A Novel Conserved Linear Neutralizing Epitope on the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein

Abstract

The continuous emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made it challenging to develop broad-spectrum prophylactic vaccines and therapeutic antibodies. Here, we have identified a broad-spectrum neutralizing antibody and its highly conserved epitope in the receptor-binding domain (RBD) of the spike protein (S) S1 subunit of SARS-CoV-2. First, nine monoclonal antibodies (MAbs) against the RBD or S1 were generated; of these, one RBD-specific MAb, 22.9-1, was selected for its broad RBD-binding abilities and neutralizing activities against SARS-CoV-2 variants. An epitope of 22.9-1 was fine-mapped with overlapping and truncated peptide fusion proteins. The core sequence of the epitope, ⁴⁰⁵D(N)EVR(S)QIAPGQ⁴¹⁴, was identified on the internal surface of the up-state RBD. The epitope was conserved in nearly all variants of concern of SARS-CoV-2. MAb 22.9-1 and its novel epitope could be beneficial for research on broad-spectrum prophylactic vaccines and therapeutic antibody drugs. IMPORTANCE The continuous emergence of new variants of SARS-CoV-2 has caused great challenge in vaccine design and therapeutic antibody development. In this study, we selected a broad-spectrum neutralizing mouse monoclonal antibody which recognized a conserved linear B-cell epitope located on the internal surface of RBD. This MAb could neutralize all variants until now. The epitope was conserved in all variants. This work provides new insights in developing broad-spectrum prophylactic vaccines and therapeutic antibodies.

Keywords: SARS-CoV-2; epitope; neutralizing antibody; receptor-binding domain; spike protein.

FIG 1

Generation and characterization of monoclonal antibodies against SARS-CoV-2. (A) A total of nine monoclonal antibodies were generated. Seven were derived from WT-S1, and two were derived from O-RBD. Heavy chain and light chain subtypes and the reactivities of the monoclonal antibodies were determined by immunofluorescence and Western blotting assays. (B) The reactivities of seven WT-S1-derived monoclonal antibodies against WT-S1 and WT-RBD were examined by ELISA. (C) Using HRP-conjugated MAb 20.8-1 as the detecting antibody, blockage of the binding of WT-S1 to MAb 20.8-1 was examined with the indicated MAbs. All five MAbs that recognized WT-RBD also recognized the same epitope. (D) Western blot detection of RBD variants that reacted with MAb 22.9-1 and MAb 2.9–4.

FIG 2

Binding of MAbs with WT-S1 and RBD variants. (A) The binding of five MAbs with the WT-S1 protein and three RBD variants was characterized by ELISA. Only MAb 22.9-1 reacted with all four tested proteins. (B) The EC₅₀ values for ELISA were calculated with GraphPad Prism. The EC₅₀ values of MAb 22.9-1 for binding with S1 and the three RBD variants ranged from 250.6 ng/mL to 276.1 ng/mL. Data from one of two independent experiments performed in triplicate are shown.

FIG 3

Neutralizing activity of monoclonal antibodies against SARS-CoV-2 pseudovirus. SARS-CoV-2 pseudovirus was incubated with 2-fold serially diluted monoclonal antibodies. The mixtures were then added to Vero E6 cells and incubated for 24 h, after which the neutralization potencies of the MAbs were evaluated (A). EC₅₀ values were calculated with GraphPad Prism (B). Among the five tested MAbs, MAb 22.9-1 showed broad neutralizing activity against all four variants. Data from one of two independent experiments performed in triplicate are shown.

FIG 4

Epitope mapping and mutation. (A) Schematic diagram of the design of truncated overlapping short peptides spanning the RBD protein. (B) The short peptides were expressed as MBP-fusion proteins. The MBP-peptide fusion proteins were probed with MAb 22.9-1 by ELISA (C) and Western blotting (D). Peptide P6 was recognized by MAb 22.9-1. (E) The peptide P6 sequence was aligned with the corresponding sequence of the spike proteins of SARS-CoV-2 variants and SARS-CoV. (F and G) All five P6 mutants were expressed as MBP-fusion proteins (F) and probed with MAb 22.9-1 by Western blot analysis (F) and ELISA (G). Data from one of two independent experiments performed in triplicate are shown.

FIG 5

Identification of the core sequence of epitope 22.9-1. Peptide P6 was sequentially truncated from the amino or carboxy terminus as depicted in the schematic diagram (A). The truncated peptides were expressed as MBP-fusion proteins (B). Then, the peptides were probed with MAb 22.9-1 by ELISA (C) and Western blotting (D). The core sequence recognized by MAb 22.9-1 was determined to be ⁴⁰⁵DEVRQIAPGQ⁴¹⁴.

FIG 6

Visualization of epitope 22.9-1 in the S protein structure model. A stabilized SARS-CoV-2 S protein with one open-state RBD (PDB ID 7VRV) adopted to visualize the epitope. The NTD and S2 subunits are shown in gray, and the RBD domains are in cyan, light green, and orange. Epitope 405 to 414 is shown in purple. (A) Top view of the S protein trimer. (B) Side view of the S protein trimer. (C) Amplification of the epitope located in the RBD region on an open-state RBD. (D) Epitope 22.9-1 within the ribbon structure of the RBD. RBM is shown in copper, epitope 22.9-1 is shown in purple, and atoms of the amino residues are labeled with a unique ID and code.