Generation of SARS-CoV-2 S1 Spike Glycoprotein Putative Antigenic Epitopes in Vitro by Intracellular Aminopeptidases

Abstract

Presentation of antigenic peptides by MHCI is central to cellular immune responses against viral pathogens. While adaptive immune responses versus SARS-CoV-2 can be of critical importance to both recovery and vaccine efficacy, how protein antigens from this pathogen are processed to generate antigenic peptides is largely unknown. Here, we analyzed the proteolytic processing of overlapping precursor peptides spanning the entire sequence of the S1 spike glycoprotein of SARS-CoV-2, by three key enzymes that generate antigenic peptides, aminopeptidases ERAP1, ERAP2, and IRAP. All enzymes generated shorter peptides with sequences suitable for binding onto HLA alleles, but with distinct specificity fingerprints. ERAP1 was the most efficient in generating peptides 8–11 residues long, the optimal length for HLA binding, while IRAP was the least efficient. The combination of ERAP1 with ERAP2 greatly limited the variability of peptide sequences produced. Less than 7% of computationally predicted epitopes were found to be produced experimentally, suggesting that aminopeptidase processing may constitute a significant filter to epitope presentation. These experimentally generated putative epitopes could be prioritized for SARS-CoV-2 immunogenicity studies and vaccine design. We furthermore propose that this in vitro trimming approach could constitute a general filtering method to enhance the prediction robustness for viral antigenic epitopes.

Immunodominance complexity: lessons yet to be learned from dominant T cell responses to SARS-COV-2

“…In the context of SARS-CoV-2, ERAPs have been proposed to play a role [ 43 ] and indeed they were shown to be important for generation of 8-11 amino acid peptides from 15 amino acid precursors [ 44 ]. Additionally, mutations in ERAP2 were shown to be associated with increased risk of death in SARS-CoV-2 infection.…”

Allotypic variation in antigen processing controls antigenic peptide generation from SARS-CoV-2 S1 spike glycoprotein

“…While in vitro enzymatic analysis of ERAP1 function has provided significant insights on the mechanism of the enzyme 30,31 , complex interactions between substrates and ERAP1's large substrate-binding cavity result in a complex landscape of poorly understood specificity determinants 30,32 . To address these complexities we previously devised a proteomic approach to concurrently analyze hundreds of trimming reactions and compared the function of different antigen processing enzymes 33 .…”

ERAP1 and ERAP2 Enzymes: A Protective Shield for RAS against COVID-19?

“…In line with this hypothesis, a missense variant in the ERAP2 gene has been linked to an increased risk of death in a small cohort of COVID-19 patients from the U.K. Biobank [55]. Furthermore, ERAP1 has been involved in the generation of peptides from the SARS-CoV2-S1 spike protein with the optimal length to bind MHC class I molecules, including the possible SARS-CoV2-protective HLA-B*15:03 allele [56,57]. Therefore, we can speculate that ERAP aminopeptidases may be actively involved in different processes during COVID-19 pathogenesis, with the first and foremost being the regulation of the RAS pathway and MHC class I antigen processing and presentation.…”

“…In line with this hypothesis, a missense variant in the ERAP2 gene has been linked to an increased risk of death in a small cohort of COVID-19 patients from the U.K. Biobank [ 55 ]. Furthermore, ERAP1 has been involved in the generation of peptides from the SARS-CoV2-S1 spike protein with the optimal length to bind MHC class I molecules, including the possible SARS-CoV2-protective HLA-B*15:03 allele [ 56 , 57 ].…”