1. Construction and Evaluation of Live- Attenuated and KBMA Listeria monocytogenes Expressing Ft Antigens as Tularemia Vaccine Candidates TVDC Tech Call May11th, 2010 1

2. Cerus/Aduro Milestones Milestone 55: Compare Cellular Immune Responses Induced by Lm-Based Tularemia Vaccines Construct vaccine candidates Measure cellular immunogenicity of live-attenuated vaccines using model epitopes Compare immunogenicity of KBMA tularemia vaccines using model epitopes 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 LVS vaccination Demonstrate that Live and KBMA Lm-IglC and/or Lm-KatG protect against an LVS challenge Demonstrate that Live and KBMA Lm-IglC and/or Lm-KatG protect against a SchuS4 challenge Milestone 57: Optimization of Vaccination Route and Regimen Compare various routes of administration including IV, IM, ID and oral Optimize dosing regimen of most potent and tolerable route Confirm optimized route and regimen provides protection against SchuS4 at UNM Milestone 59: Use Lm Platform For Delivery of Novel Ft Antigens Discovered by TVDC Clone up to 5 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 Construct multivalent vaccine candidate 2

3. MS 55: Lm Ft Vaccine Construct List 3 StrainGenetic BackgroundAntigen CassetteStatus CRS-100/LM11  actA  inlB noneSequence verified LM583  actA  inlB  uvrAB 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 BH2568  actA  inlB  uvrABprfAG155S ActAN100-KatG-C250 (@tRNA arg )Sequence verified BH2594  actA  inlB ActAN100-SL8-KatG-C250 (@tRNA arg )Sequence verified BH2596  actA  inlB  uvrABprfAG155S ActAN100-SL8-KatG-C250 (@tRNA arg )Sequence verified BH2608  actA  inlB  uvrABprfAG155S ActAN100-IglC-B8R (@ comK) ActAN100-SL8-KatG-C250 (@tRNA arg ) Sequence verified BH2683  actA  inlB  uvrABprfAG155S ActAN100-B8R-IglC-C175 (@tRNA arg )Sequence verified BH2697  actA  inlB  uvrABprfAG155S ActAN100-B8R-IglC-C175 (@comK) ActAN100-IglC-SL8 (@tRNA arg ) Sequence verified BH2699  actA  inlB  uvrABprfAG155S ActAN100-B8R-IglC-C175 (@comK) ActAN100-KatG-SL8 (@tRNA arg ) Sequence verified BH2932  actA  inlB  uvrABprfAG155S ActAN100-KatG  hyd2-SL8 Sequence verified BH2934  actA  inlB  uvrABprfAG155S ActAN100-KatG  hyd1-SL8 Sequence verified BH2936  actA  inlB  uvrABprfAG155S ActAN100-KatG  hyd1  hyd2-SL8 Sequence verified BH2938  actA  inlB  uvrABprfAG155S ActAN100-IglC(no tag)Sequence verified

4. Proposed Modification of IglC and KatG Constructs 4 KatG (∆1-23)ActAN100 SL8 1 2 Make  1,  2 and  1 +  2 strains Measure intracellular antigen expression Measure SL8 immunogenicity If expression and/or immunogenicity are improved then we will perform an LVS challenge. ActAN100 SL8 IglC actAp ActAN100 IglC actAp 1) Remove epitope tag from IglC construct 2) Remove hydrophobic regions from KatG

5. Intracellular Expression of New Antigen Constructs LnIDAntigen expression cassetteActAP60ratio 1mwm---- 2Lm11none0.041.340.03 3BH2094ActAN100-IglC-SL887.31.6054.5 4BH2292 ActAN100-IglC-B8R KatG(  SS)-SL8 83.2 1.13 1.80 45 0.62 5BH2938ActAN100-IglC123.82.3652.5 6BH2172 ActAN100-KatG(  SS)-SL8 0.260.760.34 7BH2932 ActAN100-KatG(  SS)-(  2)-SL8 15.41.212.8 8BH2934 ActAN100-KatG(  SS)-(  1)-SL8 2.20.593.7 9BH2936 ActAN100-KatG(  SS)-(  1-  2)-SL8 11.80.9312.7 10unin -.060.11.54 12345678910 62- 49- 38- 28- 17- 98- 188- DC2.4 cells infected with Lm strains at an MOI of ~10 for 8h Blot probed with  -ActA (red) and  -p60 (green, used to normalize infection level) KatG  2 expression increased 38 fold, but is still ¼ IglC level. NB837-063 5

6. Modification of KatG Improves Intracellular Expression 6 KatG (∆SS)ActAN100 SL8  1 (10aa)  2 (15aa) KatG  SSKatG  2KatG  1KatG  1  2 StrainAntigenKatGp60Ratio BH2172 KatG  SS 0.260.760.34 BH2932 KatG  2 15.41.212.8 BH2934 KatG  1 2.20.593.7 BH2936 KatG  1  2 11.80.9312.7 Deletion of KatG hydrophobic region 2 increases expression > 38 fold NB837-063

7. Removal of IglC Epitope Tag Does Not Affect Intracellular Expression 7 Lm11IglC-SL8 IglC-B8R from Bivalent strain IglC untagged AntigenIglCp60ratio Lm11 (none)0.041.340.03 ActAN100-IglC-SL887.31.6054.5 ActAN100-IglC-B8R83.21.8045 ActAN100-IglC123.82.3652.5 Deletion of epitope tag does not decrease IglC expression ActAN100 SL8 IglC actAp ActAN100 IglC actAp NB837-063

8. MS 55 Next Steps Continue to characterize new IglC and KatG constructs Compare IglC and KatG immunogenicity in mice to old strains LVS challenge after vaccination with new KatG  2 strain (MS56) Construct new bivalent strain expressing IglC + KatG  2 8

9. MS 56: Repeat of LVS Protection Study After Live and KBMA Immunization (35x IV LD 50 ) 9 Balb/c mice vaccinated IV on D0 and D28 with: 1e3 Live LVS, 2e6 Live Lm11, BH2182 (Lm-IglC), or BH2292 (Lm-IglC+KatG) 1e8 KBMA BH2182, or BH2292 Animals challenged IV one month after boost with 1.4e6 CFU LVS 20% weight loss used as cutoff for survival Live and KBMA Lm provided similar level of protection as LVS (AS09-024, Notebook 2000 pp. 138-9) LIVEKBMA

10. 25% Weight Loss Correlates Better With Survival than 20% LIVEKBMA 10 (AS09-024, Notebook 2000 pp. 138-9)

11. Animals in 2 nd Rat SchuS4 Protection Study Were Challenged Last Monday Regimens Prime → → 6 wk → Boost → 4 wk → SchuS4 IT Challenge Prime → 2 wk → Boost → 4 wk → SchuS4 IT Challenge Groups D7 Survival 1) PBS – SC1/6 2) 5x10 7 LVS (No boost) – SC6/6 3) 1x10 8 BH2182 (Lm677:IglC) – IM5/7 4) 1x10 8 BH2182 (Lm677:IglC) – ID6/7 5) 1x10 6 BH2182 (Lm677:IglC) – IM3/7 6) 1x10 8 BH2182 (Lm677:IglC) – IM 2 week p/b5/7 7) 1x10 8 BH1222 (Lm11:IglC) – IM 4/7 11

12. Draft Design for KBMA Lm Rat SchuS4 Protection Study (for Discussion) Regimens (Groups1-2) Prime→ → → → → → → →8 wk → SchuS4 IT Challenge (Groups 3-6) Prime → 4 wk → Boost → 4 wk → SchuS4 IT Challenge Groups 1) PBS - SC 2) 5x10 7 LVS (No boost) - SC 3) 1x10 8 Live BH2182 (Lm677:IglC) - IM or ID 4) 1x10 8 KBMA BH2182 (Lm677:IglC) - IM or ID 5) 2x10 9 KBMA BH2182 (Lm677:IglC) - IV 6) 2x10 9 KBMA BH2182 (Lm677:IglC) - IM or ID 12

13. MS 56 Next Steps Evaluate heterologous prime-boost regimens IglC DNA or IglC peptides as prime, followed by Lm boost. Evaluate ability of new KatG  2 strain to provide protection against lethal LVS challenge LVS protection study in C57BL/6 mice If we see protection in this mouse strain we will perform depletion studies to determine whether CD4 or CD8 T cells play a role in protection 13

14. MS 57: Regimen Optimization 14 AS09-026 Evaluate Immunogenicity of Lm vaccines administered IM with different regimens Regimens (1) 3x q1m Prime → 4 wk →Boost → 4 wk→Boost → 6d→ ELIspot (2) 2x q1mo Prime → 4 wk→Boost → 6d→ ELIspot (3) 2x q2mo Prime→ → → → → → → 8 wk→Boost → 6d→ ELIspot (4) 2x q3mo Prime→ → → → → → → → →→ → → 12wk→Boost → 6d→ ELIspot (5) 2x Lo-Hi q1mo Low dose live (2e4) Prime→ 4 wk→Boost → 6d→ ELIspot (6) 2x KBMA-Live q1mo KBMA(1e8) Prime→ 4 wk →Boost → 6d→ ELIspot Unless otherwise noted, doses are 2e6 live BH2292 (Lm-IglC-KatG)

15. MS 57: Regimen Optimization 15 AS09-026 Notebook 2000 pp. 140-141, 158 BH2292 = IglC-B8R + KatG-SL8 in Lm677 IM administration: 2e6 cfu, low=2e4 cfu, KBMA=1e8 (IglC-tag) (KatG-tag) Low-dose prime/high-dose boost regimen is rapid and may have improved safety

16. MS 57: Route and Regimen Evaluation 16 AS10-002 Evaluate potency of Live and KBMA Lm administered IM or IV by LVS protection Regimens (Groups1-4,6-8)Prime → 4 wk →Boost → 4 wk → LVS IV Challenge (Group 5)Prime → 4 wk → LVS IV Challenge Groups 1) HBSS- IV 2) 1x10 3 LVS - IV 3) 2x10 6 Live BH2182 (Lm677:IglC) - IV 4) 2x10 4 Live BH2182 (Lm677:IglC) - IV 5) 2x10 6 Live BH2182 (Lm677:IglC) - IV single vaccination 6) 1x10 8 KBMA BH2182 (Lm677:IglC) - IV 7) 2x10 6 Live BH2182 (Lm677:IglC) - IM 8) 1x10 8 KBMA BH2182 (Lm677:IglC) -IM

17. KBMA Lm IglC Provides Survival Benefit IV but Not IM 17 AS10-002, Notebook 2000 pp. 81,105,147,154 Using 25% weight loss cutoff, KBMA Lm provides benefit IV

18. MS 57 Next Steps Repeat comparison of live Lm-IglC via multiple routes followed by 15x LD 50 LVS challenge (AS10-006) IV, IM, ID Compare KBMA Lm-IglC via multiple routes followed by 15x LD 50 LVS challenge IV, IM, ID IN LVS LD 50 Determine whether low-dose prime/high-dose boost regimen provides protection against LVS challenge 18

19. MS 59: New Antigens Progress on new antigen list? 19

20. Additional Points Deliverables completed for each active milestone: MS55: Live and KBMA Lm vaccine lots delivered to UNM for testing MS57: IM route identified as potential non-IV ROA (maybe ID now) List of relevant publications from the past month: None MSCR status MS 40, 41, 42, 44: Completed and accepted by NIAID MS 46: Under NIAID review (UNM submitted to NIAID on 3/2/10) MS 55,56,57: milestones are active MS 59: milestone not started yet MS 43, 45, 47, 58: Terminated (not initiated), no MSCR to write 20

21. Action Items: 4/16/10 Justin: check which mouse strain didn’t respond to the IglC peptide pools SJL mice had extremely low responses to the peptide pools by ELISpot, their halpotype is H2-s Added to the 4/16 minutes. Barbara: ask Phil Stafford if ready to present Felgner data analysis at the 4/27 ASU tech call. (Barbara emailed Phil on 4/16/10) Discuss KBMA Rat Protection Study Overhead Rate 21

22. New Action Items: 5/11/10 Terry requested Injection stocks of untagged IglC strain for the next rat protection study Justin said he would prepare stocks next week and send them the following week The preliminary immunogenicity data comparing this strain to the original tagged version will be sent as well Terry will send UNM’s morbidity scoring system to Justin Justin will send Rick and Terry revised Rat SchuS4 challenge study outline(s) for further discussion Cerus/Aduro requested to be updated if new vaccine candidates make it onto the list 22