Transcript Anza/Cerus
Construction and Evaluation of LiveAttenuated and KBMA Listeria monocytogenes Expressing Ft Antigens as Tularemia Vaccine Candidates TVDC Tech Call Feb 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: Construct and Measure Cellular Immunogenicity of Lm-Ft Vaccines Construct epitope-tagged Lm vaccine candidates expressing IglC or KatG Measure antigen expression from live-attenuated strains in cells Prepare stocks of KBMA vaccines Characterize immunogenicity of Live-attenuated vaccine candidates Measure metabolic activity and antigen expression in cells Construct bivalent vaccine candidates Characterize immunogenicity of KBMA vaccine candidates Characterize immunogenicity of bivalent Live-attenuated vaccine candidates Work in progress Work to be initiated 3 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 pending • Membrane-targeted KatG and IglC expression cassettes were constructed • Surface-targeted KatG immunogenicity was not improved 4 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 BH2292 actAinlBuvrABprfAG155S BH2568 BH2594 BH2596 BH2608 actAinlBuvrABprfAG155S actAinlB actAinlBuvrABprfAG155S actAinlBuvrABprfAG155S BH2683 BH2697 actAinlBuvrABprfAG155S actAinlBuvrABprfAG155S BH2699 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) 5 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 Sequence verified Sequence verified Sequence verified Sequence verified Lm Bivalent Vaccine Candidates actAp construct at tRNAArg ActAN100 KatG SL8 actAp construct at comK IglC ActAN100 IglC B8R B8R BH2292 Kat G SL8 ActAN100-KatG-SL8 at tRNAArg: ActAN100 KatG SL8 IglC B8R Kat G SL8 CTD ActAN100-B8R-IglC-C175 at comK: ActAN100 BH2699 B8R IglC ActAC175 ActAN100-IglC-SL8 at tRNAArg: IglC ActAN100 SL8 CTD ActAN100-B8R-IglC-C175 at comK: ActAN100 IglC B8R BH2697 B8R IglC ActAC175 6 IglC SL8 Intracellular IglC Antigen Expression from Live Lm not Improved by Membrane Targeting Strain Antigen cassette(s) 98- Lm11 - BH2094 IglC-SL8 62- BH2182 IglC-B8R BH2292 IglC-B8R + KatG-SL8 BH2608 IglC-B8R + KatG-SL8-C250 BH2683 B8R-IglC-C175 BH2697 B8R-IglC-C175 + IglC-SL8 BH2699 B8R-IglC-C175 + KatG-SL8 4938- IglCs 281714- IglCm NB837, pp. 42-43 J774 Macrophages Infected with Lm at MOI of 10 for 7 hours 7 Semi-Quantitative Multiplex Intracellular IglC Western to Measure Antigen Expression Strain Antigen cassette(s) IglC p60 IglC/ p60 Lm11 - 0 1.3 - BH2094 IglC-SL8 46.3 1.1 42 BH2182 IglC-B8R 48.4 1.6 30.3 BH2292 IglC-B8R + KatG-SL8 21.8 1.1 19.8 IglCs 38- BH2608 IglC-B8R + KatG-SL8-C250 13.7 .8 17.1 281714- BH2683 B8R-IglC-C175 3.5 1.6 2.2 BH2697 B8R-IglC-C175 + 10.1 IglC-SL8 4.7 .5 20.2 9.4 BH2699 B8R-IglC-C175 + 15.9 KatG-SL8 1.8 8.8 9862p60 49- IglCm 8 NB837, pp. 42-44 Metabolic Activity of New KBMA Lots Metabolic activity of KBMA strains 2.5 OD 490 nm 2 1.5 BH2094 KBMA BH2683 KBMA 1 BH2182 KBMA 0.5 BH2697 KBMA 0 0 1 2 3 4 5 6 7 NB #2000, pp 68-70 Time (hrs) • Activity of ~1e7 particles as measured by MTS 9 IglC Immunogenicity from KBMA Lm Not Improved with Membrane Targeting IglC 33-17 responses AS09-027 NB2005, pp181, 202-204 IFN--SFC/2e5 splenocytes 60 unstim 33-17 1e8 KBMA particles IV 40 BH2094: IglCs-SL8 in Lm677 BH2683: IglCm-B8R in Lm677 BH2182: IglCs-B8R in Lm677 BH2697: IglCs-SL8 + IglCm-B8R in Lm677 20 0 • ELIspot performed 7 days after single IV vaccination 10 Immunogenicity to Epitope Tag Not Lower When Membrane Targeted AS09-027 NB2005, pp181, 202-204 1e8 KBMA particles IV B8R responses 200 100 0 unstim B8R 400 IFN--SFC/2e5 splenocytes IFN--SFC/2e5 splenocytes 300 SL8 responses unstim SL8 300 200 100 0 11 BH2094: IglCs-SL8 BH2683: IglCm-B8R BH2182: IglCs-B8R BH2697: IglCs-SL8 + IglCm-B8R KBMA Immune Responses Improved After Boost Vaccination IFN- SFC/2e5 splenocytes 800 IglC 33-17 responses B8R responses SL8 responses 800 unstim SL8 200 unstim B8R 600 600 150 400 400 100 200 200 50 0 0 0 unstim 33-17 AS09-019 NB2005, pp 178, 197-201 • BH2292 = Lm677: KatG-SL8 + IglC-B8R (KBMA is old lot) • KBMA-Lm primary responses low, but improve after a boost 12 MS55 Next Steps • Compare metabolic activity of old KBMA lots (>1yr, with new lots) • Improve expression of KatG from Lm by deleting hydrophobic regions? • Compare Aduro Lm-IglC immunogenicity to UCLA Lm-IglC immunogenicity IV and ID? • Aduro will also construct Lm-IglC vaccine in actA/uvrAB background 13 MS 56: Demonstrate that Lm Vaccines Induce Protective Cellular Immune Responses to Ft Antigens Construct IglC 15/11 overlapping peptide library Vaccinate mice with Lm-IglC and screen for IglC responses by ICS and ELISpot Prepare stocks of KBMA Lm vaccine Compare Lm and Ft -induced IglC specific T cell responses Compare Live and KBMA IglC responses in mice Perform LVS challenge studies to determine whether KBMA Lm vaccines protect Perform LVS challenge studies to determine whether live Lm vaccines protect Perform T cell depletion studies to determine mechanism of protection Prepare stocks of live attenuated vaccine Send KBMA Lm vaccines To UNM for SchuS4 challenge studies Send Live-attenuated Lm vaccines To UNM for SchuS4 challenge studies 14 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 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 an SchuS4 challenge in mice 15 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 ) 16 New Rat SchuS4 Protection Study Design Regimen Prime → 6 wk → Boost → 4 wk → SchuS4 IT Challenge Groups SC- PBS SC- LVS (No boost) IM- BH2182 (Lm677:IglC) IM- BH2292 (Lm677:IglC/KatG) ID- BH2292 (Lm677:IglC/KatG) IM- BH2316 (Lm11:IglC/KatG) CD8 T-cell Depletion Problem Corrected Using In-House Prepared Antibody 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 CD4 10 3 10 4 AS09-021 0.17% 10 3 10 2 0 CD8 13.3% 10 4 10 5 0 10 2 10 3 10 4 10 5 Anti-CD8 Antibody (2.43) LL 10/6/06 No depletion Anti-CD8 Antibody (2.43) ML 2004 105 105 105 104 104 104 103 103 103 102 102 102 0 0 0 0 102 CD4 103 104 105 0 102 103 104 105 0 102 103 104 105 MS56 Next Steps • T-cell depletion study ongoing(AS09-002): • Mice vaccinated with Lm-IglC or LVS • CD4, CD8, and CD4+CD8 cells depleted. • 10 LD50 LVS challenge administered • 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) • Initiate heterologus prime-boost experiments • IglC DNA, IglC Peptides as prime, followed by LM boost. 19 MS 57: Optimization of Lm Vaccination Route and Regimen Compare immunogenicity of live-attenuated Lm after vaccination by various routes using ICS and ELISpot Prepare stocks of KBMA Lm vaccine Select non-IV route Compare Live and KBMA responses in mice Perform LVS challenge studies to determine if alternative routes of administration are protective Perform LVS challenge studies to determine whether KBMA Lm vaccines protect Optimize vaccination regimen by Varying time between prime and boost UNM to performSchuS4 challenge studies after vaccination by alternate route UNM to performSchuS4 challenge studies after vaccination by alternate route and regimen 20 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 by ELIspot 21 MS57 Next Steps • Evaluate whether mice vaccinated via different routes are protected against 20 x LD50 LVS challenge (AS09-011) • Challenge this week • IM vaccination for regimen optimization (AS09-026) • Vaccinate 1x106 IM 2x Q1M, Q2M, Q3M vs. 3xQ1M, 2x104 prime, KBMA prime, • Perform ICS and ELIspot analysis • Evaluate KBMA IM by immunogenicity and LVS protection • IN LD50 22 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 List of relevant publications from the past month: None MSCR status MS 40, 41, 42, 44: Completed and accepted by NIAID MS 46: UNM reviewing MSCR (Cerus/Aduro sent edits to BG on 12/18/09) 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 24 2/9/10:Action Items 1 of 2 slides • Justin- please add arrows to images (slide 7) to show what bands are expected to be seen. (completed 2/9/10 for slides 7 &8) • Moving forward, Rick requests Justin/ Aduro to show good monovalent, KatG protection before additional future bivalent work is performed at Cerus/Aduro. This request excludes Cerus/Aduro’s ongoing, previously initiated bivalent studies in mice. Should Aduro/Cerus identify two protective proteins, a reasonable strategy may be a bivalent strain (e.g. Kat G and IglC) . • Justin plans to perform the parallel comparison of Dr. Horwitz strain and Aduro/Cerus strain with same route of vaccination 2/9/10:Action Items 2 of 2 slides • Justin will add a slide showing all the strains and their backgrounds (completed 2/9/10). • Justin: To compare the platforms in rats and confirm the IglC protection in rats, UNM and Cerus collaboratively are designing the next rat experiment. The primary focus is on the IglC monovalent strain in the next study. • Cerus/Aduro will delay the start of MS 57 till after next tech call on March 8, 2010. • Rick and Justin’s follow up call 2/10/10: For NHP vaccine efficacy studies, the tag will be removed from the IglC construct. Prior to the NHP studies, the IglC (without tag) will be confirmed in rats first. This series of experiments will be discussed at a future Cerus/Aduro technical call.