抗登革热病毒双特异性高亲和力中和抗体的构建及功能研究

发布时间：2018-10-19 18:37

【摘要】：登革热病毒和西尼罗河病毒、日本脑炎病毒、森林脑炎病毒等同属于黄病毒属，其基因组为由包膜包裹的单正链RNA，主要编码三种结构蛋白（核蛋白C、膜结合蛋白M和包膜蛋白E）和七种非结构蛋白。包膜E蛋白是成熟病毒表面的主要蛋白，提供主要的免疫源性。根据病毒包膜抗原性的不同，登革热病毒分为四种血清型（DENV1-4）。临床上登革热病毒主要引起登革热（Dengue Fever，DF）、登革热出血热（Dengue Hemorrhagic Fever，DHF）和登革休克综合征（Dengue Shock Syndrome，DSS）。每年全球约有5000万人感染登革热病毒，导致数万人死亡。但是目前临床上还没有治疗登革热感染的有效药物，主要采用相应的支持治疗。因此研制一种能够有效预防并在感染病毒后提供有效治疗的新型药物就变得十分重要。 近些年随着抗体技术的发展，利用中和抗体进行病毒感染的预防和治疗越来越受到人们的重视。抗体药物相比普通治疗方法具有高效、特异性好等优点。登革热病毒感染细胞过程中的两个关键的步骤是吸附和融合。病毒吸附至细胞表面时首先通过暴露在病毒表面E蛋白的DomainⅢ结构域同细胞表面的特定受体结合，然后通过受体介导Qg吞作用进入细胞。登革热病毒被吞入细胞后，病毒E蛋白DomainⅡ结构域顶端的疏水环介导了病毒包膜和包内小体膜的融合，从而使病毒基因组释放到细胞内。目前已有多株抗体对登革热病毒具有中和保护作用，其中文献报道单克隆抗体1A1D-2(后简称为1A1D)通过结合E蛋白DomainⅢ抑制病毒的吸附从而预防病毒感染。另同过我们前面的研究获得的单克隆抗体2A10G6（后简称2A10）通过结合E蛋白DomainⅡ而抑制病毒的融合过程。但是虽然这两种单克隆抗体都能够起到中和保护的作用，但是1A1D的应用价值主要体现在预防方面，2A10则更倾向于治疗应用，单独使用两者都不能兼顾预防和治疗。如果将两种抗体进行联合应用可以获得比单独应用一种单克隆抗体更好的效果，而且可以结合两者各自的优点，但同时也面临着安全性和费用等方面的诸多问题。 Chengbin Wu等人报道的双可变区抗体技术（Dual-Variable-DomainImmunoglobulin，DVD-Ig）为我们提供了一种新的思路，利用此技术可以将2种不同抗体的可变区构建于一株抗体分子上，使其能同时识别并结合登革热病毒的DomainⅡ和DomainⅢ连个功能域，并保持原抗体的亲和力和中和保护活性，从而简化多抗体联合应用带来的不便，并具有更高的安全性，更具有临床应用价值。同时可以通过对DVD抗体的Fc受体结合段进行改造而达到消除ADE作用的目的。 本项研究旨在利用双可变区抗体技术将已有的两种作用于不同表位的单克隆抗体的可变区构建于同一株DVD抗体上，使其具有更高的亲和力，从而提高其中和保护活性，并通过对Fc段的缺失突变以消除其ADE作用。 首先，我们通过特定的Linker将单抗1A1D和2A10的可变区序列连接，然后分别构建出DVD抗体的轻重链，装入表达载体后，共转染CHO细胞，经筛选鉴定获得稳定表达株，并大量培养纯化出足量DVD抗体。 其次，通过Western Blot和ELISA方法检测DVD抗体的体外结合活性，进一步用间接免疫荧光证实DVD-1A1D-2A10能够正常同病毒表面的E蛋白结合，并通过分别测定同EDⅠ/Ⅱ和EDⅢ的结合曲线证明四价结构对于两种可变区的结合能力没有影响，而且对于完整E蛋白的亲和力比二价抗体更高。体外蚀斑减数中和试验证明了我们构建的DVD抗体的中和保护活性比1A1D和2A10的联合应用提高了约10倍，比单一单克隆抗体更是提高了30-250倍，尤其实在抗体浓度较低的情况下的保护作用明显高于普通抗体。另外通过病毒吸附前后的感染抑制实验证实DVD抗体在吸附前和吸附后的抑制感染效果提高了2-4倍。为了进一步证实DVD抗体的中和保护作用，我们采用乳鼠体内保护模型观察其体内保护效果，结果显示DVD保护的乳鼠存活率约为其他抗体的2-5倍，说明DVD抗体在体内同样具有非常好的保护活性。同时通过体内预防和治疗实验，我们证明DVD抗体的保护效果比普通二价抗体同样提高了2-5倍，而且无论是预防还是治疗均有非常好的效果。 最后，通过将介导ADE作用的抗体Fc段缺失突变，使其ADE作用得到消除，，并且经鉴定突变对其保护活性没有影响，从而证明我们成功构建了抗多亚型登革热病毒的双特异性高亲和力中和抗体。 本研究利用两株已知分别结合E蛋白DomainⅡ和DomainⅢ的单克隆抗体构建出了一株能够同时结合这两个结构域的DVD抗体，证明了该DVD抗体在体内外均具有比普通二价抗体更好的中和保护活性，而且同时具有预防和治疗的应用价值。并且通过实验证实由于DVD抗体能够同时结合两个功能表位并且同E蛋白具有更高的亲和力而使其具有更好的中和保护活性。
[Abstract]:Dengue virus and West Nile virus, Japanese encephalitis virus and forest encephalitis virus belong to the genus Rhododendron, the genome of which is single positive strand RNA enveloped by envelope, mainly coding three structural proteins (nucleoprotein C, membrane binding protein M and envelope protein E) and seven non-structural proteins. Envelope E protein is the main protein of mature virus surface, providing primary immunity. Dengue viruses are classified into four serotypes (DENV1-4) according to different viral envelope antigens. The clinical dengue virus mainly causes dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Every year around 50 million people worldwide are infected with dengue virus, causing tens of thousands of people to die. However, there are no effective drugs for the treatment of dengue infection in clinic, and the corresponding support therapy is mainly adopted. Therefore, it is important to develop a new type of medicine which can effectively prevent and provide effective treatment after infection. In recent years, with the development of antibody technology, the prevention and treatment of virus infection by neutralizing antibody is becoming more and more popular Compared with the common treatment method, the antibody medicine has the advantages of high efficiency, good specificity and the like. An advantage is that two of the key steps in the process of dengue virus infection are adsorption and Fusion. When virus is adsorbed onto the surface of a cell, first binds to a specific receptor on the surface of the cell by exposure to the Domain III domain of the viral surface E protein, and then enters the cell surface through the receptor-mediated Qg uptake. After the dengue virus is swallowed into the cells, the hydrophobic ring at the top of the viral E protein Domain II domain mediates the fusion of the virus envelope and the small body membrane in the package, thereby releasing the viral genome to the fine It has been reported that monoclonal antibodies 1A1D-2 (hereinafter referred to as 1A1D) inhibit virus adsorption by binding to E-protein Domain III to prevent viruses. Infection. Another monoclonal antibody 2A10G6 (hereinafter referred to as 2A10) obtained from the previous study inhibits the fusion of viruses by binding to E-protein Domain II However, although both monoclonal antibodies can play a role in neutralizing protection, the application value of 1A1D is mainly embodied in prevention. The combination of the two antibodies can achieve a better effect than a single monoclonal antibody, and can combine the advantages of the two antibodies, but at the same time, it also faces many aspects such as safety and cost The dual variable region antibody technique (DVD-Ig) reported by Chanbin et al. provides a new way to construct a variable region of two different antibodies on an antibody molecule, enabling it to simultaneously identify and bind to the Domain II and Domain III of the dengue virus. has a function domain and keeps the affinity and neutralizing activity of the original antibody, thereby simplifying the inconvenience brought by the multi-antibody combined application, The value of the bed application can be eliminated by modifying the binding segment of the Fc receptor of the DVD antibody. The purpose of this study is to use a double variable region antibody technique to construct a variable region of two monoclonal antibodies that act on different table positions on the same DVD antibody so that it has a higher affinity, high in and protective activity and by deletion mutation to that Fc segment, First of all, we connect the variable region sequences of monoclonal antibodies 1A1D and 2A10 with specific Linker, then construct the light weight chain of the DVD antibody respectively. After the expression vector is loaded into the expression vector, we obtain the stable expression strain through screening and identification, and a large amount of culture can be obtained. In addition, the in vitro binding activity of DVD antibody was detected by Western blot and ELISA, and indirect immunofluorescence was used to confirm the performance of DVD-1A1C-2A10. The binding curves of ED 鈪

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