Development and Qualification of a Human IFN-gamma ELISPOT Assay for Testing T Cell Responses to an HIV gag DNA Vaccine (B203)

Abstract

Abstract Cell-mediated immunity is believed to be critical for the control and clearance of viral infections. However, frequencies of antigen-specific human T cells are rather low, quantitation of these cells requires assays that exhibit robust sensitivity, specificity, and reproducibility. In this study, a peptide-induced human IFN-gamma ELISPOT assay has been developed and qualified for testing T cell responses to an HIV gag DNA vaccine. We optimized the number of cells per well, the concentrations of HIV gag peptide pools used for stimulation, and the incubation time of cells with the stimulants. We found that overnight-cultured PBMCs, rather than immediately thawed PBMCs, yielded a greater number of spots. The assay is specific, as it detected IFN-gamma but not IL-2 or granzyme B secretion by stimulated PBMCs. The assay results were reproducible and robust across a wide range of cell concentrations. Coefficients of variation of 25% or less were observed for intra-assay replicates from samples containing more than 50 spot-forming cells. Furthermore, the use of cryopreserved PBMC samples allowed direct comparison within a single assay run of samples from multiple time points during the course of a vaccination regimen, minimizing potential run-to-run assay variability. These results indicate that the ELISPOT assay was reliable and suitable for direct ex vivo quantitation of antigen-specific T cells in HIV vaccine trials.