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Principal Investigator: Tongqing Zhou
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2020
Award: $339,649
Funding agency: National Institute of Allergy and Infectious Diseases
The SARS-CoV-2 pandemic has caused more than 29 million confirmed infections and almost 1 million deaths globally. Great efforts have been made for the development of vaccines and therapeutics. Isolation of virus-neutralizing monoclonal antibodies could be one approach to treat or prevent infection by this coronavirus. To discover novel monoclonal antibodies from the convalescent samples, we applied structure-based design coupled to efficient purification and biotinylation processes which enabled streamlined development of SARS-CoV-2 spike-ectodomain probes. Specifically, we designed constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, domains of SARC-CoV-2 spike, and a C-terminal sequence targeted by biotin ligase. The probes contained full-length spike ectodomain as well as various subregions. In addition, we also designed mutants to eliminate recognition of the ACE2 receptor to be used for sorting specific B cells. Yields of biotin-labeled probes from transient transfection ranged from 0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. We characterized antigenicity and ACE2 recognition of these probes by various methods and determined by cryo-electron microscopy the structure of the spike ectodomain probe. We also characterized antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. These probes have been distributed to laboratories inside NIH and extramural universities for sorting and characterizing new antibodies.
Recently, our collaborator David Ho's lab at Columbia University isolated sixty-one SARS-CoV-2-neutralizing monoclonal antibodies from five patients hospitalized for severe coronavirus disease 2019 (COVID-19). We provided molecular probes for epitope mapping which showed the antibodies isolated target the receptor-binding domain (RBD) and the N-terminal domain (NTD). In addition, we determined the cryo-electron microscopy structures of one antibody that targets the RBD, a second that targets the NTD, and a third that bridges two separate RBDs.
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