The ongoing COVID-19 pandemic continues to pose a global challenge due to immune evasion through a progression of mutations in the SARS-CoV-2 virus. Several vaccines are available, but the vast majority target the highly variable Spike protein which is under high evolutionary pressure. Therefore, our aim was to use immunopeptidomics and mass spectrometry analysis to uncover novel and more conserved epitopes of SARS-CoV-2 derived from various viral proteins and presented on human leukocyte antigen (HLA). Such insight could be used to design second generation vaccines with a broader antigen repertoire at its core and an extended applicability time frame. To this aim we used the B-Lymphoblastoid Cell Lines (BLCL) 9004 and 9087 which feature high HLA class I and class II expression levels and carry a number of HLA allotypes highly prevalent in the global population. BLCL were either transfected with constructs expressing SARS-CoV-2-derived proteins (N, E, nsp9) or were subjected to direct antigen delivery of SARS-CoV-2 proteins (N, nsp1/4/5/9). Harvested BLCL underwent immunoaffinity purification and peptide-HLA elution with subsequent analysis on the state-of-the-art Bruker TimsTOF Pro mass spectrometer.
We found over 200 HLA-I and HLA-II derived peptides originating from the SARS-CoV-2 virus. T cell responses were tested for 56 of the detected peptides with 5-7 HLA-matched individuals per peptide pool. We show CD8 and CD4 T cell responses against several peptides from the N, E and nsp9 proteins. One immunogenic 19-mer from Nucleoprotein was recognised by three individuals with blood collection up to 7 months post infection. Overall, immunopeptidome dataset presented here greatly reduces the otherwise theoretically predicted number of T cell epitopes (for HLA class I by a factor of 14). This work enables further evaluation of high priority epitopes that may aid vaccine development to generate a broadly targeting and mutation-resistant COVID-19 vaccine.