T cell-mediated immunity plays an important role in controlling SARS-CoV-2 infection, but the repertoire of naturally processed and presented viral epitopes on class I human leukocyte antigen (HLA-I) remains uncharacterized. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two cell lines at different times post infection using mass spectrometry. We found HLA-I peptides derived not only from canonical open reading frames (ORFs) but also from internal out-of-frame ORFs in spike and nucleocapsid not captured by current vaccines. Some peptides from out-of-frame ORFs elicited T cell responses in a humanized mouse model and individuals with COVID-19 that exceeded responses to canonical peptides, including some of the strongest epitopes reported to date. Whole-proteome analysis of infected cells revealed that early expressed viral proteins contribute more to HLA-I presentation and immunogenicity. These biological insights, as well as the discovery of out-of-frame ORF epitopes, will facilitate selection of peptides for immune monitoring and vaccine development.

Effective presentation of antigens by HLA class I molecules to CD8+ T cells is required for viral elimination and generation of long-term immunological memory. In this study, we applied a single-cell, multi-omic technology to generate the first unified ex vivo characterization of the CD8+ T cell response to SARS-CoV-2 across 4 major HLA class I alleles. We found that HLA genotype conditions key features of epitope specificity, TCR a/b sequence diversity, and the utilization of pre-existing SARS-CoV-2 reactive memory T cell pools. Single-cell transcriptomics revealed functionally diverse T cell phenotypes of SARS-CoV-2-reactive T cells, associated with both disease stage and epitope specificity. Our results show that HLA variations influence pre-existing immunity to SARS-CoV-2 and shape the immune repertoire upon subsequent viral exposure.

BackgroundHigh-risk human papillomavirus (HPV) is a primary cause of an increasing number of oropharyngeal squamous cell carcinomas (OPSCCs). The viral etiology of these cancers provides the opportunity for antigen-directed therapies that are restricted in scope compared with cancers without viral components. However, specific virally-encoded epitopes and their corresponding immune responses are not fully defined.MethodsTo understand the OPSCC immune landscape, we conducted a comprehensive single-cell analysis of HPV16+ and HPV33+ primary tumors and metastatic lymph nodes. We used single-cell analysis with encoded peptide-human leukocyte antigen (HLA) tetramers to analyze HPV16+ and HPV33+ OPSCC tumors, characterizing the ex vivo cellular responses to HPV-derived antigens presented in major Class I and Class II HLA alleles.ResultsWe identified robust cytotoxic T-cell responses to HPV16 proteins E1 and E2 that were shared across multiple patients, particularly in HLA-A*01:01 and HLA-B*08:01. Responses to E2 were associated with loss of E2 expression in at least one tumor, indicating the functional capacity of these E2-recognizing T cells and many of these interactions validated in a functional assay. Conversely, cellular responses to E6 and E7 were limited in quantity and cytotoxic capacity, and tumor E6 and E7 expression persisted.ConclusionsThese data highlight antigenicity beyond HPV16 E6 and E7 and nominate candidates for antigen-directed therapies.

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