UTILIZATION OF SOLUBLE RECEPTOR BINDING PROTEINS TO CHARACTERIZE MOLECULAR AND PHENOTYPIC FEATURES OF MEMORY B CELL AND NEUTRALIZING ANTIBODY RESPONSES IN HEPATITIS C VIRUS OR SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 INFECTION

Abstract

A goal of vaccines is to elicit a durable memory B cell response that can be recalled upon during infection. During viral infections, B- and T-cell memory responses develop against structural and nonstructural virion proteins. Antibodies against receptor binding glycoproteins on virion surfaces are associated with protection against reinfection. Therefore, it’s important to build tools to efficiently characterize an effective antibody and memory B cell response against these viral infections. Soluble antigens of the receptor binding glycoprotein envelope 2 (E2) of hepatitis C virus (HCV) or the spike receptor binding domain (S-RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were constructed with 6x-his tags on the C-terminal of each protein. E2 or S-RBD were used as baits to capture and detect memory specific B cells in HCV and SARS-CoV-2 infected individuals respectively. We used multi-dimensional flow cytometric to characterize inhibitor and activator cell surface markers. FACS sorting and in vitro stimulation followed by neutralization, binding affinity, and B cell receptor sequencing assays were used to characterize novel broadly neutralizing antibodies (bNAbs). In HCV, we detected E2 specific (E2+) memory B cells in both persistence and clearance subjects. Among E2+ mature class switched memory B cells (MBC), frequencies of resting memory B cells (rMBC) were reduced, frequencies of activated MBC (actMBC) and tissue-like MBC (tlMBC) were increased, and expression of FCRL5, an IgG receptor, was significantly upregulated. Moreover, plasma anti-E2 IgG levels were positively correlated with frequencies of E2+ rMBC and E2+ actMBC. Anti-E2 IgG levels were negatively correlated with levels of FCRL5 expression on E2+ rMBC and PD-1 expression on E2+ actMBC. We isolated and characterized 55 novel cross reactive E2 specific bNAbs from an elite HCV neutralizer who naturally cleared three infections. We detected germline precursors of these bNAbs from the earlier infections and discovered enriched amino acid substitutions in the V gene segments. We discovered that HCV specific bNAbs use a wider range of V gene segments than previously described. We characterized the first crystal structures of non-VH 1-69 bNAbs and new neutralizing epitopes in E2. In SARS-CoV-2, we detected S-RBD specific (S-RBD+) memory B cells in 13 of 14 subjects in the ambulatory mild (n= 7) or hospitalized moderate to severe disease (n= 7) cohorts. FCRL5 was significantly upregulated on S-RBD+ rMBC in all subjects. This was some of the first evidence of a durable B cell immunity after mild or severe COVID-19 disease