Engineering and Neutralization Potential of SARS-CoV-2 Spike D614G Antibody Reagents

Severe acute respiratory disease syndrome-related coronavirus 2 (SARS-CoV-2) is the etiologic agent of the COVID-19 pandemic outbreak. SARS-CoV-2 enters human cells via the binding of viral Spike (S) protein to host angiotensin-converting enzyme 2 (ACE2) surface receptors. The S receptor-binding domain (RBD) binds ACE2 receptors. Protective or neutralizing antibodies (nAbs) that interfere with S/RBD:ACE2 interaction blocks SARS-CoV-2 entry into cells and is a pivotal focus of SARS-CoV-2 preventive or therapeutic measures. Many of the currently available SARS-CoV-2 vaccines center on creating nAbs against ancestral S protein D614. The continued effectiveness of these nAbs on evolving SARS-CoV-2 variants featuring higher infectivity and mutations within the critical RBD domain is a subject of concern. The SD614G point mutation emerged in the first wave of SARS-CoV-2 variants and has expanded to dominate SARS-CoV-2 S-variants in the field. We generated a panel of diverse antibodies to native and denatured SARS-CoV-2 SD614G and S/RBD. BALB/c mice 6- to 8-weeks old were grouped into cohorts of 5 for subcutaneous immunization with each of antigens: (1) native, full-length S-ectodomain, (2) native RBD subdomain, and (3) Urea-denatured full-length S-ectodomain. A total of 33 hybridoma cell lines were isolated using semi-solid HAT selection after fusion of the splenocytes with Sp2/0-Ag14 myeloma cells. Hybridoma clones were selected and confirmed via antigen-specific ELISA. The S/RBD antibodies were purified to homogeneity using protein A/G affinity matrix. The specificity and affinity of each antibody for S/RBD peptides was determined, and the antibody neutralization potential was determined using pseudotype SARS-CoV-2 S lentivirus entry assay. Several antibodies prevented SARS-CoV-2 S protein D614 and D614G variants from virus entry using either surrogate or pseudotype viral neutralization assays. Confirmation of the antibody neutralizing capabilities will be determined using SARS-CoV-2 variant viruses in plaque assays. The differential properties of the characterized antibodies will help decipher the pathogenesis of SARS-CoV-2 variants and may be engineered to develop therapeutic interventions.