title: Interference of HCV replication by cell penetrable human monoclonal scFv specific to NS5B polymerase.
abstract: A new class of hepatitis C virus (HCV)-targeted therapeutics that is safe, broadly effective and can cope with virus mutations is needed. The HCV's NS5B is highly conserved and different from human protein, and thus it is an attractive target for anti-HCV therapeutics development. In this study, NS5B bound-phage clones selected from a human single chain variable antibody fragment (scFv) phage display library were used to transform appropriate E. coli bacteria. Two scFv inhibiting HCV polymerase activity were selected. The scFvs were linked to a cell penetrating peptide to make cell penetrable scFvs. The transbodies reduced the HCV RNA and infectious virus particles released into the culture medium and inside hepatic cells transfected with a heterologous HCV replicon. They also rescued the innate immune response of the transfected cells. Phage mimotope search and homology modeling/molecular docking revealed the NS5B subdomains and residues bound by the scFvs. The scFv mimotopes matched residues of the NS5B, which are important for nucleolin binding during HCV replication, as well as residues that interconnect the fingers and thumb domains for forming a polymerase active groove. Both scFvs docked on several residues at the thumb armadillo-like fold that could be the polymerase interactive sites of other viral/host proteins for the formation of the replication complex and replication initiation. In conclusion, human transbodies that inhibited HCV RdRp activity and HCV replication and restored the host innate immune response were produced. They are potentially future interferon-free anti-HCV candidates, particularly in combination with other cognates that are specific to NS5B epitopes and other HCV enzymes.
authors.affiliation: a Department of Microbiology and Immunology; Faculty of Veterinary Medicine ; Kasetsart University ; Bangkok , Thailand.
authors.forename: Kanyarat
authors.initials: K
authors.lastname: Thueng-in
authors.forename: Jeeraphong
authors.initials: J
authors.lastname: Thanongsaksrikul
authors.forename: Surasak
authors.initials: S
authors.lastname: Jittavisutthikul
authors.forename: Watee
authors.initials: W
authors.lastname: Seesuay
authors.forename: Monrat
authors.initials: M
authors.lastname: Chulanetra
authors.forename: Yuwaporn
authors.initials: Y
authors.lastname: Sakolvaree
authors.forename: Potjanee
authors.initials: P
authors.lastname: Srimanote
authors.forename: Wanpen
authors.initials: W
authors.lastname: Chaicumpa
mesh_terms: D045744:Cell Line, Tumor; D061505:Cell Surface Display Techniques; D002470:Cell Survival / Q000187:drug effects; D000939:Epitopes / Q000235:genetics / Q000378:metabolism; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D006526:Hepatitis C / Q000517:prevention & control / Q000821:virology; D006801:Humans; D007113:Immunity, Innate / Q000235:genetics; D050838:Interferon Regulatory Factor-3 / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008958:Models, Molecular; D009154:Mutation; D019151:Peptide Library; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D000939:Epitopes; C494232:IRF3 protein, human; D050838:Interferon Regulatory Factor-3; D019151:Peptide Library; D057127:Single-Chain Antibodies; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
keywords: ABTS, 2, 2′-Azino-di(3-ethylbenzthiazoline-6-sulfonate); AE, adverse effects; BCIP/NBT, 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium liquid substrate system; CDR(s), complementarity determining region(s); DMEM, Dulbecco's modified Eagle's medium; DNA, deoxyribonucleic acid; DTT, dithiothreitol; E. coli, Escherichia coli; ELISA, enzyme-linked immunosorbent assay; FR(s), immunoglobulin framework region(s); Fc, fragment crystallizable of immunoglobulin; HCV, hepatitis C virus; HRP, horseradish peroxidase; Hepatitis C; IFN-α, interferon-alpha; IPTG, isopropyl β-D-1-thiogalactopyranoside; IgG, immunoglobulin G; LDH, lactic dehydrogenase; NS, non-structural protein; NS5B polymerase; NS5BΔ55, recombinant C-terminally 55 amino acid deleted NS5B; NTPs, nucleotide triphosphates; Ni-NTA, nickel-nitrilotriacetic acid; OD, optical density; PBS, 0.15 molar phosphate-buffered saline, pH 7.4; PCR, polymerase chain reaction; PDB, protein data bank; PEG, pegylated; RBV, ribavirin; RFLP, restriction fragment length polymorphism; RT-PCR, reverse transcription polymerase chain reaction; RdRp, RNA-dependent RNA polymerase; SD, standard deviation; SDS, sodium dodecyl sulfate; SOC, standard-of-care; STAT-C, specifically targeted anti-viral therapy for hepatitis C; SVR, sustained virologic response; VH, variable heavy chain domain of conventional four-chain IgG; VHH, variable heavy chain domain of heavy chain antibody; VL, variable light chain domain; bp, base pairs; cDNA, complementary deoxyribonucleic acid; hepatitis C virus; human single-chain variable antibody fragments (human scFv); kDa, kilo-Daltons; mM, millimolars; nM, nanomolars; nm, nanometers; pen/PEN, penetratin gene/protein; phage display; qPCR, quantitative real-time reverse transcription polymerase chain reaction; scFv, single-chain variable antibody fragments; scfv, gene sequence coding for scFv