Predominant T-cell epitopes of SARS-CoV-2 restricted by multiple prevalent HLA-B and HLA-C allotypes in Northeast Asia

Abstract

Since the outbreak of novel coronavirus pneumonia (COVID-19), numerous T-cell epitopes in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteome have been reported. However, most of the identified CD8⁺ T-cell epitopes have been restricted primarily by HLA-A allotypes. The epitopes restricted by HLA-B and HLA-C allotypes are limited. This study focuses on the screening of T-cell epitopes restricted by 13 prevalent HLA-B and 13 prevalent HLA-C allotypes, which cover over 70% and 90% of the Chinese and Northeast Asian populations, respectively. Totally, 67 HLA-B restricted and 53 HLA-C restricted epitopes were validated as immunogenic epitopes with a herd predominance rate by peptide-PBMCs ex vivo coculture experiments using the PBMCs from convalescent Chinese cohort. In addition, 26 transfected cell lines expressing indicated HLA-B or HLA-C allotype were established, and used in the competitive peptide binding assays to define the affinities and cross-restriction of each validated epitope binding to HLA-B or HLA-C allotypes. These data will facilitate the design of T-cell-directed vaccines and SARS-CoV-2-specific T-cell detection tools tailored to the Northeast Asian population. The herd test of functionally validated T-cell epitopes, and the competitive peptide binding assay onto cell line array expressing prevalent HLA allotypes may serve as an additional criterion for selecting T-cell epitopes used in vaccine.

Keywords: HLA-B allotype; HLA-C allotype; SARS-CoV-2; T-cell epitope; vaccine.

Figure 1

The epitopes inducing CD8⁺ T-cell activation in peptides-PBMCs ex vivo cocultures. The heatmap illustrates the results of peptide-PBMCs coculture experiments. Each column represents a convalescent PBMCs sample, and each row represents an indicated epitope peptide. Each color element within the heatmap denote the fold changes of IFN-γ⁺/CD8⁺ T-cell frequency in the peptide-PBMCs coculture relative to its negative control well (PBMCs alone) (white: < 2; yellow: 2–5; cyan: 5–10; orange: >10). Elements in gray indicate no coculture experiment. (A) The cocultures of HLA-B restricted epitopes with convalescents’ PBMCs samples. (B) The cocultures of HLA-C restricted epitopes with convalescents’ PBMCs samples.

Figure 2

Herd predominance rate of 67 HLA-B restricted VEPs and 53 HLA-C restricted VEPs. Circular histogram displayed the positive rate of each VEP inducing CD8⁺ T-cell activation in the cocultures with 29–32 random convalescents’ PBMCs. (A) positive rate of each HLA-B restricted VEP as tested using 32 convalescent samples. (B) Positive rate of each HLA-C restricted VEPs as tested using 30 convalescent samples. Internal histogram exhibited the numbers of VEPs derived from different proteins.

Figure 3

The flow cytometric dot plots of intracellular IFN-γ staining in representative peptide-PBMCs cocultures. After ex vivo coculture of PBMCs with candidate epitope peptide, the cells were harvested and followed by intracellular IFN-γ staining using FITC-conjugated anti-human CD3, APC-conjugated anti-human CD8, and PE-conjugated anti-human IFN-γ antibodies. After washing, the cells were harvested and analyzed by flow cytometry to determine the frequencies of IFN-γ⁺ cells in CD3⁺/CD8⁺ populations. Negative control means PBMCs alone well. (A, B) Representative flow cytometry plots (left) and the histogram showed the frequency of IFN-γ⁺/CD3⁺/CD8⁺ T cell elicited by each epitope (right).

Figure 4

The representative flow cytometric histograms of VEPs binding to HLA-B and HLA-C allotypes in competitive peptide binding assays. The HMy2.CIR cells constantly expressing indicated HLA-B or HLA-C molecule were incubated with Cy5-labeled reference peptide and the no-labeled epitope peptide. After a defined incubation period, the unbound peptides were removed, and the relative binding affinity of the tested epitope peptide to the HLA-B or HLA-C molecule was quantified by the declined MFI of reference peptide binding to the CIR cell surface at different concentration of tested epitope peptide (5 and 15μm). In parallel, the max control well (CIR cells and Cy5-reference peptide; red solid-line peak in flow cytometry histograms) and background well (CIR cells alone; green solid-line peak in flow cytometry histograms) were performed. (A) Representative flow cytometry plots reflecting the affinity between epitopes and HLA-B4601 (left), the histogram showed the inhibition rates of each epitope at 5 and 10 μM concentrations against the Cy5-reference peptide (right) when incubated with CIR-B4601; (B) representative flow cytometry plots reflecting the affinity between epitopes and HLA-C0102 (left), the histogram showed the inhibition rates of each epitope at 5 and 10 μM concentrations against the Cy5-reference peptide (right) when incubated with CIR-C0102.