title: Cell penetrable humanized-VH/V(H)H that inhibit RNA dependent RNA polymerase (NS5B) of HCV.
abstract: NS5B is pivotal RNA dependent RNA polymerase (RdRp) of HCV and NS5B function interfering halts the virus infective cycle. This work aimed to produce cell penetrable humanized single domain antibodies (SdAb; VH/V(H)H) that interfere with the RdRp activity. Recombinant NS5BΔ55 of genotype 3a HCV with de novo RNA synthetic activity was produced and used in phage biopanning for selecting phage clones that displayed NS5BΔ55 bound VH/V(H)H from a humanized-camel VH/V(H)H display library. VH/V(H)H from E. coli transfected with four selected phage clones inhibited RdRp activity when tested by ELISA inhibition using 3'di-cytidylate 25 nucleotide directed in vitro RNA synthesis. Deduced amino acid sequences of two clones showed V(H)H hallmark and were designated V(H)H6 and V(H)H24; other clones were conventional VH, designated VH9 and VH13. All VH/V(H)H were linked molecularly to a cell penetrating peptide, penetratin. The cell penetrable VH9, VH13, V(H)H6 and V(H)H24 added to culture of Huh7 cells transfected with JHF-1 RNA of genotype 2a HCV reduced the amounts of RNA intracellularly and in culture medium implying that they inhibited the virus replication. VH/V(H)H mimotopes matched with residues scattered on the polymerase fingers, palm and thumb which were likely juxtaposed to form conformational epitopes. Molecular docking revealed that the antibodies covered the RdRp catalytic groove. The transbodies await further studies for in vivo role in inhibiting HCV replication.
authors.affiliation: Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Faculty of Science, 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: Potjanee
authors.initials: P
authors.lastname: Srimanote
authors.forename: Kunan
authors.initials: K
authors.lastname: Bangphoomi
authors.forename: Ornnuthchar
authors.initials: O
authors.lastname: Poungpair
authors.forename: Santi
authors.initials: S
authors.lastname: Maneewatch
authors.forename: Kiattawee
authors.initials: K
authors.lastname: Choowongkomon
authors.forename: Wanpen
authors.initials: W
authors.lastname: Chaicumpa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology*; D000998:Antiviral Agents / Q000737:chemistry / Q000276:immunology* / Q000494:pharmacology; D001665:Binding Sites; D002162:Camelus; D002460:Cell Line; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D016174:Hepacivirus / Q000235:genetics* / Q000276:immunology; D006801:Humans; D008969:Molecular Sequence Data; D012150:Polymorphism, Restriction Fragment Length; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000276:immunology; D016415:Sequence Alignment; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000276:immunology; D014779:Virus Replication
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D000998:Antiviral Agents; D000939:Epitopes; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase