Antibody-mediated synergy and interference in the neutralization of SARS-CoV at an epitope cluster on the spike protein

Abstract

Incomplete neutralization of virus, especially when it occurs in the presence of excess neutralizing antibody, represents a biological phenomenon that impacts greatly on antibody-mediated immune prophylaxis of viral infection and on successful vaccine design. To understand the mechanism by which a virus escapes from antibody-mediated neutralization, we have investigated the interactions of non-neutralizing and neutralizing antibodies at an epitope cluster on the spike protein of severe acute respiratory syndrome coronavirus (SARS-CoV). The epitope cluster was mapped at the C-terminus of the spike protein; it consists of structurally intertwined epitopes recognized by two neutralizing monoclonal antibodies (mAbs), 341C and 540C, and a non-neutralizing mAb, 240C. While mAb 341C binds to a mostly linear epitope composed of residues (507)PAT(509) and V(349), mAb 240C binds to an epitope that partially overlaps the former by at least two residues (P(507) and A(508)). The epitope corresponding to mAb 540C is a conformational one, involving residues L(504) and N(505). In neutralization assays, non-neutralizing 240C disrupted virus neutralization by mAb 341C and/or mAb 540C, whereas a combination of mAbs 341C and 540C blocked virus infectivity synergistically. These findings indicate that the epitope cluster on the spike protein may serve as an evolutionarily conserved platform at which a dynamic interplay between neutralizing and non-neutralizing antibodies occurs, thereby determining the outcome of SARS-CoV infection.

Fig. 1

Epitope mapping. (A) Epitope mapping by ELISA. One hundred nanograms biotin-labeled peptide WT 491–510 was added to streptavidin-coated 96-well plates in an ELISA. The X-axis indicates different mouse monoclonal antibodies and a control. The Y-axis indicates absorbance at 450 nm in ELISA. Absorbance of mAb 341C is compared with that of mAb 240C, mAb 540C and control antibody. t test results are shown above each column. p < 0.05 is considered significant. (B) Peptide 491–510 mutants. Amino acid sequences encompassing residues 491–510 (WT) and its mutations (NA > RR, P > R and TV > RR) are indicated. (C) ELISA of peptide 491–510 mutants. Biotin-labeled peptide 491–510 (100 ng/well), defined as WT, and its mutants, NA > RR, P > R and TV > RR, respectively, were added to streptavidin-coated 96-well plates. MAb 341C at 1:100 dilution that reacted with WT peptide was used as the probe The X-axis indicates peptides used in the assay. The Y-axis indicates absorbance at 450 nm, representing specific binding of mAb 341C to each individual peptide. t test results of WT and mutants comparison are shown above each column. p < 0.05 is considered significant.

Fig. 2

Epitope mapping by random peptide phage display. (A) Key residues and the core sequence consensus for antibody binding. Epitope mapping was conducted by screening two phage display libraries, C7C and 12-mer, with mAbs 240C, 341C and 540C, respectively. (B) Mapping continuous and discontinuous amino acids involved in antibody binding. Residues identified for each antibody are marked with empty squares, closed or open circles.

Fig. 3

Model for antibody interference and synergy in SARS-CoV infection (A) Exposed amino acids (depicted in purple), participating in antibody binding. (B) Steric interference between mAb 240C and mAb 341C through the proposed mechanisms of residue sharing and spatial occupancy. The residues in cyan are bound by mAb 240C, the residues in magenta are bound by mAb 341C, and the residues in brown are the shared ones for the binding of both antibodies. (C) Steric interference between mAb 240C and mAb 540C through the proposed mechanism of spatial occupancy. The residues in cyan are bound by mAb 240C, while the residues in orange are bound by mAb 540C. (D) Structural and mechanistic model for synergistic virus neutralization between mAb 341C and mAb 540C. The residues in magenta are bound by mAb 341C, while the residues in orange are bound by mAb 540C.