抗登革熱病毒雙特異性高親和力中和抗體的構建及功能研究
發(fā)布時間：2018-10-19 18:37
【摘要】：登革熱病毒和西尼羅河病毒、日本腦炎病毒、森林腦炎病毒等同屬于黃病毒屬，其基因組為由包膜包裹的單正鏈RNA，主要編碼三種結構蛋白（核蛋白C、膜結合蛋白M和包膜蛋白E）和七種非結構蛋白。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。根據(jù)病毒包膜抗原性的不同，登革熱病毒分為四種血清型（DENV1-4）。臨床上登革熱病毒主要引起登革熱（Dengue Fever，DF）、登革熱出血熱（Dengue Hemorrhagic Fever，DHF）和登革休克綜合征（Dengue Shock Syndrome，DSS）。每年全球約有5000萬人感染登革熱病毒，導致數(shù)萬人死亡。但是目前臨床上還沒有治療登革熱感染的有效藥物，主要采用相應的支持治療。因此研制一種能夠有效預防并在感染病毒后提供有效治療的新型藥物就變得十分重要。 近些年隨著抗體技術的發(fā)展，利用中和抗體進行病毒感染的預防和治療越來越受到人們的重視。抗體藥物相比普通治療方法具有高效、特異性好等優(yōu)點。登革熱病毒感染細胞過程中的兩個關鍵的步驟是吸附和融合。病毒吸附至細胞表面時首先通過暴露在病毒表面E蛋白的DomainⅢ結構域同細胞表面的特定受體結合，然后通過受體介導Qg吞作用進入細胞。登革熱病毒被吞入細胞后，病毒E蛋白DomainⅡ結構域頂端的疏水環(huán)介導了病毒包膜和包內(nèi)小體膜的融合，從而使病毒基因組釋放到細胞內(nèi)。目前已有多株抗體對登革熱病毒具有中和保護作用，其中文獻報道單克隆抗體1A1D-2(后簡稱為1A1D)通過結合E蛋白DomainⅢ抑制病毒的吸附從而預防病毒感染。另同過我們前面的研究獲得的單克隆抗體2A10G6（后簡稱2A10）通過結合E蛋白DomainⅡ而抑制病毒的融合過程。但是雖然這兩種單克隆抗體都能夠起到中和保護的作用，但是1A1D的應用價值主要體現(xiàn)在預防方面，2A10則更傾向于治療應用，單獨使用兩者都不能兼顧預防和治療。如果將兩種抗體進行聯(lián)合應用可以獲得比單獨應用一種單克隆抗體更好的效果，而且可以結合兩者各自的優(yōu)點，但同時也面臨著安全性和費用等方面的諸多問題。 Chengbin Wu等人報道的雙可變區(qū)抗體技術（Dual-Variable-DomainImmunoglobulin，DVD-Ig）為我們提供了一種新的思路，利用此技術可以將2種不同抗體的可變區(qū)構建于一株抗體分子上，使其能同時識別并結合登革熱病毒的DomainⅡ和DomainⅢ連個功能域，并保持原抗體的親和力和中和保護活性，從而簡化多抗體聯(lián)合應用帶來的不便，并具有更高的安全性，更具有臨床應用價值。同時可以通過對DVD抗體的Fc受體結合段進行改造而達到消除ADE作用的目的。 本項研究旨在利用雙可變區(qū)抗體技術將已有的兩種作用于不同表位的單克隆抗體的可變區(qū)構建于同一株DVD抗體上，使其具有更高的親和力，從而提高其中和保護活性，并通過對Fc段的缺失突變以消除其ADE作用。 首先，我們通過特定的Linker將單抗1A1D和2A10的可變區(qū)序列連接，然后分別構建出DVD抗體的輕重鏈，裝入表達載體后，共轉染CHO細胞，經(jīng)篩選鑒定獲得穩(wěn)定表達株，并大量培養(yǎng)純化出足量DVD抗體。 其次，通過Western Blot和ELISA方法檢測DVD抗體的體外結合活性，進一步用間接免疫熒光證實DVD-1A1D-2A10能夠正常同病毒表面的E蛋白結合，并通過分別測定同EDⅠ/Ⅱ和EDⅢ的結合曲線證明四價結構對于兩種可變區(qū)的結合能力沒有影響，而且對于完整E蛋白的親和力比二價抗體更高。體外蝕斑減數(shù)中和試驗證明了我們構建的DVD抗體的中和保護活性比1A1D和2A10的聯(lián)合應用提高了約10倍，比單一單克隆抗體更是提高了30-250倍，尤其實在抗體濃度較低的情況下的保護作用明顯高于普通抗體。另外通過病毒吸附前后的感染抑制實驗證實DVD抗體在吸附前和吸附后的抑制感染效果提高了2-4倍。為了進一步證實DVD抗體的中和保護作用，我們采用乳鼠體內(nèi)保護模型觀察其體內(nèi)保護效果，結果顯示DVD保護的乳鼠存活率約為其他抗體的2-5倍，說明DVD抗體在體內(nèi)同樣具有非常好的保護活性。同時通過體內(nèi)預防和治療實驗，我們證明DVD抗體的保護效果比普通二價抗體同樣提高了2-5倍，而且無論是預防還是治療均有非常好的效果。 最后，通過將介導ADE作用的抗體Fc段缺失突變，使其ADE作用得到消除，，并且經(jīng)鑒定突變對其保護活性沒有影響，從而證明我們成功構建了抗多亞型登革熱病毒的雙特異性高親和力中和抗體。 本研究利用兩株已知分別結合E蛋白DomainⅡ和DomainⅢ的單克隆抗體構建出了一株能夠同時結合這兩個結構域的DVD抗體，證明了該DVD抗體在體內(nèi)外均具有比普通二價抗體更好的中和保護活性，而且同時具有預防和治療的應用價值。并且通過實驗證實由于DVD抗體能夠同時結合兩個功能表位并且同E蛋白具有更高的親和力而使其具有更好的中和保護活性。
[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
本文編號：2282012
[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
本文編號：2282012
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