Identification of immunodominant epitopes on the membrane protein of the severe acute respiratory syndrome-associated coronavirus - PubMed (original) (raw)

Identification of immunodominant epitopes on the membrane protein of the severe acute respiratory syndrome-associated coronavirus

Yuxian He et al. J Clin Microbiol. 2005 Aug.

Abstract

Similar to other coronaviruses, the membrane (M) protein of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is a major transmembrane glycoprotein with multiple biological functions. To date, limited information is available about its antigenic properties. In this study, we identified two major immunodominant epitopes on the M protein located in the extreme N-terminal region (residues 1 to 31) and the interior C-terminal region (residues 132 to 161), respectively, by Pepscan analyses against convalescent-phase sera from SARS patients and antisera from virus-immunized mice and rabbits. Synthetic peptides M1-31 derived from the N-terminal epitope and M132-161 derived from the C-terminal epitope were highly reactive with all of the convalescent-phase sera from 40 SARS patients but not with 30 control serum samples from healthy blood donors, suggesting their potential application for serologic diagnosis of SARS. We showed that both peptides (M1-31 and M132-161) were able to induce high titers of antibody responses in the immunized rabbits, highlighting their antigenicity and immunogenicity. These findings provide important information for developing SARS diagnostics and vaccines.

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Figures

FIG. 1.

FIG. 1.

Schematic diagram of SARS-CoV M protein. The M protein contains a short N terminus in the exterior of the virion (residues 1 to 14), three transmembrane helices (residues 15 to 37, 50 to 72 and 77 to 99), and a 121-amino-acid C-terminal region inside the virus particle.

FIG. 2.

FIG. 2.

Mapping of immunodominant epitopes on the M protein of SARS-CoV by ELISA. (A) Antibodies specific for SARS-CoV in the convalescent-phase sera from 40 SARS patients were measured by commercial diagnostic kit, in which the mixture of proteins purified from viral lysates of SARS-CoV was used as coating antigen. (B) Pepscan analysis against the convalescent-phase sera from 40 SARS patients with a set of 30 overlapping peptides that span the entire sequence of the M protein as coating antigens. Sera, tested at 1:50 dilution, were considered positive when the _A_450 values were above the cutoff value (mean _A_450 value of sera from health blood donors plus three standard deviations).

FIG. 3.

FIG. 3.

Mapping of immunodominant epitopes on the M protein of SARS-CoV by Pepscan analysis against antisera from mice (A) and rabbits (B) immunized with inactivated SARS-CoV. A set of 30 overlapping peptides that span the entire sequence of the M protein were used as coating antigens in ELISA. Antisera were tested at 1:100 dilutions.

FIG. 4.

FIG. 4.

Immunoreactivity of synthetic peptides (M1-31 and M132-161) derived from the N-terminal or C-terminal immunodominant epitopes on the M protein with the convalescent-phase sera from 40 SARS patients and antisera from immunized animals. Serum samples were tested as a series of twofold dilutions by ELISA.

FIG. 5.

FIG. 5.

Detection of M protein-specific antibodies in the convalescent-phase sera of SARS patients with synthetic peptides derived from the N-terminal epitope (A) and C-terminal epitope (B), respectively. Sera from 40 SARS patients and 30 healthy blood donors were tested at 1:50 dilutions. The dashed lines represent the cutoff values (mean _A_450 value of sera from health blood donors plus three standard deviations).

FIG. 6.

FIG. 6.

Antibody responses in rabbits immunized with synthetic peptides derived from the N-terminal epitope (A and B) and C-terminal epitope (C and D), respectively. Rabbit sera were tested as a series of fourfold dilutions by ELISA.

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