Chimeric rabbit/human Fab antibodies against the hepatitis Be-antigen and their potential applications in assays, characterization, and therapy

Abstract

Hepatitis B virus (HBV) infection afflicts millions worldwide, causing cirrhosis and liver cancer. HBV e-antigen (HBeAg), a clinical marker for disease severity, is a soluble variant of the viral capsid protein. HBeAg is not required for viral replication but is implicated in establishing immune tolerance and chronic infection. The structure of recombinant e-antigen (rHBeAg) was recently determined, yet to date, the exact nature and quantitation of HBeAg still remain uncertain. Here, to further characterize HBeAg, we used phage display to produce a panel of chimeric rabbit/human monoclonal antibody fragments (both Fab and scFv) against rHBeAg. Several of the Fab/scFv, expressed in , had unprecedentedly high binding affinities ( ∼10 m) and high specificity. We used Fab/scFv in the context of an enzyme-linked immunosorbent assay (ELISA) for HBeAg quantification, which we compared with commercially available kits and verified with seroconversion panels, the WHO HBeAg standard, rHBeAg, and patient plasma samples. We found that the specificity and sensitivity are superior to those of existing commercial assays. To identify potential fine differences between rHBeAg and HBeAg, we used these Fabs in microscale immunoaffinity chromatography to purify HBeAg from individual patient plasmas. Western blotting and MS results indicated that rHBeAg and HBeAg are essentially structurally identical, although HBeAg from different patients exhibits minor carboxyl-terminal heterogeneity. We discuss several potential applications for the humanized Fab/scFv.

Structures of Hepatitis B Virus Core- and e-Antigen Immune Complexes Suggest Multi-point Inhibition

“…Previously, we identified several diverse chimeric rabbit/human Fabs against rHBeAg d using a phage display library (Zhuang et al, 2017). Sequence alignment of the complementarity determining regions (CDRs) of the 24 Fabs revealed two clusters (Fig.…”

“…These structures have shown that under oxidizing conditions the cysteine residue at position −7 forms an intramolecular disulfide bond with C61 locking rHBeAg dimers (rHBeAg d ) into an arrangement distinct from that of rHBcAg dimers (rHBcAg d ) (DiMattia et al, 2013). Analysis of HBeAg, isolated from HBV-positive patient plasma samples, by SDS-PAGE under reducing and non-reducing conditions, has confirmed the presence of the disulfide bond between C(−7) and C61 in HBeAg (Zhuang et al, 2017).…”

Capsids of hepatitis B virus e antigen with authentic C termini are stabilized by electrostatic interactions

“…The dimeric HBeAg constructs with varying C‐terminal extensions were all expressed in E. coli as previously described . All constructs contained the C48A and C107A mutations (Fig.…”

“…More recent studies with cell cultures have shown that C‐terminal processing occurs in the Golgi by furin generating an e antigen with a C‐terminal sequence extending to R154 and may involve more than one cleavage cycle . This is in contrast to HBeAg isolated from patient sera by affinity chromatography where the C terminus was identified as R151 .…”

Virus Assembly Antagonists from Redesigned Antibodies

“…In this issue of Structure, Wingfield and colleagues (Eren et al, 2018) present a new approach to fight CHB: targeting HBeAg and dimeric HBcAg. They present three crystal structures of HBeAg and HBcAg dimers in complex with a Fab fragment or an scFV that binds with sub-nanomolar affinity (Zhuang et al, 2017). The HBeAg and HBcAg monomers are very similar: an all-helix protein where long helices 3 and 4 form the intradimer interface and helix 5, along with its following loop and extended structure, has the potential to form the interdimer contacts that support capsid assembly ( Figure 1A).…”