Transcript Anza/Cerus
Construction and Evaluation of LiveAttenuated and KBMA Listeria monocytogenes Expressing Ft Antigens as Tularemia Vaccine Candidates TVDC Tech Call April 16th, 2010 1 Cerus/Aduro Milestones • Milestone 55: Compare Cellular Immune Responses Induced by Lm-Based Tularemia Vaccines • • • • 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 • • • • Construct vaccine candidates Measure cellular immunogenicity of live-attenuated vaccines using model epitopes Compare immunogenicity of KBMA tularemia vaccines using model epitopes 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 MS 55: Key Achievements • Compare Cellular Immune Responses Induced by Lm and FtBased Tularemia Vaccines • Lm expressing epitope-tagged IglC or KatG were cloned • 3 platforms: actAinlB, actAinlBuvrAB, actAinlBuvrABprfAG155S • Intracellular expression of IglC was higher than KatG • CD8 T cell responses were evaluated by B3Z assay, ICS, and ELISpot • CD8 T cell responses to SL8 were stronger when fused to IglC than KatG • prfAG155S enhanced immunogenicity of IglC-SL8 vaccine • LVS-PepO-SL8 did not induce SL8 response or boost Lm SL8 response • Bivalent strains expressing both IglC and KatG were evaluated • Intracellular expression and immunogenicity were similar to monovalent strains • KBMA Lm-IglC-SL8 primary responses were lower than live after prime • Boost improved KBMA responses • Membrane-targeted KatG and IglC expression cassettes were constructed • immunogenicity was not improved with live or KBMA 3 Proposed Modification of IglC and KatG Constructs 1) Remove epitope tag from IglC construct actAp ActAN100 actAp IglC SL8 ActAN100 IglC 2) Remove hydrophobic regions from KatG 1 2 ActAN100 KatG (∆1-23) • Design primers to make in-frame deletions of regions 1 and 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. 4 SL8 Lm Ft Vaccine Construct List Strain CRS-100/LM11 LM583 LM677 BH137 BH1222 BH2282 BH1228 BH1398 BH2094 BH2172 BH2098 BH2100 BH2180 BH2182 Genetic Background actAinlB actAinlBuvrAB actAinlBuvrABprfAG155S actAinlB actAinlB actAinlB actAinlBuvrAB actAinlBuvrAB actAinlBuvrABprfAG155S actAinlBuvrABprfAG155S actAinlB actAinlBuvrABprfAG155S actAinlB actAinlBuvrABprfAG155S Antigen Cassette none none none ActAN100-Ova ActAN100-IglC-SL8 ActAN100-KatG-SL8 ActAN100-IglC-SL8 ActAN100-KatG-SL8 ActAN100-IglC-SL8 ActAN100-KatG-SL8 ActAN100-IglC-VacQuad-SL8 ActAN100-IglC-VacQuad-SL8 ActAN100-IglC-B8R (@ comK) ActAN100-IglC-B8R (@ comK) ActAN100-IglC-B8R (@ comK) ActAN100-KatG-SL8 (@tRNAarg) ActAN100-IglC-B8R (@ comK) ActAN100-KatG-SL8 (@tRNAarg) ActAN100-KatG-C250 (@tRNAarg) ActAN100-SL8-KatG-C250 (@tRNAarg) ActAN100-SL8-KatG-C250 (@tRNAarg) ActAN100-IglC-B8R (@ comK) ActAN100-SL8-KatG-C250 (@tRNAarg) ActAN100-B8R-IglC-C175 (@tRNAarg) ActAN100-B8R-IglC-C175 (@comK) ActAN100-IglC-SL8 (@tRNAarg) ActAN100-B8R-IglC-C175 (@comK) ActAN100-KatG-SL8 (@tRNAarg) Status Sequence verified Sequence ve Sequence verified Sequence verified Sequence verified Sequence verified Sequence verified Sequence verified Sequence verified Sequence verified Sequence verified Sequence verified Sequence verified Sequence verified Remade and verified (BH2184 had point mutation in KatG) BH2316 actAinlB BH2292 actAinlBuvrABprfAG155S BH2568 BH2594 BH2596 actAinlBuvrABprfAG155S actAinlB actAinlBuvrABprfAG155S BH2608 actAinlBuvrABprfAG155S BH2683 actAinlBuvrABprfAG155S BH2697 actAinlBuvrABprfAG155S BH2699 actAinlBuvrABprfAG155S BH2932 actAinlBuvrABprfAG155S ActAN100-KatG hyd2-SL8 Sequence verified BH2934 actAinlBuvrABprfAG155S ActAN100-KatGhyd1-SL8 Sequence verified BH2936 actAinlBuvrABprfAG155S ActAN100-KatGhyd1hyd2-SL8 Sequence verified BH2938 actAinlBuvrABprfAG155S ActAN100-IglC(no tag) Sequence verified 5 Sequence verified Sequence verified Sequence verified Sequence verified Sequence verified Sequence verified Sequence verified Sequence verified MS55 Next Steps • Characterize new IglC and KatG constructs • Intracellular expression, immunogenicity, LVS challenge • If KatG immunogenicity is improved, then construct new bivalent strain expressing IglC + KatG 6 MS 56: Key Achievements • Demonstrate that Lm Vaccines Induce Protective Cellular Immune Responses to Ft Antigens • Lm-IglC induced cellular immune responses to IglC peptides in Balb/c, C57BL/6, FVBN, and C3H/HeJ mice • Responses were CD4+, CD8+, or both depending on the haplotype • IglC-specific epitopes were mapped in C57BL/6 and Balb/c mice • • • • Lm-IglC induced stronger IglC responses than LVS Lm-IglC protected 100% of mice against 10 LD50 LVS challenge Lm-IglC did not protect mice against 100 LD50 LVS challenge Live and KBMA Lm vaccine lots were produced and shipped to UNM and are being used for SchuS4 challenge studies • Lm IglC did not protect against SchuS4 challenge in mice • Lm IglC provided 75% protection against a SchuS4 challenge in rats • Lm-IglC protection against LVS challenge is T cell dependent • CD4 T cells in Balb/c mice 7 Comparison of Immune Responses Induced by BH2182 and Horwitz Lm-IglC Strains IglC pool1+2 responses unstim 33-17 20 10 40 35 30 unstim IglC pools 10 5 0 0 Spleen BH2182 AS10-005 Notebook 2000, pp. 82-84 LN Spleen LN Horwitz strain 400 IFN-g SFC/2e5 splenocytes 100 80 60 40 30 IFN-g SFC/2e5 splenocytes IFN-g SFC/2e5 splenocytes IglC 33-17 responses LLO190-201 responses unstim LLO 190 300 200 100 0 Spleen LN Spleen BH2182 LN Horwitz strain Spleen LN Spleen BH2182 LN Horwitz strain AS10-005 Both strains given ID at 5e7 CFU Spleens and lymph nodes harvested 7 days post single vx Pooled LN within a group because of low cell yield 8 AS09-023 Protection Study • Comparison of Live and KBMA IglC (BH2182) and IglC+KatG (BH2292) vaccines • Prime-boost separated by 4 weeks • Attempted 10x IV LD50 challenge after 4 weeks • Delivered ~5X LD50 dose • No Death Observed (3 negative control mice euthanized) Study number Mouse strain Route LD50 Date DOB AS09-004 Balb/c IV 4.05E+04 10/26/2009 8/9/2009 AS09-022 Balb/c IV 2.04E+04 1/11/2010 10/20/2009 AS10-014 C57BL/6 IV 3.34E+04 3/30/2010 1/1/2010 9 Age of mice Notebook # 10 weeks NB #2005, p124 12 weeks NB #2005, pp188-189 12.5 weeks NB #2000, pp112-113 AS09-023 Weights Post-Challenge 120% 80% 60% 40% 20% 0% 120% 0 Grp 1: HBSS 5 100% 10 15 Time (day) 40% 20% Grp 2: LVS 0 5 100.00% 1186 120% 0 1181 100% 652 653 80% 654 1199 60% 655 40% 656 20% 657 658 120% 15 1188 100% 1187 662 80% 490 491 60% 492 40% 493 494 20% 495 496 0% 10 Time (day) 2 4 6 8 10 12 1185 194 195 196 197 1192 198 637 638 639 Time (day) 0% Grp 5: BH2292 0 2 4 6 8 Time (day) Weight (g) Weight (g) 80.00% 60.00% 40.00% 0.00% Grp 4: BH2182 Grp 3: ANZ-100 0 2 4 6 Time (day) 8 10 12 0 120% 10 118312 100% 640 641 80% 642 643 60% 1184 644 40% 645 20% 646 647 0% Weight (g) 60% 20.00% 1197 1189 497 498 499 1182 500 191 192 193 Weight (g) Weight (g) 80% 0% 120.00% 1190 100% 353 80% 354 355 60% 484 1191 40% 485 486 20% 487 488 0% Weight (g) Weight (g) 100% Grp 6: BH2182 KBMA 2 4 6 Time (day) 10 8 10 Grp 7: BH2292 KBMA 12 0 5 10 Time (day) 15 Update on 2nd Rat SchuS4 Protection Study Design Regimens Prime → → 6 wk → Boost → 4 wk → SchuS4 IT Challenge Prime → 2 wk → Boost → 4 wk → SchuS4 IT Challenge Groups 1) PBS - SC 2) 5x107 LVS (No boost) - SC 3) 1x108 BH2182 (Lm677:IglC) - IM 4) 1x108 BH2182 (Lm677:IglC) - ID 5) 1x106 BH2182 (Lm677:IglC) - IM 6) 1x108 BH2182 (Lm677:IglC) – IM 2 week p/b week regimen 7) 1x108 BH1222 (Lm11:IglC) - IM 45 injection stock vials of Strain BH1222 (Lm actAinlB:IglC-SL8) lot number is 837-047 were shipped to UNM on 2/17/09 11 MS56 Next Steps • Repeat comparison of live and KBMA prime-boost LVS protection study (LVS challenge with 15 LD50) (AS09-024) • If KBMA protects mice against LVS challenge should we move forward with a rat study? • Evaluate heterologus prime-boost experiments • IglC DNA, IglC peptides as prime, followed by Lm boost. 12 MS 57: Key Achievements • Optimization of Lm Vaccination Route and Regimen • IV vs Oral route compared • T cell responses in spleens were higher after IV administration • Mucosal T cell responses were low, but similar after IV and oral administration • Single dose of Lm-IglC administered IM,SC,ID, and Orally induced measurable cellular immune responses, but were lower than IV • After boost vaccination, IM appears to be comparable to IV • ID and SC responses were also significantly boosted, but lower 13 Protection Against LVS Lethal Challenge by Lm-IglC may Depend on Route • 30% of the mice vaccinated ID survived • 10% of the mice vaccinated IM, IV or ID survived • IM and ID groups had a 1d increase in median survival time 14 Pilot Study to Measure IglC Immune Responses in Lungs of Vaccinated Mice IglC pool 2 responses in lung 600 IFN-g SFC/2e5 splenocytes IFN-g SFC/2e5 splenocytes IglC pool 2 responses in spleen unstim IglC pool 2 400 200 0 IN 100 unstim 80 IglC pool 2 60 40 20 0 -20 IN ID ID AS10-011 Notebook 2000 p. 90 and pp.100-102 IN = BH2182 2x either IV, IM, or SC followed by LVS challenge, then BH2182 IN ID = BH2182 2x ID followed by LVS challenge, then BH2182 ID 15 Immunogenicity in Spleen, Lung and Blood after Single Vaccination via Various Routes • Mice vaccinated with BH2182 • 5e6 IV or IM • 5e7 ID IN 16 AS10-015 Notebook 2000, p120-121 Comparison of Live and KBMA Immune Responses After IM vs. IV Delivery AS10-003 Notebook 2000 pp. 116-117 17 MS57 Next Steps • Repeat route challenge with 15x LD50 LVS (AS10-006) • IM vaccination for regimen optimization (AS09-026) • Vaccinated 1x106 IM 2x Q1M, Q2M, Q3M vs. 3xQ1M, 2x104 prime, KBMA prime, • Perform ICS and ELISpot analysis • Evaluate KBMA IM by and LVS protection (AS10-002) • IN LD50 18 MS59: New Antigens • Progress on new antigen list? 19 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 Action Items from 3/9/10 • Aduro has constructed IglC w/o the epitope tag, and will test the new IglC construct for protection in mice and later rats. • MS59 remains unopened, until a list of candidates is developed after analysis of the Felgner array and other data. 21 Action Items: 4/16/10 • Justin will check which strain didn’t respond to the iglC peptides and we’ll add the strain name to the minutes of this meeting. • 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)