omb number: 4040-0001 sf 424 (r&r) expiration date: 06/30 ... · 06/01/2015 · omb number:...

OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT11752232 Funding Opportunity Number: PA-13-302 . Received Date:2014-10-06T12:03:45.000-04:00

APPLICATION FOR FEDERAL ASSISTANCE

SF 424 (R&R)3. DATE RECEIVED BY STATE State Application Identifier

1. TYPE OF SUBMISSION* 4.a. Federal Identifier

❍ Pre-application ❍ Application ● Changed/CorrectedApplication

b. Agency Routing Number

2. DATE SUBMITTED Application Identifier c. Previous Grants.gov Tracking NumberGRANT11751828

5. APPLICANT INFORMATION Organizational DUNS*: 038415006

Legal Name*: Board of Trustees of Southern Illinois University

Department: Associate Dean for Research

Division: School of Medicine

Street1*: PO Box 19616

Street2:

City*: Springfield

County: Sangamon

State*: IL: Illinois

Province:Country*: USA: UNITED STATES

ZIP / Postal Code*: 62794-9616

Person to be contacted on matters involving this applicationPrefix: Dr. First Name*: Linda Middle Name: A. Last Name*: Toth Suffix: Ph.D

Position/Title: Associate Dean for Research


Street2:

City*: Springfield

County: Sangamon




Phone Number*: 217-545-4549 Fax Number: 217-545-0786 Email: [email protected]

6. EMPLOYER IDENTIFICATION NUMBER (EIN) or (TIN)* 376005961

7. TYPE OF APPLICANT* H: Public/State Controlled Institution of Higher Education

Other (Specify):Small Business Organization Type ❍ Women Owned ❍ Socially and Economically Disadvantaged

8. TYPE OF APPLICATION* If Revision, mark appropriate box(es).

● New ❍ Resubmission ❍ A. Increase Award ❍ B. Decrease Award ❍ C. Increase Duration

❍ Renewal ❍ Continuation ❍ Revision ❍ D. Decrease Duration ❍ E. Other (specify) :

Is this application being submitted to other agencies?* ❍Yes ●No What other Agencies?

9. NAME OF FEDERAL AGENCY*National Institutes of Health

10. CATALOG OF FEDERAL DOMESTIC ASSISTANCE NUMBERTITLE:

11. DESCRIPTIVE TITLE OF APPLICANT'S PROJECT*Essential Role of B- and T-cell Cooperation in Vaccine-Induced Protection Against HSV-2

12. PROPOSED PROJECTStart Date* Ending Date*07/01/2015 06/30/2020

13. CONGRESSIONAL DISTRICTS OF APPLICANT

IL-013

whalford

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Tracking Number: GRANT11752232 Funding Opportunity Number: PA-13-302 . Received Date:2014-10-06T12:03:45.000-04:00

SF 424 (R&R) APPLICATION FOR FEDERAL ASSISTANCE 14. PROJECT DIRECTOR/PRINCIPAL INVESTIGATOR CONTACT INFORMATIONPrefix: Dr. First Name*: William Middle Name: P Last Name*: Halford Suffix: Ph.D

Position/Title: Associate Professor

Organization Name*: Board of Trustees of Southern Illinois University

Department: MMICB

Division: School of Medicine


Street2:

City*: Springfield

County: Sangamon




Phone Number*: 217-545-4277 Fax Number: 217-545-3227 Email*: [email protected]

15. ESTIMATED PROJECT FUNDING

a. Total Federal Funds Requested* $1,504,138.00b. Total Non-Federal Funds* $0.00c. Total Federal & Non-Federal Funds* $1,504,138.00d. Estimated Program Income* $0.00

16. IS APPLICATION SUBJECT TO REVIEW BY STATEEXECUTIVE ORDER 12372 PROCESS?*

a. YES ❍ THIS PREAPPLICATION/APPLICATION WAS MADEAVAILABLE TO THE STATE EXECUTIVE ORDER 12372PROCESS FOR REVIEW ON:

DATE:

b. NO ● PROGRAM IS NOT COVERED BY E.O. 12372; OR

❍ PROGRAM HAS NOT BEEN SELECTED BY STATE FORREVIEW

17. By signing this application, I certify (1) to the statements contained in the list of certifications* and (2) that the statements hereinare true, complete and accurate to the best of my knowledge. I also provide the required assurances * and agree to comply withany resulting terms if I accept an award. I am aware that any false, fictitious, or fraudulent statements or claims may subject me tocriminal, civil, or administrative penalties. (U.S. Code, Title 18, Section 1001)

● I agree** The list of certifications and assurances, or an Internet site where you may obtain this list, is contained in the announcement or agency specific instructions.

18. SFLLL or OTHER EXPLANATORY DOCUMENTATION File Name:

19. AUTHORIZED REPRESENTATIVEPrefix: Dr. First Name*: Linda Middle Name: A. Last Name*: Toth Suffix: Ph.D

Position/Title*: Associate Dean for Research

Organization Name*: Board of Trustees of Southern Illinois University

Department: Associate Dean for Research

Division: SIU School of Medicine


Street2:

City*: Springfield

County: Sangamon




Phone Number*: 217-545-4549 Fax Number: 217-545-0786 Email*: [email protected]

Signature of Authorized Representative*Consolatrix Custeau

Date Signed*10/06/2014

20. PRE-APPLICATION File Name: Mime Type:

21. COVER LETTER ATTACHMENT File Name:1234-Cover_Letter.pdf Mime Type: application/pdf

Contact PD/PI: Halford, William, P

whalford

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424 R&R and PHS-398 SpecificTable Of Contents Page Numbers

SF 424 R&R Cover Page----------------------------------------------------------------------------------------- 1

Table of Contents------------------------------------------------------------------------- 3

Performance Sites--------------------------------------------------------------------------------------------- 4

Research & Related Other Project Information------------------------------------------------------------------ 5

Project Summary/Abstract(Description)----------------------------------------------------- 6

Project Narrative------------------------------------------------------------------------- 7

Facilities & Other Resources-------------------------------------------------------------- 8

Research & Related Senior/Key Person-------------------------------------------------------------------------- 9

PHS398 Cover Page Supplement---------------------------------------------------------------------------------- 16

PHS 398 Modular Budget---------------------------------------------------------------------------------------- 18

Personnel Justification------------------------------------------------------------------- 24

Additional Narrative Justification-------------------------------------------------------- 25

PHS 398 Research Plan----------------------------------------------------------------------------------------- 26

Specific Aims----------------------------------------------------------------------------- 27

Research Strategy------------------------------------------------------------------------- 28

Vertebrate Animals------------------------------------------------------------------------ 40

Bibliography & References Cited----------------------------------------------------------- 42

Letters Of Support------------------------------------------------------------------------ 47

Resource Sharing Plans-------------------------------------------------------------------- 50

Appendix

Number of Attachments in Appendix: 1

Table of Contents


OMB Number: 4040-0010

Expiration Date: 06/30/2016

Tracking Number: GRANT11752232 Funding Opportunity Number: PA-13-302. Received Date:2014-10-06T12:03:45.000-04:00

Project/Performance Site Location(s)

Project/Performance Site Primary Location ❍ I am submitting an application as an individual, and not on behalf of

a company, state, local or tribal government, academia, or other type of

organization.

Organization Name: Southern Illinois University School of Medicine

Duns Number: 0384150060000

Street1*:Dept of Med Microbiology, Immunology, and CellBiology

Street2: 825 North Rutledge Street

City*: Springfield

County: Sangamon


Province:

Country*: USA: UNITED STATES

Zip / Postal Code*: 62794-9626

Project/Performance Site Congressional District*: IL-013

File Name Mime Type

Additional Location(s)


Tracking Number: GRANT11752232 Funding Opportunity Number: PA-13-302. Received Date:2014-10-06T12:03:45.000-04:00

OMB Number: 4040-0001Expiration Date: 06/30/2016

RESEARCH & RELATED Other Project Information

1. Are Human Subjects Involved?* ❍ Yes ● No

1.a. If YES to Human Subjects

Is the Project Exempt from Federal regulations? ❍ Yes ❍ No

If YES, check appropriate exemption number: 1 2 3 4 5 6

If NO, is the IRB review Pending? ❍ Yes ❍ No

IRB Approval Date:

Human Subject Assurance Number

2. Are Vertebrate Animals Used?* ● Yes ❍ No

2.a. If YES to Vertebrate Animals

Is the IACUC review Pending? ❍ Yes ● No

IACUC Approval Date: 06-15-2013

Animal Welfare Assurance Number A-3209-01

3. Is proprietary/privileged information included in the application?* ❍ Yes ● No

4.a. Does this project have an actual or potential impact - positive or negative - on the environment?* ❍ Yes ● No

4.b. If yes, please explain:

4.c. If this project has an actual or potential impact on the environment, has an exemption been authorized or an

environmental assessment (EA) or environmental impact statement (EIS) been performed?

❍ Yes ❍ No

4.d. If yes, please explain:

5. Is the research performance site designated, or eligible to be designated, as a historic place?* ❍ Yes ● No

5.a. If yes, please explain:

6. Does this project involve activities outside the United States or partnership with international

collaborators?*

❍ Yes ● No

6.a. If yes, identify countries:

6.b. Optional Explanation:

Filename

7. Project Summary/Abstract* 1235-Abstract.pdf Mime Type: application/pdf

8. Project Narrative* 1236-ProjectNarrative.pdf Mime Type: application/pdf

9. Bibliography & References Cited 1237-Bibliograpy.pdf Mime Type: application/pdf

10.Facilities & Other Resources 1238-Halford_Facilities.pdf Mime Type: application/pdf

11.Equipment


Abstract Over 500 million people are infected with herpes simplex virus 2 (HSV-2), and >10 million people per year are newly infected with HSV-2. Tens of millions of these individuals endure recurrent outbreaks and/or chronic nerve pain associated with genital herpes disease. An effective HSV-2 vaccine would be invaluable in stopping this needless source of human suffering.

For the past 30 years, synthetic HSV-2 vaccine that expose vaccine recipients to 1-3% of HSV-2's proteome have been the preferred approach. This HSV-2 vaccine strategy has failed in 6 prior clinical trials years, and is the basis of 3 ongoing trials. Importantly, none of the failed HSV-2 vaccine candidates have attempted to elicit a balanced B- and T-cell response to all of HSV-2's antigens.

A live- and appropriately attenuated- HSV-2 vaccine represents a potentially simple and clinically viable solution to the world's HSV-2 epidemic, as a live HSV-2 vaccine may elicit a balanced B- and T-cell response against 99% of HSV-2's antigens. The P.I.'s laboratory has published evidence that HSV-1 and HSV-2 ICP0- mutant viruses are exquisitely sensitive to repression by the host interferon response, and thus are profoundly attenuated in normal and SCID (lymphocyte-deficient) animals. Despite being profoundly attenuated, a HSV-2 ICP0

- mutant virus still elicits ~100 times greater protection against HSV-2 genital herpes than a HSV-2 gD-2 subunit vaccine, similar in composition to the most recent candidate to fail in a human clinical trial (Belshe,et al, 2012; NEJM 366:34).

The P.I.'s published and preliminary results in HSV-2 vaccine-challenge systems suggest two important and under-recognized, principles that may explain why past HSV-2 vaccine candidates have failed; namely, (1) vaccines that contain 1% of HSV-2's proteome may not elicit 100% of the protective immunity that is attainable, and (2) optimal vaccine-induced protection against HSV-2 may require a balanced B- and T-cell response against most of HSV-2's antigens. Although the proposed studies will be performed in a HSV-2 vaccine-challenge system, it is anticipated the results will have broad implications for vaccine science as a whole.

The central hypothesis of the proposed studies is that a balanced B- and T-cell response to most of HSV-2's antigens is required to elicit optimal vaccine-induced protection against HSV-2. If this hypothesis is correct, then it would clarify what has been missing from past HSV-2 vaccines, and highlight what will be needed to obtain an effective HSV-2 vaccine in the future. A secondary hypothesis of the proposed studies is that a live HSV-2 ICP0- mutant virus represents a safe approach to elicit such a balanced B- and T-cell response to a most of HSV-2's antigens. Three Specific Aims are proposed to test these inter-related hypotheses.

Project Summary/Abstract


Narrative The preferred herpes simplex virus 2 (HSV-2) vaccine strategy has been synthetic vaccines that expose vaccine recipients to 1-3% of HSV-2's proteome. This approach has failed in 6 clinical trials spanning 25 years, and is the basis of ongoing HSV-2 vaccine trials. The proposed studies will investigate a hypothesis that the reason a live-attenuated HSV-2 ICP0- vaccine elicits ~100 times better protection against HSV-2 genital herpes is because it may elicit a balanced B- and T-cell response against 99% of HSV-2's proteins. If this hypothesis is correct, then a safe live HSV-2 ICP0- vaccine represents a superior and untested opportunity to stop the spread of HSV-2 in the human population.

Project Narrative


Halford Lab and Research Facilities at Southern Illinois University School of Medicine

1. Laboratory. The Halford Lab is housed in the Springfield Combined Laboratory Facility (SCLF) Addition, which is the newest research facility at the Southern Illinois University School of Medicine. The SCLF is a five-story building whose first three floors were occupied in Fall 2006, and the 4th and 5th floors will be completed and occupied in Spring 2008. The Medical Microbiology and Immunology department is housed in the 2nd and 3rd floors of the SCLF Addition. Dr. Halford occupies a 1000 sq ft laboratory suite in Room 2668, which contains 5 benches, 5 desks, tissue culture room, and is adequate for the proposed work. Equipment present includes three -80°C freezers, two -20°C freezers, two refrigerators, liquid N2 dewar, hybridization oven, Cyclone phosphorimager, Nanodrop spectrophotometer, 4 thermal cyclers, 5 microfuges, 2 water baths, Lauda circulating water bath, rocking platforms, bacterial incubator and rotating platform shakers, agarose gel boxes, mini-Protean 3 polyacylamide gel systems, large Protean II polyacrylamide gel systems, sequencing gel apparatuses, power supplies, vacuum blotters for nucleic acid transfer, protein blotting transfer tanks, a gel dryer, vacuum pumps, BioDoc-It gel documentation system, and all of the necessary surgical instruments and tissue homogenizers necessary for the proposed animal work. The tissue culture facilities within the laboratory include two 6-foot class II biosafety cabinets, 6 CO2 incubators, and two inverted Nikon TE2000 fluorescent microscopes. One of these microscopes is equipped with an Olympus DP72 digital camera and software which operate a Lambda SC SmartShutter™ control system that is capable of both static and time-lapse photographs for live-cell imaging of fluorescent-labeled proteins.

2. Animal. The animal facility is housed on the 1st floor of the SCLF Addition (immediately below the P.I.’s laboratory) in a new 20,000 sq. ft space which includes a surgery suite, cagewash facility, diagnostic laboratory, necropsy room, quarantine area, and infectious disease/barrier containment suites. The centralized laboratory animal care program is accredited by the AAALAC and it is directed by Teresa Liberati, D.V.M. Dr. Halford has two BSL-2 animal rooms in the facility. The facility is adequate for the proposed work.

3. Computer. Dr. Halford’s office and laboratories have four PCs, two laser jet printers, color printer, and all computers are connected to LAN for internet access, and access to three multi-use departmental printers. Software is available for word processing, photo editing, construction of slides and figures, statistical analysis, oligonucleotide design, plasmid construction, and phosphorimager analysis.

4. Office. A business manager, and three administrative assistants are dedicated to the Department of Medical Microbiology and Immunology, and are housed in a 1200 sq ft suite on the 3rd floor of the SCLF Addition. The PI has a 150 sq. ft. office in Room 2664, which is directly adjacent to his laboratory.

5. Other. Centralized facilities that are available within a two minute-walk from the Halford Lab include: Biosafety level 3 (BSL-3) laboratories. Older BSL-3 space at SIU is a 400 sq. ft laboratory that is adequate for tissue culture and laboratory benchwork. Newer BSL-3 space is a 3200 sq. ft. suite that has two 300 sq. ft. bench labs, a 250 sq. ft. lab for small animal containment, equipment storage, shower-in / shower-out, pass-through autoclave, and its own single-pass, HEPA-filtered exhaust system. This enhanced BSL-3 laboratory is shared by the SIU Medical School and the Illinois Dept of Public Health. Both facilities are certified by the CDC to meet all of the requirements for BSL-3 containment. Flow cytometry facility equipped with fluorescence-activated cell sorter (BD FACS Aria II) and two flow cytometers (BD FACSCalibur and Accuri C6). A dedicated support person, Ms. Melissa Roberts, operates the flow cytometry facility. Imaging facility equipped with a Hitachi H7000 transmission scanning electron microscope, a Hitachi S3000N Variable Pressure scanning electron microscope, Leica TCS SP5 Spectral Laser Scanning Confocal Microscope, Xenogen IVIS Lumina II, darkroom and x-ray film developer, and EM prep lab equipped with two ultramicrotomes (RMC MT-7 and Sorval MT-2B). A dedicated support person, Mr. Craig Whitworth, operates the research imaging facility. -Irradiator with a 137Cs radioactive source that is suitable for irradiating mice or cells. Molecular Core and other departmental facilities are equipped with 3 autoclaves, 2 drying ovens, Affymetrix microarray system, Alpha Innotech FluorChem 5500, Olympus BX41 microscope with digital imaging system, laser capture microdissection system, 2 Odyssey infrared imaging systems, chemiluminescence imager, Cryostat, Microtome and floating water bath, Applied Biosystems 7500 Real Time PCR System, Bio-Rad 2-D systems, Agilent Bioanalyser, two Beckman ultracentrifuges, and two Sorvall RC5 super-speed centrifuges, gamma counter, and liquid scintillation counter.

Facilities & Other Resources


BIOGRAPHICAL SKETCH Provide the following information for the Senior/key personnel and other significant contributors.

Follow this format for each person. DO NOT EXCEED FOUR PAGES.

NAME

William P. Halford, Ph.D.

eRA COMMONS USER NAME (credential, e.g., agency login)

WHALFORD

POSITION TITLE

Associate Professor Dept of Medical Microbiology and Immunology Southern Illinois University School of Medicine

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and residency training if applicable.)

INSTITUTION AND LOCATION DEGREE

(if applicable) MM/YY FIELD OF STUDY

University of California, Santa Barbara B.S. 06 / 91 Marine / Microbiology Louisiana State Univ. Med. School, New Orleans Ph.D. 12 / 96 Viral Immunology University of Pennsylvania, Philadelphia Postdoc 5 / 00 Molecular Virology Rocky Mountain Laboratories, Hamilton, MT Sabbatical 12/13 - 7/14 Cellular Immunology

A. Personal Statement I have studied herpes simplex virus (HSV) biology for the past 20 years considering aspects of latency, pathogenesis, and host immune control in vivo. One facet of my lab's work lies in utilizing molecular genetic approaches to explore how viral ICP0 regulates the latency-replication balance of HSV. A second facet of our work lies in understanding how the host immune response limits HSV replication in vivo and exploiting this knowledge to design a safe HSV-2 vaccine that is ~100-times more effective than past HSV-2 vaccine candidates. ICP0- mutants of HSV-1 and HSV-2 are exquisitely sensitive to repression by the innate interferon-/ response of animals, and hence HSV ICP0- mutant viruses establish little more than self-limited infections in mice and guinea pigs. Despite their grossly attenuated phenotype, immunization with HSV-2 ICP0- viruses elicits a highly protective immune response that is comparable in protective efficacy to recovery from a wild-type HSV-2 infection. HSV-2 ICP0- mutant viruses have two potentially important roles to play in vaccine science. First, HSV-2 ICP0- mutant viruses are a safe, live-attenuated vaccine approach that offers ~100 times greater antigenic breadth than the HSV-2 subunit vaccines that have failed in numerous clinical trials. Second, live HSV-2 ICP0- mutant vaccines elicit ~100-times greater protection against HSV-2 genital herpes than past HSV-2 vaccine candidates. As an usually robust HSV-2 vaccine, live HSV-2 ICP0- viruses represent a powerful model system to investigate the underlying principles that dictate what will be needed for a HSV-2 vaccine to elicit robust protection against HSV-2 genital herpes, and which is sustainable over a human lifetime. The P.I.'s published and preliminary results in HSV-2 vaccine-challenge systems suggest two important principles that are not widely appreciated; namely, (1) the failure of past HSV-2 vaccines may be linked to the fact they only contained 1 - 2% of HSV-2's potential antigens, and (2) optimal vaccine-induced protection against HSV-2 may require a balanced B- and T-cell response against most of HSV-2's antigens. The review of Halford, 2014 (Reference 15 below; included in the Appendix) more fully discusses the issues surrounding the safety and improved efficacy of a live-attenuated HSV-2 ICP0- viral vaccine.

B. Positions and Honors Positions and Employment 2000 - 2004 Asst Professor, Tulane University Med School, Dept of Micro & Immunology, New Orleans, LA 2004 - 2007 Assistant Professor, Montana State University, Dept of Vet Molecular Biology, Bozeman, MT 2005 - 2007 Affiliate Assistant Professor, Univ of Washington Med School, Dept of Microbiology, Seattle 2007 - Associate Professor (with tenure), Southern Illinois University School of Medicine, Department

of Medical Microbiology and Immunology, Springfield, IL. Biosketches Page 10


Other Experience and Professional Memberships 1998 – American Association of Immunologists 1996 – American Society for Virology 1996 – 2010 American Society for Microbiology

Honors 2001 Outstanding Professor, 2nd year Medical Students, Tulane University School of Medicine 2003 Charles C. Randall Lectureship, Junior Faculty Excellence Award, South Central ASM Branch 2004 Excellence in New Competitive Research Award, Tulane University 2004 Stephen L. Sacks New Investigator Award, American Herpes Foundation 2010, 2011 Teacher of the Year, Microbiology Department, So. Illinois Univ Med School, Springfield 2013-2017 Academy Scholar (for Excellence in Teaching), So Illinois Univ Med School, Springfield

C. Publications Web of Knowledge Statistics: (out of 50 total) Total citations = 1,442

1. Halford, W.P., B.M. Gebhardt, and D.J.J. Carr. 1996. Persistent cytokine expression in trigeminal ganglion latently infected with HSV 1. J. Immunol. 157: 3542-3549. PMID: 8871654.

2. Halford, W.P. and P.A. Schaffer. 2001. ICP0 is required for the efficient reactivation of HSV type 1 from neuronal latency. J. Virol. 75: 3240-3249. PMID: 11238850.

3. Härle, P., B. Sainz, D.J.J. Carr, and W.P. Halford. 2002. The immediate-early protein, ICP0, is essential for the resistance of HSV to interferon-/. Virology. 293: 295-304. PMID: 11886249.

4. Sainz, B. and W.P. Halford. 2002. Alpha/beta interferon and gamma interferon synergize to inhibit the replication of HSV type 1. J. Virol. 76: 11541-11550. PMID: 12388715.

5. Halford, W.P., J. Grace, C. Weisend, M. Soboleski, D.J.J. Carr, J.W. Balliet, Y. Imai, T.P. Margolis, and B.M. Gebhardt. 2006. ICP0 antagonizes Stat 1-dependent repression of HSV: implications for the regulation of viral latency. Virol. J. 3: 44. PMID: 16764725.

6. Liu, M., B. Rakowski, E. Gershburg, C. Weisend, O. Lucas, E. Schmidt, and W.P. Halford. 2010. ICP0 antagonizes ICP4-dependent silencing of the HSV ICP0 gene. PLoS ONE: 5:e8837. PMID: 20098619.

7. Liu, M., E.E. Schmidt, and W.P. Halford. 2010. ICP0 dismantles microtubule networks in HSV-infected cells. PLoS ONE: 5(6): e10975. PMID: 20544015.

8. Halford, W.P., R. Püschel, and B. Rakowski. 2010. HSV 2 ICP0- mutants are avirulent and immunogenic: implications for a genital herpes vaccine. PLoS ONE 5(8): e12251. PMID: 20808928.

9. Conrady C.D., Halford W.P., Carr D.J. 2011. Loss of the type I interferon pathway increases vulnerability of mice to genital herpes simplex virus 2 infection. J. Virol. 85: 1625-1633. PMID: 21147921.

10. Halford W.P., Püschel R., Gershburg E., Wilber A., Gershburg S., Rakowski B. 2011. A live-attenuated HSV-2 ICP0 virus elicits 10 to 100 times greater protection against genital herpes than a glycoprotein D subunit vaccine. PLoS ONE 6: e17748. PMID: 21412438.

11. Wuest, T., M. Zheng, S. Efstathiou, W.P. Halford, and D. Carr. 2011. The HSV-1 transactivator ICP4 drives VEGF-A dependent neovascularization. PLoS Pathogens 7:e1002278. PMID: 21998580.

12. Halford W.P., J. Geltz, and E. Gershburg. 2013. Pan-HSV-2 IgG antibody in vaccinated mice and guinea pigs correlates with protection against herpes simplex virus 2. PLoS ONE: 8:e65523. PMID: 23755244

13. Prigge, J.R., J.A. Wiley, E.A. Talago, E.M. Young, L.L. Johns, J.A. Kundert, K.M. Sonsteng, W.P. Halford, M.R. Capecchi, and E.E. Schmidt. 2013. Nuclear double-fluorescent reporter for in vivo and ex vivo analyses of biological transition. Mammalian Genome 24: 389-399. PMID: 24022199

14. Workenhe, S.T., W.P. Halford, G. Simmons, J.G Pol, B.D. Lichty, K.L. Mossman. 2014. Immunogenic HSV-mediated oncolysis shapes the adaptive anti-tumor immune response and contributes to improved therapeutic efficacy. Molecular Therapy: 22:123-31. PMID: 24343053

15. Halford, W.P. 2014. Antigenic breadth: a missing ingredient in HSV-2 subunit vaccines? Expert Rev Vaccines: 13(6): 691-710. PMID: 24837838

Biosketches Page 11


D. Research Support

Ongoing Research Support

Concept Development Award, SIU School of Medicine: $14,300 1/14 - 12/14 The Type-Specific ABVIC Assay: an Improved HSV Serological Test P.I. Halford

Completed Research Support

Louisiana Vaccine Center, Direct costs: $75,000 7/12 - 6/13 Prophylactic and Therapeutic Vaccines for Ocular HSV-1 (10% effort, Role: Co-Investigator) P.I. Hill

R21 AI81072, NIAID, Direct costs: $275,000 (+ $71,600 supplement) 7/09 - 12/12 Development of an effective genital herpes vaccine. (25% effort) P.I. Halford The goal of this study is to develop several live herpes simplex virus 2 (HSV-2) ICP0- mutant viruses that

replicate in animals, establish latent infections, are avirulent (interferon-sensitive), establish sterilizing immunity, and thus provide vaccine recipients with a safe and effective means to acquire protection against all diseases, including genital herpes, which are caused by wild-type HSV-2.

Excellence in Academic Medicine, SIU Internal Grant , Direct costs: $50,000 1/11 - 12/11 A safe and effective genital herpes vaccine. (10% effort) P.I. Halford Test the relative safety and efficacy of a live herpes simplex virus 2 (HSV-2) ICP0- mutant viral vaccine relative

to three HSV-2 vaccines that recapitulate the key properties of HSV-2 vaccines backed by pharmaceutical / biotechnology companies (i.e., Herpevac, ACAM-529, and ImmunovexHSV-2 vaccines).

Concept Development Award, SIU Internal Grant, Direct costs: $9,000 10/10 – 9/11 Development of an efficient bidirectional gene expression system. P.I. Wilber (2% effort; Role: Collaborator) Development of a transposon-based, bi-directional gene expression system that may be used to make a

wide range of stable mammalian cell lines and which may be efficiently silenced or potently induced.

Excellence in Academic Medicine, SIU Internal Grant, Direct costs: $40,000 12/08 - 11/09 Development of a live, immune-sensitive HSV-2 vaccine. (10% effort) P.I. Halford

Central Research Committee Grant, SIU Internal Grant Direct Costs: $15,000 7/08 - 12/08 Development of an effective genital herpes vaccine. (10% effort) P.I. Halford

R01 AI51414, NIAID, Direct costs: $612,500 8/03 - 1/07 Role of the LAT-ICP0 locus in regulating HSV latency. (40% effort) P.I. Halford

R21 AI51414, NIAID, Direct costs: $200,000 8/02 - 7/03 Role of the LAT-ICP0 locus in regulating HSV latency. (40% effort) P.I. Halford

Biosketches Page 12


BIOGRAPHICAL SKETCH Provide the following information for the Senior/key personnel and other significant contributors in the order listed on Form Page 2.

Follow this format for each person. DO NOT EXCEED FOUR PAGES.

NAME

Kim J. Hasenkrug

eRA COMMONS USER NAME (credential, e.g., agency login)

POSITION TITLE

Senior Investigator, Chief of the Retroviral Immunology Section, Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and residency training if applicable.)

INSTITUTION AND LOCATION DEGREE

(if applicable) MM/YY FIELD OF STUDY

College of Great Falls, Great Fall, MT Albert Einstein College of Medicine, Bronx, NY

BS MS

1977 1988

Immunology/Cell Biology

Albert Einstein College of Medicine, Bronx, NY PhD 1991 Immunology/Cell Biology

A. Personal Statement

Briefly describe why your experience and qualifications make you particularly well-suited for your role (e.g., PD/PI, mentor, participating faculty) in the project that is the subject of the application. Within this section you may, if you choose, briefly describe factors such as family care responsibilities, illness, disability, and active duty military service that may have affected your scientific advancement or productivity. Kim Hasenkrug is a Senior Investigator in the Laboratory of Persistent Viral Diseases at Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, NIH, where he has served as Chief of the Retroviral Immunology Section since 2005. He received his Ph.D. in Cell Biology and Immunology from the Albert Einstein College of Medicine in 1991 studying the structure and function of major histocompatibility complex molecules. Dr. Hasenkrug’s current research interest is the immunology of retroviral infections, which he studies in mouse models. Dr. Hasenkrug serves on the Editorial Boards of Virology and PLoS ONE, and served as a scientific advisor for the International AIDS Vaccine Initiative for many years. He has chaired sessions at numerous international symposia including Gordon Conferences, Keystone Conferences and AAI meetings and has taught retroviral immunology classes for international courses. Of specific relevance to this grant proposal, Dr. Hasenkrug has published dozens of peer-reviewed manuscripts on vaccines including the first description of the mechanisms of live attenuated vaccine protection against retroviral infection (Nature Medicine 5:189 (1999)), and elucidation of the roles of both neutralizing and non-neutralizing antibodies in vaccine protection (PNAS 92:10492-95, 1995; PNAS 101:12260 (2004)).

B. Positions and Honors

1978 - 1984 Research Assistant, McLaughlin Research Institute, Great Falls, Montana 1984 - 1986 Laboratory Supervisor, McLaughlin Research Institute, Great Falls, Montana 1986 - 1991 Pre-doctoral Fellow, Albert Einstein College of Medicine, Bronx, New York 1991 - 1992 IRTA Fellow, Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, NIH,

NIAID, Hamilton, Montana 1992 - 1998 Staff Fellow, Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, NIH, NIAID,

Hamilton, Montana 1998 - 2005 Investigator, Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, NIAID, NIH,

Hamilton, Montana 2001 - Associate Professor (Affiliate), Montana State University 2005 - Tenured Senior Investigator, Chief of the Retroviral Immunology Section, Laboratory of

Persistent Viral Diseases, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, Montana 2008 - Associate Professor (affiliate), University of Montana

Biosketches Page 13


Editorial Boards

Current Molecular Medicine 2000-2005 Virology 2005-2014 Journal of Virology 2006-2009 PLoS One 2009-2014

Advisory Committees

International AIDS Vaccine Initiative (IAVI) Vaccine Science Committee, 2003-present IAVI subcommittee on Mechanisms of Vaccine Protection by Live-attenuated Viruses, 2004-present NIH Animal Research Advisory Committee 2009-2012 NIAID Promotion and Tenure Committee 2013-present

Grant Reviews

NIAID Division of Extramural Activities Scientific Review Program Innovation Grant Program for Approaches in HIV Vaccine Research, 1998 Judge for NIH Fellow’s Award for Research Excellence, 2000 Judge for NIH Fellow’s Award for Research Excellence, 2002-2006 International AIDS Vaccine Initiative Grants Review, 2004 – 2006 Science Foundation Ireland, Principal Investigator Programme Grant Study Section 2006 The Wellcome Trust, “Vaccination strategies against retroviral infection”, 2006 Medizinische Forschungsförderung Innsbruck Research Fund, “Role of complement for the induction of T cell responses upon retroviral infection” 2006 NIH Special Emphasis Panel/Scientific Review Group 2009-10 Wellcome Trust Research Grant Review 2010, 2012

Ad hoc Journal Reviewer

AIDS, Journal of Infectious Diseases, AIDS Research and Human Retroviruses, Journal of Virology, Cellular Immunology, Nature Immunology, Immunity, Nature Medicine, Journal of Experimental Medicine, Viral Immunology, Journal of Immunology, PLoS Pathogens, Vaccine, Proceedings of the National Academy of Sciences (USA)

Keynote Address

22nd Annual Meeting of the Society for Virology. March 14-17, 2012, Essen, Germany. “The role of regulatory T cells in viral infection”

C. Selected Peer-reviewed Publications

1. Hasenkrug, K. J., Brooks, D. M., and Chesebro, B. Passive immunotherapy for retroviral disease: Influence of major histocompatibility complex type and T-cell responsiveness. Proc. Natl. Acad. Sci. (USA). 92: 10492-10495, 1995. <Go to ISI>://A1995TD89000010

2. Hasenkrug, K. J. and Chesebro, B. Immunity to retroviral infection: The Friend virus model. Proc. Natl. Acad. Sci. (USA). 94: 7811-7816. 1997. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9223268

3. Dittmer, U., Brooks, D.M., and Hasenkrug, K.J. Characterization of a live-attenuated retroviral vaccine demonstrates protection via immune mechanisms. Journal of Virology. 72: 6554-6558. 1998 http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9658099

4. Dittmer, U., Brooks, D.M., and Hasenkrug, K.J. Requirement for multiple lymphocyte subets in protection by a live attenuated vaccine against retroviral infection. Nature Medicine. 5: 189-193. 1999 http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9930867

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5. Iwashiro, Michihiro, Ron Messer, Karin E. Peterson, Ingunn M. Stromnes, Tomoharu Sugie, and Kim J. Hasenkrug. Immunosuppression by CD4+ regulatory T cells induced by chronic retroviral infection. Proceedings of the National Academy of Sciences (USA) 98: 9226-9230 2001 http://www.pnas.org/cgi/content/full/98/16/9226

6. Dittmer, Ulf, Hong He, Ronald J. Messer, Simone Schimmer, Anke R. M. Olbrich, Claes Ohlen, Philip D. Greenberg, Ingunn M. Stromnes, Michihiro Iwashiro, Shimon Sakaguchi, Leonard H. Evans, Karin E. Peterson, Guojun Yang, and Kim J. Hasenkrug. Functional impairment of CD8+ T cells by regulatory T cells during persistent retroviral infection. Immunity 20: 293-303 2004 http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15030773

7. Messer, Ronald J., Ulf Dittmer, Karin E. Peterson, and Kim J Hasenkrug. Essential role for virus-neutralizing antibodies in sterilizing immunity against Friend retrovirus infection. Proceedings of the National Academy of Sciences (USA) 101:12260-12265 2004 http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15297622

8. Koff, Wayne C., Philip R. Johnson, David I. Watkins, Dennis R. Burton, Jeffrey D. Lifson, Kim Hasenkrug, Adrian B. McDermott, Alan Schultz, Timothy J. Zamb, Rosanne Boyle and Ronald C. Desrosiers. HIV Vaccine Design: Insights from Live Attenuated SIV Vaccines. Nature Immunology 7 (1):19-23, 2006 http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16357854

9. Santiago, M. L., Montano, M., Benitez, R., Messer, R. J., Yonemoto, W., Chesebro, B., Hasenkrug, K. J.*, Greene, W. C.* (*shared senior authorship). Apobec3 encodes Rfv3, a gene influencing neutralizing antibody control of retrovirus infection. Science 321: 1343-1346, 2008 http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18772436

10. Ines Antunes, Mario Tolaini, Michihiro Iwashiro, Kagemasa Kuribayashi, David Gray, Kim J. Hasenkrug, and George Kassiotis. Suppression of retrovirus-induced T cell-mediated bone marrow pathology by regulatory T cells independently of retrovirus-specificity. Immunity 29: 782-794, 2008 http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19006695

11. Myers, Lara, Messer, Ronald J., Carmody, Aaron B., and Hasenkrug, Kim J. Tissue-Specific Abundance of Regulatory T Cells Correlates with CD8+ T Cell Dysfunction and Chronic Retrovirus Loads. Journal of Immunology 183:1636-1643, 2009

12. Dietze, Kirsten K. , Gennadiy Zelinskyy, Kathrin Gibbert, Simone Schimmer, Sandra Francois, Lara Myers, Tim Sparwasser, Kim J Hasenkrug & Ulf Dittmer.

Transient depletion of regulatory T cells in transgenic mice reactivates virus-specific CD8+ T cells and reduces chronic retroviral setpoints. Proceedings of the National Academy of Sciences (USA) 108:2420-5, 2011 http://www.ncbi.nlm.nih.gov/pubmed/21262821

13. Thorborn, Georgina, Ploquin, Mickaël J., Eksmond, Urszula, Pike, Rebecca, Bayer, Wibke, Dittmer, Ulf, Hasenkrug, Kim J. , Pepper Marion and George Kassiotis. Clonotypic composition of the CD4 + T cell response to a vectored retroviral antigen is determined by its speed. Journal of Immunology 193:1567-77 2014 http://www.ncbi.nlm.nih.gov/pubmed/25000983

14. Messer, Ronald J., Lavender, Kerry J., and Kim J. Hasenkrug. Mice of the resistant H-2b haplotype mount broad CD4+ T cell responses against 9 distinct Friend virus epitopes. Virology 456-457, 2014 http://www.ncbi.nlm.nih.gov/pubmed/24889233

15. Lavender, Kerry J., Pang, Wendy W., Messer, Ronald J., Duley, Amanda K., Race, Brent, Phillips, Katie, Scott, Dana, Peterson, Karin E., Chan, Charles K., Dittmer, Ulf, Dudek, Timothy, Allen, Todd M., Weissman, Irving L., and Hasenkrug, Kim J. BLT-humanized C57BL/6 Rag2-/-γc

-/-CD47-/- mice are resistant to GVHD and develop B and T cell immunity to HIV infection. Blood 122:4013-4020, 2013 *Faculty of 1000 article of special significance, Special commentary in Blood. http://www.ncbi.nlm.nih.gov/pubmed/24021673 D. Research Support Supported by the intramural program of the NIAID.

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SPECIFIC AIMS

Efforts to develop a herpes simplex virus 2 (HSV-2) vaccine have been ongoing for 30 years. Despite numerous HSV-2 vaccine trials, we continue to lack an effective HSV-2 vaccine. Such vaccine trial failures are representative of a growing problem for the vaccine research enterprise. About 99% of "promising" vaccine candidates have failed in billions of dollars of clinical trials spanning the past 20 years. These repeated failures have led many scientists to question whether vaccines to prevent AIDS, tuberculosis, malaria, and genital herpes are feasible, and whether scarce funding dollars should be wasted on future vaccine candidates that are likely to fail. Thus, it is imperative that the scientific community offer a real (1) explanation for why so many vaccines have failed in the past, and (2) plan for how vaccines may be made more effective in the future.

The P.I.'s work in HSV-2 vaccine-challenge systems suggests two important, but widely unappreciated, principles. First, all of the failed HSV-2 vaccines appear to lack appropriate antigenic breadth. Second, optimal vaccine-induced protection against HSV-2 may require a balanced B- and T-cell response against a wide array of HSV-2 antigens. Although the proposed studies will be performed with HSV-2, the results may have broader implications; failure to elicit a balanced B- and T-cell response against an adequate % of a pathogen's antigens may be the root cause of many HIV, TB, and Plasmodium vaccine failures as well.

Regarding the model system, the P.I. has developed a live HSV-2 ICP0 - mutant vaccine, HSV-2 0NLS, and has compared its protective efficacy to a HSV-2 glycoprotein D (gD-2) vaccine similar to Glaxo Smith Kline’s Herpevac vaccine (8, 18). Animals immunized with the live HSV-2 0NLS vaccine are up to 100-times better protected against HSV-2 than recipients of a gD-2 vaccine (Fig. 1; Ref. (38). This begs the question, why do gD-2 vaccines elicit only 1 - 2% of the protection against HSV-2 that is attainable? The HSV-2 proteome encodes ~39,100 amino acids; gD-2-based vaccines expose recipients to 0.8% of HSV-2's proteome, whereas HSV-2 0NLS encodes >99% of HSV-2's proteins (Fig. 3). As a result, the live HSV-2 0NLS vaccine elicits ~40-fold higher levels of pan-HSV-2 IgG than a gD-2 vaccine, and these IgG antibodies target 9 to 19 different HSV-2 proteins. Importantly, pan-HSV-2 IgG levels correlate with functional protection against HSV-2 (Fig. 5).

The central hypothesis of the proposed studies is that a balanced B- and T-cell response to manyHSV-2 antigens is required to elicit optimal vaccine-induced protection against HSV-2. If this hypothesis is correct, then it would clarify what has been missing from past HSV-2 vaccines, and highlight what is needed to obtain an effective HSV-2 vaccine in the future. A secondary hypothesis of the proposed studies is that a live HSV-2 ICP0- mutant virus represents a very safe approach to elicit such a balanced B- and T-cell response to most of HSV-2's antigens. Three Specific Aims are proposed to test these two inter-related hypotheses.

Specific Aim 1. To test a hypothesis that virus-specific antibodies are required for vaccine-induced protection against HSV-2.

Specific Aim 2. To test a hypothesis that complete vaccine-induced protection against HSV-2 requires the cooperative activities of antibodies and T-cells.

Specific Aim 3. To test a hypothesis that the live HSV-2 ICP0- mutant vaccine, HSV-2 0NLS, is unable to establish a reactivate-able infection in vaccine recipients.

Fig. 1. Visualization of vaccine-induced protection against HSV-2 vaginal challenge. Mice were immunized on Days 0 and 30 in their rear footpads with (from left to right) culture medium (naïve); 2.5 g gD-2+alum + 10 g MPL; or 3.106 pfu of live HSV-2 0NLS virus. On Day 130, mice were challenged with 500,000 pfu/vagina of HSV-2 MS-luciferase, and viral spread was visualized on Days 2 and 6 post-challenge in a bioluminescent imager following injection with 3 mg D-luciferin. Reproduced from Fig. 6 of Ref. (38).

Specific Aims Page 27


A. SIGNIFICANCE AND BACKGROUND A-1. HSV-2 and the need for an effective vaccine. About 3 million Americans live with recurrent genital herpes disease (33, 46, 73) More than 500 million people worldwide carry wild-type HSV-2 (14, 55), and carriers may shed HSV-2 in their genital tract on any day (80, 81). Carriers transmit new HSV-2 infections to >10 million people per year and have a 3-fold higher risk of acquiring HIV (26, 53, 70). Antiviral drugs reduce, but do not eliminate, these risks. An effective HSV-2 vaccine would eliminate these risks in the same manner that the VZV Oka vaccine has reduced the burden of disease caused by chickenpox and shingles (30, 34).

A-2. Is a HSV-2 vaccine feasible? Since the mid-1980s, much of our HSV-2 vaccine research efforts have been dedicated to testing HSV-2 glycoprotein D (gD-2)-based subunit vaccines. Such gD-2-based vaccines have failed to prevent HSV-2 genital herpes in six U.S. clinical trials (8, 20, 62, 77-79). These failures have prompted some investigators to ask if an effective HSV-2 vaccine is an attainable goal (18, 51, 64, 83). The underlying issue is not that a HSV-2 vaccine is an impossible goal, but rather that gD-2 subunit vaccines are the only HSV-2 vaccine approach that has been seriously evaluated in U.S. clinical trials (Fig. 2). While more than 14,000 individuals have been enrolled in U.S. clinical trials of gD-2-based subunit vaccines, n=0 patients have been enrolled in U.S. trials of a live-attenuated HSV-2 vaccine during the same time (Fig. 2). Live-attenuated HSV-2 viral vaccines appear to elicit far greater protection against HSV-2 genital herpes than gD-2 subunit vaccines (Fig. 1, Table 1). To date, safety concerns have limited interest in advancing a live HSV-2 vaccine to a human clinical trial. For many, the thought of immunizing a human recipient with a live, albeit attenuated form of infectious HSV-2 seems unthinkable. The human root of such concerns is understandable, but is without any basis in scientific evidence. While it is true that HSV-1 and HSV-2 ICP0 - mutant viruses are live replication-competent viruses, these viruses are so profoundly sensitive to the host interferon response that they are (1) avirulent in immunocompetent animals, and are (2) also profoundly attenuated in lymphocyte-deficient (SCID) animals (39). Aside from the evidence that live HSV-2 ICP0 - mutant viruses are avirulent, the appropriate basis for evaluating the risk of a live HSV-2 vaccine is relative to the risk posed by the current absence of a HSV-2 vaccine. During the 25 years HSV-2 subunit vaccines have been failing in U.S. clinical trials (Fig. 2), over 250 million people have been newly infected with wild-type HSV-2. I would suggest the risks associated with clinical trials of a live HSV-2 ICP0- vaccine would be preferable to the risk that >10 million people per year will continue to be newly infected with HSV-2 while HSV-2 subunit vaccines continue to fail in clinical trials (Fig. 2).

A-3. How much more effective Is a live HSV-2 vaccine vs a HSV-2 subunit vaccine? Several iterations of HSV-2 glycoprotein B (gB-2) and/or glycoprotein D (gD-2) subunit vaccines have advanced to clinical trials (8, 20, 62, 77-79). The most recent was Glaxo Smith Kline (GSK)'s Herpevac vaccine, which consisted of gD-2 and an alum/monophosphoryl lipid A (MPL) adjuvant. Over 8,000 seronegative women immunized with Herpevac acquired HSV-2 genital herpes at the same rate as >8,000 placebo-treated women (8). As the best studied HSV-2 vaccine candidate to date, we were interested to determine if a live-attenuated HSV-2 ICP0 - mutant virus, HSV-2 0NLS, would elicit protective immunity to HSV-2 that met or exceeded that elicited by the extensively studied gD-2 + alum/MPL vaccine formulation in Herpevac (8, 10-13). Comparisons of a gD-2 vaccine versus the HSV-2 0NLS vaccine supported many important conclusions, summarized as follows. First, although HSV-2 glycoprotein D (gD-2) is an excellent antigen, high antibody titers elicited against gD-2 in vaccine recipients conferred only limited protection against HSV-2. Hence, only 3 of 45 gD-2-immunized mice survived an overwhelming challenge with wild-type HSV-2, whereas 114 of 115

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Research Strategy Page 28


live HSV-2 0NLS-immunized mice survived the same overwhelming challenge (Table 1; Ref. (38). Second, mice immunized with a gD-2 subunit vaccine only modestly delay the spread of a luciferase-expressing HSV-2 challenge virus from the vagina, whereas HSV-2 0NLS-immunized mice were completely resistant to the same luciferase-expressing HSV-2 challenge virus (Fig. 1). Third, mice and guinea pigs immunized with a gD-2 subunit vaccine were 3- to 4-fold (modestly) resistant to HSV-2 vaginal shedding relative to naïve animals

(38). In contrast, animals immunized with the HSV-2 0NLS vaccine were 300- to 500-fold more resistant to HSV-2 vaginal shedding relative to naïve controls (36, 38). The full body of evidence describing the live HSV-2 0NLS vaccine may be found in the publications of Halford, et al., 2010, 2011, 2013, and 2014 (35, 36, 38, 39).

A-4. Why so different? Why should the live HSV-2 0NLS vaccine elicit up to 100-fold better protective immunity against HSV-2 than a gD-2 + alum/MPL subunit vaccine? I would suggest antigenic breadth is the most likely answer. Antigenic breadth may be the missing ingredient that explains why HSV-2 subunit vaccines have failed in prior clinical trials, and why HSV-2 0NLS elicits superior protection in side-by-side comparative testing (reviewed in Ref. 35; in Appendix). The antigenic breadth of a HSV-2 vaccine candidate may be defined as the "% of wild-type HSV-2 proteome contained, or expressed, in a vaccine." Wild-type HSV-2 is a 154-kb dsDNA virus that encodes ~39,100 amino acids distributed across 75 viral proteins. GSK's Herpevac vaccine contained 302 amino acids of gD-2, which corresponds to 0.8% of HSV-2's antigenic breadth (Fig. 3A). The 99% of HSV-2's proteome not included in Herpevac may limit its capacity to engage the entire repertoire of virus-specific B- and T-lymphocytes available in a vaccine recipient to contribute to protective immunity against HSV-2. In contrast, the live HSV-2 0NLS virus is attenuated by deletion of ~300 amino acids of ICP0, and retains the capacity to encode up to 99.3% of HSV-2's proteome (Fig. 3B). Most past and current vaccine candidates contain <2% of the HSV-2 proteome (2, 3, 8, 10-13), which raises the question, "Are the other 98% of HSV-2's proteins so inert they should be excluded from an effective HSV-2 vaccine?" This important question has not been carefully evaluated. Thus, most HSV-2 vaccines are based on a tacit assumption that immunization with <2% of HSV-2's proteome may elicit 100% of the protective immunity against HSV-2 genital herpes that is attainable.

A-5. Is 'antigenic breadth' relevant? If antigenic breadth is relevant to HSV-2 vaccines, then one would predict that recipients of the live HSV-2 0NLS vaccine should possess higher levels of IgG antibody against total HSV-2 antigen relative to animals immunized with a gD-2 subunit vaccine. To test this prediction, sera of mice immunized with a live HSV-2 0NLS or gD-2 vaccine were compared for their capacity to bind total viral antigen in HSV-2 plaques (Fig. 4). Sera of mice immunized with gD-2 contained high levels of gD-2-specific IgG (38), but the same gD-2-specific IgG only weakly bound total antigen in HSV-2-infected cells; gD-2 is only 1 of 75 viral proteins (Fig. 4). In contrast, IgG antibodies in sera of HSV-2 0NLS-immunized mice

Figure 3. Antigenic breadth of HSV-2 subunit vs live-attenuated HSV-2 vaccine. (A) The HSV-2 US6 geneencodes glycoprotein D. Herpevac was based on 302 amino acids of glycoprotein D, which corresponds to 0.8% of HSV-2's proteome. (B) The live HSV-2 0NLS vaccine is attenuated by deletion of 292 amino acids from ICP0. HSV-2 0NLSretains the capacity to encode 38,779 amino acids (99.3%) of HSV-2's proteome.

Figure 4. Mice immunized with HSV-2 0NLS possess higher levels of pan-HSV-2 IgG than mice immunized with gD-2. Immunofluorescent labeling of fixed HSV-2 plaques incubated with a 1:5,000 dilution of Day 60 serum from naive mice or mice immunized with gD-2 or HSV-2 0NLS. Levels of mouse pan HSV-2 IgG were visualized with AlexaFluor 594 goat anti-mouse IgG. Reproduced from Figure 5B of Ref. (38).



strongly bound HSV-2+ cells (Fig. 4). Flow-cytometry-based analyses revealed that serum levels of pan-HSV-2 IgG were an average 40-fold higher in mice immunized with the live HSV-2 0NLS virus relative to mice immunized with a gD-2 subunit vaccine (36). Importantly, serum levels of pan-HSV-2 IgG were predictive of an individual animal's resistance to a later vaginal challenge with wild-type HSV-2 (Fig. 5; Ref. 36). Additional, unpublished studies (not presented for lack of space) demonstrate that mice immunized with the live HSV-2 0NLS vaccine mount an IgG antibody response directed against 9 to 19 viral proteins, and only 10 5% of those antibodies are specific for gD-2. Hence, the ~100-fold increase in antigenic breadth of the live HSV-2 0NLS vaccine relative to gD-2 vaccines (Fig. 3) is not a purely theoretical consideration, but rather recipients of the HSV-2 0NLS vaccine elicit an immune response against >10 HSV-2 proteins.

A-6. Would the FDA approve a live -herpesvirus vaccine? Many scientists assume the FDA would never approve the use of a live, replicating -herpesvirus vaccine that may establish a latent infection in the peripheral nervous system of vaccine recipients. However, the FDA approved precisely such a vaccine when they approved the live-attenuated VZV Oka vaccine in the 1990s. VZV represents one of herpes simplex virus (HSV)'s closest living relatives, and thus VZV and HSV-2 share a very similar neurotropic / latent lifestyle. VZV and HSV-2 encode ~60 homologous viral proteins (19, 71), and the live VZV Oka vaccine strain is widely known (amongst virologists) to routinely establish a latent infections in vaccine recipients (48, 69). Whatever concerns may be raised about a live-attenuated HSV-2 vaccine, these same concerns apply equally to the live VZV Oka vaccine. Despite initial reservations about this live -herpesvirus vaccine, clinical experience in the first 60 million recipients of VZV Oka has been overwhelmingly positive (28, 34). The risks posed by the VZV Oka vaccine strain (7, 17, 52) have been vastly outweighed by effective control of chickenpox in school-age children, and the associated severe diseases of VZV pneumonia in naïve adults and wild-type VZV infections of the immunocompromised (30). If a clear success has been achieved with the live VZV Oka vaccine, then a similar live vaccine strategy should be equally successful in preventing the spread of HSV-2 genital herpes (30). Importantly, the live HSV-2 ICP0- virus strains discussed in this application are far safer than the VZV Oka vaccine strain (reviewed in Ref. 35; in Appendix) because HSV-2 ICP0- viruses are (1) interferon-hypersensitive and are (2) profoundly attenuated even in SCID hosts (39, 42).

A-7. Purpose of proposed studies. Evidence gathered by the P.I.'s laboratory suggests a live HSV-2 ICP0 - viral vaccine may represent a superior opportunity to stop the spread of HSV-2 genital herpes (36, 38, 39). Nonetheless, significant gaps in knowledge continue to limit enthusiasm and/or awareness of this novel HSV-2 vaccine approach. One gap in knowledge is the current lack of a cohesive explanation for why past HSV-2 subunit vaccines based on 1-2% of HSV-2's antigens have failed, and why a live HSV-2 ICP0 - viral vaccine that expresses up to 99% of HSV-2's proteome may elicit a superior and balanced B- and T-cell response against many of HSV-2's antigens. The second gap in knowledge is a prevailing belief that a live-attenuated HSV-2 vaccine would be intrinsically unsafe, despite the complete absence of any specific evidence to support such suppositions. Experiments described in three Specific Aims will address these gaps in knowledge, and will clarify why novel live HSV-2 ICP0 - mutant viruses represent a safe and highly effective means to elicit complete vaccine-induced protection against HSV-2 genital herpes. It is anticipated the results of these studies will clarify that the live HSV-2 0NLS vaccine exhibits the safety and efficacy profile a highly desirable candidate for human clinical trials.

Fig. 5. Pan-HSV-2 IgG levels correlate with protection. Mice were immunized on Days 0 and 30 with 10 g MPL+alum+2.5 g gD-2 or GFP; culture medium (naïve), 106 pfu HSV-2 0NLS, or 106 pfu HSV-2 MS (n=10 per group). Mice immunized with HSV-2 MS received 1 mg/ml acyclovir (ACV) in water from Days 0 to 20. On Day 60, blood was collected, and on Day 90 mice were challenged with 500,000 pfu per vagina of HSV-2 MS. The mean sem of HSV-2 MS shedding after challenge for each group of mouse is reflected in the y-coordinate and similarly the mean sem of the x-coordinate represents pre-challenge serum levels of pan-HSV-2 IgG. Fig. 3D from Ref. (36)

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B. INNOVATION B-1. Novel tools for HSV-2 vaccine-challenge studies. We have developed several new reagents and controls that improve upon past HSV-2 vaccine-challenge animal models, and these include: 1. Wild-type HSV-2 latently infected animals as a control for robust protection against HSV-2 challenge (38). 2. A novel panel of seven HSV-2 ICP0 - mutant viruses that exhibit a wide range of attenuation; namely,

HSV-2 0104, 0125, 0RING, 0254, 0NLS, 0MD, and 0810 (39). 3. Novel GFP+ or luciferase+ HSV-2 challenge viruses whose spread may be visualized in vivo (38, 39).

B-2. Analysis of the complex polyclonal antibody response to a live HSV-2 vaccine. We have developed a panel of new assays to analyze the antigen-specificity of the antibody response elicited by immunization with a live HSV-2 viral vaccine, and these include: 4. Immunoprecipitation-mass spec to identify HSV-2 antigens bound by IgG in 0NLS antiserum. 5. Western blots to independently confirm dominant HSV-2 antigens by MW and kinetic class. 6. Development of cell lines that express HSV-2's most dominant antigens. These methods have been used to analyze the polyvalent antibody response elicited by a live HSV-2 ICP0 - vaccine, which is directed against 9 to 19 HSV-2 proteins.

B-3. Quantification of total vaccine-induced response to HSV-2's B- and T-cell immunogens. My laboratory has demonstrated that vaccine-induced pan-HSV-2 IgG antibody correlates with protection against HSV-2 (Ref. 36; Fig. 5). An equivalent method does not exist to enumerate the total T-cell response to HSV-2 vaccines. I spent my Sabbatical Leave (7 months) in the Laboratory of Persistent Viral Diseases working with Dr. Kim Hasenkrug to adapt methods used to successfully study T-cell responses to Friend virus (44, 63, 84, 85) to analyze T-cell responses elicited by HSV-2 vaccines. This work has yielded novel methods that will be used in the proposed study to measure B- and T-cell responses to HSV-2 vaccines, and include a: 7. Novel pan-HSV-2 IgG antibody assay to gauge the total B-cell response to HSV-2 vaccines (36). 8. Novel assay to gauge the frequency of CD8+ T-cells that are activated by HSV-2 vaccines (Fig. 7B). 9. Novel IFN- ELIspot to gauge total HSV-2-specific T-cell response in vaccine recipients (Fig. 7C). Experiments performed in Specific Aims 1 and 2 will test a hypothesis that these novel metrics of the "total HSV-2-specific T-cell response" will correlate with vaccine-induced protection against HSV-2.

C. APPROACH Specific Aim 1. To test a hypothesis that virus-specific antibodies are required for vaccine-induced

protection against HSV-2.

Rationale: T-cells are critical for host control of HSV infections (23, 47, 54, 65, 66, 86). Thus, several HSV-2 vaccines have been proposed that contain only HSV-2 T-cell epitopes (15, 16, 21, 22, 49, 82). One of these, the Agenus HerpV vaccine, has advanced to human clinical trials (2). We question the wisdom of a HSV-2 vaccine that selectively boosts T-cell responses while ignoring the humoral immune response. Antibodies significantly contribute to vaccine-induced protection against HSV-2 when HSV-2 0NLS antiserum is adoptively transferred to naïve mice (Ref. 36; Fig. 9 below). Pilot studies were performed in MT mice (9, 61) as an initial test of a hypothesis that antibodies are required for vaccine-induced protection against HSV-2.

Pilot Test 1: HSV-2 0NLS vaccination of MT vs C57BL/10 mice. B-cell-deficient MT mice and wild-type C57BL/10 mice were immunized on Days 0 and 30 with HSV-2 0NLS or culture medium (mock). On Day 60, mice were vaginally challenged with wild-type HSV-2. Naïve (mock-immunized) C57BL/10 and MT mice shed high levels of HSV-2 challenge virus (Fig 6A), developed severe disease (Fig 6B), and succumbed within 7 1 days post-challenge (Fig 6C). Between Days 1 and 3 post-challenge, 0NLS-immunized C57BL/10 mice shed 300 30-fold less HSV-2 per vagina relative to naïve mice, and 100% survived disease-free for 30 days (Fig. 6A-C). HSV-2 0NLS-immunized MT mice exhibited intermediate phenotypes. At early times (Days 1-3), 0NLS-immunized MT mice shed 10- to 40-fold more HSV-2 per vagina relative to 0NLS-immunized C57BL/10 mice (red arrow in Fig. 6A). Likewise, a few 0NLS-immunized MT mice exhibited mild perivaginal



disease at Day 8 (Fig 6B). Despite eventual control of vaginal HSV-2 shedding, 7 of 8 0NLS-immunized MT mice succumbed to HSV-2 vaginal challenge (Fig. 6C). Hence, the efficacy of HSV-2 0NLS vaccine-induced protection is severely compromised in B-cell-deficient MT mice, suggesting a critical role for HSV-2-specific antibodies (Fig. 6). We hypothesize that T-cells alone are insufficient to halt the spread of HSV-2 challenge in 0NLS-immunized MT mice, but further tests will be required to validate a prediction that uncontrolled HSV-2 spread is the cause of death in 0NLS-immunized MT mice.

Pilot Test 2: HSV-2 0NLS elicits a potent T-cell response in MT mice. Lack of HSV-2-specific antibodies may explain why 0NLS-immunized MT mice remain vulnerable to HSV-2 vaginal challenge. However, it is also possible that the deficiency in MT mice extends beyond the B-cell compartment, such that the HSV-2 0NLS vaccine fails to elicit an effective T-cell response in MT mice. To address this latter possibility, novel methods were developed to measure the frequency of (1) activated CD8+ T-cells and (2) HSV-2-antigen-specific T-cells after the 2nd shot of an immunization series. These methods were developed in collaboration with Dr. Kim Hasenkrug during the P.I's sabbatical. Dr. Hasenkrug will serve as a collaborator on these studies, which are an extension of our ongoing collaboration (see Support Letter). Experiments were conducted to determine if the live HSV-2 0NLS vaccine elicits T-cell responses in C57BL/10 mice and MT mice. A U.V.-inactivated 0NLS vaccine control was added, which retains millions of HSV-2 virions per immunization but which is grossly impaired for de novo viral protein synthesis (i.e., the major source of viral peptides entering the MHC class I pathway; Ref. (24). Mice were immunized with culture medium, U.V. 0NLS, or live HSV-2 0NLS on Days 0 and 30. On Day 6 post-boost, peripheral blood was harvested from mice and activated CD8+ T-cell frequency was analyzed (Fig. 7A, B). In mock-immunized C57BL/10 and MT mice, a background of 0.4 0.1% and 0.1 0.0% of peripheral CD8+ T-cells were activated, as gauged by elevated expression of CD11a and an activation-associated glycoform of CD43 (Fig. 7A,B; Ref. 25, 85). In live HSV-2 0NLS-immunized C57BL/10 and MT mice, 1.7 0.5% of CD8+ T-cells exhibited a CD11ahi CD43hi activation phenotype which represented 4- and 15-fold increases relative to naïve controls, respectively (Fig. 7B). In contrast, immunization with U.V.-treated 0NLS activated 1.4 0.2% of CD8+ T-cells in C57BL/10 mice, but failed to elicit robust (0.5 0.1%) CD8+ T-cell activation in U.V. 0NLS-immunized MT mice (Fig. 7B). Measurements of CD11ahi / CD43hi CD8+ T-cell frequency in vaccine recipients provide no information about the CD4+ T-cell response, and do not prove that activated CD8+ T-cells are virus-specific. To address these limitations, an IFN- ELIspot assay was developed to compare HSV-2-specific T-cell responses in mice immunized with mock, U.V. 0NLS, or live 0NLS (Fig. 7C). Spleen WBC preparations were tested for their capacity to respond to three matched pairs of virus-specific versus irrelevant stimulator treatments; namely, 1. a dominant gB-2 CD8+ T-cell peptide (SSIEFARL) versus an ovalbumin (SIINFEKL) control (75); 2. HSV-2-infected JAWS II (H-2b) cells versus uninfected JAWS II cells (6, 45); or 3. direct stimulation of spleen WBCs with live HSV-2 virus versus DMSO (i.e., CD11c+ dendritic cells are HSV-2 infectable professional APCs). In C57BL/10 and MT mice, all three ELIspot stimulator pairs yielded parallel trends, but the third method was

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Fig. 6. HSV-2 0NLS vaccine-induced protection in wild-type and B-cell deficient MT mice. C57BL/10 and MT mice received right and left rear footpad immunizations of medium (mock) or 106 pfu HSV-2 0NLS on Days 0 and 30, respectively (n=8 per group). On Day 60, all mice were vaginally challenged with 500,000 pfu HSV-2 MS. A. Mean sem pfu of HSV-2 recovered in vaginal swabs at times post-challenge. B. Disease scores observed in mice on Day 8 where '0' = no disease, '4' = death, and 1-3 represent intermediate disease. C. Survival frequency of mice as a function of time post-challenge.



particularly exciting because it was not haplotype-specific, and might in principle be adapted to monitor T-cell responses to a HSV-2 vaccine in a human clinical trial. Results with this latter method are considered below. Mice in each immunization group (n=4 per) were sacrificed on Day 7 post-boost, and spleen WBCs were harvested. Triplicate samples of 105 WBCs per mouse were treated with 0.2% DMSO or 2x105 pfu HSV-2 MS. Spleen WBCs from mock-immunized C57BL/10 or MT mice did not produce IFN- in response to HSV-2 or 0.2% DMSO (Fig. 7C). Likewise, spleen WBCs of U.V. 0NLS-immunized C57BL/10 or MT mice failed to produce IFN- in response to HSV-2 or 0.2% DMSO (Fig. 7C). Spleen WBCs obtained from live HSV-2 0NLS-immunized C57BL/10 and MT mice exhibited a higher background of IFN- spot-forming cells in response to 0.2% DMSO (at Day 7 post-boost), but HSV-2 stimulation elicited a 7-fold increase over this background (150 70 and 660 160, respectively; Fig. 7C). Unlike MHC class I tetramers and peptides (43, 50, 75), this method of measuring "total T-cell response to HSV-2" is not haplotype-specific. Collectively, the data suggest that MT mice mount a T-cell response to the live HSV-2 0NLS vaccine that meets or exceeds the T-cell response of HSV-2 0NLS-immunized C57BL/10 mice (Fig. 7B, 7C). Thus, we postulate that lack of virus-specific antibodies likely accounts for defective HSV-2 0NLS vaccine-induced protection in MT mice (Fig. 6).

Expt 1-1 Does adoptive transfer of HSV-2 0NLS antiserum restore complete protection in HSV-2 0NLS-immunized MT mice?

Rationale: If antibodies play a critical role in protective immunity to HSV-2, then the T-cell-epitope Agenus HerpV vaccine (in human trials) may not elicit complete protection against genital herpes (1, 2, 67). The data above suggest HSV-2-specific T-cells alone are insufficient to protect against HSV-2 (Fig. 6, 7). To test this important hypothesis, Expt 1-1 will determine if the partial protection observed in HSV-2 0NLS-immunized MT mice may be restored to full protection by the adoptive transfer of serum antibodies from HSV-2 0NLS-immunized C57BL/10 mice.

Design: Four Control Groups of C57BL/10 mice will be immunized with 1. mock, 2. gD-2 vaccine (36, 38), 3. U.V.-inactivated HSV-2 0NLS, or 4. live HSV-2 0NLS (Table 2). Immunized C57BL/10 mice will serve as controls that empirically define "no," "partial," or "complete" vaccine-induced protection against HSV-2; and will serve as a source of C57BL/10 serum antibodies to be adoptively transferred to HSV-2 0NLS-immunized MT mice (Groups 7-10, Table 2). Control Group 6 of HSV-2 0NLS-immunized MT mice will receive no serum antibodies, and thus define the level of protective immunity to HSV-2 conferred by virus-specific T-cells alone. Group 6 and all Experimental Groups will be derived from a single cohort of age- & sex-matched MT mice that are immunized and boosted with HSV-2 0NLS at the same time. Hence, the variable under study will be the

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Fig. 7. New measures of T-cell response to HSV-2 vaccines. Mice were immunized on Days 0 and 30 with culture medium (mock), U.V-treated 0NLS, or 106 pfu live HSV-2 0NLS A. Peripheral WBCs were collected Day 6 post-boost, and analyzed by 4-color flow cytometry for Thy1.2, CD8, CD11a and an activation-assoc'd glycoform of CD43. B. Mean sem frequency of CD11ahi, CD43hi CD8+ T-cells in peripheral WBCs of C57BL/10 and MT mice (n=8/group). C. Mean sem frequency of IFN-+ cells per 106 spleen WBCs harvested Day 7 post-boost, as measured 48 h after stimulation with DMSO or 2 pfu/cell of wild-type HSV-2 (n=4 per group).



C57BL/10 serum that is adoptively transferred to 0NLS-immunized MT mice at the time of HSV-2 vaginal challenge (Table 2; Groups 7-10). Expt 1-1 will determine if the partial, T-cell-mediated-resistance observed in HSV-2 0NLS-immunized MT mice (Fig. 6) may be raised to complete protection by transfer of pooled serum from C57BL/10 mice immunized with 1. mock-, 2. gD-2-, 3. U.V.-0NLS-, or 4. live HSV-2 0NLS.

Experimental timeline: Age- and sex-matched C57BL/10 mice and/or MT mice will be immunized in their rear, right footpads on Day 0 with the indicated immunogens at 5- to 6-weeks of age (Table 2, Fig. 8). On Day 30, these mice will receive an equivalent immunization in their left, rear footpads. On Day 36, blood will be collected from mice for analysis of CD11a and CD43 activation markers on peripheral blood CD8+ T-cells (Fig. 7A, 7B). On Day 60, blood will be collected to measure pan-HSV-2 IgG levels in C57BL/10 mice (Fig. 5). On Days 88 and 92, Groups 7 - 10 will receive i.p. injections of 0.25 ml pooled serum from each C57BL/10 immunization group (Fig. 8). On Day 90 mice will be vaginally challenged with 5x105 pfu wild-type HSV-2. Between Days 90 and 120, mice will be monitored for HSV-2 vaginal shedding, pathogenesis and survival (Fig. 6A, B, and C).

Anticipated Results: gD-2-immunized animals will exhibit negligible reductions in HSV-2 shedding, and >75% will succumb to HSV-2 vaginal challenge (Table 1, Ref. 36, 38). U.V. 0NLS-immunized C57BL/10 mice will exhibit high levels of HSV-2 shedding for the first 48 hours, most will develop overt perivaginal disease, but delayed immune control will allow >90% of UV-0NLS-immunized C57BL/10 mice to survive HSV-2 challenge (data not shown). In contrast, C57BL/10 mice immunized with live HSV-2 0NLS will enjoy complete protection from HSV-2 challenge (Fig. 6). Regarding Experimental Groups 7-10 (Table 2), it is anticipated the T-cell-only-mediated resistance of HSV-2 0NLS-immunized MT mice will be fully complemented by transfer of HSV-2 0NLS antiserum (Group 10) but not by naïve serum (Group 7). It is anticipated that transfer of gD-2-antiserum or serum from UV-0NLS-immunized mice will only partially complement the defect in 0NLS-induced protection against HSV-2 in MT mice, and the degree of adoptively transferred protection will vary in proportion to the level of pan-HSV-2 IgG antibody present in the transferred serum (Fig. 5). The significance of differences between all groups of mice will be assessed by one-way ANOVA and Tukey's post hoc t-test.

Additional Experiments: Other experiments will be performed as part of Expt 1-1, but are not discussed in detail for lack of space. One set of experiments will revolve around the extent to which loss of virus-specific antibodies impairs vaccine-induced protection against wild-type HSV-2 spread to the peripheral nervous system and internal organs. These experiments will be equivalent to that presented above, but mice will be (1) sacrificed between Days 3 and 10 post-challenge to track infectious HSV-2 spread to internal organs, or (2) sacrificed on Day 30 for PCR analysis of latent HSV-2 DNA loads in dorsal root ganglia. A second set of experiments will focus on the IFN- ELIspot assay presented in Fig. 7C as a haplotype-independent method of measuring the "total HSV-2-specific T-cell response." Thus, a subset of mice in each immunization group will be sacrificed at Day 37 (Day 7 post-boost) to harvest spleen WBCs for IFN- ELIspots. Over the course of these experiments, we will determine if the frequency of T-cells stimulated to produce IFN- in response to HSV-2 (as opposed to irrelevant vaccinia-virus stimulation) correlates with protective immunity to HSV-2.

Expt 1-2 Do some subpopulations of HSV-2-specific antibodies, but not others, complement the defect in protective immunity observed in HSV-2 0NLS-immunized MT mice?

Preliminary Data: Using Western blot and immunoprecipitation-mass spectrometry (IP-mass spec), the P.I.'s laboratory has completed an unbiased analysis of the antibody response to the HSV-2 0NLS vaccine. The results of a forthcoming manuscript indicate that: 1. the live HSV-2 0NLS vaccine elicits a serum IgG antibody response against 9 to 19 viral proteins; 2. less than 20% of antibodies in HSV-2 0NLS antiserum are directed against HSV-2's 13 glycoproteins; and 3. 50 10% of HSV-2 antibodies in 0NLS antiserum are directed against two dominant antigens; infected cell proteins RR1 and ICP8.

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Rationale: HSV-2 0NLS antiserum confers significant protection against HSV-2 challenge (Fig. 5 in Ref. 36; Fig. 9 below). Thus, we anticipate live HSV-2 0NLS antiserum will strongly complement the partial protection conferred by virus-specific T-cells alone in HSV-2 0NLS-immunized MT mice (Expt 1-1). If this prediction is confirmed, then the P.I.'s laboratory will have a new model for evaluating which of many sub-species of virus-specific antibodies in 0NLS antiserum contribute to protective immunity to HSV-2. Expt 1-2 will use this new model to compare the relative importance of HSV-2 glycoprotein-B- and -D-specific antibodies as compared to the more dominant RR1- and ICP8-specific antibodies found in HSV-2 0NLS antiserum.

Design: The experimental model and measures to be collected in Expt 1-2 will be similar to Expt 1-1 (Fig. 8). However, the variable under study will be the population of virus-specific antibodies available to complement T-cell-only-mediated resistance to HSV-2 in 0NLS-immunized MT mice (Table 3). Pre-adsorption will be used to subtract antibody subsets from pooled HSV-2 0NLS antiserum using (1) fixed and permeabilized cells expressing HSV-2 antigens (Groups 4, 5, 7 & 8, Table 3), or (2) HSV-2 virion-loaded Sepharose 4A agarose (Group 6, Table 3). Mock-depleted serum will be pre-adsorbed to 6 x 107 fixed uninfected Vero cells per ml 0NLS antiserum (Group 4), whereas total HSV-2-specific antibodies will be depleted by pre-adsorbing each ml of 0NLS antiserum against 6 x 107 fixed HSV-2 infected Vero cells (Group 5). These methods are established in the P.I.'s lab, and the efficiency of depletion may be demonstrated by testing for loss of antibodies against HSV-2 Western blots and/or flow cytometry against antigen-expressing cells (36). Likewise, HSV-2 virions will be sucrose-gradient purified and coupled to CNBR-activated Sepharose4A, and used to selectively deplete antibodies against all HSV-2 glycoproteins and virion proteins, which will leave only antibodies against infected cell proteins such as RR1 and ICP8 (Group 6, Table 3). Finally, pooled 0NLS antiserum will be depleted of glycoprotein B and D-specific antibodies (Group 7) or RR1- and ICP8-specific antibodies (Group 8) by pre-adsorption to 6 x 107 fixed cells that stably overexpress gB-2 and gD-2, or RR1 and ICP8 (i.e., these cell lines have been constructed).

Experiments will be equivalent in design to Expt 1-1 (Fig. 8), but on Days 88 and 92, HSV-2 0NLS-immunized MT mice will receive i.p. injections of pre-absorbed 0NLS antiserum that has been depleted of 4. irrelevant Abs (mock), 5. total HSV-2 Abs, 6. HSV-2-virion-specific Abs, 7. gB- and gD-specific Abs, or 8. RR1- and ICP8-specific Abs (Table 3). On Day 90, all mice will be vaginally challenged with 5x105 pfu wild-type HSV-2. Between Days 90 and 120, mice will be monitored for HSV-2 vaginal shedding, pathogenesis, and survival (Fig. 6). This experiment will, for the first time, test the tacit assumption in HSV-2 vaccine science that gB-2 and gD-2 specific antibodies are the critical mediators of humoral immunity to HSV-2 (5, 59, 76).

Anticipated Results: It is anticipated that depletion of gB- and gD-specific antibodies (Group 7) will not significantly impair 0NLS antiserum's capacity to restore complete vaccine-induced protection to HSV-2 0NLS-immunized MT mice (i.e., as statistically compared to Group 4-MT mice that receive mock-depleted 0NLS antiserum). Depletion of RR1- and ICP8-specific antibodies may modestly reduce the protective effect of 0NLS antiserum because ~50% of antibodies are directed against these HSV-2 antigens (Group 8). However, 0NLS antiserum contains antibodies against up to 19 different viral proteins (not shown). Thus, we hypothesize that depletion of a subset of virus-specific antibodies from this polyclonal population is unlikely to negate the protective effect of HSV-2 0NLS antiserum shown in Fig. 9. Hence, only depletion of total HSV-2-specific antibodies in Group 5 is expected to completely ablate 0NLS antiserum's contribution to protective immunity to HSV-2. Likewise, it is anticipated that depletion of total HSV-2-virion-specific antibodies (Group 6) will produce a significant, but intermediate, effect simply because antibodies against ~10 different HSV-2 virion proteins and glycoproteins will be depleted (Table 3).



Specific Aim 2. To test a hypothesis that complete vaccine-induced protection against HSV-2 requires the cooperative activities of antibodies and T-cells.

Hypothesis: IgG molecules are one-billionth the size of T-cells, and thus may be pre-positioned in lymphatics and poised to immediately engage a viral infection. In contrast, HSV-2-specific T-cells must be recruited from the vasculature to sites of HSV-2 infection, which delays their involvement in the immune response (57, 72). Hence, virus-specific antibodies may be a central effector of adaptive immunity to HSV-2 during the first 48 hours post-infection (Fig. 6A). Moreover, virus-specific antibodies may recruit T-cells to sites of HSV-2 infection by activating the complement cascade. This hypothesis may explain why HSV-2 encodes a C3b receptor, glycoprotein C, that antagonizes complement activation (27, 56). Based on such considerations, complete vaccine-induced protection against HSV-2 may require a balanced B- and T-cell response to HSV-2's antigens.

Pilot Test 3: Transfer of 0NLS-vaccine-induced protection to naïve mice. Experiments were conducted to determine if complete HSV-2 0NLS-induced immunity could be transferred to naïve mice. Donor C57BL/10 mice (n=12 per group) were immunized on Days 0 and 30 with i. medium (naïve) or ii. 106 pfu HSV-2 0NLS. On Day 60, naïve and 0NLS-immunized mice were sacrificed to harvest serum and spleen WBCs. Fifty million spleen WBCs isolated from naïve or 0NLS-immunized donors were transferred to naïve mice by i.p. injection. After allowing one week for engraftment, recipients were treated with 0.25 ml naïve serum or 0NLS antiserum (n=6 per group), and were challenged with 105 pfu/eye HSV-2-luciferase. The expression of the luciferase reporter in challenged mice was compared on Days 4 and 6 post-challenge (Fig. 9). On Day 4, no difference was observed in luciferase expression in mice that received an adoptive transfer of 50 million naïve or 0NLS-immune WBCs (left vs right panels in Fig. 9A). In contrast, luciferase expression was consistently restricted in mice that received 0.25 ml 0NLS antiserum relative to mice that received naïve serum (top vs bottom panels in Fig. 9A). By Day 6, 0NLS antiserum or 0NLS-immune WBCs was sufficient to restrict HSV-2 MS luciferase expression; hence, only mice that received naïve serum and naïve WBCs still expressed luciferase on Day 6 (lower left of Fig. 9B).

A similar pattern of protection was observed in the duration of survival of these mice (Fig. 9C). Mice that received naïve serum and naïve WBCs succumbed by 8 1 days post-challenge (Fig. 9C). Adoptive transfer of 0NLS antiserum alone extended the duration of survival of mice to 23 5 days, and 66% survived HSV-2-luciferase challenge (Fig. 9C). Adoptive transfer of 0NLS-immune WBCs alone extended the duration of survival of mice to 19 5 days and 50% survived HSV-2-luciferase challenge (Fig. 9C). Intriguingly, mice that received 0NLS antiserum and 0NLS immune WBCs consistently survived HSV-2-luciferase challenge, and consistently exhibited little to no periocular disease (Fig. 9C). This adoptive transfer experiment was performed before the P.I.'s Sabbatical, and may be improved on many levels per superior & established methods in Dr. Hasenkrug's laboratory. Despite these technical deficiencies, these preliminary data suggest that the cooperative activities of virus-specific antibodies and virus-specific T-cells are required for complete vaccine-induced protection against HSV-2.

Fig. 9. Adoptive transfer of HSV-2 0NLS-induced immunity. Naïve recipient C57BL/10 mice (n=6/group) received an adoptive transfer of i. naïve serum (-) and naïve WBCs; ii. 0NLS antiserum (+) and naïve WBCs; iii. naïve serum (-) and 0NLS-immune WBCs; iv. 0NLS antiserum (+) and 0NLS-immune WBCs. Mice were challenged with 105 pfu per eye of HSV-2-MS-luciferase. (A, B) Luciferase expression in mice on (A) Day 4 & (B) Day 6 post-challenge. (C) Duration and frequency of survival of challenged mice; all surviving mice were sacrificed on Day 30.



Expt 2-1 What is the relative importance of virus-specific antibodies, CD4+ T-cells, and CD8+ T-cells in complete vaccine-induced protection against HSV-2 genital herpes?

Rationale: A narrative has emerged in the literature that HSV-2 vaccines based solely upon T-cell epitopes may be an efficient means to elicit protective immunity against HSV-2 (15, 16, 21, 22, 49, 82). However, this supposition is at odds with the HSV-1 immunology literature as a whole, and the HSV-2-based preliminary data presented herein (Fig. 6, 8). Experiments are proposed to empirically test two hypotheses: 1. CD4+ and CD8+ T-cells play redundant roles in protection against HSV-2 (as is true with HSV-1; Ref 29, 31, 32, 58), and 2. optimal T-cell mediated control of HSV-2 requires the presence of HSV-2-specific antibodies (Fig. 6, 8).

Design: In Expt 2-1, complete vaccine-induced protection against HSV-2 will be defined by HSV-2 0NLS-immunized C57BL/10 mice, which possess virus-specific Abs, CD4+ T-, and CD8+ T-cells (Table 4, Group 2). Group 2 and all Experimental Groups of C57BL/10 mice (Groups 5-7) will be derived from a single cohort of age- & sex-matched mice that will be immunized and boosted with HSV-2 0NLS at the same time. Hence, the primary variable under study will be the effect of depleting 5. CD4+ T-cells, 6. CD8+ T-cells, or 7. CD4+ and CD8+ T-cells just prior to HSV-2 vaginal challenge of HSV-2 0NLS-immunized C57BL/10 mice. Likewise, HSV-2 0NLS-immunized MT mice will define the level of protection conferred by virus-specific CD4+ and CD8+ T-cells alone (Group 4), and Experimental Groups 8-10 will be immunized with HSV-2 0NLS at the same time (Table 4). Thus, in 0NLS-immunized MT mice, the variable under study will be the effect of depleting 8. CD4+ T-cells, 9. CD8+ T-cells, or 10. CD4+ and CD8+ T-cells in B-cell-deficient mice that lack HSV-2-specific antibodies. Expt 2-1 will be equivalent in design to prior HSV-2 vaginal challenges with the relevant changes of T-cell depletion noted in adjacent Fig. 10. Specifically, on Days 85, 87 and 89, Groups 5 to 10 (Table 4) will receive i.p. injections of 0.25 ml containing 200 g mAb 191.1 (-CD4) and/or 200 g mAb 169.4 (-CD8). On Day 90, all mice will be vaginally challenged with 5x105 pfu wild-type HSV-2 and blood will be collected at this time to verify the efficiency of CD4 and/or CD8 T-cell depletion in each group (Fig. 7A). This protocol and the reagents are established in Dr. Hasenkrug's lab (60), and the P.I.'s pilot tests verify that >95% depletion is achieved (not shown). Between Days 90 and 120, mice will be monitored for HSV-2 vaginal shedding, pathogenesis, and survival (Fig. 6A, 6B, 6C). Comparison of the progression of HSV-2 vaginal challenge in these ten groups of mice (Table 4) will provide a body of evidence that clarifies the respective contributions of virus-specific antibodies, CD4+ T-cells, and CD8+ T-cells in vaccine-induced protection to HSV-2.

Anticipated Results & Interpretation: In HSV-2 0NLS-immunized C57BL/10 mice, depletion of CD4+ T-cells or CD8+ T-cells is expected to have a negligible effect on protection against HSV-2 vaginal challenge (Groups 5 & 6, Table 4), as these mice will retain a combination of virus-specific antibodies and at least one subset of effector T-cells. It is known in the HSV-1 immunology literature that CD4+ and CD8+ T-cells both serve as effectors of antiviral immunity (29, 31, 32, 58). In contrast, in HSV-2 0NLS-immunized MT mice, depletion of CD4+ or CD8+ T-cells is expected to significantly compromise protection against HSV-2 vaginal challenge (Groups 8 & 9, Table 4), as each T-cell subset is likely critical in animals devoid of antibody-mediated humoral immunity. Combined depletion of CD4+ and CD8+ T-cells will severely compromise protective immunity in both C57BL/10 and MT mice, but it is anticipated that depletion of CD4+ and CD8+ T-cells will render 0NLS-immunized MT mice (Group 10) as vulnerable to HSV-2 challenge as naïve MT mice (Group 3).

Fig. 10. Timeline of Experiment 2-1.

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Expt 2-2 Following an HSV-2 vaginal challenge, do virus-specific antibodies enhance T-cell recruitment to sites of HSV-2 replication in the vagina and/or dorsal root ganglia?

Rationale: Adaptive immunity to viruses is often described as a result of (1) antibody-mediated neutralization of virions and (2) CTL-mediated lysis of virus-infected cells. In contrast, virus-specific antibodies and T-cells are rarely described as serving complementary roles in a single, cooperative process that mediates antiviral immunity. Yet, the data (Fig. 6, 8) suggest that virus-specific T-cells are unable to effectively control HSV-2 infection in the absence of virus-specific antibodies. One hypothesis that may explain why this is the case is that virus-specific antibodies may play a critical role in guiding T-cells to sites of HSV-2 replication. Virus-specific T-cells cannot prevent HSV-2 replication until after they have been recruited out of the vasculature into virus-infected tissues. In naïve mice that receive pure populations of HSV-specific T-cells, artificial pro-inflammatory stimuli enhance T-cell recruitment to sites of HSV replication (57, 72). It is proposed that, in nature, virus-specific antibodies may fulfill a similar function, and recruit virus-specific T-cells to sites of HSV-2 replication. Expt 2-2 will test this important hypothesis, which may explain why a balanced B- and T-cell response is required for an effective HSV-2 vaccine.

Design: In Expt 2-2, naïve MT mice that receive adoptive transfers of antibodies and CFSE-labeled T-cells will be used to test a hypothesis that virus-specific antibodies enhance the kinetics of T-cell recruitment to sites of HSV-2 infection (Groups 4 - 7, Table 5). For these analyses, a novel Tomato+ (red)-HSV-2 virus will be constructed, similar to the "HSV-1TOM" virus made by the P.I.'s lab (4). Ex vivo imaging will be used to observe CFSE+ T-cells infiltrating sites of Tomato+ (red) HSV-2-infected cells in dissected vagina and DRG (e.g., Fig. 4 of Ref. 4). After imaging, tissues will be dissociated and flow cytometry will be used to enumerate numbers of CFSE+ Thy1.2+ CD4+ and CD8+ cells, as well as Tomato+ HSV-2 Ag+ cells at times post-challenge. Control Groups in Expt 2-2 will define the extent of HSV-2-Tomato replication and spread at times of tissue harvest in animals that possess "no," "partial," or "complete" vaccine-induced protection against HSV-2. Mock and HSV-2 0NLS-immunized mice will serve as donors of naïve serum and HSV-2 0NLS antiserum, respectively, which will be transferred to naïve MT mice by i.p. injection one day prior to HSV-2 vaginal challenge (Groups 4 - 7, Table 5). Likewise, mock and HSV-2 0NLS-immunized mice will serve as donors of naïve and immune spleen WBCs, which will be labeled with 5 M CFSE before B-cells and other WBCs are depleted using Miltenyi MACS columns. CFSE+ T-cells (>93% purity) will be adoptively transferred to naïve MT mice by i.v. injection (Groups 4 - 7). One day after adoptive transfers, all mice (Table 5) will be vaginally challenged with 5 x 105 pfu HSV-2-Tomato. Mice will be monitored for HSV-2 vaginal shedding up to the time of sacrifice; on Days 1 to 6 days post-challenge vaginas and DRG will he harvested for (1) confocal microscopic analysis of the spatial relationship between CFSE+ T-cells and Tomato+ HSV-2-infected cells and (2) flow cytometric analysis of CFSE+ T-cells infiltrating these HSV-2-infected tissues.

Anticipated Results: It is anticipated that Groups 5 and 7, which receive 0NLS-immune antiserum, will exhibit elevated numbers of CFSE+ T-cells in HSV-2-infected vaginas at 48 hours post-challenge relative to Groups 4 and 6 which lack HSV-2-specific antibodies; the significance of such differences will be evaluated by one-way ANOVA and Tukey's post-hoc t-test. In ex vivo microscopic analyses, it is anticipated that Group 7 will be unique in possessing large numbers of CFSE+ CD8+ T-cells directly apposed to Tomato+ HSV-2 cells. This prediction is based on the fact that TCR binding of MHC I-peptide complexes triggers T-cells to cease migration in virus-infected tissues, hence causing virus-specific T-cells to accumulate at cells expressing their cognate ligand (4). If virus-specific antibodies enhance T-cell recruitment to sites of HSV-2-Tomato replication, then downstream experiments will determine if this effect is C3 (complement cascade)-dependent.



Specific Aim 3. To test a hypothesis that the live HSV-2 ICP0- mutant vaccine, HSV-2 0NLS, is unable to establish a reactivate-able infection in vaccine recipients.

Rationale: Completion of Specific Aims 1 and 2 is likely to demonstrate that optimal vaccine-induced protection against HSV-2 requires a balanced B- and T-cell response against many HSV-2 antigens. If this outcome is observed, then it will be relevant to consider the possibility that the live-attenuated HSV-2 0NLS vaccine may represent an ideal means to safely elicit such robust protection against HSV-2 in the human population, and stop the spread of HSV-2 genital herpes. Expt 3-1 will test the safety-related predictions that HSV-2 0NLS (1) only establishes a self-limited infection in recipients, and (2) is incapable of later reactivation and/or spread.

Published data suggests HSV-2 0NLS replicates briefly, but fails to sustain replication and spread beyond the first 72 h post-inoculation (Fig. 8 of Ref 39). Consequently, HSV-2 0NLS-inoculated animals remain asymptomatic for >4 months (36, 38). HSV-1 and HSV-2 ICP0- mutant viruses are avirulent because they are vulnerable to repression by the innate interferon response that animals mount within hours after a viral infection (39, 42). Aside from acute virulence, another concern is that HSV-2 0NLS may establish a latent infection in vaccine recipients that later reactivates to cause disease. This is unlikely as wild-type ICP0 is necessary, and sufficient, to trigger HSV-1 reactivation in latently infected neurons (37, 40). We hypothesize that, like the HSV-1 ICP0- viruses the P.I. studied for 4 years as a postdoctoral fellow (37, 40, 41), the live HSV-2 0NLS (ICP0- mutant) vaccine strain will be unable to establish a reactivate-able latent infection.

Expt 3-1 Does a cre-labeled HSV-2 0NLS virus spread to the peripheral nervous system of ROSA26 mice, and if so, what is its capacity to spread and/or reactivate from neuronal latency?

Note: Far more is planned for Specific Aim 3 than may be articulated in the space remaining in this application.

Mapping the extent of HSV-2 0NLS-cre spread in ROSA26 mice. Dr. Stacey Efstathiou’s laboratory has developed an elegant and sensitive system that may be applied to map the distribution of neurons in the nervous system that become latently infected with HSV-1 viruses that express cre recombinase (68) (Fig. 11). Specifically, cre-expressing HSV viruses leaves a trail of permanently marked -galactosidase (-gal)+ neurons in ROSA26 reporter mice that may be visualized at the time of sacrifice (68). The basis of selective -gal expression in HSV-infected neurons is that cells of ROSA26 mice carry a silent -gal (lacZ) gene that becomes permanently de-repressed when cre recombinase excises stuffer DNA that separates a promoter and lacZ coding sequence (74). This irreversible genetic change explains why neurons that survive HSV-1 infection permanently express -gal (Fig. 11). This system will be used to determine if DRG neurons are ever infected by the HSV-2 0NLS vaccine following inoculation of the vagina or rear footpads. Specifically, cre-expressing variants of HSV-2 0NLS or HSV-2 MS will be constructed, and ROSA26 reporter mice will be inoculated with HSV-2 0NLS-cre (ICP0 –) or HSV-2 MS-cre (ICP0 +) viruses in the vagina or rear footpads. On Days 3, 6, 10, 30, 60, and 120 p.i., vaginas and DRG will be harvested to visualize the complete historical record of HSV-2 0NLS-cre spread by -gal staining (Fig. 11; n=5 mice per virus per time point); the number of DRG neurons that become infected with HSV-2 0NLS-cre versus HSV-2 MS-cre will be a focal point of analysis. This methodology is >5-fold more sensitive in detecting HSV latently infected neurons relative to in situ hybridization for HSV latency-associated transcripts (68). Dr. Efstathiou has agreed to serve as a consultant to ensure that the methods are faithfully reproduced (see Letters of Support). It is anticipated that between Days 3 and 120 p.i., the number of DRG neurons that become infected with HSV-2 0NLS-cre will be <1% than those infected by the ICP0+ virus, HSV-2 MS-cre.

Evaluating explant-induced reactivation of HSV-2 0NLS-cre in mice. The latency and reactivation potential of the HSV-2 0NLS vaccine strain will be evaluated in mice using conventional methods (37, 40). To this end, DRG will be harvested from ROSA26 mice inoculated 30 or 60 days earlier with HSV-2 0NLS-cre or HSV-2 MS-cre, and HSV-2 reactivation from explanted DRG will be compared.

Fig. 11. Cre-expressing HSV-1 viruses produce a permanent record of HSV latently infected -gal+ neurons (stained with X-Gal) in ROSA26 reporter mice, as shown on Day 147 p.i. Figure reproduced from Proenca, et al., 2008 (68) with permission of the corresponding author, Dr. Stacey Efstathiou.



Vertebrate Animals Mice to be used for experiments described in Aims 1 - 3 (n=1700 mice) will be housed in the Department of Laboratory Animal Medicine (DLAM) in the Southern Illinois University School of Medicine (SIUSOM) and all non-terminal procedures will be conducted therein. The proposed studies will be performed under currently approved SIUSOM IACUC protocols 205-13-010 and 205-13-011. The ~20,000 sq. ft. DLAM facility lies one floor below the P.I.’s laboratory, is AAALAC-accredited, and contains extensive containment facilities approved for BSL-2-level work, and is staffed by a full-time veterinarian, part-time veterinarian, and a vivarium manager who are all on call at all times. Two ~300 sq. ft. BSL-2 rooms in this facility are available to the Halford Lab, and may house up to ~800 mice at a time. The five questions about Vertebrate Animal Usage specified in NOT-OD-10-027 (March 2010) are addressed, as follows.

1. Description of proposed use of animals, including species, strains, ages, sex, number to be used:

Specific Aim 1. A total of n=300 female, wild-type C57BL/10 mice and n=300 female MT mice (6- to 8-weeks old) will be required for Experiments 1-1 and 1-2. Mice will be subcutaneously immunized twice in their right and left rear footpads on Days 0 and 30 (respectively) with the indicated immunogens. Blood will be collected from mice by retroorbital sinus bleeds using Natelson blood collecting tubes. Some experimental groups of mice will receive intraperitoneal injections of 0.25 ml mouse serum two days before and after HSV-2 challenge. HSV-2 vaginal challenge will be performed by the methods described by Halford, et al., 2011 (Ref. 38). Animals will be anaesthetized with ketamine-xylazine prior to immunization and vaginal inoculation. Following HSV-2 vaginal infection, mice will be monitored daily for moribund behavior and mice that develop symptoms of severe disease will be euthanized by CO2 inhalation.

Specific Aim 2. A total of n=300 female, wild-type C57BL/10 mice and n=400 female MT mice (6- to 8-weeks old) will be required for Expt 2-1 and 2-2. The procedures to be performed to live mice during the course of these experiments are already described in Specific Aim 1 with the exception that some groups of mice will receive i.v. injections of 30 million T-cells from naïve or HSV-2 0NLS immunized donor mice.

Specific Aim 3. A total of n=400 female ROSA26 mice (6- to 8-weeks old) will be used to assess the spread and reactivation efficiency (or lack thereof) of cre-expressing variants of HSV-2 0NLS and wild-type HSV-2 MS. The procedures to be performed to live mice during the course of these experiments are already described in Specific Aim 1.

2. Justification for the use of animals, choice of species, and numbers to be used: Mice are the only established model that allow investigation of both virological and immunological aspects of HSV-2 vaccine-challenge studies. The number of animals to be used is determined by the minimum number of replicates that is required to assure that statistical analysis will provide an objective measure of differences between treatment groups in each experiment.

3. Information on the veterinary care of the animals to be used: The animals to be used in all Specific Aims will be housed in the animal facility at the Southern Illinois University School of Medicine. Veterinarians and full time accredited animal care technicians are available 24 hours a day / 7 days a week to assist with the humane use of research animals. This facility is approved for animal research by the American Association for the Accreditation of Laboratory Animal Care.

4. Description of procedures for minimizing discomfort, distress, pain, and injury: Prior to inoculation with HSV-2, isotonic citrate buffer containing xylazine (7 mg/kg) and ketamine (100 mg/kg) will be administered i.p. to mice. The experiments proposed in this study are designed to limit the discomfort of animals. Animals will be monitored for signs of pain, discomfort or infection, and will be euthanized if a loss of mobility, weight loss, or the inability to take food or water is observed. In mouse experiments involving

Vertebrate Animals Page 40


wild-type HSV-2 MS, death is likely in unvaccinated control animals. Therefore, all mice in HSV-2 MS challenge experiments will be monitored daily between Days 1 and 30 post-challenge to ensure that any animals developing frank symptoms of disease may be euthanized at the earliest possible time at which signs of pain, ataxia, loss of water intake, or moribund behavior is noted.

5. Method of euthanasia and the reasons for its selection: The adult mice to be used in these studies will be euthanized by CO2 inhalation per the recommendations of the 2013 American Veterinary Medical Association Guidelines on Euthanasia (https://www.avma.org/KB/Policies/Documents/euthanasia.pdf).

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