Analysis of epitopes on dengue virus envelope protein recognized by monoclonal antibodies and polyclonal human sera by a high throughput assay.
Analysis of epitopes on dengue virus envelope protein recognized by monoclonal antibodies and polyclonal human sera by a high throughput assay.
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DOI：
10.1371/journal.pntd.0001447
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发表时间：
2012-01
影响因子：
3.8
通讯作者：
Wang WK
中科院分区：
文献类型：
--
作者：
Lin HE;Tsai WY;Liu IJ;Li PC;Liao MY;Tsai JJ;Wu YC;Lai CY;Lu CH;Huang JH;Chang GJ;Wu HC;Wang WK
登革病毒（DENV）的包膜蛋白（E）是中和抗体和疫苗开发的主要靶点。虽然先前关于结构域III或结构域I/II单独的研究已经报道了针对DENV E蛋白的单克隆抗体（mAb）的几个表位，但是结构域间表位的可能性以及表位与中和效力之间的关系仍然在很大程度上未被探索。我们开发了一种斑点印迹试验，通过使用67丙氨酸突变体的预测表面暴露的E残基作为一个系统的方法来识别单克隆抗体和多克隆血清识别的表位，并证实了我们的研究结果，使用捕获-ELISA测定。在测试的12种小鼠单克隆抗体中，3种识别了涉及结构域II中心界面处的残基（Q211、D215、P217）的新表位，3种识别了结构域III和结构域II的侧脊处的残基，这表明结构域间表位比以前认识到的更频繁地存在。与传统方法制备的mAb相比，新方法制备的中和mAb能够识别DENV 2和DENV 1的A链或C链/CC ′环上的多个残基，以及DENV 1的BC环和DE环、EF环/F链或G链上的多个残基。发现多克隆血清中抗E抗体的主要表位在相同或相邻单体中包括融合环和非融合残基。我们的分析对表位特异性诊断和基于表位的登革热疫苗具有意义。这种高通量的方法具有巨大的应用定位的人单克隆抗体和多克隆血清识别的结构域内和结构域间的表位，这将进一步我们的体液免疫应答DENV在表位水平的理解。登革热病毒是世界范围内虫媒病毒疾病的主要原因。包膜蛋白是中和抗体和疫苗开发的主要靶标。虽然先前的研究已经报道了包膜蛋白上的几个表位，但结构域间表位的可能性以及表位与中和效力的关系仍未被探索。我们开发了一种高通量的斑点印迹法，通过使用67丙氨酸突变体的表面暴露的包膜残基作为一种系统的方法来确定表位识别的小鼠单克隆抗体和多克隆人血清。我们的研究结果表明，域间表位的存在比以前更频繁。与传统方法制备的单克隆抗体相比，新方法制备的单克隆抗体具有独特的表位特征。此外，该测定法还可以鉴定多克隆血清中抗包膜蛋白抗体的优势表位。这些发现对未来发展表位特异性诊断和基于表位的登革疫苗具有重要意义，并增加了我们对登革病毒在表位水平上的体液免疫应答的理解。
The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies and vaccine development. While previous studies on domain III or domain I/II alone have reported several epitopes of monoclonal antibodies (mAbs) against DENV E protein, the possibility of interdomain epitopes and the relationship between epitopes and neutralizing potency remain largely unexplored. We developed a dot blot assay by using 67 alanine mutants of predicted surface-exposed E residues as a systematic approach to identify epitopes recognized by mAbs and polyclonal sera, and confirmed our findings using a capture-ELISA assay. Of the 12 mouse mAbs tested, three recognized a novel epitope involving residues (Q211, D215, P217) at the central interface of domain II, and three recognized residues at both domain III and the lateral ridge of domain II, suggesting a more frequent presence of interdomain epitopes than previously appreciated. Compared with mAbs generated by traditional protocols, the potent neutralizing mAbs generated by a new protocol recognized multiple residues in A strand or residues in C strand/CC′ loop of DENV2 and DENV1, and multiple residues in BC loop and residues in DE loop, EF loop/F strand or G strand of DENV1. The predominant epitopes of anti-E antibodies in polyclonal sera were found to include both fusion loop and non-fusion residues in the same or adjacent monomer. Our analyses have implications for epitope-specific diagnostics and epitope-based dengue vaccines. This high throughput method has tremendous application for mapping both intra and interdomain epitopes recognized by human mAbs and polyclonal sera, which would further our understanding of humoral immune responses to DENV at the epitope level. Dengue virus is the leading cause of arboviral diseases worldwide. The envelope protein is the major target of neutralizing antibodies and vaccine development. While previous studies have reported several epitopes on envelope protein, the possibility of interdomain epitopes and the relationship of epitopes to neutralizing potency remain unexplored. We developed a high throughput dot blot assay by using 67 alanine mutants of surface-exposed envelope residues as a systematic approach to identify epitopes recognized by mouse monoclonal antibodies and polyclonal human sera. Our results suggested the presence of interdomain epitopes more frequent than previously appreciated. Compared with monoclonal antibodies generated by traditional protocol, the potent neutralizing monoclonal antibodies generated by a new protocol showed several unique features of their epitopes. Moreover, the predominant epitopes of antibodies against envelope protein in polyclonal sera can be identified by this assay. These findings have implications for future development of epitope-specific diagnostics and epitope-based dengue vaccine, and add to our understanding of humoral immune responses to dengue virus at the epitope level.
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影响因子：
--
作者：
Chiou, Shyan-Song;Crill, Wayne D.;Chang, Gwong-Jen J.
通讯作者：
Chang, Gwong-Jen J.
影响因子：
30.3
作者：
Beltramello M;Williams KL;Simmons CP;Macagno A;Simonelli L;Quyen NT;Sukupolvi-Petty S;Navarro-Sanchez E;Young PR;de Silva AM;Rey FA;Varani L;Whitehead SS;Diamond MS;Harris E;Lanzavecchia A;Sallusto F
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Sallusto F
DOI：
10.4269/ajtmh.1988.38.172
发表时间：
1988-01-01
影响因子：
3.3
作者：
BURKE, DS;NISALAK, A;SCOTT, RM
通讯作者：
SCOTT, RM
DOI：
10.4269/ajtmh.1982.31.548
发表时间：
1982-01-01
影响因子：
3.3
作者：
GENTRY, MK;HENCHAL, EA;DALRYMPLE, JM
通讯作者：
DALRYMPLE, JM
影响因子：
64.8
作者：
HALSTEAD, SB;OROURKE, EJ
通讯作者：
OROURKE, EJ