Transcript Anza/Cerus
Construction and Evaluation of LiveAttenuated and KBMA Listeria monocytogenes Expressing Ft Antigens as Tularemia Vaccine Candidates TVDC Tech Call Mar 9th, 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 Lm Ft Vaccine Construct List Strain CRS-100/LM11 LM583 LM677 BH137 BH1222 BH2282 BH1228 BH1398 BH2094 BH2172 BH2098 BH2100 BH2180 BH2182 BH2316 Genetic Background actAinlB actAinlBuvrAB actAinlBuvrABprfAG155S actAinlB actAinlB actAinlB actAinlBuvrAB actAinlBuvrAB actAinlBuvrABprfAG155S actAinlBuvrABprfAG155S actAinlB actAinlBuvrABprfAG155S actAinlB actAinlBuvrABprfAG155S actAinlB 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) Sequence verified 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) BH2292 actAinlBuvrABprfAG155S BH2568 BH2594 actAinlB ActAN100-SL8-KatG-C250 (@tRNAarg) Sequence verified BH2596 actAinlBuvrABprfAG155S ActAN100-SL8-KatG-C250 (@tRNAarg) Sequence verified BH2608 actAinlBuvrABprfAG155S ActAN100-IglC-B8R (@ comK) ActAN100-SL8-KatG-C250 (@tRNAarg) Sequence verified 4 Sequence verified 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. 5 SL8 MS55 Next Steps • Characterize new IglC and KatG constructs • Intracellular expression, immunogenicity • Compare Aduro Lm-IglC immunogenicity to UCLA Lm-IglC immunogenicity 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 7 Lm Monovalent Vaccines Provide Some Protection Against SchuS4 Challenge in Rats Fisher 344 rats immunized 3 times separated by 6 weeks with: PBS, 1x107 cfu BH2172 (Lm-KatG) or 2182 (Lm-IglC), or once with 5x107 cfu LVS SC 1 month after boost, animals were challenged with ~600 cfu SchuS4 IT UNM Data provided by Terry Wu and Gloria Statom (UNM Notebook 128, pp. 119-122, 124-125, and 150 ) 8 New 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 CD4 T Cells Contribute to Lm-IglC Protection Against Lethal LVS Challenge Survival afterofLVS Challenge Survival AS09-002 BH2182 -CD4 -CD8 BH2182 -CD4 50 HBSS 0 0 5 10 days 15 100 Percent survival BH2182 BH2182 -CD8 100 Percent survival Survival afterofLVS Challenge Survival AS09-002 LVS LVS -CD4 LVS -CD8 LVS -CD4 -CD8 50 HBSS 0 0 5 10 15 days • Groups of 10 Balb/c mice were immunized 2x Q 4 weeks IV: HBSS, 2x106 Lm -IglC (BH2182), 1x103LVS • 11 weeks post boost CD4, CD8, CD4 +CD8 were administered IP @ d-6 and d-3 prior to challenge • 12 weeks post-boost 10x IV LD50 LVS challenge. • (AS09-002, Notebook 2005, pp.131, Notebook 2000, pp. 60-63). T-cell Depletion Controls Suggest Depletion was Effective No depletion Anti-CD4+ antibody (GK1.5) 10 5 10 5 8.44% 10 4 25.3% CD8 10 3 10 2 0 0 10 2 10 3 10 4 10 5 CD4 10 2 0 10 3 10 4 10 5 Anti-CD8 Antibody (2.43) ML 2004 No depletion 105 0.17% 10 3 10 2 0 105 2.16% 104 0.0014% 104 2.39% 103 CD8 13.3% 10 4 102 102 0 0 0 102 CD4 103 104 1.58% 103 105 AS09-021 Notebook #2005, pp. 173-175, 182, 186-7, 192-3 0 102 103 104 105 Review of IglC Epitope Mapping in Balb/c and C57BL/6 Mice BH2094 (actAinlBuvrAB prfA*-iglC) in Balb/c mice 180 160 Peptide #33, 34 QEYKTDEAWGIMIDL TDEAWGIMIDLSNLE CD4 140 IFNg SFC/2e5 cells 120 100 80 60 40 20 51 50 49 48 47 46 45 44 43 42 41 40 39 BH2094 (actAinlBuvrAB prfA*-iglC) in C57BL/6 mice 60 Peptide #34, 35 TDEAWGIMIDLSNLE WGIMIDLSNLELYPI CD8 50 40 30 20 10 IM08-059 51 50 49 48 47 46 45 44 43 42 41 40 39 38 37 36 35 34 33 32 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 0 1 IFNg SFC/2e5 cells 38 37 36 35 34 33 32 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 9 10 8 7 6 5 4 3 2 1 0 IglC Responses in Balb/c Mice are CD4 T Cell Restricted (but not in C57BL/6 mice) IglC pool #2 2.5 CD8 1.5 1.0 0.5 0.0 Balb/c C57BL/6 % IFN- g T cells % IFN- g T cells CD4 2.0 0.20 CD4 1.5 1.0 0.5 0.0 9 CD4 CD8 CD8 % IFN- g T cells 2.5 2.0 C57BL/6 Balb/c 33 34 0.15 0.10 0.05 0.00 34 IM08-059 35 MS56 Next Steps • Repeat comparison of live and KBMA prime-boost LVS protection study (LVS challenge with 10 LD50) (AS09-023) • If KBMA protects mice against LVS challenge should me move forward with a rat study? • Live and KBMA prime-boost LVS protection study with highdose LVS) (AS09-024) • Use less than 30X LD50 • Initiate heterologus prime-boost experiments • IglC DNA, IglC peptides as prime, followed by Lm boost. 14 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 15 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 MS57 Next Steps • Repeat route challenge with 10x 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 immunogenicity (AS10-003) and LVS protection (AS10-002) • Investigate whether ID immunization induces T cells in lymph nodes and lung • IN LD50 17 MS59: New Antigens • Reasons to Initiate MS59 • Lm IglC provides POC protection but needs improvement • KatG expresses poorly and protects only slightly • Multivalent strain likely to provide better protection in outbred populations • Potential Ft antigens: • • • • Tul4 (well characterized immunogen) ASU antigens Lipoproteins Literature searches 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 19 Status of 2/9/10 Action Items • Demonstrate good monovalent, KatG protection before additional future bivalent work is performed at Cerus/Aduro. • Cloning underway • Remove the tag from the IglC construct. • Cloning underway • Perform comparison of Dr. Horwitz strain and Aduro/Cerus strain with same route of vaccination. • ID vaccination, immunogenicity study initiated • Design the repeat rat experiment with the primary focus on the IglC monovalent strain. • Study design agreed to and initiated • Cerus/Aduro to delay the start of MS 59 till after next tech call on March 8, 2010. • Slides included in this tech call to initiate discussion Action Items: 3/9/10 • Justin: will construct IglC w/o the epitope tag, without the antibiotic resistance genes, but using the same promoter. Will test the new IglC construct for protection in mice and later rats. • Justin: MS59 will remain unopened, until a list of candidates develops after analysis of the Felgner array and other data at the end of March 2010.