Mass spectrometric identification of immunogenic SARS-CoV-2 epitopes and cognate TCRs

Abstract SARS-CoV-2 infections elicit both humoral and cellular immune responses. For the prevention and treatment of COVID19, the disease caused by SARS-CoV-2, T cell responses are important in mediating recovery and immune-protection. The identification of immunogenic epitopes that can elicit a meaningful T cell response can be elusive. Traditionally, this has been achieved using sophisticated in silico methods to predict putative epitopes; however, our previous studies find that ‘immunodominant’ SARS-CoV-2 peptides defined by such in silico methods often fail to elicit T cell responses recognizing SARS-CoV-2. We postulated that immunogenic epitopes for SARS-CoV-2 are best defined by directly analyzing peptides eluted from the peptide-MHC complex and then validating immunogenicity empirically by determining if such peptides can elicit T cells recognizing SARS-CoV-2 antigen-expressing cells. Using a tandem mass spectrometry approach, we identified epitopes of SARS-CoV-2 derived not only from structural but also non-structural genes in regions highly conserved among SARS-CoV-2 strains including recently recognized variants. We report here, for the first time, several novel SARS-CoV-2 epitopes from membrane glycol-protein (MGP) and non-structure protein-13 (NSP13) defined by mass-spectrometric analysis of MHC-eluted peptides, provide empiric evidence for their immunogenicity to induce T cell response. Significance Statement Current state of the art uses putative epitope peptides based on in silico prediction algorithms to evaluate the T cell response among COVID-19 patients. However, none of these peptides have been tested for immunogenicity, i.e. the ability to elicit a T cell response capable of recognizing endogenously presented peptide. In this study, we used MHC immune-precipitation, acid elution and tandem mass spectrometry to define the SARS-CoV-2 immunopeptidome for membrane glycol-protein and the non-structural protein. Furthermore, taking advantage of a highly robust endogenous T cell (ETC) workflow, we verify the immunogenicity of these MS-defined peptides by in vitro generation of MGP and NSP13 peptide-specific T cells and confirm T cell recognition of MGP or NSP13 endogenously expressing cell lines.