Why are validated immunogenicity assays important for HIV vaccine development?
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- Britton Tucker
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1 Why are validated immunogenicity assays important for HIV vaccine development? There is a need to compare immunogenicity of products in the pipeline, when similar or different in class when developed by more than one sponsor when tested in separate trials
2 HVTN Laboratory Program Taken a centralized approach to employ validated endpoint assays in a GLP laboratory setting to support licensure of candidate vaccines. Established a QA/QC unit, built on industry models, and an operations program to oversee these activities.
3 Moving from Assay Development to Protocol Endpoint Studies Developmental Studies Optimization and Qualification External Laboratory Scientific Advisory Committee Assay Validation Proficiency Testing Endpoints Assays Immune Monitoring Labs
4 HVTN validated assays currently used to define vaccine immunogenicity, both qualitatively and quantitatively: optimized, validated, performed in GCLP labs Antibodies: 1) binding ELISA 2) neutralizing--tzm-bl pseudovirion assay CD8+ and CD4+ T cells: 1) IFN- ELISpot 2) intracellular staining for IL-2 and IFN- by 8-color flow cytometry Other protocol support assays: 1) HIV diagnostic algorithm 2) Adenovirus serotype 5 neutralizing Ab (Merck)
5 Assay Development and QA Program Highest quality specimens from multiple clinical sites, domestic and international: cryopreservation within 8 h of venipuncture, shipping on LN2 or dry ice (M Bull et al, J Immunol Methods, in press) Validated T cell immunological assays: IFN- ELISpot (N Russell et al, J Infect Dis, 2004); 8 (14)-color ICS (H Horton et al, J Immunol Methods, in press) Assembly and proficiency testing of relevant cross-protocol and cross-clade peptides: Global and clade-specific PTE panel (F. Li et al, Vaccine 2006; U Malhotra et al, J Virol, in press)
6 Comparison of T Cell Responses in PBMC Cryopreserved within 6 vs 24 Hours After Venipuncture in 18 subjects IFN- -Secreting T Cells (ELISpot) Recognizing CMV, EBV, Flu (9-mers) and CMV (15-mers) Antigen Anticoagulant Median SFC Per 2x10 5 PBMC 6 vs 24 h P Value CEF (9-mers) ACD EDTA Hep vs vs vs 54.1 <0.0001* * CMV (15-mers) M Bull et al, J Immunol Methods, in press ACD EDTA Hep vs vs vs * <0.0001* * Immediate Blood Processing Necessary!
7 Site Affiliated Labs for PBMC Processing and Cryopreservation ~25 site affiliated labs established internationally Training Proficiency Testing All technicians undergo proficiency testing prior to specimen processing in HVTN protocols Internal Quality Control (IQC) Daily entry of PBMC yields and processing times Charts sent to HVTN Lab Ops monthly for review External Quality Control (EQC) random clinical trials samples selected for assessment of recovery and viability from each site monthly All data upon PBMC thawing for endpoint assays is compiled and analyzed by protocol and by site
8 % PBMC Viability % Viability >80% Proficiency Criteria Overall Distribution Site Number Median <66% 66% to 80% >80% 93.4% 0.5% 3.3% 96.1% (3/570)
9 Quality Monitoring of SALs Three recent HVTN phase I protocols, 10 participating SALs Variable Venipuncture to freezing time Processing time PBMC yields from fresh blood % viability (day 2 post thaw) % recovery (day 2 post thaw) Measurement 210 minutes* 163 minutes* 1.7 x 10 6 cell/ml* 93%* 71%** * median; **mean
10 T Cell Assay Validation and Proficiency Studies Assays: IFN- ELISpot, 8-color ICS Donors: Seattle control cohort of 30 HIV+, 50 HIV-; Leukapheresis every 6-12 months; blood for PBMC and sera obtained longitudinally and cryopreserved
11 Concordance of CMV responses
12 Comparison of Validated BD vs MM ELISpot Assays: MM Assay more sensitive BD kits MabTech/Millipore
13 Summary of Major Differences in the BD and MM IFN- ELISpot Assays Characteristic Plate vendor IFN- mabs Vendor Coating mab Biotinylated anti-ifn- Ab Enzyme Substrate PBMC plating Experimental wells Control wells Positivity criteria method BD assay (HVTN) BD (kit) BD (kit) pre-coated 2-3 µg/ml 2 µg/ml Streptavidin-HRP AEC 200,000 per well, 3 wells 200,000 per well, 3 wells SCHARP permutation/bootstrap MM Assay (Merck) Millipore MabTech 10 µg/ml 1 µg/ml AP-conjugated anti-biotin BCIP-NBT 100,000 per well, 2 wells 200,000 per well, 2 wells 100,000 per well, 2 wells 200,000 per well, 2 wells Merck empirical
14 CMV pp65 peptide pool responses in 16 HVTN 057 participant PBMC, comparing the BD and MM ELISpot assays
15 IFN- ELISpot Concordance Study: HVTN and Merck Labs: Interim Analysis Analysis of cryopreserved PBMC from 500 participants in phase IIB Protocol 502: 250 placebos vaccinees at Week 30 (peak immunogenicity) Predetermined criteria Overall response rate: 100% concordance in response rate Classification of calls (+ or -) at participant level: 89.9% (C.I ) Concordance also assessed for: False positive rate 2.5% for sum of all peptide pool, HVTN Lab Magnitude of positive calls fold difference in geometric mean SFC from
16 Validated 8-color staining profile for antigen-stimulated T cells (ICS) Horton et al, J Immunol Methods, in press
17 Limit of Quantitation Determined as Lowest Cytokine Response with CV 30% Example of one analysis of intra-sample CV for the high CMV responder: 60 IFN - + IL-2 + IFN - + IL CD % % CD % % Percent of undiluted CMV-stimulated cells CMV-stimulated PBMC diluted into unstimulated PBMC from same donor
18 ICS assay validation: ongoing process to improve, refine 4-colors CD3 CD4 CD8 IFN-, IL-2 8-colors CD3 CD4 CD8 Live/dead IFN- IL-2 TNF* IL-4* 14-colors CD3 CD4 CD8 Live/dead IFN- IL-2 memory/activation panel* CD57, CD27, CD45RO, CD127, CD45RA, CD28, CCR7, Tetramer, CD38, CCR5, Bcl-2, Ki67
19 erlapping \ What antigens are needed to evaluate globally relevant T cell responses? Individual Strain vs Subtype-Specific vs Global
20 Peptide Reagents Needed for Cross- Protocol, Cross-Clade Comparisons PTE: Potential T Cell Epitopes Biometric Approach: S. Self, F. Li Panel of 15 a.a. peptides whose sequences span all possible 10 a.a. T cell epitopes in HIV-1 that occur at a frequency of 15%. These sequences include all the circulating strains annotated in the Los Alamos Database (~ per subtype).
21 Percent coverage of sequences occurring at a frequency of 15% by various peptide panels (HIV-1 Env) Peptide Panel Size A B Subtype C Others* All Global PTE Global PTE HXB2 Con A Con B Con C Con M Con A+B Con A+B+C Con A+B+C+M 2.3x 1x 1x 1x 1x 1x 1x 2x 3x 4x 100% 61% 25% 70% 33% 32% 49% 75% 76% 78% 100% 56% 57% 31% 74% 27% 44% 77% 80% 80% 100% 56% 19% 31% 22% 69% 38% 37% 75% 77% 100% 55% 35% 44% 44% 37% 52% 61% 67% 70% 100% 90% 42% 55% 51% 56% 59% 75% 92% 93% *non-a+b+c
22 HVTN 054: CD4+ and CD8+ T Cell Responses at Day 28 (8-color ICS) Treatment T Cell Subset Responses 95% CI CD4 + 16/19 (84%) 60-97% Ad CD8 + 17/18 (94%) 73-99% CD4 + 11/20 (55%) 32-77% Ad CD8 + 17/20 (85%) 62-97%
23 HVTN Protocol 054 Cytokine distribution of T cell responses
24 Moving from Phase I/II to Test of Concept Phase IIB Trials Developer Merck HIV Vaccine Adenovirus* alone Gag, Pol, Nef (Clade B) NIH VRC DNA + Adenovirus* Env (clades A,B,C) Gag, Pol (clade B) *serotype 5
25 Algorithm for Defining Immune Correlates (T Cell Based Vaccines) 1) Endpoint Assay: IFN- ELISpot and/or ICS (IL-2/IFN- ) Presence, magnitude, breadth and frequency of HIV-specific T Cells 2) Further define T cell responses ELISpot/ICS: map epitopes and avidity, determine MHC restriction, cross-clade responses 3) Functional Profiling Multiparameter flow cytometry: Anti-viral cytokines and chemokines Central/effector memory phenotype Cytolytic activities Proliferative capacity Mucosal homing properties MHC-TCR interactions and avidity
26 HVTN Laboratory John Hural, Ph.D. Steve De Rosa, M.D. Helen Horton, Ph.D. Susan Zimmermann, Ph.D. Don Carter NIH VRC Gary Nabel, M.D., Ph.D. Richard Koup, M.D. Barney Graham, M.D. John Mascola, M.D. Acknowledgments NIH Division of AIDS P. D Souza I. Rodriquez-Chavez M. Johnston, J. Flores, A. Fix HVTN Leadership L Corey, M.D. Steve Self, Ph.D. Fusheng Li, Ph.D. Site PI s Merck Laboratory John Shiver, Ph.D. Dani Casimiro, Ph.D. Mike Robertson, M.D.
27 HIV Vaccines Inducing CD4+ T Cell Responses When evaluated, virtually all candidate HIV vaccine strategies induce CD4+ T cell responses Their frequency and magnitude varies with the immunogen and adjuvant used Cytokines secreted are a combination of IL-2, IFN- and TNF-_ DNA vaccines primarily generate CD4+ T cells recognizing HIV-1 Env; responses to Gag, Pol, etc., less common With adenovirus vectors, magnitude of CD4+ T cell responses less than CD8+ responses
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