Identification of presented SARS-CoV-2 HLA class I and HLA class II peptides using HLA peptidomics

Abstract

The human leukocyte antigen (HLA)-bound viral antigens serve as an immunological signature that can be selectively recognized by T cells. As viruses evolve by acquiring mutations, it is essential to identify a range of presented viral antigens. Using HLA peptidomics, we are able to identify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-derived peptides presented by highly prevalent HLA class I (HLA-I) molecules by using infected cells as well as overexpression of SARS-CoV-2 genes. We find 26 HLA-I peptides and 36 HLA class II (HLA-II) peptides. Among the identified peptides, some are shared between different cells and some are derived from out-of-frame open reading frames (ORFs). Seven of these peptides were previously shown to be immunogenic, and we identify two additional immunoreactive peptides by using HLA multimer staining. These results may aid the development of the next generation of SARS-CoV-2 vaccines based on presented viral-specific antigens that span several of the viral genes.

Keywords: HLA; Peptides; Peptidomics; SARS-CoV-2; immuno-reactive; out-of-frame-ORFs.

Graphical abstract

Figure 1

SARS-CoV-2 peptide identification pipeline Based on the selection of the most frequent HLA-I alleles in the world population, B cell lines with mono-allelic or endogenous HLA-I expression were chosen. Cells were infected with SARS-CoV-2 or transduced with SARS-CoV-2 genes. An HLA-I and HLA-II peptidome analysis revealed shared peptides and presentation hotspots presented by the B cells. Some of identified peptides were cultured with peripheral blood mononuclear cells (PBMCs) from SARS-CoV-2-infected donors, eliciting a T cell response that was detected by binding to pHLA multimers.

Figure 2

Differently presented peptide repertoire in IHW1070 B cell line after infection with SARS-CoV-2 (A) A volcano plot was used to identify the peptides that were differentially presented by the cell’s HLA-I molecules of infected compared to the uninfected control. HLA peptidomics experiments were done in three biological replicates. The red dots indicate proteins involved in immune regulation pathways, indicated in (B). (B) The table indicates the pathways that were found to be significantly enriched of the peptides that were significantly more presented after SARS-CoV-2 infection. (C) Volcano plot of proteins identified in the proteomic analysis of the cells, comparing infected and non-infected IHW01070. Proteomic experiments were done in three biological replicates. Type I interferon response proteins are marked in red, and beta proteasome subunits are marked in blue.

Figure 3

SARS-CoV-2-derived shared HLA peptides and presentation hotspots Schematic representation of all peptides with identified SARS-CoV-2 gene overexpression and infections. Each cell line is marked by a different color dot, HLA-I peptides are marked in red box, and HLA-II peptides are marked in blue box. Peptides found to be immunoreactive are marked in red, and peptides shown to bind the corresponding HLA are marked in green.

Figure 4

Schematic map of predicted and presented peptides of SARS-CoV-2 Predicted peptides are marked in black. Predicted peptides that overlapped with peptides derived from the CoV family are marked in green if they were identical or light green if similar with one substitution. Predicted peptides that were similar (with one substitution) to human peptides are marked in red. All peptides previously found to be immunogenic in different studies are marked in blue. Presented peptides, identified in this study, are marked in purple. The frequency of SNPs in the SARS-CoV-2 variants are represented in the line plot.

Figure 5

Presented SARS-CoV-2 peptides are immunogenic CD8+ T cell recognition was assessed for the identified HLA-I peptides by using fluorescent pHLA multimers. Flow cytometry plots of detected SARS-CoV-2-specific CD8+ T cell responses in COVID-19 patients or healthy non-exposed controls. The magnitude of the response is defined as the percentage of double-positive pHLA+ cells of total CD8+ cells.