SARS-CoV-2 infected cells present HLA-I peptides from canonical and out-of-frame ORFs

T cell-mediated immunity seems to play a critical role against SARS-CoV-2 infection and establishing protective memory. Yet the repertoire of viral epitopes responsible for activation of T cell responses remains mostly unknown. Identification and characterization of viral peptides presented on class I human leukocyte antigen (HLA-I) will reveal the viral signatures as seen by cytotoxic T cells that can then be harnessed for the development of effective vaccines. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two human cell lines at different times post-infection using mass spectrometry. We found HLA-I peptides derived not only from canonical ORFs, but also from internal out-of-frame ORFs in Spike and Nucleoprotein not currently captured by vaccine approaches. Whole proteome analysis revealed that expression of ubiquitination pathway proteins, and the proteasome maturation protein, POMP, were significantly altered in infected cells. Retrospective analysis of computational predictions highlighted shortcomings of in silico-only approaches in recovering the observed viral epitopes. Finally, given the experimental evidence for endogenous processing and presentation of the viral peptides we detected, we estimated that at least one HLA-A, -B, or -C allele is covered by at least one peptide for 99% of the population. These biological insights and the list of naturally presented SARS-CoV-2 peptides will facilitate data-driven selection of peptides for immune monitoring and vaccine development. RNA-seq of human lung A549 cells and human kidney HEK293T infected with SARS-CoV-2 12h, 18h and 24h post infection RNA-seq was used to evaluate viral and human transcripts abundance, and the processed file contains the % human and viral of reads in each cell line and time point.