1. 11/11/2008 KBMA Tularemia Vaccine Progress Update Nov 11 th 2008 Justin Skoble

2. Cerus-Anza Milestones Milestone 55: Compare Cellular Immune Responses Induced by Lm and Ft-Based Tularemia Vaccines Measure cellular immunogenicity of live-attenuated vaccine platforms using model epitope Compare immunogenicity of KBMA tularemia vaccine platforms using model epitope Milestone 56: Demonstrate that Lm Vaccines Induce Protective Cellular Immune Responses to Ft Antigens Measure the T-cell response to IglC induced by live and KBMA Lm expressing IglC compared with those elicited by Ftn or LVS vaccination Demonstrate that Live and KBMA Lm-IglC and Lm-KatG protect against an LVS challenge Demonstrate that Live and KBMA Lm-IglC and Lm-KatG protect against a SchuS4 challenge Milestone 57: Optimization of KBMA Lm Vaccination Route and Regimen Compare various routes of administration including IV, IM, IN, ID and oral Optimize dosing regimen of most potent and tolerable route Confirm optimized route and regimen provides protection against SchuS4 at UNM Milestone 58: Large Scale GMP-Like Production of KBMA Lm Tularemia Vaccine Optimize scalable KBMA vaccine production at 4L scale Produce up to a 30L lot of most potent vaccine under GMP-like conditions Develop quality assays to support release and stability testing of vaccine lots Perform toxicology studies using KBMA Lm platform Milestone 59: Use Lm Platform For Delivery of Novel Ft Antigens Discovered by TVDC Cerus could potentially make available the Lm platform Clone up to 10 Ft antigens identified by TVDC group into Lm expression cassettes Characterize the intracellular expression levels of various Ft antigens (and SL8 immunogenicity) Rank potency of each vaccine candidate by sharing with UNM for protection studies Determined the minimal concentration of S-59 to inactivate LVS uvrB 11/11/20082

3. Milestone 55: Summary Cellular immunogenicity of live-attenuated Lm vaccine platforms were measured Lm-expressing IglC-SL8 and Lm KatG-SL8 fusion proteins were cloned The ability of each to stimulate a CD8+ T cell response to SL8 was evaluated using a B3Z assay, ICS, and ELISpot CD8 T cell responses against SL8 were stronger when fused to IglC than KatG prfA* enhanced immunogenicity of IglC-SL8 vaccine ~ 2 fold Quadrotope tag decreased immunogenicity and will not be used ActAN100 SL8 IglC actAp ActAN100 SL8 KatG actAp Molecular constructs at tRNA Arg : ActAN100 B8R IglC actAp Molecular construct at comK: ActAN100 SL8IglC actAp A42RB8RC4LK3L 11/11/20083

4. Lm-Ft constructs ActAN100 SL8 IglC actAp ActAN100 SL8 KatG actAp Molecular constructs at tRNA Arg : ActAN100 B8R IglC actAp Molecular construct at comK: ActAN100 SL8IglC actAp A42RB8RC4LK3L StrainGenetic BackgroundAntigen CassetteStatus CRS-100/LM11  actA  inlB noneSequence verified LM677  actA  inlB  uvrABprfAG155S noneSequence verified BH137  actA  inlB ActAN100-OvaSequence verified BH1222  actA  inlB ActAN100-IglC-SL8Sequence verified BH2282  actA  inlB ActAN100-KatG-SL8Sequence verified BH1228  actA  inlB  uvrAB ActAN100-IglC-SL8Sequence verified BH1398  actA  inlB  uvrAB ActAN100-KatG-SL8Sequence verified BH2094  actA  inlB  uvrABprfAG155S ActAN100-IglC-SL8Sequence verified BH2172  actA  inlB  uvrABprfAG155S ActAN100-KatG-SL8Sequence verified BH2098  actA  inlB ActAN100-IglC-VacQuad-SL8Sequence verified BH2100  actA  inlB  uvrABprfAG155S ActAN100-IglC-VacQuad-SL8Sequence verified BH2180  actA  inlB ActAN100-IglC-B8R (@ comK)Sequence verified BH2182  actA  inlB  uvrABprfAG155S ActAN100-IglC-B8R (@ comK)Sequence verified BH2316  actA  inlB ActAN100-IglC-B8R (@ comK) ActAN100-KatG-SL8 (@tRNA arg ) Remade and verified (BH2184 had point mutation in KatG) BH2292  actA  inlB  uvrABprfAG155S ActAN100-IglC-B8R (@ comK) ActAN100-KatG-SL8 (@tRNA arg ) Sequence verified 11/11/20084

5. LaneSampleParent Insert tRNA-ArgcomKinlB 1SeeBlue Plus2 2Mock 3BH1029CRS-100NS5b 4CRS-100 5CRS207CRS-100mesothelin 6BH2292Lm 677katG-SL8iglC-B8R 7BH2182Lm 677iglC-B8R 8BH2172Lm 677katG-SL8 9BH2316CRS-100katG-SL8iglC-B8R 10BH2180CRS-100iglC-B8R 11BH2282CRS-100katG-SL8 NB2006_053 Strains Evaluated for Intracellular Expression by Multiplex IR-fluorescence Western Blot 11/11/20085

6. 1234567891011 iglC++++ katG++++ 188 62 49 38 28 < katG < iglC < p60 Overlay of 700nm and 800nm Images

7. Overlay image with boxes showing integrated intensities of selected proteins NB2006_053: 1234567891011 iglC++++ katG++++ 11/11/20087

8. Relative IglC and KatG Expression Levels Strain Insert katG*iglC*p60*iglC/p60katG/p60 iglckatG BH2292++0.2633.685.985.630.04 BH2182+030.064.017.500.00 BH2172+0.1904.760.000.04 BH2316++0.3111.023.663.010.08 BH2180+0.0530.873.618.550.01 BH2282+0.41-0.033.01-0.010.14 BH1029NS5b52.973.1216.98 ANZ207Meso0.363.130.12 11/11/20088

9. Immunogenicity of Bivalent vs Monovalent Live-attenuated Lm vaccines (ICS) IglC-tagKatG-tagLm-control C57BL/6 Mice immunized IV with 1e6 prfA* or 5e6  actA/  inlB Compared bivalent strain to dosing with ½ dose of both monovalent strains -Slight decrease in T cell frequency with bivalent strain -Decrease in immunogenicity was larger when dose decreased by ½ * p<.05, **P<.005, ***p<.005 * ** *** * * 11/11/20089

10. IglC and SL8 Responses not Induced or Boosted by LVS-PepO-SL8 IM08-090 11/11/200810 1e6 Lm-IglC administered IV, 1e4 LVSPepO-SL8 delivered ID 3 weeks between prime and boost, spleens harvested d6 post-boost LVS-pepO-SL8 does not induce measurable IglC or SL8 responses by itself Lm-induced responses not primed or boosted by LVS

11. Live-attenuated vs. KBMA primary response (ICS) 11/11/200811 1222  ActA  inlB 1228  ActA  inlB  uvrAB KBMA-Lm primary responses reduced by ~75% May improve after a boost

12. Live-attenuated vs. KBMA primary response (ELISpot) 11/11/200812 1222  ActA  inlB 1228  ActA  inlB  uvrAB

13. Milestone 55: Upcoming Experiments We will confirm that p60 expression correlates with cfu by performing an MOI dose response and perform western blot and cfu analysis in parallel We will evaluate the immunogenicity of KBMA strains after a prime and boost vaccination 11/11/200813

14. Milestone 56: Demonstrate that Lm Vaccines Induce Protective Cellular Immune Responses to Ft Antigens Measure the T-cell response to IglC induced by live and KBMA Lm expressing IglC compared with those elicited by Ftn or LVS vaccination Produce IglC overlapping peptide library 15aa overlapping by 11aa (211 amino acid long protein) Use IglC peptide library for ELISpot assays to measure the IglC-specific T cell responses induced after vaccination with live and KBMA Lm-IglC and compare to live and KBMA Ftn and LVS vaccination Demonstrate mechanism of protection induced by Lm vaccines is cellular by depletion of T cell populations and passive transfer studies Demonstrate that strains of Live and KBMA Lm-IglC-SL8 and Lm-KatG- SL8 protect against a SchuS4 challenge Produce lots of KBMA vaccine and send to UNM for testing in animal models (mice and rats) 11/11/200814

15. Previous work A single IV vaccination with Lm-IglC induces cellular immune responses to IglC in Balb/c, C57BL/6, FVBN, and C3H/HeJ mice Responses are CD4+, CD8+, or both depending on the haplotype of the mice IglC-specific CD8+ epitopes were identified in C57BL/6 and Balb/c mice Preliminary results suggest that Lm-IglC vaccine induces stronger IglC and SL8 responses than LVS-pepO-SL8 11/11/200815

16. Lm Induces Responses Against IglC Peptide by ICS and ELISpot in C57BL/6 Mice ICSELISpot Vaccination with Lm-KatG induced response against IglC peptide (33-10). GIMIDLSNL sequence not present in KatG GIMIDLS is present in Lm genome- hypothetical protein lmo0368 11/11/200816

17. Lm-IglC Vaccine Protects Balb/c mice Against Lethal LVS Challenge 11/11/200817

18. MS56 Summary Endogenous Lm antigen is cross-reactive with IglC in C57BL/6 mice Should not use this strain for further IglC immunogenicity OK to use for SL8 or B8R studies Will focus on Balb/c mice IV vaccination with LM-IglC protects 100% Balb/c against 10x LD 50 LVS challenge Lm-KatG only protects 40% of animals Vaccination with1/2 dose of both Lm strains also protected 100% as did LVS vaccination 11/11/200818

19. MS56 Next Steps Increase stringency of LVS challenge (look at 10 vs 100 LD 50 ) Anza will vaccinate mice with various live and KBMA Lm vaccines to determine whether IglC, KatG, or both protect against lethal LVS infection Entire IglC peptide library will be tested with Lm expressing an irrelevant antigen to determine if there is cross-reactivity between Lm and IglC in Balb/c mice as well Once MTA is approved, live and KBMA Lm lots will be sent to UNM for evaluation in SchuS4 challenge model. 11/11/200819

20. Milestone 57: Optimization of KBMA Lm Vaccination Route and Regimen Compare various routes of administration including IV, IM, IN, ID and oral For oral, IN, and ID administration we will first mutate the inlA gene of Lm to allow for binding of murine E-cadherin in order to mimic the human interaction We will compare the potency of the inlA gain of function mutants to our traditional platform strain Routes will be ranked by ability to induce a cellular immune response: Elispot, in vivo cytotoxity, and ICS Optimize dosing regimen of most potent and tolerable route Lm expressing IglC and/or KatG will be used Initial evaluation will be performed by immunogenicity Optimized route and regimen will be confirmed by SchuS4 protection studies at UNM 11/11/200820

21. MS57: Strain Construction for Route Optimization To facilitate route optimization, the inlA gene of our platform Lm strains has been altered to allow for binding to murine E-cadherin The sequence of the wild-type EGDe inlA gene was synthesized and the inlA gene in our platform strain was replaced (inlA WT ) in our wild-type and KBMA platform strains 2 point mutations S192N and Y369S were incorporated into the EGDe inlA sequence (inlA M ) and inserted into the chromosome of our wild-type and KBMA platform strains As published in Wollert et al., Cell 2007 StrainGenetic BackgroundAntigen CassetteStatus CRS-100  actA  inlB noneSequence verified BH2130  actA  inlBinlA WT noneSequence verified BH2164  actA  inlBinlA WT ActAN100-IglC-SL8Sequence verified BH2170  actA  inlBinlA M noneSequence verified BH2194  actA  inlBinlA M ActAN100-IglC-SL8Sequence verified BH2132  actA  inlB  uvrABprfAG155SinlA WT noneSequence verified BH2166  actA  inlB  uvrABprfAG155SinlA WT ActAN100-iglC-SL8Sequence verified BH2134  actA  inlB  uvrABprfAG155SinlA M noneSequence verified BH2168  actA  inlB  uvrABprfAG155SinlA M ActAN100-iglC-SL8Sequence verified 11/11/200821

22. inlA M Allele Does Not Increase Invasion of CaCo2 Cells 11/11/200822 CaCo2 cells are of human origin not mouse But was the cell line used in Wollert at al.

23. MS57 Previously Reported Route comparison initiated C57BL/6 mice were vaccinated with BH2164 and BH2194 IV and orally to determine whether the inlA m gain of function contributes to immunogenicity InlA Gain-of-Function mutation did not significantly enhance splenic immunogenicity either by oral or IV route 11/11/200823

24. Oral Administration Induces Lower Splenic but Equal Mucosal Immunity as IV 11/11/200824 BH1228: actAinlBuvrAB-iglC-SL8, 5e6 CFU-IV, 1e9cfu Oral

25. Repeat of (1e9) Oral Administration with inlA m vs inlA wt, Splenic responses by ELISpot 11/11/200825 No significant differences with inlA M 2194 trending higher than 2164 after oral

26. IEL Responses after Oral Administration 11/11/200826 IEL Responses to IglC and LLO epitopes were below LOD inlA m gain of function may increase SL8 mucosal immune response after oral administration, but needs repeating

27. MS57 Next Steps Mucosal immunity will be evaluated again after oral immunization to determine whether the >2fold increase in mucosal immunity seen with the inlA M strain is reproducible. An intranasal LD 50 will be performed with DVC lot16 LVS as this route of infection may be more relevant for investigation of tularemia vaccines. Murine epithelial cell line will be purchased for invasion assays

28. Action Items Barbara will contact Nancy Carr at UNM HSC Contracts and grants to urge completion of the MTA (done 11/12/08). BG offered to organize a teleconference call also. Justin- do intranasal vaccination route at some time. IV vs. IN may be different enough due to targeting differences 11/11/200828