Gastroenterology

Gastroenterology

Volume 145, Issue 2, August 2013, Pages 447-455.e4
Gastroenterology

Original Research
Full Report: Basic and Translational—Liver
Neutralizing Antibodies Induced by Cell Culture–Derived Hepatitis C Virus Protect Against Infection in Mice

https://doi.org/10.1053/j.gastro.2013.05.007Get rights and content

Background & Aims

Hepatitis C virus (HCV) infection is a major cause of liver cancer, so strategies to prevent infection are needed. A system for cell culture of infectious HCV particles (HCVcc) has recently been established; the inactivated HCVcc particles might be used as antigens in vaccine development. We aimed to confirm the potential of HCVcc as an HCV particle vaccine.

Methods

HCVcc derived from the J6/JFH-1 chimeric genome was purified from cultured cells by ultrafiltration and ultracentrifugation purification steps. Purified HCV particles were inactivated and injected into female BALB/c mice with adjuvant. Sera from immunized mice were collected and their ability to neutralize HCV was examined in naive Huh7.5.1 cells and urokinase-type plasminogen activator–severe combined immunodeficiency mice (uPA+/+-SCID mice) given transplants of human hepatocytes (humanized livers).

Results

Antibodies against HCV envelope proteins were detected in the sera of immunized mice; these sera inhibited infection of cultured cells with HCV genotypes 1a, 1b, and 2a. Immunoglobulin G purified from the sera of HCV-particle-immunized mice (iHCV-IgG) inhibited HCV infection of cultured cells. Injection of IgG from the immunized mice into uPA+/+-SCID mice with humanized livers prevented infection with the minimum infectious dose of HCV.

Conclusions

Inactivated HCV particles derived from cultured cells protect chimeric liver uPA+/+-SCID mice against HCV infection, and might be used in the development of a prophylactic vaccine.

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.

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    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.

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