Human monoclonal antibodies to SARS-coronavirus inhibit infection by different mechanisms

Abstract

SARS-CoV causes an acute infection making targeted passive immunotherapy an attractive treatment strategy. We previously generated human mAbs specific to the S1 region of SARS-CoV S protein. These mAbs bind epitopes within the receptor binding domain (RBD) or upstream of the RBD. We show that mAbs recognizing epitopes within the RBD inhibit infection by preventing viral attachment to the cellular receptor. One mAb binds upstream of the RBD and prevents viral entry by inhibiting a post-binding event. Evaluation of several mAbs demonstrated varying ability of the mAbs to select escape mutants when used individually. However, a mixture of antibodies could effectively neutralize a range of mutant viruses. These data strongly suggest that a mixture containing antibodies recognizing distinct regions and targeting more than one step in viral entry is likely to be more effective in neutralizing the virus and suppressing the generation of escape mutants, and thus potentially constitute a highly effective passive immunotherapy.

Fig. 1

The majority of the human monoclonal antibodies block receptor association. (A) Ability of RBD specific human mAbs (group 1) to inhibit receptor association of S protein was detected using a receptor binding inhibition assay. A representative member is shown for each group of mAbs. The recombinant S12–510-Fc protein was preincubated with increasing dilutions of mAbs. The mixture was added to the target VeroE6 cells and the recombinant protein binding was detected by flow cytometry following staining with an FITC-labeled anti-human IgG. (B) Comparison of the ability of N-terminal specific mAb 4D4 with RBD specific mAb 1B5 to inhibit receptor association of S12–510-Fc protein. Inhibition of receptor association was examined as described for the group 1 mAbs. Isotype: human IgG isotype control.

Fig. 2

Post-binding inhibition by mAb 4D4. Increasing concentrations of mAb 4D4 were incubated with HIV/S pseudotyped virus for 1 h at 37 °C and added to target cells in a standard neutralization protocol. Alternatively, HIV/S pseudovirus was bound to target cells at 4 °C for 1 h, and mAb 4D4 was added post-binding at increasing concentrations. Pseudotype virus entry was measured by luciferase expression in target 293T/T17 cells transiently expressing human ACE2. Statistical significance was determined by Student t-test.

Fig. 3

Effects of combinations of anti-S1 RBD specific and 4D4 mAbs on pseudotype virus entry. (A) A representative mAb from each group was mixed with mAb 4D4 at the indicated concentrations and preincubated with pseudotyped virus. Entry was measured by luciferase expression 48 h post-infection in target 293T/T17 cells transiently expressing ACE2. Statistical significance was determined by Student t-test.

Fig. 4

Combination mAbs 4D4 and 3C7 show enhanced inhibition of SARS-CoV (Urbani) infection. The mAbs 4D4 and 3C7 were analyzed for their ability to neutralize SARS-CoV (Urbani) infection in VeroE6 cells. The mAbs (alone or in combination) were preincubated with 200TCID50 SARS-CoV (Urbani) at 37 °C for 1 h and added to VeroE6 cells. Infection was measured by resulting cell death using the luciferase based Cell TiterGlo assay. Error bars represent standard deviation between duplicates within experiment.

Fig. 5

Inhibition of SARS-CoV escape mutant virus. SARS-CoV escape mutants were generated by serial passages of the virus in the presence of individual mAbs every 48 h. The neutralizing ability of mAb combination was determined by analysis of cell viability using the luciferase based Cell TiterGlo assay 48 h post-infection. (A) SARS-CoV 3C7 was used in a neutralization assay with 3H12 (group 1) and 4D4 (group 2) mAbs in combination with 3C7. (B) SARS-CoV 4D4 was used in a neutralization assay with mAbs 3H12 and 3C7 (group 1) in combination with mAb 4D4. A single outlier, in both A and B, is not included in the analysis.