1. Impact of topical ST-246® on poxvirus dermal infection, lesion severity/resolution and immune responses Principle investigator:Stacie Mosier Faculty sponsor: Dennis Hruby, PhD Program Director: Douglas Grosenbach, PhD Principle investigator:Stacie Mosier Faculty sponsor: Dennis Hruby, PhD Program Director: Douglas Grosenbach, PhD

2. Significance: Poxvirus disease is of medical significance. Currently, all troops deployed to the Middle East and Korea are required to obtain a smallpox vaccination. Outbreaks of other pox-diseases are erupting across the globe (e.g. monkeypox, cowpox, vaccinia). The US government and CDC consider smallpox a potential biological weapon. Approximately 25% of the US population is ineligible to receive the vaccine.

3. Who can NOT get the smallpox vaccine? People afflicted with atopic dermatitis or eczema. People with immune deficiency (HIV-infected, transplant recipients, genetic immune deficiencies) People receiving certain cancer therapeutics Pregnant or breast-feeding women Anyone residing with the above people! People afflicted with atopic dermatitis or eczema. People with immune deficiency (HIV-infected, transplant recipients, genetic immune deficiencies) People receiving certain cancer therapeutics Pregnant or breast-feeding women Anyone residing with the above people!

4. 90% of military personnel who obtain the smallpox vaccine will experience adverse events. 1000 out of a million will experience severe adverse events 50 out of a million will experience life- threatening adverse events. DOD, Smallpox Vaccination Program Lane JM, et al. NEJM, 1969. Lane JM, et al. J Infect Dis, 1970 Adverse events Associated with the current approved smallpox vaccine

5. Press release: MMWR Morbidity and Mortality Weekly Report May 19, 2009 Healthy military service member vaccinated Jan 13, 2009. Came into hospital 1 week later with fever; diagnosed with acute myelogenous leukemia (fast growing blood and bone marrow cancer). Battled progressive vaccinia until May 18, 2009. March 5, 2009 March 27, 2009 April 27, 2009 www.cdc.gov/mmwr/preview/ mmwrhtml/mm58e0519a1.htm

6. ST-246: Is being developed as an oral therapeutic for the treatment of smallpox. Low molecular weight, bioavailable Nontoxic Specific Shown to be efficacious in preventing systemic vaccinia and variola virus in multiple animals models (e.g. mouse, rabbit, NHP). Shown to be safe in clinical trials.

7. Systemic infection results from the formation and spread of the enveloped form of the virus, from the point of inoculation In the life-cycle, the virus is formed and released from the cell as Extra-cellular Enveloped Virus (EEV). EEV: Responsible for cell-to-cell transmission and spread throughout host. ST-246 prevents virus from obtaining envelope from host. EEV & IMV are introduced at the point of inoculation ST-246 STOP

8. Since systemic problems arise from the dissemination of the EEV at the point of inoculation: I hypothesize that a topical formulation of ST-246 applied to the inoculation site will reduce dermal infection, lesion severity and time to recovery.

9. Specific Aims: Topical Study #1 To quantitatively assess the immune responses elicited by treatment with topical formulation of ST-246 in an immune competent model. Topical Study #2 To evaluate the efficacy of the topical and/or oral treatment of ST- 246 in a model for immune deficiency

10. Procedures: Study #1 Step 1: SKH-1 mice anesthetized and vaccinated VV-WR (1x10 6 pfu/ml) Step 2: Mice treated 1x or 2x/ day with 1% topical solution ST-246 Step 3: Photograph inoculation site/ collect blood and lesion-swab samples to evaluate antibody response and viral load Step 4: At Day 30, terminate study and collect blood for immune response assays Step 5: Quantitatively assess immune response via flow cytometry, cytometric bead array, ELISA, neutralizing titer, comet-inhibition, etc.

11. Expected Outcomes: STUDY #1 ST-246 will prevent and/or reduce lesion severity. Decrease viral load. Decrease time to resolution. Immune response will not be inhibited.

12. Preliminary Data Study #1:

13. Procedures: Study #2 Step 1: Mice treated with cyclophosphamide (100mg/kg/4d) to induce immunodeficiency Step 2: Mice anesthetized and vaccinated VV-WR (1x10 6 pfu/ml) Step 3: Mice treated 1x or 2x/day with a topical 1% solution and/or oral ST-246 Step 4: Photograph inoculation site/ document lesion severity and time to resolution Step 5: Collect blood samples and organs/ verify immunodeficiency via ELISA/ measure viral load via qPCR

14. Expected Outcomes: STUDY #2 ST-246 will reduce viral load and slow viral shedding. Decrease lesion severity. Ultimately, survival in this model in unknown.

15. Preliminary Data Study #2:

16. VV-WR Tx Topical Vehicle BID Day 7

17. VV-WR Tx Topical Vehicle BID Day 11

18. VV-WR Tx Topical Vehicle BID Day 17

19. Cyclophosphamide/ VV-WR Tx Topical Vehicle BID Day 7

20. Cyclophosphamide/ VV-WR Tx Topical Vehicle BID Day 12

22. Cyclophosphamide/ VV-WR Tx Topical Vehicle BID Day 14

24. Cyclophosphamide/ VV-WR Tx Topical Vehicle BID Day 16

25. What’s the next step? Optimization of dosing and formulations. (e.g. slow-release; drug impregnated bandages) Efficacy in higher order animals. Pharmacology Toxicology Optimization of dosing and formulations. (e.g. slow-release; drug impregnated bandages) Efficacy in higher order animals. Pharmacology Toxicology

26. Acknowledgments: Howard Hughes Medical Institute Dr. Dennis Hruby Dr. Douglas Grosenbach Dr. Aklile Berhanu Mr. David King