Identification and characterization of novel neutralizing epitopes in the receptor-binding domain of SARS-CoV spike protein: revealing the critical antigenic determinants in inactivated SARS-CoV vaccine - PubMed (original) (raw)

Identification and characterization of novel neutralizing epitopes in the receptor-binding domain of SARS-CoV spike protein: revealing the critical antigenic determinants in inactivated SARS-CoV vaccine

Yuxian He et al. Vaccine. 2006.

Abstract

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is considered as a major antigen for vaccine design. We previously demonstrated that the receptor-binding domain (RBD: residues 318-510) of S protein contains multiple conformation-dependent neutralizing epitopes (Conf I to VI) and serves as a major target of SARS-CoV neutralization. Here, we further characterized the antigenic structure in the RBD by a panel of novel mAbs isolated from the mice immunized with an inactivated SARS-CoV vaccine. Ten of the RBD-specific mAbs were mapped to four distinct groups of conformational epitopes (designated Group A to D), and all of which had potent neutralizing activity against S protein-pseudotyped SARS viruses. Group A, B, C mAbs target the epitopes that may overlap with the previously characterized Conf I, III, and VI respectively, but they display different capacity to block the receptor binding. Group D mAb (S25) was directed against a unique epitope by its competitive binding. Two anti-RBD mAbs recognizing the linear epitopes (Group E) were mapped to the RBD residues 335-352 and 442-458, respectively, and none of them inhibited the receptor binding and virus entry. Surprisingly, most neutralizing epitopes (Groups A to C) could be completely disrupted by single amino acid substitutions (e.g., D429A, R441A or D454A) or by deletions of several amino acids at the N-terminal or C-terminal region of the RBD; however, the Group D epitope was not sensitive to the mutations, highlighting its importance for vaccine development. These data provide important information for understanding the antigenicity and immunogenicity of SARS-CoV, and this panel of novel mAbs can be used as tools for studying the structure of S protein and for guiding SARS vaccine design.

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Figures

Fig. 1

Fig. 1

Induction of anti-RBD antibodies in mice immunized with inactivated SARS-CoV vaccine. (A) Antibody responses against the full-length S protein. Sera were tested by ELISA at 1/100 dilution. (B) Antibody responses against the RBD of S protein. Sera were tested by ELISA at 1/100 dilution. (C) Titers of RBD-specific antibodies in the mouse sera collected after the third boost.

Fig. 2

Fig. 2

Reactivity of anti-RBD mAbs with the native and DTT-reduced RBD-Fc measured by ELISA. Antigens were coated at 1 μg/ml, and the mAbs were tested at 10 μg/ml.

Fig. 3

Fig. 3

Neutralization of SARS pseudovirus by anti-RBD mAbs. Infection of HEK293T cells expressing human ACE2 by SARS pseudovirus Tor2 was determined in the presence of serially diluted anti-RBD mAb. Percent neutralization was calculated and plotted. Numbers in parentheses indicate 50% neutralization dose (ND50) at μg/ml.

Fig. 4

Fig. 4

Epitope mapping of mAbs S29 and S33 with overlapping peptides that cover the RBD of S protein by ELISA. Each of the peptides was coated at 5 μg/ml, and the mAbs were tested at 10 μg/ml.

Fig. 5

Fig. 5

Inhibition of RBD-Fc binding to cell-associated human ACE2 expressed on 293T/ACE2 cells measured by flow cytometry. RBD-Fc was used at 1 μg/ml and mAbs were used at 50 μg/ml.

Fig. 6

Fig. 6

% Inhibition of RBD-Fc binding to cell-associated human ACE2 expressed on 293T/ACE2 cells.

Fig. 7

Fig. 7

Binding competition of S25 and Conf V mAbs. Inhibition of competing anti-RBD mAbs on the binding of biotinylated mAbs to RBD-Fc was measured by ELISA. The competing mAb was tested at 100 μg/ml, and % inhibition was calculated.

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