title: Production and characterization of high-titer serum-free cell culture grown hepatitis C virus particles of genotype 1-6.
abstract: Recently, cell culture systems producing hepatitis C virus particles (HCVcc) were developed. Establishment of serum-free culture conditions is expected to facilitate development of a whole-virus inactivated HCV vaccine. We describe generation of genotype 1-6 serum-free HCVcc (sf-HCVcc) from Huh7.5 hepatoma cells cultured in adenovirus expression medium. Compared to HCVcc, sf-HCVcc showed 0.6-2.1 log10 higher infectivity titers (4.7-6.2 log10 Focus Forming Units/mL), possibly due to increased release and specific infectivity of sf-HCVcc. In contrast to HCVcc, sf-HCVcc had a homogeneous single-peak density profile. Entry of sf-HCVcc depended on HCV co-receptors CD81, LDLr, and SR-BI, and clathrin-mediated endocytosis. HCVcc and sf-HCVcc were neutralized similarly by chronic-phase patient sera and by human monoclonal antibodies targeting conformational epitopes. Thus, we developed serum-free culture systems producing high-titer single-density sf-HCVcc, showing similar biological properties as HCVcc. This methodology has the potential to advance HCV vaccine development and to facilitate biophysical studies of HCV.
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital, Kettegaard Allé 30, 2650 Hvidovre, Denmark; Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N, Denmark.
authors.forename: Christian K
authors.initials: CK
authors.lastname: Mathiesen
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital, Kettegaard Allé 30, 2650 Hvidovre, Denmark; Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N, Denmark.
authors.forename: Tanja B
authors.initials: TB
authors.lastname: Jensen
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital, Kettegaard Allé 30, 2650 Hvidovre, Denmark; Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N, Denmark.
authors.forename: Jannick
authors.initials: J
authors.lastname: Prentoe
authors.affiliation: Department of Clinical Biochemistry, Section of Molecular Diagnostics, Aalborg University Hospital, Fredrik Bajers Vej 5, 9220 Aalborg, Denmark.
authors.forename: Henrik
authors.initials: H
authors.lastname: Krarup
authors.affiliation: CEINGE, Via Gaetano Salvatore, 486, 80145 Naples, Italy; Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, via S. Pansini 5, 80131, Naples, Italy; Okairos, viale Citta' d' Europa 279, 00144, Rome, Italy.
authors.forename: Alfredo
authors.initials: A
authors.lastname: Nicosia
authors.affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
authors.forename: Mansun
authors.initials: M
authors.lastname: Law
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital, Kettegaard Allé 30, 2650 Hvidovre, Denmark; Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N, Denmark.
authors.forename: Jens
authors.initials: J
authors.lastname: Bukh
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital, Kettegaard Allé 30, 2650 Hvidovre, Denmark; Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N, Denmark. Electronic address: judith@gottwein.eu.
authors.forename: Judith M
authors.initials: JM
authors.lastname: Gottwein
mesh_terms: D045744:Cell Line, Tumor; D002470:Cell Survival; D016895:Culture Media, Serum-Free; D015967:Gene Expression Regulation, Viral; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D014776:Virus Cultivation / Q000379:methods*
chemical_list: D016895:Culture Media, Serum-Free
keywords: Adenovirus expression medium; Biophysical characterization; Cell culture system; Genotypes; Hepatitis C virus; High-titer; Neutralization; Receptor blocking; Serum-free; Vaccine development