High-throughput identification of MHC class I binding peptides using an ultradense peptide array

Abstract

Rational vaccine development and evaluation requires identifying and measuring the magnitude of epitope-specific CD8 T cell responses. However, conventional CD8 T cell epitope discovery methods are labor-intensive and do not scale well. Here, we accelerate this process by using an ultradense peptide array as a high-throughput tool for screening peptides to identify putative novel epitopes. In a single experiment, we directly assess the binding of four common Indian rhesus macaque MHC class I molecules - Mamu-A1*001, -A1*002, -B*008, and -B*017 - to approximately 61,000 8-mer, 9-mer, and 10-mer peptides derived from the full proteomes of 82 simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV) isolates. Many epitope-specific CD8 T cell responses restricted by these four MHC molecules have already been identified in SIVmac239, providing an ideal dataset for validating the array; up to 64% of these known epitopes are found in the top 192 SIVmac239 peptides with the most intense MHC binding signals in our experiment. To assess whether the peptide array identified putative novel CD8 T cell epitopes, we validated the method by IFN-{gamma} ELISPOT assay and found three novel peptides that induced CD8 T cell responses in at least two Mamu-A1*001-positive animals; two of these were validated by ex vivo tetramer staining. This high-throughput identification of peptides that bind class I MHC will enable more efficient CD8 T cell response profiling for vaccine development, particularly for pathogens with complex proteomes where few epitope-specific responses have been defined.