6)
Regards,
BAA Coordinator
Contractor Support to DARPA/BTO PREEMPT@darpa.mil
(b) (6)
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DARPA – PREEMPT PROPOSAL OUTLINE
NOTE: 36 pages max - 12 pt. font or higher (font can be smaller for tables, charts, figures)
VOLUME I – Technical and Management Proposal
Section I – Administrative
A) Cover Page (labeled “Proposal: Volume I”) B) Official Transmittal Letter
Section II – Detailed Proposal Information
A)
1. What is the proposed work attempting to accomplish or do?
We will defuse the potential for emergence of novel bat-origin high-zoonotic risk SARS- related coronaviruses. We envisage a scenario whereby the US warfighter is deployed to a security hotspot in SE Asia. As planners choose sites for the mission, they will use an app we will design based on machine-learning models of the ecological and evolutionary potential of bat viruses to spillover. This will allow rapid assessment of the background risk of a site harboring dangerous zoonotic viruses. If there is no alternative site, a tactical forward team will deploy automated delivery technology we will develop in caves that harbor bats carrying these viruses. These devices will release broadscale immune boosting molecules and chimeric polyvalent spike protein targeted immune priming treatments to upregulate the naturally damped innate immune response of bats, and lower viral shedding from bats at the site for a few weeks or months, allowing our warfighters to execute the operation at lowered risk for spillover.
2. How is it done today? And what are the limitations?
Other than PPE, there is no available current technology to reduce the risk of exposure to novel coronaviruses from bats. Models of bat host capacity to harbor viruses, of ecological and environmental drivers of their emergence, and of the evolutionary potential of different strains to spillover are rudimentary. No vaccines or therapeutics exist for SARSr-CoVs, and exposure mitigation strategies are non-existent. SARSr-CoVs are endemic in Asian, African (1), and European bats (2) that roost in caves but forage widely at night, shedding virus in their feces and urine. The limitations of this lack of capacity are significant – we have shown evidence of recent spillover of SARSr-CoVs into people in China and have isolated strains capable of producing SARS-like illness in humanized mice that don’t respond to antibody treatment or vaccination. These viruses are a clear-and-present danger to our military and to global health security.
Executive Summary
(questions/answers below from abstract).
Commented [EA1]: Perhaps some of the more detailed information in this section could be moved to the ‘Goals and Impacts’ section

3) What is innovative in your approach?
Our group leads the world in predictive models of viral emergence. We will build on our machine-learning models of spillover hotspots, host-pathogen ecological niches and genotype- phenotype mapping by incorporating unique datasets to validate and refine hotspot risk maps of viral emergence in SE Asia and beyond. Our group has shown that bats coexist with lethal viruses by damping innate immunity pathways, likely as an evolutionary adaptation to flight. We will use this insight to design strategies, like small molecule Rig like receptor (RLR) or Toll like receptor (TLR) agonists, to upregulate bat immunity in their cave roosts, down-regulate viral replication, and reduce the risk of viral shedding and spillover (broadscale immune boosting strategy). We will complement this by treating bats with novel chimeric polyvalent recombinant spike proteins to enhance their immune response against specific, high-risk viruses (targeted immune priming strategy), especially when their immune response is boosted as above. We will design novel methods to deliver these applications remotely to reduce exposure risk during decontamination.
4) What are the key technical challenges in your approach and how do you plan to overcome these?
Modeling: Previous models have suffered from a lack of data to validate them. We have access to unique datasets that will allow us to validate our approach, including biodiversity surveys of bat caves across S. China, 10+ years of bat viral testing data in China, and 10 other countries (from NIH-NIAID and USAID EPT PREDICT work). Uniquely, we will validate our models of viral evolution/spillover risk using human serology (based on LIPS assays) in local populations that have high (~3%) seroprevalence to bat SARSr-CoVs. Identifying Immune boosting and priming treatments: Some of our approaches are novel and challenging (e.g. using CRISPRi to find the negative regulator for bat interferon production), and others are unproven in bats (e.g. Poly IC). We will begin all immune boosting and priming experiments at the beginning of the project, running them simultaneously and competitively, so that we field trial only the most efficient, cost-effective and scalable approaches.
5) Who or what will be affected and what will be the impact?
This will have direct relevance to the warfighter. Potential deployment to regions where SARSr- CoVs exist is high – countries include security hotspots in Asia (e.g. Myanmar, Bangladesh, Pakistan, Korea, Vietnam), Africa and Eastern Europe. The ability to decontaminate and defuse these viruses may prevent potentially devastating illness. These technologies could be adapted to hosts of other bat-origin CoVs (e.g. MERS-CoV, SADS-CoV) and potentially other zoonotic bat- origin viruses (Hendra, Nipah, EBOV), with benefits to livestock production, food security and global public health.
B) ExecutiveSummarySlide(mustuseprovidedtemplate) C)
Commented [EA2]: Detailed text from the ‘Executive Summary’ section may be better suited here
Goals and Impact

• Clearly describe what the team is trying to achieve and the difference it will make (qualitatively and quantitatively) if successful.
• Describe the innovative aspects of the project in the context of existing capabilities and approaches, clearly delineating the uniqueness and benefits of this project in the context of the state of the art, alternative approaches, and other projects from the past and present.
• Describe how the proposed project is revolutionary and how it significantly rises above the current state of the art.
• Describe the deliverables associated with the proposed project and any plans to commercialize the technology, transition it to a customer, or further the work.
Overview
The SARSr-CoV-bat system, and immune modulation focus: Our group’s 15 yrs work on the SARSr-CoV – Rhinolophus bat system in China has identified and isolated SARSr-CoVs with remarkable sequence identity in the spike protein to SARS-CoV (e.g. SCH014 & WIV-1). We have shown they bind and replicate efficiently in primary human lung airway cells and that chimeras with SARSr-CoV spike proteins in a SARS-CoV backbone cause SARS-like illness in humanized mice, with clinical signs that are not reduced by SARS monoclonal therapy or vaccination. We have identified a single cave site in Yunnan Province where bat SARSr-CoVs contain all the genetic components of epidemic SARS-CoV (7,8,9). We have now shown that people living up to 6 kilometers from this cave have SARSr-CoV antibodies (3% seroprevalence in 200+ cohort), suggesting active spillover, and marking these viruses as a clear-and-present danger of a new SARS-like pandemic. Our work on bat immunology suggests that bats’ unique flying ability has led to downregulated innate immune genes, and their ability to coexist with viruses such as SARSr-CoVs, henipa- and filoviruses that are lethal in many other mammals (3). We have identified bat-specific constitutively expressed bat interferon, a dampened STING-interferon production pathway (4, 5), and have identified a series of other innate immunity factors that are dampened in bats (6).
Our bat-CoV system has significant advantages for experimentation and intervention. Firstly, these viruses are fecal-orally transmitted within bat populations, so sampling can be achieved from fresh fecal pellet collection. They are BSL-3, not -4, agents, so that experimental manipulation and infection is simpler. They have frequent spillover events, making it possible to validate predictive models of spillover by sampling people. They are diverse, with frequent recombination and different strains exhibiting differential host cell binding and spillover potential. Finally, we have identified SARSr-CoV strains in a single cave in Yunnan that harbor all of the epidemic SARS-CoV genes. This specific bat population harbors an ideal evolutionary soup that could produce new human strains by high frequency RNA recombination, and thus, it presents a perfect target for next generation, technology-forward intervention strategies.
D) TechnicalPlan
• Outline and address technical challenges inherent in the approach and possible solutions for overcoming potential problems.

• Provide appropriate measurable milestones (quantitative if possible) and program metrics (see “metrics” attachment) at intermediate stages of the program to demonstrate progress, and a plan for achieving the milestones.
• Demonstrate a deep understanding of the technical challenges.
• Present a credible (even if risky) plan to achieve the program goal.
• Discuss mitigation of technical risk.
• Address TA1 and TA2 proposal content requirements (see “objectives” attachment)
TA1: Develop and validate integrated, multiscale models that quantify the likelihood a human-capable virus will emerge from an animal reservoir residing in a “hot spot” geographic region
The DEFUSE modeling and analytics team, led by Drs. Daszak, Ross, Olival, EHA, will build ecological niche models of environmental and ecological correlates and traits of cave bat communities to predict species composition of bat caves across Southern China, South and SE Asia. We will then use a series of datasets we have built to produce host-virus risk models for the region. These include our unique database of bat host-viral relationships (7); biological inventory data on all bat caves in Southern China; and modeled species distribution data for all bats. We will parameterize the model with data from three cave sites in Yunnan, China (one with high-risk SARSr-CoVs, two other control/comparison sites), including: radio- and GPS- telemetry to identify home range and additional roost sites for each bat species; inventory of bat population density, distribution and segregation and their daily, weekly and seasonal changes; viral prevalence and individual viral load; shedding of low- and high-risk SARSr-CoV strains among bat species, age classes, genders; and telemetry and mark-recapture data to assess metapopulation structure and inter-cave connectivity. We will test and validate model predictions of a cave’s viral spillover potential with data from prior PREDICT sampling in 7 other Asian countries. At the end of Yr 1, we will produce a prototype app for the warfighter that identifies the likelihood of bats harboring dangerous viral pathogens in a region. The ‘high-risk
me’ app will be updated real-time with surveillance data (e.g. field-deployable iPhone and android compatible echolocation data) from our project and others, to ground-truth and fine-tune its predictive capacity.
The Wuhan Institute of Virology team will test bat fecal, oral, blood and urogenital samples for SARSr-CoVs. We will correlate viral load data from these samples with fresh fecal pellets from individuals and from tarps laid on cave floors. We will rapidly move to fecal pellet assays to reduce roost disturbance. SARSr-CoV spike proteins will be sequenced, analyzed phylogenetically for recombination events, and high-risk viruses (spike proteins close to SARS- CoV) characterized and isolated. The UNC team will reverse-engineer spike proteins to conduct binding assay to human ACE2 (the SARS-CoV receptor). They will culture SARS-like bat coronaviruses to distinguish high-risk strains that can replicate in primary human cells and low risk strains that require exogenous enhancers. Viral spike glycoproteins that bind receptors will be inserted into SARS-CoV backbones, inoculated into human cells and humanized mice to assess capacity to cause SARS-like disease, and to be blocked by monoclonal therapies, the nucleoside analogue inhibitor GS-5734 (8) or vaccines against SARS-CoV (8,9,10,11,12,13).
The EHA modeling team will use these data to build models of risk of viral evolution and spillover. These genotype-to-phenotype machine-learning models will predict viral ability
Commented [EA3]: SDM with additional set of variables (Carlos thinks this will work)
bats near

to infect host cells based on genetic traits and results of receptor binding and mouse infection assays. Using data on diversity of spike proteins, recombinant CoVs, and flow of genes within each bat cave via bat movement and migration, we will estimate evolutionary rates, rates of recombination, and capacity to generate novel strains capable of human infection. Finally, virus-host relationship and bat home range data will be used to estimate spillover potential - extending models well beyond our field sites. We will then validate model predictions of viral spillover risk by 1) conducting spike protein-based binding and cell culture experiments, and 2) identifying spillover strains in people near our bat cave sites. Our preliminary work on this shows ~3% seroprevalence to SARSr-CoVs, using a specific ELISA (14). We will design LIPS assays to the specific high- and low- zoonotic-risk SARSr-CoVs identified in this project as we have done previously (15). We will use banked and newly collected human sera from these populations to test for presence of antibodies to the high- and low-risk SARSr-CoVs identified by our modeling. We will then model optimal strategies to maximize treatment efficacy for TA2, using stochastic simulation modeling informed by field and experimental data to characterize viral circulation dynamics in bats. We will estimate frequency and population coverage required for our intervention approaches to suppress viral spillover. We will determine the seasons, locations within a cave, and delivery methods (spray, swab, or automated cave mouth or drone) that will be most effective. Finally we will determine the time period treatment will be effective for, until re-colonization or evolution leads to return of a high-risk SARSr-CoV.
TA2: Develop scalable approaches that target and suppress the animal virus in its reservoir(s)and/or vector(s), to reduce the likelihood of virus transmission into humans.
We will evaluate two approaches to defuse SARS-related CoV spillover potential: 1) Broadscale Immune Boosting: using the unique immune damping in bats that our group has discovered, we will apply immune modulators like bat interferon to live bats, to up-regulate their naïve immunity and then assess their ability to suppress viral replication and shedding; 2) Targeted Immune Priming: building on preliminary development of polyvalent chimeric recombinant SARSr-CoV spike proteins, we will conduct application trials with live bats to assess suppression of replication and shedding of a broad range of dangerous SARS-related CoVs.
Both lines of work will begin in Yr 1 and run parallel. Prof. Linfa Wang (Duke-NUS) will lead the immune boosting work, building on his pioneering work on bat immunity (3) which shows that the long-term coexistence of bats and their viruses has led to equilibrium between viral replication and host immunity. This is likely due to down-regulation of their innate immune system as a fitness cost of flight (3). The weakened functionality of bat innate immunity factors like STING, a central DNA-interferon (IFN) sensing molecule, may allow bats to maintain an effective, but not over-response to viruses (4). A similar finding was observed for bat IFNA, which is less abundant but constitutively expressed without stimulation (5). Given high native SARSr-CoV load in bats, we aim to boost bat innate immunity through the IFN pathway, break the host-virus equilibrium to suppress bat SARSr-CoV replication and shedding.
We will trial the following, concurrently and competitively, for efficiency, cost and scalability: i) Universal bat interferon. Aerosol spraying or intranasal application of IFN or other small molecules reduces viral loads in humans, ferrets and mouse models (16, 17). Interferon has been used clinically when antiviral drugs are unavailable, e.g. against filoviruses (18). Replication of SARSr-CoV is sensitive to interferon treatments, as shown in our previous work

(16); ii) Boosting bat IFN by blocking bat-specific IFN negative regulators. Uniquely, bat IFNA is naturally constitutively expressed but cannot be induced to a high level (5), indicating a negative regulatory factor in the bat interferon production pathway. We will use CRISPRi to identify the negative regulator and then screen for compounds targeting this gene; iii) Activating dampened bat-specific IFN production pathways which include DNA-STING- dependent and ssRNA-TLR7-dependent pathways. Our work showing that mutant bat STING restores antiviral functionality suggests these pathways are important in bat-viral coexistence (4). By identifying small molecules to directly activate downstream of STING, we will activate bat interferon and promote viral clearance. A similar strategy will be applied to ssRNA-TLR7- dependent pathways; iv) Activating functional bat IFN production pathways, e.g. polyIC to TLR3- IFN pathway or 5’ppp-dsRNA to RIG-I-IFN pathway. A similar strategy has been demonstrated in a mouse model for SARS-CoV, IAV and HBV (17, 19); v) Inoculating crude coronavirus fragments to upregulate innate immune responses to specific CoVs – a partial step towards the targeted immune priming work below.
Prof. Ralph Baric (UNC) will lead the immune priming work. He will develop recombinant chimeric spike-proteins (20) from our known SARSr-CoVs, and those we characterize during project DEFUSE. The structure of the SARS-CoV spike glycoprotein has been solved and the addition of two proline residues at positions V1060P and L1061P stabilize the prefusion state of the trimer, including key neutralizing epitopes in the receptor binding domain (21). In parallel, the spike trimers or the receptor binding domain can be incorporated into alphavirus vectored or nanoparticle vaccines for delivery, either as aerosols, in baits, or as large droplet delivery vehicles (11, 22,23,24,25). We will test these in controlled lab conditions, taking the best candidate forward for testing in the field. We have built recombinant spike glycoproteins harboring structurally defined domains from SARS epidemic strains, pre-epidemic strains like SCH014 and zoonotic strains like HKU3. It is anticipated that recombinant S glycoprotein based vaccines harboring immunogenic blocks across the group 2B coronaviruses will induce broad scale immune responses that simultaneously reduce genetically heterogeneous virus burdens in bats, potentially reducing disease risk (and transmission risk to people) in these animals for longer periods (26, 27).
The immune dampening features are highly conserved in all bat species tested so far. Duke-NUS has established the only experimental breeding colony of cave bats (Eonycteris spelaea) in SE Asia. This genus is evolutionarily related to Rhinolophus spp. (the hosts of SARSr- CoVs), so we have confidence that results will be transferable. Our initial proof-of-concept tests will be in this experimental colony, extended to a small group of wild-caught Rhinolophus sinicus bats at Wuhan Institute of Zoology. We (Prof. Wang) have previous experience conducting SARS-CoV infection experiments with Rhinolophus sp. bats in the BSL-4 facility at CSIRO, AAHL (L.Wang, unpublished results).
Finally, work on a delivery method for our immune boosting and priming molecules will be overseen by Dr. Tonie Rocke at the USGS, National Wildlife Health Center who has previously developed animal vaccines through to licensure (28). Using locally acquired insectivorous bats (29, 30) we will assess delivery vehicles and methods including: 1) transdermally applied nanoparticles; 2) series of sticky edible gels that bats will groom from themselves and each other; 3) aerosolization via sprayers that could be used in cave settings; 4) automated sprays triggered by timers and movement detectors at critical cave entry points,

and 5) sprays delivered by remote controlled drone. We have already used simple gels to vaccinate bats against rabies in the lab (29), and hand delivered these containing biomarkers to vampire bats in Peru and Mexico to show they are readily consumed and transferred among bats. In our bat colony, we will trial delivery vehicles using the biomarker rhodamine B (which marks hair and whiskers upon consumption) to assess uptake. The most optimal approaches will then be tested on wild bats in our three cave sites in Yunnan Province with the most successful immunomodulators from TA2. Fieldwork will be conducted under the auspices of Dr. Yunzhi Zhang (Yunnan CDC, Consultant at EcoHealth Alliance). A small number of bats will be captured and assayed for viral load and immune function after treatment, but so as not to disturb the colony, most viral load work will be conducted on fresh fecal pellets collected daily on the cave floor. EHA has had unique access to these sites for around 10 years, under the guidance of Drs. Shi and Zhang. In year 1 of project DEFUSE, we will seek permission for experimental trials from the Provincial Forestry Department. We expect to be successful, as we have worked with the Forestry Department collaboratively for 10 years, with support of the Yunnan CDC, and we are releasing molecules that are not dangerous to people or wildlife. EHA has a proven track record of rapidly obtaining IACUC and DoD ACURO approval for bat research.
E) Management Plan
• Provide a summary of expertise of the team, including any subcontractors, and key personnel who will be doing the work. Resumes count against the page count.
• Identify a principal investigator for the project.
• Provide a clear description of the team’s organization
• Include an organization chart with the following information, as applicable: A) Programmatic relationship of team members
B) Unique capabilities of team members
C) Task responsibilities of team members
D) Teaming strategy among the team members
E) Key personnel with amount of effort to be expended by each during each year
• Provide a detailed plan for coordination including explicit guidelines for interaction among collaborators/subcontractors of the proposed effort.
• Include risk management approaches.
• Describe any formal teaming agreements that are required to execute this program.
The lead institution for Project DEFUSE is EcoHealth Alliance, New York, an international research organization focused on emerging zoonotic diseases. The PI, Dr. Peter Daszak, has 25+ years’ experience managing lab, field and modeling research projects on emerging zoonoses.

Dr. Daszak will commit 3 months annually to oversee and coordinate all project activities, and lead modeling and analytic work for TA1. Dr. Billy Karesh has 40+ years’ experience leading zoonotic and wildlife disease projects, and will commit 1 month annually to manage partnership activities and outreach. Dr. Jon Epstein, with 15 years’ experience working emerging bat zoonoses will coordinate animal trials. Dr. Kevin Olival and Dr. Noam Ross will manage and conduct the modeling and analytical approaches for this project. Support staff include field surveillance teams, modeling analysts, and consultants based in Yunnan Province, China, to oversee field trials. The EHA team has worked extensively with all other collaborators: Prof. Wang (15+ years); Dr. Shi (15+ years); Prof. Baric (5+ years) and Dr. Rocke (15+ years). Subcontracts: #1 to Prof. Ralph Baric, UNC, to oversee reverse engineering of SARSr-CoVs, BSL- 3 humanized mouse experimental infections, design and testing of immune priming treatments based on recombinant spike proteins. Assisted by senior personnel Dr. Tim Sheahan, Dr. Amy Sims, and support staff; #2 to Prof. Linfa Wang, Duke NUS, to oversee the immune boosting approach, captive bat experiments, and analyze immunological and virological responses to immune boosting treatments; #3 to Dr. Zhengli Shi, Wuhan Institute of Virology, to conduct PCR testing, viral discovery and isolation from bat samples collected in China, spike protein binding assays, and some humanized mouse work, as well as experimental trials on Rhinolophus bats. Her team will include Dr. Peng Zhou and support staff; #4 to Dr. Tonie Rocke, USGS National Wildlife Health Center, to refine delivery mechanisms for both immune boosting and immune priming treatments. With a research technician, Dr. Rocke will use a captive colony of bats at NWHC for initial trials, and oversee cave experiments in China.
Dr. Peter Daszak is President and Chief Scientist of EcoHealth Alliance, a US-based research organization focused on emerging zoonotic diseases. His >300 scientific papers include the first global map of EID hotspots (31, 32), estimates of unknown viral diversity (33), predictive models of virus-host relationships (7), and evidence of the bat origin of SARS-CoV (34, 35) and other emerging viruses (36,37,38,39). He is Chair of the NASEM Forum on Microbial Threats, and is a member of the Executive Committee and the EHA institutional lead for the $130 million USAID- EPT-PREDICT. He serves on the NRC Advisory Committee to the USGCRP, the DHS CEEZAD External Advisory Board, the WHO R&D Blueprint Pathogen Prioritization expert group, and has advised the Director for Medical Preparedness Policy on the White House National Security Staff on global health issues. Dr. Daszak won the 2000 CSIRO medal for collaborative research.
Prof. Ralph Baric is a UNC Lineberger Comprehensive Cancer Center member and Professor in the UNC-Chapel Hill Dept. of Epidemiology and Dept. of Microbiology & Immunology. His work focuses on coronaviruses as models to study the genetics of RNA virus transcription, replication, persistence, cross species transmission and pathogenesis. His group has developed a platform strategy to access the potential “pre-epidemic” risk associated with zoonotic virus cross species transmission potential and evaluation of countermeasure potential to control future outbreaks of disease (8,9,10,11,12,13).
Prof. Linfa Wang is a
Prof. Zhengli Shi has over xx years experience....

Dr. Tonie Rocke is a
Dr. Peng Zhou is a
Dr. Danielle Anderson is a
Please follow the same format and create Bios for all other personnel with Ph.D and higher. Peter Daszak will then work out how much space we have and decide who to include...
F) Capabilities
• Describe organizational experience in relevant subject area(s), existing intellectual property, specialized facilities, and any Government-furnished materials or information.
• Discuss any work in closely related research areas and previous accomplishments.
The SARSr-CoV-bat system, and immune modulation focus: Our group’s 15 yrs work on the SARSr-CoV – Rhinolophus bat system in China has identified and isolated SARSr-CoVs with remarkable sequence identity in the spike protein to SARS-CoV (e.g. SCH014 & WIV-1). We have shown they bind and replicate efficiently in primary human lung airway cells and that chimeras with SARSr-CoV spike proteins in a SARS-CoV backbone cause SARS-like illness in humanized mice, with clinical signs that are not reduced by SARS monoclonal therapy or vaccination. We have identified a single cave site in Yunnan Province where bat SARSr-CoVs contain all the genetic components of epidemic SARS-CoV (7,8,9). We have now shown that people living up to 6 kilometers from this cave have SARSr-CoV antibodies (3% seroprevalence in 200+ cohort), suggesting active spillover, and marking these viruses as a clear-and-present danger of a new SARS-like pandemic. Our work on bat immunology suggests that bats’ unique flying ability has led to downregulated innate immune genes, and their ability to coexist with viruses such as SARSr-CoVs, henipa- and filoviruses that are lethal in many other mammals (3). We have identified bat-specific constitutively expressed bat interferon, a dampened STING-interferon production pathway (4, 5), and have identified a series of other innate immunity factors that are dampened in bats (6).
G) StatementofWork(SOW)
• Provide a detailed task breakdown, citing specific tasks and their connection to the interim milestones and program metrics.
(The following information was taken from the ‘Goals and Impact’ section of the abstract we
submitted).


in
NOTE: The SOW must not include proprietary information.
• For each task/subtask, provide:
o A detailed description of the approach to be taken to accomplish each defined
task/subtask.
o Identification of the primary organization responsible for task execution (prime contractor, subcontractor(s), consultant(s), by name).
o A measurable milestone, i.e., a deliverable, demonstration, or other event/activity that marks task completion. Include quantitative metrics.
o A definition of all deliverables (e.g., data, reports, software) to be provided to the Government in support of the proposed tasks/subtasks.
Phase I:
(TA1) Task 1: Collect ecological and environmental data from Yunnan Province cave sites. Description and execution:
Preliminary Data:
Organization leading task: EcoHealth Alliance Progress Metrics:
Deliverable(s):
(TA1) Task 2: Construct niche models to predict species composition of bat caves across South and Southeast Asia
Description and execution: Preliminary Data:
Organization leading task: EcoHealth Alliance Progress Metrics:
Deliverable(s):
Each phase of the program (Phase I base and Phase II option) should be separately defined
the SOW and each task should be identified by TA (1 or 2).

(TA1) Subtask 2.1: Construct, test and validate host-pathogen risk models Description and execution:
Preliminary Data:
Organization leading task: EcoHealth Alliance Progress Metrics:
Deliverable(s):
(TA1) Subtask 2.2: Develop prototype app for the warfighter Description and execution:
Preliminary Data:
Organization leading task: EcoHealth Alliance Progress Metrics:
Deliverable(s):
(TA1) Task 3: Analyze bat samples for SARSr-CoVs Description and execution:
Preliminary Data:
Organization leading task: Wuhan Institute of Virology, Duke-NUS Progress Metrics:
Deliverable(s):
(TA1) Subtask 3.1: Characterize and isolate high-risk SARSr-CoV quasispecies Description and execution:
Preliminary Data:
Organization leading task: Wuhan Institute of Virology, Duke-NUS

Progress Metrics: Deliverable(s):
(TA1) Task 4: Develop recombinant chimeric spike proteins from characterized SARSr-CoVs
Description and execution: Preliminary Data:
Organization leading task: University of North Carolina Progress Metrics:
Deliverable(s):
(TA2) Task 5: Trial experimental approaches aimed towards ‘Broadscale Immune Boosting’ using experimental bat colonies
Description and execution: Preliminary Data:
Organization leading task: Wuhan Institute of Virology, Duke-NUS Progress Metrics:
Deliverable(s):
(TA2) Task 6: Trial experimental approaches aimed towards ‘Immune Targeting’ using experimental bat colonies
Description and execution: Preliminary Data:
Organization leading task: University of North Carolina Progress Metrics:
Deliverable(s):

(TA2) Task 7: Develop and assess delivery methods for immune boosting and priming molecules
Description and execution: Preliminary Data:
Organization leading task: USGS National Wildlife Health Center Progress Metrics:
Deliverable(s):
(TA1) Task 8: Construct genotype-to-phenotype machine learning models to predict risk of viral evolution and spillover
Description and execution: Preliminary Data:
Organization leading task: EcoHealth Alliance Progress Metrics:
Deliverable(s):
Phase II:
(TA1) Task 9: Design LIPS assays to specific high- and low- zoonotic risk SARSr-CoVs
Description and execution: Preliminary Data:
Organization leading task: Wuhan Institute of Virology, Duke-NUS Progress Metrics:
Deliverable(s):
(TA1) Task 10: Validate model predictions of viral spillover risk using data from LIPS

assays and banked human sera
Description and execution: Preliminary Data:
Organization leading task: EcoHealth Alliance Progress Metrics:
Deliverable(s):
(TA1) Task 11: Test efficacy of intervention approaches on wild-caught Rhinolophus bats
Description and execution: Preliminary Data:
Organization leading task: Wuhan Institute of Virology, Duke-NUS Progress Metrics:
Deliverable(s):
(TA1) Task 12: Construct models to maximize efficacy of intervention approaches Description and execution:
Preliminary Data:
Organization leading task: EcoHealth Alliance Progress Metrics:
Deliverable(s):
(TA2) Task 13: Deploy most effective molecule delivery methods on bat colonies of Yunnan Province caves
Description and execution: Preliminary Data:

Organization leading task: EcoHealth Alliance Progress Metrics:
Deliverable(s):
• (task name, duration, work breakdown structure element as applicable, performing organization), milestones, and the interrelationships among tasks.
NOTE: Task structure must be consistent with that in the SOW.
• Measurable milestones should be clearly articulated and defined in time relative to the start of the project.
• Indicate the types of partners (e.g., government, private industry, non-profit)
• Submit a timeline with incremental milestones toward successful engagement.
NOTE: begin transition activities during the early stages of the program (Phase I).
• Describe any potential DARPA roles.
J) PREEMPT Risk Mitigation Plan
• Provide the following:
o An assessment of potential risks to public health, agriculture, plants, animals, the
environment, and national security.
o Guidelines the proposer will follow to ensure maximal biosafety and biosecurity.
o A communication plan that addresses content, timing, and the extent of distribution of potentially sensitive dual-use information. The plan must also address how input from DARPA, other government, and community stakeholders will be taken into account in decisions regarding communication and publication of potentially sensitive dual-use information.
K) Ethical, Legal, Societal Implications (ELSI)
• Address potential ethical, legal, and societal implications of the proposed technology.
H) ScheduleandMilestones
Provide a detailed schedule showing tasks
I) PREEMPT
Transition Plan
Commented [EA4]: Description from the BAA:
PREEMPT Transition Plan
Proposers must include a PREEMPT Technology Transition Plan. Proposers must indicate the types of partners (e.g., government, private industry, non-profit) they plan to pursue and submit a timeline with incremental milestones toward successful engagement. Proposers should begin transition activities during the early stages of the program (Phase I). Awardees must include
DARPA in the development of transition relationships. If the transition plan includes a start-up company, a business development strategy must be included as well. The extent by which the proposed intellectual property (IP) rights will impede the Government’s ability to transition the technology will be considered in the proposal evaluation.

Section III – Additional Information (doesn’t count against 36 pg. limit)
A) Brief Bibliography (no page limit indicated – can be published/unpublished)
B) Up to 3 relevant papers attached (optional)

10/5/21, 3:23 PM Mail - Rocke, Tonie E - Outlook
1.
Linfa; Rocke, Tonie
Danielle Anderson (danielle.anderson@duke-nus.edu.sg); aaron.irving@duke-nus.edu.sg; Baric, Toni C;
sims0018@email.unc.edu; Luke Hamel; William B. Karesh
RE: For our DARPA PREEMPT conversations this week: HR001118S0017-PREEMPT-PA-001 Proposal Abstract
Status
Dear All,
Please see aached, a revised template for the PREEMPT Full Proposal. For the moment, please focus your aenon on Secon G (Statement of Work), as this is where technical informaon for all tasks and subtasks must be detailed. The other secons can remain as they are for now – I’ll edit these.
Please start draing your secons as indicated to expand the details of the prelim data, work plans etc. to fill out the tasks/subtasks for which your instuon has been highlighted. Duke-NUS and Wuhan (Peng and Zhengli) – I’m assuming you’re going to dra things together as possible, so I’ve put the names of both together. If this isn’t correct – please arrange among yourselves to decide who will lead which part.
Regarding the wring of each secon, keep in mind the following:
If you want to add a subtask, or change the tles, please feel free, but make a note (comment box) so we
know that you changed it – no need to use ‘track changes’
Great! Thanks.
SHI Zhengli, Ph. D
Senior Scientist & Professor
Wuhan Institute of Virology, Chinese Academy of Sciences 44 Xiao Hong Shan
430071 Wuhan, Hubei
China
Tel & Fax: (0086) 27 87197240
Email: zlshi@wh.iov.cn
Peter Daszak
2018-03-01 14:00
Zhengli Shi (zlshi@wh.iov.cn); Ralph Baric (rbaric@email.unc.edu);
(peng.zhou@wh.iov.cn); Wang
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/4
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10/5/21, 3:23 PM Mail - Rocke, Tonie E - Outlook
2. 3. 4. 5.
6. 7.
8.
Include as much detailed, technical informaon as necessary. Don't worry about the page limit. I can remove text as needed, later on.
For each task/subtask that you write, be sure to include preliminary data if applicable (e.g. no. of caves sampled, no. of samples collected, etc.)
Include any relevant figures, tables or charts – the more the beer, we can always delete/edit/shrink down later on
We think the ‘Descripon and execuon’ bullet is DARPA-speak for ‘Research Plan’ or ‘Plan of work’, i.e. where you lay out the strategy, the raonale, and the technical details of how you are going to achieve each goal.
Please put in some ideas for the bullets on ‘Progress metrics’ and ‘deliverables’. We’ll make sure we go back to these aer the first dras are collated, so that we write this in a uniform way that will appeal to DARPA.
Be sure to include any relevant references. Please use EndNote if possible; otherwise send list of references to me (Peter), cc’ing Luke Hamel. Note that some of the references are embedded as links to Pubmed webpages. I’ll be converng these back to Endnote later on, so ignore for now. We can insert as many references as we like because they’re not included in the page length.
Please send dras back to me by Wed. 3rd March (Eastern me – NYC me). Earlier if possible! As soon as they start coming in, I’ll be incorporang text, eding and adding to different secons so we have a good dra by the end of that week.
Addionally, please send me a list of all personnel you plan to include on your team, as soon as you are able. Along with names, please provide (1) Number of months to be commied to project, and (2) % effort for each member of your team (including yourself). This can be approximate right now and don’t worry about this affecng budget – it won’t - we’re going to keep that level as suggested previously...
Cheers, Peter
Peter Daszak President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4474 www.ecohealthalliance.org @PeterDaszak @EcoHealthNYC
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 2/4

10/5/21, 3:23 PM Mail - Rocke, Tonie E - Outlook
promote conservaon.
-----Original Message-----
From: Peter Daszak
Sent: Tuesday, February 27, 2018 2:14 PM
To: Zhengli Shi (zlshi@wh.iov.cn); Ralph Baric (rbaric@email.unc.edu); 周鹏 (peng.zhou@wh.iov.cn); 'Wang Linfa'; Rocke, Tonie
Cc: Danielle Anderson (danielle.anderson@duke-nus.edu.sg); 'aaron.irving@duke-nus.edu.sg'; 'antonee_baric@med.unc.edu'; 'sims0018@email.unc.edu'; Luke Hamel (hamel@ecohealthalliance.org) Subject: For our DARPA PREEMPT conversaons this week: HR001118S0017-PREEMPT-PA-001 Proposal Abstract Status
Importance: High
Dear All,
Good news from DARPA - they like our abstract and we're officially invited for a full proposal. From the aached leer, it looks like they've got a lot of proposals asking for too much $$$, but there are some clear ways we can hedge against any possible cuts. We can talk further about this, and about fleshing out the technical details on our calls this week.
I'm working on scheduling a call with the DARPA team for Thursday of Friday this week - 15 mins to go through how these bullets in the leer above will affect our full proposal. It'll just be me and Luke, but we can think about key quesons to ask them..
Re. the full proposal. Luke has taken the abstract text and started populang the full proposal framework (aached), to give us an idea of what we need to write. It's not a huge effort, but it'll have to be technically sound, but sll tell the overall 'story' that DARPA want to hear - i.e. we can provide proof-of- concept of blocking spillover based on this novel and interesng approach.
Look forward to talking with all of you. Cheers,
Peter
Peter Daszak President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4474
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 3/4

10/5/21, 3:23 PM Mail - Rocke, Tonie E - Outlook
www.ecohealthalliance.org @PeterDaszak @EcoHealthNYC
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
-----Original Message-----
From: PREEMPT [mailto:PREEMPT@darpa.mil]
Sent: Tuesday, February 27, 2018 8:51 AM
To:
Cc:
Subject: HR001118S0017-PREEMPT-PA-001 Proposal Abstract Status
(b) (6)
Thank you for your interest in the Biological Technologies Office's PREvenng EMerging Pathogenic Threats (PREEMPT) program. Please find your proposal abstract status aached.
Regards,
BAA Coordinator
Contractor Support to DARPA/BTO PREEMPT@darpa.mil
(b) (6)
(b) (6)
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 4/4

10/5/21, 3:23 PM Mail - Rocke, Tonie E - Outlook
Thanks
Will start work on this from weekend. In London for the WHO NiV meeng now. LF
Linfa (Lin-Fa) WANG, PhD FTSE
Professor & Director
Programme in Emerging Infecous Disease Duke-NUS Medical School,
8 College Road, Singapore 169857
Tel: +65 6516 8397
From: Peter Daszak [mailto:daszak@ecohealthalliance.org]
Sent: Thursday, 1 March, 2018 2:01 PM
To: Zhengli Shi (zlshi@wh.iov.cn); Ralph Baric (rbaric@email.unc.edu); 周鹏 (peng.zhou@wh.iov.cn); Wang Linfa; Rocke, Tonie
Cc: Danielle Anderson; Aaron Trent Irving; Baric, Toni C; sims0018@email.unc.edu; Luke Hamel; William B. Karesh Subject: RE: For our DARPA PREEMPT conversations this week: HR001118S0017-PREEMPT-PA-001 Proposal Abstract Status
Importance: High
Dear All,
Please see aached, a revised template for the PREEMPT Full Proposal. For the moment, please focus your aenon on Secon G (Statement of Work), as this is where technical informaon for all tasks and subtasks must be detailed. The other secons can remain as they are for now – I’ll edit these.
Please start draing your secons as indicated to expand the details of the prelim data, work plans etc. to fill out the tasks/subtasks for which your instuon has been highlighted. Duke-NUS and Wuhan (Peng and Zhengli) – I’m assuming you’re going to dra things together as possible, so I’ve put the names of both together. If this isn’t correct – please arrange among yourselves to decide who will lead which part.
Regarding the wring of each secon, keep in mind the following:
1. If you want to add a subtask, or change the tles, please feel free, but make a note (comment box) so we
know that you changed it – no need to use ‘track changes’
2. Include as much detailed, technical informaon as necessary. Don't worry about the page limit. I can
remove text as needed, later on.
3. For each task/subtask that you write, be sure to include preliminary data if applicable (e.g. no. of caves
sampled, no. of samples collected, etc.)
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/4
>gro.ecnaillahtlaehoce@hserak<hseraK.BmailliW;>gro.ecnaillahtlaehoce@lemah<
lemaHekuL;>ude.cnu.liame@8100smis<ude.cnu.liame@8100smis;>ude.cnu.dem@cirab_etteniotna<CinoT,ciraB
;>gs.ude.sun-ekud@gnivri.noraa<gnivrItnerTnoraA;>gs.ude.sun-ekud@nosredna.elleinad<nosrednAelleinaD:cC
>vog.sgsu@ekcort<
EeinoT,ekcoR;>nc.voi.hw@uohz.gnep<)nc.voi.hw@uohz.gnep(鹏周;>ude.cnu.liame@cirabr<)ude.cnu.liame@cirabr(
ciraBhplaR;>nc.voi.hw@ihslz<)nc.voi.hw@ihslz(ihSilgnehZ;>gro.ecnaillahtlaehoce@kazsad<kazsaDreteP:oT
>gs.ude.sun-ekud@gnaw.afnil<MAa5f2n2i18L10g2/1n/3auhWT
sutatS tcartsbA lasoporP 100-AP
-TPMEERP-7100S811100RH :keew siht snoitasrevnoc TPMEERP APRAD ruo roF :ER

10/5/21, 3:23 PM
4. 5.
6. 7.
8.
Mail - Rocke, Tonie E - Outlook
Include any relevant figures, tables or charts – the more the beer, we can always delete/edit/shrink down later on
We think the ‘Descripon and execuon’ bullet is DARPA-speak for ‘Research Plan’ or ‘Plan of work’, i.e. where you lay out the strategy, the raonale, and the technical details of how you are going to achieve each goal.
Please put in some ideas for the bullets on ‘Progress metrics’ and ‘deliverables’. We’ll make sure we go back to these aer the first dras are collated, so that we write this in a uniform way that will appeal to DARPA.
Be sure to include any relevant references. Please use EndNote if possible; otherwise send list of references to me (Peter), cc’ing Luke Hamel. Note that some of the references are embedded as links to Pubmed webpages. I’ll be converng these back to Endnote later on, so ignore for now. We can insert as many references as we like because they’re not included in the page length.
Please send dras back to me by Wed. 3rd March (Eastern me – NYC me). Earlier if possible! As soon as they start coming in, I’ll be incorporang text, eding and adding to different secons so we have a good dra by the end of that week.
Addionally, please send me a list of all personnel you plan to include on your team, as soon as you are able. Along with names, please provide (1) Number of months to be commied to project, and (2) % effort for each member of your team (including yourself). This can be approximate right now and don’t worry about this affecng budget – it won’t - we’re going to keep that level as suggested previously...
Cheers, Peter
Peter Daszak President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4474
www.ecohealthalliance.org
@PeterDaszak @EcoHealthNYC
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
-----Original Message-----
From: Peter Daszak
Sent: Tuesday, February 27, 2018 2:14 PM
To: Zhengli Shi (zlshi@wh.iov.cn); Ralph Baric (rbaric@email.unc.edu); 周鹏 (peng.zhou@wh.iov.cn); 'Wang Linfa'; Rocke, Tonie
Cc: Danielle Anderson (danielle.anderson@duke-nus.edu.sg); 'aaron.irving@duke-nus.edu.sg'; 'antonee_baric@med.unc.edu'; 'sims0018@email.unc.edu'; Luke Hamel (hamel@ecohealthalliance.org) Subject: For our DARPA PREEMPT conversaons this week: HR001118S0017-PREEMPT-PA-001 Proposal Abstract
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 2/4

10/5/21, 3:23 PM Mail - Rocke, Tonie E - Outlook
Status Importance: High
Dear All,
Good news from DARPA - they like our abstract and we're officially invited for a full proposal. From the aached leer, it looks like they've got a lot of proposals asking for too much $$$, but there are some clear ways we can hedge against any possible cuts. We can talk further about this, and about fleshing out the technical details on our calls this week.
I'm working on scheduling a call with the DARPA team for Thursday of Friday this week - 15 mins to go through how these bullets in the leer above will affect our full proposal. It'll just be me and Luke, but we can think about key quesons to ask them..
Re. the full proposal. Luke has taken the abstract text and started populang the full proposal framework (aached), to give us an idea of what we need to write. It's not a huge effort, but it'll have to be technically sound, but sll tell the overall 'story' that DARPA want to hear - i.e. we can provide proof-of-concept of blocking spillover based on this novel and interesng approach.
Look forward to talking with all of you. Cheers,
Peter
Peter Daszak President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4474
www.ecohealthalliance.org
@PeterDaszak @EcoHealthNYC
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
-----Original Message-----
From: PREEMPT [mailto:PREEMPT@darpa.mil]
Sent: Tuesday, February 27, 2018 8:51 AM
To:
Cc:
Subject: HR001118S0017-PREEMPT-PA-001 Proposal Abstract Status
(b) (6)
Thank you for your interest in the Biological Technologies Office's PREvenng EMerging Pathogenic Threats (PREEMPT) program. Please find your proposal abstract status aached.
(b) (6)
(b) (6)
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 3/4

10/5/21, 3:23 PM Mail - Rocke, Tonie E - Outlook
Regards,
BAA Coordinator
Contractor Support to DARPA/BTO PREEMPT@darpa.mil
Important: This email is confidential and may be privileged. If you are not the intended recipient, please delete it and notify us immediately; you should not copy or use it for any purpose, nor disclose its contents to any other person. Thank you.
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10/5/21, 3:23 PM Mail - Rocke, Tonie E - Outlook
I have included Tim in the email chain. Ralph
From: Peter Daszak [mailto:daszak@ecohealthalliance.org]
Sent: Thursday, March 1, 2018 1:01 AM
To: Zhengli Shi (zlshi@wh.iov.cn) <zlshi@wh.iov.cn>; Baric, Ralph S <rbaric@email.unc.edu>; 周鹏 (peng.zhou@wh.iov.cn) <peng.zhou@wh.iov.cn>; Wang Linfa <linfa.wang@duke-nus.edu.sg>; Rocke, Tonie <trocke@usgs.gov>
Cc: Danielle Anderson (danielle.anderson@duke-nus.edu.sg) <danielle.anderson@duke-nus.edu.sg>; aaron.irving@duke-nus.edu.sg; Baric, Toni C <antoinee_baric@med.unc.edu>; Sims, Amy C <sims0018@email.unc.edu>; Luke Hamel <hamel@ecohealthalliance.org>; William B. Karesh <karesh@ecohealthalliance.org>
Subject: RE: For our DARPA PREEMPT conversaons this week: HR001118S0017-PREEMPT-PA-001 Proposal Abstract Status
Importance: High
Dear All,
Please see aached, a revised template for the PREEMPT Full Proposal. For the moment, please focus your aenon on Secon G (Statement of Work), as this is where technical informaon for all tasks and subtasks must be detailed. The other secons can remain as they are for now – I’ll edit these.
Please start draing your secons as indicated to expand the details of the prelim data, work plans etc. to fill out the tasks/subtasks for which your instuon has been highlighted. Duke-NUS and Wuhan (Peng and Zhengli) – I’m assuming you’re going to dra things together as possible, so I’ve put the names of both together. If this isn’t correct – please arrange among yourselves to decide who will lead which part.
Regarding the wring of each secon, keep in mind the following:
1. 2. 3. 4. 5.
6.
If you want to add a subtask, or change the tles, please feel free, but make a note (comment box) so we know that you changed it – no need to use ‘track changes’
Include as much detailed, technical informaon as necessary. Don't worry about the page limit. I can remove text as needed, later on.
For each task/subtask that you write, be sure to include preliminary data if applicable (e.g. no. of caves sampled, no. of samples collected, etc.)
Include any relevant figures, tables or charts – the more the beer, we can always delete/edit/shrink down later on
We think the ‘Descripon and execuon’ bullet is DARPA-speak for ‘Research Plan’ or ‘Plan of work’, i.e. where you lay out the strategy, the raonale, and the technical details of how you are going to achieve each goal.
Please put in some ideas for the bullets on ‘Progress metrics’ and ‘deliverables’. We’ll make sure we go back to these aer the first dras are collated, so that we write this in a uniform way that will appeal to DARPA.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/4
>gro.ecnaillahtlaehoce@hserak<
hseraK.BmailliW;>gro.ecnaillahtlaehoce@lemah<lemaHekuL;>ude.cnu.liame@8100smis<CymA
,smiS;>ude.cnu.dem@cirab_etteniotna<CinoT,ciraB;>gs.ude.sun-ekud@gnivri.noraa<gs.ude.sun-ekud@gnivri.noraa
;>gs.ude.sun-ekud@nosredna.elleinad<)gs.ude.sun-ekud@nosredna.elleinad(nosrednAelleinaD:cC
>ude.cnu.liame@nahaehs<kcirtaPyhtomiT,nahaehS;>vog.sgsu@ekcort<
EeinoT,ekcoR;>gs.ude.sun-ekud@gnaw.afnil<afniLgnaW;>nc.voi.hw@uohz.gnep<)nc.voi.hw@uohz.gnep(
鹏周;>nc.voi.hw@ihslz<)nc.voi.hw@ihslz(ihSilgnehZ;>gro.ecnaillahtlaehoce@kazsad<kazsaDreteP:oT
>ude.cnu.liame@cirabr< SMhAp8l3a01R81,02c/i2r/3airBF
sutatS tcartsbA lasoporP 100-AP
-TPMEERP-7100S811100RH :keew siht snoitasrevnoc TPMEERP APRAD ruo roF :ER

10/5/21, 3:23 PM
7.
8.
Mail - Rocke, Tonie E - Outlook
Be sure to include any relevant references. Please use EndNote if possible; otherwise send list of references to me (Peter), cc’ing Luke Hamel. Note that some of the references are embedded as links to Pubmed webpages. I’ll be converng these back to Endnote later on, so ignore for now. We can insert as many references as we like because they’re not included in the page length.
Please send dras back to me by Wed. 3rd March (Eastern me – NYC me). Earlier if possible! As soon as they start coming in, I’ll be incorporang text, eding and adding to different secons so we have a good dra by the end of that week.
Addionally, please send me a list of all personnel you plan to include on your team, as soon as you are able. Along with names, please provide (1) Number of months to be commied to project, and (2) % effort for each member of your team (including yourself). This can be approximate right now and don’t worry about this affecng budget – it won’t - we’re going to keep that level as suggested previously...
Cheers, Peter
Peter Daszak President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4474
www.ecohealthalliance.org
@PeterDaszak @EcoHealthNYC
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
-----Original Message-----
From: Peter Daszak
Sent: Tuesday, February 27, 2018 2:14 PM
To: Zhengli Shi (zlshi@wh.iov.cn); Ralph Baric (rbaric@email.unc.edu); 周鹏 (peng.zhou@wh.iov.cn); 'Wang Linfa'; Rocke, Tonie
Cc: Danielle Anderson (danielle.anderson@duke-nus.edu.sg); 'aaron.irving@duke-nus.edu.sg'; 'antonee_baric@med.unc.edu'; 'sims0018@email.unc.edu'; Luke Hamel (hamel@ecohealthalliance.org) Subject: For our DARPA PREEMPT conversaons this week: HR001118S0017-PREEMPT-PA-001 Proposal Abstract Status
Importance: High
Dear All,
Good news from DARPA - they like our abstract and we're officially invited for a full proposal. From the aached leer, it looks like they've got a lot of proposals asking for too much $$$, but there are some clear ways we can hedge against any possible cuts. We can talk further about this, and about fleshing out the technical details on our calls this week.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 2/4

10/5/21, 3:23 PM Mail - Rocke, Tonie E - Outlook
I'm working on scheduling a call with the DARPA team for Thursday of Friday this week - 15 mins to go through how these bullets in the leer above will affect our full proposal. It'll just be me and Luke, but we can think about key quesons to ask them..
Re. the full proposal. Luke has taken the abstract text and started populang the full proposal framework (aached), to give us an idea of what we need to write. It's not a huge effort, but it'll have to be technically sound, but sll tell the overall 'story' that DARPA want to hear - i.e. we can provide proof-of-concept of blocking spillover based on this novel and interesng approach.
Look forward to talking with all of you. Cheers,
Peter
Peter Daszak President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4474
www.ecohealthalliance.org
@PeterDaszak @EcoHealthNYC
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
-----Original Message-----
From: PREEMPT [mailto:PREEMPT@darpa.mil]
Sent: Tuesday, February 27, 2018 8:51 AM
To:
Cc:
Subject: HR001118S0017-PREEMPT-PA-001 Proposal Abstract Status
(b) (6)
Thank you for your interest in the Biological Technologies Office's PREvenng EMerging Pathogenic Threats (PREEMPT) program. Please find your proposal abstract status aached.
Regards,
BAA Coordinator
Contractor Support to DARPA/BTO PREEMPT@darpa.mil
(b) (6)
(b) (6)
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 3/4

10/5/21, 3:23 PM Mail - Rocke, Tonie E - Outlook
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 4/4

10/5/21, 3:24 PM Mail - Rocke, Tonie E - Outlook
Call between EHA & Wuhan/Duke-NUS
**Action Items:
PD to have weekly calls w/ collaborators LH to organize these calls
LH to send an email confirming: DUNS nos., addresses and phone numbers of each collaborating institution.
The email will also provide instructions for how to sign up on Grants.gov (required for Key
Personnel of each collaborating organization)
PD to sit down with KJO, JHE and LH to determine who will be responsible for each section of the proposal. Will send to collaborators by Thu. 3/1
Collaborators to then write their respective sections and return to PD by Wed. 3/7
Peng to include section about how both Immune Boosting and Immune Targeting approaches are better than vaccination (without explicitly criticizing vaccination approach)
PD to speak with YunZhi regarding consultant field work in/around Yunnan cave
Collaborators to determine if any additional personnel required for their work (e.g. technicians, field support staff, etc.)
Collaborators to let PD know if they think anyone else should be brought on to the team -PD to determine which aspects of full proposal are baseline tasks, and which are add-ons
(by mid to late March)
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/2
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10/5/21, 3:24 PM Mail - Rocke, Tonie E - Outlook
Call between EHA & Ralph/Tim (UNC)
to draft their section and return to PD by Wed. 3/7
RB to include section stating how our project won’t drive viral evolution in negative way
PD to complete quality first draft by end of next week, Fri. 3/9
(EHA) and (UNC) to work together on UNC budget
-Once EHA has drafted modeling section of proposal, RB to provide input
to begin work on her section of the full proposal (and send to PD by Wed. 3/7) TR to cite literature referring to use of transdermal vaccine application
TR to begin drafting budget for NWHC
- TR to check with folks from F&WS to learn about their work using automatic sprayers for WNS
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct) (b) (6) (mobile)
www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics
and promote conservation.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 2/2
**Action Items:
RB and team
Jonathon Musser Amy Sims
Call between EHA & Tonie (NWHC)
**Action Items:
Tonie Rocke (TR)
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(b) (6)

PREEMPT call (Peter, Jim Gimlett of DARPA) - 2 March 2018
● Re: Issue of human subjects/samples
○ For the full proposal, we have to explicitly state that any human samples we use
will only come from previous studies, and that we have full access to these samples
■ PREEMPT will NOT fund any human sampling efforts
○ When referencing previously collected human samples (which we could have
1000s of), mention how we will triage samples to determine priority for testing, as well as how we plan to ship samples out
● Re: Number and size of DARPA awards
○ According to Jim Gimlett (Program Manager for PREEMPT), DARPA has
received many promising abstracts, and likely won’t be able to fund all of them (or at least won’t be able to fund every aspect of each project)
■ Jim is contacting other DARPA personnel to try and get additional funding for later stages of accepted proposals
● In the meantime, it’s important that we separate tasks/subtasks into ‘baseline’ tasks and ‘add-on’ tasks (as add-ons may be cut from proposed projects)
● Re: EHA collaboration with Wuhan
○ PD mentioned our existing collaboration with Wuhan, to Jim.
■ PD mentioned our standard operating procedure, and that our collaboration with Wuhan involves complete access to information and leads to research with quickly produced results
○ Jim checked in with higher ups at DARPA, and they’ve mentioned that there are no current political issues about us working in China
■ This is great news as we’ll be able to move ahead with our proposal as planned
■ Also, the fact that Jim inquired about our collaboration with Wuhan is a good sign that he’s interested in our proposal and wants to fund it.
● Re: Jim Gimlett’s response about riskiness of our proposal
○ PD asked Jim if anything about our proposal seemed too risky, or not risky
enough
■ Jim didn’t have anything to say specifically, so we should be on the right
track.
● He did mention, however, that we should show how we’ll validate
everything

○ i.e. Show that immune modulation works at every step, and show validation through modeling approaches
● Re: Concerns about host response to inocula
○ One DARPA employee mentioned the possibility of bats reacting negatively to
our inocula.
■ So we MUST show preliminary data when we write about this in our
proposal
● Show how our team has demonstrated the effectiveness of our
proposed intervention approaches - how we can reduce viral load by upregulating the immune response
○ Provide additional detail for interferon work specifically
● Some additional comments from Billy Karesh (BK):
○ The modeling approaches that we list in the proposal must be tied directly to
what the lab and field work are attempting to accomplish. In other words, we can’t just expand our modeling efforts in order to address questions from the data that we find interesting.
■ Rather, our modeling must be ‘utilitarian’. Modeling is strictly to inform our lab and field teams on how to create a more effective intervention approach (e.g. Use modeling to predict which high-risk viral strains to target in the lab)

PREEMPT call (EHA, Ralph + Time of UNC) - 2 March 2018
● to draft their section and return to PD by Wed. 3/7
○ RB to include section stating how our project won’t drive viral evolution in
negative way
● PD to complete quality first draft by end of next week, Fri. 3/9
● Jonathon Musser (EHA) and Amy Sims (UNC) to work together on UNC budget
● Once EHA has drafted modeling section of proposal, RB to provide input
● Re: Ralph Baric and team
○ Tim Sheahan (Research assistant professor at UNC)
■ Tremendous amount of experience working w/ CoV reverse genetics/ synthetic reconstruction of CoVs
■ Genetics expert. Has been looking into , in terms of
trying to find strains that can replicate in human cells
○ Amy Sims (Research associate professor)
■ Great deal of experience working w/ primary human cells and artificial construction of SARSr-CoVs to test binding to human cells. Lots of drug testing + vaccine work against SARS and MERS
■ Can also help with the subaward budget.
● Re: UNC subcontract budget
○ It will take roughly 7-10 days for RB and his team to review/get approval for the
budget.
○ Jonathon Musser (EHA) and Amy Sims (UNC) to work together on UNC budget
● Re: Modeling approaches
○ Noam Ross (NR) would like to be able to look at inputs of both sequence data as
well as ecological/host data for viruses, and make predictions about which virus traits we see when they’re in infected mice, as well as the degree to which they show up in nearby human populations
■ To understand this, it’d be helpful to know how many different viral strains (and of what risk), we’d put through the humanized mouse process in order to get an understanding of their ability to infect mice (as well as what the viral growth in those models are).
● NR interested to hear Ralph Baric’s (RB) thoughts on how many different strains we’d test over the course of project. A small handful? Dozens?
■ Ralph says it’s very budget dependent. We could look at pseudotyped viruses to see which spikes drive entry.
**Action Items:
RB and team
Commented [1]: Ralph/Tim- Please confirm this
Lineage D BetaCoVs

● For SARSr-CoVs, we know there are strains ranging from epidemic strains to those w/ 10-12% variation in spike protein, that still can infect human cells and humanized mice.
○ Within this 10-12% range, RB has tested one strain w/ 8% variation and one w/ 3%.
■ So the goal when looking for strains w/ novel variance, is to look for those w/ relatively conserved receptor binding domains (RBDs) but other variation of the spike that’s w/in the 10-12% window. 2 or 3 strains perhaps.
■ Once you have strains with greater spike variation (10-20%), there’s a reduction in capacity of viruses to grow in human cells or use human ACE2 receptor (unless RBD is conserved).
● Through recombination, it’s possible that the correct RBD could be dropped into a strain w/ 25% variation, and allow the virus to enter human cell and replicate
○ Given a 42-month time period, we’d probably screen 15-16 viruses to determine which strains we’d like to focus in on (given spike proteins), likely leaving us with 7 or 8.
● Peter (PD) thinks Noam’s modeling could involve:
○ An initial prediction of virus binding to ACE2 receptor
○ Then whether virus can replicate in human cells
○ Then focus on humanized mice and whether disease is caused in humanized
mice that looks like SARSr-CoV (This is gold standard in the end)
● Ralph thinks there are probably 5 viruses to be used as baselines - viruses that we know can replicate and use human receptor (SARS, Raccoon dog isolate, WIV16, WIV1, SCH014). These 5 capture range of 1% -12% variation in spike.
○ If you look at WIV16, WIV1 and SCH014, the RBDs that engage the receptor are fairly well conserved and can be modeled.
■ Want to model RBD, ACE2 interaction (3D protein modeling)
● RB has people who can help w/ this. Ultimately, we’re looking for
strains that conserve some sort of RBD integrity. We’re not interested in strains w/ loss of contact interface sites or deletion of RBD.
○ Knowing this will help guide modeling efforts
● We also need to model using information on protease cleavage site. ○ For SARSr-CoVs, there’s a two-step entry process.
■ 1) Virus-receptor interaction

■ 2) Either extracellular protease or Intracellular protease have to clip protein once or twice so that virus can fuse to cell membrane
● Virus can’t enter cell w/o protease. In the lab, if protease is added exogenously, strains that otherwise would not enter cell are now able
● So these are the 2 major modeling processes (the 2 screening components) ○ You screen (1) on the spike and (2) on the protease
● Some steps that RB says modeling could incorporate:
○ (1) Very conserved RBD
○ (2) Sequence variance around RBD
○ (3) Protease cleavage site
○ (4) What RBD looks like in relation to conformity to binding sites
○ (5) Overall spike variation
○ (6) RNA recombinance (maybe RBD that is capable of interfacing w/
human receptor, but has very different genetic background of the whole
spike)
● Model these to show progressively lower risk that virus could bind to and infect
cells)
● NR knows that in terms of the viruses being tested/screened, there’s a small set taken all the way to humanized mice.
○ But in terms of screening assays, are we conducting one assay to screen for spike binding, and another to screen for protease? In other words, are we testing the 2 components independently, and then based on the success of both, making a decision of how to move forward to test in our model?
■ Ralph says that the easiest way to approach this, is either w/ pseudotyped viruses or chimera viruses w/ full length spike.
● First look at receptor binding on cells, then look at virus ability to get into cells (remembering that getting into cell requires protease).
○ You’ll have spikes that bind to surface but can’t get in, and the basic approach here is to add exogenous protease to chimera virus system or pseudotyped virus to see if it can get in
● Need to make sure that RB reads the EHA modeling approach to make sure we have the right approach.
○ What we need to do is determine exactly what NR will model ■ Ralph thinks modeling is 5-step process
● (1) Find where viral sequence fits within phylogenetic tree, especially in relationship to 5 baseline viruses (SARS, raccoon dog isolate, WIV1, WIV16, SCH014

● (2) Focus on RBDs and models that predict whether viruses interact w/ mouse or human ACE2 molecules (Human would be priority, but could do mouse)
● (3) From those models, determine:
○ Where is variation?
○ How many contact residues retained?
○ Are there deletions?
■ If so, these strains get deprioritized
● (4) Look for protease cleavage sites in strains w/ SARS like spikes
○ Are sequence signatures present? ■ If yes, that’s a plus
● (5) Look at overall spike variation (recombinant molecule that’s picked up favorable RBD and dropped into variable spike protein)
○ This will predict which 8-16 strains we’ll synthesize ■ $1000/spike. So $16k?
● DARPA wants machine learning (ML) to be incorporated. They want some cool modeling approach that tells you the following:
○ When a warfighter goes to a region...
■ 1) Is it a place w/ Ebola and/or other high-risk strains that spill into
humans?
■ 2) Can we predict which species carry these viruses?
○ So we just need to make sure we include the following:
■ The species of bats that are likely to harbor viral strains very similar to
SARS virus
■ The species assemblages that are likely to drive evolution of those
strains.
○ Then all other stuff comes in when we try to validate models. We don’t need
super technical details now. DARPA just wants to make sure we have preliminary data to do the work, and that later on we can validate models and immune modulation approaches.
■ This shouldn’t be a problem as we all have preliminary data (RB, Linfa/Zhengli, TR, EHA)
● So RB thinks it will be reiterative process.
○ First look at sequences, prioritize the top 4, then test them and use the data to
refine models (perhaps using ML)
○ Then reassess the top candidates, then do another 4 (i.e. stepwise modeling),
then take 5 or 6 criterium and figure out how to prioritize which ones weight most heavily on predictive success
■ NR agrees but thinks scale is a challenge

● Due to fact that statistical strength will come from linking viral sequence to its success binding and infecting cells, we won’t really have a large dataset to work with.
○ RB says there must be 100+ spike protein variants that have been sequenced (at least). We also know rough boundaries that determine virus potential to replicate efficiently and use human receptors. So we can use these boundaries in our models as well.
■ NR thinks that even if we have enough sequences (input), our output (and ability to statistically validate models) still requires data on viruses that can successfully bind to and infect human cells. And we likely won’t find that many strains that are successful
● So we have to lean more heavily on mechanistic approach (emphasizing knowledge of RBD variation into predictive model itself), b/c we have limited set of iterations we can do
● PD says that ultimately, our final proposal is all about utilitarian modeling.
○ i.e. Modeling is strictly to inform our lab and field teams on how to create a more effective intervention approach (e.g. use modeling to predict which high-risk viral
strains to target in the lab)
○ Also, we MUST make it clear in proposal that our approach won’t drive evolution
the wrong way (e.g. drive evolution of more virulent strain that then becomes pandemic
■ This needs to be explained in detail (RB will draft this section)
● Re: ‘Immune Priming’ approach
○ Essentially, the approach is to upregulate both acquired and innate immunity
against viral spikes
○ Most juvenile bats have SARSr-CoVs, but titers drop in older adults
■ Use recombinant protein vaccine paired w/ some sort of adjuvant mixture to stimulate innate immunity and knock down titer (this should reduce virus burden in cave)
● In full proposal, stress that we’re designing simple, straightforward approach that plays on bat immunity and offers transient protection from viral spillover
● Re: Delivery method of inoculum
○ How will the recombinant spike proteins be administered?

■ RB thinks intranasal administration most effective. Perhaps as aerosolized spray
● Dextran microparticle that can be formulated either as microparticle-based vaccine or nanoparticle (so could be delivered by spray)
○ The antigen is interlaced inside matrix and can be delivered as spray.
■ Large particles go directly to macrophage for vaccine delivery. Small ones go to dendritic cells.
● Never been applied to populations like this. ○ So potentially some risk here
● Idea is to propose ‘Immune Boosting’ and ‘Immune Priming’ methods as concurrent approaches
○ Start both approaches at the beginning of the project, then have friendly competition between the two
■ Some things will work, some won’t. Focus on what works, so by end of project we have spray that warfighter sends into bat cave to transiently reduce risk of spillover
● Could be mixture of molecules (Poly IC plus RB’s recombinants, intranasal + sticky gel, etc.)
● Re: PREEMPT Transition Plan, translation plan
○ DARPA wants us to determine who a potential customer/recipient of our work
could be, to take research to next stage or apply it in way that’s useful to them
■ Potentially US Army, to use in order to reduce spillover risk to troops
■ DARPA (unlike US Army, DTRA, etc.) isn’t an end-user. They fund
projects but don’t end up using the final product
○ If we’re designing treatment, will it be publicly available? Handed over to DoD?
○ RB has some experience with this. Is working on commercial vaccine for
flavivirus, and also doing collaborative work for drugs on emerging CoV infection.
○ Some potential translation ideas for our proposal:
■ Panel of new viruses that sets DoD up to prepare for breadth of viruses, allowing for creation of vaccines.
■ Animal models that can be used to evaluate therapeutics.
■ ML programs designed to take specific features in a virus family and do a
reiterative prediction about pre-pandemic potential.
● Model will be CoV but should be able to translate, transition into
other systems where you know the surface protein (major species specificity factor) all of these proteases, etc. Also variation in RBD is known.

○ Bats that we’ll sequence will provide massive amounts of these variations that can be modeled onto ACE2 bat molecule, to see how virus has caused species to coevolve, or try to escape virus pressure.
■ i.e. interface site of ACE2 moledule in bat will show most variation, b/c virus will put selective pressure on animals, and bats w/ ability to escape, will be ones that are maintained in the population.
● This will drive variation in RBD and ability to use human receptor.
■ Another idea is...if you build chimera that broadly reduces heterogeneous pop. of SARSr-CoVs in bat cage, this might be something you’d want to develop for humans.
● RB has already generated SARS-like chimeras w/ RBD from group of bat viruses called 293 (for S1), which is 20% different than epidemic strains, and S2 region from HK3 which is 20% diff.
○ Can drive broad-based rsponese for large no. of family members (but we can test those)
○ Detailed sequence analysis could be used to engineer broad -based vaccinations for humans
● Re: Intentional wording w/in the proposal
○ In technical sections of full proposal, be careful not to confuse the reviewers (e.g.
using ‘vaccine’, ‘immune modulation’, ‘viral suppression’
■ Just talk about transiently reducing spillover, through modulation of bat
immune response
● Then when talking about translational plan, we can mention
potential vaccines for human application
■ We just want to be careful that we’re not sounding overly ambitious (e.g.
that we’ll try 6 different treatment as well as vaccines)
● Ensure that the terminology we use reflects a project that can be
completed in a 42-month period
● Re: Nanoparticle delivery method
○ Ensure that RB and TR come together when discussing potential nanoparticle
delivery of inocula.

**Action Items:
PREEMPT call (EHA, Wuhan, Duke-NUS) - 27 Feb 2018
● PD to have weekly calls w/ collaborators ○ LH to organize these calls
● LH to send an email confirming: DUNS nos., addresses and phone numbers of each collaborating institution.
○ The email will also provide instructions for how to sign up on Grants.gov (required for Key Personnel of each collaborating organization)
● PD to sit down with KJO, JHE and LH to determine who will be responsible for each section of the proposal. Will send to collaborators by Thu. 3/1
○ Collaborators to then write their respective sections and return to PD by Wed. 3/7
○ Peng to include section about how both Immune Boosting and Immune Targeting
approaches are better than vaccination (without explicitly criticizing vaccination
approach)
● PD to speak with YunZhi regarding consultant field work in/around Yunnan cave
● Collaborators to determine if any additional personnel required for their work (e.g.
technicians, field support staff, etc.)
○ Collaborators to let PD know if they think anyone else should be brought on to
the team
● PD to determine which aspects of full proposal are baseline tasks, and which are add-
ons (by mid to late March)
● Re: Timeline
○ PD sends collaborators full proposal template with assigned sections, by Thu.
3/1
○ Collaborators complete their sections and return to PD by Wed. 3/7
○ Quality first draft of full proposal, completed by Mon. 3/12
○ Send out draft budget to collaborators by Mon. 3/19 at latest
■ Budget turnarounds for each collaborating institution:
● Duke-NUS: 1-2 days
● Wuhan Univ.: 1-2 days
● UNC (Ralph): PD to ask on call
● NWHC (Tonie): PD to ask on call
○ By mid to late March, PD is going to go thru proposal, thinking carefully about timeline and budget. He’ll make sure each collaborator is doing both baseline work and additional work
● Re: Writing the full proposal
○ PD will work with KJO, JHE and LH to determine which section of the proposal
each collaborator will write

■ The plan is for each group to work on the technical aspects they’re experts on, rather than PD writing a draft of each section first
○ By Thu. 3/1, PD to send around the proposal w/ names attached to which parts we need flushing out.
○ Then, over 5-day period, collaborators can draft out their sections and return to PD by Wed. 3/7
■ PD would rather have more detailed, technical language than less. PD has no problem cutting down info.
● For each task/subtask, provide any preliminary data (e.g. no. of caves we’ve visited, no. of samples we’ve collected, data describing our work w/ Rhinolophus, etc.).
○ Include any relevant references, numbers, figures, charts
■ Please use EndNote for references, or send references as text (can be
fixed later)
■ When writing your section(s), don’t separate tasks into baseline tasks vs.
add-ons (PD will decide this)
● Re: The proposed budget
○ DARPA to fund 0-6 projects ($40 million total)
○ We have to be careful and ensure we’ve justified the money we’re asking for
■ We need to distinguish between ‘baseline’ tasks and ‘add-ons’
● This will give DARPA clear tasks to cut if funding is limited
● Given that we have a strong team, the goal is to make sure that
each collaborator is responsible for both baseline tasks and add- ons
○ This way if our proposed budget is reduced, each collaborator is still able to contribute to the project
● We may need to reconsider carrying out lab work at 4 different collaborator institutions.
○ DARPA might not fund lab work at all 4
● By mid to late March, PD is going to go thru proposal, thinking
carefully about timeline and budget. He’ll make sure each collaborator is doing both baseline work and additional work
● Re: Distinguishing our approach from vaccination approach
○ Ralph Baric’s approach is not vaccination. Rather his approach aims to boost the
immune response against specific viral proteins (potentially to include innate immune response)
■ Vaccination in bats not likely to work.
● Very limited antibodies from natural viral infection in bats
● Viruses already common in bat population - not likely to challenge
bat immune response with a vaccine

■ Peng to write section about how both Immune Boosting and Immune Targeting approaches are better than vaccination (without explicitly criticizing vaccination approach)
● Our aim is to show DARPA that we will...Develop and test a technology that’s not guaranteed to work (but is ambitious and has great potential) that allows us to reduce risk of spillover (even if only temporarily)

10/5/21, 3:26 PM
Mail - Rocke, Tonie E - Outlook
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct) (b) (6) (mobile)
www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics
and promote conservation.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/3
(b) (6)
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10/5/21, 3:26 PM Mail - Rocke, Tonie E - Outlook
Call between EHA & Wuhan/Duke-NUS
**Action Items:
PD to have weekly calls w/ collaborators LH to organize these calls
LH to send an email confirming: DUNS nos., addresses and phone numbers of each collaborating institution.
The email will also provide instructions for how to sign up on Grants.gov (required for
Key Personnel of each collaborating organization)
PD to sit down with KJO, JHE and LH to determine who will be responsible for each section of the proposal. Will send to collaborators by Thu. 3/1
Collaborators to then write their respective sections and return to PD by Wed. 3/7 Peng to include section about how both Immune Boosting and Immune Targeting approaches are better than vaccination (without explicitly criticizing vaccination approach)
PD to speak with YunZhi regarding consultant field work in/around Yunnan cave Collaborators to determine if any additional personnel required for their work (e.g. technicians, field support staff, etc.)
Collaborators to let PD know if they think anyone else should be brought on to the
team
-PD to determine which aspects of full proposal are baseline tasks, and which are add-ons (by mid to late March)
Call between EHA & Ralph/Tim (UNC)
**Action Items:
RB and team to draft their section and return to PD by Wed. 3/7
RB to include section stating how our project won’t drive viral evolution in negative
way
PD to complete quality first draft by end of next week, Fri. 3/9
Jonathon Musser (EHA) and Amy Sims (UNC) to work together on UNC budget
-Once EHA has drafted modeling section of proposal, RB to provide input
Call between EHA & Tonie (NWHC) **Action Items:
Tonie Rocke (TR) to begin work on her section of the full proposal (and send to PD by Wed. 3/7)
TR to cite literature referring to use of transdermal vaccine application TR to begin drafting budget for NWHC
- TR to check with folks from F&WS to learn about their work using automatic sprayers for WNS
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 2/3
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10/5/21, 3:26 PM
Mail - Rocke, Tonie E - Outlook
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct) (b) (6) (mobile)
www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics and promote conservation.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 3/3
(b) (6)
vog.sgsu@ekcort
1542-072-806
11735 IW ,nosidaM
.dR redeorhcS 6006
retneC htlaeH efildliW lanoitaN SGSU
ekcoR .E einoT
--
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10/5/21, 3:27 PM
Mail - Rocke, Tonie E - Outlook
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct) (b) (6) (mobile)
www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics
and promote conservation.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/1
,tseB
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)TE MP 5 - MA 11( 32/3 .irF
)TE MP 5 - MA 11( 22/3 .uhT
:3 keeW
)TE MP 5 - MA 11( 61/3 .irF
)TE MP 5 - MA 11( 51/3 .uhT
:2 keeW
)TE MP 5 - MA 11( 9/3 .uhT
)TE MP 5 - MA 11( 8/3 .uhT
:1 keeW
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;>gro.ecnaillahtlaehoce@arumhc<arumhCieskelA;>vog.sgsu.rotcartnoc@ttobbar<C)rotcartnoC(lehcaR,ttobbA:cC
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>gro.ecnaillahtlaehoce@lemah< lMPe83m2a81H02/e6/k3 euuLT
)TPMEERP( reteP htiw sllac gnimocpu gniludehcS
(b) (6)

10/5/21, 3:28 PM
Mail - Rocke, Tonie E - Outlook
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct) (b) (6) (mobile)
www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics
and promote conservation.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/2
(b) (6)
:2 keeW
)TE MP 5 - MA 11( 9/3 .uhT
)TE MP 5 - MA 11( 8/3 .uhT
:1 keeW
emiT nretsaE ni era semit lla taht eton esaelP
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rehtie no ,keew hcae llac eno evah ot si laog ehT .)rof elbaliava era uoy sa ynam sa tceles(
reteP htiw kaeps ot elbaliava era uoy )s(emit/)s(etad eht tceles ot knil siht esu esaelP
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,einoT iH
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>gro.ecnaillahtlaehoce@kazsad<kazsaDreteP.rD;>gro.ecnaillahtlaehoce@erdna<erdnAnosilA
;>gro.ecnaillahtlaehoce@arumhc<arumhCieskelA;>vog.sgsu.rotcartnoc@ttobbar<C)rotcartnoC(lehcaR,ttobbA:cC
>vog.sgsu@ekcort<EeinoT,ekcoR:oT
>gro.ecnaillahtlaehoce@lemah<MlPe23m1a810H2/7e/3kdueLW
)TPMEERP( reteP htiw sllac gnimocpu gniludehcS :eR

10/5/21, 3:28 PM
Mail - Rocke, Tonie E - Outlook
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct) (b) (6) (mobile)
www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics and promote conservation.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 2/2
(b) (6)
vog.sgsu@ekcort
1542-072-806
11735 IW ,nosidaM
.dR redeorhcS 6006
retneC htlaeH efildliW lanoitaN SGSU
ekcoR .E einoT
--
,tseB
.snoitseuq yna evah uoy fi wonk em tel esaelP
)TE MP 5 - MA 11( 32/3 .irF
)TE MP 5 - MA 11( 22/3 .uhT
:3 keeW
)TE MP 5 - MA 11( 61/3 .irF
)TE MP 5 - MA 11( 51/3 .uhT

10/5/21, 3:28 PM
Mail - Rocke, Tonie E - Outlook
Year Y1
Y2
OY1
OY .5
Total
Amount $304,500.00
$304,500.00
$304,500.00
$152,250.00
$1,065,750.00
Provide the purpose of the trip, number of trips, number of days per trip, departure and arrival destinations, number of people, estimated rental car and airfare costs, and prevailing per diem rates as determined by gsa.gov, etc.; Quotes must be supported by screenshots from travel websites.
3) Regarding 'Equipment Purchases'
Itemization with individual and total costs, including quantities, unit prices, proposed vendors (if known), and the basis of estimate (e.g., quotes, prior purchases, catalog price lists, etc.); any item that exceeds $5,000 must be supported with back-up documentation such as a copy of catalog price lists or quotes prior to purchase (NOTE: For equipment purchases, include a letter stating why the proposer cannot provide the requested resources from its own funding.
4) Regarding 'Materials'
Itemization with costs, including quantities, unit prices, proposed vendors (if known), and the basis of estimate
(e.g., quotes, prior purchases, catalog price lists, etc.); any item that exceeds $5,000 must be supported with back- up documentation such as a copy of catalog price lists or quotes prior to purchase.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/2
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erdnAnosilA;>gro.ecnaillahtlaehoce@ressum<ressuMnohtanoJ;>gro.ecnaillahtlaehoce@onaicul<onaicuLnylevE
;>gro.ecnaillahtlaehoce@arumhc<arumhCieskelA;>vog.sgsu.rotcartnoc@ttobbar<C)rotcartnoC(lehcaR,ttobbA:cC
>vog.sgsu@ekcort<EeinoT,ekcoR:oT
>gro.ecnaillahtlaehoce@lemah<MlPe30m4a810H2/7e/3kdueLW
sliated dna etalpmet tegdub TPMEERP

10/5/21, 3:28 PM
Best,
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(b) (6) (direct) (b) (6) (mobile)
www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics
and promote conservation.
Mail - Rocke, Tonie E - Outlook
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 2/2

WORKSPACE FORM
1-800-518-4726 SUPPORT@GRANTS.GOV
This Workspace form is one of the forms you need to complete prior to submitting your Application Package. This form can be completed in its entirety offline using Adobe Reader. You can save your form by clicking the "Save" button and see any errors by clicking the “Check For Errors” button. In-progress and completed forms can be uploaded at any time to Grants.gov using the Workspace feature.
When you open a form, required fields are highlighted in yellow with a red border. Optional fields and completed fields are displayed in white. If you enter invalid or incomplete information in a field, you will receive an error message. Additional instructions and FAQs about the Application Package can be found in the Grants.gov Applicants tab.
OPPORTUNITY & PACKAGE DETAILS:
Opportunity Number: Opportunity Title: Opportunity Package ID: CFDA Number:
CFDA Description: Competition ID: Competition Title: Opening Date: Closing Date: Agency:
Contact Information:
HR001118S0017
PREventing EMerging Pathogenic Threats PKG00237724
12.910
Research and Technology Development
01/19/2018
03/27/2018
DARPA - Biological Technologies Office
BAA Coordinator
PREEMPT@darpa.mil
APPLICANT & WORKSPACE DETAILS:
Workspace ID: Application Filing Name: DUNS:
Organization:
Form Name:
Form Version: SubformName: Requirement:
Download Date/Time: Form State:
FORM ACTIONS:
WS00094394
Project DEFUSE
0770900660000
ECOHEALTH ALLIANCE INC.
R & R Subaward Budget 10 YR Subform 1.4
USGS Ntl. Wildlife Health Cen Optional
Mar 06, 2018 05:28:38 PM EST Error(s)

USGS National Wildlife Health Center
Co-Investigator
View Attachment
Delete Attachment
Add Attachment
Base Salary ($)
Suffix
Rocke
Tonie
Prefix
Project Subaward/Consortium
ORGANIZATIONAL DUNS:
Budget Type:
A. Senior/Key Person
First
Project Role:
Additional Senior Key Persons:
RESEARCH & RELATED BUDGET - Budget Period 1 Enter name of Organization:
OMB Number: 4040-0001 Expiration Date: 10/31/2019
Funds Requested ($)
B. Other Personnel
Total Funds requested for all Senior Key Persons in the attached file
Total Senior/Key Person
Fringe Benefits ($)
Number of
Personnel
Project Role
Post Doctoral Associates
Graduate Students
Undergraduate Students
Secretarial/Clerical
Total Number Other Personnel
Cal.
Months Acad.
Requested Sum. Salary ($)
Funds Requested ($)
Middle Last
Cal.
Months Acad.
Sum.
Requested Salary ($)
Budget Period: 1
Start Date:
End Date:
Fringe Benefits ($)
Total Other Personnel Total Salary, Wages and Fringe Benefits (A+B)

View Attachment
Delete Attachment
Add Attachment
C. Equipment Description
List items and dollar amount for each item exceeding $5,000 Equipment item
Funds Requested ($)
Funds Requested ($)
Funds Requested ($)
Additional Equipment:
D. Travel
1. Domestic Travel Costs ( Incl. Canada, Mexico and U.S. Possessions)
2. Foreign Travel Costs
E. Participant/Trainee Support Costs
1. Tuition/Fees/Health Insurance
2. Stipends
3. Travel
4. Subsistence
5. Other
Total Travel Cost
Number of Participants/Trainees
Total Participant/Trainee Support Costs
Total funds requested for all equipment listed in the attached file Total Equipment

F. Other Direct Costs Funds Requested ($)
View Attachment
Delete Attachment
Add Attachment
1. Materials and Supplies
2. Publication Costs
3. Consultant Services
4. ADP/Computer Services
5. Subawards/Consortium/Contractual Costs
6. Equipment or Facility Rental/User Fees
7. Alterations and Renovations
8.
9.
10.
G. Direct Costs
H. Indirect Costs
Indirect Cost Type
Cognizant Federal Agency
(Agency Name, POC Name, and
POC Phone Number)
I. Total Direct and Indirect Costs
J. Fee
K. Total Costs and Fee
L. Budget Justification
Total Other Direct Costs
Total Direct Costs (A thru F)
Indirect Cost Rate (%) Indirect Cost Base ($)
Total Indirect Costs
Total Direct and Indirect Institutional Costs (G + H)
Funds Requested ($)
Funds Requested ($)
Funds Requested ($) Funds Requested ($) Funds Requested ($)
(Only attach one file.)
Total Costs and Fee (I + J)

RESEARCH & RELATED BUDGET - Cumulative Budget Totals ($)
Section A, Senior/Key Person Section B, Other Personnel Total Number Other Personnel Total Salary, Wages and Fringe Benefits (A+B) Section C, Equipment
Section D, Travel
1. Domestic
2. Foreign
Section E, Participant/Trainee Support Costs
1. Tuition/Fees/Health Insurance
2. Stipends
3. Travel
4. Subsistence
5. Other
6. Number of Participants/Trainees
Section F, Other Direct Costs
1. Materials and Supplies
2. Publication Costs
3. Consultant Services
4. ADP/Computer Services
5. Subawards/Consortium/Contractual Costs
6. Equipment or Facility Rental/User Fees
7. Alterations and Renovations
8. Other 1
9. Other 2
10. Other 3
Section G, Direct Costs (A thru F)
Section H, Indirect Costs
Section I, Total Direct and Indirect Costs (G + H)
Section J, Fee
Section K, Total Costs and Fee (I + J)

10/5/21, 3:30 PM
Mail - Rocke, Tonie E - Outlook
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct) (b) (6) (mobile)
www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics
and promote conservation.
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct) (b) (6) (mobile)
www.ecohealthalliance.org
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/3
(b) (6)
,tseB
!yadot retteb gnileef er'uoy epoH .einoT ,wonk em gnittel rof uoy knahT
:etorw >gro.ecnaillahtlaehoce@lemah< lemaH ekuL ,MP 232 ta 8102 ,7 raM ,deW nO
,tseB
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eht nihtiw knil lloP eldooD rehtona tuo dnes lliw I .)41/3( yadsendeW ro )31/3( yadseuT rehtie ,keew
txen ot llac ruo eludehcser ot gnikool era ew os ,9/3 ,.irF no noitpecer enohpllec evah t'now reteP
,einoT iH
>gro.ecnaillahtlaehoce@kazsad<kazsaDreteP.rD;>gro.ecnaillahtlaehoce@erdna<erdnAnosilA
;>gro.ecnaillahtlaehoce@arumhc<arumhCieskelA;>vog.sgsu.rotcartnoc@ttobbar<C)rotcartnoC(lehcaR,ttobbA:cC
>vog.sgsu@ekcort<EeinoT,ekcoR:oT
>gro.ecnaillahtlaehoce@lemah<MlPe41m4a810H2/7e/3kdueLW
)TPMEERP( reteP htiw sllac gnimocpu gniludehcS :eR
(b) (6)

10/5/21, 3:30 PM Mail - Rocke, Tonie E - Outlook
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics
and promote conservation.
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct) (b) (6) (mobile)
www.ecohealthalliance.org
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 2/3
,tseB
.snoitseuq yna evah uoy fi wonk em tel esaelP
)TE MP 5 - MA 11( 32/3 .irF
)TE MP 5 - MA 11( 22/3 .uhT
:3 keeW
)TE MP 5 - MA 11( 61/3 .irF
)TE MP 5 - MA 11( 51/3 .uhT
:2 keeW
)TE MP 5 - MA 11( 9/3 .uhT
)TE MP 5 - MA 11( 8/3 .uhT
:1 keeW
emiT nretsaE ni era semit lla taht eton esaelP .evoba
knil lloP eldooD eht ni raeppa taht semit/setad eht detsil ev'I ,woleB .yadirF ro yadsruhT
rehtie no ,keew hcae llac eno evah ot si laog ehT .)rof elbaliava era uoy sa ynam sa tceles(
reteP htiw kaeps ot elbaliava era uoy )s(emit/)s(etad eht tceles ot knil siht esu esaelP
.lasoporp lluf eht htiw kcart no yats ew taht erusne ot ,uoy htiw sllac ylkeew egnarra ot gnipoh si reteP
,einoT iH
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:etorw >vog.sgsu@ekcort< einoT ,ekcoR ,MP 422 ta 8102 ,7 raM ,deW nO
(b) (6)

10/5/21, 3:30 PM Mail - Rocke, Tonie E - Outlook
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics and promote conservation.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 3/3
vog.sgsu@ekcort
1542-072-806
11735 IW ,nosidaM
.dR redeorhcS 6006
retneC htlaeH efildliW lanoitaN SGSU
ekcoR .E einoT
--

10/5/21, 3:31 PM
Mail - Rocke, Tonie E - Outlook
Phone: 1-719-785-9461 Password: 9784#
Best,
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct) (b) (6) (mobile)
www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics
and promote conservation.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/1
.)woleb sliated( llac hcae rof enil ecnerefnoc citsemod eht esu esaelP
TEMA01@12/3.deW
TEMA01@41/3.deW
:setad gniwollof eht rof sllac deludehcs ev'I ,detsil evah reteP dna uoy taht ytilibaliava eht no desaB
,einoT iH
>gro.ecnaillahtlaehoce@kazsad<kazsaDreteP.rD;>gro.ecnaillahtlaehoce@erdna<erdnAnosilA
;>gro.ecnaillahtlaehoce@arumhc<arumhCieskelA;>vog.sgsu.rotcartnoc@ttobbar<C)rotcartnoC(lehcaR,ttobbA:cC
>vog.sgsu@ekcort<EeinoT,ekcoR:oT
>gro.ecnaillahtlaehoce@lemah<lMeP1m24a81H02e/9k/3uirLF
sllac TPMEERP rof setad gnimrifnoC
(b) (6)

10/5/21, 3:32 PM Mail - Rocke, Tonie E - Outlook
"Government entities must clearly demonstrate that the work [they will perform] is not otherwise available from the private sector and provide written documentation citing the specific statutory authority and contractual authority, if relevant, establishing their ability to propose to Government solicitations.” See BAA, page 18.
Essentially, this means that we will require the following items from you:
A document signed by either the NWHC director or by you (on behalf of the NWHC), stating that the NWHC has the 'contractual authority to collaborate/partner/etc. with EcoHealth Alliance in the PREEMPT proposal titled, 'Project DEFUSE.'
A statement (within the same document) describing that NWHC is the only public or private sector laboratory with the necessary skills, equipment, resources, etc...to perform the tasks listed within the full proposal. (This language should match, more or less, what you will have already listed within your technical section).
Next week, I will provide you with a template for this document. In the meantime, please communicate this requirement to your director and notify her or him that a signature will be required.
Please let me know if you have any questions. Best,
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct) (b) (6) (mobile)
www.ecohealthalliance.org
(b) (6)
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/3
.
.
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erdnAnosilA;>gro.ecnaillahtlaehoce@ressum<ressuMnohtanoJ;>gro.ecnaillahtlaehoce@onaicul<onaicuLnylevE
;>gro.ecnaillahtlaehoce@arumhc<arumhCieskelA;>vog.sgsu.rotcartnoc@ttobbar<C)rotcartnoC(lehcaR,ttobbA:cC
>vog.sgsu@ekcort<EeinoT,ekcoR:oT
>gro.ecnaillahtlaehoce@lemah<lMeP3m06a81H02e/9k/3uirLF
sliated dna etalpmet tegdub TPMEERP :eR

10/5/21, 3:32 PM Mail - Rocke, Tonie E - Outlook
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics
and promote conservation.
Year Y1
Y2
OY1
OY .5
Total
Amount $304,500.00
$304,500.00
$304,500.00
$152,250.00
$1,065,750.00
Provide the purpose of the trip, number of trips, number of days per trip, departure and arrival destinations, number of people, estimated rental car and airfare costs, and prevailing per diem rates as determined by gsa.gov, etc.; Quotes must be supported by screenshots from travel websites.
3) Regarding 'Equipment Purchases'
Itemization with individual and total costs, including quantities, unit prices, proposed vendors (if known), and the basis of estimate (e.g., quotes, prior purchases, catalog price lists, etc.); any item that exceeds $5,000 must be supported with back-up documentation such as a copy of catalog price lists or quotes prior to purchase (NOTE: For equipment purchases, include a letter stating why the proposer cannot provide the requested resources from its own funding.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 2/3
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10/5/21, 3:32 PM Mail - Rocke, Tonie E - Outlook
4) Regarding 'Materials'
Itemization with costs, including quantities, unit prices, proposed vendors (if known), and the basis of
estimate (e.g., quotes, prior purchases, catalog price lists, etc.); any item that exceeds $5,000 must be supported with back-up documentation such as a copy of catalog price lists or quotes prior to purchase.
Best,
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct) (mobile)
www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics and promote conservation.
b) (6)
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 3/3
(b) (6)
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.dR redeorhcS 6006
retneC htlaeH efildliW lanoitaN SGSU
ekcoR .E einoT
--

10/5/21, 3:32 PM
Mail - Rocke, Tonie E - Outlook
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct) (b) (6) (mobile)
www.ecohealthalliance.org
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/2
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(b) (6)

10/5/21, 3:32 PM Mail - Rocke, Tonie E - Outlook
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics
and promote conservation.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 2/2

From: Sent: To:
Cc: Subject:
Thank you, Tonie! Have an excellent weekend as well. Best,
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct) (b) (6) (mobile)
www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics and promote conservation.
On Fri, Mar 9, 2018 at 4:21 PM, Rocke, Tonie <trocke@usgs.gov> wrote:
Hello Luke and Peter: Attached is my first draft of Task 7. Ralph Baric (copied here) and I had a good chat today about viral vectors and nanoparticles. We realized much of the delivery methods would depend on his work first, so there are some gaps here and the narrative will probably change after I see what Ralph has written. At any rate, this is something to at least start with. Have a good weekend! -Tonie
On Thu, Mar 8, 2018 at 12:58 PM, Luke Hamel <hamel@ecohealthalliance.org> wrote: Hi Tonie,
Once again, please use this link, to select which date(s)/time(s) you are available to speak with Peter (select as many as you are available for). The goal is to have one call each week, on either Tuesday or Wednesday. Below, I've listed the dates/times that appear in the Doodle Poll link above. Please note that all times listed are in Eastern Time.
Week 1:
Thu. 3/13 (9 AM - 5 PM ET) Thu. 3/14 (9 AM - 5 PM ET)
Week 2:
Thu. 3/20 (9 AM - 5 PM ET) Thu. 3/21 (9 AM - 5 PM ET)
Please let me know if you have any questions. Best,
Luke Hamel <hamel@ecohealthalliance.org>
Friday, March 9, 2018 3:08 PM
Rocke, Tonie
Rachel Abbott; Aleksei Chmura; Dr. Peter Daszak; Alison Andre; Baric, Ralph S Re: Rescheduling upcoming calls with Peter (PREEMPT)
(b) (6)
1

Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(b) (6) (direct) (b) (6) (mobile) www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics and promote conservation.
--
Tonie E. Rocke
USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
608-270-2451
trocke@usgs.gov
2

From:
Sent:
To:
Cc:
Subject: Attachments:
Hello Luke and Peter: Attached is my first draft of Task 7. Ralph Baric (copied here) and I had a good chat today about viral vectors and nanoparticles. We realized much of the delivery methods would depend on his work first, so there are some gaps here and the narrative will probably change after I see what Ralph has written. At any rate, this is something to at least start with. Have a good weekend! -Tonie
On Thu, Mar 8, 2018 at 12:58 PM, Luke Hamel <hamel@ecohealthalliance.org> wrote: Hi Tonie,
Once again, please use this link, to select which date(s)/time(s) you are available to speak with Peter (select as many as you are available for). The goal is to have one call each week, on either Tuesday or Wednesday. Below, I've listed the dates/times that appear in the Doodle Poll link above. Please note that all times listed are in Eastern Time.
Week 1:
Thu. 3/13 (9 AM - 5 PM ET) Thu. 3/14 (9 AM - 5 PM ET)
Week 2:
Thu. 3/20 (9 AM - 5 PM ET) Thu. 3/21 (9 AM - 5 PM ET)
Please let me know if you have any questions. Best,
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
Rocke, Tonie <trocke@usgs.gov>
Friday, March 9, 2018 1:21 PM
Luke Hamel
Rachel Abbott; Aleksei Chmura; Dr. Peter Daszak; Alison Andre; Baric, Ralph S Re: Rescheduling upcoming calls with Peter (PREEMPT)
PREEMPT TR task 7 first draft.docx
(b) (6) (direct) (b) (6)
(mobile) www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics and promote conservation.
--
Tonie E. Rocke
1

USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
608-270-2451
trocke@usgs.gov
2

Task 7: Develop and assess delivery methods to bats for immune boosting and priming molecules
Description and execution: While work is proceeding to identify and optimize immunomodulating agents to manage SARS-Coronaviruses, we will concurrently develop and test mediums, routes, and methods of delivery to large colonies of bats. Several different approaches or combinations of approaches will be assessed to determine the most feasible and simplest method of delivery that achieves high uptake by bats, is safe for humans as well as target and non-target species, and minimizes disturbance to the colony. Sticky edible gels or pastes that bats groom from themselves and each other have been used previously to deliver pharmaceuticals to bats orally and are currently being tested as a medium for delivery of vaccines against rabies and other diseases in wild bats (see preliminary data). These may also be useful for delivering immune modulators and recombinant SARSr-CoV spike proteins to Rhinolophus bats, but may need to be combined with viral vectors (like poxvirus or adenovirus) or nanoparticles/nanoemulsions that enhance uptake through mucous membranes or transdermally after topical application.
Poxviruses in particular have been demonstrated to be effective viral vectors for delivering vaccines to wildlife (Slate et al., 2009) Freuling et al., 2013; Rocke et al., 2017). Recent laboratory studies in bats have shown that poxviruses can replicate safely at high levels in bats after oronasal administration (Stading et al., 2016)m and poxvirus vectored vaccines are immunogenic, protecting bats from rabies challenge (Stading et al 2017; see preliminary data). Poxviruses are highly safe, having been tested in a wide variety of wild and domestic animals, they allow for large inserts of foreign DNA, and they have a proven record of success. Poxviruses are good candidates for this project, but we will also consider others.
In addition to viral vectors, we will also consider methods to achieve transcutaneous delivery of the immune boosting proteins without the use of live agents. Recent advances in methods to achieve transdermal or transcutaneous delivery of drugs and vaccines have been reported. (Roberts et al., 2017). However, a major impediment to this route of vaccination is the stratum corneum, the outermost barrier layer of the skin that protects underlying layers from infection and damage. Numerous approaches have relied on mechanical methods to compromise the stratum corneum to allow the drug or vaccine to penetrate into the skin (Roberts et al., 2017). Innovations in nanotechnology show promise in being able to deliver drugs and vaccines into the deeper layers of the skin without the need for damage to the stratum corneum (Mishra et al., 2013), an important consideration. Dendritic cells and Langerhans cells, antigen-presenting cells which reside in the dermis and epidermis, can take up these transdermally delivered proteins and generate an immune response. We are currently testing poly lactic-co- glycolic acid (PLGA) as a nanoparticle to encapsulate rabies glycoprotein as a method of transcutaneous delivery of vaccine to bats. PLGA has been used previously to deliver both toll-like receptor agonists and antigens simultaneously to mice (Ebrahimian, 2017). This and other products (outlined above in Task ?) could potentially be useful with SARSr-CoV glycoproteins. Adjuvants can also be incorporated into nanoemulsions and nanoparticles to amplify the natural immune response to the vaccine antigens (Karande and Mitragotri, 2010). With SARS-CoV spike proteins, the adjuvant Matrix M1

(Isconova, Sweden) has been shown to significantly enhance the immune response in mice (Coleman et al. 2014)
In collaboration with Dr. Baric and others, we will determine the most likely immunomodulating formulations based on the results of TA2, previous animal studies and other available data and then use both laboratory and field studies to assess and optimize delivery vehicles and methods for wild bats. To reduce costs, initial studies will be conducted with locally acquired insectivorous bats (Eptesicus fuscus--big brown bats). We have successfully maintained and housed big brown bats and other insectivorous species for several experiments at our facility previously (Stading et al., 2016, 2017). We will treat bats via topical application with various test formulations that include the biomarker Rhodamine B (RB), co-house them with untreated bats, and monitor transfer between bats by collecting hair and whiskers for biomarker analysis. Rhodamine B is detectable within the hair of animals within 24 hours of consumption using a fluorescence microscope, and we have considerable experience using this biomarker for similar studies (see preliminary data).
Once we have confirmed uptake in laboratory studies, we will then assess mass delivery methods in local caves and hibernacula (using biomarker-labeled mediums but without immunomodulatory substances). We will test several different approaches including aerosolization via sprayers that could be used in cave settings and automated sprays triggered by timers and movement detectors at critical cave entry points. Within one week of application, bats will be trapped at the cave entrace using mist nets or Harp traps and hair will be collected to assess the rate of uptake via biomarker analysis. The bats will be released immediately afterward. The procedures will be tested at several different locations as it will likely take some manipulation to determine appropriate dosages for maximum uptake. After we have determined the most optimal approaches for mass delivery, we will then test them on wild bats in our three cave sites in Yunnan Province. Again, biomarker will be used to assess rates of uptake and this data can then be used in modeling studies to help determine the optimal rates of application of immunomodulating agents. Biomarker studies can also be used to assess uptake by non- target species, an important consideration in evaluating safety. Fieldwork will be conducted in collaboration with Dr. Yunzhi Zhang (Yunnan CDC, Consultant at EcoHealth Alliance).
Preliminary Data: Rocke and colleagues have developed oral vaccines and delivery methods to manage disease in free-ranging wildlife for many years, including a sylvatic plague vaccine for prairie dogs (Rocke et al., 2017), and more recently, vaccines against rabies (Stading et al., 2017) and white-nose syndrome for bats (Rocke, unpublished data). In addition to developing, testing and registering vaccines for experimental field use, vaccine delivery methods and uptake by the target species were optimized using biomarker studies prior to deployment; biomarker studies were also used to assess uptake and safety in non-target hosts (Tripp et al., 2015). A similar approach will be used to develop, test and optimize delivery methods to Rhinolophus bats in SE Asia.
To manage plague caused by Yersinia pestis in prairie dogs, a raccoon poxvirus vectored vaccine expressing plague antigens was incorporated into a peanut-butter flavored bait matrix. Rhodamine B (RB), a biomarker that dyes hair, whiskers and feces and is visible within 24 hours of consumption by animals, was included in the baits in

order to assess uptake by both target and non-target species (Figure 1). When viewed under a UV microscope at a specific wavelength, the biomarker is visible until the hair grows out (approximately 50 days in prairie dogs). Biomarker studies were initially used to assess palatability and acceptance of the bait matrix by wild prairie dogs (Tripp et al., 2014) and also used to assess bait ingestion by non-target rodents (Tripp et al., 2015). After safety was confirmed in non-targets and with the approval of USDA Center for Veterinary Biologics, a large field trial was conducted over a 3-year period that demonstrated vaccine effectiveness in four species of prairie dogs in seven western states (Rocke et al., 2017). Using biomarker analysis, we then assessed site- and individual host-level factors related to bait consumption in prairie dogs to determine those most related to increased bait consumption, including age, weight, and the availability of green vegetation. Identifying the factors that maximize the likelihood of expedient bait uptake by targeted individuals is important for developing strategies to optimize vaccine effectiveness. This will also be important in developing disease management strategies for bats.
Figure 1. Prairie dog hair and whisker samples viewed under fluorescence microscope (excitation wavelength: 540 nm, emission wavelength: 625 nm) to determine uptake of baits containing Rhodamine B. a) whiskers positive for RB uptake 20 days after bait distribution, b) hair sample positive for RB uptake 16 days after bait distribution, c and d) whiskers and hair negative for RB uptake 20 days after bait distribution (note natural dull fluorescence).
In recent years, our research team has been developing and testing vaccines and delivery methods for use in free-ranging bats. First we tested two commonly used viral vectors, modified vaccinia Ankara (MVA) and raccoon poxvirus (RCN), for their safety and replication in bats using in vivo biophotonic imaging. (Stading et al. 2017). RCN replicated to higher levels in bats than MVA, even via the oral route, and was found to be highly safe for bats (Figure 2). We then used raccoon poxvirus as a viral vector to express a novel rabies glycoprotein (mosaic or MoG) and tested the protective efficacy of this construct in bats after both oronasal and topical administration (Stading et al 2017). Both methods of application were successful, protecting nearly all of the immunized and challenged bats (Figure 3), work is now progressing to develop methods of vaccine delivery to vampire bats, one of the primary reservoirs of rabies for both humans and animals, primarily cattle, in several Latin American countries. We are also using a similar approach to develop vaccines for white-nose syndrome in bats, a devastating disease that has killed millions of insectivorous bats in North America.
a.
b.
c.
d.

MVA-luc given Days Post RCN-luc given Infection
1
3 5
Figure 2. Luminescence, indicative of viral replication of modified vaccinia Ankara (MVA) and raccoon poxvirus RCN) in Tadarida brasiliensis on days 1, 3 and 5 post- inoculation via the oronasal route.
RCN-MoG ON
RCN-MoG Topical RCN-G ON
RCN-luc

Figure 3. Results of vaccine efficacy and rabies challenge trials in Epstesicus fuscus immunized with raccoon poxvirus expressing a mosaic G protein (RCN-MoG) either oronasally (ON) or topically in comparison to RCN expressing typical G protein and RCN expressing luciferase (a negative control).
For bats a different approach is required for vaccine delivery, as in general, they are not attracted to baits. Bats, especially vampire bats, are known to practice self and mutual grooming at a high rate, and this behavior has been exploited to cull vampire bats using poisons like warfarin. The poison is applied topically to a number of bats that are released. When they return to their roost, the poison is transferred to roost-mates by contact and mutual grooming. We are exploiting this same behavior for vaccine application. Preliminary biomarker studies (without vaccine) are being conducted in vampire bats in both Mexico and Peru and also in insectivorous bats in Wisconsin. In a pilot study in Peru, we treated 50 bats from a single cave with RB-labelled glycerin jelly. Based on capture-recapture data, we estimated the population at ~200 bats, so ~25% of bats were initially marked. Upon trapping of this population a few days later, 64 bats were captured, including 19 originally marked bats (Table 1 – could be made into a figure instead). Hair was collected and examined for RB marking under a fluorescence microscope. All treated bats were positive for RB marking in addition to 39% of newly captured bats, indicating a rate of transfer of about 1.3 bats for every bat marked. Additional trials have been conducted, with transfer rates of up to 2.8 bats for every bat treated achieved at least once. These trials are being analyzed to assess factors associated with rates of transfer, e.g. sex and age of initially treated bats, time of day, etc. This data is then being used to model the rate of vaccination and impact on rabies transmission with different rates of application, prior to actual deployment of vaccine in the field.
Table 1. Marking of vampire bats a few days after application of glycerin jelly containing Rhodamine B.
All bats 64 34 25 5 58
For insectivorous bats, we are trying other approaches. Instead of hand applying the jelly to bats, we applied RB marked glycerin jelly to the entry of bat houses used by little brown bats (Myotis lucifugus). The bats became covered as they entered the houses and then consumed the material during self and mutual grooming. One week later, bats were trapped at the houses to determine the rate of uptake. Of 29 bats trapped one week post- application, 59% (17) were positive for biomarker indicating they had eaten the jelly. Thus, with additional optimization, application of vaccine to bat houses or other
Number captured
Positive
Negative
Inconclusive
% positive (w/o inc)
Recaptured marked bats
19 18 0 1 100 New bat captures 45 16 25 4 39

structures (small cave entrances) could also be a viable method of delivery. In addition, we are considering different spray applications directly to roosting bats in caves and through motion-sensing sprayers at cave entrances. Whatever the means of application, effective treatment relies on ingestion by bats, and that is easily confirmed with the use of the biomarker, RB.
Organization leading task: USGS National Wildlife Health Center Progress Metrics: Not sure exactly what format to use here
Deliverable(s):
Medium and methods to deliver immunomodulatory agents to bats. Data on uptake in insectivorous bats.
Reports, manuscripts, presentations.
Coleman CM, Liu YV, Mu H, Taylor JK, Massare M, Flyer DC, Smith GE, Frieman MB. 2014. Purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice. Vaccine 32:3169-3174.
Ebrahimian M, Hashemi M, Maleki M, Hashemitabar G, Abnous K, Ramezani M, Haghparast A. 2017. Co-delivery of dual toll-like receptor agaonists and antigen in poly(lactic-co-glycolic) acid/polyethylenimine cationic hybrid nanoparticles promote efficient in vivo immune responses. Front Immunol 8:1077.
Freuling CM, Hampson K, Selhorst T, Schro ̈der R, Meslin FX, Mettenleiter TC, Mu ̈ller T (2013) The elimination of fox rabies from Europe: determinants of success and lessons for the future. Philosophical Transactions of the Royal Society London B Biological Sciences 368(1623):20120142 (DOI: 10.1098/rstb.2012. 0142)
Karande P, Mitragotri S. 2010. Transcutaneous immunization: an overview of advantages, disease targets, vaccines, and delivery technologies. Annu Rev Chem Biomol Eng 1:175-201.
Mishra DK, Dhote V, Mishra PK. 2013. Transdermal immunization: biological framework and translational perspectives. Expert Opin Drug Deliv 10:183-200.
Roberts MS, Mohammed Y, Pastore MN, Namjoshi S, Yousef S, Alinaghi A, Haridass IN, Abd E, Leite-Silva VR, Benson HAE, Grice JE. 2017. Topical and cutaneous delivery using nanosystems. J Control Release 247:86-105.

Rocke TE, Tripp DW, Russell RE, Abbott RC, Richgels KLD, Matchett MR, Biggins DE, Griebel R, Schroeder G, Grassel SM, Pipkin DR, Cordova J, Kavalunas A, Maxfield B, Boulerice J, Miller MW. 2017. Sylvatic plague vaccine partially protects prairie dogs (Cynomys spp.) in field trials. EcoHealth DOI: 10.1007/s10393-017- 1253-x.
Slate D, Algeo TP, Nelson KM, Chipman RB, Donovan D, Blanton JD, Niezgoda M, Rupprecht CE (2009) Oral rabies vaccination in North America: opportunities, complexities, and challenges. PLoS Neglected Tropical Diseases 22 3(12):e549.doi:10.1371/journal.pntd.0000549
Stading BR, Osorio JE, Velasco-Villa A, Smotherman M, Kingstad-Bakke B, Rocke TE. Infectivity of attenuated poxvirus vaccine vectors and immunogenicity of a raccoonpox vectored rabies vaccine in the Brazilian Free-tailed bat (Tadarida brasiliensis). Vaccine. 2016;34: 5352–5358. doi:10.1016/j.vaccine.2016.08.088
Stading B, Ellison JA, Carson WC, Panayampalli SS, Rocke TE, Osorio JE. Protection of bats (Eptesicus fuscus) against rabies following topical or oronasal exporue to a recombinant raccoon poxvirus vaccine. PLoS Negl Trop Dis 11:e0005958.
Tripp DW, Rocke TE, Streich SP, Brown NL, Fernandez JR-R, Miller MW. 2014. Season and application rates affect vaccine bait consumption by prairie dogs in Colorado and Utah, USA. J Wildlife Dis 20:
Tripp DW, Rocke TE, Streich SP, Abbott RC, Osorio JE, Miller MW. 2015. Apparent field safety of a raccoon poxvirus-vectored plague vaccine in free-ranging prairie dogs, Colorado, USA. J Wildlife Dis 51:

10/5/21, 3:33 PM Mail - Rocke, Tonie E - Outlook
Its rough, but here’s a dra. One secon not included yet. Ralph
From: Rocke, Tonie [mailto:trocke@usgs.gov]
Sent: Friday, March 9, 2018 4:21 PM
To: Luke Hamel <hamel@ecohealthalliance.org>
Cc: Rachel Abbo <rabbo@usgs.gov>; Aleksei Chmura <chmura@ecohealthalliance.org>; Dr. Peter Daszak <daszak@ecohealthalliance.org>; Alison Andre <andre@ecohealthalliance.org>; Baric, Ralph S <rbaric@email.unc.edu>
Subject: Re: Rescheduling upcoming calls with Peter (PREEMPT)
Hello Luke and Peter: Attached is my first draft of Task 7. Ralph Baric (copied here) and I had a good chat today about viral vectors and nanoparticles. We realized much of the delivery methods would depend on his work first, so there are some gaps here and the narrative will probably change after I see what Ralph has written. At any rate, this is something to at least start with. Have a good weekend! - Tonie
On Thu, Mar 8, 2018 at 12:58 PM, Luke Hamel <hamel@ecohealthalliance.org> wrote: Hi Tonie,
Once again, please use this link, to select which date(s)/time(s) you are available to speak with Peter (select as many as you are available for). The goal is to have one call each week, on either Tuesday or Wednesday. Below, I've listed the dates/times that appear in the Doodle Poll link above.
Week 1:
Thu. 3/13 (9 AM - 5 PM ET) Thu. 3/14 (9 AM - 5 PM ET)
Week 2:
Thu. 3/20 (9 AM - 5 PM ET) Thu. 3/21 (9 AM - 5 PM ET)
Please let me know if you have any questions.
Best,
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct) (b) (6) (mobile) www.ecohealthalliance.org
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/2
>vog.sgsu@ekcort<EeinoT,ekcoR:oT
>ude.cnu.liame@cirabr< SMPh2p5l7a81R0,2/1c1i/r3anuBS
)TPMEERP( reteP htiw sllac gnimocpu gniludehcseR :ER
Please note that all times listed are in Eastern Time.
(b) (6)

10/5/21, 3:33 PM Mail - Rocke, Tonie E - Outlook
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics and promote conservation.
--
Tonie E. Rocke
USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
608-270-2451
trocke@usgs.gov
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 2/2

Commented [BRS1]: Need linkage to modeling group.
(TA1) Task 4: Develop recombinant chimeric spike proteins from characterized SARSr-CoVs
TA1.a. Description and execution: Our international team’s 15 yrs work experience on the SARSr-CoV – Rhinolophus bat system in China has identified and isolated SARSr-CoVs with remarkable sequence identity in the spike protein to SARS-CoV (e.g. SCH014, WIV-1, WIV-16). The pre-epidemic potential of these SARSr- CoVs is high, as our groups have shown these SARS-like bat viruses use human, bat and civet angiotensin-1 converting enzyme 2 receptors (ACE2) for entry, and importantly, bind and replicate efficiently in primary human lung airway cells, like epidemic SARS-CoV (PMC4801244, PMC4797993). Moreover, chimeras (recombinants) with SARSr-CoV spike proteins in a SARS-CoV backbone, as well as synthetically reconstructed full length SCHO14 and WIV-1 SARS-like bat viruses cause SARS-like illness in humanized mice (express human ACE2 receptor), with clinical signs that are not reduced by SARS monoclonal therapy or vaccination (PMC4801244, PMC4797993). We have identified a single cave site in Yunnan Province where bat SARSr-CoVs contain all the
genetic components of epidemic SARS-CoV (7,8,9). We have now shown that people living up to 6 kilometers from this cave have SARSr-CoV antibodies (3% seroprevalence in 200+ cohort) (PMID:29500691), suggesting active spillover, and marking these viruses as a clear-and-present danger of a new SARS-like pandemic.
TA1.b. SARSr-CoV Sample Collection and CoV Species Specificity. The Wuhan Institute of Virology team will continue to collect biodiversity surveys from SARSr-CoV viruses of bat caves across S. China. They have large, but incomplete collections of SARSr-CoVs sequences, most of which have not been evaluated for pre-epidemic potential. SARS-CoV cross species transmission is heavily regulated by S glycoprotein receptor binding domain interaction with the angiotensin 1 converting enzyme 2 receptor (ACE2). The structure of the SARS trimer prefusion spike has been solved as well as the bound SARS S RBD- ACE2 complex (PMC5223232, PMC4860016, ) Mutations in the RBD regulate SARSr-CoV capacity to replicate in human, civet, mouse and bat cell lines and animals (PMC2588415,
PMC2258931, PMID:16166518, PMC2446986). In fact, human-optimized RBD residues (Phe-442, Phe-472, Asn-479, Asp-480, and Thr-487) bind hACE2 best while civet-optimized RBD (Tyr-442, Pro-472, Arg-479, Gly- 480, and Thr-487) bind cACE2 best but also hACE2 (PMC3308800). In addition, recent data demonstrate that host proteases and S glycoprotein proteolytic processing also regulates entry and cross species transmission efficiency (PMID:16339146, PMC4151778, PMC5552337). If mismatches occur in the S-RBD-ACE2 molecules or in S proteoloytic processing, SARSr-CoV will not enter and hence cannot replicate in cells, unless this RBD- ACE2 interface is repaired by reverse genetics or mutation (PMC2588415, PMC2258931). Importantly, CoV species specific restriction events are limited after entry, as this same viral genome is replication competent when delivered by transfection or electroporation (PMC2588415, PMC2258931).Consequently, entry functions represent the key first step to evaluating the disease potential of SARSr-CoV.
Using RNAseq and Sanger sequencing methods, our groups have collected full length genome and S glycoprotein gene sequences over time and analyzed S genes analyzed phylogenetically for recombination events, and high-risk viruses by our group and others in the program (e.g., RBD-ACE2 structure modeling, receptor binding domain (RBD) sequence conservation, spike glycoprotein similarity to SARS-CoV, and protease cleavage site bioavailability, etc.). From in silico data and risk assessment modeling platforms, the primary goals in Task4 are to: i) model and down-select strains for recovery and analyses, ii) identify S glycoprotein genes which program infection in vitro using pseudotyped and chimeric viruses, iv) synthetically reconstruct a panel of high risk full length SARSr-CoV strains, v) characterize virus growth phenotypes in primary human airway cultures and vi) perform in vivo pathogenesis studies using human ACE2 transgenic mice.
Figure A. SARSr-CoV Spike Selection. (A) Virus S glycoprotein gene sequences will be phylogenetically placed into the group 2b SARS-like CoV. (B): S amino acid variation is then annotated onto the S structure and compared with the existing panel of live SARSr- CoV-predicting antigenically dissimilar strains for prioritization. (C- D) ACE2-S RBD interface residues are reviewed and structural modeling is used to predict virus-receptor interactions, noting the lack of robust HKU3 S RBD interaction with hACE2. Using the RNAseq data, modeling will also identify rare quasispecies variant strains that encode mutations in the population, which improve RBD- ACE2 interaction networks and potential potential to infect humans.

TA1.c. Preliminary Data: The Baric laboratory pioneered many of the strategic approaches (i-vi) and the SARSr- CoV reverse genetic platforms used for coronaviruses, including the use of synthetic genome design to reconstruct and recovery full length or S chimeric recombinant viruses from in silico sequence database (PMC4801244, PMC4797993, PMC2588415, PMC2258931). An example of full length recombinant SARSr-CoV reconstructed using reverse genetics in the Baric laboratory is shown in Fig A, Panel B and include human epidemic strains, civet and raccoon dog SARS-CoV strains, as well as SARSr-CoV strains (WIV16, WIV1, SHC014 and HKU3-SRBD {repaired RBD interface)}. These strains are immediately available for use in assessment of broadscale approaches to reduce population burdens of SARSr-CoV in bat cells available in the Baric, Shi and Wang laboratories (PMC2258931, PMC4801244, PMC4797993, PMC2588415, PMC4801244, PMC4797993).
TA1.d. Novel SARSr-CoV Virus Recovery. Chimeric Viruses. Our approach for testing the pre-epidemic potential of novel SARS-rCoV strains identified by sequence analyses is shown in Figure B. First, we will commercially synthesize selected SARSr-CoV S glycoprotein genes, designed for direct insertion into our full length SCH014 or WIV16 molecular clones (BSL3, not select agent, pathogenic in hACE2 transgenic mice), noting that these two SARSr-CoV are 88 and 97% identical to epidemic SARS-Urbani in the S glycoprotein. As the S gene interacts with the M glycoprotein, E protein and N protein in assembly/release, different backbone strains provide increased opportunities for recovery of viable viruses, as well as to identify potential barriers for RNA recombination between strains (PMC5708621). The chimeric viruses will be recovered in Vero cells, or in mouse cells over-expressing human, bat and civet ACE2 receptors, as receptor over-expression may support cultivation of viruses having weak RBD-ACE2 interfaces and sequence verified. Viruses will then evaluated for: i) human, civet and bat ACE2 receptor usage in vitro, ii) growth in primary human airway epithelial cells, iii) sensitivity to broadly cross neutralizing human monoclonal antibodies S215.17, S109.8, S227.14 and S230.15 and a mouse antibody (435) that recognize unique epitopes in the RBD (PMC2826557, PMID:29134417) and iv) in vivo pathogenesis studies in hACE2 transgenic
mice, using well established approaches in our laboratory (). A limited number of human SARS serum samples from the Toronto outbreak in 2003 are also available to evaluate cross neutralizing profiles using polyclonal serum (n=10), should some isolates prove highly resistant to our panel of mAB. Chimeric viruses that encode novel S genes with pre-epidemic potential (e.g., growth in HAE, use of multiple species ACE2 receptor for entry, antigenic variation, etc.) will be used to identify SARSr-CoV strains for recovery as full genome length viable viruses. We anticipate testing ~20 S genes/yr.
TA1e. Recovery of Full length SARSr-CoV. To
recover full length viruses, we will first compile the
sequence/RNAseq data from a panel of closely related
strains (e.g,<5% nucleotide variation) and compare
the full length genome sequences, scanning for unique
SNPs which might represent sequenceing errors as
previously described by our groups (PMC3497669,
PMC2583659, PMC3791741). We will identify the best consensus candidate and synthesize the genome using commercial vendors (e.g., BioBasic, etc.), as six contiguous cDNA pieces linked by unique restriction endonuclease sites that do not disturb the coding sequence, but allow for full length genome assembly. Full length genomes will be transcribed into RNA and electoration is used to recovery full length recombinant viruses (PMC3977350, PMC240733). Using the full length genomes, we will re-evaluate virus growth in primary human airway epithelial cells at low and high multiplicity of infections and in vivo in hACE2 transgenic mice, testing whether backbone genome sequence alters full length SARSr-CoV pre-epidemic or pathogenic potential in models of human infection. All experiments are performed in triplicate and the data provided to the Modeling Team for the development of risk assessment models, warfighter apps, and models to evaluate potential intervention outcomes. We anticipate recoverying 2-5 full length genomes/yr, reflecting strain differences in antigenicity, receptor usage, growth in human cells and pathogenesis.
Commented [BRS2]: Modeling team, how many you need; 60+ provide much help for modeling. I note that if we validate 1, half-a dozen additional strains with slight sequence variation also will fall in the “pre-epidemic” high risk categories so multiplies will get you into the 100’s or 1000’s of strains.
Figure B. Experimental Design. S genes of interest will be inserted into WIV16 and/or SCH014 full length molecular clones and chimeric virus rowth evaluated in primary human cells and cell lines constitutively expressing bat, civet, human and mouse ACE2 receptors. In vivo pathogenesis, hmAB cross neutralization assays and therapeutic intervention studies in vivo will prioritize strains for recovery of full-length SARSr-CoV.

TA1.f. In vivo Pathogenesis Studies. To generate a mouse model more relevant to humans, we generated a mouse that expresses human ACE2 receptor under control of HFH4, a lung ciliated epithelial cell promoter (PMC4801244). Infection of this model with wildtype SARS-CoV and WIV1 resulted in lethal disease outcomes with SARS-CoV, but minimal disease with WIV1. These data argue that WIV1 is less likely efficient at using the hACE2 receptor in vivo and hence less likely to produce severe disease outcomes in an outbreak setting. Of note, microvariation in the SARs-CoV RBD of related strains could dramatically alter these phenotypes, hence the need to evaluate the impact of low abundant, high consequence microvaration in the RBD. Briefly, groups of 10 animals will be infected intranasally with 1.0 x 104 PFU of each virus. Clinical disease (e.g., weight loss, respiratory function by Buxco plethysmography, mortality) will be followed for 6 days. One half the animals will be sacrificed at day 2 and 6 postnfection for virologic determinations, histopathology and immunohistochemistry in the lung and for 22-parameter complete blood count (CBC) in blood and BAL using the Vetscan HM5.
TA1.g. Evaluating 2ary S gene Markers for SARSr-CoV Pre-epidemic Potential. 1) Identification of high risk/low abundant variants. RNAseq will identify low abundant quasispecies variants that encode mutations in the RBD and/or residues that bind ACE2 receptor (Fig A). Low abundant mutations, especially in RBD residues that interface with ACE2 receptors, would alter risk assessment calculations as strains identified as low risk,
might actually encode high risk, but low abundant variants. To test this hypothesis, we will closely with the modeling core and Dr. Shi’s laboratory to identify highly variable residue changes in the SARSr-CoV S RBD, and use commercial gene blocks to introduce these changes singly and then in combination into the S glycoprotein gene of the parental low risk, high abundant strain parent. We will evaluate the ability of these low abundant chimeric viruses to use human, bat, civet and mouse ACE2 receptors, and more importantly, replicate efficiently in human primary cells. 2) Impact of RBD deletions on Pre-epidemic Risk Assessment. SARSr-CoV RBD sequences fall into two larger clades, heavily defined by the presence of small deletions between residues 432- 437 and 458-472, which leave the key RBD-ACE2
interface residues intact. (Fig C). To improve risk assessment, we will molecularly analyze the functional consequences of these deletions on SARSr-CoV human ACE2 receptor usage, growth in primary cells and in vivo pathogenesis. First, we will delete these regions, sequentially and then in combination, in SCH014, anticipating that the introduction of both deletions will prevent SCH014 growth in Vero and human cells. We also hypothesize that the smaller deletion may be tolerated, given it location in the RBD structure. In parallel, we will evaluate the ability of targeted recombination between high and low risk strains to restore pre-epidemic features to low risk viruses. To test this hypothesis, we will synthesize full length rs4237, a highly variable SARSr-CoV that encodes the SHC014 RBD contact interface residues at 442, 487 and 491 but also encodes mutation at 479 (N479S) and has the 432-437 and 458-472 deletions and hence, is not recoverable in vitro. Using the SCH014 backbone sequence, we will first sequentially and then in tandem repair the 432-437, 458-472 deletions in the presence and absence of the S479N. We anticipate that the S479N mutation is critical given its key role in establishing the RBD-ACE2 interface, and that restoration of the RBD deletions will significantly enhance virus recognition of hACE2 receptors and growth in Vero and HAE cultures in vitro. Together, these data will directly inform risk assessment SARSr-CoV population genetic structure obtained from difference cave ecologies. 3) S2 Proteolytic Cleave and Glycosylation Sites. In some instances, recombinant chimeric viruses may be predicted to replicate efficiently because of matched RBD-ACE2 interfaces, yet fail to replicate. After receptor binding, cell surface or endosomal proteases cleave the SARS S glycoprotein to activation fusion mediated entry (PMC4151778). Massive changes in Spike structure occur to mediate membrane fusion and entry (PMC5651768). The absence of S cleavage prevents SARS-coV entry (PMC5457962). A variety of proteases, including TMPRSS2, TMPRSS11a, HAT, trypsin and cathepsin L carry out these processes on the SARS S glycoprotein (PMC3233180, PMC3889862, PMC5479546, PMID:26206723)(Fig C). In some instances, tissue culture adaptations introduce a furin cleavage site, which can direct entry processes as well, usually by cleaving S at positions 757 and 900 in S2 of other coronaviruses, but not SARS (PMID:26206723). For SARS-CoV, a variety key cleavage sites in S have been identified including R667/S668, R678/M679 for trypsin and cathepsin L, respectively, R667 and R792 (and other unidentified sites) for TMPRSS2, and R667 for HAT. Therefore, all
Figure C. Additional Markers for High/Low Risk Strain Identification. (A). Clade 2, but not Clade 1 SARSr-CoV encode programmed deletions which likely impact ACE2 receptor usage. (B) Proteolytic and N-glycosylation sites of interest.

SARSr-CoV S gene sequences will be analyzed for the presence of these appropriately conserved proteolytic cleavage sites in S2 and for the presence of potential furin cleavage sites (R-X-[K/R]-R↓) and which can be predicted computationally (PMC3281273) . Importantly, SARr-
CoV with mismatches in proteolytic cleavage sites can be
activated by exogenous trypsin or cathepsin L (Fig D), providing
another strategy to recover non-cultivatable viruses. In instances
where clear mismatches occur in these S2 proteolytic cleavage
sites of SARSr-CoV, we will introduce the appropriate human-
specific cleavage sites and evaluate growth potential in Vero and
HAE cultures. In SARS-CoV, we will ablate several of these sites
based on pseudotype particle studies and evaluate the impact of
select SARSr-CoV changes on virus replication and pathogenesis (e.g., R667, R678, R797). Experimental outcomes from these studies will be incorporated into risk management models to identify high and low risk SARS-like CoV.
SARS S has 23 potential N-linked glycosylation sites N-linked glycosylation sites (NX(S/T; X is anything but proline) and 13 of these have been confirmed using biochemical approaches (e.g., positions: 118, 119, 227, 269, 318, 330, 357, 783, 1056, 1080, 1140, 1155, and 1176). Importantly, several N-linked glycosylation sites regulate SARS particle binding DC-SIGN/L-SIGN, alternative entry receptors for SARS-CoV infections (positions: N109, N118, N119, and especially N227, N699)( PMC1641789, PMC2168787, PMC2168787) and may protect critical sites for antibody neutralization(PMC5515730). Importantly, the emergence of human SARS-CoV from civet and raccoon dog reservoir strains was associated with the evolution of mutations that introduced two N-linked glycosylation sites that promote DC-SIGN/L-SIGN binding (N227, N699), suggesting a role in the expanding human 2003 epidemic (PMC2168787). Interesting, these N-linked glycosylation sites are absent from civet, raccoon dog strains and clade 2 SARSr-CoV, but are present in WIV1, WIV16 and SCH014 as well as human epidemic strains, supporting a potential role in host jumping (Fig C). To evaluate the role of these mutations in cross species transmission and pathogenesis, we will sequentially introduce clade 2 residues at positions N227 and N699 of SARS-CoV and SCH014 and evaluate virus growth in Vero, Huh7 cells (nonpermissive) expressing ectopically expressed DC-SIGN and HAE cultures, anticipating reduced virus growth efficiency. Using the clade 2 rs4237 molecular clone described above, we will introduce the clade I mutations that introduce N-linked glycosylation sites at positons 227 and N699 and in rs4237 RBD deletion repaired strains, evaluating virus growth efficiency on Vero, HAE or Hela cells ± ectopically expressing DC-Sign (PMC2168787). In vivo, we will evaluate pathogenesis in transgenic ACE2 mice. Experimental outcomes from these studies will be incorporated into risk management models to identify high and low risk SARS-like CoV.
Organization leading task: University of North Carolina Progress Metrics: Not sure how to do this.
Deliverable(s):
1. Methods to Produce Synthetic SARSr-CoV Virus Molecular Clones and Reverse Genetics.
a. Preliminary Data: Molecular Clones for SARSr-CoV WIV1, WIV16, SCH014 and HKU3-SRBD exist. We have demonstrated in the preliminary data that these reagents are already available.
b. Target Goals: We will generate molecular constructs for 20+ chimeric SARSr-CoV encoding different S glycoprotein genes/yr
c. Target Goals: We will generate 2-5 full length molecular clones of SARSr-CoV.
2. Methods of Recombinant virus Recovery and Characterization
a. Preliminary Data: Demonstrated recovery recombinant chimeric SARSr-CoV WIV1, WIV16,
SCH014, HKU3-SRBD, including full length recombinant viruses of WIV1, WIV16, SCH014 and
HKU3-SRBD.
b. Target Goals: We will isolate 20+ chimeric SARSr-CoV encoding novel S glycoprotein genes
c. Target Goals: We will isolate 2-5 full length SARSr-CoV/year/
i. Key Deliverables for Program-wide Success: These two key reagents position us for immediate testing of the antiviral effects of broadscale immune boosting molecules +/- immunogens on virus growth in vitro and in vivo, and on virus levels in models of
Figure D. Exogenous Trypsin Restores SARSr- CoV Growth.

Commented [BRS3]: Broadscale immune boost + chimeric immunogen
chronic SARS-CoV infection in mice.
3. Virus Phenotyping: Receptor Interactions and In Vitro Growth.
a. Preliminary Data: Cell lines encoding bat, human, civet and mouse ACE2 receptors exist and
have been validated. We have demonstrated the use of primary human airway epithelial
cultures to characterize SARSr-CoV pre-epidemic potential.
b. Target Goals: We will characterize SARSr-CoV recombinant virus growth in Vero cells,
nonpermissive cells encoding the civet, bat and human ACE2 receptors.
4. Virus Pathogenic Potential in Humans:
a. Preliminary Data: We also have transgenic human ACE2 mouse models to compare the
pathogenic potential of SARSr-CoV
b. Target Goals: We will evaluate SARSr-CoV pathogenic outcomes in hACE2 transgenic mice.
5. Virus Antigenic Variation:
a. Preliminary Data: We have robust panels of broadly cross reactive human monoclonal
antibodies against SARS and related viruses and mouse models to evaluate protection against
SARSr-CoV replication and pathogenesis.
b. We will evaluate SARS-vaccine performance against a select subset of SARSr-CoV (10), chosen
based on the overall percent of antigenic variation, coupled with distribution across the S glycoprotein structure.
6. Low Abundant High Consequence Sequence Variants:
a. We will identify the presence of low abundant, high risk SARSr-CoV, based on deep sequencing
data
7. Proteolytic Processing and Pre-epidemic Potential.
a. We will evaluate the role of proteolytic cleavage site variation on SARSr-CoV cross species
transmission and pathogenesis in vivo.
(TA2) Task 6: Trial experimental approaches aimed towards ‘Immune Targeting’ using experimental bat colonies
TA2.a. Description and execution: There is no available current technology to reduce the risk of exposure to novel coronaviruses from bats which carry zoonotic precursors to many emerging viruses including filoviruses (Ebola), Coronaviruses (SARS-CoV, MERS-CoV, etc.), paramyxoviruses (Nipha/Hendra), rhabdoviruses (rabies) and others. Unfortunately, models of bat host capacity to harbor viruses, of ecological and environmental drivers of their emergence, and of the evolutionary potential of different strains to spillover are rudimentary. No vaccines or therapeutics exist for emerging coronaviruses, filoviruses and paramyxoviruses and exposure mitigation strategies are non-existent. Using emerging coronaviruses (SARSr-CoV) as models, we will apply broadscale immune boosting strategies alone (Prof. Linfa Wang (Duke-NUS) or in the presence and absence of chimeric immunogens (targeted immune boosting strategy), designed to upregulate bat immunity in the cave roosts, down-regulate viral replication and boost adaptive immunity to high risk strains. We will use small molecule Rig like receptor (RLR) or Toll like receptor (TLR) agonists coupled with novel chimeric polyvalent recombinant spike proteins in microparticle encapsidated gels and powders for oral delivery and/or virus adjuvanted immune boosting strategies where chimeric recombinant SARSr-CoV spikes are expressed from raccoon poxvirus, which has been used extensively to devlier rabies immunogens in bats and other animals. We will design novel methods to deliver these applications remotely to reduce exposure risk during decontamination.
The Baric group has developed novel group 2b SARSr-CoV chimeric S glycoproteins that encode neutralizing domains from phylogenetically distant strains (e.g., Urbani, HKU3, BtCoV 279), which differ by ~25%. The chimeric spike programs efficient expression when introduced in the HKU3 backbone full length genome, and elicit protective immunity against multiple group 2b strains (see preliminary data). We will use this platform as a broadscale immune boosting strategy. First, we will develop robust expression systems to express SARSr-CoV chimeric spikes using ectopic expression in vitro. Then, we work with Dr. Ainslie (UNC-Pharmacy) who has developed novel microparticle delivery systems and dry

powders for aerosol release, and which encapsidate recombinant proteins and adjuvants (innate immune agonists) that will be used for parrellel broadscale immune boosting strategies ± chimeric immungens. In parallel, we will introduce
chimeric and wildtype spikes in raccoon poxvirus (RCN), in collaboration with Dr. Rocke and confirm recombinant protein expression, first in vitro and then in bats in collaboration with Dr. Shi and Dr. Wang, who have bat colonies. The goal of this aim is to develop a suite of reagents to remotely reduce exposure risk in high risk environmental settings.
TA2.b. Preliminary Data: Chimeric SARSr-CoV Spike Immunogens. Coronaviruses evolve quickly by mutation and RNA recombination, the latter provides a strategy to rapidly exchange functional motifs within the S glycoprotein and generate viruses with novel properties in terms of host range and pathogenesis (PMC237188, PMC5708621). Coronaviruses also encode neutralizing epitopes in the NTD, RBD and S2 portion of the S glycoprotein (PMID:29514901, PMC2826557, PMC2268459, PMC3256278). Given the breadth of SARSr-CoV circulating in natural settings, chimeric immunogens were designed to increase the breadth of neutralizing epitopes across the group 2b phylogenetic subgroup (PMC5708621). Using synthetic genome design and structure guided design, we fused the n-terminal NTD domain of HKU3 (1-319) with the SARS-CoV RBD (320-510) with the remaining BtCoV 279/04 S glycoprotein molecule (511-1255), introduced the chimeric S glycoprotein gene into the
HKU3 genome backbone (25% different than SARS-CoV, clade 2 virus) and recovered viable viruses (HKU3-Smix) that could replicate to titers of about 108 PFU/ml on Vero cells (Fig E). HKU3-Smix is fully neutralized by monoclonal antibodies that specifically target the SARS RBD (data not shown). In parallel, we inserted the HKU3mix S glycoprotein gene into Venezuelan equine encephalitis virus replicon vectors (VRP-Schimera) and demonstrated that VRP vaccines protect against wildtype SARS-CoV challenge and virus growth. In addition, VRP-SHKU3 and VRP-S279 both protect against HKU3mix challenge and growth in vivo (Fig E), demonstrating that neutralizing epitopes in the HKU3mix S glycoprotein are appropriately presented and provide broad cross protection
against multiple SARSr-CoV. In addition to using these immuogens as a targeted broad-based boosting strategy in bats, we will also produce a chimeric SCH014/SARS-CoV/HKU3 S gene for more focused immune targeting on known high risk strains.
In parallel, we will work with the Protein Expression Core at UNC
(https://www.med.unc.edu/csb/pep)to produce codon optimized, stabilized
and purified prefusion SARS-CoV glycoprotein ectodomains as described
by Pallesen J et al., PNAS 2016. Briefly, the chimeric S ectodomain will be
linked to a C-terminal T4 fibritin trimerization domain, an HRV3c cleavage
site, an 8xHis-Tag and a Twin-Strep-tag, and after transfection, mg quantities
will be produced in 0.5–1 L FreeStyle 293-F cells treated with kifunensine (5
μM final concentration) for 6 d. The chimeric S trimer protein will be purified
Strep-Tactin resin (IBA), treated withHRV3C protease overnight at 4 °C and
the products purified using a Superose 6 16/70 column (GE Healthcare
Biosciences) (PMC5584442). Purified recombinant protein will be used by Dr.
Rocke and Dr. Ainslie for inclusion in delivery matrices (e.g., purified
powders, dextran beads, gels) with broadscale immune agonists (adjuvants-Dr. Wang) like poly IC, TLR4 and Sting agonists and can be fine-tuned for regulated delivery by programmed degradation for time-ordered delivery (Fig F). These particles can be aerosolized, or delivered in sprays or gels to bat populations, providing new modalities for zoonotic virus disease control in wildlife populations (PMID: 28032507, PMC4267924).
TA2.c. 2nd Generation Chimeric S glycoprotein Design and Testing. Using the approach discussed above, we will also produce a chimeric SCH014 NTD/SARS-CoV-RBD/HKU3 S C terminal and generate recombinant HKU3 encoding the
Figure E. Chimeric SARSr-CoV S Glycoprotein Immunogens. (A) A chimeric S glycoprotein was synthesized which contained HKU3, SARS-CoV and BtCoV/279/04. (B) Recombinant viruses encoding the HKU3-Smix gene were viable and grew to ~108 PFU/ml in Vero Cells. (C) VRP vaccines encoding the SARS-S, BtCoV 279-S and HKU3-S protect against HKU3-Smix challenge. (D) VRP-HKU3-Smix vaccine protect against SARS-CoV lethal challenge.
Figure F. Particle Delivery Systems. Broadscale immune boosting strategies include (A) Dextran microparticles or (B) Dry Powders.

trimer spike (HKU3-SS014), for more focused immune targeting on known high risk strains with pre-epidemic potential. After sequence variation, we will evaluate virus growth in Vero and HAE cultures and the ability of SARS RBD monoclonal antibodies (S227, S230, S109) to neutralize chimeric virus infectivity (PMC2268459, PMC2826557). We will also evaluate in vivo pathogenesis in C57BL/6 mice and hACE2 transgenic mice. The recombinant HKU3-SS014 S genes will be introduced into VRP vectors and sent to Dr. Rocke for insertion into the raccoon poxvirus vaccine vector, characterization of S expression and then provided to Drs. Wang and Shi for immune boosting of bats. Recombinant HKU3-SS014 glycoprotein expression will be validated by Western blot and by vaccination of mice, allowing us to determine if the recombinant protein elicits neutralizing antibodies that protect against lethal SARS-CoV, HKU3-Smix and SCH014 challenge. In parallel, we will survey the RNAseq data for evidence of complex S glycoprotein gene RNA recombinants in the cave SARSr-CoV population genetic structure. We will synthesize 2-3 interesting recombinant S genes, insert these genes into SCHO14 or HKU3 genome backbones and VRP and characterize the viability and replicative properties of these viruses in cell culture and in mice and the VRP for S glycoprotein expression and vaccine outcomes.
TA2.d. Microparticle Performance Metrics in vitro and in Rodents and Bats.
Organization leading task: University of North Carolina Progress Metrics:
Deliverable(s):

10/5/21, 3:35 PM
Mail - Rocke, Tonie E - Outlook
Year Y1
Y2
OY1
OY .5
Total
Amount $304,500.00
$304,500.00
$304,500.00
$152,250.00
$1,065,750.00
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/3
:'stsoC tceridnI' gnidrageR )1
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yrassecen eht gnirucorp ni uoy tsissa yldalg lliw ffats AHE .snoitseuq yna evah uoy fi wonk em tel esaelP
.noissimbus tegdub lanif eht rof
deriuqer eb lliw ti ,etalpmet tegdub dehcatta eht etelpmoc ot uoy rof dedeen t'nsi liated fo level siht hguohtlA
.tegdub ruoy fo stcepsa cificeps enimreted ot nigeb uoy sa smeti gniwollof eht dnim ni peek esaelp ,yltsaL
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rof eludehcs tnemesrubsid desoporp eht dedulcni evah I ,etalpmet tegdub eht gnitelpmoc ni uoy tsissa oT
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htiw uoy edivorp ot erus eb lliw I dna ,emit retal a ta desserdda eb nac )noitacifitsuj tegduB( L noitceS .elbissop
sa yletelpmoc sa snoitces eseht tuo llif dna ,K-A snoitces no noitnetta ruoy sucof esaelp ,tnemom eht tA
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tegdub lareneg a enimreted ot AHE ta ffats ruo dna uoy htob wolla lliw ti ,retal deriuqer eb lliw taht liated
cificeps skcal etalpmet siht hguohtlA .etelpmoc ot uoy rof etalpmet tegdub TPMEERP a ,dehcatta ees esaelP
,einoT iH
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sliated dna etalpmet tegdub TPMEERP :eR

10/5/21, 3:35 PM Mail - Rocke, Tonie E - Outlook
Provide the purpose of the trip, number of trips, number of days per trip, departure and arrival destinations, number of people, estimated rental car and airfare costs, and prevailing per diem rates as determined by gsa.gov, etc.; Quotes must be supported by screenshots from travel websites.
3) Regarding 'Equipment Purchases'
Itemization with individual and total costs, including quantities, unit prices, proposed vendors (if known), and the basis of estimate (e.g., quotes, prior purchases, catalog price lists, etc.); any item that exceeds $5,000 must be supported with back-up documentation such as a copy of catalog price lists or quotes prior to purchase (NOTE: For equipment purchases, include a letter stating why the proposer cannot provide the requested resources from its own funding.
4) Regarding 'Materials'
Itemization with costs, including quantities, unit prices, proposed vendors (if known), and the basis of estimate
(e.g., quotes, prior purchases, catalog price lists, etc.); any item that exceeds $5,000 must be supported with back- up documentation such as a copy of catalog price lists or quotes prior to purchase.
Best,
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct) (b) (6) (mobile)
www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics
and promote conservation.
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 2/3
(b) (6)
:'levarT' gnidrageR )2
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edivorp ,etar a naht rehto yb detaluclac fi ,ro ,egakcap etar detaitogen RNO ro SHHD edivorp ,aimedaca
roF .setar eht etareneg ot desu stsoc esnepxe dna loop edulcni ot atad lacirotsih sraey 2 edivorp ,elbaliava
ton fI .lasoporP etaR gnicirP drawroF ro tnemeergA etaR gnicirP drawroF tnerruc edivorp ,elbaliava fI
.D.hP ,sleghciR .D .L enirehtaK
--
vog.sgsu@ekcort
1542-072-806
11735 IW ,nosidaM
.dR redeorhcS 6006
retneC htlaeH efildliW lanoitaN SGSU
ekcoR .E einoT
--

10/5/21, 3:35 PM
Mail - Rocke, Tonie E - Outlook
6006 Schroeder Rd Madison, WI 53711 (608) 270 - 2450 (office) (608) 381 - 2492 (cell) (608) 270 - 2415 (fax) krichgels@usgs.gov www.nwhc.usgs.gov
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 3/3
retneC htlaeH efildliW lanoitaN SGSU
margorP tnegA tceleS laredeF ,laiciffO elbisnopseR
hcraeseR htlaeH efildliW deilppA ,feihC hcnarB

GROSS FUNDING
$78,030.00
64.543%
46.017%
20.000%
17.919%
NET
$47,422.25
$53,438.98
$65,025.00
$66,172.54
COM
$8,497.59
$4,007.92
$4,876.88
$11,857.46
FAC
$13,749.61
$15,494.10
$3,039.27
$0.00
BUREAU
$8,360.33
$5,089.09
$5,089.10
$0.00
TOTAL BURDEN COSTS
$30,607.54
$24,591.12
$13,005.25
$11,857.46
TOTAL COM FAC

GROSS
COM
FAC
BUREAU
TOTAL BURDEN COSTS
NET FUNDING
BUR

10/5/21, 3:36 PM
Mail - Rocke, Tonie E - Outlook
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct) (b) (6) (mobile)
www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics
and promote conservation.
(b) (6)
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/2
ni esoht ot lacitnedi era taht )L-A( snoitces gniynapmocca htiw ,'2 doireP
tegduB -tegduB detaleR & hcraeseR' ,deltit si taht egap a ees neht lliw uoY
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meti enil hcae rof noitanalpxe edivorp tsum tnemucod sihT .ydaerla os enod ton evah uoy
fi ,noitacifitsuj tegdub a gnitfard nigeb esaelP :'noitacifitsuJ tegduB' ,L noitceS gnidrageR
:ekam ot ekil d'I stnemmoc lanoitidda wef a era ereht ,tegdub TPMEERP ruoy tuo gnillif era uoy sA
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,einoT iH
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)pu-wollof( tegdub TPMEERP :eR

10/5/21, 3:36 PM
Mail - Rocke, Tonie E - Outlook
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct) (b) (6) (mobile)
www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics
and promote conservation.
(b) (6)
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 2/2
,tseB
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os ,tegdub eht fo stcepsa niatrec gnidrager snoitseuq evah yam uoy taht etapicitna eW .emiT
nretsaE ,41/3 .deW yb )dedulcni 'sdoirep tegdub' evif lla htiw( tegdub ruoy nruter esaelp ,yltsaL
.sliame lla no uoy
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siht fI .smeti detaler-tegdub gnissucsid nehw tsol si gnihton erusne ot ,yltcerid laudividni
siht htiw etacinummoc ot tseisae eb lliw ti leef eW .noitutitsni ruoy ta tegdub TPMEERP
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.tnemucod eht nihtiw dnuof eb nac nottub 'doireP ddA' eht erehw
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5. raeY noitpO dna 1 raeY noitpO ,3 raeY rof sdoirep gnidda ,ssecorp siht taepeR
'.2 doireP tegduB'
ot noitamrofni dda uoy sa setadpu 'tegdub evitalumuC - tegduB detaleR &
hcraeseR' ,deltit egap eht taht eciton ll'uoY .)dedeen sa tide ro 1 doireP tegduB
morf noitamrofni revo ypoc( doirep tegdub dnoces siht tuo lliF .1 doireP tegduB

10/5/21, 3:37 PM
Mail - Rocke, Tonie E - Outlook
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct) (b) (6) (mobile)
www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics
and promote conservation.
Luke Hamel
Program Assistant
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/3
(b) (6)
,tseB
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,'1 raeY' :swollof sa era yeht dna tcejorp eht rof sdoirep 4 ylno era erehT .tcerrocni si sihT
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,einoT iH
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>gro.ecnaillahtlaehoce@lemah<MlPe10m18a10H2/3e1/k3euuLT
)pu-wollof( tegdub TPMEERP :eR

10/5/21, 3:37 PM
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct) (b) (6) (mobile)
www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics and promote conservation.
(b) (6)
Mail - Rocke, Tonie E - Outlook
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 2/3
,tseB
.elbissop
sa ylkciuq sa eussi yna evloser ot yrt lliw eW .ksa ot etatiseh ton od esaelp os ,tegdub
eht fo stcepsa niatrec gnidrager snoitseuq evah yam uoy taht etapicitna eW .emiT nretsaE
,41/3 .deW yb )dedulcni 'sdoirep tegdub' evif lla htiw( tegdub ruoy nruter esaelp ,yltsaL
.sliame lla no uoy ypoc ot erus
eb lliw ew ,noitutitsni ruoy ta tnemtraped rehtona morf enoemos si laudividni siht fI
.smeti detaler-tegdub gnissucsid nehw tsol si gnihton erusne ot ,yltcerid laudividni siht
htiw etacinummoc ot tseisae eb lliw ti leef eW .noitutitsni ruoy ta tegdub TPMEERP
eht gniganam rof elbisnopser si ohw wonk em tel esaelP :'tcatnoC fo tnioP' tegduB
.tnemucod eht nihtiw dnuof eb nac nottub 'doireP ddA' eht erehw gnithgilhgih
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,deltit egap eht taht eciton ll'uoY .)dedeen sa tide ro 1 doireP tegduB morf
noitamrofni revo ypoc( doirep tegdub dnoces siht tuo lliF .1 doireP tegduB
ni esoht ot lacitnedi era taht )L-A( snoitces gniynapmocca htiw ,'2 doireP
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nI .tcejorp desoporp eht fo raey tsrif eht ot ylno srefer '1 doireP tegduB' sihT
.'1 doireP
tegduB -tegduB detaleR dna hcraeseR' ,sdaer 2 egap fo pot eht taht ees ll'uoy ,keew
siht reilrae uoy ot tnes I etalpmet tegdub eht ot refer uoy fI :sdoirep tegdub gniddA
.noitamrofni lanoitidda htiw uoy edivorp ot yppah eb d'I ,noitces
siht gnidrager snoitseuq yna evah uoy fI .tegdub deliated ruoy nihtiw meti enil hcae rof
noitanalpxe edivorp tsum tnemucod sihT .ydaerla os enod ton evah uoy fi ,noitacifitsuj
tegdub a gnitfard nigeb esaelP :'noitacifitsuJ tegduB' ,L noitceS gnidrageR
:ekam
ot ekil d'I stnemmoc lanoitidda wef a era ereht ,tegdub TPMEERP ruoy tuo gnillif era uoy sA
,einoT iH
:etorw >gro.ecnaillahtlaehoce@lemah< lemaH ekuL ,MP 327 ta 8102 ,9 raM ,irF nO

10/5/21, 3:37 PM
Mail - Rocke, Tonie E - Outlook
Phone: 1-719-785-9461 Password: 9784#
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(direct)
(mobile) www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics
and promote conservation.
(b) (6)
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 1/1
,tseB
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>gro.ecnaillahtlaehoce@lemah<MlA4e18m81a02H/41e/3kdueLW
cihparg TPMEERP no kcabdeeF
(b) (6)

Host-Pathogen Prediction
Intervention Development
Modeling recombination and evolution
Validate models
using human serology
Simulating host-viral dynamics
Assays in humanized mice
Immune priming
Host-Pathogen Niche Modeling
Immune boosting
Machine learning genotype-phenotype mapping
Captive experiments
Field testing and deployment

From:
Sent:
To:
Subject: Attachments:
Hi Luke/Peter: In anticipation of our call tomorrow, take a look at the attached white paper and video on the link below. I think this looks like a great option for a spray device for bats, and it sounds like the material I have been working with already would work perfectly with this system. I haven't yet been able to pin them down on a price for a subcontract, but I'd like to talk to you tomorrow about this and some other budget details I am struggling with. Thanks -Tonie
---------- Forwarded message ---------- From: <Jerome.Unidad@parc.com> Date: Tue, Mar 13, 2018 at 1:53 PM Subject: Re: PARC FEA
To: trocke@usgs.gov Tonie,
Thanks for reaching out. Here’s a 1-pager on our spray technology. If you are curious about how the spray might actually look like, you can check out a video here -- https://www.parc.com/services/focus-area/amds/
We would really be interested in working with your proposal team on this. If possible, it will be more value-generating for us to be a subcontractor and to contribute more to tailoring our spray technology for the intended use case. I’d also like to mention that another aspect we could bring to the table is in transitioning out the technology into a reality, particularly towards commercialization, because we have a good history on this – particularly on the device side but also, and increasingly, in the biomedical space through other commercial partners.
Please let me know how this might turn out.
Thanks,
Jerome
From: "Rocke, Tonie" <trocke@usgs.gov>
Date: Tuesday, March 13, 2018 at 5:11 AM
To: "Unidad, Jerome <Jerome.Unidad@parc.com>" <Jerome.Unidad@parc.com> Subject: Re:
Rocke, Tonie <trocke@usgs.gov> Tuesday, March 13, 2018 12:29 PM Luke Hamel; Daszak Peter
Fwd: PARC FEA PARC_whitepaper_Biotech_v3_final.pdf
1

Hi Jerome: 1-2 CT is fine for me. I may not be in my office, however, so can I call you? Is this number good? 650-812-4209. Thanks -Tonie
On Mon, Mar 12, 2018 at 8:34 PM, <Jerome.Unidad@parc.com> wrote:
Sorry, I mixed up the schedule. I actually do have a meeting in the 1-2PM PST slot, how about 11AM-12PM PST (1-2PM
CT)? Thanks, Jerome
From: "Rocke, Tonie" <trocke@usgs.gov>
Date: Monday, March 12, 2018 at 5:25 PM
To: "Unidad, Jerome <Jerome.Unidad@parc.com>" <Jerome.Unidad@parc.com> Subject: <no subject>
Hi Jerome: I have been working on developing vaccines for use in managing disease in wild bats - e.g. rabies in vampire bats and white-nose syndrome in insectiverous bats. I am also collaborating on a PREEMPT proposal and one of my collaborators passed on your contact information and description regarding PARC's spray technology for possible application in this field. Would you have time to chat tomorrow? I'll be available anytime after noon CT. Thanks much! -Tonie
--
Tonie E. Rocke
USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
608-270-2451
trocke@usgs.gov
2

--
Tonie E. Rocke
USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
608-270-2451
trocke@usgs.gov
--
Tonie E. Rocke
USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
608-270-2451
trocke@usgs.gov
3

Innovative Solutions for Biotechnology @ PARC
Leveraging on our long history in fluid and particle manipulation and roll-to-roll processes for printing applications, the PARC hardware laboratories are currently developing innovative device solutions with potential applications in biomedicine and biotechnology at-large.
Filament Extension Atomizer (FEA)
PARC is developing a novel spray technology called the Filament Extension Atomizer (FEA) that can generate aerosol from fluids that are notoriously difficult to aerosolize due to the inherent viscosity limit with most conventional spray methods. FEA can spray fluids of a wide range of viscosities: from 1 mPa-s (the viscosity of water) up to 1000 Pa-s (the viscosity of peanut butter) – this range includes fluids or dispersions with significant (bio)macromolecular content. This macromolecular content (long chain polymers) often impart an additional resistance to aerosol/spray generation due to strain hardening or the increase of fluid viscosity as a function of extension.
T o generate aerosol from such strain hardening fluids, FEA harnesses a well- known elasto-capillary instability that generates beads-on-a-string formation (Fig. 1A) when a fluid is held in extension. As the filaments are sufficiently thinned out, droplet break-up occurs and generates free droplets (aerosol). The FEA technology implements similar mechanics in a roll-to- roll process (Fig. 1B) to massively parallelize the filament formation and break- up and continuously generate droplets. To date, we have applied this on multiple viscoelastic fluids which include polymer solutions, polymer melts, particle dispersions and other complex systems with biomolecules (Figs. 1C-E).
Fig. 1 – A. Beads-on-a-string structures in a viscoelastic fluid in extension (McKinley and Olivera, 2005), B. Multiple beads-on-a-string formations in counter-rotating rollers (FEA), FEA spraying of PEO solution (C.), hyaluronic acid (D.) and sunscreen (E.)
Consumer Scale
• Portable devices
• Small rollers (down to 10mm)
• Low throughput
• Precision fluid delivery (e.g.
bioactive doses)
• Smart, connected devices
Industrial Scale
• Large rollers (up to 100mm) • High throughput
• Unit operation in a
manufacturing line
• Particle creation (e.g. spray
drying), large area coatings
The FEA technology is inherently scalable – it can work with small rollers for consumer scale applications, tailored for precision fluid dispensing, or with large rollers for industrial scale, high throughput aerosol generation for coatings or for powder production via spray drying. Since the FEA technology applies to wide range of fluids of virtually any viscosity or composition, it allows nearly formulation-independent fluid delivery. This can have a huge impact in biomedicine and biotechnology at-large: fluids can be formulated for bio-efficacy and not for delivery with no limits on bioactive loading, even with high viscosity biomacromolecules that are notoriously hard to deliver in a controlled, reliable manner. FEA can also allow the creation of specialized particles for drug delivery with a wide range of material set and control over the particle morphology.
Fig. 2 – FEA Technology at Different Scales and Applications
PARC, 3333 Coyote Hill Road, Palo Alto, California 94304 USA +1 650 812 4000 | engage@parc.com | www.parc.com

Technical Contact:
Jerome Unidad (Jerome.Unidad@parc.com), Member of Research Staff
Copyright © 2018 Palo Alto Research Center Incorporated.
All Rights Reserved. PARC, the PARC Logo are trademarks of Palo Alto Research Center Incorporated.
A global center for commercial innovation, PARC, a Xerox company, works closely with enterprises, entrepreneurs, government program partners and other clients to discover, develop, and deliver new business opportunities. PARC was incorporated in 2002 as a wholly owned subsidiary of Xerox Corporation (NYSE: XRX).
PARC, 3333 Coyote Hill Road, Palo Alto, California 94304 USA +1 650 812 4000 | engage@parc.com | www.parc.com | page 2

From:
Sent:
To:
Subject: Attachments:
Tonie,
Thanks for reaching out. Here’s a 1-pager on our spray technology. If you are curious about how the spray might actually look like, you can check out a video here -- https://www.parc.com/services/focus-area/amds/
We would really be interested in working with your proposal team on this. If possible, it will be more value-generating for us to be a subcontractor and to contribute more to tailoring our spray technology for the intended use case. I’d also like to mention that another aspect we could bring to the table is in transitioning out the technology into a reality, particularly towards commercialization, because we have a good history on this – particularly on the device side but also, and increasingly, in the biomedical space through other commercial partners.
Please let me know how this might turn out. Thanks,
Jerome
From: "Rocke, Tonie" <trocke@usgs.gov>
Date: Tuesday, March 13, 2018 at 5:11 AM
To: "Unidad, Jerome <Jerome.Unidad@parc.com>" <Jerome.Unidad@parc.com> Subject: Re:
Hi Jerome: 1-2 CT is fine for me. I may not be in my office, however, so can I call you? Is this number good? 650-812-4209. Thanks -Tonie
On Mon, Mar 12, 2018 at 8:34 PM, <Jerome.Unidad@parc.com> wrote:
Sorry, I mixed up the schedule. I actually do have a meeting in the 1-2PM PST slot, how about 11AM-12PM PST (1-2PM
CT)? Thanks, Jerome
From: "Rocke, Tonie" <trocke@usgs.gov>
Date: Monday, March 12, 2018 at 5:25 PM
To: "Unidad, Jerome <Jerome.Unidad@parc.com>" <Jerome.Unidad@parc.com> Subject: <no subject>
Jerome.Unidad@parc.com
Tuesday, March 13, 2018 11:54 AM trocke@usgs.gov
Re: PARC FEA PARC_whitepaper_Biotech_v3_final.pdf
1

Hi Jerome: I have been working on developing vaccines for use in managing disease in wild bats - e.g. rabies in vampire bats and white-nose syndrome in insectiverous bats. I am also collaborating on a PREEMPT proposal and one of my collaborators passed on your contact information and description regarding PARC's spray technology for possible application in this field. Would you have time to chat tomorrow? I'll be available anytime after noon CT. Thanks much! -Tonie
--
Tonie E. Rocke
USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
608-270-2451
trocke@usgs.gov
--
Tonie E. Rocke
USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
608-270-2451
trocke@usgs.gov
2

Innovative Solutions for Biotechnology @ PARC
Leveraging on our long history in fluid and particle manipulation and roll-to-roll processes for printing applications, the PARC hardware laboratories are currently developing innovative device solutions with potential applications in biomedicine and biotechnology at-large.
Filament Extension Atomizer (FEA)
PARC is developing a novel spray technology called the Filament Extension Atomizer (FEA) that can generate aerosol from fluids that are notoriously difficult to aerosolize due to the inherent viscosity limit with most conventional spray methods. FEA can spray fluids of a wide range of viscosities: from 1 mPa-s (the viscosity of water) up to 1000 Pa-s (the viscosity of peanut butter) – this range includes fluids or dispersions with significant (bio)macromolecular content. This macromolecular content (long chain polymers) often impart an additional resistance to aerosol/spray generation due to strain hardening or the increase of fluid viscosity as a function of extension.
T o generate aerosol from such strain hardening fluids, FEA harnesses a well- known elasto-capillary instability that generates beads-on-a-string formation (Fig. 1A) when a fluid is held in extension. As the filaments are sufficiently thinned out, droplet break-up occurs and generates free droplets (aerosol). The FEA technology implements similar mechanics in a roll-to- roll process (Fig. 1B) to massively parallelize the filament formation and break- up and continuously generate droplets. To date, we have applied this on multiple viscoelastic fluids which include polymer solutions, polymer melts, particle dispersions and other complex systems with biomolecules (Figs. 1C-E).
Fig. 1 – A. Beads-on-a-string structures in a viscoelastic fluid in extension (McKinley and Olivera, 2005), B. Multiple beads-on-a-string formations in counter-rotating rollers (FEA), FEA spraying of PEO solution (C.), hyaluronic acid (D.) and sunscreen (E.)
Consumer Scale
• Portable devices
• Small rollers (down to 10mm)
• Low throughput
• Precision fluid delivery (e.g.
bioactive doses)
• Smart, connected devices
Industrial Scale
• Large rollers (up to 100mm) • High throughput
• Unit operation in a
manufacturing line
• Particle creation (e.g. spray
drying), large area coatings
The FEA technology is inherently scalable – it can work with small rollers for consumer scale applications, tailored for precision fluid dispensing, or with large rollers for industrial scale, high throughput aerosol generation for coatings or for powder production via spray drying. Since the FEA technology applies to wide range of fluids of virtually any viscosity or composition, it allows nearly formulation-independent fluid delivery. This can have a huge impact in biomedicine and biotechnology at-large: fluids can be formulated for bio-efficacy and not for delivery with no limits on bioactive loading, even with high viscosity biomacromolecules that are notoriously hard to deliver in a controlled, reliable manner. FEA can also allow the creation of specialized particles for drug delivery with a wide range of material set and control over the particle morphology.
Fig. 2 – FEA Technology at Different Scales and Applications
PARC, 3333 Coyote Hill Road, Palo Alto, California 94304 USA +1 650 812 4000 | engage@parc.com | www.parc.com

Technical Contact:
Jerome Unidad (Jerome.Unidad@parc.com), Member of Research Staff
Copyright © 2018 Palo Alto Research Center Incorporated.
All Rights Reserved. PARC, the PARC Logo are trademarks of Palo Alto Research Center Incorporated.
A global center for commercial innovation, PARC, a Xerox company, works closely with enterprises, entrepreneurs, government program partners and other clients to discover, develop, and deliver new business opportunities. PARC was incorporated in 2002 as a wholly owned subsidiary of Xerox Corporation (NYSE: XRX).
PARC, 3333 Coyote Hill Road, Palo Alto, California 94304 USA +1 650 812 4000 | engage@parc.com | www.parc.com | page 2

10/5/21, 3:39 PM Mail - Rocke, Tonie E - Outlook
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10/5/21, 3:39 PM Mail - Rocke, Tonie E - Outlook
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10/5/21, 3:39 PM Mail - Rocke, Tonie E - Outlook
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10/5/21, 3:39 PM Mail - Rocke, Tonie E - Outlook
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10/5/21, 3:39 PM Mail - Rocke, Tonie E - Outlook
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10/5/21, 3:40 PM Mail - Rocke, Tonie E - Outlook
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---------- egassem dedrawroF ----------

10/5/21, 3:40 PM Mail - Rocke, Tonie E - Outlook
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10/5/21, 3:40 PM Mail - Rocke, Tonie E - Outlook
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vog.sgsu@ekcort
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11735 IW ,nosidaM

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vog.sgsu@ekcort
1542-072-806

10/5/21, 3:40 PM Mail - Rocke, Tonie E - Outlook
Tonie – Thanks for all of this. Re. PARC – Billy and I would like to have a quick call with Jerome. Would you like to join?
Either way, I’ll send him an email now and try to set up a me this aernoon or tomorrow (Friday).
Boom line – this is expensive, and if we’re not exclusive with them, it’s prob beer to go with the cheaper opon.
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4474
www.ecohealthalliance.org @PeterDaszak @EcoHealthNYC
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
From: Rocke, Tonie [mailto:trocke@usgs.gov] Sent: Thursday, March 15, 2018 10:47 AM To: Billy Karesh; Peter Daszak; Luke Hamel Subject: Re: PARC FEA
A couple of other questions/comments. Are you proposing for co-PIs to meet periodically, perhaps at EHA? or elsewhere? Should we include that in our travel budgets? I think I heard yesterday someone from your shop was going to provide a budget for trips to China as well. Finally, just a heads up, I currently have a DOD SERDP grant and was caught by surprise how often they require the PI to travel to DC for progress reports and symposium (3 x last year, 2 x this year and the registration fee for each symposium is $1000), so be sure to include funds in your budget for that. Best -Tonie
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(b) (6)
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>gro.ecnaillahtlaehoce@kazsad<kMPa3z1s2a810D2/r51e/t3uehPT
AEF CRAP :ER

10/5/21, 3:40 PM Mail - Rocke, Tonie E - Outlook
On Wed, Mar 14, 2018 at 8:21 PM, Rocke, Tonie <trocke@usgs.gov> wrote: (b) (4)
Hi all: Here's PARC's proposed "lean" budget ($ ) and it may be too expensive for us at this point
(although to be honest I don't think it is overpriced). I tried to sell it as a way to illustrate the value of
their technology, but it sounds like they already have a relationship with DARPA. Perhaps I just didn't
sell it well enough. If either of you would like to try negotiating with them, feel free (bargaining is not
one of my strong suits!). One option might be to just get through #3 on their list for a total of $ and then use the prototype developed for lab testing in the field (I'm assuming this will be a hand held device), and leave the field deployable unit for another grant; or maybe at that point DARPA would fund them directly. I can discuss this idea with him tomorrow. As Billy and I discussed today in any case, the subcontract should not come from USGS as it would be charged exorbitant overhead costs. If we decide to go this route, we can reduce the NWHC budget somewhat. Let me know what you think. Thanks! - Tonie
---------- Forwarded message ---------- From: <Jerome.Unidad@parc.com> Date: Wed, Mar 14, 2018 at 5:46 PM Subject: Re: PARC FEA
To: trocke@usgs.gov Hi Tonie,
I agree based on our discussion that I think there’s a lot of interesting space for our technology for wildlife health management that we can work on together. We are certainly interested in exploring these other funding opportunities with you, particularly the WNS one which seems to be in the intermediate term.
Regarding PRE-EMPT, we understand that coming in this late that you guys probably have already fleshed out the project direction and tasks with equivalent budget and there might not be a lot of flexibility. In our proposed involvement, we would not want to change anything that you already just fleshed but rather supplement it with our spray technology. Based on preliminary estimation on how this involvement might be like, I came up with the following tasks and a lean estimate of the associated cost:
Phase I
(b) (4)
1. Development of a prototype FEA system for lab testing (Year 1) - $
2. Optimization of FEA spray conditions for PRE-EMPT fluids (Year 1) - $
3. Refinements of FEA delivery system (setup, fluid formulation, general aerosol delivery scheme)
based on preliminary lab testing (Year 2) - $
4. Preliminary design of a field-deployable FEA system (Year 2) - $
Phase II
(b) (4)
5. Fabrication and testing of field-deployable FEA systems (Year 3) - $
6. Project management and communication - $
The cost of the entire involvement as drafted is $ over the full period of 3.5 years (Phase I and II). Some of the tasks proposed above (example task 3) are included to ensure that we refine our technology continually to meet the requirements of the application at hand. We have a good working relationship with DARPA on various projects that we would want to maintain and, at the same time, we would like to take this chance to work with institutions such as yours on something with broad impact.
Please let me know what your thoughts are and whether this might work. I can make myself available for
(b) (4)
(b) (4)
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(b) (4)
(b) (4)
(b) (4)
(b) (4)

10/5/21, 3:40 PM Mail - Rocke, Tonie E - Outlook
a phone call tomorrow, as needed. Also, feel free to loop in your project PI/prime in the discussion in case it might be helpful.
Thanks, Jerome
From: "Rocke, Tonie" <trocke@usgs.gov>
Date: Wednesday, March 14, 2018 at 10:45 AM
To: "Unidad, Jerome <Jerome.Unidad@parc.com>" <Jerome.Unidad@parc.com> Subject: Re: PARC FEA
Hi Jerome: I had a good conversation with my colleagues and shared your material and video link with them. They agreed this looks like a great option for us, so we'd like to further explore how to move forward. Our PREEMPT proposal encompasses a wide variety of aims related to understanding and managing disease in bats in SE Asia, primarily in relation to SARS/coronaviruses. So a big part of the project is testing and developing the appropriate immune boosting agents. At the same time, we'd like to start developing the methods for delivery to bats via technology like your FEA you've described, first testing in laboratories and small caves - stateside, with the ultimate goal of conducting a field trial in a single cave system in China as proof of concept. So given that, what kind of budget do you think you might need to be involved, or alternatively, could you break things down for me by task, so we can see how we might structure this in stages? Honestly, we don't have alot of flexibility in the budget at the moment, but we see a ton of applications of this technology for your company in the future in managing wildlife health issues, not only with DARPA, but also DOI (white nose syndrome), USDA (bat rabies), and other government agencies, both foreign and domestic, so perhaps your company might view this as a good opportunity to test and illustrate the value of your technology. There is also another funding opportunity I am looking at for WNS that we might be able to partner on if you are interested. Let me know what you think. Also, with your permission, we'd like to include the link to the video in our proposal and perhaps pull an illustration or two for the text of the proposal with credits of course. Best regards -Tonie
On Tue, Mar 13, 2018 at 6:48 PM, <Jerome.Unidad@parc.com> wrote:
Yes, certainly split over 2-3 years. We can be very flexible on the workflow and I’d say we can structure it for maximal benefit of the project. This could mean developing an initial benchtop prototype quickly with some optimization as early as possible so that we can transition the setup to the corresponding partners (e.g. your group) to initiate the lab testing as soon as possible, and then spend the succeeding work on refining various aspects (fluid formulation for targeted spreading or bioefficacy, etc.) and maybe later on developing a field-deployable version, with motion-actuation (or timed-actuation, whatever case maybe), for Phase 2. We should be able to flesh it out very quickly depending on whatever structure you guys already have in mind.
Best,
Jerome ---------------------------------------------------------------------
Jerome Unidad, PhD
Advanced Manufacturing and Deposition Systems Hardware Systems Laboratory
PARC, A Xerox Company
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10/5/21, 3:40 PM Mail - Rocke, Tonie E - Outlook
From: Rocke, Tonie [mailto:trocke@usgs.gov]
Sent: Tuesday, March 13, 2018 12:24 PM
To: Unidad, Jerome <Jerome.Unidad@parc.com> <Jerome.Unidad@parc.com> Subject: Re: PARC FEA
Thanks Jerome. That is just what i need. The video is awesome. I'll talk things over with the PI tomorrow. In terms of a subcontract, do you think we could split it over 2-3 years? Best -Tonie
On Tue, Mar 13, 2018 at 1:53 PM, <Jerome.Unidad@parc.com> wrote: Tonie,
Thanks for reaching out. Here’s a 1-pager on our spray technology. If you are curious about how the spray might actually look like, you can check out a video here -- https://www.parc.com/services/focus-area/amds/
We would really be interested in working with your proposal team on this. If possible, it will be more value-generating for us to be a subcontractor and to contribute more to tailoring our spray technology for the intended use case. I’d also like to mention that another aspect we could bring to the table is in transitioning out the technology into a reality, particularly towards commercialization, because we have a good history on this – particularly on the device side but also, and increasingly, in the biomedical space through other commercial partners.
Please let me know how this might turn out. Thanks,
Jerome
From: "Rocke, Tonie" <trocke@usgs.gov>
Date: Tuesday, March 13, 2018 at 5:11 AM
To: "Unidad, Jerome <Jerome.Unidad@parc.com>" <Jerome.Unidad@parc.com> Subject: Re:
Hi Jerome: 1-2 CT is fine for me. I may not be in my office, however, so can I call you? Is this number good? 650-812-4209. Thanks -Tonie
On Mon, Mar 12, 2018 at 8:34 PM, <Jerome.Unidad@parc.com> wrote:
Sorry, I mixed up the schedule. I actually do have a meeting in the 1-2PM PST slot, how about
11AM-12PM PST (1-2PM CT)? Thanks,
Jerome
From: "Rocke, Tonie" <trocke@usgs.gov>
Date: Monday, March 12, 2018 at 5:25 PM
To: "Unidad, Jerome <Jerome.Unidad@parc.com>" <Jerome.Unidad@parc.com> Subject: <no subject>
Hi Jerome: I have been working on developing vaccines for use in managing disease in wild bats - e.g. rabies in vampire bats and white-nose syndrome in insectiverous bats. I am also collaborating on a PREEMPT proposal and one of my collaborators passed on your contact information and description regarding PARC's spray technology for possible application in this
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Mail - Rocke, Tonie E - Outlook
field. Would you have time to chat tomorrow? I'll be available anytime after noon CT. Thanks much! -Tonie
--
Tonie E. Rocke
USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
608-270-2451
trocke@usgs.gov
--
Tonie E. Rocke
USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
608-270-2451
trocke@usgs.gov
--
Tonie E. Rocke
USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
608-270-2451
trocke@usgs.gov
--
Tonie E. Rocke
USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
608-270-2451
trocke@usgs.gov
--
Tonie E. Rocke
USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
608-270-2451
trocke@usgs.gov
10/5/21, 3:40 PM
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10/5/21, 3:40 PM
--
Tonie E. Rocke
USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
608-270-2451
trocke@usgs.gov
Mail - Rocke, Tonie E - Outlook
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10/5/21, 3:41 PM Mail - Rocke, Tonie E - Outlook
Re. the travel – we’re working out how many trips etc., but this needs to come out of your budget. We’ll send info along to you v. soon.
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4474
www.ecohealthalliance.org @PeterDaszak @EcoHealthNYC
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
From: Rocke, Tonie [mailto:trocke@usgs.gov] Sent: Thursday, March 15, 2018 10:47 AM To: Billy Karesh; Peter Daszak; Luke Hamel Subject: Re: PARC FEA
A couple of other questions/comments. Are you proposing for co-PIs to meet periodically, perhaps at EHA? or elsewhere? Should we include that in our travel budgets? I think I heard yesterday someone from your shop was going to provide a budget for trips to China as well. Finally, just a heads up, I currently have a DOD SERDP grant and was caught by surprise how often they require the PI to travel to DC for progress reports and symposium (3 x last year, 2 x this year and the registration fee for each symposium is $1000), so be sure to include funds in your budget for that. Best -Tonie
On Wed, Mar 14, 2018 at 8:21 PM, Rocke, Tonie <trocke@usgs.gov> wrote: (b) (4)
Hi all: Here's PARC's proposed "lean" budget ($ ) and it may be too expensive for us at this point (although to be honest I don't think it is overpriced). I tried to sell it as a way to illustrate the value of their technology, but it sounds like they already have a relationship with DARPA. Perhaps I just didn't
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(b) (6)
>gro.ecnaillahtlaehoce@ybhguolliw<ybhguolliWannA:cC
>gro.ecnaillahtlaehoce@lemah<
lemaHekuL;>moc.liamg@ <hseraKylliB;>vog.sgsu@ekcort<EeinoT,ekcoR:oT
>gro.ecnaillahtlaehoce@kazsad<kMPa4z1s2a810D2/r51e/t3uehPT
APRAD rof levarT

10/5/21, 3:41 PM Mail - Rocke, Tonie E - Outlook
sell it well enough. If either of you would like to try negotiating with them, feel free (bargaining is not
one of my strong suits!). One option might be to just get through #3 on their list for a total of $ and then use the prototype developed for lab testing in the field (I'm assuming this will be a hand held device), and leave the field deployable unit for another grant; or maybe at that point DARPA would fund them directly. I can discuss this idea with him tomorrow. As Billy and I discussed today in any case, the subcontract should not come from USGS as it would be charged exorbitant overhead costs. If we decide to go this route, we can reduce the NWHC budget somewhat. Let me know what you think. Thanks! - Tonie
---------- Forwarded message ---------- From: <Jerome.Unidad@parc.com> Date: Wed, Mar 14, 2018 at 5:46 PM Subject: Re: PARC FEA
To: trocke@usgs.gov Hi Tonie,
I agree based on our discussion that I think there’s a lot of interesting space for our technology for wildlife health management that we can work on together. We are certainly interested in exploring these other funding opportunities with you, particularly the WNS one which seems to be in the intermediate term.
Regarding PRE-EMPT, we understand that coming in this late that you guys probably have already fleshed out the project direction and tasks with equivalent budget and there might not be a lot of flexibility. In our proposed involvement, we would not want to change anything that you already just fleshed but rather supplement it with our spray technology. Based on preliminary estimation on how this involvement might be like, I came up with the following tasks and a lean estimate of the associated cost:
Phase I
based on preliminary lab testing (Year 2) - $
4. Preliminary design of a field-deployable FEA system (Year 2) - $
Phase II
(b) (4)
5. Fabrication and testing of field-deployable FEA systems (Year 3) - $
6. Project management and communication - $
The cost of the entire involvement as drafted is $ over the full period of 3.5 years (Phase I and II). Some of the tasks proposed above (example task 3) are included to ensure that we refine our technology continually to meet the requirements of the application at hand. We have a good working relationship with DARPA on various projects that we would want to maintain and, at the same time, we would like to take this chance to work with institutions such as yours on something with broad impact.
Please let me know what your thoughts are and whether this might work. I can make myself available for a phone call tomorrow, as needed. Also, feel free to loop in your project PI/prime in the discussion in case it might be helpful.
Thanks,
(b) (4)
(b) (4)
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(b) (4)
(b) (4)
(b) (4)
(b) (4)
1. Development of a prototype FEA system for lab testing (Year 1) - $
2. Optimization of FEA spray conditions for PRE-EMPT fluids (Year 1) - $
3. Refinements of FEA delivery system (setup, fluid formulation, general aerosol delivery scheme)
(b) (4)

10/5/21, 3:41 PM Mail - Rocke, Tonie E - Outlook
Jerome
From: "Rocke, Tonie" <trocke@usgs.gov>
Date: Wednesday, March 14, 2018 at 10:45 AM
To: "Unidad, Jerome <Jerome.Unidad@parc.com>" <Jerome.Unidad@parc.com> Subject: Re: PARC FEA
Hi Jerome: I had a good conversation with my colleagues and shared your material and video link with them. They agreed this looks like a great option for us, so we'd like to further explore how to move forward. Our PREEMPT proposal encompasses a wide variety of aims related to understanding and managing disease in bats in SE Asia, primarily in relation to SARS/coronaviruses. So a big part of the project is testing and developing the appropriate immune boosting agents. At the same time, we'd like to start developing the methods for delivery to bats via technology like your FEA you've described, first testing in laboratories and small caves - stateside, with the ultimate goal of conducting a field trial in a single cave system in China as proof of concept. So given that, what kind of budget do you think you might need to be involved, or alternatively, could you break things down for me by task, so we can see how we might structure this in stages? Honestly, we don't have alot of flexibility in the budget at the moment, but we see a ton of applications of this technology for your company in the future in managing wildlife health issues, not only with DARPA, but also DOI (white nose syndrome), USDA (bat rabies), and other government agencies, both foreign and domestic, so perhaps your company might view this as a good opportunity to test and illustrate the value of your technology. There is also another funding opportunity I am looking at for WNS that we might be able to partner on if you are interested. Let me know what you think. Also, with your permission, we'd like to include the link to the video in our proposal and perhaps pull an illustration or two for the text of the proposal with credits of course. Best regards -Tonie
On Tue, Mar 13, 2018 at 6:48 PM, <Jerome.Unidad@parc.com> wrote:
Yes, certainly split over 2-3 years. We can be very flexible on the workflow and I’d say we can structure it for maximal benefit of the project. This could mean developing an initial benchtop prototype quickly with some optimization as early as possible so that we can transition the setup to the corresponding partners (e.g. your group) to initiate the lab testing as soon as possible, and then spend the succeeding work on refining various aspects (fluid formulation for targeted spreading or bioefficacy, etc.) and maybe later on developing a field-deployable version, with motion-actuation (or timed-actuation, whatever case maybe), for Phase 2. We should be able to flesh it out very quickly depending on whatever structure you guys already have in mind.
Best,
Jerome ---------------------------------------------------------------------
Jerome Unidad, PhD
Advanced Manufacturing and Deposition Systems Hardware Systems Laboratory
PARC, A Xerox Company
From: Rocke, Tonie [mailto:trocke@usgs.gov]
Sent: Tuesday, March 13, 2018 12:24 PM
To: Unidad, Jerome <Jerome.Unidad@parc.com> <Jerome.Unidad@parc.com> Subject: Re: PARC FEA
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10/5/21, 3:41 PM Mail - Rocke, Tonie E - Outlook
Thanks Jerome. That is just what i need. The video is awesome. I'll talk things over with the PI tomorrow. In terms of a subcontract, do you think we could split it over 2-3 years? Best -Tonie
On Tue, Mar 13, 2018 at 1:53 PM, <Jerome.Unidad@parc.com> wrote: Tonie,
Thanks for reaching out. Here’s a 1-pager on our spray technology. If you are curious about how the spray might actually look like, you can check out a video here -- https://www.parc.com/services/focus-area/amds/
We would really be interested in working with your proposal team on this. If possible, it will be more value-generating for us to be a subcontractor and to contribute more to tailoring our spray technology for the intended use case. I’d also like to mention that another aspect we could bring to the table is in transitioning out the technology into a reality, particularly towards commercialization, because we have a good history on this – particularly on the device side but also, and increasingly, in the biomedical space through other commercial partners.
Please let me know how this might turn out. Thanks,
Jerome
From: "Rocke, Tonie" <trocke@usgs.gov>
Date: Tuesday, March 13, 2018 at 5:11 AM
To: "Unidad, Jerome <Jerome.Unidad@parc.com>" <Jerome.Unidad@parc.com> Subject: Re:
Hi Jerome: 1-2 CT is fine for me. I may not be in my office, however, so can I call you? Is this number good? 650-812-4209. Thanks -Tonie
On Mon, Mar 12, 2018 at 8:34 PM, <Jerome.Unidad@parc.com> wrote:
Sorry, I mixed up the schedule. I actually do have a meeting in the 1-2PM PST slot, how about
11AM-12PM PST (1-2PM CT)? Thanks,
Jerome
From: "Rocke, Tonie" <trocke@usgs.gov>
Date: Monday, March 12, 2018 at 5:25 PM
To: "Unidad, Jerome <Jerome.Unidad@parc.com>" <Jerome.Unidad@parc.com> Subject: <no subject>
Hi Jerome: I have been working on developing vaccines for use in managing disease in wild bats - e.g. rabies in vampire bats and white-nose syndrome in insectiverous bats. I am also collaborating on a PREEMPT proposal and one of my collaborators passed on your contact information and description regarding PARC's spray technology for possible application in this field. Would you have time to chat tomorrow? I'll be available anytime after noon CT. Thanks much! -Tonie
--
Tonie E. Rocke
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10/5/21, 3:41 PM
Mail - Rocke, Tonie E - Outlook
USGS National Wildlife Health Center
6006 Schroeder Rd. Madison, WI 53711 608-270-2451 trocke@usgs.gov
--
Tonie E. Rocke
USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
608-270-2451
trocke@usgs.gov
--
Tonie E. Rocke
USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
608-270-2451
trocke@usgs.gov
--
Tonie E. Rocke
USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
608-270-2451
trocke@usgs.gov
--
Tonie E. Rocke
USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
608-270-2451
trocke@usgs.gov
--
Tonie E. Rocke
USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 5/6

10/5/21, 3:41 PM Mail - Rocke, Tonie E - Outlook
608-270-2451
trocke@usgs.gov
https://outlook.office365.com/mail/id/AAMkADUyODI5Y2E0LWY5MmEtNGNjNi04YmQ3LWVkZmU3ZWRkMTllZgBGAAAAAAAd8uDAoslFQa0tKxNiQj80... 6/6

10/5/21, 3:41 PM Mail - Rocke, Tonie E - Outlook
Tonie – re. the PARC budget, we’ll put this in our central budget, so we reduce the overhead costs as you say below. If you can reduce your NWHC budget by $50K per year, to cover us for some of the costs.
Cheers, Peter
Peter Daszak
President
EcoHealth Alliance
460 West 34th Street – 17th Floor New York, NY 10001
Tel. +1 212-380-4474
www.ecohealthalliance.org @PeterDaszak @EcoHealthNYC
EcoHealth Alliance leads cung-edge research into the crical connecons between human and wildlife health and delicate ecosystems. With this science we develop soluons that prevent pandemics and promote conservaon.
From: Rocke, Tonie [mailto:trocke@usgs.gov] Sent: Thursday, March 15, 2018 10:47 AM To: Billy Karesh; Peter Daszak; Luke Hamel Subject: Re: PARC FEA
A couple of other questions/comments. Are you proposing for co-PIs to meet periodically, perhaps at EHA? or elsewhere? Should we include that in our travel budgets? I think I heard yesterday someone from your shop was going to provide a budget for trips to China as well. Finally, just a heads up, I currently have a DOD SERDP grant and was caught by surprise how often they require the PI to travel to DC for progress reports and symposium (3 x last year, 2 x this year and the registration fee for each symposium is $1000), so be sure to include funds in your budget for that. Best -Tonie
On Wed, Mar 14, 2018 at 8:21 PM, Rocke, Tonie <trocke@usgs.gov> wrote: (b) (4)
Hi all: Here's PARC's proposed "lean" budget ($ ) and it may be too expensive for us at this point (although to be honest I don't think it is overpriced). I tried to sell it as a way to illustrate the value of their technology, but it sounds like they already have a relationship with DARPA. Perhaps I just didn't sell it well enough. If either of you would like to try negotiating with them, feel free (bargaining is not
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(b) (6)
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AEF CRAP :ER

10/5/21, 3:41 PM Mail - Rocke, Tonie E - Outlook
one of my strong suits!). One option might be to just get through #3 on their list for a total of $ and then use the prototype developed for lab testing in the field (I'm assuming this will be a hand held device), and leave the field deployable unit for another grant; or maybe at that point DARPA would fund them directly. I can discuss this idea with him tomorrow. As Billy and I discussed today in any case, the subcontract should not come from USGS as it would be charged exorbitant overhead costs. If we decide to go this route, we can reduce the NWHC budget somewhat. Let me know what you think. Thanks! - Tonie
---------- Forwarded message ---------- From: <Jerome.Unidad@parc.com> Date: Wed, Mar 14, 2018 at 5:46 PM Subject: Re: PARC FEA
To: trocke@usgs.gov Hi Tonie,
I agree based on our discussion that I think there’s a lot of interesting space for our technology for wildlife health management that we can work on together. We are certainly interested in exploring these other funding opportunities with you, particularly the WNS one which seems to be in the intermediate term.
Regarding PRE-EMPT, we understand that coming in this late that you guys probably have already fleshed out the project direction and tasks with equivalent budget and there might not be a lot of flexibility. In our proposed involvement, we would not want to change anything that you already just fleshed but rather supplement it with our spray technology. Based on preliminary estimation on how this involvement might be like, I came up with the following tasks and a lean estimate of the associated cost:
Phase I
1. Development of a prototype FEA system for lab testing (Year 1) - $(b) (4)
2. Optimization of FEA spray conditions for PRE-EMPT fluids (Year 1) - $(b) (4)
3. Refinements of FEA delivery system (setup, fluid formulation, general aerosol delivery scheme)
based on preliminary lab testing (Year 2) - $
4. Preliminary design of a field-deployable FEA system (Year 2) - $(b) (4)
Phase II
5. Fabrication and testing of field-deployable FEA systems (Year 3) - $(b) (4)
6. Project management and communication - $(b) (4)
The cost of the entire involvement as drafted is $(b) (4) over the full period of 3.5 years (Phase I and II). Some of the tasks proposed above (example task 3) are included to ensure that we refine our technology continually to meet the requirements of the application at hand. We have a good working relationship with DARPA on various projects that we would want to maintain and, at the same time, we would like to take this chance to work with institutions such as yours on something with broad impact.
Please let me know what your thoughts are and whether this might work. I can make myself available for a phone call tomorrow, as needed. Also, feel free to loop in your project PI/prime in the discussion in case it might be helpful.
Thanks,
(b) (4)
(b) (4)
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10/5/21, 3:43 PM Mail - Rocke, Tonie E - Outlook
Hi Tonie, I think I misunderstood RCN is the Raccoon poxvirus? Ralph
From: Rocke, Tonie [mailto:trocke@usgs.gov]
Sent: Tuesday, March 13, 2018 1:19 PM
To: Baric, Ralph S <rbaric@email.unc.edu>
Subject: Re: Rescheduling upcoming calls with Peter (PREEMPT)
Hi Ralph: Thanks for sending me your narrative. Just to clarify, are you proposing challenge trials in vaccinated bats at UNC? I think we should just subcontract with UW to engineer the RCN constructs. They are really skilled at it. We typically then produce the master seeds in my lab. I'm assuming those seeds would then go back to yours for challenge trials. Does that make sense? I'm trying to figure out how to prepare the budget. Same question I guess about the nanoparticles. Will you be constructing these in your lab and then testing them in bats? Thanks! -Tonie
On Sun, Mar 11, 2018 at 7:52 PM, Baric, Ralph S <rbaric@email.unc.edu> wrote: Its rough, but here’s a dra. One secon not included yet. Ralph
From: Rocke, Tonie [mailto:trocke@usgs.gov]
Sent: Friday, March 9, 2018 4:21 PM
To: Luke Hamel <hamel@ecohealthalliance.org>
Cc: Rachel Abbo <rabbo@usgs.gov>; Aleksei Chmura <chmura@ecohealthalliance.org>; Dr. Peter Daszak <daszak@ecohealthalliance.org>; Alison Andre <andre@ecohealthalliance.org>; Baric, Ralph S <rbaric@email.unc.edu>
Subject: Re: Rescheduling upcoming calls with Peter (PREEMPT)
Hello Luke and Peter: Attached is my first draft of Task 7. Ralph Baric (copied here) and I had a good chat today about viral vectors and nanoparticles. We realized much of the delivery methods would depend on his work first, so there are some gaps here and the narrative will probably change after I see what Ralph has written. At any rate, this is something to at least start with. Have a good weekend! - Tonie
On Thu, Mar 8, 2018 at 12:58 PM, Luke Hamel <hamel@ecohealthalliance.org> wrote: Hi Tonie,
Once again, please use this link, to select which date(s)/time(s) you are available to speak with Peter (select as many as you are available for). The goal is to have one call each week, on either Tuesday or Wednesday. Below, I've listed the dates/times that appear in the Doodle Poll link above. Please note that all times listed are in Eastern Time.
Week 1:
Thu. 3/13 (9 AM - 5 PM ET) Thu. 3/14 (9 AM - 5 PM ET)
Week 2:
Thu. 3/20 (9 AM - 5 PM ET) Thu. 3/21 (9 AM - 5 PM ET)
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)TPMEERP( reteP htiw sllac gnimocpu gniludehcseR :ER

10/5/21, 3:43 PM
Please let me know if you have any questions.
Best,
Luke Hamel
Program Assistant
EcoHealth Alliance
460 West 34th Street – 17th floor New York, NY 10001
(b) (6) (direct) (b) (6)
www.ecohealthalliance.org
EcoHealth Alliance leads cutting-edge scientific research into the critical connections between human and wildlife health and delicate ecosystems. With this science, we develop solutions that prevent pandemics and promote conservation.
--
Tonie E. Rocke
USGS National Wildlife Health Center 6006 Schroeder Rd.
Madison, WI 53711
608-270-2451
trocke@usgs.gov
--
Tonie E. Rocke
USGS National Wildlife Health Center 6006 Schroeder Rd.