1 LBERI Update on Animal Model Development Lovelace Respiratory Research Institute 2425 Ridgecrest Drive SE, Albuquerque, NM Sub-NIAID Tech Call 6 July 2010

2 Active Milestones #2ActiveVaccinations of study personnel- no work in June #8ActiveLVS vaccination protection of aerosol Schu4 confirmed in primates #9ActiveAerosol SOP developed for GLP transition- no work in June #10ActiveEfficacy testing of vaccine candidates (LBERI)- no work in June #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 June

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 MS8: Vaccination Practice/Challenge (n=3 scarification; n=2 subcutaneous) MS8A: Vaccination/Challenge (n=3 by scarification; n=3 by subcutaneous route; n=4 previously vaccinated; 2 SC, 2 ID) SCHU S4 Challenge 500 CFU MS8B: Vaccination/Challenge (Vaccination with Highest Dose of LVS attainable by scarification and s.c.) SCHU S4 Challenge 1000 CFU MS8C: 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 MS8D: Vaccination/2 Broth Challenge (SC Vaccination with Lot 17, compare CB vs. CAMHB) SCHU S4 Challenge 1000 CFU MS8E:Telemetery Study on Vaccinated NHP SCHU S4 Challenge 1000 CFU MS8F: Repeat Vaccination and broth challenge with additional immunology parameters

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) – MS08 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) – MS08 D – Examine if there are differences in telemetry patterns in vaccinated NHP as compared to control NHP (MS08E) – Conduct additional immunology on vaccinated NHP and expose to SCHU S4 grown in different media to build data from the broth comparison study (MS08F) 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

Animal ID Vaccine Group Culture Broth a Presented Dose (CFU) Nx Date Bacteremia Study Day a , Term A09011ControlCB2,82316-MayNG Ft A09410ControlCB44718-MayNGFt A09139ControlCB1,10717-MayNG Ft A09064Lot 17CB1,45517-MayNG A09097Lot 17CB1,4928-JunNG A09088Lot 17CB2,7668-JunNG A09094Lot 17CB1,7418-Jun-10NG A09169Lot 17CB2,17818-MayNG A09487Lot 17CB1,3448-JunNG A09164Lot 17CB2,0678-JunNG A09079ControlCAMHB98017-MayNG Ft A09518ControlCAMHB2,34118-MayNG Ft A09072ControlCAMHB1,44119-MayNGFt A09086 Lot 17CAMHB2,2669-JunNG A09066 Lot 17CAMHB1,0509-JunNG A09068 Lot 17CAMHB1,3299-JunNG A09523 Lot 17CAMHB2,0109-JunNG A08739 Lot 17CAMHB2,1509-JunNG A09140Lot 17CAMHB1,6309-JunNG A09511 Lot 17CAMHB1,8019-JunNG a CB, Chamberlain's broth; CAMHB, Cation-adjusted Mueller-Hinton broth Milestone #8 June 2010 Accomplishments Bacteremia (MS8D) NG = no growth on plates or in liquid medium Ft= growth on plates or in liquid medium

Animal ID Challenge Date Vaccine Group Culture Broth a Presented Dose (CFU) Nx Date CFU/g Tissue b SpleenLungMes. LNLiverTBLN A May-10ControlCB2,82316-May E E E E E+08 A May-10ControlCB44718-May E E E E E+07 A May-10ControlCB1,10717-May E E E E E+08 A May-10LVS Lot 17CB1,45517-May E E+07BLD 7.07E+02 A May-10LVS Lot 17CB1,4928-Jun-10BLD A May-10LVS Lot 17CB2,7668-Jun-10BLD 7.19E+02 A May-10LVS Lot 17CB1,7418-Jun-10BLD1.35E+05BLD2.54E E+05 A May-10LVS Lot 17CB2,17818-May E E+07BLD2.54E E+03 A May-10LVS Lot 17CB1,3448-Jun-10BLD3.94E+03BLD 3.37E+01 A May-10LVS Lot 17CB2,0678-Jun-10BLD3.02E+02BLD A May-10ControlCAMHB98017-May E E E E E+07 A May-10ControlCAMHB2,34118-May E E E E E+07 A May-10ControlCAMHB1,44119-May E E E E E+08 A May-10LVS Lot 17CAMHB2,2669-Jun-10BLD1.03E+03BLD 1.19E+02 A May-10LVS Lot 17CAMHB1,0509-Jun-10BLD A May-10LVS Lot 17CAMHB1,3299-Jun-10BLD3.10E+02BLD 3.59E+03 A May-10LVS Lot 17CAMHB2,0109-Jun-10BLD8.70E+02BLD 7.20E+02 A May-10LVS Lot 17CAMHB2,1509-Jun-10BLD8.00E+02BLD A May-10LVS Lot 17CAMHB1,6309-Jun-10BLD A May-10LVS Lot 17CAMHB1,8019-Jun-10BLD 5.61E+02 a CB, Chamberlain's broth; CAMHB, Cation-adjusted Mueller-Hinton broth b BLD, below limit of detection 6 Milestone #8 June 2010 Accomplishments Tissue Burden (MS8D)

BrothAnimal #Status Pres. Dose Day 0Day 3Day 5Day 7Day 10Day 14Day 21Day 28 CB A09011Ctrl2, Dead A09410Ctrl Dead A09139Ctrl1, Dead A09088Vx1, A09094Vx1, A09487Vx1, A09164Vx2, A09064Vx1, Dead A09097Vx A09169Vx2, Dead MHB A09079Ctrl Dead A09518Ctrl2, Dead A09072Ctrl1, Dead A09066Vx1, A09068Vx1, A08739Vx2, A06086Vx2, A09523Vx2, A09140Vx1, A09511Vx1, not enough sample to run CRP 7 Milestone #8 June 2010 Accomplishments CRP Levels (MS8D)

8 Milestone #8 – June 2010 Accomplishments on MS8D Completed histopathology on slides received from 8 moribund animals on MS08D Microbiology data has been qc’d Clinical pathology data qc is near completion

Milestone #8 – Plans for next month Measure IFN-γ production by PBMCs and splenocytes of TUL08D survivors by analysis of the ELISPOT plates recently removed from the ABSL3 Continue data analysis 9

10 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

11 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

Milestone #12/13 – June 2010 Accomplishments Ordered and received anti-monkey IgM-HRP antibody in order to test whether plasma from LVS-vaccinated NHPs have detectable IgM anti-LVS levels by ELISA – Will use this data to identify potential positive plasma samples and test these in the micro- agglutination assay 12

Milestone #12/13-Plans for next month Run IgM anti-LVS ELISA and repeat micro- agglutination assay if positive plasma samples can be identified Test newly received (from UNM) FF LVS using frozen PBMCs to determine whether it stimulates IFN-γ production as measured by ELISPOT or proliferation in LVS-vaccinees but not in PBMCs from naïve NHPs 13

14 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

15 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

Milestone #21 – June 2010 Accomplishments Measured IFN-  intracellular staining in fresh PBMCs from one naïve NHP (A07993) PBMC (4x10 5 ) stimulated 20 h with media only or UNM prep ffLVS (1.25x10 4, 5x10 4 or 2x10 5 cfu/well) Stained with antibodies to detect CD3-, CD8-, CD14-, CD16- and CD56-positive cells 16

Milestone #21-Data Interpretation No antigen dependent IFN-  response in CD4 (CD3+CD8-) or CD8 (CD3+CD8+) T cells, CD14+ (monocytes) or CD3-CD16+ (NK cells). Background IFN-  staining for T cells was < 0.5% IFN-  +, CD14+ < 0.8% and NK cells 0.1%. CD56+ cells (monocytes in macaques?) showed an increase from 10% IFN-  + CD56+ cells in media control to approx 17 % IFN-  + in the wells with the two highest antigen concentrations. 17

Test additional fresh PBMCs from naïve NHPs in the ICS assay if they become available (excess from other studies conducted in the laboratory) OR draw blood from TVDC NHPs specifically for this assay before they are placed on upcoming studies 18 Milestone #21 - Plans for next month

Action Items 19 LBERI needs discussion on LD 50 exposure days: 1 or 2 Status of NIAID review of MS08E protocol Status of NIAID review of MS08F protocol

20 Additional Points Deliverables completed for each active milestone: No deliverables were completed during the month of June List of relevant publications from the past month Bob Sherwood submitted abstract “Characterization of a Nonhuman Primate Cynomolgus Macaque Model of Pulmonary Tularemia for Vaccine Screening” For presentation at the 2010 Chemical and Biological Defense Science and Technology Conference, November 2010, Orlando, Florida. Sponsored by DTRA. MSCR status MS 3: NIAID accepted on 3/9/10 as Final. 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 reviewed 6/15/10 (LBERI will revise by end July 2010) MS 8 a: LBERI writing and due to UNM TBD, once revised MS11 is submitted MS 8 b: LBERI writing and due to UNM TBD, once revised MS11 is submitted MS 8 c: LBERI writing and due to UNM 7/23-27/10

Action Items (1 of 2) UNM suggested that LBERI use the MSXX format rather than “round” or and “Tul 08X” (Note LBERI will use the MSXX format for future tech calls and reports) Julie Wilder- Will gather the data for comparing the UNM FF LVS stimulation vs DVC FF LVS stimulation and will to Barbara (completed 7/7/10) Michelle Valderas: will check the IACUC to determine whether the NHP blood draws for Julie Wilder could occur before the NHP are assigned to a study protocol and before the study protocol is approved by NIAID. LBERI will write the LD50 amendment for all aerosol exposures on 1 day for all 12 NHP. Patrick :Will send the reviewed amended version to LBERI on 7/7. (NIAID canceled this MS7 study 7/8/10) 21

Action Items (2 of 2) LBERI (Trevor Brasel) should keep with Chamberlains grown SCHU S4 on the MS9, since all of LBERI’s work from the beginning of the TVD contract is with SCHU S4 grown in Chamberlains. Patrick Sanz: Tul 08E: vaccinated/telemetry: will review and get it back to LBERI by end of week on 7/9. Please to Barbara and Bob Sherwood, as Michelle Valderas is on annual leave from 7/9 to 7/16. Patrick Sanz: Tul 08 F: immuno/ potential two broth comparison for the challenge: Patrick will get back to UNM on the turnaround time on this one. 22