Development and characterization of a severe acute respiratory syndrome-associated coronavirus-neutralizing human monoclonal antibody that provides effective immunoprophylaxis in mice

Abstract

Background: Severe acute respiratory syndrome (SARS) remains a significant public health concern after the epidemic in 2003. Human monoclonal antibodies (MAbs) that neutralize SARS-associated coronavirus (SARS-CoV) could provide protection for exposed individuals.

Methods: Transgenic mice with human immunoglobulin genes were immunized with the recombinant major surface (S) glycoprotein ectodomain of SARS-CoV. Epitopes of 2 neutralizing MAbs derived from these mice were mapped and evaluated in a murine model of SARS-CoV infection.

Results: Both MAbs bound to S glycoprotein expressed on transfected cells but differed in their ability to block binding of S glycoprotein to Vero E6 cells. Immunoprecipitation analysis revealed 2 antibody-binding epitopes: one MAb (201) bound within the receptor-binding domain at aa 490-510, and the other MAb (68) bound externally to the domain at aa 130-150. Mice that received 40 mg/kg of either MAb prior to challenge with SARS-CoV were completely protected from virus replication in the lungs, and doses as low as 1.6 mg/kg offered significant protection.

Conclusions: Two neutralizing epitopes were defined for MAbs to SARS-CoV S glycoprotein. Antibodies to both epitopes protected mice against SARS-CoV challenge. Clinical trials are planned to test MAb 201, a fully human MAb specific for the epitope within the receptor-binding region.

Figure 3.

Epitope mapping by immunoprecipitation. Truncated fragments of surface (S) glycoprotein of severe acute respiratory syndrome (SARS)—associated coronavirus (right) were tagged with 6-histidine (His) epitopes and were immunoprecipitated with monoclonal antibody (MAb) 68 (top left), MAb 201 (middle left), or nickel-nitrilotriacetic acid (Ni-NTA) agarose (bottom left); resolved by SDS-PAGE; transferred to solid support; and then visualized using anti-His (C-term) antibody, followed by a 1:5000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG, treatment with enhanced chemiluminescence reagent, and autoradiography. In the right panel, epitopes are depicted for MAb 201 (490–510 [blackened boxes]) and MAb 68 (130–150 [shaded boxes]).

Figure 1.

In vitro characterization of monoclonal antibodies (MAbs) 201 and 68. A, Results of flow-cytometry analysis of human epithelial kidney—293T/17 cells expressing full-length surface (S) glycoprotein (S₁₂₅₅) of severe acute respiratory syndrome (SARS)-associated coronavirus. Cells were stained with either MAb 201 (circles) or MAb 68 (squares), followed by phycoerythrin-labeled goat anti-human IgG. B, Results for Vero E6 cells incubated with 10 nmol/L of a c-Myc (human proto-oncogene) epitope-tagged protein consisting of the first 590 aa of S glycoprotein (S₅₉₀), in the presence of various concentrations of MAb 201 (circles) or MAb 68 (squares). S glycoprotein binding was detected via the epitope tag using the murine anti-c-Myc antibody 9E10, followed by phycoerythrin-labeled goat anti-mouse IgG. Reduction in fluorescent intensity mediated by the MAb was calculated and plotted.

Figure 2.

Patterns of inhibition of cytopathic effects (CPEs) in microneutralization assays. A, Results of infection of Vero E6 cells with severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV), preincubated with either monoclonal antibody (MAb) 201 (circles) or MAb 68 (squares) at various concentrations. After 3 days of incubation, CellTiter96 reagent was added to measure the metabolic activity of cells. Protective effect was calculated as described in Materials and Methods and plotted. B, Dilution series for each MAb incubated with SARS-CoV and subsequently added to Vero E6 cells in triplicate. Three days postinfection, individual wells were graded by the percentage of cells involved in the CPE, as shown in the key (right). C, Photographs demonstrating results of microscopic examination of infected Vero E6 cells. This experiment was identical to that described for panel B, with the exception that the virus inoculum was 3000 TCID₅₀/well, instead of the standard 100 TCID₅₀/well. Arrows indicate small foci of CPE observed in the presence of MAb 68 at 150 nmol/L but not of MAb 201 at 150 nmol/L. More-extensive CPE was seen in the presence of both MAbs at the lowest concentration of 9.4 nmol/L.

Figure 4.

Efficacy of immunoprophylaxis in mice challenged with severe acute respiratory syndrome (SARS)—associated coronavirus (SARS-CoV). Mice (4 in each group) were treated with the indicated monoclonal antibody (MAb), at estimated doses of 40, 8, and 1.6 mg/kg, 1 day prior to intranasal challenge with 10⁵ TCID₅₀ of SARS-CoV. Two days after infection, lung tissue (A) or nasal turbinate tissue (B) was harvested and homogenized, and in vitro virus-titration assays were performed. Virus titers are shown as mean log₁₀ TCID₅₀ per gram of tissue, with standard errors. Comparisons of the results between groups were made by use of the nonparametric, 2-tailed Mann-Whitney test. *P > .05, compared with control. The limit of detection of the assay is indicated (A, dashed line).