The membrane protein of severe acute respiratory syndrome coronavirus acts as a dominant immunogen revealed by a clustering region of novel functionally and structurally defined cytotoxic T-lymphocyte epitopes

Abstract

Background: Severe acute respiratory syndrome coronavirus (SARS-CoV), which emerged with highly contagious and life-threatening characteristics in 2002, remains a potential risk for future outbreaks. Membrane (M) and envelope (E) proteins are major structural proteins of the SARS-CoV. The M protein has been determined as a protective antigen in humoral responses. However, its potential roles in stimulating cellular immunity remain elusive.

Methods: In this study, a panel of peptides derived from M and E proteins were tested by in vitro refolding, T2 cell-binding assays, and responses stimulated by cytotoxic T-lymphocyte (CTL) epitopes in HLA-A2.1/K(b) transgenic mice and human peripheral blood mononuclear cells (PBMCs).

Results: A nonameric epitope Mn2 and a decameric epitope Md3 derived from the M protein were identified and used for the evaluation of M protein-specific immunity. Responses stimulated by M protein-specific CTL epitopes have been found in the PBMCs of donors who had recovered from SARS infection. Additionally, the transmembrane domain of the M protein may contain a T cell epitope cluster revealed by the immunogenic and structural analysis of a panel of truncated peptides overlapping with Mn2 and Md3.

Conclusions: The M protein of SARS-CoV holds dominant cellular immunogenicity. This, together with previous reports of a strong humoral response against the M protein, may help to further explain the immunogenicity of SARS and serves as potential targets for SARS-CoV vaccine design.

Table 1

NOTE. FI, fluorescent index; SIV, simian immunodeficiency virus. a Estimated half-time of dissociation (T1/2) of HLA-A*0201 peptide complexes calculated using the BIMAS program (http://bimas.dcrt.nih.gov/molbio/hla_bind/index.html). b FI = (mean fluorescein isothiocyanate [FITC] fluorescence with the given peptide—mean FITC fluorescence without peptide)/(mean FITC fluorescence without peptide). Fl ⩾ 1 represents the high-affinity peptide; FI⩾1 represents the low-affinity peptides.

Figure 1

Binding affinity of the synthesized peptides to HLA-A*0201 molecules. A, The refolding results were analyzed by fast-performance liquid chromatography Superdex 200 10/300 GL gel-filtration chromatography, which was calibrated with the standard protein marker, showing that the predicted molecular weight of the protein coming out with 15.2 mL eluting buffer would be 45 kDa, coincident with the molecular mass of the human leukocyte antigen (HLA) complex. A positive control (PC) was performed by the HLA-A*0201-restricted epitope Flu, and the negative control (NC) peptide was CM9 (Table 1). B, In the T2 cell-binding assay, the distribution histograms of cell populations are presented with different fluorescence intensity of fluorescein isothiocyanate (FITC)-anti-HLA-A2 staining of T2 cells cocultured with PC (Flu), NC (CM9), and high-affinity (Fl ⩾ 1) peptides Mn1, Mn2, Md2, Md3, and En4. The results are representative of 3 independent experiments.

Figure 2

Detection of peptide-specific CD8⁺ T cells in HLA-A2.1/K^b transgenic mice. A, Fresh splenocytes from dead mice inoculated with peptides that had double-positive results with both refolding and T2 cellbinding assays were manipulated with the enzyme-linked immunospot (ELISPOT) assay. The bars represent the quantity of spot-forming cells (SFCs) in 10⁵ splenocytes. The results of peptides Mn2 and Md3 consistent with the positive control Flu and the nonspecific stimulus phytohemagglutinin (PHA) have a statistically significant difference compared with the results from peptides Md2 and En4 (P < .05). B, Survey of the peptide-specific CD8⁺ T cells induced in HLA-A2.1/K^b transgenic mice inoculated with DNA plasmid. The mean numbers of the SFCs in 10⁵ Mn2- and Md3- stimulated splenocytes from mice inoculated with pD3-M, represented by the gray bars, are significantly higher than the white bar, which represents the SFC numbers of Mn2- and Md3-stimulated splenocytes from mice inoculated with pcDNA3, according to the statistics. There is no statistically significant difference between the PHA-stimulated splenocytes of these 2 groups.

Figure 3

Measurement of peptide-specific CD8⁺ T cells from peripheral blood mononuclear cells (PBMCs) of donors recovered from severe acute respiratory syndrome (SARS) by enzyme-linked immunospot (ELISPOT) assay. The thawed PBMCs were manipulated with the ELISPOT assay kit. Shaded bars represent the mean numbers of spot-forming cells (SFCs) in the PBMCs of HLA-A2⁺ recovered donors, whereas white bars represent the PBMCs from HLA-A2+ healthy donors. The former differs statistically from the latter when stimulated with the peptides. Nevertheless, no difference has been shown between them when stimulated with nonspecific stimulus phytohemagglutinin (PHA).

Figure 4

Frequency of Mn2- and Md3-specific CD8⁺ T cells. A, In the tetramer staining, the splenocytes from mice inoculated with peptides were cultured in vitro for 10 days. Next, the cells were stained with phycoerythrin (PE)-labeled HLA-A*0201/peptide chimeric tetramer together with fluorescein isothiocyanate (FITC)-labeled anti-mouse CD8 antibody. Top left: Mn2-inoculated mice stained with Mn2 tetramer. Top right: Phosphatebuffered saline (PBS)-inoculated mice stained with Mn2 tetramer. Bottom left: Md3-inoculated mice stained with Md3 tetramer. Bottom right: PBS-inoculated mice stained with Md3 tetramer. The results are representatives of 3 independent experiments. B, Mn2-specific (top left) and Md3-specific (bottom left) CD8⁺ T cells were detected from the thawed peripheral blood mononuclear cells (PBMCs) of HLA-A2⁺ donors recovered from severe acute respiratory syndrome, by staining with fluorescein isothiocyanate-labeled anti-CD8 mAb along with PE-labeled HLA-A*0201/peptide tetramers. No specific CD8⁺ T cells were stained with Mn2 (top right) or Md3 tetramers among HLA-A2⁺ healthy donors (bottom right).

Table 2

This table is available in its entirety in the online version of Journal of Infectious Diseases.

Figure 5

Structures of HLA-A*0201 complexed with Mn2 (GLMWLSYFV) and Md3 (TLACFVLAAV). Structural overviews of HLA-A2 with peptides Mn2 (A) and Md3 (B) demonstrate that both have 2 molecules in 1 asymmetric unit. 2Fo-Fc electron density for Mn2 (C) and Md3 (D) peptides are shown as violet and blue wire (contoured at 1.0 δ) viewed in profile through the HLA-A2 α2 helix. The carbon atoms are shown as yellow and green for Mn2 and Md3, respectively. E, Comparison of Mn2 (yellow) and Md3 (green) peptide structures by superimposition of the second residues from the N-terminal residues (Leu) and the C-terminal residues (Val). F, General side chain orientation for the 2 peptides as viewed in profile from the peptide N terminus toward the C terminus (up is toward the T cell receptor; down is toward the floor of the peptide binding groove; left is toward the a1 helix domain; right is toward the α2 helix domain). The figure was generated using the PyMOL program (http://www.pymol.org/).

Figure 6

Schematic diagram of severe acute respiratory syndrome coronavirus (SARS-CoV) membrane (M) protein on the virion envelope, addressing the immune epitopes. The M protein comprises a domain spanning a triple membrane of ∼80 amino acid residues, a short Nterminus protruding out of the membrane, and a long cytoplasmic Cterminus. Md3 (green) and Mn2 (yellow) are completely included in the second and the third transmembrane domain, respectively. The identified B cell epitopes that could be recognized by antibodies are also represented in the diagram (cyan). The predicted structure of the M protein is based on previous studies [48].