title: Optimization and qualification of an 8-color intracellular cytokine staining assay for quantifying T cell responses in rhesus macaques for pre-clinical vaccine studies.
abstract: Vaccination and SIV challenge of macaque species is the best animal model for evaluating candidate HIV vaccines in pre-clinical studies. As such, robust assays optimized for use in nonhuman primates are necessary for reliable ex vivo measurement of immune responses and identification of potential immune correlates of protection. We optimized and qualified an 8-color intracellular cytokine staining assay for the measurement of IFNγ, IL-2, and TNF from viable CD4 and CD8 T cells from cryopreserved rhesus macaque PBMC stimulated with peptides. After optimization, five laboratories tested assay performance using the same reagents and PBMC samples; similar results were obtained despite the use of flow cytometers with different configurations. The 8-color assay was then subjected to a pre-qualification study to quantify specificity and precision. These data were used to set positivity thresholds and to design the qualification protocol. Upon completion of the qualification study, the assay was shown to be highly reproducible with low inter-aliquot, inter-day, and inter-operator variability according to the qualification criteria with an overall variability of 20-40% for each outcome measurement. Thus, the 8-color ICS assay was formally qualified according to the ICH guidelines Q2 (R1) for specificity and precision indicating that it is considered a standardized/robust assay acceptable for use in pre-clinical trial immunogenicity testing.
authors.affiliation: Nonhuman Primate Immunogenicity Core, Vaccine Research Center, NIAID, NIH, Bethesda, MD 20892, USA.
authors.forename: Mitzi M
authors.initials: MM
authors.lastname: Donaldson
authors.forename: Shing-Fen
authors.initials: SF
authors.lastname: Kao
authors.forename: Leila
authors.initials: L
authors.lastname: Eslamizar
authors.forename: Connie
authors.initials: C
authors.lastname: Gee
authors.forename: Gerrit
authors.initials: G
authors.lastname: Koopman
authors.forename: Michelle
authors.initials: M
authors.lastname: Lifton
authors.forename: Joern E
authors.initials: JE
authors.lastname: Schmitz
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Sylwester
authors.forename: Aaron
authors.initials: A
authors.lastname: Wilson
authors.forename: Natalie
authors.initials: N
authors.lastname: Hawkins
authors.forename: Steve G
authors.initials: SG
authors.lastname: Self
authors.forename: Mario
authors.initials: M
authors.lastname: Roederer
authors.forename: Kathryn E
authors.initials: KE
authors.lastname: Foulds
mesh_terms: D016915:AIDS Vaccines / Q000008:administration & dosage*; D000818:Animals; D002469:Cell Separation; D003116:Color; D016207:Cytokines / Q000032:analysis*; D004195:Disease Models, Animal; D004353:Drug Evaluation, Preclinical; D005434:Flow Cytometry / Q000379:methods; D017408:Guidelines as Topic; D006801:Humans; D042541:Intracellular Space; D008253:Macaca mulatta; D015588:Observer Variation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016097:Simian Acquired Immunodeficiency Syndrome / Q000276:immunology*; D015302:Simian Immunodeficiency Virus / Q000276:immunology*; D013194:Staining and Labeling / Q000379:methods*; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D016915:AIDS Vaccines; D016207:Cytokines