LAUSR

ABSTRACT Hepatitis B virus (HBV) envelopes as well as empty subviral particles carry in their lipid membranes the small (S), middle (M), and large (L) surface proteins, collectively known as hepatitis B surface antigen (HBsAg). Due to their common S domain all three proteins share a surface‐exposed hydrophilic antigenic loop (AGL) with a complex disulfide bridge‐dependent structure. The AGL is critical for HBV infectivity and virion secretion, and thus represents a major target for neutralizing antibodies. Previously, a human monoclonal antibody (mAb) targeting a conformational epitope in the AGL, IgG12, exhibited 1000‐fold higher neutralizing activity than hepatitis B immune globulin (HBIG). Here we designed a single‐chain variable fragment (scFv) homolog of IgG12, G12‐scFv, which could be efficiently produced in soluble form in the cytoplasm of E. coli SHuffle cells. Independent in vitro assays verified specific binding of G12‐scFv to a conformational S epitope shared with IgG12. Despite 20‐fold lower affinity, G12‐scFv but not an irrelevant scFv potently neutralized HBV infection of susceptible hepatoma cells (IC50=1.8nM). Strikingly, low concentrations of G12‐scFv blocked virion secretion from HBV producing cells (IC50=1.25nM) without disturbing intracellular viral replication, whereas extracellular HBsAg was reduced only at >100‐fold higher though still nontoxic concentration. The inhibitory effects correlated with S binding specificity and presumably also G12‐scFv internalization into cells. Together these data suggest G12‐scFv as a highly specific yet easily accessible novel tool for basic, diagnostic, and possibly future therapeutic applications. HIGHLIGHTSG12‐scFv was solubly expressed in the cytoplasm of E. coli SHuffle T7 strain.G12‐scFv exerted target‐specific high‐affinity binding to S protein (Kd=10.9nM).G12‐scFv potently neutralized HBV infection of a susceptible cell line (IC50=1.8nM).Low concentrations of G12‐scFv interfered selectively with virion rather than SVP secretion (IC50=1.25nM).Anti‐virion effect of G12‐scFv depended on S‐binding specificity and presumably correlated with cell internalization.