COVID-19 has become endemic and continues to pose a global challenge due to immune evasion through a progression of mutations in the SARS-CoV-2 viral genome. 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 the mass spectrometry-based approach of immunopeptidomics to uncover novel and more conserved epitopes of SARS-CoV-2 derived from various viral proteins and presented on prevalent human leukocyte antigen (HLA) allotypes. Such insight will facilitate the design of second-generation vaccines with a broader antigen repertoire at its core and duration of protection. To this aim we used the B-Lymphoblastoid Cell Lines (BLCL) 9004 and 9087 which feature robust 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, Nsp8, Nsp9) or were subjected to direct antigen delivery of SARS-CoV-2 proteins (N, Nsp1/4/5/9). Harvested BLCL underwent immunoaffinity purification to isolate peptide-HLA complexes with subsequent data dependent acquisition on the TimsTOF Pro mass spectrometer used to interrogate their antigenic peptide cargo.
We found 248 unique HLA-I and HLA-II derived peptides originating from SARS-CoV-2 (12 from E, 71 from N, 28 from Nsp1, 19 from Nsp4, 73 from Nsp8 and 45 peptides from Nsp9). We next tested immune cell responses against 56 of the detected peptides with 5-7 HLA-matched COVID convalescent 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 the conserved Nucleoprotein was recognised by three individuals with blood collection up to 7 months post infection indicating long-lasting T cell immunity. Overall, the immunopeptidome dataset presented here provides experimental evidence for high priority peptide epitopes may guide vaccine development to generate a mutation-resistant COVID-19 vaccine targeting a broad range of SARS-CoV-2 proteins.