Engineering and Neutralization Potential of SARS-CoV-2 Spike D614G Antibody Reagents
World Microbe Forum
Virtual EventJune 21, 2021
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.
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Hui-Chen (Jane) Foreman, PhD
Scientist, BEI Resources
Jane is a Molecular Immunologist and Virologist with experience in both in vitro cell-based assays and in vivo murine-based disease models. She obtained a PhD degree from the University of Chicago, where she helped elucidate the importance of HSP90 complexes in regulating transcription factor activation. She then pursued her postdoctoral training at SUNY Stony Brook, investigating host-pathogen interactions between murine gamma herpesvirus 68 and immune signaling (IRF and STAT). She joined ATCC about 3 1/2 years ago as a Scientist in the Immunology unit. She collaborated with the SBC unit to successfully fulfill a one-year contract under Mid-BRR to identify the sequence of the immunoglobulin variable region of select M. tuberculosis hybridomas. This work earned a one-year continuation, where she delivered validation of those sequences. She also participates in the ATCC Internal Research and Development (IRAD) program. Herein, she has generated a novel microbe with a unique genomic marker using CRISPR technology. The editing strategy is being patented and expanded to generate new microbial products for publication, commercialization, and grant applications in support of AFS initiatives.
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