title: Inhibition of HCV replication by humanized-single domain transbodies to NS4B.
abstract: NS4B of hepatitis C virus (HCV) initiates membrane web formation, binds RNA and other HCV proteins for viral replication complex (RC) formation, hydrolyses NTP, and inhibits innate anti-viral immunity. Thus, NS4B is an attractive target of a novel anti-HCV agent. In this study, humanized-nanobodies (VHs/VHHs) that bound to recombinant NS4B were produced by means of phage display technology. The nanobodies were linked molecularly to a cell penetrating peptide, penetratin (PEN), for making them cell penetrable (become transbodies). Human hepatic (Huh7) cells transfected with HCV JFH1-RNA that were treated with transbodies from four Escherichia coli clones (PEN-VHH7, PEN-VHH9, PEN-VH33, and PEN-VH43) had significant reduction of HCV RNA amounts in their culture fluids and intracellularly when compared to the transfected cells treated with control transbody and medium alone. The results were supported by the HCV foci assay. The transbody treated-transfected cells also had upregulation of the studied innate cytokine genes, IRF3, IFNβ and IL-28b. The transbodies have high potential for testing further as a novel anti-HCV agent, either alone, adjunct of existing anti-HCV agents/remedies, or in combination with their cognates specific to other HCV enzymes/proteins.
authors.affiliation: Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand; Center of Research Excellence on Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
authors.forename: Kittirat
authors.initials: K
authors.lastname: Glab-Ampai
authors.affiliation: Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand; Center of Research Excellence on Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
authors.forename: Aijaz Ahmad
authors.initials: AA
authors.lastname: Malik
authors.affiliation: Center of Research Excellence on Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
authors.forename: Monrat
authors.initials: M
authors.lastname: Chulanetra
authors.affiliation: Graduate Program in Biomedical Science, Faculty of Allied Health Sciences, Thammasat University, Pathumthani 12120, Thailand.
authors.forename: Jeeraphong
authors.initials: J
authors.lastname: Thanongsaksrikul
authors.affiliation: Center of Research Excellence on Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand; School of Pathology, Institute of Medicine, Suranaree University of Technology, Nakhonratchaseema Province 30000, Thailand.
authors.forename: Kanyarat
authors.initials: K
authors.lastname: Thueng-In
authors.affiliation: Graduate Program in Biomedical Science, Faculty of Allied Health Sciences, Thammasat University, Pathumthani 12120, Thailand.
authors.forename: Potjanee
authors.initials: P
authors.lastname: Srimanote
authors.affiliation: Graduate Program in Biomedical Science, Faculty of Allied Health Sciences, Thammasat University, Pathumthani 12120, Thailand.
authors.forename: Pongsri
authors.initials: P
authors.lastname: Tongtawe
authors.affiliation: Center of Research Excellence on Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand; Graduate Program in Biomedical Science, Faculty of Allied Health Sciences, Thammasat University, Pathumthani 12120, Thailand. Electronic address: wanpen.cha@mahidol.ac.th.
authors.forename: Wanpen
authors.initials: W
authors.lastname: Chaicumpa
mesh_terms: D061067:Antibodies, Monoclonal, Humanized / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics; D000914:Antibodies, Viral / Q000008:administration & dosage* / Q000737:chemistry / Q000235:genetics; D000998:Antiviral Agents / Q000008:administration & dosage / Q000737:chemistry; D002352:Carrier Proteins / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics; D002460:Cell Line; D061505:Cell Surface Display Techniques; D057846:Cell-Penetrating Peptides / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics; D003198:Computer Simulation; D016174:Hepacivirus / Q000235:genetics / Q000276:immunology* / Q000502:physiology*; D006801:Humans; D007113:Immunity, Innate / Q000235:genetics; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D061905:Single-Domain Antibodies / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000276:immunology* / Q000502:physiology*; D014779:Virus Replication / Q000235:genetics / Q000276:immunology* / Q000502:physiology*
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D000914:Antibodies, Viral; D000998:Antiviral Agents; D002352:Carrier Proteins; D057846:Cell-Penetrating Peptides; C080107:NS4B protein, flavivirus; D011994:Recombinant Proteins; D061905:Single-Domain Antibodies; D017361:Viral Nonstructural Proteins; C414409:penetratin
keywords: Direct acting anti-HCV; Hepatitis C virus; Humanized-nanobody; NS4B; Transbody; qRT-PCR