Epitope mapping and biological function analysis of antibodies produced by immunization of mice with an inactivated Chinese isolate of severe acute respiratory syndrome-associated coronavirus (SARS-CoV)

Abstract

Inactivated severe acute respiratory syndrome-associated coronavirus (SARS-CoV) has been tested as a candidate vaccine against the re-emergence of SARS. In order to understand the efficacy and safety of this approach, it is important to know the antibody specificities generated with inactivated SARS-CoV. In the current study, a panel of twelve monoclonal antibodies (mAbs) was established by immunizing Balb/c mice with the inactivated BJ01 strain of SARS-CoV isolated from the lung tissue of a SARS-infected Chinese patient. These mAbs could recognize SARS-CoV-infected cells by immunofluorescence analysis (IFA). Seven of them were mapped to the specific segments of recombinant spike (S) protein: six on S1 subunit (aa 12-798) and one on S2 subunit (aa 797-1192). High neutralizing titers against SARS-CoV were detected with two mAbs (1A5 and 2C5) targeting at a subdomain of S protein (aa 310-535), consistent with the previous report that this segment of S protein contains the major neutralizing domain. Some of these S-specific mAbs were able to recognize cleaved products of S protein in SARS-CoV-infected Vero E6 cells. None of the remaining five mAbs could recognize either of the recombinant S, N, M, or E antigens by ELISA. This study demonstrated that the inactivated SARS-CoV was able to preserve the immunogenicity of S protein including its major neutralizing domain. The relative ease with which these mAbs were generated against SARS-CoV virions further supports that subunit vaccination with S constructs may also be able to protect animals and perhaps humans. It is somewhat unexpected that no N-specific mAbs were identified albeit anti-N IgG was easily identified in SARS-CoV-infected patients. The availability of this panel of mAbs also provided potentially useful agents with applications in therapy, diagnosis, and basic research of SARS-CoV.

Fig. 1

Epitope mapping of mAbs to the S protein by ELISA. MAbs were added at 1:100,000 dilution to each well against either the recombinant full-length S protein (bars in shade) expressed from transiently transfected 293T cells or the vector DNA-transfected 293T cells (bars in blank). “+” and “−” were control sera from New Zealand rabbits immunized with either the full-length S DNA vaccine or the empty vector DNA, respectively. The OD_450nm values less than 0.2 are considered non-specific binding to the full-length S antigen in this ELISA assay.

Fig. 2

Designs of recombinant S segments used in this study. Schematic representation of the entire Spike protein is shown on top, including its natural leader and its transmembrane domain close to the C terminal tail. Predicated N-glycosylation sites are marked by asterisks and the ACE2 receptor binding domain is also noted. DNA plasmids expressing different segments of the S protein were shown in the lower part of the figure with their amino acid residue numbers marked.

Fig. 3

Epitope mapping of mAb 2A3 using specific recombinant S antigens expressed in 293T transfected cells. (A) Detection of various recombinant S proteins as the coating antigens by ELISA. The OD_450nm values in ELISA less than 0.2 are considered non-specific binding to S antigens. (B) Detection of S and its subdomains by Western blot analysis. Samples included uninfected Vero E6 cell lysate (Vero E6), SARS-CoV-infected Vero E6 cell lysate (SARS-CoV), and recombinant S proteins (S, S1, S1.1, S1.2, and S2) as labeled.

Fig. 4

Detection of SARS-CoV-associated S protein with mAb 3A3 by Western blot analysis. Samples loaded included uninfected Vero E6 lysate and SARS-CoV-infected Vero E6 lysate. Full length S protein (S), the dimmer form of S (di-S), and processed S protein (S′) are indicated.

Fig. 5

Epitope mapping of mAb 2D4. (A) Western blot analysis of mAb 2D4 with the following samples: uninfected Vero E6 cells, SARS-CoV-infected Vero E6 lysate, recombinant S proteins (S, S1 and S2) expressed from transiently transfected 293T cells, and the control 293T cells transfected with vector DNA. (B) ELISA with mAb 2D4 at 1:500 dilution against different recombinant S antigens (S1.1, S1.1a, S1.1b, S1.2, and S2) expressed from transiently transfected 293T cells and control 293T cells transfected with vector DNA. OD_450nm values less than 0.1 are considered non-specific binding in this ELISA assay.

Fig. 6

Neutralizing activities of mAbs against the Urbani strain of SARS-CoV as measured by neutral red staining in infected Vero E6 cells. Neutralizing activities are plotted as the percent inhibition of viral infection against a particular rabbit serum dilution based on the geometric means from triplet wells. The 50% inhibition (IC50) or 75% inhibition (IC75) levels are marked.