Mapping of antigenic sites on the nucleocapsid protein of the severe acute respiratory syndrome coronavirus

Abstract

Antigenic sites on the nucleocapsid (N) protein of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) were mapped by Pepscan analysis with overlapping peptides that span the N protein sequence. Two major immunodominant epitopes located in the C-terminal region (amino acids [aa] 362 to 412) and middle region (aa 153 to 178) reacted with more than 75% of sera from SARS patients. Several minor immunodominant epitopes were reactive with about 50% of the SARS sera. Antisera from mice immunized with inactivated SARS-CoV recognized the two major immunodominant epitopes and one antigenic site located adjacent to the N-terminal region (aa 76 to 101), which did not react with the sera from SARS patients. Several monoclonal antibodies against SARS-CoV bound to the N- or C-terminal antigenic sites. These results suggest that the above antigenic sites on the N protein are important in eliciting humoral immune response against SARS-CoV in humans and animals and can be used as antigens for developing diagnostic tests.

FIG. 1.

Detection of anti-N Abs in convalescent-phase sera from SARS patients by ELISA. Recombinant SARS-CoV N protein was used for coating plates, and the sera from 42 SARS patients and 30 healthy blood donors were tested at a 1:50 dilution. Sera were considered positive when the optical density values were above the cutoff value (mean absorbance at 405 nm of sera from healthy blood donors plus 3 standard deviations).

FIG. 2.

Mapping of the immunodominant epitopes on the SARS-CoV N protein by Pepscan analysis against antisera from SARS patients. A set of overlapping peptides that covers the entire N protein sequence was used for the coating plate, and the sera (1:50 dilution) from 42 SARS patients and 30 healthy blood donors were tested by ELISA. Sera were considered positive when the optical density values were above the cutoff value (mean optical density of absorbance at 405 nm of sera from healthy blood donors plus 3 standard deviations). The positive rate of SARS sera for each peptide was calculated.

FIG. 3.

Anti-N Ab responses in the sera of mice immunized with inactivated SARS-CoV. Two immune serum samples and one normal mouse serum sample at a 1:100 dilution were tested against the recombinant N protein by ELISA.

FIG. 4.

Mapping of the antigenic sites on the SARS-CoV N protein by Pepscan analysis against mouse antisera. The reactivity of the mouse antisera with the overlapping peptides that cover the entire sequences of the N protein was determined by ELISA. Mouse sera were tested at a 1:100 dilution.

FIG. 5.

Epitope mapping of anti-N MAbs. Reactivity of anti-N MAbs with the recombinant N protein and peptides derived from the N protein was determined by ELISA. (A) The recombinant N protein; (B) the peptide corresponding to aa 388 to 404; (C) the peptide corresponding to aa 76 to 93; (D) the peptide corresponding to aa 84 to 101. Pooled sera from two mice immunized with inactivated SARS-CoV were used as the positive control (P), while a normal mouse serum was used as the negative control (N). The MAbs were tested at a concentration of 10 μg/ml, and the control sera were tested at a 1:100 dilution.