Identification of an antigenic determinant on the S2 domain of the severe acute respiratory syndrome coronavirus spike glycoprotein capable of inducing neutralizing antibodies

Abstract

Severe acute respiratory syndrome (SARS) is a life-threatening disease caused by a newly identified coronavirus (CoV), SARS-CoV. The spike (S) glycoprotein of CoV is the major structural protein responsible for induction of host immune response and virus neutralization by antibodies. Hence, knowledge of neutralization determinants on the S protein is helpful for designing protective vaccines. To analyze the antigenic structure of the SARS-CoV S2 domain, the carboxyl-terminal half of the S protein, we first used sera from convalescent SARS patients to test the antigenicity of 12 overlapping fragments spanning the entire S2 and identified two antigenic determinants (Leu 803 to Ala 828 and Pro 1061 to Ser 1093). To determine whether neutralizing antibodies can be elicited by these two determinants, we immunized animals and found that both of them could induce the S2-specific antisera. In some animals, however, only one determinant (Leu 803 to Ala 828) was able to induce the antisera with the binding ability to the native S protein and the neutralizing activity to the SARS-CoV pseudovirus. This determinant is highly conserved across different SARS-CoV isolates. Identification of a conserved antigenic determinant on the S2 domain of the SARS-CoV S protein, which has the potential for inducing neutralizing antibodies, has implications in the development of effective vaccines against SARS-CoV.

FIG. 1.

Location and expression of the partially overlapping fragments covering the S2 domain. (A) Schematic representation of the S2 domain (represented by the thick top line) and the twelve fragments F1 to F12 and their amino acid locations on the S protein. The dashed box defines the major antigenic determinant (Leu 803 to Ala 828) described in this paper. (B) SDS-PAGE of the E. coli-expressed and Ni-NTA-purified Trx fusion fragments F1 to F12 (lanes 1 to 12).

FIG. 2.

Identification of the antigenic determinants on the S2 domain. A total of 15 patients' sera and 2 healthy human sera were used in ELISA to analyze the antigenicity of F1 to F12. In each group, 17 bars represent the 2 healthy human sera and the 15 different patients' sera, respectively, from left to right. The serum samples were diluted 1:800.

FIG. 3.

Western blot analysis of polyclonal antisera from animals immunized with the Trx-fusion proteins. R31 and M31 represent the antisera of rabbit 1 and mouse 1 immunized by Trx-F3; R91 and M91 represent the antisera of rabbit 1 and mouse 1 immunized by Trx-F9. 28kD, 33kD, 35kD, and 81kD represent the molecular masses of GST (lanes 1, 4, 7, and 10), GST-F3 (lanes 2 and 8), GST-F9 (lanes 5 and 11), and GST-S2 (lanes 3, 6, 9, and 12) protein, respectively. Molecular mass standards are shown on the left. The antisera from other immunized animals showed results similar to those obtained with antisera R31 and M31 or antisera R91 and M91.

FIG. 4.

Ability of the antisera to bind to the cell surface-expressed S protein. The S-expressing SP2/0 cells (SP2/0-S) and the normal control cells (SP2/0) submitted to flow cytometric analysis were stained with the healthy human serum (healthy) and the patient serum (patient) (A); the antisera from two rabbits (R31 and R32) and four mice (M31 to M34) immunized with Trx-F3 (B); or the antisera from two rabbits (R91 and R92) and four mice (M91 to M94) immunized with Trx-F9 (C). Data of flow cytometry were analyzed with Summit version 3.1 software (Dako Cytomation).

FIG. 5.

Neutralization assay of pseudotype viruses. (A) The neutralizing activity of SARS patient serum and normal human serum (diluted 1:100) for HIV/SARS or HIV/VSV-G pseudovirus. (B) The neutralizing activity of the animal sera (1:10 to 1:320 diluted) for HIV/SARS pseudovirus. R31 and R32, antisera from the two rabbits immunized with Trx-F3; M31 to M34, antisera from the four mice immunized with Trx-F3; R91 and R92, antisera from the two rabbits immunized with Trx-F9; M91 to M94, antisera from the four mice immunized with Trx-F9; RC and MC, the preimmune sera from rabbits and mice. (C) The neutralizing activity of the two neutralizing antisera (M34 and R31) and their corresponding preimmune sera (MC and RC) (diluted 1:40) for HIV/VSV-G pseudovirus. All results of neutralization assays represent four repeated experiments.

FIG. 6.

Comparison of the amino acid sequences between antigenic determinant I (26 amino acids; represented by the blank box) on the SARS-CoV S2 domain and the immunodominant domain (68 amino acids; represented by the shaded box) on the MHV-A59 S2 subunit (7).