Identification Of Conserved HLA-A～*0201-restricted Epitopes From The Nucleocapsid Protein And The Spike Protein Of SARS-CoV

Severe acute respiratory syndrome (SARS) is a serious threat to public health and the economy on a global scale because of its communicability, associated mortality, and the potential for pandemic spread. The SARS-associated coronavirus (SARS-CoV) has been identified as the etiological agent for SARS. Although the SARS outbreak is now under control, the likelihood of possible human and animal reservoirs suggest that this virus will continue to pose a worldwide public health threat.Little information about the pathogenesis of SARS is available at present. This has significantly impeded the progress for designing more effective strategies for the prevention and treatment of SARS. Due to the complexity of immune response to SARS-CoV and that to the other coronaviruses in hosts, the structural and immunogenic properties of the structural protein of SARS-CoV must be ascertained experimentally.In coronavirus infections, such as MHV, BoCV, PEDV, TCoV and IBV, T cells are critical to protection against illness. Both CD4 and CD8 T cells (T helper and cytotoxic T cells or CTL, respectively) are involved. But, both cell-mediated and humoral immune responses have been associated with exacerbations of disease in some coronavirus infections. The S protein of feline infectious peritonitis virus (FIPV), a coronavirus, induces antibodies in the immunized animals that do not neutralize, but rather enhance infection after virus challenge. The CD8+ T-cell epitope existing in the surface glycoprotein of murine hepatitis virus caused an immunopathological reactione. In addition to the S protein, coronaviruses also contain other proteins, which may induce humoral or cellular immune responses that exacerbate the pathology in some coronavirus infections.CD8 CTL has been implicated as playing a role in recovery or clearance of virus during animal coronavirus and SARS-CoV infection. But, immune responses againstSARS-CoV do cause pathological damage to the tissues of the host. Some proteins in SARS-CoV may induce harmful immune and/or inflammatory responses, a potential cause of SARS pathogenesis.Epitopes are the important antigenic elements of virus structural proteins, which are functional in inducing antibody production and cell-mediated immunity against viruses. Identification of epitopes from structural proteins of SARS-CoV could provide the basis for the development of immunity-based treatment for SARS and understanding of the mechanisms underlying SARS-CoV immunopathogenesis.Previous studies of SARS coronavirus showed that, the N protein and S protein of SARS-CoV are important immunogens for T lymphocytes and may play a concernful role in immunity-mediated virus elimination. In this study, we focused on the N protein and S protein of the SARS-CoV as the target antigens for our reseach of mapping epitopes, and identified virus-specific cytotoxic T lymphocyte epitopes from conserved regions of two proteins.Since HLA-A*0201 is expressed in all major ethnicities in the 39-46% range. Furthermore, coronaviruses are especially prone to mutation and recombination. In the study, the epitopes identified are HLA-A*0201 -restricted CTL epitopes and selected from conserved regions of N protein and S protein, respectivly. Thus the epitopes may be "broad spectrum" over a large number of strains of SARS-CoV and have great population coverage. We propose that this epitope may be of practical significance for the development of immunotherapy for SARS and characterization of the mechanisms underlying SARS-CoV immunopathogenesis.In the study, four candidate peptides from conserved regions of N protein were designed, based on a HLA-A*0201 binding peptide prediction system and a proteosomal cleavage site prediction system, and tested for their binding affinity to HLA-A*0201 molecules using T2 cells. The binding affinities were expressed as fluorescence index (FI) defined as (MFI sample-MFI background) / (MFI background). The MFI background represented the value without peptide. The four candidate peptides and their FI are: 1.9 for N(223-23i)LLLDRLNQL> 1.1 for Na6O-i68)LQLPQGTTL> 1.6forN(332-340)LTYHGAIKLand 0.8 for N(304-3i2)QIAQFAPSA, respectively. Candidate peptides were then assessed for their capacity to elicit in vitro specific immune responses mediated by cytotoxic T lymphocytes(CTLs) from peripheral blood lymphocytes sourced from healthy HLA-A2+ donors. In this study, SARS-CoV N protein-derived peptides, N(223-23i) and N(332-340), induced specific CTLs specifically released interferon-gamma and lysed target cells (T2 cells loaded with corresponding peptides) upon stimulation with peptides-pulsed autologous dendritic cells, respectively. These results show the existence of functional anti-N protein CTL precursors within the peripheral T cell repertoire of healthy donors, and indicate N protein-derived peptides, N(223-23i) and N032-340), as potential epitopes for the development of vaccination strategies. To further address whether the immunogenic candidate peptide is naturally processed and presented, HLA-A*0201 molecules transgenic mice immunized with pCI-neo(N) plasmid containing a full-length cDNA encoding the SARS-CoV N protein. To examine CTL level in DNA-immunized mice, splenocytes were collected from immunized mice and restimulated in vitro for an additional 6 days. The cytokine determination and cytotoxicity assays were performed to examine the CTL level. J(A2/kb) cells loaded with the corresponding peptides were used as targets in cytotoxicity assays. CTLs from immunized mice were able to lyse peptide N(223-23i> and/or N(332-34o>-pulsed J(A2/kb) cells. Furthermore, Bulk CTLs from immunized mice responding to peptide N(223-23i) and/or N(332-34o> showed different IFN-y production. But the CTL responses induced by N(223-23i) were more efficiently than by N(332-340)- HLA-A*0201-N223-23i tetramer staining revealed the presence of significant populations of N223.23i-specific CTLs in N223-23iⁱnduced CD8+ T cells. According to the experimental results, N(223-23i) was a HLA-A*0201-resteicted CTL epitopes of N proteinIn the study, eight candidate peptides from conserved regions of S protein were designed, based on a HLA-A*0201 binding peptide prediction and a proteosomal cleavage site prediction system, and tested for their binding affinity to HLA-A*0201 molecules using T2 cells. The eight candidate peptides and their FI are: 0.5 for S(2-io)FIFLLFLTL, 0.8 for S(85i-859)MIAAYTAAL, 0.4 for S(404-4i2)VIADYNYKL, 0.3 for S(2o8.2i6)DLPSGFNTL, 1.1 for S(94o-948)ALNTLVKQL, 1.1 for Sai74-n82)NLNESLIDL, 1.2 for S(673.68i)SIVAYTMSL and 1.5 for S(958-966)VLNDILSRL, respectively. Candidate peptides were then assessed for their capacity to elicit in vitro specific immune responses mediated by cytotoxic T lymphocytes (CTLs) from peripheral blood lymphocytes sourced from healthy HLA-A2+ donors. In this study, SARS-CoV S protein-derived peptides, S(in4.ii82), S(673-68i) andS(958-966), induced specific CTLs specifically released interferon-gamma and lysed target cells (T2 cells loaded with corresponding peptides) upon stimulation with peptides-pulsed autologous dendritic cells, respectively. These results show the existence of functional anti-S protein CTL precursors within the peripheral T cell repertoire of healthy donors, and indicate S protein-derived peptides, S(H74-n82). S(673-68i) and S(95g₉66)> as potential epitopes for the development of vaccination strategies. To further address whether the immunogenic candidate peptide is naturally processed and presented, HLA-A*0201 molecules transgenic mice immunized with S/pVAXl plasmid containing a full-length cDNA encoding the SARS-CoV S protein. To examine CTL level in DNA-immunized mice, splenocytes were collected from immunized mice and restimulated in vitro for an additional 6 days. The cytokine determination and cytotoxicity assays were performed to examine the CTL level. J(A2/kb) cells loaded with the corresponding peptides were used as targets in cytotoxicity assays. CTLs from immunized mice were able to lyse peptide S(95g.966)-pulsed J(A2/kb) cells. Furthermore, Bulk CTLs from immunized mice responding to peptide S(95g₉66) showed great IFN-y production. According to the experimental results, S(95g₉66) was a novel HLA-A*0201-resteicted CTL epitopes of S protein.In conclusion, our study has identified two HLA-A*0201 -restricted epitope from conserved regions of N protein and S protein of SARS-CoV, respectively. We propose that the newly identified epitopes could be used for evaluation of SARS-CoV-specific CD8+ T-cell responses during the course of SARS infection and treatment. Furthermore, the epitopes should help in the characterization of mechanisms of virus control and immunopathology in SARS-CoV infection, and may be relevant to the development of ethnically unbiased, widely applicable immunotherapeutic approaches for this disease.