A conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in Middle East respiratory syndrome coronavirus spike protein

Abstract

Prophylactic and therapeutic strategies are urgently needed to combat infections caused by the newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV). Here, we have developed a neutralizing monoclonal antibody (MAb), designated Mersmab1, which potently blocks MERS-CoV entry into human cells. Biochemical assays reveal that Mersmab1 specifically binds to the receptor-binding domain (RBD) of the MERS-CoV spike protein and thereby competitively blocks the binding of the RBD to its cellular receptor, dipeptidyl peptidase 4 (DPP4). Furthermore, alanine scanning of the RBD has identified several residues at the DPP4-binding surface that serve as neutralizing epitopes for Mersmab1. These results suggest that if humanized, Mersmab1 could potentially function as a therapeutic antibody for treating and preventing MERS-CoV infections. Additionally, Mersmab1 may facilitate studies of the conformation and antigenicity of MERS-CoV RBD and thus will guide rational design of MERS-CoV subunit vaccines.

Importance: MERS-CoV is spreading in the human population and causing severe respiratory diseases with over 40% fatality. No vaccine is currently available to prevent MERS-CoV infections. Here, we have produced a neutralizing monoclonal antibody with the capacity to effectively block MERS-CoV entry into permissive human cells. If humanized, this antibody may be used as a prophylactic and therapeutic agent against MERS-CoV infections. Specifically, when given to a person (e.g., a patient's family member or a health care worker) either before or after exposure to MERS-CoV, the humanized antibody may prevent or inhibit MERS-CoV infection, thereby stopping the spread of MERS-CoV in humans. This antibody can also serve as a useful tool to guide the design of effective MERS-CoV vaccines.

FIG 1

Mersmab1 inhibited MERS-CoV spike-mediated pseudovirus entry into DPP4-expressing Huh-7 cells and neutralized MERS-CoV infection in both Vero E6 and Calu-3 cells. An anti-SARS-CoV RBD MAb, 33G4, was included as a control. (A) Selected anti-MERS-CoV MAbs (Mersmab1, Mersmab2, Mersmab3, and Mersmab10) were tested for their inhibition of MERS-CoV spike-mediated pseudovirus entry into DPP4-expressing Huh-7 cells. The data are presented as mean percentages of inhibition ± standard deviations (SD) (n = 4 experiments). (B) Anti-MERS-CoV MAbs were tested for their neutralizing activity against infection by authentic MERS-CoV (EMC strain) in Vero E6 cells. The neutralization capability of MAbs was characterized using a 50% neutralization dose (ND₅₀), which was defined as the concentration of the MAb that reduced CPE by 50%. The data are presented as mean ND₅₀s ± SD (n = 3). (C) Mersmab1 neutralized MERS-CoV infection in Calu-3 cells. A standard microneutralization assay was used to assess the potency of Mersmab1 in neutralizing MERS-CoV infection. Levels of CPE were scored as follows: CPE0 (no CPE), CPE1 (5 to 10%), CPE2 (10 to 25%), CPE3 (25 to 50%), and CPE4 (>50%).

FIG 2

Mersmab1 blocked the binding between MERS-CoV RBD and its receptor, DPP4. (A) AlphaScreen assay was performed to detect whether Mersmab1 could block the binding between recombinant MERS-CoV RBD and recombinant DPP4. MERS-CoV RBD-Fc (0.01 μg/ml) was mixed with DPP4-His (0.27 μg/ml) in the presence of Mersmab1-Fab. (B) Flow cytometry assay was carried out to detect whether Mersmab1 could block the binding between MERS-CoV RBD and host cells expressing DPP4 on their surface. Gray shading, Huh-7 cell control; red line, binding of MERS-CoV RBD-Fc (MERS RBD, 0.5 μg/ml) to Huh-7 cells; blue line, Mersmab1 inhibits MERS RBD (0.5 μg/ml) binding of Huh-7 cells; green line, anti-SARS-CoV MAb 33G4 (0.5 μg/ml) control. (C) Flow cytometry assay shows that Mersmab1 inhibits the binding between MERS-CoV RBD and DPP4 in a dose-dependent fashion. The data in panels A and C are presented as means ± SD (n = 4) and those in panel C as percentages of inhibition.

FIG 3

Mersmab1 recognizes MERS-CoV spike protein RBD in a conformation-dependent manner. (A) AlphaScreen assay was performed to detect the binding between Mersmab1 and MERS-CoV RBD. MERS-CoV RBD-His (2.8 μg/ml) was mixed with Mersmab1 (0.5 μg/ml). SARS-CoV 33G4 MAb and SARS-CoV RBD protein were used as controls. (B) ELISA was carried out to detect the binding between Mersmab1 (containing mouse IgG Fc) and MERS-CoV spike protein fragments (containing human IgG Fc). Recombinant human Fc (hFc), SARS-CoV RBD, and anti-SARS-CoV MAb 33G4 were used as controls. (C) ELISA was performed to identify Mersmab1 IgG subtypes using antibodies that target mouse IgG1, IgG2a, IgG2b, and IgG3, respectively. (D) ELISA was used to detect the binding between Mersmab1 and MERS-CoV S1 protein fragments in the presence or absence of DTT. An anti-Fc MAb (Sigma) was used as the control. All data are presented as means ± SD (n = 4). A450, absorbance at 450 nm.

FIG 4

Mapping of recognizing epitopes of Mersmab1 in MERS-CoV RBD. (A) Expression levels of mutant MERS-CoV RBDs in the 293T cell culture supernatant were detected by SDS-PAGE (stained by Coomassie blue) (top) and Western blotting (recognized by anti-MERS-CoV-S1 polyclonal antibodies) (bottom). Protein molecular mass markers (kDa) are indicated on the left. (B) ELISA was performed to detect the binding of Mersmab1 to mutant MERS-CoV RBD proteins. (C) The R511A mutation in the MERS-CoV spike protein slightly reduced MERS-CoV spike-mediated pseudovirus entry into DPP4-expressing Huh-7 cells. (D) R511A mutation in the MERS-CoV spike protein significantly reduced the inhibitory effect of Mersmab1 on MERS-CoV spike-mediated pseudovirus entry. All data are presented as means ± SD (n = 2). The MERS-CoV RBD protein wild type was used as the control for panels A and B, while the pseudovirus wild type was included as the control for panels C and D.

FIG 5

Structural analysis of the recognizing epitopes of anti-MERS-CoV RBD and anti-SARS-CoV RBD MAbs. (A) Crystal structure of MERS-CoV RBD. The core structure is in cyan, and the RBM is in pink. Critical residues at the RBD-DPP4 binding interface are in green (Protein Data Bank [PDB] accession no. 4KQZ). (B) Crystal structure of MERS-CoV RBD (cyan) complexed with its receptor, human DPP4 (yellow) (PDB accession no. 4KR0). (C) Crystal structure of SARS-CoV RBD complexed with anti-SARS-CoV MAb m396 Fab (PDB accession no. 2DD8). The light chain and heavy chain of the MAb are in yellow and green, respectively. (D) Crystal structure of SARS-CoV RBD complexed with anti-SARS-CoV MAb F26G19 Fab (PDB accession no. 3BGF). (E) Crystal structure of SARS-CoV RBD complexed with anti-SARS-CoV MAb 80R scFv (PDB accession no. 2GHW). C_H, C_L, V_H, and V_L, constant heavy, constant light, variable heavy, and variable light chain domains, respectively. scFV, single-chain variable fragment.