1. Slide 1 LBERI Update on Animal Model Development Lovelace Respiratory Research Institute 2425 Ridgecrest Drive SE, Albuquerque, NM 87108 Sub-NIAID Tech Call 8 April 2008

2. Slide 2 Milestones #2ActiveVaccinations of study personnel #3Active - Optimization of bioaerosol methods #4Active - Confirmation of aerosol in vivo in NHP efficacy studies in primates #7Active - SCHU S4 LD50 in primates #12/13Active -Assays for detecting relevant immune responses in animals and humans #21Active- Correlates of protection- in vitro assay or other readout of effector function of Ft developed for multiple species

3. Slide 3 Milestone #2 – Vaccinations of Study Personnel  Vaccinations completed –32 LBERI scientists and staff received the LVS vaccination between 9/11/07 and 1/8/08  All have completed the 1 month post-vaccination follow-up –4 UNM scientists and staff received the LVS vaccination on 3/18/08  The 1 week post-vaccination follow-up was completed  The 1 month post-vaccination follow-up is pending –1 LBERI staff received the LVS vaccination on 4/1/2008 –Dr. Lyons received UNM IRB approval to allow blood draws on the vaccinated LBERI and UNM scientists after their LVS vaccinations. The LBERI and UNM scientists and staff will be offered the opportunity to volunteer to donate bloods for the development of immunoassays, approximately 2 months after receiving the LVS vaccination

4. Slide 4 Milestone #2 – Vaccinations of Personnel Plans for this month  4 UNM personnel awaiting 1 month follow-up  Continue follow-up with blood draws from vaccinated LBERI and UNM personnel for the development of immunoassays  1 UNM personnel may be vaccinated on 4/29/08  USAMRIID is temporarily closing the LVS vaccinations until a new protocol is in place (after 4/29)

5. Slide 5 MS#3 – Flow Diagram MS 3: Bioaerosol Development Red: completed Green: in progress Blue: steps in the milestone MicropumpAeromistCollison Nebulizer Order & receive instrument Set up instrument Order & receive instrument Set up instrument Order & receive instrument Set up instrument Lyophilized LVS Fresh LVS Frozen LVS Fresh LVS Frozen LVS Fresh LVS Fresh Schu4 Frozen Schu4 Down Select for SCHU S4 Generator Prepare bioaerosol SOP and write MS completion report

6. Slide 6 Milestone #3 – Bioaerosol Development Accomplishments  Completed Collison, Sparging Generator, Micropump and Aeromist LVS testing (fresh vs. frozen)  Tested additional technologies (e.g., ultrasonic, Aeroeclipse II, others) with BG spores; F. tularensis testing not pursued due to difficult setup, practicality and/or no significant differences vs. Collison  Completed frozen and fresh SCHU S4 testing with the Aeromist and Collison  Performed pathogenicity study of LVS and SCHU S4 bioaerosols in mice.

7. Slide 7 SCHU S4 Bioaerosol Data to Date  No bioaerosol optimization testing was performed in March 2008  LBERI is experiencing SCHU S4 growth issues in Chamberlains –Have pinpointed problem to Chamberlains broth as cultures grow as expected on BCGA and QC results are normal –Working in conjunction with UNM personnel to remedy problem  Requesting UNM-produced Chamberlains lot –Is it worthwhile to explore media optimization for SCHU S4? This was performed for LVS, but not for SCHU S4.

8. Slide 8 Milestone #3 – Bioaerosol Development Plans for this month  Continue MS Completion report  Determine problem(s) with SCHU S4 growth in Chamberlains as soon as possible

9. Slide 9 MS#4 – Flow Diagram MS 4: NHP Aerosol Confirmation Red: completed Green: in progress Blue: steps in the milestone Aerosol Challenge Approach Vaccinated NHP Challenges Vaccinated NHPs available; awaiting completion of naïve and LD50 challenges Naïve NHP Challenges Cohort 1 (n=2) Cohort 2 (n=2); to follow mouse challenges MS 3 Mouse Challenges Cohort 1 Cohort 2 Cohort 3 NOTE: Chamberlains growth problems have halted Cohort 3 mouse challenges two times

10. Slide 10 MS#4 Overview  Mouse studies –Objectives:  To verify that aerosolized SCHU S4 are virulent in mice (concern that aerosol method may alter virulence)  To determine SCHU S4 deposition as to related to the bioaerosol parameters; this will influence NHP exposure procedures  Naïve NHP studies –Objectives:  To verify that aerosolized SCHU S4 are virulent in NHP (similar concern as described for mice)  Vaccinated NHP studies –Purpose is to demonstrate protection in NHP previously vaccinated with LVS –This study will follow determination of LD50 aerosol dose

11. Slide 11 Milestone #4 – Confirmation of Aerosol in vivo in NHP  Pathology report completed (see following 2 slides) for 2 naïve primates infected with SCHU S4 in December 2007.

12. Slide 12 Pulmonary histopathology from cynomolgus macaques exposed to aerosols of F. tularensis SCHU S4. A, C, and E are from A04339, and B, D, and F are from A04344. Arrowheads (A, B) indicate primary foci of pyogranulomatous to necrotizing bronchopneumonia. Small arrows (C) indicate smaller foci of pyogranulmatous to necrotizing embolic pneumonia detected only in A04339. Large arrow (E) indicates a colony of small coccobacilli.

13. Slide 13 Histopathology of the tracheobronchial lymph node, spleen and lung from a cynomolgus macaque (A04339) exposed to aerosols of F. tularensis. Arrowheads in the lymph node (A) and spleen (B) indicate foci of pyogranulomatous to necrotizing inflammation. Small arrows indicate foci of nasal turbinate ulceration with fibrinosuppurative rhinitis in the nasal cavity (C).

14. Slide 14 Milestone #4 – Confirmation of Aerosol in vivo in NHP Plans for next month  Further investigate SCHU S4 deposition –BALB/c mice –Aeromist vs. Collison –48h Chamberlains broth  Growth problems encountered –High and low doses  1 x 10 7 and 10 8 CFU/mL in generator suspension; should result in countable colonies upon culture –n=5 per group followed by immediate Nx and lung culture on selective media  Complete Micro report  Schedule follow-on NHP SCHU S4 bioaerosol challenge (n=2); late April/early May

15. Slide 15 MS#7 – Flow Diagram MS 7: NHP SCHU S4 LD50/ED50 Red: completed Green: in progress Blue: steps in the milestone Round 1 (n=4 NHP each dose) x CFU (TBD) Round 2 (n=4 NHP each dose) y CFU (TBD) Round 3 (n=4 NHP each dose) x CFU (TBD) y CFU (TBD) LD50/ED50 Determination 5000 CFU Delivered (Based on virulence study) 500 CFU 50,000 CFU

16. Slide 16 MS#7 Overview  Objectives: –To determine the SCHU S4 LD 50 aerosol dose in NHP –To observe and document the natural history of disease through clinical observations and in-life/post-life sample (blood and tissue) analyses

17. Slide 17 MS#7 Tentative Endpoints  The endpoints for each set of exposures will be clinical observations, temperature monitoring, body weight records, gross necropsy, and viable bacterial blood/tissue cultures

18. Slide 18 Milestone #7 – Confirmation of Aerosol in vivo in NHP Plans for next month  Confirm LD50 dosing/challenge scheme  Confirm endpoints and schedule appropriate personnel  Confirm SCHU S4 bioaerosol method –Aeromist vs. Collison  Initiate ABSL-3 move-in and challenge dates –Attempt late May/early June for 1 st cohort

19. Slide 19 Milestone #12/13 – Immune Responses in Animals and Humans Immunoassay Development and Comparisons in Animal Models Red: completed Green: in progress Blue: steps in the milestone Choose PBMC Freezing Method Choose PBMC Purification Method Method chosen: Purdue ListServ Test whether method results in loss of B cells Cerus Proliferation assay: Works for Con A and LVS IFN  ELISPOT Develop Immunoassay methodologies Plasma IgG ELISA Phenotype Blood and PBMCs Plasma IgA ELISA IFN  Intracellular Staining

20. Slide 20 Update on responsiveness of non-LVS-vaccinated NHPs to LVS antigens ex vivo Issue:  Non-vaccinated NHPs often respond to FF LVS by secreting IFN  as detected by the ELISPOT assay?  We do not know how consistent these responses are because we rarely bleed the non-LVS vaccinated NHPs more than once.  It is important to determine whether the responses we observe are consistent so that we can be confident of their responses when we screen them before vaccination or use in other protocols.

21. Slide 21 An example of consistent responses: A04260 A04260 TUL 26 PBMCs plated at 1.33 x 10 6 /ml; TUL31 PBMCs plated at 1 x 10 6 /ml Tul 26 1/14/08 ; Tul 31 3/27/08 NT

22. Slide 22 An example of inconsistent responses: A04168 A04168 All plated at 1.33 x 106/ml Tul 21 11/26/07; Tul 23 12/4/07; Tul 31 3/27/08 NT

23. Slide 23 Proliferation is consistent even if IFNγ secretion is not A04168 NT Tul 21 11/26/07; Tul 23 12/4/07

24. Slide 24 A semi-consistent response: A05477 A05477 All cells plated at 1.33 x 106/ml; Is FF and HK LVS mixed up in TUL 21? Tul 21 11/26/07; Tul 23 12/4/07; Tul 31 3/27/08 NT

25. Slide 25 Proliferation is consistent: A05477 NT Tul 21 11/26/07; Tul 23 12/4/07

26. Slide 26 MS 12/13: Plans for the next month  Continue to test PBMCs frozen and thawed using the Cerus protocol  Continue to work on optimizing the IFNγ ELISPOT assay such that non-LVS vaccinated NHPs have low background responses and LVS-vaccinated NHPs have antigen-specific responses. –Test higher doses of HK LVS and lower doses of FF LVS –Test a range of doses of HK- and FF-SCHU S4 –Prescreen newly purchased NHPS for dose responsiveness to LVS and Schu 4 antigen preparations. –Test the same range of antigen doses on LVS-vaccinated NHPs to determine minimal antigen preparations dose that provides sensitive and specific response in IFNγ ELISPOT assay and proliferation assay.

27. Slide 27 MS 21 (LBERI/UNM) Correlates of protection: in vitro assay or other readout of effector function of Ft developed for multiple species  Work out the conditions to detect intracellular IFNγ production by flow cytometry in LVS-vaccinated NHPs –Order needed reagents (anti-CD28)  Moving the flow assay to Correlates of Protection MS 21 because this is not meant as a routine assay but will lead to a more mechanistic understanding

28. Slide 28 Action Items  Trevor: will try growing SCHU S4 on BCGA plates first and then innoculate into liquid Chamberlains (completed 4/21/08)  Kristin will provide the USARMIID growth conditions to UNM  Trevor will write the MS3 MSCR and associated SOPs  Bob share technicium particle aerosol study at next LBERI tech call or at the site visit on 4/24/08  Julie W- tell Barbara the number of NHP to be vaccinated on the natural history study  Vicki- try to obtain clinical LVS lot prior to next NHP vaccination at LBERI  Kristin: has USAMRIID histopathology photos that she could share with Julie Hutt, from NHP with tularemia showing riddling of white foci in spleen  Kristin can share the clinical signs detected at USAMRIID NHP infected with SCHU S4  Bob: will schedule NHP rooms longer and get IACUC amended for 35 days so NHP studies can go as long as the USAMRIID NHP studies.  Vicki/Freyja: NIAID should supply both SCHU S4 and LVS O antigen mutants to LBERI to test impact on background in Elispot assay with non vaccinated NHP. Will send to UNM.  Julie: Under MS21, will try to develop the triple positive functional T cell intra cellular flow assay in NHP