Analysis of SARS-CoV-2 Variant-Specific Serum Antibodies Post-Vaccination Utilizing Immortalized Human Hepatocyte-like Cells (HLC) to Assess Development of Protective Immunity

Abstract

AbstractBackgroundOur previous studies demonstrated that SARS-CoV-2 spike proteins could bind to hepatocytes via the asialoglycoprotein receptor-1 (ASGR-1) facilitating direct infection by the SARS-CoV-2 virus. Immortalized E12-HLC expressed the phenotypic and biological properties of primary human hepatocytes, including their ability to bind spike proteins via ASGR-1 with exception of the spike 1 protein. This binding could be inhibited by spike protein-specific monoclonal antibodies. We used the same spike-blocking analysis to determine if post-vaccination serum was capable of blocking spike protein binding to HLC. Samples collected from subjects prior to, and post-vaccination were quantified for anti-variant-specific antibody (original wild type, alpha (α), beta (β), gamma (γ) and delta (δ) variants) by a flow cytometry based immunofluorescent assay. Inhibition of variant spike protein binding to HLC and AT-2 (as a known model for spike 1 binding to the ACE-2 receptor) was analyzed by confocal microscopy. This study was designed to investigate the ability of post-vaccination antibodies to mediate immunity to spike S2, and to validate the utility of the E12-HLC in analyzing that immunity.MethodsSerum was collected from 10 individuals pre- and post-vaccination with the J&J, Moderna or Pfizer vaccines. The serum samples were quantified for variant-specific antibodies in a flow cytometry-based immunofluorescent assay utilizing beads coated with biotinylated variant spike proteins (α, β, γ, δ). Presence of variant-specific antibodies was visualized by anti-human IgG-Alexa 488. Inhibition of spike protein binding to cells was analyzed by immunofluorescent confocal analysis. Biotinylated variant spike proteins were preincubated with serum samples and then tested for binding to target cells. Binding was visualized by Streptavidin-Alexa 594. Results were compared to binding of unblocked spike variants.ResultsAll variant spike proteins tested bound to both the HLC and AT-2 cells. Pre-vaccination serum samples had no detectable reactivity to any of the variant spike proteins and were unable to inhibit binding of the variant spike proteins to either target cell. Post-vaccination serum samples demonstrated a progression of SARS-CoV-2 antibody levels from low early post-vaccination levels to higher levels at 2.5 months after vaccination. Concurrently, serum samples taken at those different timeframes demonstrated that serum obtained from shortly after vaccination were not as effective in blocking spike protein as serum obtained after 2.5 months post-vaccination. Antibody concentrations were not necessarily associated with better blocking of spike protein binding as spike variant-specific serum antibody concentrations varied significantly between subjects and within each subject. It was also demonstrated that vaccination with all the various available vaccines stimulated antibodies that inhibited binding of the available variant spike proteins to both HLC and AT-2 cells.ConclusionHLC, along with AT-2 cells, provides a useful platform to study the development of protective antibodies that prevent the binding SARS-CoV-2 spike proteins to target cells. It was shown that vaccination with the three available vaccines all elicited serum antibodies that were protective against binding of each of the variant spike proteins to both AT-2 and HLC cells. This study suggests that analysis of immune serum to block spike binding to target cells may be a more useful technique to assess protective immunity than quantitation of gross antibody alone.