T-cell epitopes in severe acute respiratory syndrome (SARS) coronavirus spike protein elicit a specific T-cell immune response in patients who recover from SARS

Abstract

The immunogenicity of HLA-A2-restricted T-cell epitopes in the S protein of the Severe acute respiratory syndrome coronavirus (SARS-CoV) and of human coronavirus strain 229e (HCoV-229e) was analyzed for the elicitation of a T-cell immune response in donors who had fully recovered from SARS-CoV infection. We employed online database analysis to compare the differences in the amino acid sequences of the homologous T epitopes of HCoV-229e and SARS-CoV. The identified T-cell epitope peptides were synthesized, and their binding affinities for HLA-A2 were validated and compared in the T2 cell system. The immunogenicity of all these peptides was assessed by using T cells obtained from donors who had fully recovered from SARS-CoV infection and from healthy donors with no history of SARS-CoV infection. HLA-A2 typing by indirect immunofluorescent antibody staining showed that 51.6% of SARS-CoV-infected patients were HLA-A2 positive. Online database analysis and the T2 cell binding test disclosed that the number of HLA-A2-restricted immunogenic epitopes of the S protein of SARS-CoV was decreased or even lost in comparison with the homologous sequences of the S protein of HCoV-229e. Among the peptides used in the study, the affinity of peptides from HCoV-229e (H77 and H881) and peptides from SARS-CoV (S978 and S1203) for binding to HLA-A2 was higher than that of other sequences. The gamma interferon (IFN-gamma) release Elispot assay revealed that only SARS-CoV-specific peptides S1203 and S978 induced a high frequency of IFN-gamma-secreting T-cell response in HLA-A2(+) donors who had fully recovered from SARS-CoV infection; such a T-cell epitope-specific response was not observed in HLA-A2(+) healthy donors or in HLA-A2(-) donors who had been infected with SARS-CoV after full recovery. Thus, T-cell epitopes S1203 and S978 are immunogenic and elicit an overt specific T-cell response in HLA-A2(+) SARS-CoV-infected patients.

FIG. 1.

Alignment of S protein of HCoV-229e and SARS-CoV analyzed by Clustal W (1.82). *, same amino acid residue. : , related amino acid residue. The underlined sequences are the epitopes and their homologous sequences on HCoV-229e.

FIG. 2.

Structural model constructed by computer analysis. The S978 (A) and S1203 (B) epitopes were used in this study. They had a similar structure, with leucine or isoleucine in position 2 and valine or leucine in position 9 as well as in the positive control, the influenza virus peptide 58-66. (C and D) Complex formed by HLA-A2- and SARS-specific epitopes S978 and S1203, which is shown in yellow. S978 and S1203 adapt to combine into the antigen peptide binding site of HLA-A2.

FIG. 3.

Elispot assay. (A) Spots in the Elispot assay with PBMCs obtained from HLA-A2⁺ donors who had fully recovered from SARS for 1 month. The peptides used in the Elispot assay are marked. PBMCs from SARS patients could be activated by epitopes from SARS virus (S978 and S1203), and PBMCs could be activated slightly by the influenza virus peptide epitope 58-66. PBMCs did not secrete IFN-γ without the peptide or when the sequences homologous to S978 and S1203 from HCoV-229e were added to the culture system. (B) The spots are a measure of IFN-γ secretion from PBMCs. The black bar shows the PBMCs from 12 patients who had recovered from SARS, and the open bars show the PBMCs from eight healthy HLA-A2⁺ donors. SARS-CoV-specific peptides S1203 and S978 induced much greater IFN-γ secretion in the PBMCs from donors who had recovered from SARS than in those from healthy donors. Two peptides from HCoV-229e induced moderate IFN-γ-secreting cells in the PBMC from patients who had recovered from SARS infection. Their homologous sequences, non-HLA-A2 associated peptides, induced lower IFN-γ secreting cells or did not induce any IFN-γ-secreting cells in PBMCs from patients who had recovered from SARS, healthy donors,or the nonpeptide group. As a positive control peptide, influenza virus peptide 58-66 induced moderate levels of IFN-γ-secreting cells in HLA-A2-positive PBMCs both from patients who had recovered from SARS and from healthy donors.