Monoclonal Anti-Claudin 1 Antibodies Prevent Hepatitis C Virus Infection of Primary Human Hepatocytes

doi:10.1053/j.gastro.2010.05.073

Gastroenterology

Volume 139, Issue 3, September 2010, Pages 953-964.e4

https://doi.org/10.1053/j.gastro.2010.05.073 Get rights and content

Background & Aims

Hepatitis C virus (HCV) infection is a challenge to prevent and treat because of the rapid development of drug resistance and escape. Viral entry is required for initiation, spread, and maintenance of infection, making it an attractive target for antiviral strategies. The tight junction protein claudin-1 (CLDN1) has been shown to be required for entry of HCV into the cell.

Methods

Using genetic immunization, we produced 6 monoclonal antibodies against the host entry factor CLDN1. The effects of antibodies on HCV infection were analyzed in human cell lines and primary human hepatocytes.

Results

Competition and binding studies demonstrated that antibodies interacted with conformational epitopes of the first extracellular loop of CLDN1; binding of these antibodies required the motif W(30)-GLW(51)-C(54)-C(64) and residues in the N-terminal third of CLDN1. The monoclonal antibodies against CLDN1 efficiently inhibited infection by HCV of all major genotypes as well as highly variable HCV quasispecies isolated from individual patients. Furthermore, antibodies efficiently blocked cell entry of highly infectious escape variants of HCV that were resistant to neutralizing antibodies.

Conclusions

Monoclonal antibodies against the HCV entry factor CLDN1 might be used to prevent HCV infection, such as after liver transplantation, and might also restrain virus spread in chronically infected patients.

Section snippets

Primary Hepatocytes and Cell Lines

Culture of primary human hepatocytes (PHH),¹⁸ human hepatoma cell lines (Huh7.5.1),¹⁹ and HepG2,²⁰ human embryonic kidney cell lines (HEK293T),⁹ and BOSC23, and Chinese hamster ovary (CHO) cells¹⁸ has been described.

Production and Screening of anti-CLDN1 mAbs

Anti-CLDN1 mAbs were raised by genetic immunization of Wistar rats using a eukaryotic expression vector encoding the full-length human CLDN1 complementary DNA according to proprietary GENOVAC technology (GENOVAC GmbH, Freiburg, Germany). Following completion of immunization,

Production of anti-CLDN1 mAbs With High Affinity to CLDN1 Expressed on Human Hepatocytes

To explore CLDN1 as a target for antiviral strategies, we generated anti-CLDN1 mAbs by genetic immunization using a full-length CLDN1 complementary DNA expression vector. Genetic immunization generated 6 mAbs that reacted with native human CLDN1 expressed on BOSC23 and CHO cells, showing negligible staining of parental cells (Figure 1A and B). Analyses of the antibody isotypes revealed IgG2a and IgG2b with a κ light chain. All of the mAbs bound to the surface of Huh7.5.1 cells and PHH (Figure 1C

Discussion

Here we demonstrate, for the first time, the successful production of anti-CLDN1 mAbs, which broadly inhibit HCV infection including patient-derived escape variants that are resistant to autologous host neutralizing responses. Because of the absence of preventive strategies for reinfection and limited efficacy and tolerability of interferon-based antiviral therapies in liver transplant recipients, there is a major medical need for the development of novel approaches preventing HCV infection of

Acknowledgments

The authors thank F. V. Chisari (The Scripps Research Institute, La Jolla, CA) for the gift of Huh7.5.1 cells; R. Bartenschlager (University of Heidelberg, Germany), F.-L. Cosset (Inserm U758, ENS Lyon, France), and J. Ball (University of Nottingham, UK) for providing plasmids for production of HCVcc and HCVpp; KaLy-Cell (Illkirch, France) for providing primary human hepatocytes; M. Parnot and M. Bastien-Valle (all Inserm U748, Strasbourg, France) for excellent technical assistance; J.

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: Conflicts of interest The authors disclose no conflicts. Inserm, the University of Strasbourg and Genovac have filed a patent application on monoclonal anti-Claudin 1 antibodies for the inhibition of hepatitis C virus infection.

: Funding Supported by Inserm, University of Strasbourg, the chair of excellence program of the Agence Nationale de la Recherche France (ANR-05-CEXC-008), GENOVAC, the Bundesministerium für Wirtschaft und Technologie (BMWi: Pro-INNO II), and by the French Ministry for Research and Education.

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Basic—Liver, Pancreas, and Biliary TractMonoclonal Anti-Claudin 1 Antibodies Prevent Hepatitis C Virus Infection of Primary Human Hepatocytes

Background & Aims

Methods

Results

Conclusions

Section snippets

Primary Hepatocytes and Cell Lines

Production and Screening of anti-CLDN1 mAbs

Production of anti-CLDN1 mAbs With High Affinity to CLDN1 Expressed on Human Hepatocytes

Discussion

Acknowledgments

J Hepatol

J Biol Chem

Gastroenterology

J Immunol Methods

Best Pract Res Clin Gastroenterol

Lancet

Lancet

Hepatitis C virus infection

N Engl J Med

Telaprevir and peginterferon with or without ribavirin for chronic HCV infection

N Engl J Med

Hepatitis C and liver transplantation

Nature

New and experimental therapies for HCV

Nat Rev Gastroenterol Hepatol

Hepatitis C virus entry: possible targets for therapy

Gut

Host neutralizing responses and pathogenesis of hepatitis C virus infection

Hepatology

Spontaneous control of primary hepatitis C virus infection and immunity against persistent reinfection

Gastroenterology

Rapid induction of virus-neutralizing antibodies and viral clearance in a single-source outbreak of hepatitis C

Proc Natl Acad Sci U S A

Binding of hepatitis C virus to CD81

Science

The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus

EMBO J

Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry

Nature

Basic—Liver, Pancreas, and Biliary Tract
Monoclonal Anti-Claudin 1 Antibodies Prevent Hepatitis C Virus Infection of Primary Human Hepatocytes