Identification of SARS-CoV RBD-targeting monoclonal antibodies with cross-reactive or neutralizing activity against SARS-CoV-2

Abstract

SARS-CoV-2-caused COVID-19 cases are growing globally, calling for developing effective therapeutics to control the current pandemic. SARS-CoV-2 and SARS-CoV recognize angiotensin-converting enzyme 2 (ACE2) receptor via the receptor-binding domain (RBD). Here, we identified six SARS-CoV RBD-specific neutralizing monoclonal antibodies (nAbs) that cross-reacted with SARS-CoV-2 RBD, two of which, 18F3 and 7B11, neutralized SARS-CoV-2 infection. 18F3 recognized conserved epitopes on SARS-CoV and SARS-CoV-2 RBDs, whereas 7B11 recognized epitopes on SARS-CoV RBD not fully conserved in SARS-CoV-2 RBD. The 18F3-recognizing epitopes on RBD did not overlap with the ACE2-binding sites, whereas those recognized by 7B11 were close to the ACE2-binding sites, explaining why 7B11 could, but 18F3 could not, block SARS-CoV or SARS-CoV-2 RBD binding to ACE2 receptor. Our study provides an alternative approach to prevent SARS-CoV-2 infection using anti-SARS-CoV nAbs.

Keywords: COVID-19; Cross-neutralization; Neutralizing monoclonal antibodies; Receptor-binding domain; SARS-CoV; SARS-CoV-2.

Fig. 1

Schematic map of SARS-CoV RBD-specific mAbs in cross-reacting with SARS-CoV-2 RBD in the S protein, cross-neutralizing against SARS-CoV-2 S protein-mediated viral entry, and inhibiting the SARS-CoV-2 RBD-ACE2 binding. Anti-SARS-CoV-RBD mAbs bound to SARS-CoV-2 RBD in the S protein. Some of these mAbs directly neutralized SARS-CoV-2 infection before its entry to host cells expressing ACE2 receptor, or blocked the binding of RBD to ACE2 receptor on the cell membrane.

Fig. 2

Detection of cross-reactivity of SARS-CoV RBD-specific mAbs with SARS-CoV-2 RBD protein and their cross-neutralizing activity against SARS-CoV-2 infection. (A) Cross-reactivity of anti-SARS-CoV RBD mAbs with SARS-CoV-2 RBD protein. (B) Reactivity of these mAbs with SARS-CoV RBD protein was included as control. The binding of SARS-CoV RBD-specific mouse mAbs to SARS-CoV-2 RBD (A) or SARS-CoV RBD (B) protein was detected by ELISA. The data are presented as mean A450 value ± standard deviation of the mean (s.e.m.) (n = 3). (C) Cross-neutralizing activity of anti-SARS-CoV RBD mAbs against SARS-CoV-2 infection. (D) Neutralizing activity of these mAbs against SARS-CoV infection was used as control. Neutralization against SARS-CoV-2 (C) or SARS-CoV (D) infection was detected by a pseudovirus neutralization assay in human ACE2 receptor-expressing 293T (hACE2/293T) cells. The percent (%) pseudovirus neutralization was calculated based on the relative luciferase unit (RLU) of pseudovirus infection with and without respective mAbs. The data are presented as mean neutralization (%) ± s. e.m. (n = 3). For (A–D), SARS-CoV RBD-immunized mouse sera were included as positive control (Pos con) since we have confirmed their cross-reactivity with SARS-CoV-2 RBD in previous studies (Tai et al., 2020), and MERS-CoV RBD-specific mAb was used as negative control (Neg con). Each mAb was detected at 10 and 1 μg/ml, respectively. The experiments were repeated twice and obtained similar results.

Fig. 3

Identification of epitopes of SARS-CoV RBD-targeting nAbs by ELISA. The two (18F3 and 7B11) SARS-CoV RBD-specific nAbs with cross-neutralizing activity against SARS-CoV-2 were detected for their potential recognition of epitopes on SARS-CoV RBD. SARS-CoV RBD-targeting nAbs (33G4 and 13B6) without cross-neutralizing activity were included as controls. The plates were coated with SARS-CoV RBD wild type (WT) or each mutant protein (1 μg/ml), and the mAbs at serial dilutions were added for binding. Arrows represent mAbs that did not bind to mutant proteins at the indicated amino acid residues on SARS-CoV RBD, which are epitopes recognized by the mAbs tested. The data are presented as mean A450 ± s. e.m. (n = 2). The experiments were repeated once and obtained similar results.

Fig. 4

Mapping of epitopes of SARS-CoV RBD-specific mAbs on SARS-CoV or SARS-CoV-2 RBD and their inhibition to the binding of SARS-CoV or SARS-CoV-2 RBD to ACE2 receptor. (A) Amino acid alignment of SARS-CoV RBD (residues 318–510) and SARS-CoV-2 RBD (residues 331–524) in S proteins and mapping of epitopes recognized by SARS-CoV RBD-specific nAbs with (18F3 and 7B11) and without (13B6) cross-neutralizing activity against SARS-CoV-2 infection. The alignment was performed using Clustal Omega. The identified mAb epitopes on SARS-CoV RBD and the residues corresponding to SARS-CoV-2 RBD were labeled in red with respective mAbs shown underneath. Letters in cyan represent receptor ACE2-binding residues and their locations in SARS-CoV or SARS-CoV-2 RBD (Li et al., 2005; Yan et al., 2020). (B) Inhibition of SARS-CoV RBD-specific mAbs for the binding of SARS-CoV or SARS-CoV-2 RBD to ACE2 receptor by flow cytometry analysis. Percent (%) inhibition was calculated based on the relative fluorescence intensity with or without respective mAbs at 10 and 1 μg/ml, respectively. SARS-CoV or SARS-CoV-2 RBD (2 μg/ml) was used for the binding to hACE2/293T cells. The data are expressed as mean inhibition (%) ± s. e.m. (n = 3). The experiments were repeated twice and obtained similar results. (C) Representative flow cytometry images of SARS-CoV RBD-specific mAbs (10 μg/ml) in inhibition of the binding between SARS-CoV or SARS-CoV-2 RBD and ACE2 receptor. The binding of SARS-CoV or SARS-CoV-2 RBD (2 μg/ml) to hACE2/293T cells is shown in red line, and the blockage of this binding by mAbs (33G4, 13B6, 18F3, and 7B11) is shown in blue line. hIgG-Fc protein (background control) was shown in gray shade.