Ⅰ型登革病毒E蛋白Ⅲ区抗体的制备、鉴定及禽流感H5N1病毒血凝素基因在昆虫细胞中的表达
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摘要
Ⅰ型登革病毒E蛋白Ⅲ区抗体的制备、鉴定
登革病毒(DENV)属黄病毒属成员,分4个血清型(DENV-1～4),均可引起登革热、登革出血热(dengue hemorrhagic fever,DHF)和登革休克综合征(dengue shock syndrome,DSS)。DENV广泛流行于全世界100多个国家和地区,据WHO估计,目前全球每年约有5千万～1亿人感染DENV。机体在感染了某一型DENV后,会对该型病毒持续产生保护性免疫应答,但是却无法阻止其他型登革病毒的再次感染,反而还会因为抗体的存在,对其他型的病毒起到增强感染的作用,从而导致严重DHF或DSS。这种现象被称为抗体依赖性增强作用(antibody dependent enhancement,ADE),主要认为是由于体内已经存在的抗体与不同型别的病毒交叉结合后,更容易与细胞的Fc受体结合,促进了病毒的感染。
DENV E蛋白是病毒最大的结构蛋白与主要糖蛋白,在登革病毒的致病机理及诱发机体的抗感染免疫中发挥重要的作用,E蛋白包含三个结构域,Ⅰ区(DomainⅠ,DⅠ),Ⅱ区(DomainⅡ,DⅡ)和Ⅲ区(DomainⅢ,DⅢ),其中Ⅲ区是一个相对独立和完整的区域,文献报导,经胰蛋白处理的E蛋白能够分离到完整的Ⅲ区片段。该区是病毒与细胞结合的主要区域,在感染中发挥作用,同时也是中和抗体的主要靶标,并且针对DⅢ的特异性抗体是病毒附着宿主细胞的最强拮抗剂。
由于目前DENV感染尚无有效的防治措施,登革疫苗也难以同时针对四型DENV,所以制备具有中和活性的抗体对深入研究DENV抗原表位和疫苗研制有着重要意义。针对这四型DENV的抗体被大致分为型特异性中和抗体,交叉非中和抗体和交叉中和抗体。目前认为交叉非中和抗体和处于亚中和浓度的抗体,均能导致ADE效应的发生。而Yamanaka等人最新的研究认为,在机体补体水平处于正常状态下,中和抗体在体内可以发挥中和作用,但补体水平下降,则表现出增强感染作用。因此,找到仅具有中和作用的抗体和如何避免ADE效应的产生,成为制备保护性抗体的关键。鉴于EDⅢ蛋白在诱发中和抗体中起到的重要作用,我们选取重组DENV-1 EDⅢ蛋白作为免疫原制备小鼠单克隆抗体和兔多抗血清,旨在进一步分析病毒型特异抗体和交叉抗体究竟识别哪些抗原优势表位,以及这些抗体是否具有型特异性中和活性或交叉中和活性。型特异性抗体所识别的优势抗原表位,可用于对登革病毒的感染进行对应的分型诊断,而型特异性的中和抗体所识别的表位,则可用做四价登革疫苗的组分之一;交叉抗体所识别的表位,在疫苗的组分中去除可以防止诱导产生ADE效应,而同时具有四型病毒交叉中和活性的抗体所识别的表位,则有望直接用于研制亚单位疫苗,诱导出同时针对四型登革病毒的保护性抗体;此外,高效价中和单抗的获得还可以用于发展成为治疗性的抗体,用于特异性治疗登革病的急性发作。
本项研究工作在论文中的前三部分中进行论述:
第一部分:DENV-1 EDⅢ单克隆抗体的制备
通过酵母表达系统表达并纯化了四型DENV EDⅢ重组蛋白,使用重组DENV-1 EDⅢ蛋白免疫BALB/c小鼠和新西兰兔,共制备了24株针对该重组蛋白的杂交瘤细胞株和兔免疫血清,Ig亚类鉴定21株为IgG1,2株为IgG2a,1株为IgG2b。24株单抗中,有8株单抗特异性结合DENV-1 EDⅢ蛋白,而另外16株单抗和兔免疫血清则与另外三型登革病毒E蛋白有不同程度的交叉反应。
第二部分:针对DENV-1 EDⅢ单克隆抗体识别位点分析及PRNT中和活性测定
我们在前期获得24株针对DENV-1 EDⅢ蛋白单克隆抗体和一份兔免疫血清的基础上,对单抗所识别的抗原结合区域进行了测定,发现这些抗体至少识别三个抗原结合区域,分别命名为位点Ⅰ、位点Ⅱ和位点Ⅲ。经噬斑减少中和试验(Plaque Reduction Neutralization Test,PRNT)测定,24株单抗腹水有23株具有中和活性。识别位点Ⅰ的13株单抗,不仅与各型DENV EDⅢ抗原有交叉反应,也在不同程度上表现出交叉的中和活性,并且抗体与EDⅢ蛋白的交叉反应与交叉中和活性没有必然联系。这13株交叉单抗中,有4株同时对四型登革病毒均具有中和活性;识别位点Ⅲ的7株抗体,除9E18A8和9F26A1,对DENV-1都表现出了很高的中和活性,PRNT中和效价最高可达1:16384。并且在这7株抗体中,9C23A3,9F26A1和9F3A具有高效价的针对DENV-1的中和活性具有型特异性;免疫兔血清仅对DENV-1和DENV-3具有中和活性,并且24株单抗中也有14株同时对DENV-1和DENV-3表现出中和活性,说明DENV1 EDⅢ和DENV-3 EDⅢ之间存在交叉的可以诱导中和抗体的优势抗原表位,容易引起机体产生交叉保护性抗体。
第三部分:评价NS1抗原捕获ELISA用于测定DENV-1 EDⅢ单克隆抗体的中和活性的可行性
通过抗体中和DENV-1后再感染C6/36细胞,利用NS1抗原捕获ELISA检测细胞培养上清中NS1的水平,从而评价抗体对DENV-1的中和效应。实验结果显示23株对DENV-1具有PRNT中和活性的单抗中,NS1抗原捕获ELISA仅能测出14株具有中和活性,而两种方法均能检测到多抗兔血清有中和活性。NS1抗原捕获ELISA测定出的抗体中和效价远低于PRNT试验测定出的结果;识别位点Ⅲ的那些具有高效价PRNT中和活性的抗体,NS1抗原捕获ELISA也测定出具有较高的中和活性。
综合以上三部分的研究结果,本项研究的结论如下:
1成功制备了24株针对该重组蛋白的杂交瘤细胞株和1份兔免疫血清,8株单抗特异性结合DENV-1 EDⅢ蛋白,而另外16株单抗和兔免疫血清则与另外三型登革病毒蛋白有不同程度的交叉反应。这些抗体的获得,为筛选针对EDⅢ的中和抗体和抗体所对应的优势抗原表位奠定了基础。
2 24株针对DENV-1 EDⅢ单抗,至少识别三个抗原结合区域,位点Ⅰ是四型DENV的交叉抗原识别区域,识别该位点的抗体能够与至少两型以上的DENV EDⅢ结合,同时对DENV还表现出交叉中和活性,交叉抗体的获得对进一步研究ADE效应和研究同时诱导对四型DENV均具有中和作用的交叉抗原表位具有重要意义;位点Ⅲ诱导产生抗体大多仅与DENV-1 EDⅢ特异性结合,同时对DENV-1表现出极高的中和活性,推测该位点在DENV-1感染细胞的过程中具有重要的作用,对于该位点的进一步研究,可以深入理解DENV在感染过程中的机制,而这个位点也有望用作DENV四价疫苗的组分之一,型特异性的中和抗体还可以发展成为特异针对DENV-1的治疗性抗体。
3 DENV-1 EDⅢ免疫产生的兔多抗血清对DENV-3的感染具有交叉保护性,而对DENV-2和DENV-4无保护性。提示使用DENV-1 EDⅢ作为亚单位疫苗,不能对DENV-2和DENV-4起到有效保护。
4 DENV-1 NS1抗原捕获ELISA用于测定抗体的中和活性敏感性较差,无法代替传统的PRNT中和试验;但由于ELISA简单、快速、高通量的特点,可以用于批量筛选一些中和活性强的抗体,作为PRNT试验的补充。
禽流感H5N1病毒血凝素基因在昆虫细胞中的表达
禽流感是由A型流感病毒引起的一种禽类的传染性疾病综合征,该疾病对全世界的家禽市场和社会公共卫生环境都造成了严重的危害。A型流感病毒含8个反义RNA片断,共编码10种蛋白,核衣壳蛋白(Nucleocapsid protein,NP)、基质蛋白(Matrix Protein,M)、血凝素(hemagglutinin,HA)、神经氨酸酶(Neuraminidase,NA)是四种重要的宿主免疫反应的靶蛋白。其中HA是病毒表面最多的糖蛋白,也是变异最大的基因,根据抗原性不同可进一步分为16个亚型(H1～H16);HA与宿主细胞表面受体的唾液酸残基末端的结合,可以调节病毒和细胞膜的融合;HA还是病毒主要的保护性抗原,抗HA的抗体对病毒的感染具有中和作用。由于禽流感病毒的抗原性和致病性很大程度上决定于HA基因的变异情况,所以简单、高效制备具有接近天然性质的血凝素抗原对研究HA功能和活性,制备亚单位疫苗以及研发诊断、治疗性抗体都将具有十分重要意义。本研究选用杆状病毒-昆虫细胞真核表达系统,以流行范围广、致病能力强的H5N1禽流感病毒血凝素H5抗原作为靶标,建立一种稳定、快速并能大量获取具有良好抗原性和生物活性的HA重组蛋白的方法,以期用于HA功能和生物学特性的研究,为进一步获取特异性针对该抗原的抗体,建立起快速、简便、特异性强的诊断方法奠定基础。
本项研究工作在论文中的第四部分中进行论述:
第四部分:禽流感病毒H5N1血凝素基因在昆虫细胞中的表达及鉴定
构建杆状病毒重组质粒Bacmid/H5,转染Sf9昆虫细胞后,利用免疫荧光鉴定重组his-H5蛋白成功表达,通过Ni-NTA亲和树脂纯化后的重组his-H5蛋白纯度达到90%以上,经Western-blot证实该重组蛋白与禽流感病毒H5标准血清产生特异性的免疫反应,并且在血凝试验中显示出1:128的血凝效价。
本项研究结论如下:
采用的杆状病毒-昆虫细胞表达体系成功高效表达并纯化了A型流感病毒重组血凝素his-H5蛋白,Western-blot实验证实该重组蛋白具有良好的抗原性;血凝试验证明该蛋白保留了天然状态下结构,具有良好的血凝活性。该重组蛋白有望用于HA功能和生物学活性的进一步研究,也将为下一步亚单位疫苗、中和抗体以及特异性诊断试剂的研发奠定基础。
Dengue virus is a member of the Flavivirus genus in the Flaviviridae family and has four distinct serotypes(DENV-1,DENV-2,DENV-3 and DENV-4).Infection with any of the four serotypes of DV causes a spectrum of clinical features ranging from asymptomatic infections,undifferentiated fever,and classical dengue fever to life-threatening manifestations such as dengue hemorrhagic fever(DHF) and dengue shock syndrome(DSS).The diease is endemic in more than 100 countries and is found virtually throughout the tropics and cause an estimated 50-100 million illnesses annually,including 250 000-500 000 cases of dengue haemorrhagic fever.After being infected by one serotype dengue virus,an individual develops long-lived immunity to homologous serotypes,but antibodies to one serotype are not sufficient to protect against other serotypes,subsequent heterologous infections may lead to the developing DHF or DSS.This is known as antibody dependent enhancement(ADE) for it is postulated that subneutralizing cross-reactive antibodies may facilitate viral infection through Fc receptor by binding with the virus. Antibodies induced by dengue virus infections can be roughly divided into type-specific neutralizing antibodies,cross-reactive nonneutralizing antibodies,and cross-neutralizing antibodies.It is thought that cross-reactive nonneutralizing antibodies and neutralizing antibodies in sub-neutralizing concentration will both lead to ADE.Yamanaka et al.reported that antibodies showed enhancing activities or neutralizing activities depended on the levels of complement in vitro and complement levels were considered an additional factor involved in the in vivo ADE phenomenon: neutralizing at a normal level of complement,but increaseing infetion at a low level.
Envelope(E) protein is the the major protein on the surface of DENV virions,it can mediate receptor binding and membrane fusion.Antibodies against E protein long-lastingly exist in patients' sera,and most importantly,confers protective immunity.E protein contains three domains:a central domain(DⅠ),a dimerization domain(DⅡ),and an immunoglobulin(Ig)-like domain(DⅢ).It had been found many type- and subtype- specific neutralizing epitopes were mapped to domainⅢ,and antibodies to this domain were the most powerful blockers to virus infection.
At present,however,there is no specific treatment available for DENV infections and no vaccine that is effective against all four DENV serotypes,so preparation of antibodies with neutralizing abilities is meaningful for the study of antigen epitopes and the production of vaccine.For the great importance of EDⅢin inducing neutralizing antibodies,we chose the recombinant DENV-1 EDⅢprotein as the antigen to immunize five BALB/c mice and one New Zealand rabbit for producing monoclonal antibodies and rabbit hyperimmune serum and tried to find out which epitopes were serotype specific and which were cross-reactive.We also tried to find out andibodies recoganized these epitopes,which had serotype specific neutralizing abilities and which had cross-neutralizing abilities.These will give guidelines for designing a vaccine without inducing ADE.
This study consists of three parts,as follows:
Ⅰ.Preparation and characterization of antibodies to DENV-1 EDⅢ
A total of 24 hybridoma cell lines that stably produced MAbs were initially established on the basis of their strong positive reactivity with the rDENV1- EDⅢprotein.The immunoglobulin isotype determinations revealed that the 24 MAbs comprised IgG2a(n=2),IgG2b(n=1),and IgG1(n=21).The serotype specificity of the MAbs was further identified by testing their reactivity to the four rDENV EDⅢproteins by ELISA and the four serotypes DENV in the IFA.Of the 24 MAbs,8 MAbs specifically reacted to DENV1 with no cross-reactivities to the other three DENV serotypes in either the ELISA or the IFA.The remaining 16 MAbs and the rabbit hyperimmune serum had cross-reactivities to the four DENV serotypes in different cross-reactivity patterns.
Ⅱ.To study the DENV-1 EDⅢepitopes recoganized by these antibodies and detect their neutralizing abilities to DENV with Plaque Reduction Neutralization Test.
The distinct binding epitopes of the 24 DENV EDⅢMAbs were determined by competition ELISA using recombinant DENV1-EDⅢas the immobilized antigen. The results showed that the 24 MAbs bound at least to three different epitopes on the DENV-1 EDⅢprotein and 13 MAbs recognized eptiopeⅠshowed a cross-reactivity with other DENV serotypes in ELISA and IFA.Each of the 24 hybridoma cell lines were injected intraperitoneally in BALB/c mice to produce ascitic fluids. Neutralization ability to DENV-1～4 of these ascetic fluids were detected by Plaque Reduction Neutralization Test(PRNT).The results indicated that 23 MAbs had neutralizing activities to DENV,from which 13 cross-reactive MAbs recognized eptiopeⅠalso showed cross-neutralizing abilities to DENV,but there was no certain relationship between the two.Four MAbs of the 13 could neutralize all four dengue-serotypes;The 7 MAbs recoganized epitopeⅢshowed great neutralizing abilities to DENV-1,except 9E18A8 and 9F26A1.Three MAbs from the greatly neutralizing antibodies were serotype-specifc and could be developed as therapeutic antibodies for DENV-1;The rabbit hyperimmune serum neutralizd only DENV-1 and DENV-3,which indicated,that DENV-1 EDⅢand DENV-3 EDⅢshared crossly protective epitopes.
Ⅲ.To evaluate the neutralizing abilities of anti-DENV antibodies with DENV-1 NS1 antigen capture ELISA.
DENV-1 neutralized firstly by antibodies were used to infect C6/36 cell,the DENV-1 NS 1 antigen capture ELISA was used to evaluate the neutralizing abilities of these antibodies by detecting the NS1 secreted from the the infected cell.The results indicated that only 14 MAbs of the 23 neutralizing MAbs detected by PRNT had neutralizing abilities,while the rabbit hyperimmune serum had neutralizing ability detected by both methods.Neutralizing titers detected by NS1 capture ELISA were far lower than that detected by PRNT.Antibodies recoganized epitopeⅢshowed greater neutralizing titers than other antibodie in both methods.
Conclusions:
1.A total of 24 hybridoma cell lines that stably produced MAbs were initially established on the basis of their strong positive reactivities with the rDENV1- EDⅢprotein.Eight MAbs specifically reacted to DENV1 with no cross-reactivities to the other three DENV serotypes in either the ELISA or the IFA.The remaining 16 MAbs and the rabbit hyperimmune serum had cross-reactivities to the four DENV serotypes in different cross-reactivity patterns.These obtained antibodies can be used to study their neutralizing ablilities and are potential tools for studing the mechanism of reduced or enhanced virus infection.
2.The 24 MAbs bound to at least three different epitopes on the DENV-1 EDⅢprotein and MAbs recognized eptiopeⅠshowed a cross-reactivities with other DENV serotypes in ELISA and IFA and cross-neutralizing abilities to DENV in PRNT.This could be used to study common epitopes of DENV,which is meaningful for developing DENV vaccine;Most of the MAbs recoganized epitopeⅢshowed serotype specificities and great neutralizing abilities to DENV-1.It was supposed that epitopeⅢplayed a important role in virus infection and cell binding,so this epitope could be used as one of the potential candidates of tetravalent vaccine.
3.The rabbit hyperimmune serum to DENV-1 EDⅢcould protected cells against DENV-1 and only crossly protected cells from being infected by DENV-3,it indicated that DENV-1 EDⅢwould not generate sufficient protection for DENV-2 and DENV-4 as a subunit vaccine.
4.DENV-1 NS1 antigen capture ELISA was not sensitive enough for evaluating antibodies' neutralizing abilities.But it could be used for screening the antibodies with high neutralizing abilities as a supplement to PRNT.
登革病毒(DENV)属黄病毒属成员,分4个血清型(DENV-1～4),均可引起登革热、登革出血热(dengue hemorrhagic fever,DHF)和登革休克综合征(dengue shock syndrome,DSS)。DENV广泛流行于全世界100多个国家和地区,据WHO估计,目前全球每年约有5千万～1亿人感染DENV。机体在感染了某一型DENV后,会对该型病毒持续产生保护性免疫应答,但是却无法阻止其他型登革病毒的再次感染,反而还会因为抗体的存在,对其他型的病毒起到增强感染的作用,从而导致严重DHF或DSS。这种现象被称为抗体依赖性增强作用(antibody dependent enhancement,ADE),主要认为是由于体内已经存在的抗体与不同型别的病毒交叉结合后,更容易与细胞的Fc受体结合,促进了病毒的感染。
DENV E蛋白是病毒最大的结构蛋白与主要糖蛋白,在登革病毒的致病机理及诱发机体的抗感染免疫中发挥重要的作用,E蛋白包含三个结构域,Ⅰ区(DomainⅠ,DⅠ),Ⅱ区(DomainⅡ,DⅡ)和Ⅲ区(DomainⅢ,DⅢ),其中Ⅲ区是一个相对独立和完整的区域,文献报导,经胰蛋白处理的E蛋白能够分离到完整的Ⅲ区片段。该区是病毒与细胞结合的主要区域,在感染中发挥作用,同时也是中和抗体的主要靶标,并且针对DⅢ的特异性抗体是病毒附着宿主细胞的最强拮抗剂。
由于目前DENV感染尚无有效的防治措施,登革疫苗也难以同时针对四型DENV,所以制备具有中和活性的抗体对深入研究DENV抗原表位和疫苗研制有着重要意义。针对这四型DENV的抗体被大致分为型特异性中和抗体,交叉非中和抗体和交叉中和抗体。目前认为交叉非中和抗体和处于亚中和浓度的抗体,均能导致ADE效应的发生。而Yamanaka等人最新的研究认为,在机体补体水平处于正常状态下,中和抗体在体内可以发挥中和作用,但补体水平下降,则表现出增强感染作用。因此,找到仅具有中和作用的抗体和如何避免ADE效应的产生,成为制备保护性抗体的关键。鉴于EDⅢ蛋白在诱发中和抗体中起到的重要作用,我们选取重组DENV-1 EDⅢ蛋白作为免疫原制备小鼠单克隆抗体和兔多抗血清,旨在进一步分析病毒型特异抗体和交叉抗体究竟识别哪些抗原优势表位,以及这些抗体是否具有型特异性中和活性或交叉中和活性。型特异性抗体所识别的优势抗原表位,可用于对登革病毒的感染进行对应的分型诊断,而型特异性的中和抗体所识别的表位,则可用做四价登革疫苗的组分之一;交叉抗体所识别的表位,在疫苗的组分中去除可以防止诱导产生ADE效应,而同时具有四型病毒交叉中和活性的抗体所识别的表位,则有望直接用于研制亚单位疫苗,诱导出同时针对四型登革病毒的保护性抗体;此外,高效价中和单抗的获得还可以用于发展成为治疗性的抗体,用于特异性治疗登革病的急性发作。
本项研究工作在论文中的前三部分中进行论述:
第一部分:DENV-1 EDⅢ单克隆抗体的制备
通过酵母表达系统表达并纯化了四型DENV EDⅢ重组蛋白,使用重组DENV-1 EDⅢ蛋白免疫BALB/c小鼠和新西兰兔,共制备了24株针对该重组蛋白的杂交瘤细胞株和兔免疫血清,Ig亚类鉴定21株为IgG1,2株为IgG2a,1株为IgG2b。24株单抗中,有8株单抗特异性结合DENV-1 EDⅢ蛋白,而另外16株单抗和兔免疫血清则与另外三型登革病毒E蛋白有不同程度的交叉反应。
第二部分:针对DENV-1 EDⅢ单克隆抗体识别位点分析及PRNT中和活性测定
我们在前期获得24株针对DENV-1 EDⅢ蛋白单克隆抗体和一份兔免疫血清的基础上,对单抗所识别的抗原结合区域进行了测定,发现这些抗体至少识别三个抗原结合区域,分别命名为位点Ⅰ、位点Ⅱ和位点Ⅲ。经噬斑减少中和试验(Plaque Reduction Neutralization Test,PRNT)测定,24株单抗腹水有23株具有中和活性。识别位点Ⅰ的13株单抗,不仅与各型DENV EDⅢ抗原有交叉反应,也在不同程度上表现出交叉的中和活性,并且抗体与EDⅢ蛋白的交叉反应与交叉中和活性没有必然联系。这13株交叉单抗中,有4株同时对四型登革病毒均具有中和活性;识别位点Ⅲ的7株抗体,除9E18A8和9F26A1,对DENV-1都表现出了很高的中和活性,PRNT中和效价最高可达1:16384。并且在这7株抗体中,9C23A3,9F26A1和9F3A具有高效价的针对DENV-1的中和活性具有型特异性;免疫兔血清仅对DENV-1和DENV-3具有中和活性,并且24株单抗中也有14株同时对DENV-1和DENV-3表现出中和活性,说明DENV1 EDⅢ和DENV-3 EDⅢ之间存在交叉的可以诱导中和抗体的优势抗原表位,容易引起机体产生交叉保护性抗体。
第三部分:评价NS1抗原捕获ELISA用于测定DENV-1 EDⅢ单克隆抗体的中和活性的可行性
通过抗体中和DENV-1后再感染C6/36细胞,利用NS1抗原捕获ELISA检测细胞培养上清中NS1的水平,从而评价抗体对DENV-1的中和效应。实验结果显示23株对DENV-1具有PRNT中和活性的单抗中,NS1抗原捕获ELISA仅能测出14株具有中和活性,而两种方法均能检测到多抗兔血清有中和活性。NS1抗原捕获ELISA测定出的抗体中和效价远低于PRNT试验测定出的结果;识别位点Ⅲ的那些具有高效价PRNT中和活性的抗体,NS1抗原捕获ELISA也测定出具有较高的中和活性。
综合以上三部分的研究结果,本项研究的结论如下:
1成功制备了24株针对该重组蛋白的杂交瘤细胞株和1份兔免疫血清,8株单抗特异性结合DENV-1 EDⅢ蛋白,而另外16株单抗和兔免疫血清则与另外三型登革病毒蛋白有不同程度的交叉反应。这些抗体的获得,为筛选针对EDⅢ的中和抗体和抗体所对应的优势抗原表位奠定了基础。
2 24株针对DENV-1 EDⅢ单抗,至少识别三个抗原结合区域,位点Ⅰ是四型DENV的交叉抗原识别区域,识别该位点的抗体能够与至少两型以上的DENV EDⅢ结合,同时对DENV还表现出交叉中和活性,交叉抗体的获得对进一步研究ADE效应和研究同时诱导对四型DENV均具有中和作用的交叉抗原表位具有重要意义;位点Ⅲ诱导产生抗体大多仅与DENV-1 EDⅢ特异性结合,同时对DENV-1表现出极高的中和活性,推测该位点在DENV-1感染细胞的过程中具有重要的作用,对于该位点的进一步研究,可以深入理解DENV在感染过程中的机制,而这个位点也有望用作DENV四价疫苗的组分之一,型特异性的中和抗体还可以发展成为特异针对DENV-1的治疗性抗体。
3 DENV-1 EDⅢ免疫产生的兔多抗血清对DENV-3的感染具有交叉保护性,而对DENV-2和DENV-4无保护性。提示使用DENV-1 EDⅢ作为亚单位疫苗,不能对DENV-2和DENV-4起到有效保护。
4 DENV-1 NS1抗原捕获ELISA用于测定抗体的中和活性敏感性较差,无法代替传统的PRNT中和试验;但由于ELISA简单、快速、高通量的特点,可以用于批量筛选一些中和活性强的抗体,作为PRNT试验的补充。
禽流感H5N1病毒血凝素基因在昆虫细胞中的表达
禽流感是由A型流感病毒引起的一种禽类的传染性疾病综合征,该疾病对全世界的家禽市场和社会公共卫生环境都造成了严重的危害。A型流感病毒含8个反义RNA片断,共编码10种蛋白,核衣壳蛋白(Nucleocapsid protein,NP)、基质蛋白(Matrix Protein,M)、血凝素(hemagglutinin,HA)、神经氨酸酶(Neuraminidase,NA)是四种重要的宿主免疫反应的靶蛋白。其中HA是病毒表面最多的糖蛋白,也是变异最大的基因,根据抗原性不同可进一步分为16个亚型(H1～H16);HA与宿主细胞表面受体的唾液酸残基末端的结合,可以调节病毒和细胞膜的融合;HA还是病毒主要的保护性抗原,抗HA的抗体对病毒的感染具有中和作用。由于禽流感病毒的抗原性和致病性很大程度上决定于HA基因的变异情况,所以简单、高效制备具有接近天然性质的血凝素抗原对研究HA功能和活性,制备亚单位疫苗以及研发诊断、治疗性抗体都将具有十分重要意义。本研究选用杆状病毒-昆虫细胞真核表达系统,以流行范围广、致病能力强的H5N1禽流感病毒血凝素H5抗原作为靶标,建立一种稳定、快速并能大量获取具有良好抗原性和生物活性的HA重组蛋白的方法,以期用于HA功能和生物学特性的研究,为进一步获取特异性针对该抗原的抗体,建立起快速、简便、特异性强的诊断方法奠定基础。
本项研究工作在论文中的第四部分中进行论述:
第四部分:禽流感病毒H5N1血凝素基因在昆虫细胞中的表达及鉴定
构建杆状病毒重组质粒Bacmid/H5,转染Sf9昆虫细胞后,利用免疫荧光鉴定重组his-H5蛋白成功表达,通过Ni-NTA亲和树脂纯化后的重组his-H5蛋白纯度达到90%以上,经Western-blot证实该重组蛋白与禽流感病毒H5标准血清产生特异性的免疫反应,并且在血凝试验中显示出1:128的血凝效价。
本项研究结论如下:
采用的杆状病毒-昆虫细胞表达体系成功高效表达并纯化了A型流感病毒重组血凝素his-H5蛋白,Western-blot实验证实该重组蛋白具有良好的抗原性;血凝试验证明该蛋白保留了天然状态下结构,具有良好的血凝活性。该重组蛋白有望用于HA功能和生物学活性的进一步研究,也将为下一步亚单位疫苗、中和抗体以及特异性诊断试剂的研发奠定基础。
Dengue virus is a member of the Flavivirus genus in the Flaviviridae family and has four distinct serotypes(DENV-1,DENV-2,DENV-3 and DENV-4).Infection with any of the four serotypes of DV causes a spectrum of clinical features ranging from asymptomatic infections,undifferentiated fever,and classical dengue fever to life-threatening manifestations such as dengue hemorrhagic fever(DHF) and dengue shock syndrome(DSS).The diease is endemic in more than 100 countries and is found virtually throughout the tropics and cause an estimated 50-100 million illnesses annually,including 250 000-500 000 cases of dengue haemorrhagic fever.After being infected by one serotype dengue virus,an individual develops long-lived immunity to homologous serotypes,but antibodies to one serotype are not sufficient to protect against other serotypes,subsequent heterologous infections may lead to the developing DHF or DSS.This is known as antibody dependent enhancement(ADE) for it is postulated that subneutralizing cross-reactive antibodies may facilitate viral infection through Fc receptor by binding with the virus. Antibodies induced by dengue virus infections can be roughly divided into type-specific neutralizing antibodies,cross-reactive nonneutralizing antibodies,and cross-neutralizing antibodies.It is thought that cross-reactive nonneutralizing antibodies and neutralizing antibodies in sub-neutralizing concentration will both lead to ADE.Yamanaka et al.reported that antibodies showed enhancing activities or neutralizing activities depended on the levels of complement in vitro and complement levels were considered an additional factor involved in the in vivo ADE phenomenon: neutralizing at a normal level of complement,but increaseing infetion at a low level.
Envelope(E) protein is the the major protein on the surface of DENV virions,it can mediate receptor binding and membrane fusion.Antibodies against E protein long-lastingly exist in patients' sera,and most importantly,confers protective immunity.E protein contains three domains:a central domain(DⅠ),a dimerization domain(DⅡ),and an immunoglobulin(Ig)-like domain(DⅢ).It had been found many type- and subtype- specific neutralizing epitopes were mapped to domainⅢ,and antibodies to this domain were the most powerful blockers to virus infection.
At present,however,there is no specific treatment available for DENV infections and no vaccine that is effective against all four DENV serotypes,so preparation of antibodies with neutralizing abilities is meaningful for the study of antigen epitopes and the production of vaccine.For the great importance of EDⅢin inducing neutralizing antibodies,we chose the recombinant DENV-1 EDⅢprotein as the antigen to immunize five BALB/c mice and one New Zealand rabbit for producing monoclonal antibodies and rabbit hyperimmune serum and tried to find out which epitopes were serotype specific and which were cross-reactive.We also tried to find out andibodies recoganized these epitopes,which had serotype specific neutralizing abilities and which had cross-neutralizing abilities.These will give guidelines for designing a vaccine without inducing ADE.
This study consists of three parts,as follows:
Ⅰ.Preparation and characterization of antibodies to DENV-1 EDⅢ
A total of 24 hybridoma cell lines that stably produced MAbs were initially established on the basis of their strong positive reactivity with the rDENV1- EDⅢprotein.The immunoglobulin isotype determinations revealed that the 24 MAbs comprised IgG2a(n=2),IgG2b(n=1),and IgG1(n=21).The serotype specificity of the MAbs was further identified by testing their reactivity to the four rDENV EDⅢproteins by ELISA and the four serotypes DENV in the IFA.Of the 24 MAbs,8 MAbs specifically reacted to DENV1 with no cross-reactivities to the other three DENV serotypes in either the ELISA or the IFA.The remaining 16 MAbs and the rabbit hyperimmune serum had cross-reactivities to the four DENV serotypes in different cross-reactivity patterns.
Ⅱ.To study the DENV-1 EDⅢepitopes recoganized by these antibodies and detect their neutralizing abilities to DENV with Plaque Reduction Neutralization Test.
The distinct binding epitopes of the 24 DENV EDⅢMAbs were determined by competition ELISA using recombinant DENV1-EDⅢas the immobilized antigen. The results showed that the 24 MAbs bound at least to three different epitopes on the DENV-1 EDⅢprotein and 13 MAbs recognized eptiopeⅠshowed a cross-reactivity with other DENV serotypes in ELISA and IFA.Each of the 24 hybridoma cell lines were injected intraperitoneally in BALB/c mice to produce ascitic fluids. Neutralization ability to DENV-1～4 of these ascetic fluids were detected by Plaque Reduction Neutralization Test(PRNT).The results indicated that 23 MAbs had neutralizing activities to DENV,from which 13 cross-reactive MAbs recognized eptiopeⅠalso showed cross-neutralizing abilities to DENV,but there was no certain relationship between the two.Four MAbs of the 13 could neutralize all four dengue-serotypes;The 7 MAbs recoganized epitopeⅢshowed great neutralizing abilities to DENV-1,except 9E18A8 and 9F26A1.Three MAbs from the greatly neutralizing antibodies were serotype-specifc and could be developed as therapeutic antibodies for DENV-1;The rabbit hyperimmune serum neutralizd only DENV-1 and DENV-3,which indicated,that DENV-1 EDⅢand DENV-3 EDⅢshared crossly protective epitopes.
Ⅲ.To evaluate the neutralizing abilities of anti-DENV antibodies with DENV-1 NS1 antigen capture ELISA.
DENV-1 neutralized firstly by antibodies were used to infect C6/36 cell,the DENV-1 NS 1 antigen capture ELISA was used to evaluate the neutralizing abilities of these antibodies by detecting the NS1 secreted from the the infected cell.The results indicated that only 14 MAbs of the 23 neutralizing MAbs detected by PRNT had neutralizing abilities,while the rabbit hyperimmune serum had neutralizing ability detected by both methods.Neutralizing titers detected by NS1 capture ELISA were far lower than that detected by PRNT.Antibodies recoganized epitopeⅢshowed greater neutralizing titers than other antibodie in both methods.
Conclusions:
1.A total of 24 hybridoma cell lines that stably produced MAbs were initially established on the basis of their strong positive reactivities with the rDENV1- EDⅢprotein.Eight MAbs specifically reacted to DENV1 with no cross-reactivities to the other three DENV serotypes in either the ELISA or the IFA.The remaining 16 MAbs and the rabbit hyperimmune serum had cross-reactivities to the four DENV serotypes in different cross-reactivity patterns.These obtained antibodies can be used to study their neutralizing ablilities and are potential tools for studing the mechanism of reduced or enhanced virus infection.
2.The 24 MAbs bound to at least three different epitopes on the DENV-1 EDⅢprotein and MAbs recognized eptiopeⅠshowed a cross-reactivities with other DENV serotypes in ELISA and IFA and cross-neutralizing abilities to DENV in PRNT.This could be used to study common epitopes of DENV,which is meaningful for developing DENV vaccine;Most of the MAbs recoganized epitopeⅢshowed serotype specificities and great neutralizing abilities to DENV-1.It was supposed that epitopeⅢplayed a important role in virus infection and cell binding,so this epitope could be used as one of the potential candidates of tetravalent vaccine.
3.The rabbit hyperimmune serum to DENV-1 EDⅢcould protected cells against DENV-1 and only crossly protected cells from being infected by DENV-3,it indicated that DENV-1 EDⅢwould not generate sufficient protection for DENV-2 and DENV-4 as a subunit vaccine.
4.DENV-1 NS1 antigen capture ELISA was not sensitive enough for evaluating antibodies' neutralizing abilities.But it could be used for screening the antibodies with high neutralizing abilities as a supplement to PRNT.
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[35] Roehrig JT, Hombach J,Barrett AD. Guidelines for Plaque-Reduction Neutralization Testing of Human Antibodies to Dengue Viruses[J]. Viral Immunol, 2008, 21(2): 123-32.
[36] Lambeth CR, White LJ, Johnston RE, et al. Flow cytometry-based assay for titrating dengue virus[J]. J Clin Microbiol, 2005, 43(7): 3267-72.
[37] Ludert JE, Mosso C, Ceballos-Olvera I, et al. Use of a commercial enzyme immunoassay to monitor dengue virus replication in cultured cells[J]. Virol J,2008,5:51.
[38] Flamand M, Megret F, Mathieu M, et al. Dengue virus type 1 nonstructural glycoprotein NS1 is secreted from mammalian cells as a soluble hexamer in a glycosylation-dependent fashion[J]. J Virol, 1999, 73(7): 6104-10.
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[1]Gibbons RV,Vaughn DW.Dengue:an escalating problem[J].BMJ,2002,324(7353):1563-6.
[2]Shu PY,Huang JH.Current advances in dengue diagnosis[J].Clin Diagn Lab Immunol,2004,11(4):642-50.
[3]Mackenzie JS,Gubler DJ,Petersen LR.Emerging flaviviruses:the spread and resurgence of Japanese encephalitis,West Nile and dengue viruses[J].Nat Med,2004,10(12 Suppl):S98-109.
[4]Whitehead SS,Blaney JE,Durbin AP,et al.Prospects for a dengue virus vaccine[J].Nat Rev Microbiol,2007,5(7):518-28.
[5]Halstead SB.Pathogenesis of dengue:challenges to molecular biology[J].Science,1988,239(4839):476-81.
[6]Halstead SB.Dengue hemorrhagic fever:two infections and antibody dependent enhancement,a brief history and personal memoir[J].Rev Cubana Med Trop,2002,54(3):171-9.
[7]Lindenbach BD,Thiel HJ,Rice CM:Flaviviridae:the viruses and their replication,D.M.Knipe,P.M.Howley,editor,Fields Virology,Philadelphia:Lippincott Williams & Wilkins,2007:1103-13.
[8]Nowak T,Wengler G.Analysis of disulfides present in the membrane proteins of the West Nile flavivirus[J].Virology,1987,156(1):127-37.
[9]Winkler G,Heinz FX,Kunz C.Studies on the glycosylation of flavivirus E proteins and the role of carbohydrate in antigenic structure[J].Virology,1987,159(2):237-43.
[10]Lorenz IC,Allison SL,Heinz FX,et al.Folding and dimerization of tick-borne encephalitis virus envelope proteins prM and E in the endoplasmic reticulum[J].J Virol,2002,76(11):5480-91.
[11] Konishi E,Mason PW. Proper maturation of the Japanese encephalitis virus envelope glycoprotein requires cosynthesis with the premembrane protein[J]. J Virol, 1993, 67(3): 1672-5.
[12] Zhang Y, Corver J, Chipman PR, et al. Structures of immature flavivirus particles[J]. EMBO J, 2003, 22(11): 2604-13.
[13] Rey FA, Heinz FX, Mandl C, et al. The envelope glycoprotein from tick-borne encephalitis virus at 2 Aresolution[J]. Nature, 1995, 375(6529): 291-8.
[14] Modis Y, Ogata S, Clements D, et al. Variable surface epitopes in the crystal structure of dengue virus type 3 envelope glycoprotein[J]. J Virol, 2005, 79(2):1223-31.
[15] Kawano H, Rostapshov V, Rosen L, et al. Genetic determinants of dengue type 4 virus neurovirulence for mice[J]. J Virol, 1993, 67(11): 6567-75.
[16] Roehrig JT, Volpe KE, Squires J, et al. Contribution of disulfide bridging to epitope expression of the dengue type 2 virus envelope glycoprotein[J]. J Virol, 2004, 78(5): 2648-52.
[17] Rey FA. Dengue virus envelope glycoprotein structure: new insight into its interactions during viral entry[J]. Proc Natl Acad Sci U S A, 2003, 100(12):6899-901.
[18] Modis Y, Ogata S, Clements D, et al. A ligand-binding pocket in the dengue virus envelope glycoprotein[J]. Proc Natl Acad Sci U S A, 2003, 100(12):6986-91.
[19] Mukhopadhyay S, Kuhn RJ,Rossmann MG. A structural perspective of the flavivirus life cycle[J]. Nat Rev Microbiol, 2005, 3(1): 13-22.
[20] Allison SL, Schalich J, Stiasny K, et al. Oligomeric rearrangement of tick-borne encephalitis virus envelope proteins induced by an acidic pH[J]. J Virol, 1995,69(2): 695-700.
[21] Stiasny K, Allison SL, Mandl CW, et al. Role of metastability and acidic pH in membrane fusion by tick-borne encephalitis virus[J]. J Virol, 2001, 75(16):7392-8.
[22] Stiasny K, Allison SL, Marchler-Bauer A, et al. Structural requirements for low-pH-induced rearrangements in the envelope glycoprotein of tick-borne encephalitis virus[J]. J Virol, 1996, 70(11): 8142-7.
[23] Allison SL, Schalich J, Stiasny K, et al. Mutational evidence for an internal fusion peptide in flavivirus envelope protein E[J]. J Virol, 2001, 75(9):4268-75.
[24] Bray M, Men R, Tokimatsu I, et al. Genetic determinants responsible for acquisition of dengue type 2 virus mouse neurovirulence[J]. J Virol, 1998,72(2): 1647-51.
[25] Gualano RC, Pryor MJ, Cauchi MR, et al. Identification of a major determinant of mouse neurovirulence of dengue virus type 2 using stably cloned genomic-length cDNA[J]. J Gen Virol, 1998, 79 ( Pt 3 ): 437-46.
[26] Chen Y, Maguire T, Hileman RE, et al. Dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate[J]. Nat Med, 1997, 3(8):866-71.
[27] Hung JJ, Hsieh MT, Young MJ, et al. An external loop region of domain Ⅲ of dengue virus type 2 envelope protein is involved in serotype-specific binding to mosquito but not mammalian cells[J]. J Virol, 2004, 78(1): 378-88.
[28] Hurrelbrink RJ,McMinn PC. Attenuation of Murray Valley encephalitis virus by site-directed mutagenesis of the hinge and putative receptor-binding regions of the envelope protein[J]. J Virol, 2001, 75(16): 7692-702.
[29] Lin B, Parrish CR, Murray JM, et al. Localization of a neutralizing epitope on the envelope protein of dengue virus type 2[J]. Virology, 1994, 202(2):885-90.
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[31] Mandl CW, Guirakhoo F, Holzmann H, et al. Antigenic structure of the flavivirus envelope protein E at the molecular level, using tick-borne encephalitis virus as a model[J]. J Virol, 1989, 63(2): 564-71.
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[1]Gibbons RV,Vaughn DW.Dengue:an escalating problem[J].BMJ,2002,324(7353):1563-6.
[2]Shu PY,Huang JH.Current advances in dengue diagnosis[J].Clin Diagn Lab Immunol,2004,11(4):642-50.
[3]Mackenzie JS,Gubler DJ,Petersen LR.Emerging flaviviruses:the spread and resurgence of Japanese encephalitis,West Nile and dengue viruses[J].Nat Med,2004,10(12 Suppl):S98-109.
[4]Whitehead SS,Blaney JE,Durbin AP,et al.Prospects for a dengue virus vaccine[J].Nat Rev Microbiol,2007,5(7):518-28.
[5]Halstead SB.Pathogenesis of dengue:challenges to molecular biology[J].Science,1988,239(4839):476-81.
[6]Halstead SB.Dengue hemorrhagic fever:two infections and antibody dependent enhancement,a brief history and personal memoir[J].Rev Cubana Med Trop,2002,54(3):171-9.
[7]Lindenbach BD,Thiel HJ,Rice CM:Flaviviridae:the viruses and their replication,D.M.Knipe,P.M.Howley,editor,Fields Virology,Philadelphia:Lippincott Williams & Wilkins,2007:1103-13.
[8]Nowak T,Wengler G.Analysis of disulfides present in the membrane proteins of the West Nile flavivirus[J].Virology,1987,156(1):127-37.
[9]Winkler G,Heinz FX,Kunz C.Studies on the glycosylation of flavivirus E proteins and the role of carbohydrate in antigenic structure[J].Virology,1987,159(2):237-43.
[10]Lorenz IC,Allison SL,Heinz FX,et al.Folding and dimerization of tick-borne encephalitis virus envelope proteins prM and E in the endoplasmic reticulum[J].J Virol,2002,76(11):5480-91.
[11] Konishi E,Mason PW. Proper maturation of the Japanese encephalitis virus envelope glycoprotein requires cosynthesis with the premembrane protein[J]. J Virol, 1993, 67(3): 1672-5.
[12] Zhang Y, Corver J, Chipman PR, et al. Structures of immature flavivirus particles[J]. EMBO J, 2003, 22(11): 2604-13.
[13] Rey FA, Heinz FX, Mandl C, et al. The envelope glycoprotein from tick-borne encephalitis virus at 2 Aresolution[J]. Nature, 1995, 375(6529): 291-8.
[14] Modis Y, Ogata S, Clements D, et al. Variable surface epitopes in the crystal structure of dengue virus type 3 envelope glycoprotein[J]. J Virol, 2005, 79(2):1223-31.
[15] Kawano H, Rostapshov V, Rosen L, et al. Genetic determinants of dengue type 4 virus neurovirulence for mice[J]. J Virol, 1993, 67(11): 6567-75.
[16] Roehrig JT, Volpe KE, Squires J, et al. Contribution of disulfide bridging to epitope expression of the dengue type 2 virus envelope glycoprotein[J]. J Virol, 2004, 78(5): 2648-52.
[17] Rey FA. Dengue virus envelope glycoprotein structure: new insight into its interactions during viral entry[J]. Proc Natl Acad Sci U S A, 2003, 100(12):6899-901.
[18] Modis Y, Ogata S, Clements D, et al. A ligand-binding pocket in the dengue virus envelope glycoprotein[J]. Proc Natl Acad Sci U S A, 2003, 100(12):6986-91.
[19] Mukhopadhyay S, Kuhn RJ,Rossmann MG. A structural perspective of the flavivirus life cycle[J]. Nat Rev Microbiol, 2005, 3(1): 13-22.
[20] Allison SL, Schalich J, Stiasny K, et al. Oligomeric rearrangement of tick-borne encephalitis virus envelope proteins induced by an acidic pH[J]. J Virol, 1995,69(2): 695-700.
[21] Stiasny K, Allison SL, Mandl CW, et al. Role of metastability and acidic pH in membrane fusion by tick-borne encephalitis virus[J]. J Virol, 2001, 75(16):7392-8.
[22] Stiasny K, Allison SL, Marchler-Bauer A, et al. Structural requirements for low-pH-induced rearrangements in the envelope glycoprotein of tick-borne encephalitis virus[J]. J Virol, 1996, 70(11): 8142-7.
[23] Allison SL, Schalich J, Stiasny K, et al. Mutational evidence for an internal fusion peptide in flavivirus envelope protein E[J]. J Virol, 2001, 75(9):4268-75.
[24] Bray M, Men R, Tokimatsu I, et al. Genetic determinants responsible for acquisition of dengue type 2 virus mouse neurovirulence[J]. J Virol, 1998,72(2): 1647-51.
[25] Gualano RC, Pryor MJ, Cauchi MR, et al. Identification of a major determinant of mouse neurovirulence of dengue virus type 2 using stably cloned genomic-length cDNA[J]. J Gen Virol, 1998, 79 ( Pt 3 ): 437-46.
[26] Chen Y, Maguire T, Hileman RE, et al. Dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate[J]. Nat Med, 1997, 3(8):866-71.
[27] Hung JJ, Hsieh MT, Young MJ, et al. An external loop region of domain Ⅲ of dengue virus type 2 envelope protein is involved in serotype-specific binding to mosquito but not mammalian cells[J]. J Virol, 2004, 78(1): 378-88.
[28] Hurrelbrink RJ,McMinn PC. Attenuation of Murray Valley encephalitis virus by site-directed mutagenesis of the hinge and putative receptor-binding regions of the envelope protein[J]. J Virol, 2001, 75(16): 7692-702.
[29] Lin B, Parrish CR, Murray JM, et al. Localization of a neutralizing epitope on the envelope protein of dengue virus type 2[J]. Virology, 1994, 202(2):885-90.
[30] Wengler G. An analysis of the antibody response against West Nile virus E protein purified by SDS-PAGE indicates that this protein does not contain sequential epitopes for efficient induction of neutralizing antibodies[J]. J Gen Virol, 1989, 70( Pt 4 ): 987-92.
[31] Mandl CW, Guirakhoo F, Holzmann H, et al. Antigenic structure of the flavivirus envelope protein E at the molecular level, using tick-borne encephalitis virus as a model[J]. J Virol, 1989, 63(2): 564-71.
[32] Roehrig JT, Bolin RA,Kelly RG. Monoclonal antibody mapping of the envelope glycoprotein of the dengue 2 virus, Jamaica[J]. Virology, 1998,246(2): 317-28.
[33] Crill WD,Roehrig JT. Monoclonal antibodies that bind to domain Ⅲ of dengue virus E glycoprotein are the most efficient blockers of virus adsorption to Vero cells[J]. J Virol, 2001, 75(16): 7769-73.
[34] Wengler G. Cell-associated West Nile flavivirus is covered with E+pre-M protein heterodimers which are destroyed and reorganized by proteolytic cleavage during virus release[J]. J Virol, 1989, 63(6): 2521-6.
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