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Selection of 2 subtype C gp120 proteins for Phase 2b proof-of-concept trials in RSA

Selection of 2 subtype C gp120 proteins for Phase 2b proof-of-concept trials in RSA. Susan Barnett Criteria for Selection of Envs for Upcoming HIV Vaccine Trials HVTN Full Group Meeting Washington D.C. June 2, 2011. Background Subtype C gp120s selection process within P5 framework.

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Selection of 2 subtype C gp120 proteins for Phase 2b proof-of-concept trials in RSA

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  1. Selection of 2 subtype C gp120 proteinsfor Phase 2b proof-of-concept trials in RSA Susan Barnett Criteria for Selection of Envs for Upcoming HIV Vaccine Trials HVTN Full Group Meeting Washington D.C. June 2, 2011

  2. BackgroundSubtype C gp120s selection process within P5 framework • Pox Protein Public Private Partnership (P5) • Goal: To confirm and extend the results of RV144 • Partners: BMGF, NIAID, HVTN, US MHRP, sanofi-pasteur, Novartis Vaccines • Sub C vaccines for Phase 2B South Africa (2013) • ALVAC prime will contain the ZM96 gp120-TM or gp140 • Bivalent (C/C) gp120 protein boost in MF59 adjuvant • Large scale production of gp120 proteins required (50k doses each) • gp120 monomers selected as boosts • Comparability with RV144 • Superior productivity and homogeneity • Env Selection Working Group formed to advise P5

  3. Selection of subtype C gp120 candidatesEnv proteins from 23 mostly early subtype C strains evaluated Gp120s from 23 primary sub C HIV-1’s Sequence, antigenicity, immunogenicity, 4500 neut assays (Tiers 1 &2), expression levels, integrity, stability, stable CHO lines, homogeneity of product (% monomer), etc. 2 gp120s for a bivalent boost for Ph2b POC in RSA

  4. Short-list of subtype C HIV-1 from southern Africagp120s from these strains evaluated at NVD as potential candidates • FTM – Female to Male; MTF – Male to Female • Fiebig – Stages in primary HIV infection based on presence or absence of different plasma markers • CAP45 = CAP45.2.00.G3J; CAP84 = CAP84.2.00.32J; CAP239 = CAP239.2.00.G3J

  5. Primary criteria for gp120 boost selectionMandatory criteria for advancment of candidates • Genetically representative of southern African subtype C HIV-1 strains (at least 70% aa identity) • Sufficient expression & secretion in stable CHO K1 cell lines for scaleable production • Homogeneity, identity, & integrity of protein • Monomeric (>50%), binds CD4, reactive with sub C sera • Immunogenic in small animals eliciting high titer binding Abs and neutralizing Abs

  6. Secondary criteria for Env selectionProposed (but not mandatory) based on emerging results Presence of a4b7 binding sites, specific binding to a4b7 Directly test binding of CHO-produced gp120s (J. Arthos) Demonstration of Ab that block a4b7 binding in immune sera Env binding (“antigenicity”) to known neutralizing or ADCC mAbs (CD4bs, CD4i, V2, V3, quarternary) Recognition of epitopes in immune sera similar to those seen RV144 vaccinee sera mAb competition ELISA (recognition of known neut & ADCC epitopes)

  7. Sequence analysis – Phylogeny & presence of motifsPhylogeny (PHYML protein-based) & Integrin 47 motif Analysis by Bette Korber Lack of Integrin 47 motif TV1 ZM249.PL1 1086.C Du156.12 CAP45 Du422.1 CAP84 CAP239 • Jim Arthos tested the binding of 293HEK produced gp120s - as expected, they did not bind Integrin 47 • CHO-produced gp120s to be tested for 47-binding

  8. All gp120s bind to regional sub C HIV+ sera (RSA)Binding of gp120s to purified anti-subtype C IgG (low-µM affinity) by SPR Binding affinity in µM to purified IgG from SubC HIV-positive donors from RSA gp120s

  9. Antigenicity of 293HEK produced subtype C gp120sSummary of SPR analysis • All subtype C gp120 Envs bind sCD4, b12 (except TV1 – weak binding) and undergo CD4-induced conformation change

  10. Immunogenicity evaluationsSmall animal studies performed with 293HEK produced gp120s • Immunogenicity studies conducted in guinea pigs & rabbits with 25 mcg doses of proteins in MF59 • Assays performed with 2 wk post-3rd & 2 wk post-4th sera • ELISA binding Abs, avidities (Novartis) • MAb competition ELISA to map epitope recognition (J. Heeney lab) • Mab competition assay to map epitope recognition (B. Haynes lab) • Virus neutralization in TZM-bl and A3R5 (D. Montefiori lab)

  11. Immunogenicity: High titer Abs in rabbitsELISA Ab titers >1,000,000; high avidity • 25µg gp120 (+MF59) immunized, 5 rabbits/group, at weeks 0, 4, 12 & 24 – sera analyzed 2weeks-post 2nd, 3rd & 4th immunizations for Ab-binding & viral-neutralization

  12. Subtype C gp120 Immunization in Rabbits2wp4 neutralization breadth (ID50) TZM assay (Tier 1& 2)

  13. Weighted means analysis of virus neutralization (D. Montefiori; P. Gilbert) Goal to summarize titers across species, time points, and assays to provide a ranking of gp120 immunogens (>4000 assays performed) Neutralization titers combined across 6 panels Guinea Pig, post 3rd and 4th, TZM-bl Rabbit, post 3rd and 4th, TZM-bl Rabbit, post 3rd and 4th, A3R5 For each immunogen computed a weighted mean (and 95% CI): Area under the magnitude-breadth curve (AUC-MB) was computed for each animal across viruses assayed within a panel Average AUC-MB across animals within a panel Calculate the weighted average of mean AUC-MB values Rank immunogensby weighted means.

  14. Within panel mean area under curve- magnitude breadth (AUC-MB) analysis Tier 1, 6 viruses TZM-bl Tier 1, 6 viruses TZM-bl Tier 1B (n=1) plus Tier 2 (n=5)

  15. Summary of virus neutralization resultsWeighted means and 95% CIs

  16. Immunogenicity: mAb competitionCompetition of rabbit sera mAbs A32 & CH01 and sCD4 (B. Haynes lab) • TV1 and ZM249.PL1 gp120 immunized rabbit sera shows the highest competition for mAb A32 (similar to results in guinea pigs)

  17. Monomer/dimer quantification excludes ZM249 Purified protein from suspension cultures of stable CHO lines dimer monomer ZM249M.PL1 M 29-35% CAP239 M 67-84% monomer dimer monomer TV1 M 89-100% dimer

  18. Expression & antigenicity of 1086.C gp120Transmitted founder virus from female in Malawi AHI cohort (B. Haynes) • Purified 1086.C gp120 monomers were more antigenic than 1086C trimers with regards to CD4BS, V2, A32/C1, quaternary epitopes, 2G12 CHO-epitope. • CHO-produced 1086.C gp120 binds 47 integrin Marker 1086C 63521 MN 250 150 dimer monomer 100 75 50 37 25 Non-reducing SDS-PAGE

  19. Comparative immunogenicity in macaques of trimeric gp140s from 1086.c and TV1 in MF59 6 Tier 2 sub C HIV (A3R5 assay) Key data for 1086.C gp120 protein in MF59 adjuvant In macaque study expected in June.

  20. Summary of subtype C gp120 candidatesUpdated since ESWG meeting on March 15, 2011

  21. Summary and conclusion • Evaluations to date of sub C gp120 candidates indicate that TV1 & CAP239 are viable candidates • Met all criteria of sequence, antigenicity, immunogenicity • Expression levels & monomer fraction in CHO cell lines • Strains are phylogenetically & phenotypically complementary • Critical data for 1086.C gp120 will be available shortly (immunogenicity, expression & monomer in stable CHO) • Selection of 2 final candidates now is critical to maintaining timelines for large scale manufacturing for Phase 2b proof of concept trials in RSA

  22. Acknowledgements Pox Protein Public Private Partnership (P5) Nina Russell (BMGF) Francine McCutchan (BMGF) Jose Esparza (BMGF) Susan Barnett (NVD) Niranjan Kanesa-thasan (NVD) Jim Tartaglia (s-p) Sanjay Garunathan (s-p) Jerome Kim (MHRP) Nelson Michael (MHRP) James Kublin (HVTN) Larry Corey (HVTN) Alan Fix (NIAID) Katherine Kripke (NIAID) Peggy Johnston (NIAID) Song Ding (CHUV) Giuseppe Pantaleo (CHUV) Jim Lane (NIAID) Siobhan Malone (BMGH) Env Selection WG (ESWG) Michael Pensiero (NIAID) Francine McCutchan (BMGF) Jim Tartaglia (s-p) David Montefiori (Duke) Georgia Tomaras (Duke) Bart Haynes (Duke) Rob O’Connell (MHRP) Jose Esparza (BMGF) Julie McElrath (HVTN) Carolyn Williamson (UCT) Contributors to ESWG Celia Labranche (Duke) Robert Parks (Duke) Larry Liao (Duke) Sydeaka Watson (Baylor) Bette Korber (LANL) Alan DeCamp (SHARP/VISC) YouYi Fong (SHARP/VISC) Peter Gilbert (SHARP/VISC) Vijay Mehra (NIAID) Patricia D’Souza (NIAID) Jim Bradac (NIAID) NIH Grants & Contracts AI066287, HHSN266200500007C

  23. AcknowledgementsNovartis Vaccines and Diagnostics, Inc. Research Team Avishek Nandi Samuel Stephenson Karin Hartog Harold Legg Susan Hilt Yide Sun Karen Matsuoka Mark Wininger Frank Situ Jimna Cisto Priyanka Ramesh Pampi Sarkar DeeAnn Martinez-Guzman Luis Brito Manmohan Singh Kaustuv Banerjee Gib Otten Carlo Zambonelli Antu Dey Indresh Srivastava (now at VRC) Andrea Carfi HIV Global ProgramTeam Emanuela Palla Niranjan Kanesa-Thasan  Fred Porter Mary Wu Susan Barnett. Project Management & Contracts Brian Burke Laurie Peltier Charles Gerken Randy Deck Legal, & IP Maureen Rogers Helen Lee Regina Bautista Marcus Dawson Vaccine Research Leadership Jeffrey Ulmer Christian Mandl Rino Rappuoli

  24. Back up slides

  25. Env-pseudotyped viruses assayed in TZM-bl cells Tier 1 Clade Tier 2 Clade MW965.26 C TV1.21* C MN.3 B Du156.12* C SF162.LS B Du422.1* C Bx08 B ZM197M.PB7 C SS1196.1 B CAP45.2.00.G3 C SHIV-Bal.P4 B ZM249M.PL1* C ZM214M.PL15 C Du172.17 C Ce1086_B2* C 6535.3 B QH0692.42 B *Vaccine strains

  26. Env.IMC.LucR viruses assayed in A3R5 cells Virus Clade Tier CAP45.2.00.G3 C 1B Ce1086_B2* C 2 Du151.2 C 2 Ce1176_A3 C 2 Ce2010_F5 C 2 Du422.1* C 2 *Vaccine strains

  27. Aggregate NAb Response Against Tier 1 Viruses (n=6) in Rabbits and Macaques (TZM-bl assay) post 4th post 3rd post 4th Geometric mean titer Rabbits Macaques

  28. Aggregate NAb Response Against Subtype C Viruses (n=6) in Rabbits and Macaques (A3R5 assay) post 4th post 3rd post 4th Geometric mean titer Rabbits Macaques

  29. Aggregate NAb Response Against Subtype C Tier 2 Viruses (n=6) in TZM-blcells (post-3rd in rabbits) Autologous positive

  30. Summary of results (Mar 15, 2011) Subtype C gp120s (HEK293T) rank-ordered based on specific criteria * Based on neutralization, Ab binding titers, avidity and mAb cross-competition

  31. Potential N-linked Glycosylation (PNLG) in V1/V2 180 140 160 Du156.12: NCVTYN----NSMNSSAT--YNNS---MNGEIKNCSFNTTTELRDKKQKVYALFYRTDVVPLNNNN-NNS--EYILIN Du422.1: NCKNVN----ISANANATATLNSS---MNGEIKNCSFNTTTELRDKKQKVYALFYKPDVVPLNGGE-HNETGEYILIN EF203960: RCT--N----ATIN--------GS---LTEEVKNCSFNITTELRDKKQKAYALFYRPDVVPLNKNSPSGNSSEYILIN EF203963: NCT--N----ANISVTSVSNDSRN---TNEEIKNCSFNTTTEIRDKKQQVYALFYRSDVAPLSK----ANSSEYILIN EF203983: NCST-----------NITKANVTS-----EEMRSCSFNITTELRDKKQKVNALFYKLDIVPINESSS---SSEYRLVN TV1c8.2: NCTDTNVTGNRRTVTGNSTNNTNGTGIYNIEEMKNCSFNATTELRDKKHKEYALFYRLDIVPLNENSD—NFT-YRLIN ZM249M.PL1: NCNNV------NVTHNSTYNNTEG-----EQIKNCSFNITTELRDKKQKVYALFYKLDILPLNGNND---SNEYRLIN ZM96: NCTEVNTRNVNNSVVNNTTNVNNSMNGDMKNCSFNITTELKDKKKNVYALFYKLDIVSLNETDDSETGNSSKYYRLIN • PNLG highlighted in red • a4b7 binding site in blue • Cathepsin site in all sequences • Most CladeC Envshave PNLG at 158 & 160 (for PG9 & PG16 recognition). .

  32. Rabbit studies with CHO derived sub C gp120s (1)Objective: To evaluate immunogenicity of monomeric and dimeric gp120s

  33. Rabbit studies with CHO derived sub C gp120s (2)Objective: To evaluate bivalent-immunization using CHO-produced gp120s

  34. DNA & NYVAC prime- Env protein boost in NHPStudies to support research Ph2B POC trials in RSA (G. Pantaleo) NYVAC-C-KC is a highly attenuated replication competent strain. NYVAC-C is replication incompetent. NYVAC vectors have ZM96 gp140 insert.

  35. Elicitation of virus neutralizing Abs in NHPTV1 gp120 NAbs titers comparable to TV1 gp140 trimer Tier 1 viruses assayed in TZM-bl cells

  36. Elicitation of virus neutralizing Abs in NHPTV1 gp120 NAbs titers comparable to TV1 gp140 trimer Tier 2 viruses assayed in A3R5 cells

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