Examining CD8⁺ and CD4⁺ T cell responses after primary Yellow Fever vaccination in a cohort of 210 volunteers, we have identified and tetramer-validated 92 CD8⁺ and 50 CD4⁺ T cell epitopes, many inducing strong and prevalent (i.e., immunodominant) T cell responses. Restricted by 40 and 14 HLA-class I and II allotypes, respectively, these responses have wide population coverage and might be of considerable academic, diagnostic and therapeutic interest. The broad coverage of epitopes and HLA overcame the otherwise confounding effects of HLA diversity and non-HLA background providing the first evidence of T cell immunodomination in humans. Also, double-staining of CD4⁺ T cells with tetramers representing the same HLA-binding core, albeit with different flanking regions, demonstrated an extensive diversification of the specificities of many CD4⁺ T cell responses. We suggest that this could reduce the risk of pathogen escape, and that multi-tetramer staining is required to reveal the true magnitude and diversity of CD4⁺ T cell responses. Our T cell epitope discovery approach uses a combination of (1) overlapping peptides representing the entire Yellow Fever virus proteome to search for peptides containing CD4⁺ and/or CD8⁺ T cell epitopes, (2) predictors of peptide-HLA binding to suggest epitopes and their restricting HLA allotypes, (3) generation of peptide-HLA tetramers to identify T cell epitopes, and (4) analysis of ex vivo T cell responses to validate the same. This approach is systematic, exhaustive, and can be done in any individual of any HLA haplotype. It is all-inclusive in the sense that it includes all protein antigens and peptide epitopes, and encompasses both CD4⁺ and CD8⁺ T cell epitopes. It is efficient and, importantly, reduces the false discovery rate. The unbiased nature of the T cell epitope discovery approach presented here should support the refinement of future peptide-HLA class I and II predictors and tetramer technologies, which eventually should cover all HLA class I and II isotypes. We believe that future investigations of emerging pathogens (e.g., SARS-CoV-2) should include population-wide T cell epitope discovery using blood samples from patients, convalescents and/or long-term survivors, who might all hold important information on T cell epitopes and responses.

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