HVTN Laboratory Program: Immunogenicity and Research Assays
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1 HVTN Laboratory Program: Immunogenicity and Research Assays Erica Andersen-Nissen, PhD Director, Cape Town HVTN Immunology Laboratory Considerations for a Pan-African HIV Vaccine Development Agenda Kigali, Rwanda March 2015
2 Overview Overview of the HVTN Laboratory Center Validated assay examples ICS BAMA Research assay examples Innate responses/systems biology Mucosal
3 HVTN Laboratory Center: Key Players NIAID Division of AIDS Clinical Vaccine Branch Cross Network HIV Activities HIV Vaccine Enterprise Non-HIV Vaccine Activities Scientific Leadership Julie McElrath, PI and Director Nicole Frahm, Associate Director, FHCRC Lab Science Georgia Tomaras, Associate Director, Duke Lab Science John Hural, Associate Director, LC Operations Key Investigators Stephen DeRosa, David Montefiori Erica Andersen-Nissen, Lynn Morris Lab Sciences Advisory Board HVTN Leadership and Operations Center HVTN Statistical and Data Management Center Cellular & Innate Studies Laboratory Center Operations Humoral Studies Fred Hutchinson Cancer Research Center Cape Town HVTN Laboratory Specialized Working Groups Virus & Host Genetics, Systems Biology, Mucosal Immunity, HIV Diagnostics, TB Immunology, NHP HIV Diagnostics Repositories QA/QC Site Affiliated Labs Protocol Support Labs Viral Genetics, Host Genetics, Anti-Vector Analysis Duke University NICD
4 Expansion of the HVTN Core Lab Network: South Africa Cellular Responses Humoral Responses Lynn Morris s Group NICD, Johannesburg CHIL, Cape Town Cape Town HVTN Immunology Laboratory (CHIL)
5 Validated Endpoints Assays Assays are critical for decisions to advance vaccine candidates Performed under GCLP conditions Formally validated using industry standard processes Enable cross-protocol comparisons between regimens Intracellular cytokine staining (ICS) Establishing concordance Analysis of HVTN 097 T cell responses Binding Antibodies Measuring breadth of binding Ab responses in HVTN 097
6 Intracellular Cytokine Staining (ICS) Assay RV144 Ab Staining Panel
7 ICS Assay Concordance: Seattle/Cape Town Background subtracted correlations of positive ICS responses (Percent T cells expressing IFN-γ and/or IL-2 after peptide stimulation) CMV ENV CD4 CD8 Cape Town Seattle Colors indicate data from different HVTN trials
8 Binding Antibody Multiplex Assay (BAMA) ~100 Antigens in a Single Sample Validated for Clinical Trials Small volume allows interrogation of mucosal samples Tomaras et al. (2008) J. Virol Haynes et al. (2012) NEJM Yates, Tomaras (2013) Nature Mucosal Immunology Eckels et al BMC Bioinformatics Yates, Tomaras (2014) Science Trans. Medicine Georgia Tomaras, Duke University
9 V1-V2 Breadth Immune Monitoring Assays New well-characterized cross-clade antigen panels to cover subtypes AE, A, B, BC, and C from diverse geographic regions (Malawi, China, Thailand, South Africa, US, India, Zambia, Uganda, Trinidad, Kenya). Envelope Panel: 8 gp120, and 8 gp140 gp70 V1-V2 Panel: 16 gp70 V1-V2 Yates, DeCamp, Alam, Pinter, Korber, Montefiori, Tomaras and BMGF et al. In Prep Georgia Tomaras, Duke University
10 Research Assays: Innate/Systems Biology How do innate responses to vaccination predict adaptive responses? Vaccina.on ICS Assay (T cell responses) Weeks: Innate.me points (hours): Whole blood (enumeration of cell populations) - Serum (cytokine concentrations) - Cells (gene expression profiling) Identify innate responses that predict adaptive responses Erica Andersen-Nissen
11 Change in expression of over 200 genes at 72 hrs post vaccination with MRKAD5 predicts CD8+ T cell ICS responses to HIV Gag at 4 weeks SH2D1B/EAT-2: expressed in APC; Increases cellular immune responses (Aldhamen et al., 2011) 11 3/25/15 11 Zak, Andersen-Nissen et al, PNAS 2012
12 Analysing the contribution of innate cells to the vaccine response -What can we learn about the role of different cell types in the innate response to HIV vaccines? -How will this help us to better target certain responses? Average Log2(Fold change) > 0.5 P<0.05 for at least 1 cell type n=7 Erica Andersen-Nissen
13 Research Assays: Mucosal Immunology HIV transmission in mucosal tissues Several antibody functions may contribute to HIV protection: Neutralization Blocking virus interaction with non-canonical receptors (e.g. DC-SIGN) Complement activation Phagocytosis Antibody-dependent, cellular cytotoxicity (ADCC) many depend on Fc receptors (FcRs) McElrath & Haynes, Immunity 2010 relative distribution of FcR-bearing effector cells in mucosa is not well defined. Rena Astronomo, FHCRC
14 Ex vivo mucosal challenge assay: Examine Ab inhibition of viral infection Trim tissue, cut into explants and wash with cold PBS Culture overnight w/ PHA, IL-2, -7 & -15 Add virus pre-incubated with Ab OR Ab and virus simultaneously Wash out virus 2-3 mm vaginal explants Culture up to 21 days collecting media or tissue for readout Rena Astronomo, FHCRC
15 Assay Development and Standardization: Control Cohorts are Invaluable! HIV positive and healthy HIV negative volunteers Scheduled visits as well as flexibility for additional asneeded visits Collect: Blood (whole blood, PBMC, plasma, serum) Leukapheresis Cervical/vaginal/rectal fluid Cytobrush, semen Cervical/vaginal biopsies Rectal/Sigmoid colon biopsies
16 Please Come Visit CHIL!
17 Acknowledgements HVTN Lab Center Julie McElrath Georgia Tomaras Nicole Frahm Steve DeRosa John Hural HVTN 097 Protocol Team Glenda Grey Nicole Grunenberg Ying Huang Niya Gu Volunteers Rena Astronomo Dan Zak Alan Aderem Linda- Gail Bekker Lyne\e Denny Carolyn Williamson Funders /DAIDS Patricia D Souza Mary Marovitch Carl Dieffenbach Siobhan Malone Glenda Gray Michelle Mulder
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