Discovery of human antibody specific to the spike glycoprotein subunit 2 of SARS-CoV-2 and characterization of its broad-spectrum neutralizing activity.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve and mutate during transmission, making the development of effective broad-spectrum antibodies for the prevention and treatment of SARS-CoV-2 infections of great significance. In this study, peripheral blood mononuclear cells were isolated from 15 convalescent coronavirus disease 2019 (COVID-19) patients to construct a human-derived single-chain variable fragment (scFv) antibody library against SARS-CoV-2. Monoclonal antibodies targeting the conserved structural region of the spike glycoprotein subunit 2 (S2) protein with broad-spectrum neutralizing activity were subsequently screened. METHODS: The cDNA of the peripheral blood mononuclear cells from 15 convalescent COVID-19 patients was used to amplify the human-derived antibody heavy chain variable region (VH) and light chain variable region (VL) via polymerase chain reaction (PCR). The VH and VL genes were randomly assembled into scFvs using overlap extension PCR. The constructed scFv gene library was cloned into the phage display vector pComb3XSS and electroporated into XL1-Blue cells to generate a human SARS-CoV-2 scFv antibody library. Then monoclonal antibodies specific to the SARS-CoV-2 S2 domain were screened by phage display. Gene sequencing and recombinant human immunoglobulin G (IgG) monoclonal antibody expression were performed to produce human IgG antibody. The antibody was subsequently evaluated for its binding ability and neutralizing activity against different viral subtypes. Furthermore, the fragment antigen-binding region of the selected antibody was modeled using structure prediction tools to support subsequent structural analysis. RESULTS: The library size of the constructed human-derived SARS-CoV-2 scFv library is 1.6 × 10⁷ CFU. The S2-specific full-length IgG antibody was selected and expressed, which demonstrated neutralizing activity against both the SARS-CoV-2 wild type strain and the Omicron variant. CONCLUSIONS: A human single-chain antibody library against SARS-CoV-2 was successfully constructed. From this library, a human antibody targeting the highly conserved S2 region of SARS-CoV-2 was selected, which exhibited broad-spectrum neutralizing activity. This antibody shows potent in vitro neutralizing activity and offers a preliminary molecular basis for the subsequent development of SARS-CoV-2 therapeutics and broad-spectrum vaccines.