新型抗人肠道病毒71型单克隆抗体中和保护作用的结构基础及机制

发布时间：2018-01-22 05:00

本文关键词： 人肠道病毒71型中和保护抗体空间表位冷冻电镜机制　出处：《第二军医大学》2016年博士论文　论文类型：学位论文

【摘要】：人肠道病毒71型(enterovirus 71)属于小RNA病毒科肠道病毒属,是手足口病最常见的病原体。自1969年被发现以来,EV71已经在全世界范围内,尤其是亚太地区引起了数十次手足口病的大流行,造成了数千万人感染,数万患者产生严重的并发症。我国是手足口病疫情的高发区,2009年以来,该疾病的发病人数和死亡人数连续7年位居丙类传染病之首。尽管现在EV71的疫苗完成三期临床试验准备上市,但是对于重症患者,目前尚无有效的治疗策略,因此研制抗病毒的特效药物迫在眉睫。EV71病毒颗粒是一个直径约300埃的正二十面体结构,成熟病毒衣壳由VP1、VP2、VP3和VP4组成,其中VP2、VP3和VP1的大部分区域位于病毒衣壳外部,VP4和VP1的N端位于衣壳的内部。研究表明,在脱衣壳的过程中,病毒构象会发生改变,5次轴处附近的“峡谷”发生坍塌形成小孔,疏水口袋内的鞘氨醇分子释放;2次轴处的VP2的反向平行α螺旋对会像两侧平移,形成直径超过9埃的“孔洞”,埋藏在2次轴正下方的VP1 N端会迁移到病毒外部,参与核酸释放通道的形成。因此筛选针对不同结合表位的单克隆抗体有助于进一步阐述病毒脱衣壳的生物学机制。本课题采用灭活病毒和重组衣壳蛋白分别免疫Balb/C小鼠,运用经典的杂交瘤筛选技术,获得了15株可以特异性结合EV71病毒的单克隆抗体,其中包括6株中和保护抗体。动物保护实验证明,在10ug/g乳鼠体重抗体的使用剂量下,6G5、12B6、2B6、2G12的保护效率均能达到100%,在2ug/g的抗体剂量下,6G5的保护率仍能维持100%,12B6也能达到80%的保护,高于目前文献所报道的中和保护性单抗。针对目前EV71可以引起无菌性脑膜炎(asepic meningitis)、脑炎(encephalitis)、脑脊髓炎(encephalomyelitis)和急性弛缓性麻痹(acute flaccid paralysis,AFP)等严重的神经系统并发症,我们同时也在神经细胞上测试了15株单克隆抗体的中和保护效果,发现与人横纹肌瘤细胞相比,在相同滴度病毒的攻击下,2B6的保护效果有了大幅度的提高。究其原因,推测是因为EV71在肌肉组织和神经系统不同的入侵机制所导致。根据体内外保护功能和识别表位的异同可以将中和保护抗体分为3类进行研究,第一类2B6、2G12可以结合VP1-GHloop上的215-KQEKD-219,第二类7G6、12H3识别VP1 N端的4-VADVI-8,第三类6G5、12B6为空间表位,为鉴定6G5所识别表位,我们运用冷冻电镜技术,解析6G5Fab与三种不同病毒生理周期的复合物结构,包括6G5Fab-天然成熟病毒,6G5Fab-空心病毒和6G5Fab-皱缩的空心病毒,分辨率分别达到了5.6埃、4.9埃和5.0埃。6G5识别位点位于病毒颗粒的2次轴和3次轴之间,分布在VP2和VP3的交汇处,包括VP2上135-139位的LDTKL,223-226位的GADG,296-298位的GAT和VP3上73-81位的VSAQAGKGE,144-147位的KDRA,209-212位的PNTA。这些构成了一个由“凹槽”“把手”和“峡谷”组成的复杂的结合区域。通过突变6G5可变区上与病毒表位相互作用的关键氨基酸,发现组成该空间表位的6个功能域均参与了6G5与病毒的结合。随后我们深入研究了三类抗体的中和保护机制,发现2B6的结合通过降低VP1-GHloop的柔性来稳定5次轴附近“峡谷”底部的结构,从而抑制病毒的构象变化,影响病毒的脱颗粒进程;7G6可以阻碍病毒核酸通道的形成,抑制病毒核酸的释放;最有趣的是6G5的保护机制,它可以欺骗病毒,发挥“类受体”的功能,提前介导细胞的脱衣壳进程,诱导病毒基因组的释放,并且抑制135S脱衣壳中间体的形成。基于以上研究成果我们认为6G5的结合位点是一个全新的中和表位,其保护机制为抗病毒的治疗策略带来了全新的思路,本研究成果也有助于深入理解抗EV71病毒侵染的中和保护机制,并为抗体药物研发打下了坚实的基础。
[Abstract]:Human enterovirus 71 (enterovirus 71) belongs to a small RNA virus, enterovirus, is the most common pathogen of HFMD. Since 1969 it is found that EV71 has been around the world, especially the Asia Pacific region has attracted dozens of HFMD pandemic, resulting in tens of millions of people have a serious infection. The complications of tens of thousands of patients. And China is the epidemic of HFMD incidence area, since 2009, the incidence of the disease and the death toll for 7 consecutive years ranked first in class C infectious disease. Although the vaccine EV71 completed phase three clinical trials for listing, but in severe cases, there is no effective treatment strategies, therefore to develop powerful antiviral drugs of imminent.EV71 virus particles is a diameter of about 300 angstrom is twenty structure, mature capsid by VP1, VP2, VP3 and VP4, VP2, VP3 and VP1 of the region a In the viral capsid external, internal VP4 and VP1 N terminal is in the capsid. The results show that in the process of uncoating of the virus conformation changes, 5 axis near the "Canyon" collapsed to form small molecules within the hydrophobic pocket, sphingosine release; the reverse VP2 2 axis parallel alpha the spiral like both sides of pan, formed more than 9 in diameter of the "hole", buried in the VP1 N 2 axis just below the end of the virus will migrate to the outside, in nucleic acid release channel. So screening for biological mechanism of different binding epitope of the monoclonal antibody is helpful to further elaborate the virus uncoating. The subject of inactivated virus and recombinant capsid protein Balb/C mice were immunized using classical hybridoma screening technique, obtained 15 strains of monoclonal antibody could bind to the EV71 virus, including 6 strains of neutralizing antibody of animal protection. Experiments show that in the dose of 10ug/g rat weight antibody, protection efficiency of 6G5,12B6,2B6,2G12 can reach 100%, the antibody dose of 2ug/g, 6G5 and the protection rate could still maintain 100%, 12B6 can achieve 80% protection, higher than the current literature and protective monoclonal antibody. The EV71 can cause sterility meningitis, encephalitis (Asepic meningitis) (encephalitis), encephalomyelitis (encephalomyelitis) and acute flaccid paralysis (acute flaccid, paralysis, AFP) and severe neurological complications, and protective effect on nerve cells we also tested 15 strains of monoclonal antibodies, compared with human rhabdomyoma cells in the same titer of virus attacks, the protective effect of 2B6 has been greatly improved. The reason is that because the EV71 resulted in the invasion mechanism of muscle tissue and the nervous system is not the same. According to the in vivo protection function and the similarities and differences between epitopes can be divided into the study and protection of antibody of 3 kinds, the first kind of 2B6,2G12 can be combined with VP1-GHloop 215-KQEKD-219, second 7G6,12H3 VP1 N recognition terminal 4-VADVI-8 third class 6G5,12B6 space for the identification of epitopes, 6G5 epitopes, we use electron cryomicroscopy composite structure, analysis of 6G5Fab and three different kinds of viruses of the menstrual cycle, including 6G5Fab- natural mature virus, 6G5Fab- virus and 6G5Fab- virus hollow hollow shrunken, the resolution reached 5.6, between 4.9 and 5.6G5 recognition sites located on viral particles 2 axis and 3 axis, at the intersection of VP2 distribution and VP3, including VP2 135-139 of the LDTKL, 223-226 GADG, 296-298 GAT and VP3 73-81 a bit VSAQAGKGE, 144-147 KDRA, 209-212 PNTA. which consists of a groove"" Handle complex binding region "and" Canyon "composition. The key amino acid epitope interaction with virus by mutation of 6G5 variable region on the combination of the 6 functional domains found the spatial epitopes were involved in 6G5 and virus. And then we deeply study the protective mechanism of three kinds of antibodies, found structure the combination of 2B6 by reducing the flexibility of VP1-GHloop to stabilize near 5 axis at the bottom of the canyon, conformational changes which inhibit the virus, the virus removal effect of particle formation process; 7G6 can block the viral nucleic acid channel, inhibiting viral nucleic acid release; the most interesting is the protective mechanism of 6G5, it can deceive the virus. Play a" receptor "function, uncoating process advance mediated cell, induced by the viral genome and inhibition of 135S release, the formation of uncoating intermediates. The above results we believe that the combination of 6G5 based on The site is a new neutralizing epitope. Its protection mechanism has brought a whole new train of thought for the strategy of antiviral therapy. This research achievement is also helpful to understand the neutralization protection mechanism of anti EV71 virus infection, and lay a solid foundation for the development of antibody drugs.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392

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