Hepatitis C virus (HCV) entry into host cells is a complex process requiring multiple host factors, including claudin-1 (CLDN1).Safe and effective therapeutic entry inhibitors need to be developed. We isolated a human hepatic Huh7.5.1-derived cell mutant that is nonpermissive to HCV, and comparative microarray analysis showed that the mutant was CLDN1 defective. Four hybridomas were obtained, which produced monoclonal antibodies (MAbs) that interacted with the parental Huh7.5.1 cell but not with the CLDN1-defective mutant. All MAbs produced by these hybridomas specifically bound to human CLDN1 with a very high affinity and prevented HCV infection of Huh7.5.1 cells in a dose-dependent manner, without apparent cytotoxicity. Two selected MAbs also inhibited HCV infection of human liver-chimeric mice without significant adverse effects. CLDN1 may be a potential target to prevent HCV infection in vivo. Anti-CLDN1 MAbs may hence be promising candidates as novel anti-HCV agents. IMPORTANCESafe and effective therapeutic entry inhibitors against hepatitis C virus (HCV) are very useful for combination therapies with other anti-HCV drugs, such as direct-acting antivirals. In this study, we first showed an effective strategy for developing functional monoclonal antibodies (MAbs) against extracellular domains of a multimembrane-spanning target protein, claudin-1 (CLDN1), by using parental cells expressing the intact target membrane protein and target-defective cells. The established MAbs against CLDN1, which had a very high affinity for intact CLDN1, efficiently inhibited in vitro and in vivo HCV infections. These anti-CLDN1 MAbs are promising leads for novel entry inhibitors against HCV. W orldwide, 170 million people are infected with hepatitis C virus (HCV), which is a major cause of liver cirrhosis and hepatocellular carcinoma. Thus, overcoming HCV infection is an important global health care issue (1). HCV is an enveloped, positive-sense, single-stranded RNA virus in the Flaviviridae family (2). Recent clinical research using direct-acting antivirals that target HCV enzymes, such as sofosbuvir and simeprevir, has provided new insights into combination therapy with inhibitors of multiple targets (3-5).Preventing viral entry into hepatocytes is an attractive target for anti-HCV agents, but strategies for preventing HCV entry into host cells are clinically unavailable (6). Host factors involved in initiating infection include heparan sulfate (7), low-density lipoprotein receptor (8), CD81 (9), scavenger receptor class B type I (SRBI) (10), claudin-1 (CLDN1) (11), occludin (12, 13), epidermal growth factor receptor (EGFR) (14), and Niemann-Pick C1-like 1 (15). Among these, CLDN1 is considered a potent target because it is essential for HCV entry into cells via interaction with CD81 and for cell-to-cell HCV transmission (16,17). Anti-CLDN1 antibodies (Abs) that inhibit HCV infection in vitro were reported by Baumert et al. (18,19) and Hötzel et al. (20), but a CLDN1 binder that prevents HCV infection in vivo has not...

It is well known that occludin (OCLN) is involved in hepatitis C virus (HCV) entry into hepatocytes, but there has been no conclusive evidence that OCLN is essential for HCV infection. In this study, we first established an OCLN-knockout cell line derived from human hepatic Huh7.5.1-8 cells using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 system, in which two independent targeting plasmids expressing single-guide RNAs were used. One established cell clone, named OKH-4, had the OCLN gene truncated in the N-terminal region, and a complete defect of the OCLN protein was shown using immunoblot analysis. Infection of OKH-4 cells with various genotypes of HCV was abolished, and exogenous expression of the OCLN protein in OKH-4 cells completely reversed permissiveness to HCV infection. In addition, using a co-culture system of HCV-infected Huh7. 5.1-8 cells with OKH-4 cells, we showed that OCLN is also critical for cell-to-cell HCV transmission. Thus, we concluded that OCLN is essential for HCV infection of human hepatic cells. Further experiments using HCV genomic RNA-transfected OKH-4 cells or HCV subgenomic replicon-harboring OKH-4 cells suggested that OCLN is mainly involvedin the entry step of the HCV life cycle. It was also demonstrated that the second extracellular loop of OCLN, especially the two cysteine residues, is critical for HCV infection of hepatic cells. OKH-4 cells may be a useful tool for understanding not only the entire mechanism of HCV entry, but also the biological functions of OCLN.

Hepatitis C virus (HCV) entry into host cells is a multistep process requiring various host factors, including the tight junction protein occludin (OCLN), which has been shown to be essential for HCV infection in cell culture systems. However, it remains unclear whether OCLN is an effective and safe target for HCV therapy, owing to the lack of binders that can recognize the intact extracellular loop domains of OCLN and prevent HCV infection. In this study, we successfully generated four rat anti-OCLN monoclonal antibodies (mAbs) by genetic immunization method and unique cell differential screening. These four mAbs bound to human OCLN with a very high affinity (Kd< 1 nM). One mAb recognized the second loop of human and mouse OCLN, whereas the three other mAbs recognized the first loop of human OCLN. All mAbs inhibited HCV infection in Huh7.5.1-8 cells in a dose-dependent manner, without apparent cytotoxicity. Additionally, the anti-OCLN mAbs prevented both cell-free HCV infection and cell-to-cell HCV transmission. Kinetic studies with anti-OCLN and anti-claudin-1 (CLDN1) mAbs demonstrated that OCLN interacts with HCV after CLDN1 in the internalization step. Two selected mAbs completely inhibited HCV infection in human liver chimeric mice, without apparent adverse effects. Therefore, OCLN would be an appropriate host target for anti-HCV entry inhibitors, and anti-OCLN mAbs may be promising candidates for novel anti-HCV agents, particularly in combination with direct-acting HCV antiviral agents. Hepatitis C virus (HCV) entry into host cells is thought to be a very complex process involving various host entry factors, such as the tight junction proteins claudin-1 and occludin (OCLN). In this study, we developed novel functional monoclonal antibodies (mAbs) that recognize intact extracellular domains of OCLN, which is essential for HCV entry into host cells. The established mAbs against OCLN, which had a very high affinity and selectivity for intact OCLN, strongly inhibited HCV infection both and Using these anti-OCLN mAbs, we found that OCLN is necessary for the later stages of HCV entry. These anti-OCLN mAbs are likely to be very useful for understanding the OCLN-mediated HCV entry mechanism and might be promising candidates for novel HCV entry inhibitors.

No abstract