Inhibition Of Hepatitis C Virus Infection By Anti-claudin1 Antibody In Vitro

BACKGROUND & OBJECTIVEHepatitis C virus (HCV) is a major cause of liver disease, including liver cirrhosis and hepatocellular carcinoma. With an estimated 170 million infected individuals, HCV has a major impact on public health. A vaccine protecting against HCV infection is not available, and current standard therapy, which is pegylated interferon-a combined with ribavirin, is characterized by limited efficacy, high costs, and substantial side effects. As a consequence, the number of patients presenting with the long-term sequelae of chronic hepatitis C, including hepatocellular carcinoma (HCC), is expected to increase further over the next 20 years. So, more effective therapies and prophylactic measurements are urgently needed for HCV.HCV is an enveloped single-stranded RNA virus of positive polarity that is a member of the genus Hepacivirus within the family Flaviviridae. It is composed of a 5’-non-coding region (NCR), which includes an internal ribosome entry site (IRES), an open reading frame that encodes structural and non-structural proteins, and a 3’-NCR. The structural proteins, which form the viral particle, include the core protein and the envelope glycoproteins E1 and E2. The non-structural proteins include the p7 ion channel, the NS2-3 protease, the NS3 serine protease and RNA helicase, the NS4A polypeptide, the NS4B and NS5A proteins and the NS5B RNA-dependent RNA polymerase (RdRp).Viral entry is the first step of virus-host cell interactions leading to productive infection. HCV entry is believed to be a highly orchestrated process involving several host cell factors including SR-BI, CD81, CLDN1 and OCLN, thereby offering multiple novel targets for antiviral therapy. Among the host entry factors, tight junction protein CLDN1 is a promising antiviral target since it is essential for HCV entry and to date there is no evidence for CLDN1-independent HCV entry. Furthermore, CLDN1 has been suggested to play an important role in HCV cell-cell transmission.Viruses can disseminate within a host by two mechanisms:release of cell-free virions or direct passage between infected and uninfected cells. In general, direct cell-cell transfer is considered more rapid and efficient than cell-free spread because it obviates rate-limiting early steps in the virus life cycle, such as virion attachment. Recent developments have allowed HCV to be propagated in cell cultures [cell culture-grown hepatitis C virus (HCVcc)], allowing studies on viral transmission. HCV infection of hepatoma cells results in focal areas of infection that are potentiated by cell-cell contact, and this suggests localized transmission between adjacent cells. Recently, it has been demonstrated that HCV can be transmited in vitro by at least two routes, cell-free virus infection and direct transfer between cells, with the latter offering a novel route for evading neutralizing Abs. And it was indicated that HCV cell-cell transmission is dependent on CLDN1 expression and CD81-independent routes of cell-cell transmission exist.METHODSHCVpp and HCVcc were established as models intro. Anti-CLDN1 antibody monoclonal and polyclonal antibodies were produced by genetic immunization. Anti-CLDN1 mAb was used to prevent HCV infection in HCV cell culture. Inhibition of HCV spread by the polyclonal antibody against claudin-1 and anti-HCV sera from HCV patients was assessed by the established HCV spread assay.RESULTSWe successfully produced anti-CLDN1 polyclonal and monoclonal antibodies. Anti-CLDN1 mAb bound to the extracellular loops of human CLDN1 expressed on the cell surface with high affinity and showed no toxicity to hepatoma cells (Huh7.5.1). In vitro, it inhibited HCV infection in a dose dependent manner and was effective to HCV infection 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. The combination of anti envelope and anti-CLDN1 antibodies resulted in an additive/synergistic effect on the inhibition of HCV infection. To investigate anti-CLDN1 antibodies’ inhibition on HCV spread, HCV spread assay was successfully established and used to evaluate the inhibitory effects of the antibody against claudin-1 and HCV patients’ sera on HCV spread. Our data showed that both were efficient to inhibit HCV spread by cell-free virus. The antibody against claudin-1 efficiently inhibited HCV spread through cell-cell, while anti-HCV sera from HCV patients only had slight effect on it.CONCLUSIONIn accord with others’report, HCV is able to evade neutralizing antibodies from HCV patients’sera. Antibody against CLDN1 can efficiently inhibit HCV entry and viral spread in vitro. It is indicated that Abs against HCV receptors, especially anti-CLDN1 Abs, may constitute a novel antiviral approach to prevent HCV infection and might restrain virus spread in chronically infected patients.