DEFINING IMMUNOGENIC PEPTIDES TARGETING SARS-CoV-2 VARIANTS of CONCERN

ABSTRACT

Background: Identifying Severe Acute Respiratory Syndrome Coronavirus Type 2 (SARS-CoV-2) specific T-cell epitope-derived peptides that are also found within variants of concern (VOC) is critical for measuring the duration of cellular immunity induced by the virus and COVID-19 vaccines. Therefore, we assessed whether the peptides selected from topologically important regions of SARS-CoV-2 proteins avoid major mutations of VOC and induce T-cell immune response. Methods: We selected 32 peptides within topologically important regions of SARS-CoV-2 Spike (S) and Nucleocapsid (NC) proteins by applying an insilico pipeline to 607 viral sequences in 2019. To determine if these peptides avoid VOC mutations, we analyzed S and NC protein regions derived from 1.7 x 106 viral genomic sequences compiled from Mar 2020-Aug 2021. We identified α-, β-and δ-VOC mutations found within >1% of the S and NC protein sequences. These mutations were compared to the peptides. To determine T-cell immune response to these selected peptides as a pool, we assessed interferon-γ (IFN-γ), interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α) and CD107a/b (degranulation marker) production within peripheral blood mononuclear cell (PBMC) samples derived from COVID-19 post-recovery donors (n=23) by employing dual color FluoroSpot and intracellular cytokine staining (ICS). Results: We found 88% of S protein-derived peptides did not contain mutations of α-, β-and δ-VOC. All peptides from S protein (n=25) avoided known T-cell escape mutations. Of the 7 NC-derived peptides, three contained the L139F mutation found within α-and δ-VOC, however, this mutation was observed within <2% NC protein sequences. A peptide pool containing our 32 selected peptides elicited an immune response within PBMCs from 17/23 COVID-19 post-recovery donors. FluoroSpot analysis revealed IFN-γ and IL-2 production to our peptide pool was similar/higher compared to the commercial S and NC peptide pools. The response of CD4 and CD8 T-cells to our peptide pool was polyfunctional expressing ≥2 markers within most of the donors when ICS was performed, with IFN-γ and TNF-α being the main cytokines produced. Conclusion: Applying an immunoinformatics pipeline allowed us to select peptides from the S and NC proteins which avoid the majority of mutations found within the α-, β-and δ-VOC. Our peptide pool elicited a polyfunctional T-cell response making it an ideal candidate for assessing the duration of cellular immunity induced by SARS-CoV-2 variants and vaccines.