Claudin一1抗体应用于抑制丙型肝炎感染的体外研究
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摘要
背景和目的
丙型肝炎是一种常见的由丙型肝炎病毒引起的肝脏疾病，常导致肝硬化和肝细胞癌。全世界约有1.7亿人感染丙型肝炎病毒，丙型肝炎已成为一个对公共健康有重大影响的疾病。目前尚无针对丙型肝炎的保护性疫苗，目前的标准治疗方法-长效干扰素联合利巴韦林疗效有限，费用昂贵，副作用大。在今后20年内，由丙型肝炎引起的肝硬化和肝癌患者数量将会继续增加。因此，目前迫切需要针对丙型肝炎更为有效的预防措施和治疗方法。
丙型肝炎病毒是一种单链包膜RNA病毒，属于黄病毒科嗜肝病毒属，由5’非编码区（包括内部核糖进入位点），开放读码框（编码结构蛋白和非结构蛋白）和3’编码区构成。构成病毒颗粒的结构蛋白包括核心蛋白和包膜糖蛋白E1和E2。非结构蛋白包括P7离子通道，NS2-3蛋白酶，NS3丝氨酸蛋白酶和RNA解旋酶，NS4A多肽，NS4B和NS5A蛋白以及NS5B-RNA依赖性RNA聚合酶。
病毒入侵是病毒-宿主细胞相互作用产生感染的第一步。HCV病毒入侵是多因素参与的复杂过程，包括宿主细胞因素：SR-BI, CD81, CLDN1和OCLN等，为抗病毒治疗提供了多个新的靶点。在这些HCV受体中，紧密连接蛋白-CLDN1是一个非常有前景的抗病毒靶点，它对HCV入侵至关重要，且目前未发现CLDN1非依赖性HCV入侵。而且，CLDN1在病毒细胞间传播中发挥重要作用。
病毒可以以两种方式在宿主细胞间传播：将病毒释放至细胞外或直接将病毒由感染细胞传递给非感染细胞。通常，病毒在细胞间的直接传递更为迅速有效，因为这一过程省去了病毒粘附等病毒生命周期中的限速步骤。最近的研究成功建立了HCV细胞培养模型，使病毒能在细胞培养中传播，该模型为研究病毒传播创造了条件。HCV感染肝癌细胞常呈现为相邻细胞间的灶状感染，提示病毒在相邻细胞间传播。新近研究表明，HCV在体外以两种方式传播-细胞外病毒感染或病毒在细胞间的直接传播，后者可以逃避中和抗体的攻击。这种HCV细胞间的直接传播依赖CLDN1的表达，但存在CD81非依赖型细胞间传播。
方法
在体外建立HCVpp及HCVcc模型。通过基因免疫制备anti-CLDN1多克隆及单克隆抗体。应用anti-CLDN1单克隆抗体在HCV细胞培养中预防HCV感染。在HCV传播试验中应用anti-CLDN1多克隆抗体及HCV患者血清抑制HCV传播。
结果
我们成功制备了CLDN1多克隆及单克隆抗体。抗-CLDN1抗体与人源性CLDN1外环在细胞高亲和性结合并对肝癌细胞(Huh7.5.1)及肝原代细胞无细胞毒性。在体外，该抗体剂量依赖性抑制HCV主要基因型。我们还发现抗-CLDN1抗体有效抑制HCV通过细胞外病毒以及通过细胞间进行传播。
结论
在体外模型中，抗-CLDN1抗体可以抑制HCV入侵和病毒的传播。针对HCV受体的抗体，尤其是抗-CLDN1抗体，可望成为新的抗病毒治疗方法应用于预防HCV感染以及限制病毒在慢性HCV感染者中的传播。
BACKGROUND & OBJECTIVE
Hepatitis C virus (HCV) is a major cause of liver disease, including liver cirrhosis andhepatocellular carcinoma. With an estimated 170 million infected individuals, HCV has amajor impact on public health. A vaccine protecting against HCV infection is not available,and current standard therapy, which is pegylated interferon- combined with ribavirin, ischaracterized 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 20years. So, more effective therapies and prophylactic measurements are urgently needed forHCV.
HCV is an enveloped single-stranded RNA virus of positive polarity that is a memberof the genus Hepacivirus within the family Flaviviridae. It is composed of a 5'-non-codingregion (NCR), which includes an internal ribosome entry site (IRES), an open readingframe that encodes structural and non-structural proteins, and a 3'-NCR. The structuralproteins, which form the viral particle, include the core protein and the envelopeglycoproteins E1 and E2. The non-structural proteins include the p7 ion channel, the NS2-3protease, the NS3 serine protease and RNA helicase, the NS4A polypeptide, the NS4B andNS5A 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 hostcell factors including SR-BI, CD81, CLDN1 and OCLN, thereby offering multiple noveltargets for antiviral therapy. Among the host entry factors, tight junction protein CLDN1 isa promising antiviral target since it is essential for HCV entry and to date there is noevidence for CLDN1-independent HCV entry. Furthermore, CLDN1 has been suggested toplay an important role in HCV cell-cell transmission.
Viruses can disseminate within a host by two mechanisms: release of cell-free virionsor direct passage between infected and uninfected cells. In general, direct cell-cell transferis considered more rapid and efficient than cell-free spread because it obviates rate-limitingearly steps in the virus life cycle, such as virion attachment. Recent developments haveallowed HCV to be propagated in cell culture [cell culture-grown hepatitis C virus(HCVcc)], allowing studies on viral transmission. HCV infection of hepatoma cells resultsin focal areas of infection that are potentiated by cell-cell contact, and this suggestslocalized transmission between adjacent cells. Recently, it has been demonstrated that HCVcan be transmited in vitro by at least two routes, cell-free virus infection and direct transferbetween cells, with the latter offering a novel route for evading neutralizing Abs. And it wasindicated that HCV cell-cell transmission is dependent on CLDN1 expression andCD81-independent routes of cell-cell transmission exist.
METHODS
HCVcc was established as a model in vitro. Anti-CLDN1 antibody monoclonal andpolyclonal antibodies were produced by genetic immunization. Anti-CLDN1 mAb wasused to prevent HCV infection in HCV cell culture. Inhibition of HCV spread by thepolyclonal antibody against claudin-1 and anti-HCV sera from HCV patients was assessedby the established HCV spread assay.
RESULTS
We successfully produced anti-CLDN1 polyclonal and monoclonal antibodies. Anti-CLDN1 mAb bound to the extracellular loops of human CLDN1 expressed on the cellsurface with high affinity and showed no toxicity to hepatoma cells (Huh7.5.1) and primaryhepatocytes. In vitro, it inhibited HCV infection in a dose dependent manner and waseffective to HCV infection of several genotypes. Our data showed that anti-CLDN1antibody was promising to inhibit HCV spread and disemination.
CONCLUSION
Antibody against CLDN1 can efficiently inhibit HCV entry and viral spread in vitro. Itis indicated that Abs against HCV receptors, especially anti-CLDN1 Abs, may constitute anovel antiviral approach to prevent HCV infection and might restrain virus spread inchronically infected patients.
丙型肝炎是一种常见的由丙型肝炎病毒引起的肝脏疾病，常导致肝硬化和肝细胞癌。全世界约有1.7亿人感染丙型肝炎病毒，丙型肝炎已成为一个对公共健康有重大影响的疾病。目前尚无针对丙型肝炎的保护性疫苗，目前的标准治疗方法-长效干扰素联合利巴韦林疗效有限，费用昂贵，副作用大。在今后20年内，由丙型肝炎引起的肝硬化和肝癌患者数量将会继续增加。因此，目前迫切需要针对丙型肝炎更为有效的预防措施和治疗方法。
丙型肝炎病毒是一种单链包膜RNA病毒，属于黄病毒科嗜肝病毒属，由5’非编码区（包括内部核糖进入位点），开放读码框（编码结构蛋白和非结构蛋白）和3’编码区构成。构成病毒颗粒的结构蛋白包括核心蛋白和包膜糖蛋白E1和E2。非结构蛋白包括P7离子通道，NS2-3蛋白酶，NS3丝氨酸蛋白酶和RNA解旋酶，NS4A多肽，NS4B和NS5A蛋白以及NS5B-RNA依赖性RNA聚合酶。
病毒入侵是病毒-宿主细胞相互作用产生感染的第一步。HCV病毒入侵是多因素参与的复杂过程，包括宿主细胞因素：SR-BI, CD81, CLDN1和OCLN等，为抗病毒治疗提供了多个新的靶点。在这些HCV受体中，紧密连接蛋白-CLDN1是一个非常有前景的抗病毒靶点，它对HCV入侵至关重要，且目前未发现CLDN1非依赖性HCV入侵。而且，CLDN1在病毒细胞间传播中发挥重要作用。
病毒可以以两种方式在宿主细胞间传播：将病毒释放至细胞外或直接将病毒由感染细胞传递给非感染细胞。通常，病毒在细胞间的直接传递更为迅速有效，因为这一过程省去了病毒粘附等病毒生命周期中的限速步骤。最近的研究成功建立了HCV细胞培养模型，使病毒能在细胞培养中传播，该模型为研究病毒传播创造了条件。HCV感染肝癌细胞常呈现为相邻细胞间的灶状感染，提示病毒在相邻细胞间传播。新近研究表明，HCV在体外以两种方式传播-细胞外病毒感染或病毒在细胞间的直接传播，后者可以逃避中和抗体的攻击。这种HCV细胞间的直接传播依赖CLDN1的表达，但存在CD81非依赖型细胞间传播。
方法
在体外建立HCVpp及HCVcc模型。通过基因免疫制备anti-CLDN1多克隆及单克隆抗体。应用anti-CLDN1单克隆抗体在HCV细胞培养中预防HCV感染。在HCV传播试验中应用anti-CLDN1多克隆抗体及HCV患者血清抑制HCV传播。
结果
我们成功制备了CLDN1多克隆及单克隆抗体。抗-CLDN1抗体与人源性CLDN1外环在细胞高亲和性结合并对肝癌细胞(Huh7.5.1)及肝原代细胞无细胞毒性。在体外，该抗体剂量依赖性抑制HCV主要基因型。我们还发现抗-CLDN1抗体有效抑制HCV通过细胞外病毒以及通过细胞间进行传播。
结论
在体外模型中，抗-CLDN1抗体可以抑制HCV入侵和病毒的传播。针对HCV受体的抗体，尤其是抗-CLDN1抗体，可望成为新的抗病毒治疗方法应用于预防HCV感染以及限制病毒在慢性HCV感染者中的传播。
BACKGROUND & OBJECTIVE
Hepatitis C virus (HCV) is a major cause of liver disease, including liver cirrhosis andhepatocellular carcinoma. With an estimated 170 million infected individuals, HCV has amajor impact on public health. A vaccine protecting against HCV infection is not available,and current standard therapy, which is pegylated interferon- combined with ribavirin, ischaracterized 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 20years. So, more effective therapies and prophylactic measurements are urgently needed forHCV.
HCV is an enveloped single-stranded RNA virus of positive polarity that is a memberof the genus Hepacivirus within the family Flaviviridae. It is composed of a 5'-non-codingregion (NCR), which includes an internal ribosome entry site (IRES), an open readingframe that encodes structural and non-structural proteins, and a 3'-NCR. The structuralproteins, which form the viral particle, include the core protein and the envelopeglycoproteins E1 and E2. The non-structural proteins include the p7 ion channel, the NS2-3protease, the NS3 serine protease and RNA helicase, the NS4A polypeptide, the NS4B andNS5A 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 hostcell factors including SR-BI, CD81, CLDN1 and OCLN, thereby offering multiple noveltargets for antiviral therapy. Among the host entry factors, tight junction protein CLDN1 isa promising antiviral target since it is essential for HCV entry and to date there is noevidence for CLDN1-independent HCV entry. Furthermore, CLDN1 has been suggested toplay an important role in HCV cell-cell transmission.
Viruses can disseminate within a host by two mechanisms: release of cell-free virionsor direct passage between infected and uninfected cells. In general, direct cell-cell transferis considered more rapid and efficient than cell-free spread because it obviates rate-limitingearly steps in the virus life cycle, such as virion attachment. Recent developments haveallowed HCV to be propagated in cell culture [cell culture-grown hepatitis C virus(HCVcc)], allowing studies on viral transmission. HCV infection of hepatoma cells resultsin focal areas of infection that are potentiated by cell-cell contact, and this suggestslocalized transmission between adjacent cells. Recently, it has been demonstrated that HCVcan be transmited in vitro by at least two routes, cell-free virus infection and direct transferbetween cells, with the latter offering a novel route for evading neutralizing Abs. And it wasindicated that HCV cell-cell transmission is dependent on CLDN1 expression andCD81-independent routes of cell-cell transmission exist.
METHODS
HCVcc was established as a model in vitro. Anti-CLDN1 antibody monoclonal andpolyclonal antibodies were produced by genetic immunization. Anti-CLDN1 mAb wasused to prevent HCV infection in HCV cell culture. Inhibition of HCV spread by thepolyclonal antibody against claudin-1 and anti-HCV sera from HCV patients was assessedby the established HCV spread assay.
RESULTS
We successfully produced anti-CLDN1 polyclonal and monoclonal antibodies. Anti-CLDN1 mAb bound to the extracellular loops of human CLDN1 expressed on the cellsurface with high affinity and showed no toxicity to hepatoma cells (Huh7.5.1) and primaryhepatocytes. In vitro, it inhibited HCV infection in a dose dependent manner and waseffective to HCV infection of several genotypes. Our data showed that anti-CLDN1antibody was promising to inhibit HCV spread and disemination.
CONCLUSION
Antibody against CLDN1 can efficiently inhibit HCV entry and viral spread in vitro. Itis indicated that Abs against HCV receptors, especially anti-CLDN1 Abs, may constitute anovel antiviral approach to prevent HCV infection and might restrain virus spread inchronically infected patients.
引文
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