Monoclonal Antibodies against Occludin Completely Prevented Hepatitis C Virus Infection in a Mouse Model
Monoclonal Antibodies against Occludin Completely Prevented Hepatitis C Virus Infection in a Mouse Model
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针对 Occludin 的单克隆抗体完全预防小鼠模型中的丙型肝炎病毒感染
DOI：
10.1128/jvi.02258-17
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发表时间：
2018
影响因子：
5.4
通讯作者：
Fukasawa Masayoshi
中科院分区：
文献类型：
--
作者：
Shimizu Yoshimi;Shirasago Yoshitaka;Kondoh Masuo;Suzuki Tetsuro;Wakita Takaji;Hanada Kentaro;Yagi Kiyohito;Fukasawa Masayoshi
丙型肝炎病毒（HCV）进入宿主细胞是一个多步骤的过程，需要各种宿主因子，包括紧密连接蛋白闭合蛋白（OCLN），它已被证明是必要的HCV感染在体外细胞培养系统。然而，目前还不清楚OCLN是否是HCV治疗的有效和安全的靶点，因为缺乏可以识别OCLN的完整细胞外环结构域并预防HCV感染的结合剂。本研究采用基因免疫法和独特的细胞差异筛选法成功制备了4株大鼠抗OCLN单克隆抗体。这四种MAb以非常高的亲和力（抗体解离常数<1 nM）结合人OCLN。一种单克隆抗体识别人和小鼠OCLN的第二环，而其他三种单克隆抗体识别人OCLN的第一环。所有单克隆抗体均能抑制HCV感染Huh 7.5.1 -8细胞，且呈剂量依赖性，无明显细胞毒性。此外，抗OCLN单克隆抗体可预防无细胞HCV感染和细胞间HCV传播。用抗OCLN和抗claudin-1（CLDN 1）单克隆抗体进行的动力学研究表明，在内化步骤中，OCLN在CLDN 1之后与HCV相互作用。两个选定的单克隆抗体完全抑制HCV感染的人肝嵌合小鼠没有明显的不良反应。因此，OCLN将是抗HCV进入抑制剂的合适宿主靶标，并且抗OCLN单克隆抗体可能是新型抗HCV剂的有希望的候选者，特别是与直接作用的HCV抗病毒剂组合。重要的是HCV进入宿主细胞被认为是一个非常复杂的过程，涉及各种宿主进入因子，例如紧密连接蛋白claudin-1和OCLN。在这项研究中，我们开发了新的功能单克隆抗体，识别完整的OCLN，这是必要的HCV进入宿主细胞的胞外结构域。所建立的单克隆抗体对完整的OCLN具有很高的亲和力和选择性，在体外和体内均能有效抑制HCV感染。使用这些抗OCLN单克隆抗体，我们发现OCLN对于HCV进入的后期阶段是必需的。这些抗OCLN单克隆抗体对于理解OCLN介导的HCV进入机制可能非常有用，并且可能是新型HCV进入抑制剂的有希望的候选者。
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 inin vitrocell 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 the genetic immunization method and unique cell differential screening. These four MAbs bound to human OCLN with a very high affinity (antibody dissociation constant of <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.IMPORTANCEHCV 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 infection bothin vitroandin 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.