Yoshitaka Shirasago, Masuo Kondoh, Kentaro Hanada, Takaji Wakita, Masayoshi Fukasawa, Tetsuro Suzuki, Yoshimi Shimizu, Kiyohito Yagi
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 in vitro 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.IMPORTANCE 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 in vitro and in vivo 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.
