登革病毒包膜E蛋白Ⅲ区抗体中和表位与功能研究
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摘要
登革病毒(DENV)是引起人类疾病的蚊传播病毒,它能导致无症状感染、登革热(DF)和更严重的登革出血热(DHF)或登革休克综合征(DSS)。登革疾病的负担在热带和亚热地区日益增加,特别是在南美、东南亚和加勒比地区,在非洲和中东的报道也越来越多。而在我国南方地区,几乎每年都发生DF流行。登革疾病的流行形势越来越严峻,其致病机理还没有完全弄清楚,但是病毒、宿主的免疫史和个体的遗传背景在登革严重疾病的发生中都起着重要作用。其中,病毒本身的作用很关键。任何一种血清型DENV造成的初次感染都可能使个体在感染不同于初次感染的异型血清型(heterotypic serotype) DENV时,病毒载量增加、潜伏期缩短,从而引起DHF/DSS的机率也大大增加。这与引起再次感染的病毒血清型、基因型、病毒序列以及初次与再次感染的间隔时间都有关系。
为应对日益增加的登革疾病,很多研究机构都在加紧研发登革疫苗。目前,已经有几类疫苗在进行临床前的评价,包括减毒活疫苗、灭活病毒疫苗、基于重组蛋白的亚单位疫苗和裸DNA疫苗等。由于采用基于减毒活疫苗的传统方法,还没有研制出可批准使用的疫苗,研究者更多地将目光转向重组亚单位疫苗。尽管研究登革疫苗是可行的,但是其研发还是面临着相当严峻的挑战：(1)登革疫苗必须能对4种血清型DENV的感染同时提供保护作用,因而需要研制四价疫苗；(2)登革疫苗要能提供长期的保护。因为有研究报道,初次感染20年以后的再感染时仍会发生DHF：(3)还没有合适的复制登革疾病的动物模型；(4)尽管中和抗体的保护性作用已广为接受,但其与实际保护效果的关系仍需确定；(5)在DENV的传播模式和病毒株改变时,登革疫苗需要重新评价。因此,要研制出可靠的登革疫苗,就要解决两方面的难题：一是疫苗的效果,主要指疫苗的保护性；二是疫苗的安全性问题。
DENV的包膜由脂质双层构成,含有两类包膜相关蛋白：膜蛋白(envelope,E)和M蛋白(membrane, M)。E蛋白通过结合到细胞受体而参与病毒的吸附及穿入后与内吞体的融合。E蛋白含有3个不同的结构域：结构域I(EDⅠ)、EDⅡ、 EDⅢ。EDⅢ在病毒颗粒表面是暴露且可及的,而且,重组EDⅢ也能抑制病毒的感染性。多项研究发现,EDⅢ含有单一血清型特异性(type-specific, TS)表位、针对2-3种血清型DENV的亚复合体反应(sub-complex reactive, sCR)表位以及同时针对4种血清型DENV的复合体反应(complex reactive, CR)表位,多株鼠源单克隆抗体也分别定位到这些表位。研究表明,许多TS和sCR鼠单抗是针对DENV单抗中起主要中和及保护作用的单抗。由此,登革病毒EDⅢ蛋白也成为亚单位疫苗很有前景的备选靶标。另外,最近有关人源单抗的研究认为,针对EDⅢ的单抗仅占人免疫血清中抗DENV总抗体中的很小一部分,而且EDⅢ上的表位不是人抗DENV中和抗体的主要靶标。尽管如此,鼠源单抗的抗原性表位及其中和活性谱仍有许多方面未研究清楚,更加深入的研究对加深了解体液免疫反应的复杂性、改善登革疫苗的免疫策略或研究治疗性抗体均有不可替代的作用。
对EDⅢ单抗的抗原性表位和中和活性的研究可加深对疫苗效果的研究,但是,还有一个难题就是疫苗的安全性问题。前已述及,任何一种血清型登革病毒引起初次感染后,由不同血清型引起再次感染时,登革疾病加重的机率大大增加,其背后最主要的机制就是抗体依赖的增强作用(antibody-dependent enhancement, ADE),即先前存在交叉反应弱中和抗体能与登革病毒结合,增强表达FcγR受体的细胞的感染水平。最近,Dejnirattisai W.等的研究成果也呈现出ADE发生的复杂性,即抗E蛋白抗体和抗前膜(precursor membrane, prM)抗体均能增强DENV感染。要研制能提供长期保护的四价疫苗,还没有合适的登革疾病的动物模型对登革疫苗进行安全性评价,因此,登革疫苗的体外评价显得格外重要。
因此,在本实验室前期研究中,利用登革病毒重组EDⅢ蛋白免疫小鼠,制备了107株与DENV重组EDⅢ反应的单抗。本研究中,我们首先采用酶联免疫吸附试验(ELISA)和间接免疫荧光(IFA)测定法对107单抗的结合特异性(binding specificity)进行检测,随后,我们利用已建立的简单高通量的微中和试验(ELISPOT-MNT)法对筛选出来的94株单抗的中和活性谱进行全面的检测和分析。在此基础上,对94株单抗的抗原性位点进行鉴定分析,以深入认识重组EDⅢ免疫诱导产生单抗的抗原性位点及其与体外保护性作用的关系。然后,我们创新性地建立了简单高通量的测定DENV ADE效应的方法学,以进行疫苗安全性的评价和登革疾病的风险评估。
由此,本研究的主要目的有三个：第一,对107株单抗的结合特异性和体外保护性作用进行鉴定；第二,对筛选出来的94株单抗识别的抗原性位点进行分析；第三,建立一种简单高通量的测定登革抗体ADE活性的新方法,实现对登革疫苗体外安全性评价。
由此,本研究共分为如下三个部分的内容：
第一部分登革病毒包膜E蛋白Ⅲ区(EDⅢ)单克隆抗体的结合特异性和中和活性鉴定
本实验室在前期工作中,采用毕赤酵母表达的4种血清型DENV的重组EDⅢ作为免疫原,单独或混合免疫BALB/c小鼠,制备了107株单抗。本研究中,我们对这些单抗针对不同血清型的结合特异性或交叉反应性、中和活性进行了详细鉴定。首先,ELISA和IFA测定结果显示,有94株单抗对重组EDⅢ或DENV感染的C6/36细胞的结合呈现特异性或交叉反应性,包括42株针对单一血清型DENV的TS单抗、26株针对2-3种血清型的sCR单抗及23株针对4种血清型的CR单抗,还有3株黄病毒交叉反应单抗。但是,ELISA和IFA的鉴定结果并不完全相符,推测96孔板上包被的重组EDⅢ蛋白的构象与感染C6/36细胞的登革病毒表面E蛋白的构象的差异可能造成了这种不一致。
随后,我们借助已建立的ELISPOT-MNT微中和试验对以上单抗进行体外保护性测定。根据单抗对病毒的50%抑制浓度(50%inhibition concentration, IC50)的大小,将单抗分为三类：强(IC50≤1μg/ml),中等(1μg/ml50μg/ml)。DENV-1TS单抗(17株)、DENV-4TS单抗(14株)对相应的血清型病毒显示出强、中、无的复杂中和活性；DENV-2TS单抗(7株)对DENV-2均无中和活性；DENV-3TS单抗(3株)对DENV-3均显强中和活性。sCR单抗(26株)与CR单抗(23株)的中和活性谱均较复杂,即单抗对不同血清型DENV出现强、中或无中和活性。还有3株黄病毒交叉反应单抗均无中和活性。以上结果表明,单抗的结合特性与中和活性并不平行,即结合特异性相似的单抗可能显示出不同的中和活性谱,我们分析以上差异可能是由于单抗对孤立的重组EDⅢ蛋白与天然病毒粒子表面展示的E蛋白的识别有差异。以上研究明确了众多EDⅢ免疫单抗的结合能力和体外中和能力,为后续DENV EDⅢ单抗的抗原性位点鉴定、深入的结构研究和全面的功能测定奠定了良好的基础。
第二部分登革病毒包膜E蛋白Ⅲ区(EDⅢ)单克隆抗体的表位鉴定
DENV的EDⅢ区被认为是研究亚单位疫苗有前景的抗原性区域。但是,EDⅢ上的抗原性位点及其与EDⅢ免疫诱导产生单抗的体外保护作用的关系还没有完全研究清楚。由此,本研究在94株单抗的结合特性及中和活性鉴定基础上,对其抗原性位点进行详细的鉴定。
首先,我们对94株单抗进行合成重叠多肽扫描分析,发现12株单抗(2株DENV-2TS单抗、5株sCR单抗、1株CR单抗和2株黄病毒交叉反应单抗)分别特异性地识别几个不同的线性表位,但这12株单抗均无中和活性。相比之下,23株CR单抗中有15株(约占2/3)特异性地识别相同的aa310-319序列,进一步的序列比对和残基替换证实aa309-320表位是各血清型DENV间高度保守且免疫优势的复合体表位。
值得注意的是,15株CR单抗与4个血清型DENV的重组EDⅢ或感染的C6/36细胞呈现较强交叉反应,但是,它们对4个型DENV具有复杂的中和活性谱,大多数只显示弱或中等中和活性。如何解释这些单抗的结合交叉反应性与中和能力的差异呢?我们继续采用位点定向突变和酵母表面展示分析,发现ABloop上的Q316和H317是该表位的关键残基；而且,三维建模分析表明,该表位在EDⅢ表面充分暴露,但在E蛋白二聚体和三聚体表面的可及性差,特别是在成熟病毒粒子表面可及性更差。可以认为,重组EDⅢ作为免疫原可诱导产生针对在病毒粒子表面不暴露的单抗,因此这些单抗的中和活性较弱。通过研究,我们合理地解释了定位至EDⅢ AB loop的弱中和活性的交叉反应单抗与重组EDⅢ免疫方式的关系,加深了对EDⅢ亚单位疫苗免疫策略的认识,为后续改进基于EDⅢ的疫苗免疫方案提供了依据。
第三部分建立简易、高通量的方法评价登革病毒抗体的功能
开发登革疫苗需要尽可能减少潜在的ADE风险,从而要求针对DENV的4个血清型获得均衡的保护性抗体反应。在缺乏合适的登革疾病动物模型的情况下,有必要开发简单、可靠且高通量的方法用于评价登革热疾病的风险和疫苗的安全性。
在本部分研究中,我们采用表达Fcy受体的K562细胞株和已知具有增强活性的单抗,创新性地建立了通过“NS1抗原捕获ELISA"检测培养上清中NS1抗原的简单高通量的登革抗体的ADE检测方法(ELISA-ADE方法)。为评价该方法,我们同时采用2种方法(即新建立的NS1抗原定量方法和传统的病毒滴度测定方法)检测抗体的ADE活性。发现两种方法的动力学相当,线性相关分析证明,这2种方法具有很好的一致性(R=0.938,P=0.000)。随后,我们还采用同为96孔板检测和读板的ELISA-ADE法和已经建立的ELISPOT-MNT微中和试验评价2株黄病毒交叉反应鼠单抗(4G2和2A10G6)和4份DENV-1感染患者的恢复期血清的增强活性和中和抗体活性。结果表明,新方法可获得特征性的剂量反应ADE图谱,而且增强效应发生在亚中和浓度,ADE效应与经ELISPOT-MNT测定获得的中和效应具有合理的相互关系。
本研究新建立的简单高通量的ELISA-ADE法和已建立ELISPOT-MNT检测方法对DENV的增强和中和抗体活性的功能性评价非常有价值。它们能在大规模研究中进行DENV疫苗的安全性和有效性的评价,同时加速阐明疫苗免疫和登革疾病进展过程中的疾病风险及相关机制。小结
通过以上三个部分的研究,本课题在以下三个方面取得研究成果和创新：
一、本研究详细通过详细鉴定,明确了重组EDⅢ免疫制备的107株单抗中的94株针对不同血清型的结合特异性及交叉反应性。随后,我们借助已建立的ELISPOT-MNT微中和试验对以上单抗进行体外保护性测定。42株DENVTS单抗中,有约一半对单一血清型DENV显示较强的中和活性,26株sCR单抗中约1/3及23株CR单抗中约2/3对不同血清型DENV表现出强、中等或弱的复杂活性谱。我们通过重组EDⅢ蛋白与天然病毒粒子表面EDⅢ蛋白的构象性差异,合理地解释了单抗的结合能力与体外中和能力方面的不一致。通过以上研究,我们获得了有价值的强中和活性单抗(如本实验室其它研究者进行的3E31、2D73等单抗的晶体结构与功能研究),为后续EDⅢ单抗的抗原性位点鉴定、深入的结构研究、全面的功能测定和治疗性单抗的研究奠定了良好的基础。
二、本部分研究在以上研究定基础上,对94单抗的抗原性位点进行详细的鉴定。通过合成重叠多肽扫描分析、序列比对、氨基酸替换分析等,我们发现23株CR单抗中有15株(约占2/3)特异性地识别相同的高度保守且免疫优势的DENV复合体表位(aa310-319序列),这是我们首次发现CR单抗识别这一表位具有免疫优势。我们继续采用位点定向突变和酵母表面展示分析,发现该表位的关键残基定位于AB loop上的Q316和H317。结合三维建模分析,我们认为,重组EDⅢ作为免疫原可诱导产生针对在病毒粒子表面表位不暴露的单抗,这些单抗对EDⅢ的结合能力强但中和活性较弱,合理地解释了定位至AB loop弱中和活性的交叉反应单抗与重组EDⅢ免疫方式的关系,加深了对EDⅢ亚单位疫苗免疫策略的认识,为后续改进基于EDⅢ的疫苗免疫方案提供了依据。
三、在本部分研究中,我们用表达Fcy受体的K562细胞株和已知具有增强活性的单抗,在国内外首次创新性地建立了通过NS1抗原捕获ELISA定量检测培养上清中NS1抗原的ADE检测方法(ELISA-ADE方法),可对登革抗体的增强活性进行简单、快速、高通量的检测,同时避免了其它方法繁琐、费时的检测程序及对复杂仪器的要求。我们首次提出,采用同为96孔板检测的简单高通量的ELISA-ADE法和已建立ELISPOT-MNT检测方法对登革单抗和多克隆免疫血清的增强和中和抗体活性进行检测和功能性评价,这将有助于大规模研究中进行登革疫苗的安全性和有效性的评价,同时有助于评价和阐明疫苗免疫和登革疾病进展过程中的疾病风险及相关机制。
Dengue virus (DENV) is a mosquito-borne virus that causes human dengue diseases, ranging from asymptomatic infections, dengue fever (DF) to more severe dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). The burden of dengue diseases is on the rise in tropical and subtropical areas, especially in South America, South-east Asia and Caribbean regions. There are also more and more reports of dengue diseases in Africa and the Middle East. In Southern China, the epidemics of DF occur almost each year. The rigorous situations of dengue epidemic arouse much concern, however, its pathogenesis is far from elucidation. It is accepted that virus itself, immunity history of the host and individual genetic background all play important roles in the progress of the severe dengue diseases. Among these factors, the role that dengue virus itself plays is critical. The primary infection caused by any of the four serotypes of DENV probably predisposes individuals to display increased viral loads, shorten latent period and accordingly elevated risk of progressing into DHF/DSS when the individual is infected by a serotype different from that causing primary infection. The severity of diseases are associated with the serotype, genotype, virus sequences of DENV causing secondary infection and as well as the interval between the primary and secondary infection.
To deal with the ever-increasing dengue diseases, many research organizations speed up their efforts in developing effective dengue vaccine candidates. At present, there are already several kinds of vaccines that are at preclinical levels, including live-attenuated vaccines, inactivated virus vaccines, subunit vaccines based on recombinant proteins and naked DNA constructs. Since there is no licensed vaccine available on the basis of traditional live attenuated vaccines, researchers focused more on immune strategy of recombinant subunit vaccines. Though developing dengue vaccine candidates is feasible, considerable challenges present:(1) Dengue vaccines must provide protection against infections caused by all four serotypes of DENV simultaneously, thereby making necessary development of tetravalent vaccines;(2) Dengue vaccines can provide long-term protection, because there is report that DHF developed when re-infection occurred20years after the primary infection;(3) There is still no suitable animal models to replicate dengue diseases;(4) Though the in vitro protective role of neutralizing antibodies is widely accepted, their relationship with practical protection still needs determination;(5) At the time of changes of transmission patterns and viral strains of DENV, dengue vaccines need re-evaluation. Thus, to develop reliable dengue vaccines, it is essential to address two issues:one is the efficacy of vaccines, namely the protection of vaccines, and the other is safety issue of vaccines.
The envelope of DENV consists of lipid bilayer, containing two kinds of envelope-associated proteins:envelop protein (E) and membrane protein (M). E protein involves viral attachment, and fusion with endosome after entry via binding to cellular receptors. E protein contains three different domains, domain Ⅰ (EDⅠ), EDⅡ and EDⅢ. EDⅢ is surface exposed on the virion and accessible, and recombinant EDIII can inhibit the infectivity of the virus. A line of evidences demonstrated that EDIII contains type-specific (TS) epitopes against a single DENV serotype, sub-complex reactive (sCR) epitopes against two or three DENV serotypes, and complex reactive epitopes (CR) against all four serotypes. A number of murine monoclonal antibodies (mAbs) localize to these epitopes. Many studies show that some TS and sCR murine mAbs are mainly neutralizing and protective mAbs against DENV, accordingly, EDIII protein of DENV has become a promising candidate target of subunit vaccines. In addition, mAbs against EDIII are shown recently to take up a small part in the total DENV-specific antibodies in human immune sera, and the epitopes on EDIII are not the main targets of human neutralizing antibodies against DENV. In spite of this, the antigenic sites of murine mAbs and their neutralizing activity spectrum are far from making clear. Further studies are essential to gain deeper insight into the complexity of humoral immune reactions, the optimization of immune strategy of dengue vaccines and the study of therapeutic antibody.
The studies of antigenic epitopes and neutralizing potency of mAbs directed against EDIII of DENV are instrumental to efficacy of dengue vaccines, but safety issue of dengue vaccines is another difficulty. As mentioned above, after the primary infection by any of the four serotypes, the risk of development into severe dengue diseases is significantly increased in the case of secondary infection caused by a heterotypic serotype. One of the main underlying mechanisms is antibody-dependent enhancement (ADE). Pre-existing cross-reactive weakly neutralizing antibodies bind to virus and enhance the infectious level of cells bearing Fc gamma receptor (FcyR). Recently, the study of Dejnirattisai W. and colleagues present the complexity of ADE because both anti-E and anti-prM (precursor membrane) antibodies can enhance DENV infection. Since it is urgent to develop tetravalent vaccines that provide long-term protection and there is no appropriate animal models for the evaluation of safety issues of dengue vaccines, the evaluation of dengue vaccines in vitro appears critically important.
One hundred and seven strains of mAbs were produced using recombinant EDⅢ protein as immunogens to immune BALB/c mice in previous studies of our laboratory. In the current study, we first identified the binding specificity or cross-reactivity of107mAbs utilizing enzyme-linked immuno-sorbent assay (ELISA) and immunofluorescence assay (IFA), followed by the measurement of the neutralizing activities of94strains mAbs selected using established enzyme-linked immuno-spot mini-neutralizing test (ELISPOT-MNT). On the basis of these, we identified and analyzed the antigenic sites of94mAbs to know further the antigenic sites recognized by mAbs obtained by immune of recombinant EDⅢ and their relationship with protective roles of these mAbs in vitro. Then, we develop a simple and high-throughput alternative assay for ADE determination to assess the safety issue of dengue vaccines and the risk of dengue diseases.
Therefore, the three main aims of this study are as follows:First, to identify the binding specificity or cross-reactivity and neutralizing activities of107mAbs. Second, to analyze the antigenic sites recognized by tested mAbs in this study. Third, to develop a simple and high-throughput alternative assay for ADE measurement to realize the evaluation of the safety issue of dengue vaccines in vitro.
This study is composed of three parts:
Part Ⅰ Characterization of binding capacity/and neutralizing activities of monoclonal antibodies against E protein domain Ⅲ of Dengue virus
In previous studies of our laboratory, we produced107strains of mAbs by expressing recombinant EDⅢ of each DENV serotype by Pichia pastoris, and immunizing BALB/c mice intraperitoneally with each of the recombinant EDⅢ proteins alone, or a combination of recombinant EDⅢs of all four serotypes. In this study, characterized in detail the binding specificity or cross-reactivity with and neutralizing activities of these mAbs against different serotypes of DENVs. ELISA and IFA showed that94strains of mAbs exhibited binding specificity or cross-reactivity with recombinant EDⅢ proteins and DENV-infected C6/36cells, including42strains of TS mAbs,26strains of sCRs mAbs,23strains of CR mAbs and3strains of flavivirus cross-reactive mAbs. Of note, the binding specificity or cross-reactivity identified by IFA was not completely consistent with that identified by ELISA, probably due to the conformational discrepancy between coated recombinant EDⅢ protein and surface-exposed E protein on the infecting DENV virions.
Then, with the enzyme-linked immunospot based mini-neutralization test (ELISPOT-MNT) established previously, we determined the neutralizing activities in vitro of these mAbs against all four DENV serotypes. According to the50%inhibition concentration (IC50), mAbs were designated strong (IC50≤1μg/ml), moderate (IC50between1and50μg/ml) and weak (IC50>50μg/ml) neutralizing activities. Seventeen DENV-1TS mAbs and14DENV-2TS mAbs showed strongly, moderately or weakly neutralizing activity towards a single serotype. All of the7DENV-2TS mAbs displayed no neutralizing activity against DENV-2. All of the3DENV-3TS mAbs exhibited strong neutralizing activity against DENV-3. The panel of26sCR mAbs and23CR mAbs displayed a varied repertoire of neutralizing activity towards the four DENV serotypes, with strongly, moderately or weakly neutralizing activity towards different serotypes. It was found that the binding specificity or cross-reactivity and neutralizing potency was not always consistent. Such inconsistence may due to the conformational difference between isolated recombinant EDIII and E protein displayed on the surface of natural viral particles. Our results identified clearly the binding specificity or cross-reactivity and in vitro neutralizing capacity of our panel of mAbs, laying a good foundation for further study of antigenic sites of these mAbs, deeper structural studies and full functional determination.
Part II Characterization of epitopes in envelope protein domain III of dengue virus recognized by monoclonal antibodies
E protein domain Ⅲ (EDⅢ) is considered a promising region for subunit vaccine candidates. However, the antigenic sites in the EDⅢ and their relationship with protective roles in vitro of mAbs elicited using recombinant EDⅢ as immunogens have not been clearly defined.
In this study, based on the binding specificity or cross-reactivity with and neutralizing activities of94mAbs against different serotypes of DENVs, we characterized in detail the antigenic sites recognized by these mAbs. First, through pepscan using synthetic overlapping peptides, we found that12mAbs consisting of2DENV-2type-specific mAbs,5sub-complex reactive mAbs, one complex reactive mAb and2flavivirus cross-reactive mAbs specifically recognized different linear epitopes and display no neutralizing activity against any serotype. In contrast, we observed that two third (15of23) complex-reactive mAbs predominantly recognized the same amino acid sequence309-320. Further sequence alignment amino acid substitution analysis demonstrated that aa309-320is a DENV complex reactive epitope that is immunodominant and highly conserved epitope across different serotypes of dengue virus.
Of note, all15complex-reactive mAbs exhibited significant cross-reactivity with coated EDIII from all DENV serotypes in ELISA and also with DENV-1,-2,-3, and-4infected C6/36cells in IFA. However, neutralization tests indicated that the majority of these15mAbs were either moderate or weak neutralizers. How to account for such difference between significant cross-reactivity and neutralizing capacity exhibited by these15mAbs? Through further identification of this epitope by site-directed mutagenesis and yeast surface display mapping, two residues on the AB loop, Q316and H317, were discovered to be critical. Three-dimensional modeling analysis of the310-319epitope in the crystal structures of the DENV E protein suggests that this antigenic site is surface-exposed on EDIII but less accessible on the surface of the E protein dimer and trimer, especially in tightly packed E protein on the surface of the mature virion. It is concluded that EDIII as immunogen may elicit mAbs to the epitope that is not exposed on the virion surface, therefore contributing inefficiently to their neutralization potency. With this study, we reasonably provide explanation for the relationship between poorly neutralizing cross-reactive antibody response to AB loop of EDIII and EDIII prime-boost strategy. We thus gained deeper insight into the EDIII-based subunit immune strategy and provided data for further exploration of optimized EDIII-based vaccination regimens.
Part Ⅲ Development of simple high-throughput assays to evaluate the function of antibodies against dengue virus
Development of dengue vaccines entails minimizing potential antibody-dependent enhancement (ADE) and achieving an even protective antibody response against all four dengue virus (DENV) serotypes. In the absence of appropriate animal models, it is necessary to develop reliable and high-throughput platforms to determine the immune status of the disease process and vaccine efficacy,
In this study, using FcyR-expressing K562cells and a known enhancing antibody, we creatively developed a simple high-throughput ADE assay of enhancing activity of DENV-specific antibodies by quantitative measurement of NS1antigen in the culture supernatant using NS1capture ELISA. To evaluate this method, enhancing activity was measured simultaneously by two methods, the newly developed increased NS1antigen quantitation and traditional enhanced virus titration. The kinetics of NS1production were compatible with the results of the virus titration assay. Linear correlation analysis demonstrated a good agreement (R=0.938, P=0.000) between NS1and infectious viral titer determination. Then, enhancing and neutralizing antibody activities of two flaviviral cross-reactive murine monoclonal antibodies (mAbs)(4G2and2A10G6) and four primary DENV-1infected human sera were evaluated and analysed by ELISA-ADE and an available enzyme-linked immunospot-based micro-neutralization test (ELISPOT-MNT) both in96-well formats and for easy readout. It was shown that characteristic ADE profiles in a dose-dependent manner and the enhancements occurred at sub-neutralizing concentrations, displaying reasonable associations with the neutralizing effect measured via ELISPOT-MNT.
The newly developed simple high-throughput ELISA-ADE and ELISPOT-MNT assays are valuable for the functional evaluation of DENV enhancing and neutralizing antibody activities. These assays can significantly facilitate evaluations of the safety and efficacy of DENV vaccine candidates in large-scale studies and promote the elucidation of immune status during vaccination and dengue disease progression.
Summarization:
1. In this study, we characterized in detail the binding capacity with different serotypes of DENV and found that94mAbs exhibited binding specificity or cross-reactivity with recombinant EDIII protein and/or DENV-infected C6/36cells. Then, with the established ELISPOT-MNT, we determined the neutralizing activities in vitro of these mAbs against all four DENV serotypes. Half of the42DENV-1type-specific mAbs showed strongly neutralizing activity towards a single serotype. One third of26sub-complex reactive mAbs and two third of23complex reactive mAbs displayed a varied repertoire of neutralizing activity towards the four DENV serotypes, with strongly, moderately or weakly neutralizing activity towards different serotypes. Conformational difference between isolated recombinant EDIII and E protein displayed on the surface of natural viral particles reasonably account for the inconsistence between binding and neutralizing capacity. With this study, we obtained strongly neutralizing mAbs of interest (e.g. mAbs3E31and2D73was used for crystal structure and functional research by other investigator in our laboratory). Our results laid a good foundation for further study of antigenic sites of these mAbs, deeper structural and functional determination, and therapeutic mAbs.
2. In this study, on the basis of the study above, we characterized in detail the antigenic sites recognized by these mAbs. Through pepscan, sequence alignment and amino acid substitution, we found that two third (15of23) complex-reactive mAbs predominantly recognized the same amino acid sequence309-320. This is the first report of immunodominance of highly conserved DENV complex reactive epitope aa310-319. Further identification of this epitope by site-directed mutagenesis and yeast surface display mapping, two residues on the AB loop, Q316and H317, were discovered to be critical. With three-dimensional modeling analysis, we concluded that EDIII as immunogen may elicit mAbs to epitope that is not exposed on the virion surface, therefore contributing inefficiently to their neutralization potency. With this study, we reasonably provide explanation for the relationship between poorly neutralizing cross-reactive antibody response to AB loop of EDIII and EDIII prime-boost strategy. We thus gained deeper insight into the EDIII-based subunit immune strategy and provided data for further exploration of optimized EDIII-based vaccination regimens.
3. In this study, we developed a simple high-throughput ADE assay (ELISA-ADE) of enhancing activity of DENV-specific antibodies by quantitative measurement of NS1antigen in the culture supernatant using NS1capture ELISA using FcyR-expressing K562cells and a known enhancing antibody. This new assay allows for simple, rapid and high-throughput detection of enhancement activities, while avoiding cumbersome, time-consuming procedures and complex equipment. We present for the first time the newly developed simple high-throughput ELISA-ADE and ELISPOT-MNT assays for the functional evaluation of DENV enhancing and neutralizing antibody activities of mAbs and polyclonal immune sera. These assays can significantly facilitate evaluations of the safety and efficacy of DENV vaccine candidates in large-scale studies and promote the elucidation of immune status during vaccination and dengue disease progression.
为应对日益增加的登革疾病,很多研究机构都在加紧研发登革疫苗。目前,已经有几类疫苗在进行临床前的评价,包括减毒活疫苗、灭活病毒疫苗、基于重组蛋白的亚单位疫苗和裸DNA疫苗等。由于采用基于减毒活疫苗的传统方法,还没有研制出可批准使用的疫苗,研究者更多地将目光转向重组亚单位疫苗。尽管研究登革疫苗是可行的,但是其研发还是面临着相当严峻的挑战：(1)登革疫苗必须能对4种血清型DENV的感染同时提供保护作用,因而需要研制四价疫苗；(2)登革疫苗要能提供长期的保护。因为有研究报道,初次感染20年以后的再感染时仍会发生DHF：(3)还没有合适的复制登革疾病的动物模型；(4)尽管中和抗体的保护性作用已广为接受,但其与实际保护效果的关系仍需确定；(5)在DENV的传播模式和病毒株改变时,登革疫苗需要重新评价。因此,要研制出可靠的登革疫苗,就要解决两方面的难题：一是疫苗的效果,主要指疫苗的保护性；二是疫苗的安全性问题。
DENV的包膜由脂质双层构成,含有两类包膜相关蛋白：膜蛋白(envelope,E)和M蛋白(membrane, M)。E蛋白通过结合到细胞受体而参与病毒的吸附及穿入后与内吞体的融合。E蛋白含有3个不同的结构域：结构域I(EDⅠ)、EDⅡ、 EDⅢ。EDⅢ在病毒颗粒表面是暴露且可及的,而且,重组EDⅢ也能抑制病毒的感染性。多项研究发现,EDⅢ含有单一血清型特异性(type-specific, TS)表位、针对2-3种血清型DENV的亚复合体反应(sub-complex reactive, sCR)表位以及同时针对4种血清型DENV的复合体反应(complex reactive, CR)表位,多株鼠源单克隆抗体也分别定位到这些表位。研究表明,许多TS和sCR鼠单抗是针对DENV单抗中起主要中和及保护作用的单抗。由此,登革病毒EDⅢ蛋白也成为亚单位疫苗很有前景的备选靶标。另外,最近有关人源单抗的研究认为,针对EDⅢ的单抗仅占人免疫血清中抗DENV总抗体中的很小一部分,而且EDⅢ上的表位不是人抗DENV中和抗体的主要靶标。尽管如此,鼠源单抗的抗原性表位及其中和活性谱仍有许多方面未研究清楚,更加深入的研究对加深了解体液免疫反应的复杂性、改善登革疫苗的免疫策略或研究治疗性抗体均有不可替代的作用。
对EDⅢ单抗的抗原性表位和中和活性的研究可加深对疫苗效果的研究,但是,还有一个难题就是疫苗的安全性问题。前已述及,任何一种血清型登革病毒引起初次感染后,由不同血清型引起再次感染时,登革疾病加重的机率大大增加,其背后最主要的机制就是抗体依赖的增强作用(antibody-dependent enhancement, ADE),即先前存在交叉反应弱中和抗体能与登革病毒结合,增强表达FcγR受体的细胞的感染水平。最近,Dejnirattisai W.等的研究成果也呈现出ADE发生的复杂性,即抗E蛋白抗体和抗前膜(precursor membrane, prM)抗体均能增强DENV感染。要研制能提供长期保护的四价疫苗,还没有合适的登革疾病的动物模型对登革疫苗进行安全性评价,因此,登革疫苗的体外评价显得格外重要。
因此,在本实验室前期研究中,利用登革病毒重组EDⅢ蛋白免疫小鼠,制备了107株与DENV重组EDⅢ反应的单抗。本研究中,我们首先采用酶联免疫吸附试验(ELISA)和间接免疫荧光(IFA)测定法对107单抗的结合特异性(binding specificity)进行检测,随后,我们利用已建立的简单高通量的微中和试验(ELISPOT-MNT)法对筛选出来的94株单抗的中和活性谱进行全面的检测和分析。在此基础上,对94株单抗的抗原性位点进行鉴定分析,以深入认识重组EDⅢ免疫诱导产生单抗的抗原性位点及其与体外保护性作用的关系。然后,我们创新性地建立了简单高通量的测定DENV ADE效应的方法学,以进行疫苗安全性的评价和登革疾病的风险评估。
由此,本研究的主要目的有三个：第一,对107株单抗的结合特异性和体外保护性作用进行鉴定；第二,对筛选出来的94株单抗识别的抗原性位点进行分析；第三,建立一种简单高通量的测定登革抗体ADE活性的新方法,实现对登革疫苗体外安全性评价。
由此,本研究共分为如下三个部分的内容：
第一部分登革病毒包膜E蛋白Ⅲ区(EDⅢ)单克隆抗体的结合特异性和中和活性鉴定
本实验室在前期工作中,采用毕赤酵母表达的4种血清型DENV的重组EDⅢ作为免疫原,单独或混合免疫BALB/c小鼠,制备了107株单抗。本研究中,我们对这些单抗针对不同血清型的结合特异性或交叉反应性、中和活性进行了详细鉴定。首先,ELISA和IFA测定结果显示,有94株单抗对重组EDⅢ或DENV感染的C6/36细胞的结合呈现特异性或交叉反应性,包括42株针对单一血清型DENV的TS单抗、26株针对2-3种血清型的sCR单抗及23株针对4种血清型的CR单抗,还有3株黄病毒交叉反应单抗。但是,ELISA和IFA的鉴定结果并不完全相符,推测96孔板上包被的重组EDⅢ蛋白的构象与感染C6/36细胞的登革病毒表面E蛋白的构象的差异可能造成了这种不一致。
随后,我们借助已建立的ELISPOT-MNT微中和试验对以上单抗进行体外保护性测定。根据单抗对病毒的50%抑制浓度(50%inhibition concentration, IC50)的大小,将单抗分为三类：强(IC50≤1μg/ml),中等(1μg/ml
第二部分登革病毒包膜E蛋白Ⅲ区(EDⅢ)单克隆抗体的表位鉴定
DENV的EDⅢ区被认为是研究亚单位疫苗有前景的抗原性区域。但是,EDⅢ上的抗原性位点及其与EDⅢ免疫诱导产生单抗的体外保护作用的关系还没有完全研究清楚。由此,本研究在94株单抗的结合特性及中和活性鉴定基础上,对其抗原性位点进行详细的鉴定。
首先,我们对94株单抗进行合成重叠多肽扫描分析,发现12株单抗(2株DENV-2TS单抗、5株sCR单抗、1株CR单抗和2株黄病毒交叉反应单抗)分别特异性地识别几个不同的线性表位,但这12株单抗均无中和活性。相比之下,23株CR单抗中有15株(约占2/3)特异性地识别相同的aa310-319序列,进一步的序列比对和残基替换证实aa309-320表位是各血清型DENV间高度保守且免疫优势的复合体表位。
值得注意的是,15株CR单抗与4个血清型DENV的重组EDⅢ或感染的C6/36细胞呈现较强交叉反应,但是,它们对4个型DENV具有复杂的中和活性谱,大多数只显示弱或中等中和活性。如何解释这些单抗的结合交叉反应性与中和能力的差异呢?我们继续采用位点定向突变和酵母表面展示分析,发现ABloop上的Q316和H317是该表位的关键残基；而且,三维建模分析表明,该表位在EDⅢ表面充分暴露,但在E蛋白二聚体和三聚体表面的可及性差,特别是在成熟病毒粒子表面可及性更差。可以认为,重组EDⅢ作为免疫原可诱导产生针对在病毒粒子表面不暴露的单抗,因此这些单抗的中和活性较弱。通过研究,我们合理地解释了定位至EDⅢ AB loop的弱中和活性的交叉反应单抗与重组EDⅢ免疫方式的关系,加深了对EDⅢ亚单位疫苗免疫策略的认识,为后续改进基于EDⅢ的疫苗免疫方案提供了依据。
第三部分建立简易、高通量的方法评价登革病毒抗体的功能
开发登革疫苗需要尽可能减少潜在的ADE风险,从而要求针对DENV的4个血清型获得均衡的保护性抗体反应。在缺乏合适的登革疾病动物模型的情况下,有必要开发简单、可靠且高通量的方法用于评价登革热疾病的风险和疫苗的安全性。
在本部分研究中,我们采用表达Fcy受体的K562细胞株和已知具有增强活性的单抗,创新性地建立了通过“NS1抗原捕获ELISA"检测培养上清中NS1抗原的简单高通量的登革抗体的ADE检测方法(ELISA-ADE方法)。为评价该方法,我们同时采用2种方法(即新建立的NS1抗原定量方法和传统的病毒滴度测定方法)检测抗体的ADE活性。发现两种方法的动力学相当,线性相关分析证明,这2种方法具有很好的一致性(R=0.938,P=0.000)。随后,我们还采用同为96孔板检测和读板的ELISA-ADE法和已经建立的ELISPOT-MNT微中和试验评价2株黄病毒交叉反应鼠单抗(4G2和2A10G6)和4份DENV-1感染患者的恢复期血清的增强活性和中和抗体活性。结果表明,新方法可获得特征性的剂量反应ADE图谱,而且增强效应发生在亚中和浓度,ADE效应与经ELISPOT-MNT测定获得的中和效应具有合理的相互关系。
本研究新建立的简单高通量的ELISA-ADE法和已建立ELISPOT-MNT检测方法对DENV的增强和中和抗体活性的功能性评价非常有价值。它们能在大规模研究中进行DENV疫苗的安全性和有效性的评价,同时加速阐明疫苗免疫和登革疾病进展过程中的疾病风险及相关机制。小结
通过以上三个部分的研究,本课题在以下三个方面取得研究成果和创新：
一、本研究详细通过详细鉴定,明确了重组EDⅢ免疫制备的107株单抗中的94株针对不同血清型的结合特异性及交叉反应性。随后,我们借助已建立的ELISPOT-MNT微中和试验对以上单抗进行体外保护性测定。42株DENVTS单抗中,有约一半对单一血清型DENV显示较强的中和活性,26株sCR单抗中约1/3及23株CR单抗中约2/3对不同血清型DENV表现出强、中等或弱的复杂活性谱。我们通过重组EDⅢ蛋白与天然病毒粒子表面EDⅢ蛋白的构象性差异,合理地解释了单抗的结合能力与体外中和能力方面的不一致。通过以上研究,我们获得了有价值的强中和活性单抗(如本实验室其它研究者进行的3E31、2D73等单抗的晶体结构与功能研究),为后续EDⅢ单抗的抗原性位点鉴定、深入的结构研究、全面的功能测定和治疗性单抗的研究奠定了良好的基础。
二、本部分研究在以上研究定基础上,对94单抗的抗原性位点进行详细的鉴定。通过合成重叠多肽扫描分析、序列比对、氨基酸替换分析等,我们发现23株CR单抗中有15株(约占2/3)特异性地识别相同的高度保守且免疫优势的DENV复合体表位(aa310-319序列),这是我们首次发现CR单抗识别这一表位具有免疫优势。我们继续采用位点定向突变和酵母表面展示分析,发现该表位的关键残基定位于AB loop上的Q316和H317。结合三维建模分析,我们认为,重组EDⅢ作为免疫原可诱导产生针对在病毒粒子表面表位不暴露的单抗,这些单抗对EDⅢ的结合能力强但中和活性较弱,合理地解释了定位至AB loop弱中和活性的交叉反应单抗与重组EDⅢ免疫方式的关系,加深了对EDⅢ亚单位疫苗免疫策略的认识,为后续改进基于EDⅢ的疫苗免疫方案提供了依据。
三、在本部分研究中,我们用表达Fcy受体的K562细胞株和已知具有增强活性的单抗,在国内外首次创新性地建立了通过NS1抗原捕获ELISA定量检测培养上清中NS1抗原的ADE检测方法(ELISA-ADE方法),可对登革抗体的增强活性进行简单、快速、高通量的检测,同时避免了其它方法繁琐、费时的检测程序及对复杂仪器的要求。我们首次提出,采用同为96孔板检测的简单高通量的ELISA-ADE法和已建立ELISPOT-MNT检测方法对登革单抗和多克隆免疫血清的增强和中和抗体活性进行检测和功能性评价,这将有助于大规模研究中进行登革疫苗的安全性和有效性的评价,同时有助于评价和阐明疫苗免疫和登革疾病进展过程中的疾病风险及相关机制。
Dengue virus (DENV) is a mosquito-borne virus that causes human dengue diseases, ranging from asymptomatic infections, dengue fever (DF) to more severe dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). The burden of dengue diseases is on the rise in tropical and subtropical areas, especially in South America, South-east Asia and Caribbean regions. There are also more and more reports of dengue diseases in Africa and the Middle East. In Southern China, the epidemics of DF occur almost each year. The rigorous situations of dengue epidemic arouse much concern, however, its pathogenesis is far from elucidation. It is accepted that virus itself, immunity history of the host and individual genetic background all play important roles in the progress of the severe dengue diseases. Among these factors, the role that dengue virus itself plays is critical. The primary infection caused by any of the four serotypes of DENV probably predisposes individuals to display increased viral loads, shorten latent period and accordingly elevated risk of progressing into DHF/DSS when the individual is infected by a serotype different from that causing primary infection. The severity of diseases are associated with the serotype, genotype, virus sequences of DENV causing secondary infection and as well as the interval between the primary and secondary infection.
To deal with the ever-increasing dengue diseases, many research organizations speed up their efforts in developing effective dengue vaccine candidates. At present, there are already several kinds of vaccines that are at preclinical levels, including live-attenuated vaccines, inactivated virus vaccines, subunit vaccines based on recombinant proteins and naked DNA constructs. Since there is no licensed vaccine available on the basis of traditional live attenuated vaccines, researchers focused more on immune strategy of recombinant subunit vaccines. Though developing dengue vaccine candidates is feasible, considerable challenges present:(1) Dengue vaccines must provide protection against infections caused by all four serotypes of DENV simultaneously, thereby making necessary development of tetravalent vaccines;(2) Dengue vaccines can provide long-term protection, because there is report that DHF developed when re-infection occurred20years after the primary infection;(3) There is still no suitable animal models to replicate dengue diseases;(4) Though the in vitro protective role of neutralizing antibodies is widely accepted, their relationship with practical protection still needs determination;(5) At the time of changes of transmission patterns and viral strains of DENV, dengue vaccines need re-evaluation. Thus, to develop reliable dengue vaccines, it is essential to address two issues:one is the efficacy of vaccines, namely the protection of vaccines, and the other is safety issue of vaccines.
The envelope of DENV consists of lipid bilayer, containing two kinds of envelope-associated proteins:envelop protein (E) and membrane protein (M). E protein involves viral attachment, and fusion with endosome after entry via binding to cellular receptors. E protein contains three different domains, domain Ⅰ (EDⅠ), EDⅡ and EDⅢ. EDⅢ is surface exposed on the virion and accessible, and recombinant EDIII can inhibit the infectivity of the virus. A line of evidences demonstrated that EDIII contains type-specific (TS) epitopes against a single DENV serotype, sub-complex reactive (sCR) epitopes against two or three DENV serotypes, and complex reactive epitopes (CR) against all four serotypes. A number of murine monoclonal antibodies (mAbs) localize to these epitopes. Many studies show that some TS and sCR murine mAbs are mainly neutralizing and protective mAbs against DENV, accordingly, EDIII protein of DENV has become a promising candidate target of subunit vaccines. In addition, mAbs against EDIII are shown recently to take up a small part in the total DENV-specific antibodies in human immune sera, and the epitopes on EDIII are not the main targets of human neutralizing antibodies against DENV. In spite of this, the antigenic sites of murine mAbs and their neutralizing activity spectrum are far from making clear. Further studies are essential to gain deeper insight into the complexity of humoral immune reactions, the optimization of immune strategy of dengue vaccines and the study of therapeutic antibody.
The studies of antigenic epitopes and neutralizing potency of mAbs directed against EDIII of DENV are instrumental to efficacy of dengue vaccines, but safety issue of dengue vaccines is another difficulty. As mentioned above, after the primary infection by any of the four serotypes, the risk of development into severe dengue diseases is significantly increased in the case of secondary infection caused by a heterotypic serotype. One of the main underlying mechanisms is antibody-dependent enhancement (ADE). Pre-existing cross-reactive weakly neutralizing antibodies bind to virus and enhance the infectious level of cells bearing Fc gamma receptor (FcyR). Recently, the study of Dejnirattisai W. and colleagues present the complexity of ADE because both anti-E and anti-prM (precursor membrane) antibodies can enhance DENV infection. Since it is urgent to develop tetravalent vaccines that provide long-term protection and there is no appropriate animal models for the evaluation of safety issues of dengue vaccines, the evaluation of dengue vaccines in vitro appears critically important.
One hundred and seven strains of mAbs were produced using recombinant EDⅢ protein as immunogens to immune BALB/c mice in previous studies of our laboratory. In the current study, we first identified the binding specificity or cross-reactivity of107mAbs utilizing enzyme-linked immuno-sorbent assay (ELISA) and immunofluorescence assay (IFA), followed by the measurement of the neutralizing activities of94strains mAbs selected using established enzyme-linked immuno-spot mini-neutralizing test (ELISPOT-MNT). On the basis of these, we identified and analyzed the antigenic sites of94mAbs to know further the antigenic sites recognized by mAbs obtained by immune of recombinant EDⅢ and their relationship with protective roles of these mAbs in vitro. Then, we develop a simple and high-throughput alternative assay for ADE determination to assess the safety issue of dengue vaccines and the risk of dengue diseases.
Therefore, the three main aims of this study are as follows:First, to identify the binding specificity or cross-reactivity and neutralizing activities of107mAbs. Second, to analyze the antigenic sites recognized by tested mAbs in this study. Third, to develop a simple and high-throughput alternative assay for ADE measurement to realize the evaluation of the safety issue of dengue vaccines in vitro.
This study is composed of three parts:
Part Ⅰ Characterization of binding capacity/and neutralizing activities of monoclonal antibodies against E protein domain Ⅲ of Dengue virus
In previous studies of our laboratory, we produced107strains of mAbs by expressing recombinant EDⅢ of each DENV serotype by Pichia pastoris, and immunizing BALB/c mice intraperitoneally with each of the recombinant EDⅢ proteins alone, or a combination of recombinant EDⅢs of all four serotypes. In this study, characterized in detail the binding specificity or cross-reactivity with and neutralizing activities of these mAbs against different serotypes of DENVs. ELISA and IFA showed that94strains of mAbs exhibited binding specificity or cross-reactivity with recombinant EDⅢ proteins and DENV-infected C6/36cells, including42strains of TS mAbs,26strains of sCRs mAbs,23strains of CR mAbs and3strains of flavivirus cross-reactive mAbs. Of note, the binding specificity or cross-reactivity identified by IFA was not completely consistent with that identified by ELISA, probably due to the conformational discrepancy between coated recombinant EDⅢ protein and surface-exposed E protein on the infecting DENV virions.
Then, with the enzyme-linked immunospot based mini-neutralization test (ELISPOT-MNT) established previously, we determined the neutralizing activities in vitro of these mAbs against all four DENV serotypes. According to the50%inhibition concentration (IC50), mAbs were designated strong (IC50≤1μg/ml), moderate (IC50between1and50μg/ml) and weak (IC50>50μg/ml) neutralizing activities. Seventeen DENV-1TS mAbs and14DENV-2TS mAbs showed strongly, moderately or weakly neutralizing activity towards a single serotype. All of the7DENV-2TS mAbs displayed no neutralizing activity against DENV-2. All of the3DENV-3TS mAbs exhibited strong neutralizing activity against DENV-3. The panel of26sCR mAbs and23CR mAbs displayed a varied repertoire of neutralizing activity towards the four DENV serotypes, with strongly, moderately or weakly neutralizing activity towards different serotypes. It was found that the binding specificity or cross-reactivity and neutralizing potency was not always consistent. Such inconsistence may due to the conformational difference between isolated recombinant EDIII and E protein displayed on the surface of natural viral particles. Our results identified clearly the binding specificity or cross-reactivity and in vitro neutralizing capacity of our panel of mAbs, laying a good foundation for further study of antigenic sites of these mAbs, deeper structural studies and full functional determination.
Part II Characterization of epitopes in envelope protein domain III of dengue virus recognized by monoclonal antibodies
E protein domain Ⅲ (EDⅢ) is considered a promising region for subunit vaccine candidates. However, the antigenic sites in the EDⅢ and their relationship with protective roles in vitro of mAbs elicited using recombinant EDⅢ as immunogens have not been clearly defined.
In this study, based on the binding specificity or cross-reactivity with and neutralizing activities of94mAbs against different serotypes of DENVs, we characterized in detail the antigenic sites recognized by these mAbs. First, through pepscan using synthetic overlapping peptides, we found that12mAbs consisting of2DENV-2type-specific mAbs,5sub-complex reactive mAbs, one complex reactive mAb and2flavivirus cross-reactive mAbs specifically recognized different linear epitopes and display no neutralizing activity against any serotype. In contrast, we observed that two third (15of23) complex-reactive mAbs predominantly recognized the same amino acid sequence309-320. Further sequence alignment amino acid substitution analysis demonstrated that aa309-320is a DENV complex reactive epitope that is immunodominant and highly conserved epitope across different serotypes of dengue virus.
Of note, all15complex-reactive mAbs exhibited significant cross-reactivity with coated EDIII from all DENV serotypes in ELISA and also with DENV-1,-2,-3, and-4infected C6/36cells in IFA. However, neutralization tests indicated that the majority of these15mAbs were either moderate or weak neutralizers. How to account for such difference between significant cross-reactivity and neutralizing capacity exhibited by these15mAbs? Through further identification of this epitope by site-directed mutagenesis and yeast surface display mapping, two residues on the AB loop, Q316and H317, were discovered to be critical. Three-dimensional modeling analysis of the310-319epitope in the crystal structures of the DENV E protein suggests that this antigenic site is surface-exposed on EDIII but less accessible on the surface of the E protein dimer and trimer, especially in tightly packed E protein on the surface of the mature virion. It is concluded that EDIII as immunogen may elicit mAbs to the epitope that is not exposed on the virion surface, therefore contributing inefficiently to their neutralization potency. With this study, we reasonably provide explanation for the relationship between poorly neutralizing cross-reactive antibody response to AB loop of EDIII and EDIII prime-boost strategy. We thus gained deeper insight into the EDIII-based subunit immune strategy and provided data for further exploration of optimized EDIII-based vaccination regimens.
Part Ⅲ Development of simple high-throughput assays to evaluate the function of antibodies against dengue virus
Development of dengue vaccines entails minimizing potential antibody-dependent enhancement (ADE) and achieving an even protective antibody response against all four dengue virus (DENV) serotypes. In the absence of appropriate animal models, it is necessary to develop reliable and high-throughput platforms to determine the immune status of the disease process and vaccine efficacy,
In this study, using FcyR-expressing K562cells and a known enhancing antibody, we creatively developed a simple high-throughput ADE assay of enhancing activity of DENV-specific antibodies by quantitative measurement of NS1antigen in the culture supernatant using NS1capture ELISA. To evaluate this method, enhancing activity was measured simultaneously by two methods, the newly developed increased NS1antigen quantitation and traditional enhanced virus titration. The kinetics of NS1production were compatible with the results of the virus titration assay. Linear correlation analysis demonstrated a good agreement (R=0.938, P=0.000) between NS1and infectious viral titer determination. Then, enhancing and neutralizing antibody activities of two flaviviral cross-reactive murine monoclonal antibodies (mAbs)(4G2and2A10G6) and four primary DENV-1infected human sera were evaluated and analysed by ELISA-ADE and an available enzyme-linked immunospot-based micro-neutralization test (ELISPOT-MNT) both in96-well formats and for easy readout. It was shown that characteristic ADE profiles in a dose-dependent manner and the enhancements occurred at sub-neutralizing concentrations, displaying reasonable associations with the neutralizing effect measured via ELISPOT-MNT.
The newly developed simple high-throughput ELISA-ADE and ELISPOT-MNT assays are valuable for the functional evaluation of DENV enhancing and neutralizing antibody activities. These assays can significantly facilitate evaluations of the safety and efficacy of DENV vaccine candidates in large-scale studies and promote the elucidation of immune status during vaccination and dengue disease progression.
Summarization:
1. In this study, we characterized in detail the binding capacity with different serotypes of DENV and found that94mAbs exhibited binding specificity or cross-reactivity with recombinant EDIII protein and/or DENV-infected C6/36cells. Then, with the established ELISPOT-MNT, we determined the neutralizing activities in vitro of these mAbs against all four DENV serotypes. Half of the42DENV-1type-specific mAbs showed strongly neutralizing activity towards a single serotype. One third of26sub-complex reactive mAbs and two third of23complex reactive mAbs displayed a varied repertoire of neutralizing activity towards the four DENV serotypes, with strongly, moderately or weakly neutralizing activity towards different serotypes. Conformational difference between isolated recombinant EDIII and E protein displayed on the surface of natural viral particles reasonably account for the inconsistence between binding and neutralizing capacity. With this study, we obtained strongly neutralizing mAbs of interest (e.g. mAbs3E31and2D73was used for crystal structure and functional research by other investigator in our laboratory). Our results laid a good foundation for further study of antigenic sites of these mAbs, deeper structural and functional determination, and therapeutic mAbs.
2. In this study, on the basis of the study above, we characterized in detail the antigenic sites recognized by these mAbs. Through pepscan, sequence alignment and amino acid substitution, we found that two third (15of23) complex-reactive mAbs predominantly recognized the same amino acid sequence309-320. This is the first report of immunodominance of highly conserved DENV complex reactive epitope aa310-319. Further identification of this epitope by site-directed mutagenesis and yeast surface display mapping, two residues on the AB loop, Q316and H317, were discovered to be critical. With three-dimensional modeling analysis, we concluded that EDIII as immunogen may elicit mAbs to epitope that is not exposed on the virion surface, therefore contributing inefficiently to their neutralization potency. With this study, we reasonably provide explanation for the relationship between poorly neutralizing cross-reactive antibody response to AB loop of EDIII and EDIII prime-boost strategy. We thus gained deeper insight into the EDIII-based subunit immune strategy and provided data for further exploration of optimized EDIII-based vaccination regimens.
3. In this study, we developed a simple high-throughput ADE assay (ELISA-ADE) of enhancing activity of DENV-specific antibodies by quantitative measurement of NS1antigen in the culture supernatant using NS1capture ELISA using FcyR-expressing K562cells and a known enhancing antibody. This new assay allows for simple, rapid and high-throughput detection of enhancement activities, while avoiding cumbersome, time-consuming procedures and complex equipment. We present for the first time the newly developed simple high-throughput ELISA-ADE and ELISPOT-MNT assays for the functional evaluation of DENV enhancing and neutralizing antibody activities of mAbs and polyclonal immune sera. These assays can significantly facilitate evaluations of the safety and efficacy of DENV vaccine candidates in large-scale studies and promote the elucidation of immune status during vaccination and dengue disease progression.
引文
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