title: Humanized-VHH transbodies that inhibit HCV protease and replication.
abstract: There is a need for safe and broadly effective anti-HCV agents that can cope with genetic multiplicity and mutations of the virus. In this study, humanized-camel VHHs to genotype 3a HCV serine protease were produced and were linked molecularly to a cell penetrating peptide, penetratin (PEN). Human hepatic (Huh7) cells transfected with the JFH-1 RNA of HCV genotype 2a and treated with the cell penetrable nanobodies (transbodies) had a marked reduction of the HCV RNA intracellularly and in their culture fluids, less HCV foci inside the cells and less amounts of HCV core antigen in culture supernatants compared with the infected cells cultured in the medium alone. The PEN-VHH-treated-transfected cells also had up-regulation of the genes coding for the host innate immune response (TRIF, TRAF3, IRF3, IL-28B and IFN-β), indicating that the cell penetrable nanobodies rescued the host innate immune response from the HCV mediated-suppression. Computerized intermolecular docking revealed that the VHHs bound to residues of the protease catalytic triad, oxyanion loop and/or the NS3 N-terminal portion important for non-covalent binding of the NS4A protease cofactor protein. The so-produced transbodies have high potential for testing further as a candidate for safe, broadly effective and virus mutation tolerable anti-HCV agents.
authors.affiliation: Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. toeyku61@hotmail.com.|Laboratory for Research and Technology Development, Department of Parasitology and Center of Excellence on Therapeutic Proteins and Antibody Engineering, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. toeyku61@hotmail.com.
authors.forename: Surasak
authors.initials: S
authors.lastname: Jittavisutthikul
authors.affiliation: Laboratory for Research and Technology Development, Department of Parasitology and Center of Excellence on Therapeutic Proteins and Antibody Engineering, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. srimanote_p@y7mail.com.|Graduate Program in Biomedical Science, Faculty of Allied Health Sciences, Thammasat University, Pathum-thani 12120, Thailand. srimanote_p@y7mail.com.
authors.forename: Jeeraphong
authors.initials: J
authors.lastname: Thanongsaksrikul
authors.affiliation: Laboratory for Research and Technology Development, Department of Parasitology and Center of Excellence on Therapeutic Proteins and Antibody Engineering, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. mam_mt41@hotmail.com.|Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand. mam_mt41@hotmail.com.
authors.forename: Kanyarat
authors.initials: K
authors.lastname: Thueng-In
authors.affiliation: Laboratory for Research and Technology Development, Department of Parasitology and Center of Excellence on Therapeutic Proteins and Antibody Engineering, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. monchu82@gmail.com.
authors.forename: Monrat
authors.initials: M
authors.lastname: Chulanetra
authors.affiliation: Graduate Program in Biomedical Science, Faculty of Allied Health Sciences, Thammasat University, Pathum-thani 12120, Thailand. srimanote_p@y7mail.com.
authors.forename: Potjanee
authors.initials: P
authors.lastname: Srimanote
authors.affiliation: Laboratory for Research and Technology Development, Department of Parasitology and Center of Excellence on Therapeutic Proteins and Antibody Engineering, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. watee.see@gmail.com.
authors.forename: Watee
authors.initials: W
authors.lastname: Seesuay
authors.affiliation: Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. ajaz_me@hotmail.com.|Laboratory for Research and Technology Development, Department of Parasitology and Center of Excellence on Therapeutic Proteins and Antibody Engineering, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. ajaz_me@hotmail.com.
authors.forename: Aijaz Ahmad
authors.initials: AA
authors.lastname: Malik
authors.affiliation: Laboratory for Research and Technology Development, Department of Parasitology and Center of Excellence on Therapeutic Proteins and Antibody Engineering, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. srimanote_p@y7mail.com.|Graduate Program in Biomedical Science, Faculty of Allied Health Sciences, Thammasat University, Pathum-thani 12120, Thailand. srimanote_p@y7mail.com.
authors.forename: Wanpen
authors.initials: W
authors.lastname: Chaicumpa
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002162:Camelus; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D057846:Cell-Penetrating Peptides; D016207:Cytokines / Q000096:biosynthesis; D004337:Drug Carriers / Q000378:metabolism; D020869:Gene Expression Profiling; D016174:Hepacivirus / Q000187:drug effects* / Q000276:immunology / Q000502:physiology*; D018937:Hepatitis C Antibodies / Q000235:genetics / Q000494:pharmacology*; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D011485:Protein Binding; D012367:RNA, Viral / Q000032:analysis; D017422:Sequence Analysis, DNA; D061905:Single-Domain Antibodies / Q000235:genetics / Q000494:pharmacology*; D014162:Transfection; D014758:Viral Core Proteins / Q000032:analysis; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors*; D014779:Virus Replication / Q000187:drug effects*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000998:Antiviral Agents; D002352:Carrier Proteins; D057846:Cell-Penetrating Peptides; D016207:Cytokines; D004337:Drug Carriers; D018937:Hepatitis C Antibodies; D012367:RNA, Viral; D061905:Single-Domain Antibodies; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C069130:nucleocapsid protein, Hepatitis C virus; C414409:penetratin