title: Optimization and qualification of a multiplex bead array to assess cytokine and chemokine production by vaccine-specific cells.
abstract: The magnitude and functional phenotype (e.g. proliferation, immune stimulation) of vaccine-induced T-cell responses are likely to be critical in defining responses that can control pathogenic challenge. Current multi-parameter flow cytometric techniques may not be sufficient to measure all of these different functions, since characterizing T-cell responses by flow cytometry is presently limited to concurrent measurement of at most 10 cytokines/chemokines. Here, we describe extensive studies conducted using standardized GCLP procedures to optimize and qualitatively/quantitatively qualify a multiplex bead array (MBA) performed on supernatant collected from stimulated peripheral blood mononuclear cells (PBMC) to assess 12 cytokines and chemokines of interest. Our optimized MBA shows good precision (intra-assay, inter-day, inter-technician; coefficients of variation <30%) and linearity for most of the analytes studied. We also developed positivity criteria that allow us to define a response as positive or negative with a high degree of confidence. In conclusion, we provide a detailed description of the qualification of an MBA, which permits quantitative and qualitative evaluation of vaccine-induced immunogenicity and analysis of immune correlates of protection. This assay provides an excellent complement to the existing repertoire of assays for assessing immunogenicity in HIV vaccine clinical trials.
authors.affiliation: Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
authors.forename: Olivier D
authors.initials: OD
authors.lastname: Defawe
authors.forename: Youyi
authors.initials: Y
authors.lastname: Fong
authors.forename: Evgenia
authors.initials: E
authors.lastname: Vasilyeva
authors.forename: Melissa
authors.initials: M
authors.lastname: Pickett
authors.forename: Donald K
authors.initials: DK
authors.lastname: Carter
authors.forename: Erin
authors.initials: E
authors.lastname: Gabriel
authors.forename: Supachai
authors.initials: S
authors.lastname: Rerks-Ngarm
authors.forename: Sorachai
authors.initials: S
authors.lastname: Nitayaphan
authors.forename: Nicole
authors.initials: N
authors.lastname: Frahm
authors.forename: M Juliana
authors.initials: MJ
authors.lastname: McElrath
authors.forename: Stephen C
authors.initials: SC
authors.lastname: De Rosa
mesh_terms: D016915:AIDS Vaccines / Q000276:immunology*; D018925:Chemokines / Q000096:biosynthesis* / Q000276:immunology; D016207:Cytokines / Q000096:biosynthesis* / Q000276:immunology; D005434:Flow Cytometry; D015488:HIV Antigens / Q000276:immunology; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007963:Leukocytes, Mononuclear / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism*
chemical_list: D016915:AIDS Vaccines; D018925:Chemokines; D016207:Cytokines; D015488:HIV Antigens