【作者】 李洁；

【导师】 车小燕；

【作者基本信息】 南方医科大学 ， 临床检验诊断学， 2013， 博士

【摘要】 登革病毒(Dengue Virus, DENV)是一种通过蚊子(以埃及伊蚊和白纹伊蚊为主)传播的病毒,属于黄病毒属的黄病毒科。DENV含有四个血清型：DENV1, DENV2, DENV3及DENV4。各种血清型的DENV之间存在60-70%的同源序列。人类感染了DENV的其中任何一种血清型后将引发从轻度自限性登革热(Dengue Fever, DF)到重度甚至死亡的登革出血热(Dengue Hemorrhagic Fever, DHF)及登革休克综合征(Dengue Shock Syndrome, DSS)。目前,对于登革热的研究已经持续进行了60多年,仍然没有安全有效的疫苗及治疗药物得以认证使用,因而近年来登革热已逐渐成为影响全球热带及亚热带地区的主要公共卫生问题。每年世界上大约有5000万-1亿人感染DENV,其中大约50万人会出现严重的并发症从而引发2.5万人死亡。机体初次感染任一血清型的DENV所诱发的抗体能够中和同一血清型的再次感染,但是当机体再次感染其他三种不同的血清型时,此异型中和抗体会促进Fcγ受体阳性细胞内病毒的摄入及复制,引发抗体依赖增强作用(antibody-dependent enhancement, ADE),从而诱发登革热严重的并发症如DHF/DSS等。ADE作用的存在是登革热疫苗研发的主要障碍。因此,如果能够对抗体诱导中和作用的机制及抗原位点有了全面深入的理解,对于疫苗的研发策略以及临床试验中疫苗效果及安全性的评估有着非常重要的意义。DENV是一种有包膜的单股正链RNA病毒,基因组长度大约11kb,编码3个结构蛋白和7个非结构蛋白。三种结构蛋白分别是衣壳蛋白(capsid protein, C),膜蛋白(membrane protein, M)和包膜蛋白(envelope protein, E)。7种非结构蛋白分别为NS1-NS2a-NS2b-NS3-NS4a-NS4b-NS5。E蛋白是成熟DENV颗粒表面的主要结构蛋白,作为宿主体液免疫应答的主要抗原靶标,在受体的结合及病毒与细胞膜融合中起着关键性的作用。在病毒进入宿主细胞的过程中,内涵图环境的酸性化触发了病毒表面的E蛋白二聚体发生构象变化成为三聚体,该变化诱发了病毒与宿主细胞膜的融合,最终使得DENV的RNA基因组释放进入细胞质并启动了病毒的感染。目前人们已经通过结晶学(Crystallography)技术对虫媒病毒及蜱传脑炎的E蛋白进行了结构学分析,病毒颗粒表面的E蛋白含有3个结构区：位于中心的Ⅰ区(EDⅠ)；含有融合环(fusion peptide, FP)的延展Ⅱ区(EDⅡ)；以及呈免疫球蛋白样折叠的含有10个β-strands (A-G及AxCxDx)的EDⅢ。其中EDⅢ作为一个独立暴露于病毒表面的区域,成为了制备保护性单克隆抗体的重要抗原靶标,相关研究亦表明EDⅢ含有非常重要的中和抗原表位。在本实验室的前期工作中,我们用杂交瘤技术制备了一组抗DENV1-4EDⅢ的单抗,用酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)、免疫荧光试验(immunofluorescence assay, IFA)和蛋白印迹实验(western blot,WB)对这些单抗的免疫反应性进行了分析,在此基础上,借助已经建立的中和实验方法学enzyme-linked immunospot based micro-neutralization test (ELISPOT-MNT)对单抗的中和活性进行检测,其中2株具备针对DENV1-4较高交叉中和活性的单抗3E31及2D73被选用进行下一步的功能和结构学分析以期能够对这2株中和抗体的中和作用机制进行全面深入的了解。由此,本研究的目的主要有三个,一、寻求一种能够精确滴定DENV的新方法为后续的实验做好准备；二、通过一些功能实验对交叉中和抗体进行分析；三、借助结晶学技术,从结构上分析2株交叉中和抗体的中和作用机制,为登革热疫苗及治疗性药物的研发提供帮助。由此,本研究分为以下三个方面的内容：第一部分：建立基于ELISA的TCID50方法学(TCID50-ELISA)滴定DENV在对于DENV的研究中,常常因为不能准确地滴定DENV而受到了阻碍,目前人们建立的DENV滴定方法包含噬斑实验(plaque assay, PA),半数组织感染量测定(tissue cultureinfectious dose-50assay, TCID50),荧光焦点实验以及基于免疫荧光的荧光激活细胞分选术(FACS)。作为DENV滴定的金标准,PA及TCID50方法均受到了病毒株的传代及细胞系的种类的限制,很多DENV临床分离株并不能在单层细胞上形成噬斑或者肉眼可见的细胞病变效应(cytopathic effect, CPE),影响了滴定结果的判断。另外,这两种方法均需要实验者每天借助显微镜进行人工检测,耗时耗力。而相比之下荧光焦点实验及FACS能够更精确快速地进行病毒滴定,缺点是该两种方法需要具备丰富经验的实验者及较好的实验室条件。因而,在我们的研究的最初,为了后续实验的顺利进行,试图建立一种全新的DENV滴定方法学以克服传统方法的缺陷。NS1蛋白是DENV的一种多功能糖蛋白,也是DENV复制的主要标志,其主要以分泌及膜表达两种方式存在,其中分泌的NS1被认为和上清中DENV的滴度相关。本实验室在前期的研究中建立了一种NS1抗原捕获ELISA方法,在本研究中,我们在传统的TCID50方法学的基础上,借助已经建立的NS1抗原捕获ELISA检测病毒感染上清中的NS1蛋白,以替代传统方法学中的肉眼观察CPE,经过分析比较后证实这种TCID50-ELISA方法具备较好的准确性和重复性,且操作简便,能够替代传统的TCID50-CPE及PA方法学进行DENV的滴定。第二部分：四型交叉抗DENV EDⅢ单克隆抗体的功能鉴定在本实验室的前期工作中,借助毕赤酵母真核表达体系表达DENV1-4EDⅢ重组蛋白,随后利用杂交瘤单克隆抗体技术制备了一组抗DENV1-4EDⅢ的单抗,并对该组抗体的血清型、交叉反应性及中和活性进行了鉴定。最终发现其中2株交叉反应性单抗具备针对DENV四个血清型较高的中和活性,命名为3E31及2D73。在本研究中,我们通过一系列相关实验对3E31及2D73的功能进行全面的鉴定。首先我们用表面等离子共振技术(Surface Plasmon Resonance, SPR)对2株抗体与DENV1-4EDIII及DENV1-4E的亲和力进行了进一步的动态分析。SPR不仅可以检测出抗EDⅢ单抗与EDⅢ及E蛋白抗原动态相互作用情况下的平衡解离常数(equilibrium dissociation constant, KD),而且可以得到EDⅢ单抗和抗原的结合常数(association constant,Ka)及解离常数(dissociation constant, Kd),以及表示抗体可及性(accessibility)的单抗活性百分数。另外Ka及Kd值也为下一步抗原抗体共结晶提供了指导意义。结果显示这2株单抗的亲和力很高均达到了nM水平,且针对四个血清型EDⅢ抗原的亲和力无明显差异,结合中和实验的结果,并未发现两者之间存在相互关系。更为重要的是,在膜融合抑制实验中,3E31显示出了抑制融合作用,而2D73却表现出了显著的膜融合增强作用,这一现象到目前为止从未被报道过。最后,我们对这2株交叉中和单抗的ADE活性进行了鉴定,结果显示它们显示出了不同的ADE活性,单抗3E31不会引发针对四个血清型DENV的感染增强作用,而单抗2D73却能够在DENV2, DENV3及DENV4的感染后引发ADE作用。基于以上的功能鉴定结果,我们推测单抗3E31及2D73的中和作用机制可能不同。第三部分：结晶学技术分析四型交叉抗DENV EDⅢ中和抗体的中和作用机制为了对单抗2D73及3E31的中和作用机制进行更深入的探讨,将抗体的Fab段与E蛋白Ⅲ区抗原进行共结晶,获取晶体后通过X-ray衍射后分别获取了2组高分辨率的结晶学数据,分辨率分别为2.2A及2.0A。通过软件对数据进行三维立体结构学分析,最终确定该2株单抗结合的抗原表位。结果显示这2株单抗识别了2个不同但稍有重叠的表位。单抗3E31的抗原表位主要集中于ABloop及E strand。其中形成氢键及盐桥的氨基酸在DENV四个血清型中完全保守,这解释了该单抗具备四型交叉中和能力。单抗2D73识别的表位主要集中于Astrand及G strand,这些氨基酸同样在DENV四个血清型中高度保守。通过与目前已发表的其他相关结构进行比较后发现,3E31识别的表位与单抗2H12相同,而2D73的表位则与单抗4E11、1A1D-2所识别的表位非常接近,人们把这类单抗称为"A strand"单抗。随后,通过对2D73表位及3E31表位在完整DENV颗粒表面的融合前E蛋白二聚体表面暴露情况进行分析,发现3E31表位完全隐藏于病毒颗粒内部,而2D73表位仅有极小部分暴露在外。那么该2株抗体是如何结合完整病毒并进行中和作用的?而当E蛋白二聚体经过构象变化转变为三聚体后,3E31表位仍然隐藏,2D73表位却大部分暴露,该结果与功能研究中的膜融合抑制实验结果相呼应。结合功能实验的结果,我们最终对2株四型交叉中和单抗3E31及2D73的中和作用机制进行了深入明了的阐述,并进一步为抗DENV疫苗及治疗性药物的研发提供了新的思路,同时也为理解ADE的发生机制提供了帮助。小结通过以上三部分的研究结果发现,本研究的具有以下三个创新之处,其中包括：一、利用我们建立的DENV特异性NSl抗原捕获ELISA,建立了一种新的基于传统TCID50测定的TCID50-ELISA方法学,在6天时间内对DENV1-4进行精确的滴定。TCID50-ELISA方法不仅显示出了与传统的噬斑实验及TCID50结果的一致性,还具备较好的重复性。原因在于我们通过检测NS1蛋白替代CPE的观察,从而排除了不同操作者及实验室带来的主观差异。另外,TCID50-ELISA方法还克服了传统的噬斑实验及TCID50-CPE方法学的缺点,能够同时运用于C6/36, Vero E6, BHK-21及Vero cells等不同的敏感细胞株,且不受细胞株状态的影响。另外,TCID50-ELISA方法亦能够对临床分离株进行精确的滴定。基于很好的准确度和重复性,TCID50-ELISA可以替代传统的方法对DENV进行滴定,对登革热的研究起到了促进作用。二、在本研究中,我们通过一些功能实验对2株抗EDⅢ交叉反应中和抗体3E31及2D73进行了鉴定。2株单抗均能中和DENV四个血清型的感染,且单抗与DENV结合的亲和力及中和能力呈温度依赖性。这两株单抗在膜融合抑制实验及ADE实验中均表现出了不同的作用,提示二者的中和作用机制可能并不相同。本研究发现抗病毒中和抗体2D73能够在病毒与细胞膜融合过程中起到增强作用,据我们所知,过去并未出现相关报道。这一新发现为后续人们对抗病毒中和抗体的功能研究提供了新思路。另外,研究发现中和单抗3E31不具备ADE活性,提示此抗体具备成为免疫治疗性抗体的潜在可能性。三、为了对中和单抗3E31及2D73的中和作用机制的结构学基础进行研究,本研究中,我们通过结晶学技术将单抗的Fab段与EDⅢ蛋白进行抗原抗体的共结晶。通过X-ray对晶体进行衍射后,收集高分辨率的数据进行三维立体晶体结构的数据分析。根据最终解析的抗原抗体结合的晶体结构,我们对单抗3E31和2D73所识别的抗原表位有了清晰立体的了解。随后,我们通过模型模拟分析这2个表位分别在融合前E蛋白二聚体全长及融合后三聚体的暴露程度,以及单抗在等病毒生命周期内的不同形态下(未成熟及成熟颗粒)的结合情况。通过以上的结构分析,结合前期的功能研究,我们对单抗3E31和2D73的中和作用机制及ADE发生机制进行了深入的探讨,弥补了目前人们对这两方面理解的不足,对登革热研究中有效疫苗的研发和评估非常关键。更多还原

【Abstract】 Dengue virus (DENV) is a member of the family Flaviviridae, genus Flavivirus, which is closely related to other important human pathogens such as yellow fever (YF), West Nile (WN), Japanese encephalitis (JE) and tick-borne encephalitis (TBE). There are four circulating serotypes of dengue (DENV1, DENV2, DENV3, DENV4) that share approximately60-70%sequence identity. Human infection with each serotype of DENV may result in a spectrum of clinical disease, ranging from an acute, debilitating, self-limited febrile illness (DF) to a life-threatening hemorrhagic (DHF) and capillary leak syndrome (DSS). Worldwide, an estimated50million infections with dengue occur annually, with approximately500,000cases of severe dengue and20,000deaths. Considerable research efforts for over60years have sought to develop an effective vaccine strategy, however neither effective nor antiviral treatment is approved for human use, and only supportive therapy is available. Infection results in life-long protective immunity against the serotype responsible, however risk of severe disease is increased in the case of secondary infection with a heterologous serotype in a process known as antibody dependent enhancement (ADE) of disease. ADE is thought to occur due to the presence of weakly neutralizing cross-reactive antibodies from a primary response to one serotype, which dominate the human immune response and facilitate viral entry into Fcγ-receptor-positive cells during a secondary infection with a different serotype. ADE is thought to be the major obstacle for the development of dengue vaccine. A clearer understanding of the mechanisms of antibody-mediated neutralization and the key antigenic sites the antibodies recognizewillassist in the design of new vaccine strategies and in assessing the efficacy and safety of those already in clinical trials.DENV is an enveloped virus with a single-stranded, positive-sense RNA genome that encodes three structural proteins (C, prM/M, and E) and seven non-structural proteins (NS1-NS2a-NS2b-NS3-NS4a-NS4b-NS5). The E protein is the major component of the mature virion surface and along with prM, found on immature particles, is the primary antigen targeted by the host’s antibody response. During cell entry, the conformational change in the surface-arrayed dimeric E subunits that drives virus and host-cell membrane fusion is triggered by the low pH of endosomal compartments, allowing release of the RNA genome into the cytoplasm to initiate infection. The virion surface ectodomain (sE) comprises three structural domains, a centrally-located domain I (EDI), an extended EDII containing the fusion loop (fusion peptide, FP), and EDⅢ with an immunoglobulin-like fold comprising ten β-strands (A-G and AXCXDX), which is exposed at the surface of the viral particle, forms an attractive antigen for raising protective monoclonal antibodies (MAb).In our previous work, a panel of monoclonal antibodies (MAbs) raised against dengue envelope protein domain III were produced by hybridoma technique and characterized by using enzyme linked immunosorbent assay (ELISA), immunofluorescence assay (IFA) and western blot (WB). Then the neutralizing activity of these MAbs were determined by enzyme-linked immunospot based micro-neutralization test (ELISPOT-MNT), which is established previously. In this study, two MAbs3E31and2D73that cross-react with the four serotypes of dengue and neutralize all four dengue serotypes have been characterized by further analysis of function and structure.Thus, there are three major objectives for our current study:first of all, to develop a new method for accurate titration of dengue virus; secondly, to characterize the anti-EDIII cross-reactive neutralizing MAbs by functional assays; finally, to obtain structural insight into the neutralization mechanisms of our MAbs by X-ray crystallography and thereby to lay the foundation for the development of dengue vaccine and therapeutic drugs.Thereby, this study is divided into three parts:I. Development of ELISA-based TCID50assay (TCID50-ELISA) for titrating DENVResearch on DENV is often hindered by inefficient and inaccurate or costly viral titration methods. Hence, a simple and efficient assay for accurate titration of DENV in infected cultures would greatly facilitate dengue research, vaccine development, and laboratory detection. To date, a variety of methods for titrating DENV have been developed, including classical assays, the plaque assay and the tissue culture infectious dose-50assay (TCID50), and immunofluorescence-based assays such as fluorescence-activated cell sorting (FACS) assay and fluorescent focus assay. As a standard method for titrating DENV, however, the plaque or TCID50assays have their disadvantages, as they are limited to some strains and passages of the virus, and a few cell lines. Most primary clinical isolates do not form clear plaques or have a visible cytopathic effect (CPE) on cell monolayers. Furthermore, both of these assays require manual microscope examination daily, which is time consuming and labour intensive. FACS and fluorescent focus assays can provide more rapid and accurate quantitation of DENV than the traditional plaque assay. However, each of these techniques requires experienced technicians and sophisticated laboratories, hindering its application in most laboratories lacking sophisticated equipment. Therefore, at the beginning of our research, we set to establish a new method for titrating dengue virus to overcome the disadvantages of classical methods. As we know, nonstructural protein1(NS1), a multifunctional glycoprotein in dengue virus, is highly conserved for all serotypes of DENV and is strongly immunogenic. Some of the NS1protein is expressed as a soluble secreted form, which has been implicated to contribute to dengue viral propagation and the amount secreted is closely related to dengue viral titer. In our previous study, we established a dengue NS1antigen capture enzyme-linked immunosorbent assay (ELISA). In the present study, a novel TCID50assay was developed, which employs this dengue NS1antigen capture ELISA instead of observation of CPE in the classical assay. The novel TCID50-ELISA method described here provides a more reliable and more accurate alternative compared to the plaque assay and TCID50-CPE for titration of dengue virus.Ⅱ. Function analysis of cross-reactive MAbs against envelope protein domain Ⅲ of dengue virusIn our previous work, we expressed DENV1-4EDⅢ recombinant protein by using yeast system. Then, a panel of anti-EDⅢ MAbs were produced from BALB/c mice immunized with recombinant EDⅢ from DENV1-4. Furthermore, the serotype specificity and cross-reactivity of these MAbs were determined by indirect ELISA and indirect immunofluorescent assay (IFA). As the aim of this study is to develop antibodies against all four DENV serotypes,3E31and2D73were selected for further studies because they were found to neutralize the infection of all four dengue serotypes. In this study, MAb3E31and2D73were further characterized by a serial of functional assays such as surface plasmon resonance (SPR), membrane fusion inhibition assay, temperature-dependent assay and ADE assay. SPR can not only provide the affinity data (equilibrium dissociation constant, KD), but also with the association constant (Ka) and dissociation constant (Kd) of the binding of EDⅢ MAbs to EDⅢ protein, as well as the percentage activitiy, which represent the accessibility of the protein. In addition, the SPR data also provide insight into the next co-crystallization of antibody and antigen. The SPR results revealed that the affinity of two MAbs could reach nM and there is no significant difference between the binding affinities for DENV1, DENV2, DENV3and DENV4. Additionally, the data did not show any relationship with the neutralization capacity. For the fusion assay, to our surprise, while3E31inhibited fusion,2D73enhanced fusion in a dose-dependent manner. To our knowledge, this is the first time fusion enhancing antibodies have been described for any virus. Finally, the ADE activity of MAb3E31and2D73were eveluated. We have observed that3E31does not induce ADE in Fcy-receptor-expressing cells. By contrast, binding by2D73led to ADE in DENV2, DENV3and DENV4but not DENV1. On the basis of the functional analysis data, we proposed that the neutralization mechanism of MAb3E31and MAb2D73might be different.Ⅲ. Structure analysis of the neutralization mechanism of anti-EDⅢ cross-reactive MAbs using X-ray crystallography.To understand the structural basis of neutralization by our mAbs, crystal structures of the complexes between recombinant DENV4EDⅢ and Fab fragments from3E31and2D73were determined at2.2A and2.0A resolution, respectively. The crystal structures were analyzed by software and the3E31epitope and2D73epitope were finally defined. The MAbs recognize two distinct, slightly overlapping epitopes on EDⅢ. For3E31, the epitope comprises the AB loop and β-strand E. Residues that form hydrogen bonds with the Fab and are strictly conserved in DENV1-4, consistent with the neutralization assay, which showed that MAb blocked virus infection of all four serotypes. For2D73, the epitope cluster on β-strands A and β-strand G. The key epitope residues are again highly conserved. In addition, antibody:EDⅢ structures that show binding to this region have been reported with the3E31epitope being similar to that recognized by mAb2H12and the2D73epitope being similar to those described for the A-strand-specific mAbs1A1D-2and4E11. Moreover, to investigate the accessibility of these anti-EDIII mAb epitopes on native forms of the E protein, we superimposed the crystal structures of our Fab:EDⅢ complexes onto both dimeric pre-fusion and trimeric post-fusion sE structures, as well as E proteins in the context of immature and mature whole virions. The results revealed that both epitopes are partially hidden in the mature virion dimer, which raises the question of how the MAbs could bind to any of the E-DⅢ epitopes. Location of the epitope on the post-fusion trimer structure suggests that fusion inhibition observed for3E31is most likely due to a steric blockade of E trimer formation. By contrast, the availability of the2D73epitope in the post-fusion trimer suggests that it could remain bound during the conformational transition from dimer to trimer and possibly shifting the equilibrium to trimer formation and this way activate fusion. In combination with the functional data and crystal structures, we finally determine the neutralization mechanisms and reveal stark differences between the two antibodies3E31and2D73, a finding that has far reaching consequences in vaccine and therapeutic drugs development and understanding the role of antibodies in ADE.Summarization:1. Using the DENV group NS1antigen capture ELISA established previously, we developed a novel TCID50-ELISA based on the classical TCID50. With this new method, DENV1-4could be accurately titrated at day6. Virus titers obtained by TCID50-ELISA were comparable to those obtained by the plaque assay and by the traditional TCID50-cytopathic effect (CPE) test (TCID50-CPE), with a better reproducibility. Because the observation of the CPE was replaced by determination of NS1protein, which can eliminate subjective variations between operators and laboratories. Moreover, this TCID50-ELISA assay overcomes the flaws inherent in the plaque assay and the TCID50-CPE assay. For instance, the TCID50-ELISA assay showed a wider application to C6/36, Vero E6, BHK-21, and Vero cells compared with other titration methods. Finally, the TCID50-ELISA assay has been used successfully for clinically DENV isolates, which might not be assessable by other titration methods. Based on its reliability and ease of execution, the TCID50-ELISA test represents a promising assay for titration of DENV, and will facilitate dengue research.2. In this study, some functional approaches were carried out to characterize two cross-reactive neutralizing MAbs raised against DENV EDIII. Both MAbs could block the infection of all four dengue serotypes. Additionally, the affinity and neutralization capacity against dengue viral particle are temperature sensitive. Furthermore, the activity of3E31and2D73in membrane fusion and ADE are totally different, indicating that the neutralization mechanism of them might be different. We found that2D73enhanced the membrane fusion, to our knowledge this is the first time fusion enhancing antibodies have been described for any virus. This new finding provides insight into the functional study of anti-viral neutralization antibodies. Finally, MAb2D73showed no ADE activity, highlighting its potential as immunotherapeutics.3. To understand the structural basis of neutralization by3E31and2D73, crystal structures of the complexes between recombinant DENV4EDIII and Fab fragments from3E31and2D73were determined by crystallography technique. The crystals were diffracted by X-ray diffraction and then two data sets with high resolution were collected. According to the solved3D crystal structures, the epitopes recognized by MAb3E31and2D73were defined accurately. Moreover, we superimposed the crystal structures of our Fab:EDIII complexes onto both dimeric pre-fusion and trimeric post-fusion sE structures, as well as E proteins in the context of immature and mature whole virions to investigate the accessibility of these anti-EDIII mAb epitopes on native forms of the E protein. In conculsion, the results of our combined structural and functional studies uncover two distinct mechanisms of neutralization used by our MAbs and possibly other recently reported MAbs targeting EDIII, and may further people’s understanding of cross-neutralization as well as the structural basis for ADE, critical for the design of effective vaccines in the future.更多还原