1. 111 Progress Report 11/05/2008 TVDC team – UNM Prepared by Terry Wu

2. 222 Active Milestones Active Milestones: 5, 11, 12/13, 14, 17, 19, 21, 27, 35 Today’s presentation will include: 5.Small animal models of tularemia 14.Assays in vaccinated humans 27.Testing of ivt proteins for ASU (ASU MS 26) 35.RNA and genomic DNA isolations for ASU

3. 33 Milestone 5 – flow diagram Small animal models Fischer 344 rats BALB/c mice Guinea pigs SCHU S4 LD 50 i.n. (i.t.) LVS LD 50 s.c., i.d. i.n. LVS vaccination Clearance of vaccination strain SCHU S4 challenge (in/it) Clinical signs and survival Model selection SCHU S4 LD 50 i.n. (i.t.) LVS LD 50 s.c., i.d. i.n. LVS vaccination Clearance of vaccination strain SCHU S4 challenge (in/it) Clinical signs and survival SCHU S4 LD 50 i.n. (i.t.) LVS LD 50 s.c., i.d. i.n. LVS vaccination Clearance of vaccination strain SCHU S4 challenge (in/it) Clinical signs and survival Blue: Steps in the milestone Red: Completed Green: In progress Statistical analyses Robustness of Fischer 344 rat model

4. 4 Bronchopneumonia (Rats) vs. Vasculitis (Mice)

5. Histological Comparison of Mice, Rats, and NHP infected i.t. with SCHU S4 Infect i.t. with 1000 SCHU S4 Collect organs and tissues –NHP (days 1, 4, & 7) –Mice and rats (days 1, 2, 3, 4 & 7) –Lungs, LALN, spleen, liver, GI tract, mesenteric LN, thymus, nasal cavity, etc 5

6. Quantum Dots Reduced SCHU S4 Virulence in vivo 6

7. 7 Additional Plans Wrap up manuscript describing the Fischer 344 rat model Complete MSCR – almost done with first rough draft 7

8. 888 MS 14: Assays in Vaccinated Humans Assay to measure activation of macrophage killing mechanisms in humans Macrophage T cells Determine the approximate yield of macrophages from whole blood (1-200 ml max) Determine and optimize cell number and MOI Positive control w/ IFN  Blue: Steps in the milestone Red: Completed Green: In progress Isolate & differentiate monocytes to macrophages (buffy coat) Determine the approximate yield of PBMC and T cells from whole blood Test PBMC Test purified T cells in vitro expansion? Repeat w/ human vaccinee

9. Functional Correlate of Protection 9 immune T cell mm F F mm F F X X naive T cell mm F F F F F F Mouse assay optimized for LVS and SCHU S4 Working on rat and human assays

10. 10 Cell Yield from Whole Blood From 100 ml of whole blood –1.1 x 10 8 PBMC –8.5 x 10 6 monocytes –3.3 x 10 6 macrophages after 6 day culture For comparison, we have been using ~10 7 murine macrophage per well in the 24 well plate Scale down the number of macrophages per well while maintaining assay sensitivity

11. 11 MS14: Goals for Human Correlate of Protection Assay Reasonable scale Rapid setup and readout High sensitivity High specificity Ideal to use whole blood or PBMC

12. 12

13. MS 14: Plans Optimize infection of human monocytes Determine whether monocytes would be a good target 13

14. 14 MS 17: Characterization of Fischer 344 Rat Fischer 344 rats Humoral immunity Cell mediated immunity. LVS vaccination Passive transfer of serum Protection against i.t SCHU challenge Blue: Steps in the milestone Red: Completed Green: In progress Production of ascites fluid for CD4 and CD8 depletion In vivo depletion Pretection against i.t. SCHU SCHU challenge Active vs passive immunizatioin Purchase and culture hybridoma cell lines

15. 15 Passive Immunization Protects Against i.t. SCHU S4 challenge (240 CFU) Treatment Survival Ratio (No. alive/total) Unvaccinated-0/6 LVS vaccinated-6/6 Normal rat serum1.5 ml0/6 1.0 ml0/6 0.5 ml0/6 0.25 ml0/6 Immune rat serum1.5 ml6/6 1.0 ml6/6 0.5 ml5/6 0.25 ml6/6

16. 16 Passive Immunization Protects Against i.t. SCHU S4 challenge (0.25 ml & 217 CFU)

17. Passively Immunized Rats Develop Enhanced Immunity after Recovering from SCHU S4 infection (2 x 10 4 CFU) 17 Naive LVS IRS/SCHU S4 LVS/SCHU S4

18. Kinetics of SCHU S4 Proliferation and Dissemination in Actively- and Passively-immunized Rats 18

19. 19 MS17: Plans Repeat growth kinetics experiment in actively and passively immunized rats Compare the histopathology Define the role of T cells in passive immunity

20. 20 Milestone 27 – flow diagram SOP for detecting T cell stimulation with ivt proteins and peptide T cell proliferation IFN  ELISpot assay Assay optimization using ivt proteins Production Assay development (UNM) Production of ivt proteins & peptide library (ASU) Blue: Steps in the milestone Red: Completed Green: In progress Identification of stimulatory proteins & peptides Screening (UNM)

21. 21 MS27: Plans Vaccinated NHP with LVS Meeting with ASU over complexity of peptide pool. –2065 linear expression element constructs –1.5 x 10 8 LN cells (conservative estimate) –3 peptides/well in single wells –8 peptides/well in duplicates –Splenocytes Boost vaccinated NHP with LVS Screen10 days after boost

22. 22 Gray: (sub )milestone title Red: completed Green: in progress Milestone 35 – flow diagram Printing and testing GDP confirmed Printing arraysGDP Confirmation Comparions of substrate Poly-L Lysine vs Corning Ultragaps Compare TIGR PFGR Arrays to in house arrays RNA shipped 1/29/2007 Testing of linear amplification of procaryotic Transcripts (LAPT) process and dilution testing of Schu S4 RNA with and without mouse lung RNA MS-33 (ASU) Optimization of RNA isolation and microarray conditions Infect BALB/c mice i.n. with SCHU S4 Isolate RNA from LVS and SCHU S4 Isolate RNA from infected lungs Isolate total RNA from LVS and SCHU S4 Isolate eukaryotic and prokaryotic RNA Ship to ASU Blue: Steps in the milestone Red: Completed Green: In progress

23. 23 MS 35 – Work Accomplished Prepared RNA from Fischer 344 rat lungs 1, 3, 5, 7, and 24 h after infection with SCHU S4 Agilent analyzer down Will ship when analyzed

24. Action Items: The next UNM Tech call is scheduled on 12/5/08 friday noon-1pm MT (2-3pm Eastern). Freyja, Terry, Rick and Kristin agreed to the new date. This replaces the UNM Tech call originally scheduled on 12/3/08. Terry will send rat manuscript draft to Barbara and Barbara will email to NIAID for approval to send to the journal. (completed, including Freyja’s review; Kristin also received it) UNM/ASU scheduled a teleconference for 11/6/08 1pm (completed) to discuss details of polypeptide pooling for testing at UNM with vaccinated and boosted NHP lymph node and spleen cells 24