doi: 10

doi: 10.1128/JVI.01062-13. the three other MAbs acknowledged the first loop of human OCLN. All MAbs inhibited HCV contamination in Huh7.5.1-8 cells in a dose-dependent manner without apparent cytotoxicity. Additionally, the anti-OCLN MAbs prevented both cell-free HCV contamination and ITSN2 cell-to-cell HCV transmission. Kinetic studies with anti-OCLN and anti-claudin-1 (CLDN1) MAbs exhibited that OCLN interacts with HCV after CLDN1 in the internalization step. Two selected MAbs completely inhibited HCV contamination 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 brokers, particularly in combination with direct-acting HCV antiviral brokers. IMPORTANCE 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 OCLN. In this study, we developed novel functional MAbs that recognize intact extracellular domains of OCLN, which is essential for HCV entry into host cells. The established MAbs against OCLN, which had very high affinity and selectivity for intact OCLN, strongly inhibited HCV contamination both and family that possesses a single-stranded, positive-sense RNA genome. An estimated 185 million people are infected with HCV worldwide (1). Persistent HCV contamination can result in liver cirrhosis and hepatocellular carcinomas (2). The recent development of direct-acting antiviral brokers (DAAs) against HCV has markedly improved the outcome of antiviral treatments without serious side effects. The latest generation of DAA therapies is not prone to drug resistance; however, extensive and long-term use of DAAs might cause the emergence of drug-resistant viruses, which could be a major obstacle in successful pharmacological treatment of HCV in the future. Conversely, host-targeting brokers exhibit a high genetic barrier to drug resistance and thus may be candidates for next-generation HCV therapies, even though there is some concern regarding adverse effects. Although the detailed mechanism remains unclear, HCV entry into hepatocytes is usually a multistep process involving various host entry factors such as the low-density lipoprotein receptor (LDL-R) (3), Neferine glycosaminoglycans (GAGs) (4), the high-density lipoprotein receptor scavenger receptor class B type I (SR-BI) (5), the tetraspanin cluster of differentiation 81 (CD81) (6), the cholesterol transporter Niemann-Pick disease type C1 like 1 (7), epidermal growth factor receptor (8), and the tight junction (TJ) proteins claudin-1 (CLDN1) (9) and occludin (OCLN) (10). We previously showed that both CLDN1 and OCLN are essential for HCV contamination of human hepatic cells using would be essential for HCV contamination (13). HCV entry inhibitors targeting host CD81, SR-BI, CLDN1, Niemann-Pick disease type Neferine C1 like 1, and epidermal growth factor receptor exhibit broad pangenomic activities (12, 14,C19). Further, Colpitts et al. reported that anti-CLDN1 monoclonal antibodies (MAbs) inhibited contamination of hepatic cells with DAA-resistant strains of HCV and showed synergistic inhibition with current DAAs (20). From the genetic studies, knockout mice were found to have defects in development and fertility (21, 22), and knockout mice died within 1 day of birth with wrinkled skin (23), whereas knockout mice showed no apparent abnormal phenotypes (24). Hence, among the host entry factors, OCLN may be a promising target for novel host-targeting anti-HCV brokers. However, the lack of OCLN-specific binders has hindered the development of OCLN-targeting drugs against HCV contamination. In this study, we created anti-human OCLN (hOCLN) MAbs that recognize the intact extracellular loop domains of OCLN using DNA immunization methods and screening of differential cell binding. The anti-hOCLN MAbs prevented both and HCV infections without apparent adverse effects. Based on these results, we propose the use of OCLN-targeting brokers as potential anti-HCV drugs and the usefulness of our anti-hOCLN MAbs for understanding HCV entry mechanisms mediated by OCLN. RESULTS Development and characterization of MAbs against Neferine extracellular domains of hOCLN. To.