1. 1 LBERI Update on Animal Model Development Lovelace Respiratory Research Institute 2425 Ridgecrest Drive SE, Albuquerque, NM 87108 Sub-NIAID Tech Call 4 May 2010

2. 2 Active Milestones #2ActiveVaccinations of study personnel- no work in April #8ActiveLVS vaccination protection of aerosol Schu4 confirmed in primates #9ActiveAerosol SOP developed for GLP transition- no work in April #10ActiveEfficacy testing of vaccine candidates (LBERI)- no work in April #11ActiveIn Vivo GLP NHP model efficacy SOPs and efficacy testing of vaccine candidates- no work in April #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 #29 ActiveAnalysis of T cells from lymph nodes and T cell epitopes- no work in April

3. 3 MS#8 – LVS Vaccinated NHP Challenged with SCHU S4 L VS Vaccinated NHP Challenged with SCHU S4 Red: completed Green: in progress Blue: steps in the milestone Round 1 Vaccination Practice/Challenge (n=3 scarification; n=2 subcutaneous) Round 2 Vaccination/Challenge (n=3 by scarification; n=3 by subcutaneous route; n=4 previously vaccinated; 2 SC, 2 ID) SCHU S4 Challenge 500 CFU Round 3 Vaccination/Challenge (Vaccination with Highest Dose of LVS attainable by scarification and s.c.) SCHU S4 Challenge 1000 CFU Round 4 Vaccination/Challenge (Vaccination with Lot 16 n=3; Lot 17 n=3; Lot 20 n=8; Lot 4 n=8) SCHU S4 Challenge 1000 CFU Round 5 Vaccination/2 Broth Challenge (SC Vaccination with Lot 17, compare CB vs. CAMHB) SCHU S4 Challenge 1000 CFU Vaccination/Challenge Non-Telemetered Natural History Study SCHU S4 Challenge 1000 CFU Vaccination/Challenge Telemetered Natural History Study SCHU S4 Challenge 1000 CFU

4. 4 Milestone #8 - Objective and Endpoints Describe the natural history of aerosol delivered SCHU S4 infection in NHPs that have been previously vaccinated with LVS – Compare two different methods of vaccination (scarification and subcutaneous) – TUL08 A and B – Compare 4 different LVS lots as vaccines (all delivered by subcutaneous route) – TUL08 C – Compare SCHU S4 growth media to see if it has an effect on virulence (Chamberlain’s broth vs. Mueller-Hinton broth) – TUL08 D Endpoints – histopathology – bacterial CFUs of internal organs (lung, spleen, liver, kidneys, and lymph nodes) – records of clinical symptoms post-infection – clinical chemistry and hematology during infection

5. 5 Milestone #8 – April 2010 Accomplishments Vaccinated NHP for Tul08D – Vaccinated NHP over 2 days using Lot 17. In all cases save 1 NHP, vaccinations were given within 30 minutes of preparation. – Average post-vaccination titers were 8.75 x 10 8 and 2.10 x 10 8 CFU/mL for Days 1 and 2, respectively. – Pre and post titers on Day 2 differed by 2 logs, unlike day 1 where no drop was observed – NHP were re-randomized to prevent any issues associated with differing doses Performed practice bioaerosols (n=6) using SCHU S4 grown in LRRI- prepared supplemented Mueller-Hinton broth (MH) – Primary goal was to evaluate bioaerosol efficacy (spray factors) for SCHU S4 grown in MH and compare to historical data for SCHU S4 cultured in Chamberlain’s broth – Culture procedures mimicked those established for SCHU S4 grown in Chamberlain’s broth – At 24h, bacteria were harvested, normalized to OD 600 of 0.200 and diluted An OD 600 of 0.200 ± 0.01 was defined as 1.4 x 10 9 CFU/mL Three (3) generator suspension concentrations were targeted: – 1 x 10 6 CFU/mL – 5 x 10 6 CFU/mL – 1 x 10 7 CFU/mL [see data next slide]

6. 6 Milestone #8 – April 2010 Accomplishments Date F. tularensis Strain Growth Protocol NebulizerCFU/mL CFU/LCFU/3.5L Post- Spray Factor Brand Operating Pressure OutputTarget Pre- Actual Post- Actual 23-Apr-10SCHU S4 1. 48h BCGA 2. Normalized to OD600 of 0.100 3. Inoculate 100 mL of MH w/ 400  L 4. Incubate 24 at 37°C, 200 rpm, in the dark 5. Harvest and dilute (no centrifugation) Collison22.0 psig7.511 L/min1.00E+066.75E+055.10E+055.05E+011.77E+029.90E-08 Collison22.0 psig7.511 L/min1.00E+068.65E+054.75E+056.46E+012.26E+021.36E-07 Collison22.0 psig7.511 L/min5.00E+061.78E+061.48E+062.87E+021.00E+031.94E-07 Collison22.0 psig7.511 L/min5.00E+064.95E+061.10E+062.46E+028.61E+022.24E-07 Collison22.0 psig7.511 L/min1.00E+07 Suspected error 5.45E+062.10E+027.36E+023.86E-08 Collison22.0 psig7.511 L/min1.00E+075.30E+066.00E+064.54E+021.59E+037.56E-08

7. Milestone #8 – Data Interpretation Actual generator suspensions were lower than targets, though still within an acceptable range – OD600 estimate was inaccurate Actual concentration of normalized suspension was 7.57 x 10 7 CFU/mL (well below the estimated concentration of 1.4 x 10 9 CFU/mL) – Data indicate the need to further evaluate SCHU S4 growth in MH, particularly with respect to obtained concentrations at 24h CFU/L concentrations were optimal for Sprays 3-6 – Calculated CFU in 3.5L (target for nonhuman primate exposure) were close to 1,000 CFU for these four bioaerosol runs This is similar to what is observed with SCHU S4, particularly with respect to the starting generator suspension concentrations; essentially, we can preliminarily conclude that SCHU S4 grown in MH behaves similarly as a bioaerosol when compared to SCHU S4 cultured in Chamberlain’s broth 7

8. 8 Milestone #8 – Plans for next month The next set of practice bioaerosols are scheduled for early May 2010 – The objective is to mimic procedures for the actual nonhuman primate aerosol challenges and obtain enough replicates to demonstrate that 1,000 CFU presented can be achieved with SCHU S4 grown in MH – Prior to this, we are conducting additional MH cultures in order to better understand our starting concentration at 24h and obtain more titer data from OD 600 -normalized suspensions Challenges are set for May 11 and 12 for Tul08D

9. Milestone #8 – Plans MS8E and MS8F Tul08E – disease course in vaccinated and challenged NHP, with and without telemeters. – Points for discussion: Draft Study protocol consistency with annual meeting presentation Timelines – for study inception and serial sacrifices Telemetry issues Tul08F – Duration study – Some NHP at supplier are measles titer negative even after vaccination. Do we want these NHP in order to stick to our timeline, or would we rather wait and have measles titer positive animals. 9

10. 10 Milestone #12/13 – Immune Responses in Animals and Humans Immunoassay Development and Comparisons in Animal Models Red: completed Green: In progress Yellow: on hold; restart if necessary Blue: steps in the milestone Choose PBMC Freezing Method Choose PBMC Purification Method Method chosen: Purdue ListServ Cerus Proliferation assay IFN  ELISPOT Develop Immunoassay methodologies Plasma IgG ELISA Plasma IgA ELISA Determine protein:CFU relationship in FF and HK LVS antigens Microagglutination assay

11. 11 Milestone #12/13 - Objective and Endpoints Develop immunoassays that reliably distinguish LVS-vaccinated from non- vaccinated NHPs – Thus far can rely on IgG anti-LVS ELISA for this purpose Develop immunoassays that may distinguish NHPs which survive SCHU S4 aerosol challenge from those that do not – Thus far have not been able to refine our assays to function as such – IgG anti-LVS plasma levels do not correlate with protection – IFNγ production or proliferation by bulk PBMCs in response to LVS or SCHU S4 antigens also appear not to distinguish those NHPs which have been protected from SCHU S4-induced mortality but more data mining may be in order (i.e. only examining responsiveness early post- LVS vaccination or late; only looking at specific types of stimuli (FF vs. HK))

12. Milestone #12/13 – April 2010 Accomplishments 12 Attempt to create Bradford standard curves with bovine gamma globulin standard set, ready-to-use from Fermentas and sample/lysis buffer from Sjostedt lab, Umea, versus PBS as dilution buffer.

13. 13 Milestone #12/13 – April 2010 Accomplishments SubstanceLysis buffer from Sjostedt lab Compatibility chart for Bradford reagent (Fermentas), ready-to-use thiourea2 M1 M urea7 M5 M CHAPS4 %5% ASB-140.5%Not indicated or ASB- C7Bz0, 0.25% DTT1.0%10mM Beta-mercaptoethanol1%1 M Protease inhibitor0.5x

14. Milestone #12/13 – Data Interpretation Bradford assay will not be a good assay for determination of protein content in fixed F.t. preparations. LBERI is developing an ELISA method according to results at UNM and have ordered secondary antibodies to detect mouse  -fopA and  -dnaK binding to plate-bound F.t preparations. 14

15. 15 Milestone #12/13 – Plans for next month Run ELISA described in previous slide.

16. 16 MS #21 – Correlates of protection Establish assays of effector function that detect correlates of protection Establish conditions to detect intracellular cytokines in NHP PBMCs Confirm response in LVS-vaccinated NHPs Confirm low response in non- LVS-vaccinated NHPs Establish conditions to detect growth of LVS in NHP PBMCs Confirm reduced growth in LVS- vaccinated NHPs Confirm growth in non- LVS-vaccinated NHPs Red: completed Green: In progress Blue: steps in the milestone

17. 17 Milestone #21 - Objective and Endpoints Develop immunoassays of effector function that can detect correlates of protection from SCHU S4 aerosol-induced morbidity – Developing two assays for this purpose thus far: – 1) Intracellular cytokine staining; Perhaps IFNγ production by particular cell types will distinguish NHPs which are protected vs. those that are not Perhaps the presence of particular cells that are producing more than one cytokine (ex. IFNγ, TNFα and IL-2) will predict protection – 2) inhibition of in vitro growth of SCHU S4 by some cells contained in the PBMC preparations of protected NHPs vs. those that succumb to SCHU S4 aerosol

18. Milestone #21 – April 2010 Accomplishments – In vitro growth assay NHP PBMC + F.t. 18 For both NHPs tested in this experiment, LVS bacteria were kept at a steady-state in pre-vaccination PBMC over 72 h in vitro, whereas post vaccination PBMC eliminated bacteria over 72 h. The assay is now ready to move to B3 and be tested with NHP PBMC and SCHUS4.

19. Milestone #21-Data Interpretation For both NHPs tested in this experiment, LVS bacteria were kept at a steady-state in pre-vaccination PBMC over 72 h in vitro, whereas post vaccination PBMC eliminated bacteria over 72 h. 19

20. Milestone #21 – April 2010 Accomplishments: Intracellular staining of IFN-  in NHP PBMC 3 experiments: 20 h stimulation ffLVS/hkLVS/PHA FcR block:  -CD32 Surface stain: CD3,CD8,CD14,CD16, CD56 Intracellular stain: IFN-  All  freshly prepared PBMC IFN  RESPONSE NHP IDPost vacc. CD3+CD8- (CD4 T cells) CD3+CD8+CD14+CD3-CD16+CD56+ ffLVS CD56+ hkLVS 1) A09068d 7 + - --+ - 1) A09088d 7 ---- +? - 2) A09088d 14- + -- +? + 3) A09523d 14 + + + - + - Only ffLVS, no hkLVS response in T cells To both ff and hk 20

21. MS #21 – April 2010 Accomplishments: ICS IFN-  in NHP PBMC – A09068 and A09088 day 7 post vacc. 21 01.25x10 4 cfu/well5x10 4 2x10 5 A09068 ff LVS 0.6% 143 144 0.5 0.4 127 0.5 153 0.6 147 0.5 160 1.9 0.9 151 166

22. MS #21 – April 2010 Accomplishments: ICS IFN-  in NHP PBMC – A09088 day 14 post vacc. 22 01.25x10 4 cfu/well5x10 4 2x10 5 99 0.5 104 0.7 103 111 0.5 0.7 104 109 0.9 0.8 1.2 122 ff LVS A09088

23. MS #21 – April 2010 Accomplishments: ICS IFN-  in NHP PBMC – A09523 day 14 post vacc. 23 01.25x10 4 cfu/well5x10 4 2x10 5 A09523 ff LVS 0.6 104 106 0.7 1.6 1.2 128 1.3 142 1.2 163 1.6 136 135 1.2 1.5 ff LVS A09523 CD4+ cells CD8+ cells 71 0.5 75 0.31.7 76 1.2 91 1.3 114 133 1.3 105 1.4 105 108

24. MS #21 – April 2010 Accomplishments: ICS IFN-  in NHP PBMC – A09523 day 14 post vacc. 24

25. Milestone #21-Data Interpretation The production of IFN-  by NHP PBMCs appears to be quite variable and different individuals have different PBMC subpopulations that respond to F. tularensis antigen restimulation and respond at different time points post vaccination and to which antigen preparations, hk or ff. Generally, T cells do not appear to respond to hk LVS, only ffLVS at the time points tested. 25

26. Move in vitro growth assay into LBERI B3 facility and start using SCHUS4. Attempt to test more time points post vaccination of IFN-  ICS from the same NHP individuals with freshly prepared cells. If enough cells, test CD4 staining to confirm that CD3+CD8- cells are indeed CD4 T cells. 26 Milestone #21 - Plans for next month

27. 27 Additional Points Deliverables completed for each active milestone: No deliverables were completed during the month of April List of relevant publications from the past month None MSCR status MS 3: NIAID reviewing MSCR (UNM sent to NIAID on 2/18/2010 ) MS 4: NIAID reviewing MSCR (UNM sent to NIAID on 3/23/2010 ) MS 7: NIAID reviewing MSCR (UNM sent to NIAID on 4/7/2010 ) MS 11: NIAID reviewing MSCR (UNM sent to NIAID on 3/24/2010 ) MS 8 a: LBERI writing and due to UNM 5/14/10 MS 8 b: LBERI writing and due to UNM 6/30/10 MS 8 c: LBERI writing and due to UNM 5/4/10

28. Action Items (1 of 3) Trevor: will report on the new SCHU S4 MH growth and aerosol data from the week of 5/4 as soon as available. (DONE 5/7/10) Trevor will provide the CFU data including plate counts for the SCHU S4 growth pre and post aerosol (DONE 5/7/10) Trevor will report on the flow cytometer data for measuring the starting bacterial concentrations, as they set up this instrument NIAID wants an LBERI LD 50 study with more NHP numbers and with lower aerosol doses (5 CFU and 25 CFU) of SCHU S4. NIAID wants the MS 11: natural history data separated into two reports, for the telemetry and the non telemetry studies. NIAID wants all the data related to the serial sacrifices. 28

29. Action Items (2 of 3) NIAID wants more than 2 weeks to review and approve a specific study protocol. After the study protocol is approved, then UNM can order NHP. NIAID does not want NHP ordered before the study protocols are formally approved, with all details of the study protocol. NIAID wants the duration of the experiments, with the date that the study will start ( dates for vaccination, implants, challenge and then last sacrifice). NIAID wants to know exact length and complexity of the study. NIAID does not want to order the study MS8E. Kristy will look for the impact of lead placement in the wave forms during reanalysis of the respiratory, heart rate and temperature telemetry data. Kristy will give high priority to the telemetry data reanalysis and will present at next month’s LBERI meeting or as soon as available. 29

30. Action Items (3 of 3) UNM/LBERI propose next set of experiments in writing to NIAID and then get NIAID’s approval. NIAID requested more low dose LD50 on MS7, a repeat of the telemetry NHP on MS11 and more Lot 17 LVS vaccination/challenge NHP. UNM/LBERI will write several NHP study rationales and send to Patrick, Ed, and Kristin for review. LBERI will better integrate the interpretations of the pathology report into the MS7 MSCR. LBERI will incorporate new animals’ data in 2010 for LD50 before rewriting the MS7 LD 50 study MSCR LBERI will perform next fresh bloods PBMC infections with LVS and use a different Y scale for bacterial growth measurements. Cecilia will write a summary of each flow cytometry experiment design, the experiment results and the analysis for each of the flow cytometry slides for the June LBERI technical call and all future LBERI slide sets and discuss with Julie before submitting. 30