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We obtained anticoagulant blood samples from 5 convalescent patients (Supplementary Table S1) with different HLA haplotypes for peptides screening. A total of 57 overlapping peptides of SARS-CoV-2 N protein were designed in this study. ELISpot experiment was performed with PBMCs of convalescent patients under the stimulation of different overlapping peptides. Within the positive overlapping peptides that could stimulate T cells to secrete IFN-γ, peptide nCoV-N49 showed the highest response intensity. Subsequently, potential CD8+ T cell epitopes from nCoV-N49 were predicted according to HLA haplotype of the positive convalescent patient (Table 1). The epitope named N25 (KTFPPTEPK) with HLA-A*1101 restriction was verified as positive by ELISpot.
Peptide nCoV-N49 Predicted epitope Position HLA typing Affinity (nmol/L) Affinity (nmol/L) LLNKHIDAYKTFPPTEPK KTFPPTEPK† 361–369 A*1101 6.28 0.01 LLNKHIDAYK 352–361 A*1101 149.83 0.9 YKTFPPTEPK 360–369 A*1101 19.13 0.1 Abbreviations: SARS-CoV-2=severe acute respiratory syndrome coronavirus type 2; HLA=human leukocyte antigen.
† The peptide was named N25.Table 1. Predicted CD8+ T cell epitopes derived from long peptide nCoV-N49 of SARS-CoV-2.
We selected one HLA-A*1101-positive convalescent patient (ID:11) for intracellular cytokine staining based on flow cytometry. The in vitro cultured PBMCs were harvested; after being stimulated with N25 epitope for 10 h, the PBMCs were stained with fluorescent antibodies. The result showed that PBMCs from the convalescent donor could secrete detectable IFN-γ after being stimulated by N25 epitope. The proportion of IFN-γ-positive CD8+ T cells was 1.30%, which was higher than 0.22% of the control sample without epitope stimulation (Figure 1A). Then the cultured PBMCs with the HLA-A*1101/N25 tetramer were stained and analyzed by flow cytometry, immediately. The N25 tetramer staining showed 3.59% of the CD8+ T cells were specific for peptide N25 in the convalescent patient, and the control tetramer was detected as 0.24% (Figure 1B).
Figure 1.The SARS-CoV-2-specific CD8+ T cells detection in COVID-19 convalescents using the peptide-based IFN-γ-secreting intracellular cytokine staining and tetramer staining in flow cytometry. (A) Intracellular cytokine (IFN-γ) staining, negative control (left), positive sample (right); (B) Detection of SARS-CoV-2-specific CD8+ T cells using N25/HLA- A*1101 tetramer.Abbreviations: SARS-CoV-2=severe acute respiratory syndrome coronavirus type 2; COVID-19=coronavirus disease 2019; HLA=human leukocyte antigen.
To confirm the binding feature of peptide N25 with HLA-A*1101, in vitro folding assay was conducted. The heavy and light chains of HLA-A*1101 molecule were expressed in the form of inclusion bodies by the expression system of Escherichia coli. Then the heavy and light chain proteins were renatured with epitope N25 in vitro. The results showed peptide N25 could help to form the stable HLA-A*1101 complex under the assistance of N25 and that the purified complex eluted at 16.5 mL during purification by chromatography column. The purified complex had the highest peak that could represent efficient renaturation. The formation of the HLA-A*1101/N25 complex was also verified by identification of two protein bends: HLA-A*1101 and β2 microglobulin in SDS-PAGE (Figure 2A).
Figure 2.The HLA-A*1101-binding of newly identified SARS-CoV-2 T-cell epitope and the structural conformation of the peptide presented by HLA-A*1101. (A) The abilities of peptide N25 presented by HLA-A*1101 were evaluated using in vitro refolding and SDS-PAGE. M: protein ladder; P1: Peak 1, presents the heavy chain aggregates; P2: Peak 2, presents the N25/HLA-A*1101 complex; P3: Peak 3, presents the light chain. (B) Crystal of N25/HLA-A*1101 complex. (C) The overall conformation of the structurally-defined peptide N25 was shown with green sticks. The carbon, nitrogen, and oxygen atoms were shown in green, blue, and red, respectively. The α1 helix and β-sheets of HLA-A*1101 were shown in grey stripes. (D) Alignment of the SARS-CoV-2 N25 peptide amino acid sequence with other sarbecoviruses.Abbreviations: SARS-CoV-2=severe acute respiratory syndrome coronavirus type 2; HLA=human leukocyte antigen; SDS-PAGE=Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis.
To show whether N25 was a HLA-A*1101-restricted T-cell epitope with the classic peptide conformation, the crystal structure of HLA-A*1101/N25 complex was determined. The HLA-A*1101/N25 complex protein was purified, and then used to screen the protein crystals by the crystal screening kit (Crystal Screen, Hampton Research, California, USA) at a concentration of 15 mg/mL. On Day 7, the HLA-A*1101/N25 complex was observed to grow one rhombus crystal at 18 ℃ using the buffer solution 2.0 M Ammonium sulfate of CS I kit (Figure 2B). The crystal was sent to Shanghai Synchrotron Radiation Facility and the structure was determined by X-ray crystallography to 1.5 Å resolution (Supplementary Table S2). As other HLA-A*1101-presented peptides, the residues at position 2 (P2) and C-terminus (PΩ) of peptide N25 act as anchor residues (8-9). The ability of MHC to bind peptides is mainly determined by the interaction between the residues at P2 and PΩ of peptide with the B and F pocket of MHC molecule, respectively (10). According to our previous studies, the HLA-A*1101 molecule preferred serine, threonine, or aliphatic amino acids at the second residue of peptide, while the PΩ of HLA-A*1101-binding peptide was usually lysine (11). N25 epitope fits this pattern very well with the P2 residue threonine in pocket B, and the PΩ residue lysine into pocket F, showing a stable bind to the HLA-A*1101 molecule (Figure 2C). In addition to the primary anchors, i.e. threonine at P2 and lysine at the PΩ positions, the P6 residue threonine also acts as a secondary anchor residue for N25, with the side chain inserting into the peptide binding groove of HLA-A*1101. Except these three residues, other residues of N25 are accessible for T-cell recognition.
The sequence conservation of the peptide N25 among the sarbecoviruses such as SARS-CoV and bat coronavirus RaTG13, etc. (Figure 2D) was also analyzed. N25 is completely conserved among sarbecoviruses. This indicated that the peptide N25 that was identified in our study can be used for the evaluation of the T-cell immunity for all these sarbecoviruses and may also act as a candidate for the T cell-based vaccine for these viruses.
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