Pooled-Peptide Epitope Mapping Strategies Are Efficient and Highly Sensitive: An Evaluation of Methods for Identifying Human T Cell Epitope Specificities in Large-Scale HIV Vaccine Efficacy Trials

<div><p>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.</p></div

Correlation of responses to peptide pools and the peptides they contain.

<p>Rank-based correlations were assessed between the magnitudes of T-cell responses to positive peptide pools that contain a single positive peptide and the individual response to the positive peptide (A, one dot per pool, dashes indicate line of unity) and positive pools containing >1 positive peptide and the sum of the individual responses to the positive peptides they contain (B). Positive pools containing no positive 15-mer and >1 false negative 15-mer were plotted against the sum of the negative peptides they contained (C). 95% confidence intervals and p-value were computed using a participant-based bootstrap (n = 15 responders).</p

Evaluation of epitope mapping strategies.

<p>Epitope mapping strategies were assessed based on the number of positive 15-mers (A) and the number of epitopes (B) that were identified. By definition, all 15-mers were identified by the Test-all strategy. Epitopes detected using an optimal peptide were not necessarily identified by the Test-all strategy, but may have been detected by a pool strategy if all pools containing the epitope were positive. Potential benefits were explored of using computational HLA binding predictors to identify the epitope-containing 15-mer within a positive mini-pool (C, n = 20 pools) and to identify the optimal peptide within a positive 15-mer (D, n = 25 peptides). Ranks of the 15-mers in the mini-pool or the optimal peptides within the 15-mer were based on predicted HLA binding with each participant’s alleles. Observed average (line) and 95% confidence interval (dashed lines) are illustrated relative to a distribution of averages computed using random rankings.</p

T-cell responses to small pools of 15-mer peptides.

<p>ELISpot responses to peptide pools were categorized based first on their positivity (A, positive; B, negative; C, response magnitudes, one dot indicates the mean of a triplicate assay) and subsequently on whether or not they contained a positive 15-mer or a 15-mer containing the sequence of a positive optimal peptide.</p

Vaccine primed IFN-γ ELISpot responses to HIV Gag peptides.

<p>Peptides derived from a consensus clade B Gag sequence were used individually to detect T-cell responses in participants (n = 15 vaccine responders) of the Step HIV vaccine trial. Responses were quantified in units of spot forming cells per million (SFC/M) using an IFN-γ ELISpot assay (A; one dot indicates the mean of a triplicate assay). Optimal length peptides were also used to further define the epitopes. Negative peptides containing the sequence of a positive optimal peptide were labeled as false-negative. Breadth was the minimal number of epitopes that explained the responses of each participant (B). Breadth was occasionally greater than the number of 15-mer responses when responses were detected to optimal peptides, but not the parent 15-mer peptide. The magnitudes of responses to 15-mer peptides and those that confirmed their positivity were plotted to assess their similarity and their correlation (C; dashes illustrate line of equality). Magnitude of response to a 15-mer peptide differed depending on the relative position of the epitope containing optimal peptide (D).</p