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

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.

Neurofascin and Compact Myelin Antigen-Specific T Cell Response Pattern in Chronic Inflammatory Demyelinating Polyneuropathy Subtypes

“…The Elispot technique can be easily used with frozen and subsequently thawed PBMCs. ELISPOT appears to be one of the fast growing applications in biomedical research such as in vaccine development ( 29 ), HIV research ( 30 ), and cancer and allergy research ( 31 ), most of them in multicenter trials. Furthermore, comparative B and T cell ELISPOT assays are useful in the process control of kidney transplant recipients ( 32 ).…”

Utilizing Computational Machine Learning Tools to Understand Immunogenic Breadth in the Context of a CD8 T-Cell Mediated HIV Response

“…To confirm that the selected subset of predicted peptides were recognized by anti-HIV specific T-cells, IFNγ ELISPOT assays were performed using a 3D Matrix approach described elsewhere (Fiore-Gartland et al 2016). The peptides were evaluated in 32 HIV+ volunteers to determine the contribution of individual HLA and input sequences and correlate these metrics to observed Tcell responses.…”

<strong>Selective HLA Restriction Permits the Evaluation and Interpretation of Immunogenic Breadth at Comparable Levels to Autologous HLA</strong>

“…To understand immunogenic breadth at the population level requires the linkage of the experimental target inputs of the immunoassay to the population from which they are derived. How this linkage is defined is not always straight forward, although it has been made for some disease models (Seaman et al 2010) and techniques (Fiore-Gartland et al 2016). Our interest is in designing and developing an HIV CD8 T-cell restricted candidate vaccine that accounts for the relationships and linkages between an individual's immune repertoire and the prevalent HIV transmission sequences as key parameters to assess to ensure immunogenic breadth.…”