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. 2022 Jan 18:12:764949.
doi: 10.3389/fimmu.2021.764949. eCollection 2021.

CD8+ T-Cell Epitope Variations Suggest a Potential Antigen HLA-A2 Binding Deficiency for Spike Protein of SARS-CoV-2

Congling Qiu  1   2   3 , Chanchan Xiao  2   3 , Zhigang Wang  1 , Guodong Zhu  3   4 , Lipeng Mao  2   3 , Xiongfei Chen  5 , Lijuan Gao  2   3 , Jieping Deng  2   3 , Jun Su  1 , Huanxing Su  3   6 , Evandro Fei Fang  3   7 , Zhang-Jin Zhang  3   8 , Jikai Zhang  9 , Caojun Xie  5 , Jun Yuan  5 , Oscar Junhong Luo  3   10 , Li An Huang  1 , Pengcheng Wang  2   3 , Guobing Chen  1   2   3
Affiliations

CD8+ T-Cell Epitope Variations Suggest a Potential Antigen HLA-A2 Binding Deficiency for Spike Protein of SARS-CoV-2

Congling Qiu et al. Front Immunol. .

Abstract

We identified SARS-CoV-2 specific antigen epitopes by HLA-A2 binding affinity analysis and characterized their ability to activate T cells. As the pandemic continues, variations in SARS-CoV-2 virus strains have been found in many countries. In this study, we directly assess the immune response to SARS-CoV-2 epitope variants. We first predicted potential HLA-A*02:01-restricted CD8+ T-cell epitopes of SARS-CoV-2. Using the T2 cell model, HLA-A*02:01-restricted T-cell epitopes were screened for their binding affinity and ability to activate T cells. Subsequently, we examined the identified epitope variations and analyzed their impact on immune response. Here, we identified specific HLA-A2-restricted T-cell epitopes in the spike protein of SARS-CoV-2. Seven epitope peptides were confirmed to bind with HLA-A*02:01 and potentially be presented by antigen-presenting cells to induce host immune responses. Tetramers containing these peptides could interact with specific CD8+ T cells from convalescent COVID-19 patients, and one dominant epitope (n-Sp1) was defined. These epitopes could activate and generate epitope-specific T cells in vitro, and those activated T cells showed cytolytic activity toward target cells. Meanwhile, n-Sp1 epitope variant 5L>F significantly decreased the proportion of specific T-cell activation; n-Sp1 epitope 8L>V variant showed significantly reduced binding to HLA-A*02:01 and decreased proportion of n-Sp1-specific CD8+ T cell, which potentially contributes to the immune escape of SARS-CoV-2. Our data indicate that the variation of a dominant epitope will cause the deficiency of HLA-A*02:01 binding and T-cell activation, which subsequently requires the formation of a new CD8+ T-cell immune response in COVID-19 patients.

Keywords: CD8+ T-cell epitope; SARS-CoV-2; antigen presentation deficiency; spike protein; variations.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Identification of HLA-A2-restricted T-cell epitopes in SARS-CoV-2 Spike protein. Establishment of T2 binding assay. T2 cells were incubated with a series of concentrations (0 µM, 0.625 µM, 1.25 µM, 2.5 µM, 5 µM, 10 µM, and 20 µM) of positive control (Influenza A M1 peptide M58-66 GILGFVFTL) and negative control (Zika virus P30-38 GLQRLGYVL) peptides. (A) The stabilized HLA-A2 was detected with anti-HLA-A2 fluorescent antibody flow cytometry staining. (B, C) Screening of SARS-CoV-2 epitopes in T2 cells. Predicted peptides named as n-Sp1-15 were synthesized and 10 µM of each peptide was incubated with T2 cells. (B) The binding of peptides on T2 cells was measured with anti-HLA-A2 staining flow cytometry. (C) Values indicate the MFI of stabilized HLA-A2. Screening of SARS-CoV-2 epitopes with ELISA. Peptide exchange assay was performed with coated UV-sensitive peptide/MHC complex and given peptides. (D) The binding capability was measured by an anti-HLA-A2 ELISA assay. Blank, no peptides; Neg ctrl, negative control, peptide Zika virus peptide P30-38 GLQRLGYVL; Pos ctrl, positive control, influenza A M1 peptide M58-66 GILGFVFTL; HLA ctrl, UV-sensitive peptide without UV irradiation; UV ctrl, UV-sensitive peptide with UV irradiation. (E, F) Measurement of peptide-specific CD8+ T cells in HLA-A2+ convalescent COVID-19 patients and healthy donors by flow cytometry. (E) shows a representative FACS plot and the percentage of tetramer-positive cells is shown in (F) Healthy donors, n = 4; convalescent COVID-19 patients, n = 10. Ctrl: tetramer with UV-sensitive peptide; Pa, convalescent COVID-19 patients; ND, healthy donors.
Figure 2
Figure 2
Activation of CD8+ T cell by peptides on SARS-CoV-2 Spike protein. (A, B) Mitomycin pretreated T2 cells were loaded with n-Sp1, 2, 6, 7, 11, 13, and 14 peptides and incubated with CD8+ T cell from healthy donors at a 1:1 ratio. Activation, cytotoxicity, and generation of epitope-specific CD8+ T cells were evaluated. Expression level of the T-cell activation marker CD69 was evaluated with flow cytometry after 16 h of stimulation. (B) Values indicate the percentage of CD69+CD8+ T cells, n = 3. (C, D) Expression level of the T-cell activation marker CD137 was evaluated with flow cytometry after 16 h stimulation. (D) Values indicate the percentage of CD137+CD8+ T cells. n = 3. (E, F) Epitope-specific CD8+ T-cell-mediated cytotoxicity was evaluated after 7 days of culture. The remaining CFSE-labeled T2 cells were calculated as survived target cells. (F) Values indicate the percentage of surviving T2 cells, n = 3. Apoptosis of T2 cells after 7 days culture. (G, H) Epitope-stimulated T-cell-mediated T2 apoptosis was calculated as the proportion of CFSE+AnnexinV+ cells. (H) Values indicate the percentage of CFSE+Annexin V+ T cells, n = 3. (I, J) The expression of IFN-γ after epitope stimulation for 7 days. IFN-γ was measured with intracellular staining flow cytometry. (J) Values indicate the percentage of IFN-γ+CD8+ T cells, n = 6. (K, L) Epitope-specific CD8+ T-cell generation after 7 days of stimulation. Stimulated CD8+ T cells were stained with given epitope-based tetramer and measured with flow cytometry. (L) Values indicate the percentage of tetramer+ cells, n = 5. Day 0 ctrl: staining before stimulation; T2 ctrl: T2 without peptide loading; Pos ctrl: positive control, T2 loaded with influenza A M1 peptide M58-66 GILGFVFTL. Neg ctrl, negative control; T2 loaded with Zika virus P30-38 GLQRLGYVL peptides. (M, N) A representative dot plot showing the measurement of peptide specific CD8+ T cells in HLA-A2+ non-vaccinated and vaccinated donors by flow cytometry. (N) Values indicate the percentage of n-Sp1 tetramer+CD8+ T cells, n = 5 of non-vaccinated donors and n = 16 of vaccinated donors. The data are represented as the mean ± SD in three independence experiments, ***p ≤ 0.001.
Figure 3
Figure 3
Immune response alteration in epitope mutants of SARS-CoV-2. (A) Variation frequency of epitopes on SARS-CoV-2 Spike protein. All SARS-CoV-2 virus sequences were collected from GISAID database, and the amino acid mutations in each epitope were calculated. (B) List of the epitope mutants for subsequent experiments. Comparison of epitope mutant binding to HLA-A2 in T2 cells. Wild-type and mutated epitopes were synthesized and incubated with T2 cells. (C, D) The peptide binding on T2 cells was measured with anti-HLA-A2 staining flow cytometry. (D) Values indicate the stabilized HLA-A2. Blank: no peptides; Neg ctrl, negative control; Zika virus peptide (P30-38 GLQRLGYVL), Pos ctrl: positive control; influenza A M1 peptide (M58-66 GILGFVFTL). (E) Comparison of epitope mutant binding to HLA-A2 by ELISA assay. Peptide exchange assay was performed with coated UV-sensitive peptide/MHC complex and given peptides. The binding capability was measured with anti-HLA-A2 ELISA assay. Blank: no peptides; HLA ctrl: UV-sensitive peptide without UV irradiation; Neg ctrl: negative control, Zika virus peptide (P30-38 GLQRLGYVL); Pos ctrl, positive control; influenza A M1 peptide (M58-66 GILGFVFTL); UV ctrl, UV-sensitive peptide with UV irradiation. (F, G) Measurement of n-Sp1- and n-Sp1-m1-specific CD8+ T cells in HLA-A2 convalescent COVID-19 patients with flow cytometry. (G) Values indicate the percentage of tetramer+ T cells, n = 3. Ctrl, tetramer with UV-sensitive peptide. The data are represented as the mean ± SD in three independence experiments, **p ≤ 0.01; ****p ≤ 0.001.
Figure 4
Figure 4
Comparison of T-cell activation and function by n-Sp1 mutants. (A–D) Mitomycin pretreated T2 cells were loaded with n-Sp2, 6, 7, 11, 13, 14, plus n-Sp1 (n-Sp1 mix), n-Sp1m1 (n-Sp1m1 mix), n-Sp1m2 (n-Sp2 mix), or n-Sp1m3 (n-Sp3 mix) peptides, respectively. Next, pretreated T2 cells were co-cultured with CD8+ T cells from healthy donors at a 1:1 ratio. Expression level of T-cell activation marker CD69 and CD137 was evaluated with flow cytometry after 16 h of stimulation. (A, C) are the representative plots of data in bar graphs (B, D), n = 4. Generation of n-Sp1- and n-Sp1m1-specific CD8+ T cells in the same subjects. (E, F) The epitope-specific CD8+ T cells from the same subjects were measured with corresponding epitope peptide-based tetramer after 7 days of stimulation. (E) is the representative plot for (F). (G, H) The epitope simulation condition and tetramer information are labeled above and below the dot plot, respectively. n = 3. n-Sp1 tetramer could not recognize mutant-stimulated CD8+ T cells in the same subjects. n-Sp1 tetramer was used to stain n-Sp1 or n-Sp1 mutant peptide-stimulated CD8+ T cells from the same subjects after 7 days. (G) is the representative plot for (H). The epitope simulation condition and the tetramer information were labeled above and below the dot plot, respectively. n = 3. T2 ctrl: T2 cells without peptide loading; Neg ctrl: T2 cells loaded with Zika virus peptide P30-38 GLQRLGYVL; Pos ctrl: T2 cells loaded with influenza A M1 peptide M58-66 GILGFVFTL. The data are represented as the mean ± SD in three independence experiments, ***p ≤ 0.001.
Figure 5
Figure 5
Molecular mechanism of epitope mutant presentation on HLA-A2. (A) GalaxyPepDock was used for molecular docking to demonstrate the pMHC structure of n-Sp1 and n-Sp1-m1. Summaries of molecular docking, including the templates used for docking. The first 4 characters of the template were PDB ID number. The models were ranked by the similarity scores and combined with the same parameters in one line. (B) Simulated crystal structure of pMHC were presented for n-Sp1 and n-Sp1-m1. The gray stick represented epitope peptide. (C) Comparison of pMHC structure between n-Sp1 and n-Sp1-m1. The HLA-A2 structure was shown as ribbon diagrams and n-Sp1 and n-Sp1-m1 mutation sites were shown as green and red sticks, respectively. The angle altered amino acids were indicated as F3 and P8 at position 4 and 8, respectively.
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