构建登革病毒NS1抗原拓扑图及建立ELISPOT微中和试验快速高通量评价登革病毒抗体中和活性
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摘要
登革病毒(dengue virus, DENV)属于黄病毒科(Flavivirade)黄病毒属(Flavivirus),主要的传播途径是通过蚊虫叮咬敏感的脊椎动物宿主。DENV根据血清抗原反应分可为四个血清型,分别为DENV-1、DENV-2、DENV-3和DENV-4型。各型病毒感染后均可引起的症状主要有登革热(dengue fever, DF)、登革出血热(dengue hemorrhagic fever, DHF)和登革休克综合征(dengue shock syndrome, DSS)。
登革病毒的传播在早期较为局限,主要在热带和亚热带地区。但随着世界经济的一体化和各国之间的交流增强,登革病毒的传播范围日益扩大,目前在非洲、美洲、东南亚和西大平洋地区一百多个国家和地区均出现过登革病毒的流行。调查数据显示,全世界每年约有0.5-1亿人感染登革病毒,出现登革出血热症状的严重病例达50万人,2-2.5万人死亡,其中大多为儿童,死亡率为5%。因此,登革病毒的感染已成为全球重要的公共卫生问题,如何有效控制感染是解决这一问题的关键。
人体在感染登革病毒后,可以无症状,也可只出现类似感冒症状,可自愈,稍重的是出现DF症状,这些病例主要出现在初次感染的患者,儿童患者多需要住院治疗。有相当部分二次感染的患者可以出现严重的病症DHF/DSS。目前还没有一个特异性的治疗方法,患者只能对症治疗,也缺乏有效的疫苗来防止感染。根据目前的研究结果显示,出现DHF和DSS的主要原因是抗体依赖增强(antibody-dependent inhancment, ADE)效应,而其根源是登革病毒存在四个不同的血清型。研究认为,病人感染其中一型病毒后,免疫系统会产生众多对各种血清型病毒有交叉反应的抗体,这些抗体短期内有一定的交叉保护性作用,但并不具有长期保护性作用。相反的,这些与其他血清型病毒有交叉反应性的非中和抗体或者是一些亚中和浓度抗体可能就是引起DHF/DSS的重要原因。这些抗体与病毒颗粒结构蛋白结合后,不能阻止病毒进入细胞,而其Fc部分会与某些细胞(如单核细胞或树突状细胞)膜上的Fc受体结合,这种结合有助于登革热病毒进入细胞,增强细胞对病毒的摄取及病毒在细胞内的复制,导致感染的增强,从而促使登革的严重疾病的发生。对于防止登革疾病传播的疫苗研发中,如果仅对一型病毒进行抗感染保护,会在病人感染异型病毒时,产生的ADE效应将会影响病人的生命。因此,各种类型的疫苗是否是引起ADE效应是登革防疫研究中一个关键点。
登革病毒从首次分离至今,已有六十余年,但对其感染后疾病的防治还存在较大困难。其中的最大障碍是对登革病毒感染后的机体免疫机制不明,研究的针对性不能集中。登革病毒的基因组结构是单股正链RNA分子,长约11000nt,编码三个结构蛋白,分别为衣壳蛋白(C)、膜蛋白(M)和包膜蛋白(E),七个非结构蛋白(non-structure protein, NS),分别为NS1、NS2A、NS2B、 NS3、NS4A、NS4B和NS5。结构蛋白中的E蛋白与病毒感染有关,主要是与靶细胞表面的病毒受体结合,参与病毒与细胞之间的膜融合。在非结构蛋白中,虽然它们并不是成熟病毒颗粒的组成成份,但从目前的研究成果看来,它们在病毒感染与机体的免疫过程中起重要的作用。其中特别受到重视的是NS1蛋白,它是登革病毒中主要的非结构蛋白,通常有三个形式存在,一为分泌型,可以直接由感染的细胞分泌到胞外,可用于登革病毒早期诊断指标。二为胞内型,主要存在于细胞内生物腔膜上,三为膜型,存在于感染细胞膜上,它的功能推测可以通过细胞毒作用,直接参与病毒的清除,另外,也可能在病毒RNA的复制中起一定作用,但因为对其的蛋白结构未完成了解,与抗体的结合形式不清楚,所以其真正功能还需进一步研究。在抗原蛋白的结构未完全阐明之前,可以利用各种技术分析预测它们的二级结构,如冷冻电镜、酵母展示、点突变和多肽结合位点分析等。本研究利用合成的NS1重叠多肽与148株NS1单克隆抗体进行结合反应,可以得到单抗的线性结合位点。依据这些单抗的结合位点的特性,如一个单抗与多个位点结合,表明这些位点的物理位置是相近的,从而可以构建出NS1抗原蛋白的拓扑图。同时,我们也用流式细胞术,筛查各抗体与感染后细胞的表达的NS1蛋白抗原的结合类型,了解有哪些抗体是可以与感染细胞膜表面抗原进行结合的,从而可以为解释NS1蛋白在登革病毒感染免疫机制中的作用奠定基础。
疫苗是一个有效预防感染性疾病的方式。如前所述,登革病毒存在四个不同的血清型,病人在二次感染异型病毒时,会产生严重的疾病,因此,疫苗的研制除了要有良好的中和保护作用外,还必须验证是否存在ADE效应。从目前的研究来看,无论是单一价的疫苗或多价疫苗,能同时符合这两个要求的疫苗还没有面世。在疫苗的研究中,中和实验是病毒研究中应用相当普遍的技术。传统的方法是蚀斑减少中和实验(plaque reducing neutralization test, PRNT),但这种方法费时费力,对结果的判断较为主观,许多研究者在开发一些可以替代它的方法。包括有基于流式细胞术的方法、基于ELISA技术的方法等。这些方法虽然是可以省时省力,但也存在一些问题,如病毒用量远比PRNT大。另外,在实际应用中,并不是所有的病毒特别是一些临床分离株,重复性较差。新近有研究者结合前期开发主要用于原位检测细胞因子分泌的技术----基于酶联免疫斑点分析(enzyme-linked immunospot-based microneutralization assay, ELISPOT)的微中和实验。本研究将从实际应用出发,建立和评价这一种技术,并应用这一方法检测临床收集的29例1型登革病毒感染患者恢复期血清的中和效价。本研究共分为如下三个部分：
第一部分：分析登革病毒NS1抗体结合位点构建NS1拓扑图
登革病毒NS1蛋白在登革病毒感染后免疫机制中的作用越来越受到重视,主要原因是它不是病毒颗粒的组成成份,可以避免引起ADE效应。它有多种表达形式,其中在感染细胞膜表面表达的部分,研究认为可能介导细胞杀伤功能而在病毒清除中起重要作用。研究结果显示,NS1蛋白应有三个区域,分别为RⅠ、RⅡ和RⅢ,但因其结构尚未解晰,对于它的真正分区功能未能从理论或实验中得到充分的证明。本研究合成DENV-1NS1的重叠多肽(每条多肽与上一多肽重叠5个氨基酸,最后一条重叠8个氨基酸,共35条),检测它们与148株NS1单克隆抗体的结合情况,分析它们的结合位点。检测结果表明,148株抗体中,有82株抗体可以与多肽进行结合,其中有七株抗体具有结合两个区域,具体为1A11A9、2E8A5与RI区的1肽和RⅡ22肽结合,6D14A4与RⅠ区的1肽、14肽和RⅡ区的22肽结合,1F32A1与RI区的1肽和RⅡ区的31肽结合,7E1A4和7E9A2与RI区的5肽和RⅢ区的27肽结合,1B7A1与RⅡ区的22肽和RⅢ区的32肽结合,三个区域相互之间有密切连接位点。利用以上多肽结合的位点,根据单抗结合位点在物理位置上的相近性,我们构建出NS1抗原蛋白的二级结构模拟图。另外,研究证明WNV的NS1抗体可以通过抗体依赖细胞杀伤功能对感染细胞进行吞噬清除,其基础是此抗体须与N在细胞表面表达NS1进行结合。我们用流式细胞术分析了以上148株抗体与感染病毒后细胞膜表面的NS1蛋白结合情况。结果显示,分别有92株、87株、75株和91株抗体与同型病毒感染后细胞膜表面的NS1蛋白结合。按照单抗与多肽的结合位点结果,针对RⅡ区结合的单抗,有82%(18/22)的抗体可以与DENV1-4任一型或多型病毒感染后细胞膜上抗原结合,而针对结合RI和RⅢ的抗体,分别有60%(29/48)和54%(6/11)的抗体可与膜抗原结合。这一结合模式可能与膜型NS1蛋白在感染细胞膜上嵌合形式有关。我们的研究结果与前期对黄病毒NS1的研究数据基本一致。这些数据可为研究NS1的功能和NS1抗体应用打下坚实基础。
第二部分：酶联免疫斑点微中和实验方法检测登革病毒抗体中和活性方法的建立与评价
目前中和实验的金标准方法是PRNT方法,这一方法在六孔板中进行,在病毒感染细胞后,需要在7-10天的时间孵育来形成可以用于检测的蚀斑。如果要完成大量的中和实验标本,这一方法将相当耗时而且结果判断主观性强。微中和实验是一个应用较多的高通量检测方法,这些实验是在96孔板中完成。本研究将评价一个新近开发的基于ELISPOT技术的微中和实验方法,并建立用于检测抗体或血清对登革病毒的中和活性的方法学。按照该法的基本原理是病毒感染细胞后,细胞表面可在较短时间内表达NS1蛋白,将细胞固定处理后,利用抗NS1的抗体可以建立ELISA检测方法,最后用固体原位显色方法可以通过斑点的形成指示病毒对细胞的感染情况。选用VERO E6细胞为病毒感染的靶细胞,通过对病毒加入量和登革各型病毒在感染后的孵育时间的条件优化,建立了ELISPOT方法检测登革各型病毒的方法学。方法学建立后,我们对33株抗登革病毒EDIII单克隆抗体针对四个血清型的中和活性检测,然后再从中选择十株单抗进行PRNT检测它们针对各型病毒的中和活性,两种检测方法应用相同病毒株、抗体稀释梯度进行,并对检测结果进行对比分析。结果显示,基于ELISPOT方法可以在96孔板中加入200PFUs病毒的情况下,形成的斑点清晰可辨,登革病毒四个血清型有不同的感染后孵育时间,DENV-1为4天,DENV-2和DENV-4为2天,DENV-3为3天。对比两种方法,它们之间的相关性较好,r=0.863,P<0.001；如果以PRNT方法为标准,我们将其结果与ELISPOT方法结果进行卡方检验(McNemar),结果显示ELISPOT与PRNT结果无显著性差别(P<1.000)。ELISPOT微中和实验检测这10株单抗针对四型登革病毒的中和效价的敏感性和特异性分别为95.6%(22/23)and88.24%(15/17)。如此可见,基于ELISPOT的微中和实验可以应用于四型登革病毒的中和活性实验检测,而且这种方法省时省力,适合进行批量筛查。
第三部分Ⅰ型登革病毒感染患者康复期血清中和效价分析
对登革病毒引起的疾病目前还未有特异高效的治疗方法或药物,因目前也没有一个能良好复制登革病毒感染后症状的动物模型,使疫苗的研制存在一定的困难。人体在自然感染登革病毒后,免疫状态的维持可以从一个侧面了解机体的抗病毒机制。本研究收集了29例在2006年感染1登革病毒的患者恢复期血清(2010年获取),利用本实验室建立和评价的ELISPOT微中和实验方法,检测它们针对四型登革病毒的中和效价,并对结果进行对比。结果显示,大部分血清对四型病毒有交叉中和活性,对四型登革病毒的中和效价(IC50,血清稀释度倒数)进行非参数检验(Kruska-Wallis H),结果显示,四组中和活性有显著性差异(X2=21.134,P<0.001),其中对同型(DENV-1)的中和活性最高,对DENV-4的中和活性最低。结果表明,机体在感染一种血清型病毒后,对四型病毒均有可能存在一定的四型交叉中和活性,但对同型病毒的中和活性强,而对其他型病的中和活性较弱。这些弱中和活性的交叉抗体,可能会在机体受二次异型病毒感染时引起ADE效应。
小结：
综上所述,本研究在以下方面取得成果：
1、我们合成DENV-1NS1的重叠多肽35条,与148株NS1单克隆抗体进行结合,分析它们的结合位点,有七株抗体的结合位点在不同的两个区域,根据这些位点的物理位置相近性,成功构建出NS1蛋白抗原结构二级结构模拟图,结果与前期对黄病毒NS1蛋白的研究结果一致。研究结果可以在蛋白结晶之前增加对它基本结构的了解。我们还应用流式分析术,筛查了148株单抗与感染细胞膜型NS1抗原结合情况,得到抗体对各型病毒感染分泌于膜上NS1抗原的结合模式。结果显示,分别有92、87、75和91株抗体与同型病毒感染后细胞膜表面的NS1蛋白结合,针对RⅡ区结合的单抗,有82%的抗体可以与DENV1-4任一型或多型病毒感染后细胞膜上抗原结合(18/22),而针对结合RⅠ和RⅢ的抗体,有60%(29/48)和54%(6/11)的抗体可与膜抗原结合,这些结合模式可能与NS1蛋白在感染细胞膜上的嵌合模式有关。
2、建立了基于ELISPOT技术的微中和实验方法检测平台,可以为登革病毒和其他相关病毒提供快速高通量的中和效价测定支撑。同时,我们也对此方法进行评价,对比PRNT方法,方法的特异性和敏感性高,与PRNT有良好的相关性,而且结果判断更加客观,非常适合批量处理的高通量检测实验。
3、从29例1型病毒感染三年后的恢复期病人血清的中和活性检测结果中,获得了它们对同型和异型病毒的中和效价。通过统计分析,对同型病毒的中和活性远强于异型病毒的活性,这可以验证目前的有关初次感染后可以长期存在对同型病毒强的中和保护抗体而对异型病毒有弱中和抗体的理论。
Dengue virus (DENV) is a RNA virus of the family Flaviviridae; genus Flavivirus and primarily transmitted by Aedes mosquitoes among vertebrates. DENV is classified four serotypes according to their reaction to antigen, DENV1-4, each of which is capable of causing same clinical manifestations including dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS).
The transmission of DENV is limited in early mainly in tropical and subtropical regions. But now DENV is endemic in over100countries and regiaons in Africa, America, Southeast Asia and the West Pacific region along with the world economics integration and internetional exchange. Evidence shows that each year around the world50-100million people infected with DENV and there are about0.5million patients would be emergence with DHF or DSS,20-25thousand death cases, mostly children, mortality is about5%. Therefore, DENV infection has become an vital public health problem worldwide, how to effectively control the infection is the key to deal with this problem.
The people who infected by DENV would be asymptomatic or develop flu-like symptoms, self-healing or mild symptoms DF, which appeared mainly in the initial infection patients but most of children need hospital treatment. However, sever symptoms including DHF and DSS would emergence in second infection patients. There is no specific treatment, patients can only accept symptomatic treatment, and no an effective vaccine to prevent DENV infection. Most researchs suggest that antibody-dependent inhancment (ADE) is the main cause of DHF and DSS because there are four different dengue virus serotypes. Studies show that there are various antibodies in sera of DENV infection patients and they have a short-term cross-reactive with all four serotypes. But the cross-reactive antibodies do not have long-term protection function. Instead, they would potentially cause the DHF/DSS. Their binding to virus particle can't block but to enhance virus enter cells through Fc receptor-mediated entry pathway if the cells (such as monocytes and dendritic cells) express Fc receptor, which cotributes to the uptaking of virus and viral replication in cells, leading to infection enhancement and severe dengue disease occurrence. Vaccine is the most effective way to prevent dengue disease transmission, but if the vaccine can only protect one of serotypes of virus, the ADE would occurrence when the patient is infected with the other different serotype virus, which affects the life of the patient. Therefore, the key point in the development of various types of vaccines is to avoid the ADE.
First stain of DENV had been isolated more than sixty years ago, but there are not any effective methods to prevent and cure the diseases caused by DENV, Which is the biggest obstacle to dengue virus infection of immune mechanism is unknown and there is not a focus to study. Dengue virus genomic structure is a single stranded positive-sense RNA molecules,11000nt, encoding three structural proteins, capsid protein (C), membrane protein (M) and envelope protein (E), and seven nonstructural proteins (non-structure protein, NS), NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. The E protein contributes to viral infection through binding the surface of the target cells with the virus receptor, which causes the fusion between viral and cellular membrane. Although the nonstructural proteins are not the composition of mature virus particles, but newly researchs show that they have important function during virus infection and immune process. NS1is the main non-structural protein whichs have three forms. First is the secreted NSl (sNS1), secreted out of infected cells and contributes to the early diagnosis of DENV infection. Second is intracellular form which mainly presents in the intracellular biological membranes. The last form is membrane asociated NS1(mNS1) which existences on the infected cell membrane. The function of mNS1is not clear now, researchs show it maybe involved in viral clearance through cytotoxicity and contributed to viral RNA replication. And because the structure of NS1and the binding with antibody is unknow, so many further researchs would be performed in the futrue. Various technical analysis to predict secondary structure of protein before the crystal structrue accomplishment, such as electron cryo-microscopy, yeast display, point mutation and the peptide binding site analysis. This study will use a panel of148monoantibodies araised from NS1protein to bind the overlapping peptides of NS1and analysis the linear binding sites of mAbs. If an antibody has two binding sites in different domains, the sites would be near in physical location, which would contribute to draw the topography of DENV NS1protein. At the same time, in order to have some knowledge if our panel mAbs can binding the mNS1, we also screen all mAbs binding patterm using flow cytometry, which would produce important foundation data to explained the mNS1fuction in the immune mechanisms of DENV infection.
Vaccine is the most effective way to prevent the infectious disease. So as in DENV transmittion, but DENV have four different serotypes, which would cause ADE effect in secondary infection and severe diseases would occurance. Thus the value of vaccine against DENV is not only the protection but also no ADE effect. Unfortunately, up to day, there is no a satisfied vaccine development, either a single valence or multivalent vaccince. Neutralization test is most important technology during vaccine research. The traditional method of neutralization is plaque reduction neutralization test (PRNT), but it is time-consuming, laborious and lack of objective judgment. Many researchers want to develop a method to replace it including based on flow cytometric method, the based on ELISA method. Although these methods can save time and labor, but still exist some problems, such as need more virus dosage than PRNT, don't be available for some clinical isolation virus and have poor repeatability. Recently, researchs develop a new microneutralization to detecte neutralization of antibody beaed on enzyme-linked immunospot-based microneutralization assay (ELISPOT). Our study would evaluation of this method and establish a platform to detecte the neutralization activity of the antibodies againsts DENV and a panel of29Convalescent sera originate the patients who infected by DENV-1over three years ago.
This research is divided into three parts as flowing:
Part I:Draw the topography of DENV NS1protein according to analysis the binding sites of their monoantibodies
More and more researchs about DENV infectionimmune mechanism focus on NS1protein because it can avoid the ADE. NS1has three species and the mNS1maybe induce the cy to toxic function and contribute to the viral clearance. Some researchs' results showed that NS1protein had three regions, RI, RII and RIII, but the exact function of each domain is still not proved because the structure don't be developed yet. Our research focues on the binding site of a panel of148mAbs to the NS1according to detection the binding between mAbs and overlapping peptides of NS1(total35peptides and every peptide overlap5aa with upper peptide except the last overlap8aa). The results show that a total of82mAbs can bind to peptides of NS1,7of82mAbs have two sites in different domain,1A11A9and2E8A5binds to NO.1peptide in RⅠ domain and NO.22peptide in RⅡ domain,6D14A4binds to NO.1and14peptide in RⅠ domain and NO.22peptide in RⅡ domain,1F32A1binds to NO.1peptide in RⅠ domain and NO.31peptide in RⅡ,7E1A4and7E9A2binds to NO.5peptide in RⅠ domain and NO.27peptide in RⅢ domain,1B7A1binds to NO.22peptide in RⅡ and NO.32peptide in RⅢ domain, which indicates that there is a close connection among three domain. We draw the secondary structure simulation diagram of DENV NS1protein according to the binding sites because the different binding sites of an antibody would be near in physical location. Some researchs indicated that antibodies to WNV NS1protein can clear infected cell through antibody dependent cell cytotoxicity, which is based on the binding mNS1antigen. We used flow cytometry to analysis the binding patterns of the panel of148mAbs. The results show that92,87,75and91mAbs can bind to mNS1of hemeotype DENV-1, DENV-2, DENV-3and DENV-4respectively infection cells. And82%(18/22) mAbs to RⅡ domain and60%(29/48) mAbs to RⅠ can bind to mNS1. Our study results are similar to the former research on the NS1protein of Flavivirus. These data would contribute to the study of NS1function and the mAbs application research.
Part Ⅱ:Establish and Evaluation of a Micro-neutralization Test Based on ELISPOT
PRNT is the gold standard method for neutralization test; this method is performed in6-well format and required for7-10days incubation to form visible plaques. If there are a large number of neutralization test specimens to perform, it would be quite time-consuming and labor-intensive using PRNT. Micro neutralization test is performed in96-well format and widely used in high throughput screening. This research evaluates a newly developed microneutralization test based on ELISPOT (ELISPOT-MNT). For estabilish the methodology, we used VERO E6cell as the target cell of DEVN infection and optimize the virus dosage and the incubation time after DENV infection. After the methodology being established, the neutralization activity of33mAbs, arised from the domain III of the DENV envelope protein (EDIII) against four serotype of DENV, was performed using the method. A panel of10from33mAbs was selected to evaluate this method using PRNT. The same virus stain and mAbs dilution gradient were used in two methods and compare the results. The results show that the spots are clear with the virus dosage200pfus per well of96-well plate when the infected cell incubate for2-4days. The incubation time is4days for DENV-1, DENV-2and DENV-4for2days, DENV-3for3days. The comparison of two methods show that there is a good relationship between them(r=0.863, P<0.001). McNemar's association test was used to measure the disagreement between the two assay methods. There was no significant (P=1.000) disagreement between them.. The method showed a sensitivity of95.6%(22/23) and a specificity of88.24%(15/17) when these10MAbs were tested against four DENV serotype strains and used the PRNT as the reference. Therefore, ELISPOT-MNT shows potential as a primary method for screening neutralization activities of large numbers of antibodies.
Part Ⅲ The Pattern of Neutralization in Convalescent Sera from the Patients with DENV-1Infection
There are not any specific and efficient treatments or drugs for the diseases caused by DENV. Presently, the complaints of dengue can not be well replicated in animal model, which hamper the vaccine research. We can learn some about the antivital mechanism from the immune status of human body after naturally infected with DENY We used the ELISPOT-MNT to detecte the neutralization activity to four DENV serotypes of29sera from the patients infected with DENV-1in2006and compared the results. The results showed that most of sera have cross-neutralization activity to four serotype DENVs. The mean values(IC50, the recip of serum dilution) of DENV-1and DENV-2,-3and-4were analysised by kruska-Wallis H test with SPSS13.0, the result showed that there is significant difference among four DENV serotypes (X2=21.134, P<0.001), the value of DENV-1is the highest and DENV-4is the lowest. Our study indicates that there is cross-neutralization activity to four serotype DENVs in patient primary infection of one serotype DENV, but the strength to heterotype is lower than homeotype. The weak cross neutralization antibodies maybe contribute to the ADE effect when the second infection of different serotype DENV occurrence. Summarization:
1, We sythesize35overlapping peptides of DENV-1NS1that be used to bind to a panel of148mAbs against NS1and anslysis the binding site. There are seven mAbs binding to different domain of NS1, which is the base to draw a secondary structure simulation diagram of DENV NS1protein. The results are similar to to the former research on the NS1protein of Flavivirus. This research would contribute to learning the profile of NS1protein before the crystal constructure developed. We also have screened the binding mode to mNS1of148mAbs and obtained the profile of them using flow cytometric. The results show that92,87,75and91mAbs can bind to mNS1of hemeotype DENV-1, DENV-2, DENV-3and DENV-4respectively infection cells. And82%(18/22) mAbs to RII domain and60%(29/48) mAbs to RI can bind to mNS1.
2, The microneutralization test based on the ELISPOT was developed to detecte the neutralization activity of antibody or immune sera against DENV. It is a time-saving and high-throughput method, which would be the support of the virus nerutralization research. We also evaluated this method compared with PRNT. The results show that it has high specificity(95.6%), high sensitivity(88.24%), good correlation with PRNT(r=0.863, P<0.001) and more objective judgement of result than PRNT. ELISPOT-MNT is availabe for batch processing.
3, We obtain the neutralization titer profiles against homeotypic and heterotypic DENV of29convalescent sera from patients with DENV-1infection three years ago.The statistical analysis indicates that the neutralization activity against homeotypic virus is far stronger than heterotypic virus, which verifies the theory that antibodies arise primary infection can strongly protecte the challenge of hemeotypic virus but weak to heterotypic virus.
登革病毒的传播在早期较为局限,主要在热带和亚热带地区。但随着世界经济的一体化和各国之间的交流增强,登革病毒的传播范围日益扩大,目前在非洲、美洲、东南亚和西大平洋地区一百多个国家和地区均出现过登革病毒的流行。调查数据显示,全世界每年约有0.5-1亿人感染登革病毒,出现登革出血热症状的严重病例达50万人,2-2.5万人死亡,其中大多为儿童,死亡率为5%。因此,登革病毒的感染已成为全球重要的公共卫生问题,如何有效控制感染是解决这一问题的关键。
人体在感染登革病毒后,可以无症状,也可只出现类似感冒症状,可自愈,稍重的是出现DF症状,这些病例主要出现在初次感染的患者,儿童患者多需要住院治疗。有相当部分二次感染的患者可以出现严重的病症DHF/DSS。目前还没有一个特异性的治疗方法,患者只能对症治疗,也缺乏有效的疫苗来防止感染。根据目前的研究结果显示,出现DHF和DSS的主要原因是抗体依赖增强(antibody-dependent inhancment, ADE)效应,而其根源是登革病毒存在四个不同的血清型。研究认为,病人感染其中一型病毒后,免疫系统会产生众多对各种血清型病毒有交叉反应的抗体,这些抗体短期内有一定的交叉保护性作用,但并不具有长期保护性作用。相反的,这些与其他血清型病毒有交叉反应性的非中和抗体或者是一些亚中和浓度抗体可能就是引起DHF/DSS的重要原因。这些抗体与病毒颗粒结构蛋白结合后,不能阻止病毒进入细胞,而其Fc部分会与某些细胞(如单核细胞或树突状细胞)膜上的Fc受体结合,这种结合有助于登革热病毒进入细胞,增强细胞对病毒的摄取及病毒在细胞内的复制,导致感染的增强,从而促使登革的严重疾病的发生。对于防止登革疾病传播的疫苗研发中,如果仅对一型病毒进行抗感染保护,会在病人感染异型病毒时,产生的ADE效应将会影响病人的生命。因此,各种类型的疫苗是否是引起ADE效应是登革防疫研究中一个关键点。
登革病毒从首次分离至今,已有六十余年,但对其感染后疾病的防治还存在较大困难。其中的最大障碍是对登革病毒感染后的机体免疫机制不明,研究的针对性不能集中。登革病毒的基因组结构是单股正链RNA分子,长约11000nt,编码三个结构蛋白,分别为衣壳蛋白(C)、膜蛋白(M)和包膜蛋白(E),七个非结构蛋白(non-structure protein, NS),分别为NS1、NS2A、NS2B、 NS3、NS4A、NS4B和NS5。结构蛋白中的E蛋白与病毒感染有关,主要是与靶细胞表面的病毒受体结合,参与病毒与细胞之间的膜融合。在非结构蛋白中,虽然它们并不是成熟病毒颗粒的组成成份,但从目前的研究成果看来,它们在病毒感染与机体的免疫过程中起重要的作用。其中特别受到重视的是NS1蛋白,它是登革病毒中主要的非结构蛋白,通常有三个形式存在,一为分泌型,可以直接由感染的细胞分泌到胞外,可用于登革病毒早期诊断指标。二为胞内型,主要存在于细胞内生物腔膜上,三为膜型,存在于感染细胞膜上,它的功能推测可以通过细胞毒作用,直接参与病毒的清除,另外,也可能在病毒RNA的复制中起一定作用,但因为对其的蛋白结构未完成了解,与抗体的结合形式不清楚,所以其真正功能还需进一步研究。在抗原蛋白的结构未完全阐明之前,可以利用各种技术分析预测它们的二级结构,如冷冻电镜、酵母展示、点突变和多肽结合位点分析等。本研究利用合成的NS1重叠多肽与148株NS1单克隆抗体进行结合反应,可以得到单抗的线性结合位点。依据这些单抗的结合位点的特性,如一个单抗与多个位点结合,表明这些位点的物理位置是相近的,从而可以构建出NS1抗原蛋白的拓扑图。同时,我们也用流式细胞术,筛查各抗体与感染后细胞的表达的NS1蛋白抗原的结合类型,了解有哪些抗体是可以与感染细胞膜表面抗原进行结合的,从而可以为解释NS1蛋白在登革病毒感染免疫机制中的作用奠定基础。
疫苗是一个有效预防感染性疾病的方式。如前所述,登革病毒存在四个不同的血清型,病人在二次感染异型病毒时,会产生严重的疾病,因此,疫苗的研制除了要有良好的中和保护作用外,还必须验证是否存在ADE效应。从目前的研究来看,无论是单一价的疫苗或多价疫苗,能同时符合这两个要求的疫苗还没有面世。在疫苗的研究中,中和实验是病毒研究中应用相当普遍的技术。传统的方法是蚀斑减少中和实验(plaque reducing neutralization test, PRNT),但这种方法费时费力,对结果的判断较为主观,许多研究者在开发一些可以替代它的方法。包括有基于流式细胞术的方法、基于ELISA技术的方法等。这些方法虽然是可以省时省力,但也存在一些问题,如病毒用量远比PRNT大。另外,在实际应用中,并不是所有的病毒特别是一些临床分离株,重复性较差。新近有研究者结合前期开发主要用于原位检测细胞因子分泌的技术----基于酶联免疫斑点分析(enzyme-linked immunospot-based microneutralization assay, ELISPOT)的微中和实验。本研究将从实际应用出发,建立和评价这一种技术,并应用这一方法检测临床收集的29例1型登革病毒感染患者恢复期血清的中和效价。本研究共分为如下三个部分：
第一部分：分析登革病毒NS1抗体结合位点构建NS1拓扑图
登革病毒NS1蛋白在登革病毒感染后免疫机制中的作用越来越受到重视,主要原因是它不是病毒颗粒的组成成份,可以避免引起ADE效应。它有多种表达形式,其中在感染细胞膜表面表达的部分,研究认为可能介导细胞杀伤功能而在病毒清除中起重要作用。研究结果显示,NS1蛋白应有三个区域,分别为RⅠ、RⅡ和RⅢ,但因其结构尚未解晰,对于它的真正分区功能未能从理论或实验中得到充分的证明。本研究合成DENV-1NS1的重叠多肽(每条多肽与上一多肽重叠5个氨基酸,最后一条重叠8个氨基酸,共35条),检测它们与148株NS1单克隆抗体的结合情况,分析它们的结合位点。检测结果表明,148株抗体中,有82株抗体可以与多肽进行结合,其中有七株抗体具有结合两个区域,具体为1A11A9、2E8A5与RI区的1肽和RⅡ22肽结合,6D14A4与RⅠ区的1肽、14肽和RⅡ区的22肽结合,1F32A1与RI区的1肽和RⅡ区的31肽结合,7E1A4和7E9A2与RI区的5肽和RⅢ区的27肽结合,1B7A1与RⅡ区的22肽和RⅢ区的32肽结合,三个区域相互之间有密切连接位点。利用以上多肽结合的位点,根据单抗结合位点在物理位置上的相近性,我们构建出NS1抗原蛋白的二级结构模拟图。另外,研究证明WNV的NS1抗体可以通过抗体依赖细胞杀伤功能对感染细胞进行吞噬清除,其基础是此抗体须与N在细胞表面表达NS1进行结合。我们用流式细胞术分析了以上148株抗体与感染病毒后细胞膜表面的NS1蛋白结合情况。结果显示,分别有92株、87株、75株和91株抗体与同型病毒感染后细胞膜表面的NS1蛋白结合。按照单抗与多肽的结合位点结果,针对RⅡ区结合的单抗,有82%(18/22)的抗体可以与DENV1-4任一型或多型病毒感染后细胞膜上抗原结合,而针对结合RI和RⅢ的抗体,分别有60%(29/48)和54%(6/11)的抗体可与膜抗原结合。这一结合模式可能与膜型NS1蛋白在感染细胞膜上嵌合形式有关。我们的研究结果与前期对黄病毒NS1的研究数据基本一致。这些数据可为研究NS1的功能和NS1抗体应用打下坚实基础。
第二部分：酶联免疫斑点微中和实验方法检测登革病毒抗体中和活性方法的建立与评价
目前中和实验的金标准方法是PRNT方法,这一方法在六孔板中进行,在病毒感染细胞后,需要在7-10天的时间孵育来形成可以用于检测的蚀斑。如果要完成大量的中和实验标本,这一方法将相当耗时而且结果判断主观性强。微中和实验是一个应用较多的高通量检测方法,这些实验是在96孔板中完成。本研究将评价一个新近开发的基于ELISPOT技术的微中和实验方法,并建立用于检测抗体或血清对登革病毒的中和活性的方法学。按照该法的基本原理是病毒感染细胞后,细胞表面可在较短时间内表达NS1蛋白,将细胞固定处理后,利用抗NS1的抗体可以建立ELISA检测方法,最后用固体原位显色方法可以通过斑点的形成指示病毒对细胞的感染情况。选用VERO E6细胞为病毒感染的靶细胞,通过对病毒加入量和登革各型病毒在感染后的孵育时间的条件优化,建立了ELISPOT方法检测登革各型病毒的方法学。方法学建立后,我们对33株抗登革病毒EDIII单克隆抗体针对四个血清型的中和活性检测,然后再从中选择十株单抗进行PRNT检测它们针对各型病毒的中和活性,两种检测方法应用相同病毒株、抗体稀释梯度进行,并对检测结果进行对比分析。结果显示,基于ELISPOT方法可以在96孔板中加入200PFUs病毒的情况下,形成的斑点清晰可辨,登革病毒四个血清型有不同的感染后孵育时间,DENV-1为4天,DENV-2和DENV-4为2天,DENV-3为3天。对比两种方法,它们之间的相关性较好,r=0.863,P<0.001；如果以PRNT方法为标准,我们将其结果与ELISPOT方法结果进行卡方检验(McNemar),结果显示ELISPOT与PRNT结果无显著性差别(P<1.000)。ELISPOT微中和实验检测这10株单抗针对四型登革病毒的中和效价的敏感性和特异性分别为95.6%(22/23)and88.24%(15/17)。如此可见,基于ELISPOT的微中和实验可以应用于四型登革病毒的中和活性实验检测,而且这种方法省时省力,适合进行批量筛查。
第三部分Ⅰ型登革病毒感染患者康复期血清中和效价分析
对登革病毒引起的疾病目前还未有特异高效的治疗方法或药物,因目前也没有一个能良好复制登革病毒感染后症状的动物模型,使疫苗的研制存在一定的困难。人体在自然感染登革病毒后,免疫状态的维持可以从一个侧面了解机体的抗病毒机制。本研究收集了29例在2006年感染1登革病毒的患者恢复期血清(2010年获取),利用本实验室建立和评价的ELISPOT微中和实验方法,检测它们针对四型登革病毒的中和效价,并对结果进行对比。结果显示,大部分血清对四型病毒有交叉中和活性,对四型登革病毒的中和效价(IC50,血清稀释度倒数)进行非参数检验(Kruska-Wallis H),结果显示,四组中和活性有显著性差异(X2=21.134,P<0.001),其中对同型(DENV-1)的中和活性最高,对DENV-4的中和活性最低。结果表明,机体在感染一种血清型病毒后,对四型病毒均有可能存在一定的四型交叉中和活性,但对同型病毒的中和活性强,而对其他型病的中和活性较弱。这些弱中和活性的交叉抗体,可能会在机体受二次异型病毒感染时引起ADE效应。
小结：
综上所述,本研究在以下方面取得成果：
1、我们合成DENV-1NS1的重叠多肽35条,与148株NS1单克隆抗体进行结合,分析它们的结合位点,有七株抗体的结合位点在不同的两个区域,根据这些位点的物理位置相近性,成功构建出NS1蛋白抗原结构二级结构模拟图,结果与前期对黄病毒NS1蛋白的研究结果一致。研究结果可以在蛋白结晶之前增加对它基本结构的了解。我们还应用流式分析术,筛查了148株单抗与感染细胞膜型NS1抗原结合情况,得到抗体对各型病毒感染分泌于膜上NS1抗原的结合模式。结果显示,分别有92、87、75和91株抗体与同型病毒感染后细胞膜表面的NS1蛋白结合,针对RⅡ区结合的单抗,有82%的抗体可以与DENV1-4任一型或多型病毒感染后细胞膜上抗原结合(18/22),而针对结合RⅠ和RⅢ的抗体,有60%(29/48)和54%(6/11)的抗体可与膜抗原结合,这些结合模式可能与NS1蛋白在感染细胞膜上的嵌合模式有关。
2、建立了基于ELISPOT技术的微中和实验方法检测平台,可以为登革病毒和其他相关病毒提供快速高通量的中和效价测定支撑。同时,我们也对此方法进行评价,对比PRNT方法,方法的特异性和敏感性高,与PRNT有良好的相关性,而且结果判断更加客观,非常适合批量处理的高通量检测实验。
3、从29例1型病毒感染三年后的恢复期病人血清的中和活性检测结果中,获得了它们对同型和异型病毒的中和效价。通过统计分析,对同型病毒的中和活性远强于异型病毒的活性,这可以验证目前的有关初次感染后可以长期存在对同型病毒强的中和保护抗体而对异型病毒有弱中和抗体的理论。
Dengue virus (DENV) is a RNA virus of the family Flaviviridae; genus Flavivirus and primarily transmitted by Aedes mosquitoes among vertebrates. DENV is classified four serotypes according to their reaction to antigen, DENV1-4, each of which is capable of causing same clinical manifestations including dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS).
The transmission of DENV is limited in early mainly in tropical and subtropical regions. But now DENV is endemic in over100countries and regiaons in Africa, America, Southeast Asia and the West Pacific region along with the world economics integration and internetional exchange. Evidence shows that each year around the world50-100million people infected with DENV and there are about0.5million patients would be emergence with DHF or DSS,20-25thousand death cases, mostly children, mortality is about5%. Therefore, DENV infection has become an vital public health problem worldwide, how to effectively control the infection is the key to deal with this problem.
The people who infected by DENV would be asymptomatic or develop flu-like symptoms, self-healing or mild symptoms DF, which appeared mainly in the initial infection patients but most of children need hospital treatment. However, sever symptoms including DHF and DSS would emergence in second infection patients. There is no specific treatment, patients can only accept symptomatic treatment, and no an effective vaccine to prevent DENV infection. Most researchs suggest that antibody-dependent inhancment (ADE) is the main cause of DHF and DSS because there are four different dengue virus serotypes. Studies show that there are various antibodies in sera of DENV infection patients and they have a short-term cross-reactive with all four serotypes. But the cross-reactive antibodies do not have long-term protection function. Instead, they would potentially cause the DHF/DSS. Their binding to virus particle can't block but to enhance virus enter cells through Fc receptor-mediated entry pathway if the cells (such as monocytes and dendritic cells) express Fc receptor, which cotributes to the uptaking of virus and viral replication in cells, leading to infection enhancement and severe dengue disease occurrence. Vaccine is the most effective way to prevent dengue disease transmission, but if the vaccine can only protect one of serotypes of virus, the ADE would occurrence when the patient is infected with the other different serotype virus, which affects the life of the patient. Therefore, the key point in the development of various types of vaccines is to avoid the ADE.
First stain of DENV had been isolated more than sixty years ago, but there are not any effective methods to prevent and cure the diseases caused by DENV, Which is the biggest obstacle to dengue virus infection of immune mechanism is unknown and there is not a focus to study. Dengue virus genomic structure is a single stranded positive-sense RNA molecules,11000nt, encoding three structural proteins, capsid protein (C), membrane protein (M) and envelope protein (E), and seven nonstructural proteins (non-structure protein, NS), NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. The E protein contributes to viral infection through binding the surface of the target cells with the virus receptor, which causes the fusion between viral and cellular membrane. Although the nonstructural proteins are not the composition of mature virus particles, but newly researchs show that they have important function during virus infection and immune process. NS1is the main non-structural protein whichs have three forms. First is the secreted NSl (sNS1), secreted out of infected cells and contributes to the early diagnosis of DENV infection. Second is intracellular form which mainly presents in the intracellular biological membranes. The last form is membrane asociated NS1(mNS1) which existences on the infected cell membrane. The function of mNS1is not clear now, researchs show it maybe involved in viral clearance through cytotoxicity and contributed to viral RNA replication. And because the structure of NS1and the binding with antibody is unknow, so many further researchs would be performed in the futrue. Various technical analysis to predict secondary structure of protein before the crystal structrue accomplishment, such as electron cryo-microscopy, yeast display, point mutation and the peptide binding site analysis. This study will use a panel of148monoantibodies araised from NS1protein to bind the overlapping peptides of NS1and analysis the linear binding sites of mAbs. If an antibody has two binding sites in different domains, the sites would be near in physical location, which would contribute to draw the topography of DENV NS1protein. At the same time, in order to have some knowledge if our panel mAbs can binding the mNS1, we also screen all mAbs binding patterm using flow cytometry, which would produce important foundation data to explained the mNS1fuction in the immune mechanisms of DENV infection.
Vaccine is the most effective way to prevent the infectious disease. So as in DENV transmittion, but DENV have four different serotypes, which would cause ADE effect in secondary infection and severe diseases would occurance. Thus the value of vaccine against DENV is not only the protection but also no ADE effect. Unfortunately, up to day, there is no a satisfied vaccine development, either a single valence or multivalent vaccince. Neutralization test is most important technology during vaccine research. The traditional method of neutralization is plaque reduction neutralization test (PRNT), but it is time-consuming, laborious and lack of objective judgment. Many researchers want to develop a method to replace it including based on flow cytometric method, the based on ELISA method. Although these methods can save time and labor, but still exist some problems, such as need more virus dosage than PRNT, don't be available for some clinical isolation virus and have poor repeatability. Recently, researchs develop a new microneutralization to detecte neutralization of antibody beaed on enzyme-linked immunospot-based microneutralization assay (ELISPOT). Our study would evaluation of this method and establish a platform to detecte the neutralization activity of the antibodies againsts DENV and a panel of29Convalescent sera originate the patients who infected by DENV-1over three years ago.
This research is divided into three parts as flowing:
Part I:Draw the topography of DENV NS1protein according to analysis the binding sites of their monoantibodies
More and more researchs about DENV infectionimmune mechanism focus on NS1protein because it can avoid the ADE. NS1has three species and the mNS1maybe induce the cy to toxic function and contribute to the viral clearance. Some researchs' results showed that NS1protein had three regions, RI, RII and RIII, but the exact function of each domain is still not proved because the structure don't be developed yet. Our research focues on the binding site of a panel of148mAbs to the NS1according to detection the binding between mAbs and overlapping peptides of NS1(total35peptides and every peptide overlap5aa with upper peptide except the last overlap8aa). The results show that a total of82mAbs can bind to peptides of NS1,7of82mAbs have two sites in different domain,1A11A9and2E8A5binds to NO.1peptide in RⅠ domain and NO.22peptide in RⅡ domain,6D14A4binds to NO.1and14peptide in RⅠ domain and NO.22peptide in RⅡ domain,1F32A1binds to NO.1peptide in RⅠ domain and NO.31peptide in RⅡ,7E1A4and7E9A2binds to NO.5peptide in RⅠ domain and NO.27peptide in RⅢ domain,1B7A1binds to NO.22peptide in RⅡ and NO.32peptide in RⅢ domain, which indicates that there is a close connection among three domain. We draw the secondary structure simulation diagram of DENV NS1protein according to the binding sites because the different binding sites of an antibody would be near in physical location. Some researchs indicated that antibodies to WNV NS1protein can clear infected cell through antibody dependent cell cytotoxicity, which is based on the binding mNS1antigen. We used flow cytometry to analysis the binding patterns of the panel of148mAbs. The results show that92,87,75and91mAbs can bind to mNS1of hemeotype DENV-1, DENV-2, DENV-3and DENV-4respectively infection cells. And82%(18/22) mAbs to RⅡ domain and60%(29/48) mAbs to RⅠ can bind to mNS1. Our study results are similar to the former research on the NS1protein of Flavivirus. These data would contribute to the study of NS1function and the mAbs application research.
Part Ⅱ:Establish and Evaluation of a Micro-neutralization Test Based on ELISPOT
PRNT is the gold standard method for neutralization test; this method is performed in6-well format and required for7-10days incubation to form visible plaques. If there are a large number of neutralization test specimens to perform, it would be quite time-consuming and labor-intensive using PRNT. Micro neutralization test is performed in96-well format and widely used in high throughput screening. This research evaluates a newly developed microneutralization test based on ELISPOT (ELISPOT-MNT). For estabilish the methodology, we used VERO E6cell as the target cell of DEVN infection and optimize the virus dosage and the incubation time after DENV infection. After the methodology being established, the neutralization activity of33mAbs, arised from the domain III of the DENV envelope protein (EDIII) against four serotype of DENV, was performed using the method. A panel of10from33mAbs was selected to evaluate this method using PRNT. The same virus stain and mAbs dilution gradient were used in two methods and compare the results. The results show that the spots are clear with the virus dosage200pfus per well of96-well plate when the infected cell incubate for2-4days. The incubation time is4days for DENV-1, DENV-2and DENV-4for2days, DENV-3for3days. The comparison of two methods show that there is a good relationship between them(r=0.863, P<0.001). McNemar's association test was used to measure the disagreement between the two assay methods. There was no significant (P=1.000) disagreement between them.. The method showed a sensitivity of95.6%(22/23) and a specificity of88.24%(15/17) when these10MAbs were tested against four DENV serotype strains and used the PRNT as the reference. Therefore, ELISPOT-MNT shows potential as a primary method for screening neutralization activities of large numbers of antibodies.
Part Ⅲ The Pattern of Neutralization in Convalescent Sera from the Patients with DENV-1Infection
There are not any specific and efficient treatments or drugs for the diseases caused by DENV. Presently, the complaints of dengue can not be well replicated in animal model, which hamper the vaccine research. We can learn some about the antivital mechanism from the immune status of human body after naturally infected with DENY We used the ELISPOT-MNT to detecte the neutralization activity to four DENV serotypes of29sera from the patients infected with DENV-1in2006and compared the results. The results showed that most of sera have cross-neutralization activity to four serotype DENVs. The mean values(IC50, the recip of serum dilution) of DENV-1and DENV-2,-3and-4were analysised by kruska-Wallis H test with SPSS13.0, the result showed that there is significant difference among four DENV serotypes (X2=21.134, P<0.001), the value of DENV-1is the highest and DENV-4is the lowest. Our study indicates that there is cross-neutralization activity to four serotype DENVs in patient primary infection of one serotype DENV, but the strength to heterotype is lower than homeotype. The weak cross neutralization antibodies maybe contribute to the ADE effect when the second infection of different serotype DENV occurrence. Summarization:
1, We sythesize35overlapping peptides of DENV-1NS1that be used to bind to a panel of148mAbs against NS1and anslysis the binding site. There are seven mAbs binding to different domain of NS1, which is the base to draw a secondary structure simulation diagram of DENV NS1protein. The results are similar to to the former research on the NS1protein of Flavivirus. This research would contribute to learning the profile of NS1protein before the crystal constructure developed. We also have screened the binding mode to mNS1of148mAbs and obtained the profile of them using flow cytometric. The results show that92,87,75and91mAbs can bind to mNS1of hemeotype DENV-1, DENV-2, DENV-3and DENV-4respectively infection cells. And82%(18/22) mAbs to RII domain and60%(29/48) mAbs to RI can bind to mNS1.
2, The microneutralization test based on the ELISPOT was developed to detecte the neutralization activity of antibody or immune sera against DENV. It is a time-saving and high-throughput method, which would be the support of the virus nerutralization research. We also evaluated this method compared with PRNT. The results show that it has high specificity(95.6%), high sensitivity(88.24%), good correlation with PRNT(r=0.863, P<0.001) and more objective judgement of result than PRNT. ELISPOT-MNT is availabe for batch processing.
3, We obtain the neutralization titer profiles against homeotypic and heterotypic DENV of29convalescent sera from patients with DENV-1infection three years ago.The statistical analysis indicates that the neutralization activity against homeotypic virus is far stronger than heterotypic virus, which verifies the theory that antibodies arise primary infection can strongly protecte the challenge of hemeotypic virus but weak to heterotypic virus.
引文
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[3]Scott B Halstead. Dengue. Lancet 2007,370:1644-1652.
[4]秦鄂德,秦成峰,姜涛.2008,登革病毒与登革病毒病.北京：科学出版社.
[5]Chaturvedi, U.C., R. Shrivastava and R. Nagar, Dengue vaccines:problems and prospects. Indian J Med Res,2005.121(5):p.639-652.
[6]Falgout B., Chanock R., and Lai C.J. Proper processing of dengue virus nonstructural glycoprotein NS1 requires the N-terminal hydrophobic signal sequence and the downstream nonstructural protein NS2a. J Virol 1989, 63(5):1852-1860.
[7]Brandt W.E., Cardiff R.D., and Russell P.K. Dengue virions and antigens in brain and serum of infected mice. J Virol,1970,6(4),500-506.
[8]David A Muller and Paul R Young.The Many Faces of the Flavivirus Non-structural Glycoprotein NS1. Molecular Virology and Control of Flaviviruses. Pei-Yong Shi(Ed.). Caister Academic Press (Norfolk, UK),2012, p51-75.
[9]Muller DA, Landsberg MJ, Bletchly C, et al. Structure of the Dengue Virus Glycoprotein NS1 by Electron Microscopy and Single Particle Analysis. J Gen Virol.2012,93(Pt 4):771-779.
[10]Irina Gutschea, Fasseli Coulibalya, James E. Voss, et al. Secreted dengue virus nonstructural protein NS1 is an atypical barrel-shaped high-density lipoprotein. Proc Natl Acad Sci,2011,108(19):8003-8008.
[11]Kyung Min Chung, Grant E. Nybakken, Bruce S. Thompson,et al. Antibodies against West Nile Virus Nonstructural Protein NS1 Prevent Lethal Infection through Fcy-Receptor-Dependent and -Independent Mechanisms. Journal of Virology,2006,80(3):1340-1351.
[12]Ding X, Hu D, Chen Y, et al. Full serotype-and group-specific NS1 capture enzyme-liked immunosorbent assay for rapid differential diagnosis of dengue virus infection. Clin Vaccine Immunol,2011,18(3):430-434.
[13]Kumarasamy V, Chua SK, Hassan Z, et al. Evaluating the sensitivity of a commercial dengue NS1 antigen-capture ELISA for early diagnosis of acute dengue virus infection. Singapore Med J,2007,48(7):669-673.
[14]Shrivastava A, Dash PK, Tripathi NK, et al. Evaluation of a commercial Dengue NS1 enzyme-linked immunosorbent assay for early diagnosis of dengue infection. Indian J Med Microbiol,2011,29(1):51-55.
[15]D.H. Libraty, P.R. Young, D. Pickering, et al. High circulating levels of the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever. J Infect Dis,2002, 186(8):1165-1168.
[16]Vianney Tricou,Hang TT Vu, Nhu VN Quynh,et al.Comparison of two dengue NS1 rapid tests for sensitivity, specificity and relationship to viraemia and antibody responses. BMC Infectious Diseases 2010,10:142
[17]Shi-Wei Lin, Yung-Chun Chuang, Yee-Shin Lin, Et al. Dengue virus nonstructural protein NS1 binds to prothrombin/thrombin and inhibits prothrombin activation. Journal of Infection,2012,64(3):325-334.
[18]Panisadee Avirutnan, Lijuan Zhang, Nuntaya Punyadee, et al. Secreted NS1 of Dengue Virus Attaches to the Surface of Cells via Interactions with Heparan Sulfate and Chondroitin Sulfate E. PLoS Pathogens,2007,3 (11):e183.
[19]Amorim JH, Diniz MO, Cariri FA, et al. Protective immunity to DENV2 after immunization with a recombinant NS1 protein using a genetically detoxified heat-labile toxin as an adjuvant.Vaccine,2012,30(5):837-845.
[20]Andrew K. I. Falconar and Fernando Martinez.The NS1 Glycoprotein Can Generate Dramatic Antibody-Enhanced Dengue Viral Replication in Normal Out-Bred Mice Resulting in Lethal Multi-Organ Disease. PLoS One.2011, 6(6):e21024.
[21]Kanesa-Thasan N, Sun W, Kim-Ahn G, Van Albert S, Putnak JR, King A, et al.Safety and immonogenicity of attenuated dengue virus vaccines (Aventis Pasteur) in human volunteers. Vaccine 2001,19:3179-3188.
[22]Edelman R, Wasserman SS, Bodison SA, Putnak RJ, Eckels KH, Tang D, et al. Phase I trial of 16 formulations of a tetravalent live-attenuated dengue vaccine. Am J Trop Med Hyg,2003,69:48-60.
[23]Blaney JE, Durbin AP, Murphy BR, et al. Development of a live attenuated dengue virus vaccine using reverse genetics. Virol Immunol,2006,19:10-32.
[24]Rothman AL. Dengue:defining protective versus pathology immunity. J Clin Invest,2004,113:948-951.
[25]Yauch, L.E. and S. Shresta, Mouse models of dengue virus infection and disease. Antiviral Res,2008,80(2):87-93.
[26]Clements DE, Coller BA, Lieberman MM, et al. Development of a recombinant tetravalent dengue virus vaccine:immunogenicity and efficacy studies in mice and monkeys. Vaccine,2010,28(15):2705-2715.
[27]Alan L. Rothman. Immunity to dengue virus:a tale of original antigenic sin and tropical cytokine storms. Nature Reviews Immunology,2011,11(8):532-543.
[28]Muylaert L.R., Chembers T.J., Galler R., et al. Mutagenesis of the N-linked glycosylation sites of the yellow fever virus NS1 protein:effects on virus replication and mouse neurovirulence. Virology,1996,222(1):159-168.
[29]Michael S. Diamond, Theodore C. Pierson and Daved H. Fremont. The Structural Immunology of Antibody Protection against West Nile Virus. Immunol Rev.2008,225:212-225.
[2]Scott B Halstead. Dengue. Lancet 2007,370:1644-1652.
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[13]Vianney Tricou, Nguyet Nguyen Minh, Jeremy Farrar, et al. Simmons Kinetics of Viremia and NS1 Antigenemia Are Shaped by Immune Status and Virus Serotype in Adults with Dengue. Plos Neglect Trop D,2011, 5(9):e1309.
[14]Muller DA, Landsberg MJ, Bletchly C, et al. Structure of the Dengue Virus Glycoprotein NS1 by Electron Microscopy and Single Particle Analysis. J Gen Virol,2012,93(4):771-779.
[15]Irina Gutschea, Fasseli Coulibalya, James E. Voss, et al. Secreted dengue virus nonstructural protein NS1 is an atypical barrel-shaped high-density lipoprotein. Proc Natl Acad Sci,2011,108(19):8003-8008.
[16]Kyung Min Chung, Grant E. Nybakken, Bruce S. Thompson,et al. Antibodies against West Nile Virus Nonstructural Protein NS1 Prevent Lethal Infection through Fcy-Receptor-Dependent and -Independent Mechanisms. Journal of Virology,2006,80(3):1340-1351.
[17]Michael S. Diamond, Theodore C. Pierson and Daved H. Fremont. The structural immunology of antibody protection against West Nile virus. Immunological Reviews.2008,225:212-225.
[18]Russell PK, Nisalak A. Dengue virus identification by the plaque reduction neutralization test. J Immunol.1967,99:291-296.
[19]World Health Organization Department of Immunization Vaccines Biologicals.2007. Guidelines for plaque-reduction neutralization testing of human antibodies to dengue viruses. World Health Organization, Geneva, Switzerland. WHO/IVB/07.07. http://whqlibdoc.who.int/hq/2007 /who_ivb_07.07_eng.pdf.
[20]Thomas SJ, Ananda Nisalak, Kathryn B. Anderson et al. Dengue plaque reduction neutralization test (PRNT) in primary and secondary dengue virus infections:how alterations in assay conditions impact performance. Am J Trop Med Hyg,2009,81:825-833.
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[1]Kerrie Vaughan, Jason Greenbaum, Martin Blythe, et al. Meta-analysis of All Immune Epitope Data in the Flavivirus Genus:Inventory of Current Immune Epitope Data Status in the Context of Virus Immunity and Immunopathology. Viral Immunology,2010,23(3):259-284.
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[3]Vianney Tricou, Nguyet Nguyen Minh, Jeremy Farrar. Kinetics of Viremia and NS1 Antigenemia Are Shaped by Immune Status and Virus Serotype in Adults with Dengue. Plos Neglect Trop D.2011,5(9):e1309.
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[6]Kyung Min Chung, Grant E. Nybakken, Bruce S. Thompson, et al. Antibodies against West Nile Virus Nonstructural Protein NS1 Prevent Lethal Infection through Fcγ Receptor-Dependent and -Independent Mechanisms. Journal of Virology,2006,80(3):1340-1351.
[7]David A. Muller, Michael J. Landsberg, Cheryl Bletchly, et al. Structure of the Dengue Virus Glycoprotein NS1 by Electron Microscopy and Single Particle Analysis. J Gen Virol,2012,93(pt4):771-779.
[8]Irina Gutschea, Fasseli Coulibalya, James E. Voss, et al. Secreted dengue virus nonstructural protein NS1 is an atypical barrel-shaped high-density lipoprotein. Proc Natl Acad Sci,2011,108(19):8003-8008.
[9]Kyung Min Chung, Grant E. Nybakken, Bruce S. Thompson,et al. Antibodies against West Nile Virus Nonstructural Protein NS1 Prevent Lethal Infection through Fcy-Receptor-Dependent and -Independent Mechanisms. Journal of Virology,2006,80(3):1340-1351.
[10]Brian R. Murphy and Stephen S. Whitehead. Immune Response to Dengue Virus and Prospects for a Vaccine. Annu Rev Immunol,2011,29:587-619.
[11]Rey FA, Heinz FX, Mandl C, et al. The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution. Nature,1995,375(6529):291-298.
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[18]Shrivastava A, Dash PK, Tripathi NK, et al. Evaluation of a commercial Dengue NS1 enzyme-linked immunosorbent assay for early diagnosis of dengue infection. Indian J Med Microbiol,2011,29(1):51-55.
[19]Muylaert L.R., Chembers T.J., Galler R., et al. Mutagenesis of the N-linked glycosylation sites of the yellow fever virus NS1 protein:effects on virus replication and mouse neurovirulence. Virology,1996,222(1):159-168.
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[22]Amorim JH, Diniz MO, Cariri FA, et al. Protective immunity to DENV2 after immunization with a recombinant NS1 protein using a genetically detoxified heat-labile toxin as an adjuvant.Vaccine,2012,30(5):837-845.
[23]D.H. Libraty, P.R. Young, D. Pickering, et al. High circulating levels of the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever. J Infect Dis, 2002,186(8):1165-1168.
[24]Shi-Wei Lin, Yung-Chun Chuang, Yee-Shin Lin, et al. Dengue virus nonstructural protein NS1 binds to prothrombin/thrombin and inhibits prothrombin activation. Journal of Infection,2012,64(3):325-334.
[25]Chiou-Feng Lin, Huan-Yao Lei, Ai-Li Shiau, et al. Endothelial Cell Apoptosis Induced by Antibodies Against Dengue Virus Nonstructural Protein 1 Via Production of Nitric Oxide. J Immunol,2002,169(2):657-664.
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[1]George R, Lum LCS. Clinical spectrum of dengue infection. In Gubler DJ and Kuno G (ed), Dengue and dengue hemorrhagic fever,3rd ed. CAB International Press, London, United Kingdom.1997, p 89-114.
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[4]Whitehead SS, Blaney JE, Durbin AP, et al. Prospects for a dengue virus vaccine. Nature Reviews Microbiology,2007,5(7):518-528.
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[8]Shanaka WW, Alcena DC, Rose RC, et al. An automated Dengue virus microneutralization plaque assay performed in human Fc_ receptor-expressing CV-1 cells. Am J Trop Med Hyg,2009,80(1):61-65.
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[14]World Health Organization Department of Immunization Vaccines Biologicals.2007. Guidelines for plaque-reduction neutralization testing of human antibodies to dengue viruses. World Health Organization, Geneva, Switzerland. WHO/IVB/07.07. http://whqlibdoc.who.int/hq/2007/who_ivb_07.07_eng.pdf.
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[1]Scott B Halstead. Dengue.Lancet,2007,370:1644-1652.
[2]Halstead SB. Dengue hemorrhagic fever:two infections and antibody dependent enhancement, a brief history and personal memoir. Rev Cuba Med Trop.2002,54(3):171-179.
[3]Halstead SB. Pathogenesis of dengue:challenges to molecular biology. Science, 1988,239(4839):476-481.
[4]Chaturvedi, U.C., R. Shrivastava and R. Nagar. Dengue vaccines:problems and prospects. Indian J Med Res,2005,121(5):639-652.
[5]Yauch, L.E. and S. Shresta. Mouse models of dengue virus infection and disease. Antiviral Res,2008,80(2):87-93.
[6]Clements DE, Coller BA, Lieberman MM, et al. Development of a recombinant tetravalent dengue virus vaccine:immunogenicity and efficacy studies in mice and monkeys. Vaccine,2010,28(15):2705-2715.
[7]Hu D, Di B, Ding X, et al. Kinetics of non-structural protein 1, IgM and IgG antibodies in dengue type 1 primary infection. Virol J,2011,8:47.
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[15]Sujan Shresta. Role of Complement in Dengue Virus Infection:Protection or Pathogenesis. Mbio,2012,3(1):e00003-12.
[1]Lindenbach BD, Thiel HJ, Rice CM.2007. Flaviviridae:the viruses and their replication, p 1103-1113. In Knipe DM and Howley PM (ed), Fields virology, 5th ed. Lippincott Williams & Wilkins, Philadelphia, PA.
[2]Murphy, B.R. and S.S. Whitehead, Immune response to dengue virus and prospects for a vaccine. Annu Rev Immunol,2011.29:p.587-619.
[3]Scott B Halstead. Dengue. Lancet 2007,370:1644-1652.
[4]秦鄂德,秦成峰,姜涛.2008,登革病毒与登革病毒病.北京：科学出版社.
[5]Chaturvedi, U.C., R. Shrivastava and R. Nagar, Dengue vaccines:problems and prospects. Indian J Med Res,2005.121(5):p.639-652.
[6]Falgout B., Chanock R., and Lai C.J. Proper processing of dengue virus nonstructural glycoprotein NS1 requires the N-terminal hydrophobic signal sequence and the downstream nonstructural protein NS2a. J Virol 1989, 63(5):1852-1860.
[7]Brandt W.E., Cardiff R.D., and Russell P.K. Dengue virions and antigens in brain and serum of infected mice. J Virol,1970,6(4),500-506.
[8]David A Muller and Paul R Young.The Many Faces of the Flavivirus Non-structural Glycoprotein NS1. Molecular Virology and Control of Flaviviruses. Pei-Yong Shi(Ed.). Caister Academic Press (Norfolk, UK),2012, p51-75.
[9]Muller DA, Landsberg MJ, Bletchly C, et al. Structure of the Dengue Virus Glycoprotein NS1 by Electron Microscopy and Single Particle Analysis. J Gen Virol.2012,93(Pt 4):771-779.
[10]Irina Gutschea, Fasseli Coulibalya, James E. Voss, et al. Secreted dengue virus nonstructural protein NS1 is an atypical barrel-shaped high-density lipoprotein. Proc Natl Acad Sci,2011,108(19):8003-8008.
[11]Kyung Min Chung, Grant E. Nybakken, Bruce S. Thompson,et al. Antibodies against West Nile Virus Nonstructural Protein NS1 Prevent Lethal Infection through Fcy-Receptor-Dependent and -Independent Mechanisms. Journal of Virology,2006,80(3):1340-1351.
[12]Ding X, Hu D, Chen Y, et al. Full serotype-and group-specific NS1 capture enzyme-liked immunosorbent assay for rapid differential diagnosis of dengue virus infection. Clin Vaccine Immunol,2011,18(3):430-434.
[13]Kumarasamy V, Chua SK, Hassan Z, et al. Evaluating the sensitivity of a commercial dengue NS1 antigen-capture ELISA for early diagnosis of acute dengue virus infection. Singapore Med J,2007,48(7):669-673.
[14]Shrivastava A, Dash PK, Tripathi NK, et al. Evaluation of a commercial Dengue NS1 enzyme-linked immunosorbent assay for early diagnosis of dengue infection. Indian J Med Microbiol,2011,29(1):51-55.
[15]D.H. Libraty, P.R. Young, D. Pickering, et al. High circulating levels of the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever. J Infect Dis,2002, 186(8):1165-1168.
[16]Vianney Tricou,Hang TT Vu, Nhu VN Quynh,et al.Comparison of two dengue NS1 rapid tests for sensitivity, specificity and relationship to viraemia and antibody responses. BMC Infectious Diseases 2010,10:142
[17]Shi-Wei Lin, Yung-Chun Chuang, Yee-Shin Lin, Et al. Dengue virus nonstructural protein NS1 binds to prothrombin/thrombin and inhibits prothrombin activation. Journal of Infection,2012,64(3):325-334.
[18]Panisadee Avirutnan, Lijuan Zhang, Nuntaya Punyadee, et al. Secreted NS1 of Dengue Virus Attaches to the Surface of Cells via Interactions with Heparan Sulfate and Chondroitin Sulfate E. PLoS Pathogens,2007,3 (11):e183.
[19]Amorim JH, Diniz MO, Cariri FA, et al. Protective immunity to DENV2 after immunization with a recombinant NS1 protein using a genetically detoxified heat-labile toxin as an adjuvant.Vaccine,2012,30(5):837-845.
[20]Andrew K. I. Falconar and Fernando Martinez.The NS1 Glycoprotein Can Generate Dramatic Antibody-Enhanced Dengue Viral Replication in Normal Out-Bred Mice Resulting in Lethal Multi-Organ Disease. PLoS One.2011, 6(6):e21024.
[21]Kanesa-Thasan N, Sun W, Kim-Ahn G, Van Albert S, Putnak JR, King A, et al.Safety and immonogenicity of attenuated dengue virus vaccines (Aventis Pasteur) in human volunteers. Vaccine 2001,19:3179-3188.
[22]Edelman R, Wasserman SS, Bodison SA, Putnak RJ, Eckels KH, Tang D, et al. Phase I trial of 16 formulations of a tetravalent live-attenuated dengue vaccine. Am J Trop Med Hyg,2003,69:48-60.
[23]Blaney JE, Durbin AP, Murphy BR, et al. Development of a live attenuated dengue virus vaccine using reverse genetics. Virol Immunol,2006,19:10-32.
[24]Rothman AL. Dengue:defining protective versus pathology immunity. J Clin Invest,2004,113:948-951.
[25]Yauch, L.E. and S. Shresta, Mouse models of dengue virus infection and disease. Antiviral Res,2008,80(2):87-93.
[26]Clements DE, Coller BA, Lieberman MM, et al. Development of a recombinant tetravalent dengue virus vaccine:immunogenicity and efficacy studies in mice and monkeys. Vaccine,2010,28(15):2705-2715.
[27]Alan L. Rothman. Immunity to dengue virus:a tale of original antigenic sin and tropical cytokine storms. Nature Reviews Immunology,2011,11(8):532-543.
[28]Muylaert L.R., Chembers T.J., Galler R., et al. Mutagenesis of the N-linked glycosylation sites of the yellow fever virus NS1 protein:effects on virus replication and mouse neurovirulence. Virology,1996,222(1):159-168.
[29]Michael S. Diamond, Theodore C. Pierson and Daved H. Fremont. The Structural Immunology of Antibody Protection against West Nile Virus. Immunol Rev.2008,225:212-225.