Aug 20, 2008, Page 1 KBMA Tularemia Vaccine Progress Update Aug 20 th 2008

July 8, 2008, Page 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

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 Aug 20, 2008, Page 3 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

Milestone 55: Upcoming Experiments We will construct the bivalent IglC/KatG expressing Lm strain on the KBMA background We will evaluate the immunogenicity of the bivalent Live attenuated Lm strain expressing IglC-B8R and KatG-SL8 fusion proteins We will compare the immunogenicity of each monovalent strain with the bivalent strains We will produce a lot of live Ftn-PepO-SL8 The SL8 responses induced by Ft-PepO-SL8 and LM-iglC- SL8 will be compared Aug 20, 2008, Page 4

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) Aug 20, 2008, Page 5

Pool 1 : Peptide #s 1-26 Pool 2: Peptide #s peptides (2 pools) Balb/cH2-d C57BL/6H2-b C3H/HeJH2-k FVB/NJH2-q SJL/JH2-s Lm  actA  inlB  uvrAB prfA* IglC-SL8 (BH2094) 1x 10 6 cfu IV ELISpot with peptide library 7 days IM NB#2000 pp11-14 Search for IglC Immune Responses after Vaccination with Lm-IglC VaccinationMice Aug 20, 2008, Page 6 Harvest spleens

IM IglC responses Balb/c C57BL/6 FVBN C3H SJL unstim iglC pool1 iglC pool2 IFN-  SFC per 2e5 splenocytes LLO pool-specific response Balb/c C57BL/6 FVBN C3H SJL unstim LLO pool IFN-  SFC per 2e5 splenocytes Responses to IglC Peptide Pools in All Strains of Mice by IFN-  ELIspot IglC responses were detectable in all 5 strains of mice (weak with SJL) Responses were mostly to pool2 peptides Responses were strongest in FVBN Mice Aug 20, 2008, Page 7

IM IglC Immune Responses Were Also Detected by ICS Responses to IglC pool1 peptides were weak in all 5 strains of mice Responses to IglC pool2 peptides were CD4 (Balb/c, FVBN), CD8 (C57BL/6) or both (C3H/HeJ), SJL had very weak responses Responses were strongest in FVBN Mice Aug 20, 2008, Page 8 iglC pool1 Balb/c C57BL/6 FVB/N C3H/HeJ SJL CD4 CD8 % IFN-  T cells iglC pool2 Balb/c C57BL/6 FVB/N C3H/HeJ SJL CD4 CD8 % IFN-  T cells LLO pool Balb/c C57BL/6 FVB/N C3H/HeJ SJL CD4 CD8 % IFN-  T cells

Mapping IglC Responses in Balb/c and C57BL/6 mice IM Peptide #33, 34 QEYKTDEAWGIMIDL TDEAWGIMIDLSNLE CD4+ Peptide #34, 35 TDEAWGIMIDLSNLE WGIMIDLSNLELYPI CD8+ Peptide #9 NCRLFIDSLTIAGEK ? Balb/c C57BL/6

IM Mapping IglC Responses in FVBN and C3H/HeJ mice Peptide #37, 38 NLELYPISAKAFSIS YPISAKAFSISIEPT CD4+ Peptide #33, 34 QEYKTDEAWGIMIDL TDEAWGIMIDLSNLE CD8+ Peptide #23,24 ITLGVLIKSNVRTKI VLIKSNVRTKIEEKV CD4+ Peptide #24 VLIKSNVRTKIEEKV Peptide #35, 36 WGIMIDLSNLELYPI IDLSNLELYPISAKA CD4+ C3H/HeJ FVB/NJ

IM Mapping IglC Responses in SJL mice Aug 20, 2008, Page 11 Peptide #37, 38 NLELYPISAKAFSIS YPISAKAFSISIEPT Peptide #46 DGLTTSQGSLPVCCA Peptide #50 STDKGVAKIGYIAAA SJL

IM ICS with Individual Peptides Balb/c response to peptide 34 is CD4 C57BL/6 responses to peptides 34 and 35 are CD8 Robust FVBN response to peptides 37 and 38 are CD4 Aug 20, 2008, Page 12 Balb/c unstim 34 LLO 91 LLO pool % IFN-  T cells C57BL/6 unstim 3435 SL8 LLO 190 LLO pool CD4 CD8 % IFN-  T cells FVBN unstim 3738 LLO pool % IFN-  T cells CD4 CD8 CD4 CD8

IM C3H/HeJ responses to peptides 23 and 24 are CD4 C3H/HeJ responses to peptides 33 and 34 are CD8 C3H/HeJ responses to peptides 35 and 36 are CD4 SJL responses to peptides 37 and 38 are weak ICS with Individual Peptides Aug 20, 2008, Page 13 C3H unstim LLO pool % IFN-  T cells CD4 CD8 SJL unstim LLO pool % IFN-  T cells CD4 CD8

MS56 Summary A single IV vaccination with Lm-IglC induces a cellular immune response 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 Aug 20, 2008, Page 14

MS56 Next Steps Finer mapping of optimal IglC peptides in regions of potential CD8 responses (because 9mers may stimulate a stronger response than 15mers) We will order seven 9mers (overlapping by 8) spanning peptide # 9 to determine whether the weak response in Balb/c mice in that region is a secondary region of immunogenicity We will order fourteen 9mers (overlapping by 8) that span peptides #33-35 to map the optimal CD8+ epitopes in that region in C57Bl/6 and C3H/HeJ mice. Comparison of Lm-IglC and Ft vaccines: Direct comparison of the celllular immune response to an endogenous Ft antigen by ELIspot and ICS Aug 20, 2008, Page 15

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 Aug 20, 2008, Page 16

Milestone 57: Progress 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 BH2164  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 Virulence, immunogenicity, and cellular infectivity of inlA WT and inlA M - expressing strains will be evaluated in the coming month/months Aug 20, 2008, Page 17

Anza QA performed in past 6 months All pipetmen are calibrated twice per year (March and Sept) All incubators, refrigeration equipment, and freezers are calibrated once per year (Sept) All BSCs, and fume hoods are calibrated once per year (Oct) All centrifuges are checked and calibrated once per year (var) Spectrophotometer, thermometers, balances, are calibrated once per year (var) All freezers are on backup power and are alarmed (via protection 1), but we are investigating a new wireless system that is connected through the web Aug 20, 2008, Page 18

Action Items Barbara: Add Dr. Meredith Leong to TVDC routing lists (done 8/21/08) Terry will contact Justin for the peptide library pools (done 8/21/08) Justin: use IV route with the construct strain first rather than optimizing many different routes Justin: will test iglC strain or mix the two, and test now with iv vaccination first, to determine whether it elicits protection. Get information sooner rather than later. Anza/Cerus can send them to UNM as soon as MTA is completed. Barbara: joining Anza/UNM teleconference on 8/21 regarding multiparty MTA with UCLA Justin: Anza will move sites in January 2009 Aug 20, 2008, Page 19