An E. coli-produced single-chain variable fragment (scFv) targeting hepatitis B virus surface protein potently inhibited virion secretion

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 1,000-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=1.8 nM). Strikingly, low concentrations of G12-scFv blocked virion secretion from HBV producing cells (IC50=1.25 nM) 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.

A 33-residue peptide tag increases solubility and stability of Escherichia coli produced single-chain antibody fragments

“…Plasmid pET28a-His-G12-scFv-HA expresses a G12-scFv protein with a His tag on the N terminus and a HA tag at the C-terminus 18 . Into this plasmid, the P17 tag coding sequence was inserted downstream of the HA tag sequence, with the resulting plasmid pET28a-His-G12-scFv-HA-P17 encoding the same protein but now with the additional P17 tag at the C-terminus.…”

“…While the periplasmic space, in contrast, provides an oxidative environment and contains thiol-disulfide oxidoreductases such as DsbA and DsbB 17 , recombinant expression yields are mostly very low. Recently, soluble expression of some scFvs has been achieved in an engineered E. coli strain (termed SHuffle) which features a more oxidative and disulfide-promoting cytoplasmic environment due to inactivation of the gor and trxB pathways and the constitutive expression of the disulfide-bond isomerase DsbC 18 – 21 . Also, other E. coli strains co-expressing a redox-active enzyme (e.g., Erv1p, DsbB or VKOR) plus a disulfide-bond isomerase like DsbC or PDI have become available 22 – 25 .…”

“…This is predominantly mediated by the association of a 33-amino acid (aa) P17 protein segment (named P17 peptide or P17 tag hereafter) with cell-surface low density lipoprotein receptor-related protein (LRP) 33 . We previously generated an scFv of mAb G12 18 , 34 , which targets the surface protein of hepatitis B virus (HBV), a virus with pronounced liver tropism. To improve hepatocyte targeting and uptake of G12-scFv 18 , here we attach the P17 tag to its C-terminus.…”

“…We previously generated an scFv of mAb G12 18 , 34 , which targets the surface protein of hepatitis B virus (HBV), a virus with pronounced liver tropism. To improve hepatocyte targeting and uptake of G12-scFv 18 , here we attach the P17 tag to its C-terminus. When expressed in the E. coli SHuffle strain, the G12-scFv-P17 fusion protein displays much higher solubility than the untagged G12-scFv.…”

A promising cancer diagnosis and treatment strategy: targeted cancer therapy and imaging based on antibody fragment

“…All antibody fragments retain antigen-specific [26]. Meanwhile, current antibody fragment preparation techniques are relatively complete and there are many methods to choose from, such as phage display technology, yeast display technology [27][28][29].…”