Production and characterization of high-titer serum-free cell culture grown hepatitis C virus particles of genotype 1-6

Christian K Mathiesen; Tanja B Jensen; Jannick Prentoe; Henrik Krarup; Alfredo Nicosia; Mansun Law; Jens Bukh; Judith M Gottwein

doi:10.1016/j.virol.2014.03.021

Production and characterization of high-titer serum-free cell culture grown hepatitis C virus particles of genotype 1-6

Virology. 2014 Jun:458-459:190-208. doi: 10.1016/j.virol.2014.03.021. Epub 2014 May 24.

Authors

Christian K Mathiesen¹, Tanja B Jensen¹, Jannick Prentoe¹, Henrik Krarup², Alfredo Nicosia³, Mansun Law⁴, Jens Bukh¹, Judith M Gottwein⁵

Affiliations

¹ 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.
² Department of Clinical Biochemistry, Section of Molecular Diagnostics, Aalborg University Hospital, Fredrik Bajers Vej 5, 9220 Aalborg, Denmark.
³ 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.
⁴ Department of Immunology and Microbial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
⁵ 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.

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.

Keywords: Adenovirus expression medium; Biophysical characterization; Cell culture system; Genotypes; Hepatitis C virus; High-titer; Neutralization; Receptor blocking; Serum-free; Vaccine development.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Cell Line, Tumor
Cell Survival
Culture Media, Serum-Free
Gene Expression Regulation, Viral
Genotype*
Hepacivirus / genetics*
Hepacivirus / metabolism*
Humans
Virus Cultivation / methods*

Substances

Culture Media, Serum-Free

Abstract

Publication types

MeSH terms

Substances

Grants and funding