无感染增强的广谱抗登革病毒中和抗体的构建及机制研究

发布时间：2018-05-11 02:36

本文选题：登革病毒 + E蛋白　；参考：《第二军医大学》2011年博士论文

【摘要】：登革病毒是黄病毒属、有包膜的单正链RNA病毒,根据其包膜的抗原性不同,分为四种血清型(DENV1-4),主要以埃及伊蚊和白纹伊蚊为传播媒介,广泛流行于热带和亚热带地区。临床上主要引起登革热(Dengue Fever,DF)、登革出血热(Dengue hemorrhagic fever,DHF)和登革休克综合征(dengue shock syndrome,DSS)。其中,DF是自限性发热性疾病;而DHF/DSS则引发血管通透性显著增加,导致血浆渗漏引发休克,严重威胁患者的生命。世界上约有半数的人口生活在登革热疫区,每年有超过5000万的感染病例,其中有50万人发展为严重的登革出血热和登革休克综合征。但是,临床上缺乏预防和治疗DENV感染的有效疫苗和药物,主要以对症支持治疗为主。基于临床上针对DENV感染无有效防治手段的现状,治疗性抗体策略获得了广泛关注。大量体内、外实验证明:中和抗体能有效阻止DENV的感染,不仅能发挥病毒感染前的预防作用,而且在病毒感染后的一段时期内,依然能发挥治疗病毒感染的作用。 DENV基因组编码3个结构蛋白(核蛋白C,膜结合蛋白M和包膜蛋白E)和7种非结构蛋白( NS1、NS2a、NS2b、NS3、NS4a、NS4b和NS5)。其中,包膜E蛋白是病毒的主要结构蛋白,位于成熟病毒颗粒表面,排列成平行二聚体结构,构成病毒颗粒的主要突起。E蛋白在空间结构上形成3个不同的结构域(ED1,ED2和ED3),其中,第3个结构域(ED3)介导了病毒与靶细胞的吸附,包含最重要的中和表位。但是,E蛋白上中和性表位知之甚少,同一血清型登革病毒内不同基因型病毒株之间的差异对抗体保护作用的影响也还有待于进一步分析。这些问题都阻碍了登革病毒致病机理的阐明及防治手段的开发。因此,确定登革病毒E蛋白的中和性表位,阐明抗体中和作用的机制,进一步探讨病毒致病性相关的关键氨基酸残基,不仅能促进对其致病机制的了解,也为登革热的防治提供重要信息;无论从DENV发病机理,还是研发疫苗和治疗药物,都具有重要意义。在登革病毒致病机制及其抗体治疗策略的研究过程中,人们发现交叉反应性非中和抗体以及亚中和浓度的抗体能够通过抗体的Fc段产生抗体依赖的感染增强作用(ADE),进而促进疾病的进展,这种现象阻碍了疫苗和单克隆抗体在登革热及其他相关病毒防治中的应用。登革热常发生几种血清型的交叉感染,因此,抗体要应用于治疗,必须能同时对四种血清型病毒均能产生有效的保护。现有的强中和活性的单克隆抗体,多为单个型或只针对少数几个型的单克隆抗体,虽也有多株交叉反应性抗体,但针对不同血清型病毒的保护效价不一;而几种抗体的混合应用又存在成分复杂以及需进行多次临床试验的问题。双可变区抗体技术( Dual-Variable-DomainImmunoglobulin, DVD-Ig)可以通过将2株不同抗体可变区构建于一株抗体分子上并保持原有抗体的亲和力和保护活性,大大简化了多株抗体混合应用带来的麻烦;获得的抗体可进一步经过基因工程改造,将抗体的Fc断改造以消除ADE。在针对四种血清型登革病毒的抗体治疗过程中,通过将只针对几个型的具有强中和活性的单克隆抗体进行重新构建,可获得同时针对四种血清型登革病毒具有广谱中和活性的双可变区抗体。本项研究旨在利用杂交瘤筛选技术筛选获得具有中和活性的单克隆抗体,验证其体内外中和活性,进一步分析登革病毒ED3蛋白的中和表位,明确了抗体结合的关键位点,初步阐明其中和作用的机制;进一步采用基因工程方法构建嵌合抗体及无ADE活性的广谱中和性抗体。研究主要包括四个部分: 一、登革病毒ED3区特异单克隆抗体的制备及功能鉴定我们以重组串联的四种血清型登革病毒ED3蛋白作为免疫原,采用常规方法建立分泌针对登革病毒ED3区的杂交瘤细胞系。最终获得了14株登革病毒ED3区特异单抗(1B12、1E12、1G6、1H8、2F9、2G9、2H12、3A10、3H12、4H10、5C10、6E1、6H7和7G5)。为了对这14株单抗所针对的病毒进行鉴定,我们以四种血清型病毒感染的BHK细胞制备的抗原片对其进行免疫荧光检测。结果证实,这14株杂交瘤细胞所分泌的单抗均特异性针对登革病毒,对其他黄病毒属成员不具有交叉反应;14株单抗对不同血清型登革病毒的交叉反应测定结果显示,1B12、1E12、2H12、3A10、5C10、6H7和7G5是登革1型病毒特异的单抗;1H8和2G9为登革3型病毒特异单抗;1G6为登革4型病毒特异单抗;2F9可交叉结合登革1、3型病毒;3H12和4H10可交叉结合1-3型登革病毒;6E1可交叉结合四种血清型病毒。上述结果表明,我们获得了ED3结构域特异的针对四种血清型登革病毒的单抗,为ED3蛋白的抗原表位分析、登革病毒的诊断以及新型疫苗和抗病毒药物研究提供重要信息和工具。采用蚀斑中和减少试验检测这14株登革病毒特异单抗的体外中和活性,获得一株登革4型病毒特异的中和抗体;其次,对体外有中和活性的单抗采用乳鼠颅内保护模型观察其体内保护作用。结果显示,其中一株特异性针对登革4型病毒的单抗1G6具有较强的体外中和活性和体内保护作用,单一50μg剂量的抗体可以可在登革4型病毒感染4h和24h后分别使90%和30%的小鼠存活,表明它对登革病毒的感染具有一定治疗作用。为了确定中和抗体的作用机制,我们采用吸附前后蚀斑实验测定其在病毒感染周期中发挥作用的方式。结果显示,1G6主要通过抑制登革病毒与靶细胞的吸附来发挥中和作用;当病毒与靶细胞吸附之后,其中和效价大大降低。二、中和表位的筛选与鉴定为了分析登革4型病毒特异性中和抗体的中和表位,我们首先采用噬菌体随机12肽库对单抗1G6抗原表位进行筛选获得了其一致表位序列,并进行了每5个氨基酸的系列缺失突变进一步证实了肽库的筛选结果。序列比对结果显示,该单抗表位所对应的多肽序列位于登革4型病毒E蛋白第3结构域387LTLH390区域。在一级结构上,登革4型病毒E蛋白第388和390位氨基酸与登革1-3型病毒ED3蛋白存在不同;从已有结晶的三维结构上分析,发现,位于第390位的组氨酸,含有一个与其它型登革病毒所不同的咪唑环结构。为了确定单抗1G6与E蛋白结合的关键位点,我们进一步进行了定点突变并以间接ELISA方式测定不同氨基酸位点的突变对抗体结合力的影响。结果显示,当T388G和H390G单独突变时,单抗1G6与ED3蛋白的结合力有显著下降;当二者联合突变时,其结合力完全丧失,表明登革病毒E蛋白T388和H390位是1G6与其结合的关键位点。三、嵌合抗体的构建及其生物学特性为了减弱鼠源抗体的异源反应及下一步构建具有广谱中和活性的双可变区抗体,首先用5’RACE法获得了鼠源抗体1G6的轻重链可变区序列,并全基因合成了具有交叉中和登革1-3型病毒的抗体1A1D-2的轻重链可变区序列。在此基础上,将轻重链可变区分别与人重链(IgG1)和轻链(κ)恒定区连接构成全长人-鼠嵌合抗体后分别装入真核表达载体pcDNA3.1(+),构建了嵌合抗体轻重链的表达载体,共转染CHO细胞,经G418压力筛选获得可稳定分泌人-鼠嵌合抗体的稳转细胞株。实验证明,该抗体具有与亲本鼠源抗体相同的抗原结合活性和体外中和活性,为下一步构建双可变区中和抗体奠定了基础。四、无ADE活性的双可变区抗体的构建及功能鉴定采用基因工程技术,以overlap PCR的方式将单抗1A1D-2和1G6的轻重链可变区序列分别以9个氨基酸的linker进行前后连接,构建成DVD1A1D-1G6-VL、DVD1G6-1A1D-VL和DVD1A1D-1G6-VH、DVD1G6-1A1D-VH片段,分别与人IgG 1的轻重链恒定区连接后装入表达载体表达载体pcDNA3.1(+),共转染CHO细胞,G418筛选获得2种结构的DVD抗体稳转细胞株,无血清大量培养及抗体纯化,体外结合实验证实,DVD1A1D-1G6的结合活性要优于DVD1G6-1A1D。体内外实验证明,DVD1A1D-1G6抗体保持了双价抗体的体内外中和活性,实现了抗病毒谱的扩大,具有抗四种血清型登革病毒活性。进一步将介导ADE效应的Fc段缺失突变后,其ADE活性消失,同时Fc段的突变对其中和活性无明显影响,表明我们成功构建了具有广谱抗登革病毒的无ADE中和抗体。本研究筛选获得了14株登革病毒特异单克隆抗体,其中一株新型登革4型病毒特异性中和抗体,发现了新的E蛋白功能表位,为阐明E蛋白的结构与功能、登革病毒致病和免疫的分子机理奠定了理论基础;进一步构建获得了无ADE活性的可同时中和四种血清型登革病毒的双可变区抗体,为研制抗登革病毒新型抗体药物作出了探索和提供了重要信息。
[Abstract]:Dengue virus (dengue virus) is the genus of the genus yellows, with coated single positive chain RNA virus. According to its membrane antigenicity, it is divided into four serotypes (DENV1-4). It is mainly used as the medium of Aedes aegypti and Aedes albopictus, widely prevalent in the tropics and subtropics. It mainly causes Dengue Fever (DF) and dengue haemorrhagic fever (Dengue hemorrhagic). Fever, DHF) and dengue shock syndrome (dengue shock syndrome, DSS). Among them, DF is a self limiting febrile disease, and DHF/DSS causes a significant increase in vascular permeability, resulting in shock caused by plasma leakage and a serious threat to the lives of patients. About half of the population of the world lives in dengue fever epidemic areas, with more than 50 million cases of infection each year, 500 thousand of them developed into severe dengue hemorrhagic fever and dengue shock syndrome. However, the clinical lack of effective vaccines and drugs for the prevention and treatment of DENV infection is mainly due to symptomatic support therapy. Based on the clinical status of the non effective prevention and treatment of DENV infection, the therapeutic antibody strategy has received extensive attention. The experiments have proved that neutralizing antibodies can effectively prevent DENV infection, not only play a preventive role before the virus infection, but also play a role in the treatment of virus infection in a period after the virus infection.
The DENV genome encodes 3 structural proteins (nuclear protein C, membrane binding protein M and envelope protein E) and 7 non structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5). Among them, the envelope protein is the main structural protein of the virus, located on the surface of the mature virus particles and arranged in a parallel two polymer structure, which constitutes the main protuberance of the virus particles in the air. 3 different domains (ED1, ED2 and ED3) are formed in the inter structure, of which third domains (ED3) mediate the adsorption of the virus and the target cells, including the most important neutralization epitopes.
However, little is known about the neutralization epitopes on the E protein, and the impact of different genotypes in the same serotype dengue virus on the protective effect of the antibody still remains to be further analyzed. These problems have hindered the elucidation of the pathogenesis of dengue virus and the development of the means of prevention and control. Therefore, the neutralization of the E protein of dengue virus is determined. The sexual epitopes, which elucidate the mechanism of antibody neutralization, further explore the key amino acid residues associated with viral pathogenicity, not only promote the understanding of its pathogenesis, but also provide important information for the prevention and control of dengue fever. It is of great significance in the pathogenesis of DENV, and for the development of vaccines and treatment drugs.
In the study of the pathogenesis of dengue virus and its antibody therapy strategy, it is found that cross reactive non neutralizing antibodies and subneutralizing antibodies can produce antibody dependent infection enhancement (ADE) through the Fc segment of the antibody, thereby promoting the progression of the disease. This phenomenon hinders the vaccine and monoclonal antibodies in dengue fever. And other related virus prevention and control applications. Dengue fever often occurs several serotypes of cross infection, therefore, the antibody must be used for treatment, must be able to produce effective protection of four serotype viruses. There are multiple cross reactive antibodies, but the protective titers for different serotype viruses are different, and the mixed application of several antibodies has complex components and many clinical trials need to be carried out. Dual-Variable-DomainImmunoglobulin (DVD-Ig) can be used to construct a variable region of 2 different antibodies in one. The antibody molecules of the plant maintain the affinity and protective activity of the original antibody, which greatly simplifies the trouble caused by the mixed application of multiple antibodies. The obtained antibodies can be further transformed by genetic engineering, and the Fc of the antibody is transformed to eliminate ADE. in the treatment of four serotype dengue viruses by targeting only a few types. The monoclonal antibodies with strong neutralization activity were rebuilt to obtain double variable region antibody against four serotype dengue viruses with broad spectrum and activity.
The aim of this study is to screen the neutralization activity of monoclonal antibodies with hybridoma screening technique, to verify the neutralization activity in vivo and in vitro and to further analyze the neutralization epitopes of the ED3 protein of dengue virus, and to clarify the key sites of antibody binding. A broad spectrum neutralizing antibody with no ADE activity is included. The study mainly consists of four parts.
Preparation and functional identification of specific monoclonal antibodies against dengue virus ED3 region
The hybridoma cell lines secreted against dengue virus (dengue virus) ED3 region were established with four serotypes of serotype dengue virus ED3 protein as immunogen, and 14 dengue virus ED3 specific monoclonal antibodies (1B12,1E12,1G6,1H8,2F9,2G9,2H12,3A10,3H12,4H10,5C10,6E1,6H7 and 7G5) were finally obtained.
In order to identify the virus targeted by these 14 monoclonal antibodies, we detected the immunofluorescence of the antigen prepared by the BHK cells infected by four serotype viruses. The results showed that the monoclonal antibodies secreted by these 14 hybridoma cells were specific against dengue virus and did not cross reaction to other members of the genus YhV; 14 monoclonal antibodies were against the virus. The cross reaction determination of different serotype dengue virus showed that 1B12,1E12,2H12,3A10,5C10,6H7 and 7G5 were specific monoclonal antibodies of dengue type 1 virus; 1H8 and 2G9 were dengue type 3 specific monoclonal antibodies; 1G6 was dengue type 4 virus specific monoclonal antibody; 2F9 could be crossed with dengue virus 1,3 virus; 3H12 and 4H10 can be cross combined with type 1-3 dengue virus; 6E1 intersecting These results suggest that we have obtained ED3 domain specific monoclonal antibodies against four serotype dengue viruses, which provide important information and tools for the antigen epitope analysis of ED3 protein, the diagnosis of dengue virus, and the research of new vaccines and antiviral drugs.
The neutralization activity of the 14 dengue virus specific monoclonal antibodies was detected by plaque neutralization and reduction test, and a specific neutralizing antibody of dengue type 4 virus was obtained. Secondly, the protective effect of the neutralization activity in vitro on the internal protection model of the milk rat was observed. The results showed that one of them was specific against dengue type 4 virus. The monoclonal antibody 1G6 has strong in vitro neutralization activity and in vivo protective effect. A single 50 g dose of antibody can survive dengue type 4 virus infection of 4H and 24h, respectively. It shows that it has a certain therapeutic effect on the infection of dengue virus.
In order to determine the mechanism of neutralizing antibody, we used the plaque assay before and after adsorption to determine its role in the virus infection cycle. The results showed that 1G6 played a neutralization effect mainly by inhibiting the adsorption of dengue virus and target cells, and when the virus was adsorbed with the target cells, the potency of the virus was greatly reduced.
Two, screening and identification of neutralization epitopes
In order to analyze the neutralization epitopes of dengue type 4 virus specific neutralizing antibodies, we first screened the epitopes of mAb 1G6 epitopes by using phage random 12 peptide library, and carried out a series of deletion mutations for every 5 amino acids to further confirm the screening results of the peptide library. Sequence alignment results showed that the monoclonal antibody table was a monoclonal antibody table. The corresponding polypeptide sequence is located in the 387LTLH390 region of the dengue type 4 virus E protein third domain. In the first order structure, the dengue type 4 virus E protein 388th and 390th amino acids are different from the dengue type 1-3 virus ED3 protein; from the three dimensional structure of the existing crystallization, it is found that the 390th bits of histidine, which is located in one of the other types, is found. The structure of the imidazole ring of the leather virus.
In order to determine the key site of the combination of monoclonal antibody 1G6 and E protein, we further carried out site directed mutagenesis and measured the effect of mutation of different amino acid sites on antibody binding power in indirect ELISA. The results showed that when T388G and H390G were mutated separately, the binding power of the monoclonal antibody 1G6 and the ED3 protein decreased significantly; when the two combined mutation, The complete loss of binding capacity indicates that the T388 and H390 sites of dengue virus E protein are the key sites for 1G6 binding.
Three, construction of chimeric antibody and its biological characteristics
In order to weaken the heterologous reaction of mouse antibody and construct a double variable region antibody with broad-spectrum and neutralization activity in the next step, the light heavy chain variable region sequence of mouse source antibody 1G6 was first obtained by 5 'RACE method, and the whole gene was synthesized with light and heavy chain variable region sequences with the antibody 1A1D-2 of the cross neutralization and dengue type 1-3 virus. The heavy chain variable regions were connected to the constant region of human heavy chain (IgG1) and light chain (kappa) to form a full-length human mouse chimeric antibody, respectively, to be loaded into the eukaryotic expression vector pcDNA3.1 (+) respectively. The expression vector of the heavy chain of chimeric antibody was constructed, and the CHO cells were transfected together. The stable secreting human mouse chimeric antibody was obtained by G418 pressure screening. The experiment proved that The antibody has the same antigen binding activity and in vitro neutralization activity as the parent mouse antibody, which lays the foundation for the construction of neutralizing antibodies in two variable regions.
Four, construction and functional identification of double variable region antibody without ADE activity.
Using gene engineering technology, the light and heavy chain variable region sequences of mAb 1A1D-2 and 1G6 were connected with 9 amino acid linker, respectively, and constructed into DVD1A1D-1G6-VL, DVD1G6-1A1D-VL and DVD1A1D-1G6-VH, DVD1G6-1A1D-VH fragments. They were connected to the light and heavy chain constant region of the human IgG 1 and loaded into the expression vector. The body pcDNA3.1 (+), CO transfected CHO cells, G418 screened to obtain 2 kinds of DVD antibody stable cell lines, no serum-free culture and antibody purification. In vitro binding experiment proved that the binding activity of DVD1A1D-1G6 was better than that of DVD1G6-1A1D. in vivo and in vitro. The DVD1A1D-1G6 antibody held the neutralization activity of the bivalent antibody in vivo and in vivo, and realized the resistance to disease. The activity of the four serotype dengue virus has been expanded, and the ADE activity of the Fc segment, which mediates the deletion of the ADE effect, disappears, and the mutation of the Fc segment has no obvious effect on the activity and activity, indicating that we successfully constructed a broad-spectrum anti dengue virus without ADE neutralization antibody.
In this study, we screened 14 dengue specific monoclonal antibodies, one of which was a new type of dengue type 4 virus specific neutralizing antibody, and found a new functional epitope of E protein. It laid the theoretical basis for elucidating the structure and function of E protein and the molecular mechanism of dengue virus disease and immunization; further construction obtained the same activity without ADE. The double variable region antibodies of four dengue viruses were neutralized and neutralized, which provided important information for developing anti dengue virus new 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】：2011
【分类号】：R392

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