Abstract
The interferon gamma, enzyme-linked immunospot (IFN-γ ELISpot) assay is widely used to identify viral antigen-specific T cells is frequently employed to quantify T cell responses in HIV vaccine studies. It can be used to define T cell epitope specificities using panels of peptide antigens, but with sample and cost constraints there is a critical need to improve the efficiency of epitope mapping for large and variable pathogens. We evaluated two epitope mapping strategies, based on group testing, for their ability to identify vaccine-induced T-cells from participants in the Step HIV-1 vaccine efficacy trial, and compared the findings to an approach of assaying each peptide individually. The group testing strategies reduced the number of assays required by >7-fold without significantly altering the accuracy of T-cell breadth estimates. Assays of small pools containing 7–30 peptides were highly sensitive and effective at detecting single positive peptides as well as summating responses to multiple peptides. Also, assays with a single 15-mer peptide, containing an identified epitope, did not always elicit a response providing validation that 15-mer peptides are not optimal antigens for detecting CD8+ T cells. Our findings further validate pooling-based epitope mapping strategies, which are critical for characterizing vaccine-induced T-cell responses and more broadly for informing iterative vaccine design. We also show ways to improve their application with computational peptide:MHC binding predictors that can accurately identify the optimal epitope within a 15-mer peptide and within a pool of 15-mer peptides.
Highlights
Measures of immunogenicity are an integral part of iterative and rational vaccine design [1,2]
The enzyme-linked immunospot (ELISpot) assay can be used to quantify the frequency of antigen-specific T cells via the secretion of interferon gamma (IFN-γ) following brief ex vivo stimulation with one or multiple peptide antigens [3,4]
The magnitude readout of the assay is the frequency of cytokine-secreting cells, and IFN-γ itself is not necessarily linked to CD8+/CD4+ T cell cytolytic activity [5], ELISpot responses identify antigen-specific T cells and have previously been associated with endpoints in vaccine clinical trials [6,7,8,9,10,11]
Summary
Measures of immunogenicity are an integral part of iterative and rational vaccine design [1,2]. One of the main advantages of the ELISpot assay over flow-cytometric assays (e.g., intracellular cytokine staining [ICS] [12] or tetramer sorting) is the ability to efficiently screen a wide array of peptide antigens covering the entire set of vaccine immunogens, effectively mapping the specificity of T-cell responses [13] Another benefit is that extensive effort has gone towards standardization of the assay across labs to reduce variability [14,15,16], and towards statistical method development, which has improved positivity calls [2,17,18,19,20,21,22]. Most candidate HIV vaccines have elicited HIV-specific T-cell responses [29,30,31,32,33] including the recombinant adenovirus-vectored vaccine tested in the Step Study, in which vaccine recipient responders targeting at least three Gag epitopes had lower viral load compared to non-responders [11]
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