Original ResearchFull Report: Basic and Translational—LiverNeutralizing Antibodies Induced by Cell Culture–Derived Hepatitis C Virus Protect Against Infection in Mice
Section snippets
Cell Culture
Huh7 and Huh7.5.1 cells2 (a generous gift from Dr Francis V. Chisari) were cultured in 5% CO2 at 37°C in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (DMEM-10) as previously reported.1
Plasmids
pJ6/JFH1 was generated by replacing the JFH-1 structural region with that of the J6CF strain as previously reported.6
HCV Particle Purification
J6/JFH-1 chimeric HCVcc was purified by ultrafiltration and ultracentrifugation. The methods for HCVcc production and purification were as described in the Supplementary
HCV Particle Purification
HCV particles for mouse immunization were obtained using the cell culture system described in the Materials and Methods section. J6/JFH-1 chimeric HCV was secreted into culture medium supplemented with 2% fetal bovine serum, which was collected and centrifuged; the supernatant was concentrated by ultrafiltration using a 500-kilodalton cut-off membrane. The concentrated culture fluid was diafiltered using phosphate-buffered saline. These purification procedures removed approximately 90% of the
Discussion
There is no vaccine available for clinical use against HCV infection. Development of an HCV cell culture system has facilitated an understanding of the HCV life cycle, and the production of HCV prophylactic and therapeutic drugs. HCVcc mimics the native HCV particle, and thus has potential for use as an HCV-particle vaccine. In the present study, we established an efficient method for HCV particle production and purification, which allowed evaluation of the HCV particle as a vaccine candidate.
Acknowledgments
The Huh7.5.1 cell line was a kind gift from Dr Francis V. Chisari; AP33 was a generous gift from Genentech, Inc; and hepatitis C virus complementary DNA of the H77 and J6CF strains were kindly provided by Dr Robert Purcell and Dr Jens Bukh. The authors thank Dr Junichi Tanabe for his much appreciated contribution to this work.
References (39)
- et al.
Characterization of infectious hepatitis C virus from liver-derived cell lines
Biochem Biophys Res Commun
(2008) - et al.
Specific inhibition of hepatitis C virus expression by antisense oligodeoxynucleotides
J Biol Chem
(1994) - et al.
Hepatitis C virus: an infectious molecular clone of a second major genotype (2a) and lack of viability of intertypic 1a and 2a chimeras
Virology
(1999) - et al.
Progress in the development of preventive and therapeutic vaccines for hepatitis C virus
J Hepatol
(2011) - et al.
A pilot study of therapeutic vaccination with envelope protein E1 in 35 patients with chronic hepatitis C
Hepatology
(2003) - et al.
A candidate vaccine based on the hepatitis C E1 protein: tolerability and immunogenicity in healthy volunteers
Vaccine
(2004) - et al.
Whole recombinant yeast-based immunotherapy induces potent T cell responses targeting HCV NS3 and core proteins
Vaccine
(2007) - et al.
Safety and immunogenicity of HCV E1E2 vaccine adjuvanted with MF59 administered to healthy adults
Vaccine
(2010) - et al.
Therapeutic vaccine IC41 as late add-on to standard treatment in patients with chronic hepatitis C
Vaccine
(2009) - et al.
Immunogenicity and safety of different injection routes and schedules of IC41, a hepatitis C virus (HCV) peptide vaccine
Vaccine
(2010)
Hepatitis C virus envelope glycoprotein immunization of rodents elicits cross-reactive neutralizing antibodies
Vaccine
Uptake and presentation of hepatitis C virus-like particles by human dendritic cells
Blood
Production and characterization of HCV particles from serum-free culture
Vaccine
Production of infectious hepatitis C virus in tissue culture from a cloned viral genome
Nat Med
Robust hepatitis C virus infection in vitro
Proc Natl Acad Sci U S A
Complete replication of hepatitis C virus in cell culture
Science
Hepatitis C virus replication in mice with chimeric human livers
Nat Med
Vaccination for hepatitis C virus: closing in on an evasive target
Expert Rev Vaccines
The NS3 helicase and NS5B-to-3'X regions are important for efficient hepatitis C virus strain JFH-1 replication in Huh7 cells
J Virol
Cited by (0)
Conflicts of interest The authors disclose no conflicts.
Funding Supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science; from the Ministry of Health, Labour and Welfare of Japan; from the Ministry of Education, Culture, Sports, Science and Technology of Japan; from the National Institute of Biomedical Innovation; and from the Research on Health Sciences Focusing on Drug Innovation from the Japan Health Sciences Foundation.