登革病毒非结构蛋白1和包膜蛋白Ⅲ区B细胞表位研究
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摘要
由登革病毒(dengue virus,DENV)所致的登革热是热带和亚热带地区主要的公共卫生问题,在我国南方地区,特别是东南沿海各省几乎每年均有登革热流行。DENV有4种血清型依次为DENV-1、-2、-3和-4。任何一种血清型的感染均可引起一系列的临床症状,包括隐匿性感染、典型登革热(DF)以及危胁生命的登革出血热(DHF)或登革休克综合征(DSS)。初次感染DENV后,对于同型病毒的再次感染可产生长久的免疫力,但Ⅰ～Ⅳ型DENV之间缺乏交叉免疫保护作用,因而生活在疫区的每个人一生中都有可能面临Ⅰ～Ⅳ型DENV感染的威胁。流行病学调查结果显示再次感染异型DENV是导致DENV感染患者发生DHF/DSS的重要危险因素,由于在一个地区存在不同血清型DENV的交替流行,人群普遍易感,这就更增加了DHF/DSS发生的可能性,也是目前研制特异性登革疫苗的主要障碍。
当前人类对登革热的防治面临着许多难题,没有特异性的治疗药物和安全有效的疫苗,但及时采取临床救治措施可以大大减少DHF/DSS的发病率和死亡率,由于多数DENV感染者早期缺乏特异的临床表现,仅有发热、寒战等流感样症状,难以跟其它发热疾病和出血热疾病区分,必须依赖实验室的诊断加以确认。然而,当前实验室诊断存在众多问题：病毒分离培养和RT-PCR等检测技术影响因素多,需要昂贵仪器、专门的操作场地及熟练技术人员,不利于推广；黄病毒属病毒间抗体具有广泛的交叉反应性,血清学检测抗体的方法难以区分是黄病毒属疫苗接种的结果还是DENV感染结果。因此,研制疫苗和早期诊断免疫试剂是目前登革热防治的迫切任务。针对当前DENV感染防治中存在的问题,本课题旨在鉴定DENV蛋白中可作为诊断靶标和疫苗候选蛋白的B细胞表位,为研制DENV感染诊断试剂和疫苗的奠定基础。
DENV非结构蛋白1(NS1)具有良好的免疫原性,能够诱导产生保护性的体液免疫和细胞免疫,是DENV疫苗研制的靶标。NS1上既有血清型特异性表位,同时也存在Ⅰ～Ⅳ型DENV交叉反应性表位,是一种理想的早期分型诊断标志物。DENV包膜(E)蛋白中第三功能区(EⅢ)是一个相对独立和完整的区域,暴露于病毒颗粒表面,是E蛋白中诱导中和抗体的主要区域,针对EⅢ的特异性抗体是拮抗病毒与宿主细胞结合的最强拮抗剂,因此,EⅢ是DENV特异性亚单位疫苗的主要候选。了解病毒蛋白表位及其如何诱导保护性和致病性免疫应答将有助于研制有效疫苗和诊断试剂。本研究首先采用Ⅰ～Ⅳ型重组DENV NS1蛋白和对应的同型DENV分别免疫小鼠制备针对Ⅰ～Ⅳ型DENV NS1的交叉性单抗、每种血清型特异性单抗和动物免疫血清,这些小鼠单抗和免疫血清同覆盖DENV-1 NS1蛋白的重叠多肽反应,鉴定DENV-1 NS1上的B细胞表位和优势表位；接着采用毕赤酵母表达的重组DENV-1 EⅢ免疫小鼠,制备针对Ⅰ～Ⅳ型DENV EⅢ的交叉性中和单抗和DENV-1血清型特异性中和单抗,采用针对DENV-1 EⅢ中和单抗同覆盖DENV-1 EⅢ蛋白的重叠多肽反应鉴定DENV-IE蛋白EⅢ上中和B细胞表位。以上研究为研制DENV感染的诊断试剂和疫苗提供依据。
本研究的目的在于：(1)全面分析DENV-1 NS1蛋白的B细胞表位；(2)精确定位DENV-1E蛋白EⅢ中和B细胞表位。这些DENV蛋白上的B细胞表位为研制DENV诊断试剂和疫苗奠定基础。
本研究分为3个部分：
第一部分：DENV NS1单抗、DENV-1 EⅢ单抗以及免疫动物血清的制备与鉴定
首先采用Ⅰ～Ⅳ型DENV NS1重组蛋白及其对应同型DENV分别免疫Balb／c小鼠,制备鉴定针对Ⅰ～Ⅳ型DENV NS1的交叉反应单抗、每种血清型特异性单抗和对应的动物血清。从9只DENV-1 NS1免疫小鼠,4只DENV-2 NS1免疫小鼠,4只DENV-3 NS1免疫小鼠,10只DENV-4 NS1免疫小鼠中共获得抗DENV NS1单抗149株和对应的27只NS1小鼠免疫血清,这些单抗Ig亚类大多数为IgG1,其中25株DENV-1 NS1血清型特异性单抗,20株DENV-2 NS1血清型特异性单抗,15株DENV-3 NS1血清型特异性单抗和15株DENV-4 NS1血清型特异性单抗,其它74株交叉反应性单抗以不同的方式同Ⅰ～Ⅳ型DENVNS1反应。
接着采用DENV-1 EⅢ重组蛋白为免疫原,免疫Balb/c小鼠,制备抗DENV-1EⅢ单抗,鉴定单抗的免疫学特性、中和活性和交叉反应性。共制备抗DENV-1EⅢ单抗37株,36株单抗具有中和活性,这些单抗亚类大多数为IgG1。这些中和单抗中,其中17株DENV-1血清型特异性单抗,9株Ⅰ～Ⅳ型DENV共同交叉反应性单抗,其它10株交叉反应性单抗以不同的方式同Ⅰ～Ⅳ型DENV EⅢ反应。
这些单抗和动物免疫血清为下一步研究蛋白的B细胞表位奠定了基础。
第二部分：DENV-1 NS1 B细胞表位的鉴定
1.49株来源于Ⅰ～Ⅳ型DENV NS1的针对Ⅰ～Ⅳ型DENV NS1的交叉单抗、每种血清型特异性单抗和对应的27只小鼠免疫血清与一组覆盖DENV-1 NS1蛋白的重叠15肽反应,分析DENV-1 NS1的B细胞表位。在25株DENV-1血清型特异性单抗中大多数单抗同DENV-1 NS1的3个区域反应,对应DENV-1 NS1氨基酸残基序列依次位于第1-15、71-85和338-352。蛋白序列比对分析表明以上3个DENV-1血清型特异性抗体识别表位在黄病毒属病毒或Ⅰ～Ⅳ型DENV无共同序列,在目前已报道的DENV-1分离株中高度保守,提示这3个区域是DENV-1血清型特异性B细胞表位。Ⅰ～Ⅳ型DENV共同交叉单抗鉴定DENV-1NS1上5个Ⅰ～Ⅳ型DENV共同交叉B细胞表位,位于DENV-1 NS1氨基酸残基第21-35、111-125、191-205、261-275和291-305。蛋白序列比对分析表明以上Ⅰ～Ⅳ型DENV共同交叉B细胞表位中的25VHTWTEQYKFQ35、112KYSWKSWGKAK122、193AVHADMGYWIES204、266GPWHLGKLE274和294RGPSLRTTT302在已报道的Ⅰ～Ⅳ型DENV分离株中高度保守,提示这5个表位是Ⅰ～Ⅳ型DENV共同交叉B细胞表位。DENV-1 NS1免疫小鼠血清反应结果表明位于DENV-1 NS1氨基酸残基第1-15和21-35的2区域具有高度的免疫反应性,提示以上2区域是DENV-1 NS1上优势B细胞表位,DENV-1 NS1第1-15是优势DENV-1血清型特异性免疫B细胞表位。DENV NS1上氨基酸残基第111-125、191-205和261-275在每种血清型DENV NS1免疫动物血清中均表现出强的免疫原性,提示以上3个区域是DENV NS1上优势Ⅰ～Ⅳ型DENV共同交叉B细胞表位。这些NS1上免疫优势的DENV-1血清型特异性B细胞表位和Ⅰ～Ⅳ型DENV共同交叉B细胞表位可能有助于研制DENV疫苗和诊断试剂。
第三部分：DENV-1 EⅢ中和B细胞表位鉴定
36株针对DENV-1 EⅢ中和单抗同两组分别覆盖DENV-1 EⅢ的重叠反应多肽(12肽和16肽)反应定位DENV-1 EⅢ上的诱导中和抗体的B细胞表位。17株DENV-1血清型特异性中和单抗中2株分别同DENV-1 EⅢ的2个区域反应,位于DENV-1 E蛋白氨基酸残基序列第3129-348和381-392。蛋白序列比对分析表明以上两区域黄病毒属病毒或Ⅰ～Ⅳ型DENV无共同序列,仅DENV-1 E蛋白氨基酸残基第381—392在目前已报道的DENV-1分离株中高度保守,提示DENV-1 E蛋白氨基酸残基第333-348是DENV-1分离株特异性中和B细胞表位,第381-392是DENV-1血清型特异性中和B细胞表位。9株Ⅰ～Ⅳ型DENV共同交叉中和单抗中7株同DENV-1 EⅢ的1个区域反应,位于DENV-1 E蛋白氨基酸残基序列第309-320,蛋白序列比对分析表明DENV-1 E蛋白中310KEVAETQHGT319在已报道的Ⅰ～Ⅳ型DENV分离株中高度保守,氨基酸残基替代发现DENV-1中E309、V312、A313和V320不影响蛋白抗原性,提示该位点是Ⅰ～Ⅳ型DENV共同交叉中和B细胞表位。这些DENV-1 E蛋白EⅢ上中和B细胞表位可能有助于研制新的DENV亚单位疫苗。
小结
综合以上三个部分的研究结果,本研究的发现和创新点如下：
一、采用一组来源于Ⅰ～Ⅳ型DENV NS1的每种血清型特异性单抗、交叉单抗和免疫血清全面分析DENV-1 NS1的B细胞表位。精确定位3个DENV-1NS1血清型特异性B细胞表位分别位于NS1蛋白氨基酸残基第1-15、71-85和338-352位氨基酸。蛋白序列比对分析表明这3个DENV-1血清型特异性B细胞表位在已报道的DENV-1分离株中高度保守,其中aa1-15在DENV-1 NS1免疫小鼠血清中有很强的抗原性,是免疫优势DENV-1 NS1血清型特异性B细胞表位。精确定位Ⅰ-Ⅳ型DENV NS1共同交叉表位的依次位于NS1蛋白氨基酸残基第21-35、111-125、191-205、261-275和291-305位氨基酸。以上5个Ⅰ-Ⅳ型DENV NS1共同交叉表位中25VHTWTEQYKFQ35、112KYSWKSWGKAK122, 193AVHADMGYWIES204、266GPWHLGKLE274和294RGPSLRTTT302在已报道的Ⅰ-Ⅳ型DENV分离株中高度保守,其中区域111-125aa、191-205aa和261-275aa在各型DENY-1 NS1免疫血清中有强的抗原反应性,区域aa21-35在DENV-2 NS1免疫小鼠血清和DENV-1 NS1免疫小鼠血清中具有很强的抗原反应性。这些新发现的NS1血清型特异性和Ⅰ～Ⅳ型共同交叉的B细胞表位将有助于DENV疫苗和分型诊断试剂盒的研制。
二、采用一组来源于DENV-1 E蛋白EⅢ的中和单抗分析DENV-1 E蛋白EⅢ诱导中和抗体的B细胞表位。精确定位1个DENV-1血清型特异性中和B细胞表位,位于DENV-1 E蛋白氨基酸残基第381-392位氨基酸,蛋白序列比对分析表明该表位在已报道的DENV-1分离株中高度保守。精确定位1个Ⅰ～Ⅳ型DENV共同交叉中和B细胞表位,位于DENV-1 E蛋白氨基酸残基第309-320,蛋白序列比对分析表明该表位中310KEVAETQHGT319在已报道的Ⅰ～Ⅳ型DENV分离株中高度保守,E309、V312、A313和V320氨基酸残基替换不影响蛋白抗原性。这些新发现中和表位将有助于DENV亚单位疫苗研制。
Dengue, caused by dengue virus (DENV); is a major public health problem in over 100 tropical and subtropical countries and occurres almost every year in southern China, especially in the southeast coastal provinces. DENV is classified into four serotypes, DENV-1,-2,-3, and -4. An infection with any of these serotypes can cause clinical manifestations including dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). Subsequent heterologous infections may increase the risk of the developing DHF or DSS. Since protection against heterologous serotype infections is both partial and transient, people who live in epidemic areas may be susceptible to four DENV infections in their lifetime. Thus, the cocirculation of virus serotypes in a community is the most common risk factor associated with the emergence of DHF or DSS, the most severe forms of dengue. Since no vaccine is available that elicits long-term protective immunity against all four DENV serotypes and early identification of multiple DENV infections in the acute phase of the illness is important for reducing morbidity and mortality associated with DHF and DSS, as well as for epidemiological control in areas where multiple flaviviruses are endemic. No single diagnostic assay in isolation is adequately sensitive and specific enough to diagnose all acute cases of dengue. DENVspecific RT-PCR and virus isolation relies heavily on experienced technicians and specialized laboratory equipment, and sometimes provides false-positive results due to contamination. DENV serology is a simple and robust approach to diagnosis, but this method is not sensitive in the very early stages of disease and strictly and lacks specificity because of cross-reactivity with other flaviviruses. So the development of a DENV vaccine and an early diagnostic method remain a global public health challenge. A better understanding of how viral components correlate with immune protection and pathogenic determinants is likely to contribute to the development of an effective vaccine and diagnostic method. In the present study, we mapped the B-cell epitopes on viral proteins which correlate with protection and diagnostic marker.
Immunization with NS1 or passive administration of anti-NS1 MAbs can protect mice against a lethal virus challenge. NS1 has also been identified as being both a group-specific and serotype-specific determinant and as an early serotyping diagnostic marker. EIII of Envelope (E) protein is a relative independence and integrity of the region, which can be isolated after trypsin treatment of the E protein, and on the surface of DENV virions. It can mediate receptor binding and membrane fusion. Antibodies against EIII of E protein is the most powerful blockers to virus infection. This region has been identified as a candidate targets for vaccine development. A better understanding of how viral components correlate with immune protection and pathogenic determinants is likely to contribute to the development of an effective vaccine. In this study, we produced MAbs against those proteins. The B cell epitopes on those DENV proteins were mapped by those MAbs and antisera from small animals immunized by those DENV proteins, respectively, using competition binding assays and Pepscan analysis. This may be useful for developing an early diagnostic method and designing DENV vaccines.
The purpose of this study is to:(1) comprehensively analysis the B cell epitopes of DENV-1 NS1 protein; (2) precisely map the neutralizing B cell epitopes in EⅢof the envelope protein of DENV-1; which are useful for DENV vaccine and early diagnosis.
The research is divided into three parts:
Ⅰ. Production and characterization of monoclonal antibodies and hyperimmune serum against DENV NS1 protein or DENV-1 EIII
A combination of purified recombinant NS1 protein derived from each DENV serotype and with the corresponding DENV was used to immunize BALB/c mice for the production of hybridomas and hyperimmune serum, respectively. A total of 149 hybridoma cell lines from 27 mice immunized with the four DENV serotypes (9 mice with DENV-1 NS1,4 mice with DENV-2 NS1,4 mice with DENV-3 NS1, and 10 mice with DENV-4 NS1) that stably produced MAbs were established on the basis of their strong reactivity with both recombinant NS1 protein and DENV-infected cell lysates as antigen in the ELISA. Immunofluorescence assays (IFA) showed that all MAbs recognized the native NS1 antigen in DENV-infected cells. Most of the MAbs were identified as IgG1 isotype. The serotype specificity and cross-reactivity of the MAbs was further characterized by testing their reactivity with each virus serotype by ELISA and Western blot analysis. Of 149 MAbs,25 MAbs,20 MAbs,15 MAbs, and 15 MAbs reacted exclusively with the DENV-1 NS1, DENV-2 NS1, DENV-3 NS1, and DENV-4 NS1 proteins, respectively, in ELISA plus Western blotting or IFA assays. The remaining 74 MAbs showed various patterns of cross-reactivity with the four DENV serotypes.
The purified recombinant DENV-1 EIII also was used to immunize BALB/c mice for the production of hybridomas and hyperimmune serum. A total of 37 hybridoma cell lines that stably produced MAbs were established on the basis of their strong reactivity with both recombinant DENV-1 EIII protein and DENV-1-infected cell lysates as antigen in the ELISA. IFA showed that all MAbs recognized the native E antigen in DENV-infected cells. Most of the MAbs were identified as IgGl isotype. The serotype specificity and cross-reactivity of the MAbs was further characterized by testing their reactivity with each virus serotype by ELISA and Western blot analysis. The neutralization efficiency to DENV-1,2,3,4 of 37 MAbs against EIII of DENV-1 was characterized by Plaque Reduction Neutralization Test (PRNT).36 MAbs can neutralize DENV infectivity. Of those 36 MAbs,17 MAbs reacted exclusively with the DENV-1 E protein in ELISA plus Western blotting or IFA assays. 9 MAbs reacted exclusively with four DENV serotypes in ELISA plus Western blotting or IFA assays.The remaining 10 MAbs showed various patterns of cross-reactivity with the four DENV serotypes.
These MAbs provides a good tool for mapping B-cell epitopes of those DENV proteins.
Ⅱ. Comprehensively mapping of B-cell epitopes of nonstructural protein 1 from DENV-1
We mapped B-cell linear epitopes on NS1 using 149 monoclonal antibodies with DENV serotype specificity and cross-reactivity as well as antisera from 27 mice immunized with the four DENV serotypes. Epitope recognition analysis was performed using a set of 15-mer sequential overlapping peptides that spanned the entire NS1 protein from DENV-1. Among the 25 DENV-1 serotype-specific MAbs, most of those MAbs showed a strong reaction with three regions of NS1 that are DENV-1 serotype-specific epitopes, namely amino acid residues 1-15,71-85, and 338-352. We compared NS1 sequences in different members of the flavivirus family by aligning NS1 residues 1-15,71-85,111-125, and 338-352 from DENV-1,-2,-3,-4, WNV, YFV, and JEV. Alignment revealed that the amino acid sequences of peptide 1 (residues 1-15), peptide 8 (residues 71-125), and peptide 35 (residues 338-352) were completely conserved among DENV-1 serotypes, but different among the flaviviruses, suggesting that three regions of NS1, i.e. peptide 1 (residues 1-15), peptide 8 (residues 71-125), and peptide 35 (residues 338-352), were DENV-1 serotype specific epitopes. We also identified five group-specific B-cell epitopes, namely amino acid residues 21-35,111-125,191-205,261-275, and 291-305, that were highly conserved among isolates of the four DENV serotypes. NS1 sequences from different members of the flavivirus family were compared by aligning NS1 residues 21-35,111-125,191-205,261-275, and 291-305 from DENV-1,-2,-3,-4, WNV, YFV, and JEV. This comparison revealed that the amino acid sequences in these regions were conserved among the four DENV serotypes, but different among the other flaviviruses (WNV, YFV, and JEV) and the amino acid sequences 25VHTWTEQYKFQ35,112KYSWKSWGKAK122,193AVHADMGYWIES204, 266GPWHLGKLE274, and 294RGPSLRTTT302 were similar among the serotypes, indicated that these regions of NS1 are group-specific B-cell epitopes. Peptide 1 (residues 1-15) showed high immunoreactivity, i.e. a high mean absorbance/cut-off ratio, with sera from DENV-1 NS1-immunized mice. This suggests that the region of NS1 comprising residues 1-15 is a serotype-specific immunodominant epitope. Peptide 3 (residues 21-35) reacted with sera from DENV-1-and DENV-2-immunized mice; the high mean absorbance/cut-off ratio was different from that of sera from DENV-3-and DENV-4-immunized mice, suggesting that this region is a DENV-1 and DENV-2 serotype-specific immunodominant epitope. Peptides 12 (residues 111-125), 20 (residues 191-205), and 27 (residues 261-275) had high cross-immunoreactivity with sera from mice immunized with each of the four DENV serotypes. This suggests that the three regions are group-specific immunodominant epitopes among the four DENV serotypes. Taken together, these data suggest that the most immunoreactive epitopes on the NS1 protein involve amino acid residues 1-15,21-35,111-125, 191-205, and 261-275. These novel immunodominant serotype-and group-specific B-cell epitopes of DENV NS1 may aid the development of new dengue vaccines and diagnostic assays.
Ⅲ. Characterization of neutralizing B cell epitopes on envelope protein domainⅢof DENV-1
The neutralizing B-cell epitopes on EⅢof DENV-1 E protein were mapped by 36 neutralizing MAbs against DENV-1 EⅢ. Epitope recognition analysis was performed using two sets of sequential overlapping peptides (16-mer and 12-mer) that spanned the entire EⅢfrom DENV-1 E protein, respectively. Among the 17 DENV-1 serotype-specific neutralizing MAbs, two of those MAbs reacted two regions of DENV-1 E that were DENV-1 serotype-specific neutralizing B-cell epitopes, namely amino acid residues 329-348, and 381-392. We compared E sequences in different members of the flavivirus family by aligning E protein residues 329-348, and 381-392 from DENV-1,-2,-3,-4, WNV, YFV, and JEV. Alignment revealed that only the amino acid sequence of 381-392 was completely conserved among DENV-1 serotypes, but different among the flaviviruses, suggesting that E protein amino acid sequence of 381-392 was a DENV-1 serotype specific neutralizing B-cell epitope. Among the 9 DENV group-specific neutralizing MAbs,7 of those MAbs reacted one region of DENV-1 E that was a DENV group-specific neutralizing epitope, namely amino acid residues 309-320. E sequences from different members of the flavivirus family were compared by aligning E residues 309-320 from DENV-1,-2,-3,-4, WNV, YFV, and JEV. This comparison revealed that the amino acid sequences in these regions were conserved among the four DENV serotypes, but different among the other flaviviruses (WNV, YFV, and JEV) and the amino acid sequences 310KEVAETQHGT319 was similar among the serotypes, indicated that these regions of E is group-specific neutralizing B-cell epitopes. We further defined contact residues on E protein residues 309-320 of a panel of those MAbs and found that substitution of residues E309, V312, A313 and V320 in DENV-2,-3,-4 isolates, were antigenically silent. These novel neutralizing B-cell epitopes of DENV E may aid the development of dengue vaccines.
Summarization:
We identified several immunodominant B-cell epitopes on NS1 using a panel of MAbs and antisera raised in mice against the four DENV serotypes. We identified three regions of NS1 that are DENV-1 serotype-specific B-cell epitopes, namely amino acid residues 1-15,71-85, and 338-352. We compared NS1 sequences in different members of the flavivirus family by aligning NS1 residues 1-15,71-85, 111-125, and 338-352 from DENV-1,-2,-3,-4, WNV, YFV, and JEV. Alignment revealed that the amino acid sequences of peptide 1 (residues 1-15), peptide 8 (residues 71-125), and peptide 35 (residues 338-352) were completely conserved among DENV-1 serotypes, but different among the flaviviruses, suggesting that three regions of NS1, i.e. peptide 1 (residues 1-15), peptide 8 (residues 71-125), and peptide 35 (residues 338-352), were DENV-1 serotype specific B-cell epitopes. Peptide 1 (residues 1-15) showed high immunoreactivity with sera from DENV-1 NS1-immunized mice. This suggests that the region of NS1 comprising residues 1-15 is a serotype-specific immunodominant B-cell epitope. We also identified five group-specific B-cell epitopes, namely amino acid residues residues 21-35,111-125, 191-205,261-275,and 291-305. We compared NS1 sequences in different members of the flavivirus family by aligning NS1 residues 21-35,111-125,191-205, 261-275, and 291-305 from DENV-1,-2,-3,-4, WNV, YFV, and JEV. Alignment revealed that the amino acid sequences in these regions were conserved among the four DENV serotypes, but different among the other flaviviruses (WNV, YFV, and JEV) and the amino acid sequences 25VHTWTEQYKFQ35,112KYSWKSWGKAK122, 193AVHADMGYWIES204,266GPWHLGKLE274, and 294RGPSLRTTT302 were similar among the serotypes, indicated that these regions of NS1 were highly conserved among the four DENV serotypes and were group-specific B-cell epitopes. Peptides 12 (residues 111-125),20 (residues 191-205), and 27 (residues 261-275) had high cross-immunoreactivity with sera from mice immunized with each of the four DENV serotypes. This suggests that the three regions are group-specific B-cell immunodominant epitopes among the four DENV serotypes. These novel immunodominant serotype-and group-specific B-cell epitopes of DENV NS1 may aid the development of new dengue vaccines and diagnostic assays.
We also characterized the neutralizing B-cell epitopes on EⅢof DENV-1 E protein using a panel of neutralizing MAbs against EⅢof DENV-1 E protein. We identified E residues 381-392 that is a DENV-1 serotype-specific neutralizing epitope. We compared E sequences in different members of the flavivirus family by aligning E residues 381-392 from DENV-1,-2,-3,-4, WNV, YFV, and JEV. Alignment revealed that the amino acid sequences were completely conserved among DENV-1 serotypes, but different among the flaviviruses, suggesting that E residues 381-392 was a DENV-1 serotype specific neutralizing B-cell epitope. We also identified a group-specific neutralizing B-cell epitope, namely-amino acid residues residues 309-320 on E protein. We compared E sequences in different members of the flavivirus family by aligning E residues 309-320 from DENV-1,-2,-3,-4, WNV, YFV, and JEV. Alignment revealed that the amino acid sequences in the region were conserved among the four DENV serotypes, but different among the other flaviviruses (WNV, YFV, and JEV) and the amino acid sequences 310KEVAETQHGT319 was similar among the serotypes, indicated that these regions of E protein was highly conserved among the four DENV serotypes and was a group-specific neutralizing B-cell epitope. We further defined contact residues on residues 309-320 of a panel of those group-specific neutralizing MAbs and found that substitution of residues E309, V312, A313 and V320 in DENV-2,-3,-4 isolates, were antigenically silent. Those neutralizing B-cell epitopes of DENV E protein may aid the development of dengue vaccines.
当前人类对登革热的防治面临着许多难题,没有特异性的治疗药物和安全有效的疫苗,但及时采取临床救治措施可以大大减少DHF/DSS的发病率和死亡率,由于多数DENV感染者早期缺乏特异的临床表现,仅有发热、寒战等流感样症状,难以跟其它发热疾病和出血热疾病区分,必须依赖实验室的诊断加以确认。然而,当前实验室诊断存在众多问题：病毒分离培养和RT-PCR等检测技术影响因素多,需要昂贵仪器、专门的操作场地及熟练技术人员,不利于推广；黄病毒属病毒间抗体具有广泛的交叉反应性,血清学检测抗体的方法难以区分是黄病毒属疫苗接种的结果还是DENV感染结果。因此,研制疫苗和早期诊断免疫试剂是目前登革热防治的迫切任务。针对当前DENV感染防治中存在的问题,本课题旨在鉴定DENV蛋白中可作为诊断靶标和疫苗候选蛋白的B细胞表位,为研制DENV感染诊断试剂和疫苗的奠定基础。
DENV非结构蛋白1(NS1)具有良好的免疫原性,能够诱导产生保护性的体液免疫和细胞免疫,是DENV疫苗研制的靶标。NS1上既有血清型特异性表位,同时也存在Ⅰ～Ⅳ型DENV交叉反应性表位,是一种理想的早期分型诊断标志物。DENV包膜(E)蛋白中第三功能区(EⅢ)是一个相对独立和完整的区域,暴露于病毒颗粒表面,是E蛋白中诱导中和抗体的主要区域,针对EⅢ的特异性抗体是拮抗病毒与宿主细胞结合的最强拮抗剂,因此,EⅢ是DENV特异性亚单位疫苗的主要候选。了解病毒蛋白表位及其如何诱导保护性和致病性免疫应答将有助于研制有效疫苗和诊断试剂。本研究首先采用Ⅰ～Ⅳ型重组DENV NS1蛋白和对应的同型DENV分别免疫小鼠制备针对Ⅰ～Ⅳ型DENV NS1的交叉性单抗、每种血清型特异性单抗和动物免疫血清,这些小鼠单抗和免疫血清同覆盖DENV-1 NS1蛋白的重叠多肽反应,鉴定DENV-1 NS1上的B细胞表位和优势表位；接着采用毕赤酵母表达的重组DENV-1 EⅢ免疫小鼠,制备针对Ⅰ～Ⅳ型DENV EⅢ的交叉性中和单抗和DENV-1血清型特异性中和单抗,采用针对DENV-1 EⅢ中和单抗同覆盖DENV-1 EⅢ蛋白的重叠多肽反应鉴定DENV-IE蛋白EⅢ上中和B细胞表位。以上研究为研制DENV感染的诊断试剂和疫苗提供依据。
本研究的目的在于：(1)全面分析DENV-1 NS1蛋白的B细胞表位；(2)精确定位DENV-1E蛋白EⅢ中和B细胞表位。这些DENV蛋白上的B细胞表位为研制DENV诊断试剂和疫苗奠定基础。
本研究分为3个部分：
第一部分：DENV NS1单抗、DENV-1 EⅢ单抗以及免疫动物血清的制备与鉴定
首先采用Ⅰ～Ⅳ型DENV NS1重组蛋白及其对应同型DENV分别免疫Balb／c小鼠,制备鉴定针对Ⅰ～Ⅳ型DENV NS1的交叉反应单抗、每种血清型特异性单抗和对应的动物血清。从9只DENV-1 NS1免疫小鼠,4只DENV-2 NS1免疫小鼠,4只DENV-3 NS1免疫小鼠,10只DENV-4 NS1免疫小鼠中共获得抗DENV NS1单抗149株和对应的27只NS1小鼠免疫血清,这些单抗Ig亚类大多数为IgG1,其中25株DENV-1 NS1血清型特异性单抗,20株DENV-2 NS1血清型特异性单抗,15株DENV-3 NS1血清型特异性单抗和15株DENV-4 NS1血清型特异性单抗,其它74株交叉反应性单抗以不同的方式同Ⅰ～Ⅳ型DENVNS1反应。
接着采用DENV-1 EⅢ重组蛋白为免疫原,免疫Balb/c小鼠,制备抗DENV-1EⅢ单抗,鉴定单抗的免疫学特性、中和活性和交叉反应性。共制备抗DENV-1EⅢ单抗37株,36株单抗具有中和活性,这些单抗亚类大多数为IgG1。这些中和单抗中,其中17株DENV-1血清型特异性单抗,9株Ⅰ～Ⅳ型DENV共同交叉反应性单抗,其它10株交叉反应性单抗以不同的方式同Ⅰ～Ⅳ型DENV EⅢ反应。
这些单抗和动物免疫血清为下一步研究蛋白的B细胞表位奠定了基础。
第二部分：DENV-1 NS1 B细胞表位的鉴定
1.49株来源于Ⅰ～Ⅳ型DENV NS1的针对Ⅰ～Ⅳ型DENV NS1的交叉单抗、每种血清型特异性单抗和对应的27只小鼠免疫血清与一组覆盖DENV-1 NS1蛋白的重叠15肽反应,分析DENV-1 NS1的B细胞表位。在25株DENV-1血清型特异性单抗中大多数单抗同DENV-1 NS1的3个区域反应,对应DENV-1 NS1氨基酸残基序列依次位于第1-15、71-85和338-352。蛋白序列比对分析表明以上3个DENV-1血清型特异性抗体识别表位在黄病毒属病毒或Ⅰ～Ⅳ型DENV无共同序列,在目前已报道的DENV-1分离株中高度保守,提示这3个区域是DENV-1血清型特异性B细胞表位。Ⅰ～Ⅳ型DENV共同交叉单抗鉴定DENV-1NS1上5个Ⅰ～Ⅳ型DENV共同交叉B细胞表位,位于DENV-1 NS1氨基酸残基第21-35、111-125、191-205、261-275和291-305。蛋白序列比对分析表明以上Ⅰ～Ⅳ型DENV共同交叉B细胞表位中的25VHTWTEQYKFQ35、112KYSWKSWGKAK122、193AVHADMGYWIES204、266GPWHLGKLE274和294RGPSLRTTT302在已报道的Ⅰ～Ⅳ型DENV分离株中高度保守,提示这5个表位是Ⅰ～Ⅳ型DENV共同交叉B细胞表位。DENV-1 NS1免疫小鼠血清反应结果表明位于DENV-1 NS1氨基酸残基第1-15和21-35的2区域具有高度的免疫反应性,提示以上2区域是DENV-1 NS1上优势B细胞表位,DENV-1 NS1第1-15是优势DENV-1血清型特异性免疫B细胞表位。DENV NS1上氨基酸残基第111-125、191-205和261-275在每种血清型DENV NS1免疫动物血清中均表现出强的免疫原性,提示以上3个区域是DENV NS1上优势Ⅰ～Ⅳ型DENV共同交叉B细胞表位。这些NS1上免疫优势的DENV-1血清型特异性B细胞表位和Ⅰ～Ⅳ型DENV共同交叉B细胞表位可能有助于研制DENV疫苗和诊断试剂。
第三部分：DENV-1 EⅢ中和B细胞表位鉴定
36株针对DENV-1 EⅢ中和单抗同两组分别覆盖DENV-1 EⅢ的重叠反应多肽(12肽和16肽)反应定位DENV-1 EⅢ上的诱导中和抗体的B细胞表位。17株DENV-1血清型特异性中和单抗中2株分别同DENV-1 EⅢ的2个区域反应,位于DENV-1 E蛋白氨基酸残基序列第3129-348和381-392。蛋白序列比对分析表明以上两区域黄病毒属病毒或Ⅰ～Ⅳ型DENV无共同序列,仅DENV-1 E蛋白氨基酸残基第381—392在目前已报道的DENV-1分离株中高度保守,提示DENV-1 E蛋白氨基酸残基第333-348是DENV-1分离株特异性中和B细胞表位,第381-392是DENV-1血清型特异性中和B细胞表位。9株Ⅰ～Ⅳ型DENV共同交叉中和单抗中7株同DENV-1 EⅢ的1个区域反应,位于DENV-1 E蛋白氨基酸残基序列第309-320,蛋白序列比对分析表明DENV-1 E蛋白中310KEVAETQHGT319在已报道的Ⅰ～Ⅳ型DENV分离株中高度保守,氨基酸残基替代发现DENV-1中E309、V312、A313和V320不影响蛋白抗原性,提示该位点是Ⅰ～Ⅳ型DENV共同交叉中和B细胞表位。这些DENV-1 E蛋白EⅢ上中和B细胞表位可能有助于研制新的DENV亚单位疫苗。
小结
综合以上三个部分的研究结果,本研究的发现和创新点如下：
一、采用一组来源于Ⅰ～Ⅳ型DENV NS1的每种血清型特异性单抗、交叉单抗和免疫血清全面分析DENV-1 NS1的B细胞表位。精确定位3个DENV-1NS1血清型特异性B细胞表位分别位于NS1蛋白氨基酸残基第1-15、71-85和338-352位氨基酸。蛋白序列比对分析表明这3个DENV-1血清型特异性B细胞表位在已报道的DENV-1分离株中高度保守,其中aa1-15在DENV-1 NS1免疫小鼠血清中有很强的抗原性,是免疫优势DENV-1 NS1血清型特异性B细胞表位。精确定位Ⅰ-Ⅳ型DENV NS1共同交叉表位的依次位于NS1蛋白氨基酸残基第21-35、111-125、191-205、261-275和291-305位氨基酸。以上5个Ⅰ-Ⅳ型DENV NS1共同交叉表位中25VHTWTEQYKFQ35、112KYSWKSWGKAK122, 193AVHADMGYWIES204、266GPWHLGKLE274和294RGPSLRTTT302在已报道的Ⅰ-Ⅳ型DENV分离株中高度保守,其中区域111-125aa、191-205aa和261-275aa在各型DENY-1 NS1免疫血清中有强的抗原反应性,区域aa21-35在DENV-2 NS1免疫小鼠血清和DENV-1 NS1免疫小鼠血清中具有很强的抗原反应性。这些新发现的NS1血清型特异性和Ⅰ～Ⅳ型共同交叉的B细胞表位将有助于DENV疫苗和分型诊断试剂盒的研制。
二、采用一组来源于DENV-1 E蛋白EⅢ的中和单抗分析DENV-1 E蛋白EⅢ诱导中和抗体的B细胞表位。精确定位1个DENV-1血清型特异性中和B细胞表位,位于DENV-1 E蛋白氨基酸残基第381-392位氨基酸,蛋白序列比对分析表明该表位在已报道的DENV-1分离株中高度保守。精确定位1个Ⅰ～Ⅳ型DENV共同交叉中和B细胞表位,位于DENV-1 E蛋白氨基酸残基第309-320,蛋白序列比对分析表明该表位中310KEVAETQHGT319在已报道的Ⅰ～Ⅳ型DENV分离株中高度保守,E309、V312、A313和V320氨基酸残基替换不影响蛋白抗原性。这些新发现中和表位将有助于DENV亚单位疫苗研制。
Dengue, caused by dengue virus (DENV); is a major public health problem in over 100 tropical and subtropical countries and occurres almost every year in southern China, especially in the southeast coastal provinces. DENV is classified into four serotypes, DENV-1,-2,-3, and -4. An infection with any of these serotypes can cause clinical manifestations including dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). Subsequent heterologous infections may increase the risk of the developing DHF or DSS. Since protection against heterologous serotype infections is both partial and transient, people who live in epidemic areas may be susceptible to four DENV infections in their lifetime. Thus, the cocirculation of virus serotypes in a community is the most common risk factor associated with the emergence of DHF or DSS, the most severe forms of dengue. Since no vaccine is available that elicits long-term protective immunity against all four DENV serotypes and early identification of multiple DENV infections in the acute phase of the illness is important for reducing morbidity and mortality associated with DHF and DSS, as well as for epidemiological control in areas where multiple flaviviruses are endemic. No single diagnostic assay in isolation is adequately sensitive and specific enough to diagnose all acute cases of dengue. DENVspecific RT-PCR and virus isolation relies heavily on experienced technicians and specialized laboratory equipment, and sometimes provides false-positive results due to contamination. DENV serology is a simple and robust approach to diagnosis, but this method is not sensitive in the very early stages of disease and strictly and lacks specificity because of cross-reactivity with other flaviviruses. So the development of a DENV vaccine and an early diagnostic method remain a global public health challenge. A better understanding of how viral components correlate with immune protection and pathogenic determinants is likely to contribute to the development of an effective vaccine and diagnostic method. In the present study, we mapped the B-cell epitopes on viral proteins which correlate with protection and diagnostic marker.
Immunization with NS1 or passive administration of anti-NS1 MAbs can protect mice against a lethal virus challenge. NS1 has also been identified as being both a group-specific and serotype-specific determinant and as an early serotyping diagnostic marker. EIII of Envelope (E) protein is a relative independence and integrity of the region, which can be isolated after trypsin treatment of the E protein, and on the surface of DENV virions. It can mediate receptor binding and membrane fusion. Antibodies against EIII of E protein is the most powerful blockers to virus infection. This region has been identified as a candidate targets for vaccine development. A better understanding of how viral components correlate with immune protection and pathogenic determinants is likely to contribute to the development of an effective vaccine. In this study, we produced MAbs against those proteins. The B cell epitopes on those DENV proteins were mapped by those MAbs and antisera from small animals immunized by those DENV proteins, respectively, using competition binding assays and Pepscan analysis. This may be useful for developing an early diagnostic method and designing DENV vaccines.
The purpose of this study is to:(1) comprehensively analysis the B cell epitopes of DENV-1 NS1 protein; (2) precisely map the neutralizing B cell epitopes in EⅢof the envelope protein of DENV-1; which are useful for DENV vaccine and early diagnosis.
The research is divided into three parts:
Ⅰ. Production and characterization of monoclonal antibodies and hyperimmune serum against DENV NS1 protein or DENV-1 EIII
A combination of purified recombinant NS1 protein derived from each DENV serotype and with the corresponding DENV was used to immunize BALB/c mice for the production of hybridomas and hyperimmune serum, respectively. A total of 149 hybridoma cell lines from 27 mice immunized with the four DENV serotypes (9 mice with DENV-1 NS1,4 mice with DENV-2 NS1,4 mice with DENV-3 NS1, and 10 mice with DENV-4 NS1) that stably produced MAbs were established on the basis of their strong reactivity with both recombinant NS1 protein and DENV-infected cell lysates as antigen in the ELISA. Immunofluorescence assays (IFA) showed that all MAbs recognized the native NS1 antigen in DENV-infected cells. Most of the MAbs were identified as IgG1 isotype. The serotype specificity and cross-reactivity of the MAbs was further characterized by testing their reactivity with each virus serotype by ELISA and Western blot analysis. Of 149 MAbs,25 MAbs,20 MAbs,15 MAbs, and 15 MAbs reacted exclusively with the DENV-1 NS1, DENV-2 NS1, DENV-3 NS1, and DENV-4 NS1 proteins, respectively, in ELISA plus Western blotting or IFA assays. The remaining 74 MAbs showed various patterns of cross-reactivity with the four DENV serotypes.
The purified recombinant DENV-1 EIII also was used to immunize BALB/c mice for the production of hybridomas and hyperimmune serum. A total of 37 hybridoma cell lines that stably produced MAbs were established on the basis of their strong reactivity with both recombinant DENV-1 EIII protein and DENV-1-infected cell lysates as antigen in the ELISA. IFA showed that all MAbs recognized the native E antigen in DENV-infected cells. Most of the MAbs were identified as IgGl isotype. The serotype specificity and cross-reactivity of the MAbs was further characterized by testing their reactivity with each virus serotype by ELISA and Western blot analysis. The neutralization efficiency to DENV-1,2,3,4 of 37 MAbs against EIII of DENV-1 was characterized by Plaque Reduction Neutralization Test (PRNT).36 MAbs can neutralize DENV infectivity. Of those 36 MAbs,17 MAbs reacted exclusively with the DENV-1 E protein in ELISA plus Western blotting or IFA assays. 9 MAbs reacted exclusively with four DENV serotypes in ELISA plus Western blotting or IFA assays.The remaining 10 MAbs showed various patterns of cross-reactivity with the four DENV serotypes.
These MAbs provides a good tool for mapping B-cell epitopes of those DENV proteins.
Ⅱ. Comprehensively mapping of B-cell epitopes of nonstructural protein 1 from DENV-1
We mapped B-cell linear epitopes on NS1 using 149 monoclonal antibodies with DENV serotype specificity and cross-reactivity as well as antisera from 27 mice immunized with the four DENV serotypes. Epitope recognition analysis was performed using a set of 15-mer sequential overlapping peptides that spanned the entire NS1 protein from DENV-1. Among the 25 DENV-1 serotype-specific MAbs, most of those MAbs showed a strong reaction with three regions of NS1 that are DENV-1 serotype-specific epitopes, namely amino acid residues 1-15,71-85, and 338-352. We compared NS1 sequences in different members of the flavivirus family by aligning NS1 residues 1-15,71-85,111-125, and 338-352 from DENV-1,-2,-3,-4, WNV, YFV, and JEV. Alignment revealed that the amino acid sequences of peptide 1 (residues 1-15), peptide 8 (residues 71-125), and peptide 35 (residues 338-352) were completely conserved among DENV-1 serotypes, but different among the flaviviruses, suggesting that three regions of NS1, i.e. peptide 1 (residues 1-15), peptide 8 (residues 71-125), and peptide 35 (residues 338-352), were DENV-1 serotype specific epitopes. We also identified five group-specific B-cell epitopes, namely amino acid residues 21-35,111-125,191-205,261-275, and 291-305, that were highly conserved among isolates of the four DENV serotypes. NS1 sequences from different members of the flavivirus family were compared by aligning NS1 residues 21-35,111-125,191-205,261-275, and 291-305 from DENV-1,-2,-3,-4, WNV, YFV, and JEV. This comparison revealed that the amino acid sequences in these regions were conserved among the four DENV serotypes, but different among the other flaviviruses (WNV, YFV, and JEV) and the amino acid sequences 25VHTWTEQYKFQ35,112KYSWKSWGKAK122,193AVHADMGYWIES204, 266GPWHLGKLE274, and 294RGPSLRTTT302 were similar among the serotypes, indicated that these regions of NS1 are group-specific B-cell epitopes. Peptide 1 (residues 1-15) showed high immunoreactivity, i.e. a high mean absorbance/cut-off ratio, with sera from DENV-1 NS1-immunized mice. This suggests that the region of NS1 comprising residues 1-15 is a serotype-specific immunodominant epitope. Peptide 3 (residues 21-35) reacted with sera from DENV-1-and DENV-2-immunized mice; the high mean absorbance/cut-off ratio was different from that of sera from DENV-3-and DENV-4-immunized mice, suggesting that this region is a DENV-1 and DENV-2 serotype-specific immunodominant epitope. Peptides 12 (residues 111-125), 20 (residues 191-205), and 27 (residues 261-275) had high cross-immunoreactivity with sera from mice immunized with each of the four DENV serotypes. This suggests that the three regions are group-specific immunodominant epitopes among the four DENV serotypes. Taken together, these data suggest that the most immunoreactive epitopes on the NS1 protein involve amino acid residues 1-15,21-35,111-125, 191-205, and 261-275. These novel immunodominant serotype-and group-specific B-cell epitopes of DENV NS1 may aid the development of new dengue vaccines and diagnostic assays.
Ⅲ. Characterization of neutralizing B cell epitopes on envelope protein domainⅢof DENV-1
The neutralizing B-cell epitopes on EⅢof DENV-1 E protein were mapped by 36 neutralizing MAbs against DENV-1 EⅢ. Epitope recognition analysis was performed using two sets of sequential overlapping peptides (16-mer and 12-mer) that spanned the entire EⅢfrom DENV-1 E protein, respectively. Among the 17 DENV-1 serotype-specific neutralizing MAbs, two of those MAbs reacted two regions of DENV-1 E that were DENV-1 serotype-specific neutralizing B-cell epitopes, namely amino acid residues 329-348, and 381-392. We compared E sequences in different members of the flavivirus family by aligning E protein residues 329-348, and 381-392 from DENV-1,-2,-3,-4, WNV, YFV, and JEV. Alignment revealed that only the amino acid sequence of 381-392 was completely conserved among DENV-1 serotypes, but different among the flaviviruses, suggesting that E protein amino acid sequence of 381-392 was a DENV-1 serotype specific neutralizing B-cell epitope. Among the 9 DENV group-specific neutralizing MAbs,7 of those MAbs reacted one region of DENV-1 E that was a DENV group-specific neutralizing epitope, namely amino acid residues 309-320. E sequences from different members of the flavivirus family were compared by aligning E residues 309-320 from DENV-1,-2,-3,-4, WNV, YFV, and JEV. This comparison revealed that the amino acid sequences in these regions were conserved among the four DENV serotypes, but different among the other flaviviruses (WNV, YFV, and JEV) and the amino acid sequences 310KEVAETQHGT319 was similar among the serotypes, indicated that these regions of E is group-specific neutralizing B-cell epitopes. We further defined contact residues on E protein residues 309-320 of a panel of those MAbs and found that substitution of residues E309, V312, A313 and V320 in DENV-2,-3,-4 isolates, were antigenically silent. These novel neutralizing B-cell epitopes of DENV E may aid the development of dengue vaccines.
Summarization:
We identified several immunodominant B-cell epitopes on NS1 using a panel of MAbs and antisera raised in mice against the four DENV serotypes. We identified three regions of NS1 that are DENV-1 serotype-specific B-cell epitopes, namely amino acid residues 1-15,71-85, and 338-352. We compared NS1 sequences in different members of the flavivirus family by aligning NS1 residues 1-15,71-85, 111-125, and 338-352 from DENV-1,-2,-3,-4, WNV, YFV, and JEV. Alignment revealed that the amino acid sequences of peptide 1 (residues 1-15), peptide 8 (residues 71-125), and peptide 35 (residues 338-352) were completely conserved among DENV-1 serotypes, but different among the flaviviruses, suggesting that three regions of NS1, i.e. peptide 1 (residues 1-15), peptide 8 (residues 71-125), and peptide 35 (residues 338-352), were DENV-1 serotype specific B-cell epitopes. Peptide 1 (residues 1-15) showed high immunoreactivity with sera from DENV-1 NS1-immunized mice. This suggests that the region of NS1 comprising residues 1-15 is a serotype-specific immunodominant B-cell epitope. We also identified five group-specific B-cell epitopes, namely amino acid residues residues 21-35,111-125, 191-205,261-275,and 291-305. We compared NS1 sequences in different members of the flavivirus family by aligning NS1 residues 21-35,111-125,191-205, 261-275, and 291-305 from DENV-1,-2,-3,-4, WNV, YFV, and JEV. Alignment revealed that the amino acid sequences in these regions were conserved among the four DENV serotypes, but different among the other flaviviruses (WNV, YFV, and JEV) and the amino acid sequences 25VHTWTEQYKFQ35,112KYSWKSWGKAK122, 193AVHADMGYWIES204,266GPWHLGKLE274, and 294RGPSLRTTT302 were similar among the serotypes, indicated that these regions of NS1 were highly conserved among the four DENV serotypes and were group-specific B-cell epitopes. Peptides 12 (residues 111-125),20 (residues 191-205), and 27 (residues 261-275) had high cross-immunoreactivity with sera from mice immunized with each of the four DENV serotypes. This suggests that the three regions are group-specific B-cell immunodominant epitopes among the four DENV serotypes. These novel immunodominant serotype-and group-specific B-cell epitopes of DENV NS1 may aid the development of new dengue vaccines and diagnostic assays.
We also characterized the neutralizing B-cell epitopes on EⅢof DENV-1 E protein using a panel of neutralizing MAbs against EⅢof DENV-1 E protein. We identified E residues 381-392 that is a DENV-1 serotype-specific neutralizing epitope. We compared E sequences in different members of the flavivirus family by aligning E residues 381-392 from DENV-1,-2,-3,-4, WNV, YFV, and JEV. Alignment revealed that the amino acid sequences were completely conserved among DENV-1 serotypes, but different among the flaviviruses, suggesting that E residues 381-392 was a DENV-1 serotype specific neutralizing B-cell epitope. We also identified a group-specific neutralizing B-cell epitope, namely-amino acid residues residues 309-320 on E protein. We compared E sequences in different members of the flavivirus family by aligning E residues 309-320 from DENV-1,-2,-3,-4, WNV, YFV, and JEV. Alignment revealed that the amino acid sequences in the region were conserved among the four DENV serotypes, but different among the other flaviviruses (WNV, YFV, and JEV) and the amino acid sequences 310KEVAETQHGT319 was similar among the serotypes, indicated that these regions of E protein was highly conserved among the four DENV serotypes and was a group-specific neutralizing B-cell epitope. We further defined contact residues on residues 309-320 of a panel of those group-specific neutralizing MAbs and found that substitution of residues E309, V312, A313 and V320 in DENV-2,-3,-4 isolates, were antigenically silent. Those neutralizing B-cell epitopes of DENV E protein may aid the development of dengue vaccines.
引文
[1]Mackenzie JS, Gubler DJ, Petersen LR. Emerging flaviviruses:the spread and resurgence of Japanese encephalitis, West Nile and dengue viruses. Nat Med 2004;10:S98-109
[2]梁文佳,何剑峰,罗会明等广东省2001-2006年登革热流行病学分析.华南预防医学2007,5：4-7.
[3]黄春文,陈敏红,陈艳,等.福州市2004～2008年登革热疫情监测分析.中国卫生检验杂志.2009,19(8)：1870-1871.
[4]George R, Lum L.C.S.1997. Clinical spectrum of dengue infection,in:Gubler, D.J., Kuno, G. (ed.), Dengue and dengue hemorrhagic fever. CAB International, London, United Kingdom,pp.89-113.89-114
[5]Kanakaratne N, Wahala WM, Messer WB, et al. Severe dengue epidemics in Sri Lanka,2003-2006. Emerg Infect Dis 2009;15:192-199
[6]Kurane I, Ennis FE. Immunity and immunopathology in dengue virus infections. Semin Immunol 1992;4:121-127
[7]Gubler DJ. Dengue and dengue hemorrhagic fever. Clin Microbiol Rev 1998; 11:480-496
[8]WHO. Dengue and dengue haemorrhagic fever.2009
[9]De Paula SO, Fonseca BA. Dengue:a review of the laboratory tests a clinician must know to achieve a correct diagnosis. Braz J Infect Dis 2004;8:390-398
[10]WHO. Dengue:Guidelines for diagnosis, treatment, prevention and control. New Edition 2009
[11]WHO. Dengue haemorrhagic fever:diagnosis, treatment and control. Handbook of the World Health Organization.Geneva:WHO,2000, pp.1-84.
[12]WHO Report of the Scientific Working Group meeting on Dengue Report on Dengue. Report on dengue.Geneva:WHO,2007, pp.1-150.
[13]Qiu LW, Di B, Wen K, et al. Development of an antigen capture immunoassay based on monoclonal antibodies specific for dengue virus serotype 2 nonstructural protein 1 for early and rapid identification of dengue virus serotype 2 infections. Clin Vaccine Immunol 2009; 16:88-95
[14]Lindenbach BD, and C. M. Rice. Flaviviridae:the viruses and their replication, p.991-1041. In D. M. Knipe, P. M. Howley, and D. E. Griffin (ed.), Fields virology. Lippincott William & Wilkins, Philadelphia, PA.2001.
[15]Costa SM, Azevedo AS, Paes MV, et al. DNA vaccines against dengue virus based on the nsl gene:the influence of different signal sequences on the protein expression and its correlation to the immune response elicited in mice. Virology 2007;358:413-423
[16]Costa SM, Freire MS, Alves AM, Alves AM. DNA vaccine against the non-structural 1 protein (NS1) of dengue 2 virus. Vaccine 2006;24:4562-4564
[17]Chung KM, Nybakken GE, Thompson BS, et al. Antibodies against West Nile Virus nonstructural protein NS1 prevent lethal infection through Fc gamma receptor-dependent and-independent mechanisms. J Virol 2006;80:1340-1351
[18]Falgout B, Bray M, Schlesinger JJ, Lai CJ. Immunization of mice with recombinant vaccinia virus expressing authentic dengue virus nonstructural protein NS1 protects against lethal dengue virus encephalitis. J Virol 1990; 64:4356-4363
[19]Henchal EA, Henchal LS, Schlesinger JJ. Synergistic interactions of anti-NS1 monoclonal antibodies protect passively immunized mice from lethal challengewith dengue 2 virus. J Gen Virol 1988;69 (Pt:8):2101-2107
[20]Schlesinger JJ, Brandriss MW, Walsh EE. Protection of mice against dengue 2 virus encephalitis by immunization with the dengue 2 virus non-structural glycoprotein NS1. J Gen Virol 1987;68 (Pt 3):853-857
[21]Chang HH, Shyu HF, Wang YM, et al. Facilitation of cell adhesion by immobilized dengue viral nonstructural protein 1 (NS1):arginine-glycine-aspartic acid structural mimicry within the dengue viral NS1 antigen. J Infect Dis 2002; 186:743-751
[22]Chen MC, Lin CF, Lei HY, et al. Deletion of the C-terminal region of dengue virus nonstructural protein 1 (NS1) abolishes anti-NS1-mediated platelet dysfunction and bleeding tendency. J Immunol 2009; 183:1797-1803
[23]Falconar AK. The dengue virus nonstructural-1 protein (NS1) generates antibodies to common epitopes on human blood clotting, integrin/adhesin proteins and binds to human endothelial cells:potential implications in haemorrhagic fever pathogenesis. Arch Virol 1997;142:897-916
[24]Sun DS, King CC, Huang HS, et al. Antiplatelet autoantibodies elicited by dengue virus non-structural protein 1 cause thrombocytopenia and mortality in mice. J Thromb Haemost 2007;5:2291-2299
[25]Alcon-LePoder S, Drouet MT, Roux P, et al. The secreted form of dengue virus nonstructural protein NS1 is endocytosed by hepatocytes and accumulates in late endosomes:implications for viral infectivity. J Virol 2005;79:11403-11411
[26]Kurosu T, Chaichana P, Yamate M, Anantapreecha S, Ikuta K. Secreted complement regulatory protein clusterin interacts with dengue virus nonstructural protein 1. Biochem Biophys Res Commun 2007;362:1051-1056
[27]Lin CF, Chiu SC, Hsiao YL, et al. Expression of cytokine, chemokine, and adhesion molecules during endothelial cell activation induced by antibodies against dengue virus nonstructural protein 1. J Immunol 2005; 174:395-403
[28]Lin CF, Lei HY, Shiau AL, et al. Antibodies from dengue patient sera cross-react with endothelial cells and induce damage. J Med Virol 2003;69: 82-90
[29]Lin CF, Lei HY, Shiau AL, et al. Endothelial cell apoptosis induced by antibodies against dengue virus nonstructural protein 1 via production of nitric oxide. J Immunol 2002; 169:657-664
[30]Lin CF, Wan SW, Chen MC, et al. Liver injury caused by antibodies against dengue virus nonstructural protein 1 in a murine model. Lab Invest 2008;88:1079-1089
[31]Avirutnan P, Zhang L, Punyadee N, et al. Secreted NS1 of dengue virus attaches to the surface of cells via interactions with heparan sulfate and chondroitin sulfate E. PLoS Pathog 2007;3:e183
[32]Falconar AK, Young PR. Production of dimer-specific and dengue virus group cross-reactive mouse monoclonal antibodies to the dengue 2 virus non-structural glycoprotein NS1. J Gen Virol 1991;72 (Pt 4):961-965
[33]Henchal EA, Henchal LS, Thaisomboonsuk BK. Topological mapping of unique epitopes on the dengue-2 virus NS1 protein using monoclonal antibodies. J Gen Virol 1987;68 (Pt 3):845-851
[34]Shu PY, Chen LK, Chang SF, et al. Dengue NS1-specific antibody responses: isotype distribution and serotyping in patients with Dengue fever and Dengue hemorrhagic fever. J Med Virol 2000;62:224-232
[35]Shu PY, Chen LK, Chang SF, et al. Potential application of nonstructural protein NS1 serotype-specific immunoglobulin G enzyme-linked immunosorbent assay in the seroepidemiologic study of dengue virus infection:correlation of results with those of the plaque reduction neutralization test. J Clin Microbiol 2002;40:1840-1844
[36]Xu H, Di B, Pan YX, et al. Serotype 1-specific monoclonal antibody-based antigen capture immunoassay for detection of circulating nonstructural protein NS1:Implications for early diagnosis and serotyping of dengue virus infections. J Clin Microbiol 2006;44:2872-2878
[37]Young PR, Hilditch PA, Bletchly C, Halloran W. An antigen capture enzyme-linked immunosorbent assay reveals high levels of the dengue virus protein NS1 in the-sera-of infected patients. J Clin Microbiol 2000;38:1053-1057
[38]Lai CY, Tsai WY, Lin SR, et al. Antibodies to envelope glycoprotein of dengue virus during the natural course of infection are predominantly cross-reactive and recognize epitopes containing highly conserved residues at the fusion loop of domain Ⅱ. J Virol 2008;82:6631-6643
[39]Mason PW, Dalrymple JM, Gentry MK, et al. Molecular characterization of a neutralizing domain of the Japanese encephalitis virus structural glycoprotein. J Gen Virol 1989;70 (Pt 8):2037-2049
[40]Kuhn RJ, Zhang W, Rossmann MG, et al. Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell 2002; 108:717-725
[41]Lindenbach BD, Thiel HJ, Rice CM. Flaviviridae:the viruses and their replication. In:Knipe DM, Howley PM eds, Fields Virology.5th Edition ed. Philadelphia:Lippincott Williams & Wilkins; 2007:1103-1113
[42]Crill WD, Roehrig JT. Monoclonal antibodies that bind to domain III of dengue virus E glycoprotein are the most efficient blockers of virus adsorption to Vero cells. J Virol 2001;75:7769-7773
[43]Falconar AK, Young PR, Miles MA. Precise location of sequential dengue virus subcomplex and complex B cell epitopes on the nonstructural-1 glycoprotein. Arch Virol 1994;137:315-326
[44]Wu HC, Huang YL, Chao TT, et al. Identification of B-cell epitope of dengue virus type 1 and its application in diagnosis of patients. J Clin Microbiol 2001;39:977-982
[45]Wu HC, Jung MY, Chiu CY, et al. Identification of a dengue virus type 2 (DEN-2) serotype-specific B-cell epitope and detection of DEN-2-immunized animal serum samples using an epitope-based peptide antigen. J Gen Virol 2003;84:2771-2779
[46]Thullier P, Demangel C, Bedouelle H, et al. Mapping of a dengue virus neutralizing epitope critical for the infectivity of all serotypes:insight into the neutralization mechanism. J Gen Virol 2001;82:.1885-1892
[47]Beasley DW, Aaskov JG. Epitopes on the dengue 1 virus envelope protein recognized by neutralizing IgM monoclonal antibodies. Virology 2001;279: 447-458
[48]da Silva AN, Nascimento EJ, Cordeiro MT, et al. Identification of continuous human B-cell epitopes in the envelope glycoprotein of dengue virus type 3 (DENV-3). PLoS One 2009;4:e7425
[49]Roehrig JT, Bolin RA, Kelly RG Monoclonal antibody mapping of the envelope glycoprotein of the dengue 2 virus, Jamaica. Virology 1998;246: 317-328
[50]晋晶,潘玉先,丁细霞等。 Ⅲ型登革病毒NS1单克隆抗体的制备及鉴定.广东医学2007,1726-1729
[1]Xu H, Di B, Pan YX, et al. Serotype 1-specific monoclonal antibody-based antigen capture immunoassay for detection of circulating nonstructural protein NS1:Implications for early diagnosis and serotyping of dengue virus infections. J Clin Microbiol 2006;44:2872-2878
[2]Qiu LW, Di B, Wen K, et al. Development of an antigen capture immunoassay based on monoclonal antibodies specific for dengue virus serotype 2 nonstructural protein 1 for early and rapid identification of dengue virus serotype 2 infections. Clin Vaccine Immunol 2009;16:88-95.
[3]晋晶,潘玉先,丁细霞等。 Ⅲ型登革病毒NS1单克隆抗体的制备及鉴定.广东医学2007,1726-1729
[4]温坤。Ⅰ型登革病毒E蛋白Ⅲ区抗体的制备、鉴定及禽流感H5N1病毒血凝素基因在昆虫细胞中的表达。南方医科大学硕士论文2009
[1]Falconar AK, Young PR, Miles MA. Precise location of sequential dengue virus subcomplex and complex B cell epitopes on the nonstructural-1 glycoprotein. Arch Virol 1994; 137:315-326
[2]Green S, Kurane I, Pincus S, Paoletti E, Ennis FA. Recognition of dengue virus NS1-NS2a proteins by human CD4+ cytotoxic T lymphocyte clones. Virology 1997;234:383-386
[3]Mathew A, Kurane I, Green S, et al. Predominance of HLA-restricted cytotoxic T-lymphocyte responses to serotype-cross-reactive epitopes on nonstructural proteins following natural secondary dengue virus infection. J Virol 1998;72:3999-4004
[4]Carter JM, Loomis-Price L. B cell epitope mapping using synthetic peptides. Curr Protoc Immunol 2004;Chapter 9:Unit 9 4
[5]Huang JH, Wey JJ, Sun YC, et al. Antibody responses to an immunodominant nonstructural 1 synthetic peptide in patients with dengue fever and dengue hemorrhagic fever. J Med Virol 1999;57:1-8
[6]Wu HC, Huang YL, Chao TT, et al. Identification of B-cell epitope of dengue virus type 1 and its application in diagnosis of patients. J Clin Microbiol 2001;39:977-982
[7]Westaway EG, Blok,.J..,1997.Taxonomy and evolutionary relationships of flaviviruses, in:Gubler, D.J., Kuno, G (Eds),.Dengue and dengue hemorrhagic fever.CAB International, London, United Kingdom,pp.147-173.
[8]Xu H, Di B, Pan YX, et al. Serotype 1-specific monoclonal antibody-based antigen capture immunoassay for detection of circulating nonstructural protein NS1:Implications for early diagnosis and serotyping of dengue virus infections. J Clin Microbiol 2006;44:2872-2878
[9]Qiu LW, Di B, Wen K, et al. Development of an antigen capture immunoassay based on monoclonal antibodies specific for dengue virus serotype 2 nonstructural protein 1 for early and rapid identification of dengue virus serotype 2 infections. Clin Vaccine Immunol 2009; 16:88-95
[10]Chen MC, Lin CF, Lei HY, et al. Deletion of the C-terminal region of dengue virus nonstructural protein 1 (NS1) abolishes anti-NS1-mediated platelet dysfunction and bleeding tendency. J Immunol 2009;183:1797-1803
[11]Lin CF, Chiu SC, Hsiao YL, et al. Expression of cytokine, chemokine, and adhesion molecules during endothelial cell activation induced by antibodies against dengue virus nonstructural protein 1. J Immunol 2005;174:395-403
[12]Sun DS, King CC, Huang HS, et al. Antiplatelet autoantibodies elicited by dengue virus non-structural protein 1 cause thrombocytopenia and mortality in mice. J Thromb Haemost 2007;5:2291-2299
[1]Kuhn RJ, Zhang W, Rossmann MG, et al. Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell 2002; 108: 717-725
[2]Jaiswal S, Khanna N, Swaminathan S. High-level expression and one-step purification of recombinant dengue virus type 2 envelope domain Ⅲ protein in Escherichia coli. Protein Expr Purif 2004;33:80-91
[3]Chin JF, Chu JJ, Ng ML. The envelope glycoprotein domain Ⅲ of dengue virus serotypes 1 and 2 inhibit virus entry. Microbes Infect 2007;9:1-6
[4]Thullier P, Demangel C, Bedouelle H, et al. Mapping of a dengue virus neutralizing epitope critical for the infectivity of all serotypes:insight into the neutralization mechanism. J Gen Virol 2001;82:1885-1892
[5]Lin B, Parrish CR, Murray JM, Wright PJ. Localization of a neutralizing epitope on the envelope protein of dengue virus type 2. Virology 1994;202: 885-890
[6]Roehrig JT, Bolin RA, Kelly RG Monoclonal antibody mapping of the envelope glycoprotein of the dengue 2 virus, Jamaica. Virology 1998;246:317-328
[7]Beasley DW, Aaskov JG Epitopes on the dengue 1 virus envelope protein recognized by neutralizing IgM monoclonal antibodies. Virology 2001;279: 447-458
[8]Crill WD, Roehrig JT. Monoclonal antibodies that bind to domain III of dengue virus E glycoprotein are the most efficient blockers of virus adsorption to Vero cells. J Virol 2001;75:7769-7773
[9]Roehrig JT. Antigenic structure of flavivirus proteins. Adv Virus Res 2003;59:141-175
[10]Thullier P, Lafaye P, Megret F, et al. A recombinant Fab neutralizes dengue virus in vitro. J Biotechnol 1999;69:183-190
[11]Lisova O, Hardy F, Petit V, Bedouelle H. Mapping to completeness and transplantation of a group-specific, discontinuous, neutralizing epitope in the envelope protein of dengue virus. J Gen Virol 2007;88:2387-2397
[12]Hung JJ, Hsieh MT, Young MJ, et al. An external loop region of domain III of dengue virus type 2 envelope protein is involved in serotype-specific binding
[1]Wallis TP, Huang CY, Nimkar SB, et al. Determination of the disulfide bond arrangement of dengue virus NS1 protein. The Journal of biological chemistry 2004;279:20729-20741
[2]Takada A, Kawaoka Y. Antibody-dependent enhancement of viral infection: molecular mechanisms and in vivo implications. Reviews in medical virology 2003;13:387-398
[3]Halstead SB. Dengue. Lancet 2007;370:1644-1652
[4]Young PR, Hilditch PA, Bletchly C, et al. An antigen capture enzyme-linked immunosorbent assay reveals high levels of the dengue virus protein NS1 in the sera of infected patients. Journal of clinical microbiology 2000;38:1053-1057
[5]Libraty DH, Young PR, Pickering D, et al. High circulating levels of the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever. The Journal of infectious diseases 2002; 186:1165-1168
[6]Kuno G, Vorndam AV, Gubler DJ, Gomez I. Study of anti-dengue NS1 antibody by western blot. Journal of medical virology 1990;32:102-108
[7]Falkler WA, Jr., Diwan AR, Halstead SB. Human antibody to dengue soluble complement-fixing (SCF) antigens. J Immunol 1973;111:1804-1809
[8]Chuansumrit A, Chaiyaratana W, Pongthanapisith V, et al. The use of dengue nonstructural protein 1 antigen for the early diagnosis during the febrile stage in patients with dengue infection. The Pediatric infectious disease journal 2008;27:43-48
[9]Xu H, Di B, Pan YX, et al. Serotype 1-specific monoclonal antibody-based antigen capture immunoassay for detection of circulating nonstructural protein NS1:Implications for early diagnosis and serotyping of dengue virus infections. Journal of clinical microbiology 2006;44:2872-2878
[10]Falconar AK. The dengue virus nonstructural-1 protein (NS1) generates antibodies to common epitopes on human blood clotting, integrin/adhesin proteins and binds to human endothelial cells:potential implications in haemorrhagic fever pathogenesis. Archives of virology 1997; 142:897-916
[11]Falconar—AK. Antibody responses are generated to immunodominant ELK/KLE-type motifs on the nonstructural-1 glycoprotein during live dengue virus infections in mice and humans:implications for diagnosis, pathogenesis, and vaccine design. Clin Vaccine Immunol 2007;14:493-504
[12]Lin CF, Lei HY, Shiau AL, et al. Endothelial cell apoptosis induced by antibodies against dengue virus nonstructural protein 1 via production of nitric oxide. J Immunol 2002; 169:657-664
[13]Lin CF, Lei HY, Shiau AL, et al. Antibodies from dengue patient sera cross-react with endothelial cells and induce damage. Journal of medical virology 2003;69:82-90
[14]Alcon-LePoder S, Drouet MT, Roux P, et al. The secreted form of dengue virus nonstructural protein NS1 is endocytosed by hepatocytes and accumulates in late endosomes:implications for viral infectivity. Journal of virology 2005;79:11403-11411
[15]Avirutnan P, Zhang L, Punyadee N, 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:e183
[16]Avirutnan P, Malasit P, Seliger B, Bhakdi S, Husmann M. Dengue virus infection of human endothelial cells leads to chemokine production, complement activation, and apoptosis. J Immunol 1998;161:6338-6346
[17]Winkler G, Maxwell SE, Ruemmler C, Stollar V. Newly synthesized dengue-2 virus nonstructural protein NS1 is a soluble protein but becomes partially hydrophobic and membrane-associated after dimerization. Virology 1989; 171: 302-305
[18]Avirutnan P, Punyadee N, Noisakran S, et al. Vascular leakage in severe dengue virus infections:a potential role for the nonstructural viral protein NS1 and complement. The Journal of infectious diseases 2006; 193:1078-1088
[19]Kurosu T, Chaichana P, Yamate M, Anantapreecha S, Ikuta K. Secreted complement regulatory protein clusterin interacts with dengue virus nonstructural protein 1. Biochemical and biophysical research communications 2007;362:1051-1056
[1]WHO.2009. Dengue and dengue haemorrhagic fever.
[2]Rey, F. A., F. X. Heinz, C. Mandl, et al. The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution. Nature 1995; 375:291-298.
[3]Modis, Y., S. Ogata, D. Clements et al.. A ligand-binding pocket in the dengue virus envelope glycoprotein. Proc Natl Acad Sci USA.2003; 100:6986-6991.
[4]Rey, F. A. Dengue virus envelope glycoprotein structure:new insight into its interactions during viral entry. Proc Natl Acad Sci U S A.2003; 100:6899-6901.
[5]AbuBakar, S., A. Azmi, N. Mohamed-Saad et al. Antibody responses of dengue fever patients to dengue 2 (New Guinea C strain) viral proteins. Malays JPathol.1997; 19:41-51.
[6]Crill, W. D., H. R. Hughes, M. J. Delorey, et al. Humoral immune responses of dengue fever patients using epitope-specific serotype-2 virus-like particle antigens. PLoS One.2009; 4:e4991.
[7]Lai, C. Y., W. Y. Tsai, S. R. Lin, et al. Antibodies to envelope glycoprotein of dengue virus during the natural course of infection are predominantly cross-reactive and recognize epitopes containing highly conserved residues at the fusion loop of domain II. J Virol.2008; 82:6631-6643.
[8]Churdboonchart, V., N. Bhamarapravati, S. Peampramprecha, et al. Antibodies against dengue viral proteins in primary and secondary dengue hemorrhagic fever. Am J Trop Med Hyg.1991; 44:481-493.
[9]Deubel, V., M. Bordier, F. Megret, et al. Processing, secretion, and immunoreactivity of carboxy terminally truncated dengue-2 virus envelope proteins expressed in insect cells by recombinant baculoviruses. Virology. 1991; 180:442-447.
[10]Aaskov, J. G, H. M. Geysen, and T. J. Mason. Serologically defined linear epitopes in the envelope protein of dengue 2 (Jamaica strain 1409). Arch Virol. 1989; 105:209-221.
[11]Innis, B. L., V. Thirawuth, and C. Hemachudha. Identification of continuous-epitopes of the envelope glycoprotein of dengue type 2 virus. Am J Trop Med Hyg.1989; 40:676-687.
[12]Henchal, E. A., J. M. McCown, D. S. Burke, et al. Epitopic analysis of antigenic determinants on the surface of dengue-2 virions using monoclonal antibodies. Am J Trop Med Hyg.1985; 34:162-169.
[13]Simantini, E., and K. Banerjee. Epitope mapping of dengue 1 virus E glycoprotein using monoclonal antibodies. Arch Viro.l 1995; 140:1257-1273.
[14]Mason, P. W., M. U. Zugel, A. R. Semproni, et al. The antigenic structure of dengue type 1 virus envelope and NS1 proteins expressed in Escherichia coli. J Gen Virol.1990; 71 (Pt9):2107-2114.
[15]Roehrig, J. T., A. J. Johnson, A. R. Hunt, et al. Antibodies to dengue 2 virus E-glycoprotein synthetic peptides identify antigenic conformation. Virology. 1990; 177:668-675.
[16]Megret, F., J. P. Hugnot, A. Falconar, et al. Use of recombinant fusion proteins and monoclonal antibodies to define linear and discontinuous antigenic sites on the dengue virus envelope glycoprotein. Virology.1992; 187:480-491.
[17]Lin, B., C. R. Parrish, J. M. Murray, et al. Localization of a neutralizing epitope on the envelope protein of dengue virus type 2. Virology.1994; 202: 885-890.
[18]Trirawatanapong, T., B. Chandran, R. Putnak, et al. Mapping of a region of dengue virus type-2 glycoprotein required for binding by a neutralizing monoclonal antibody. Gene 1992; 116:139-150.
[19]Hiramatsu, K., M. Tadano, R. Men, et al. Mutational analysis of a neutralization epitope on the dengue type 2 virus (DEN2) envelope protein: monoclonal antibody resistant DEN2/DEN4 chimeras exhibit reduced mouse neurovirulence. Virology.1996; 224:437-445.
[20]Roehrig, J. T., R. A. Bolin, and R. G. Kelly. Monoclonal antibody mapping of the envelope glycoprotein of the dengue 2 virus, Jamaica. Virology.1998; 246:317-328.
[21]Lai, C. J., A. P. Goncalvez, R. Men, et al. Epitope determinants of a chimpanzee dengue virus type 4 (DENV-4)-neutralizing antibody and protection against DENV-4 challenge in mice and rhesus monkeys by passively transferred humanized antibody. J Virol.2007; 81:12766-12774.
[22]Stiasny, K., S. Kiermayr, H. Holzmann, et al. Cryptic properties of a cluster of dominant flavivirus cross-reactive antigenic sites. J Virol.2006; 80:9557-9568.
[23]Goncalvez, A. P., R. H. Purcell, and C. J. Lai. Epitope determinants of a chimpanzee Fab antibody that efficiently cross-neutralizes dengue type 1 and type 2 viruses map to inside and in close proximity to fusion loop of the dengue type 2 virus envelope glycoprotein. J Virol.2004; 78:12919-12928.
[24]Goncalvez, A. P., R. Men, C. Wernly, et al. Chimpanzee Fab fragments and a derived humanized immunoglobulin G1 antibody that efficiently cross-neutralize dengue type 1 and type 2 viruses. J Virol.2004; 78:12910-12918.
[25]Goncalvez, A. P., R. E. Engle, M. St Claire, et al. Monoclonal antibody-mediated enhancement of dengue virus infection in vitro and in vivo and strategies for prevention. Proc Natl Acad Sci USA.2007; 104:9422-9427.
[26]Hung, J. J., M. T. Hsieh, M. J. Young, et al. An external loop region of domain III of dengue virus type 2 envelope protein is involved in serotype-specific binding to mosquito but not mammalian cells. J Virol.2004; 78:378-388.
[27]Crill, W. D., and J. T. Roehrig. Monoclonal antibodies that bind to domain III of dengue virus E glycoprotein are the most efficient blockers of virus adsorption to Vero cells. J Virol.2001; 75:7769-7773.
[28]Thullier, P., C. Demangel, H. Bedouelle, et al. Mapping of a dengue virus neutralizing epitope critical for the infectivity of all serotypes:insight into the neutralization mechanism. J Gen Virol.2001; 82:1885-1892.
[29]Lisova, O., F. Hardy, V. Petit, et al. Mapping to completeness and transplantation of a group-specific, discontinuous, neutralizing epitope in the envelope protein of dengue virus. JGen Virol. 2007; 88:2387-2397.
[30]Rajamanonmani, R., C. Nkenfou, P. Clancy, et al. On a mouse monoclonal antibody that neutralizes all four dengue virus serotypes. J Gen Virol.2009; 90:799-809
[31]Falconar, A. K.1999. Identification of an epitope on the dengue virus membrane (M) protein defined by cross-protective monoclonal antibodies: design of an improved epitope sequence based on common determinants present in both envelope (E and M) proteins. Arch Virol 144:2313-2330.
[32]Gromowski, G D., and A. D. Barrett. Characterization of an antigenic site that contains a dominant, type-specific neutralization determinant on the envelope protein domain Ⅲ (ED3) of dengue 2 virus. Virology.2007; 366:349-360.
[33]Gromowski, G. D., N. D. Barrett, and A. D. Barrett. Characterization of dengue virus complex-specific neutralizing epitopes on envelope protein domain III of dengue 2 virus. J Virol.2008; 82:8828-8837.
[34]Matsui, K., G. D. Gromowski, L. Li, et al. Characterization of dengue complex-reactive epitopes on dengue 3 virus envelope protein domain III. Virology.2009; 384:16-20.
[35]Serafin, I. L., and J. G Aaskov. Identification of epitopes on the envelope (E) protein of dengue 2 and dengue 3 viruses using monoclonal antibodies. Arch Virol.2001; 146:2469-2479.
[36]Markoff, L. J., B. L. Innis, R. Houghten, et al. Development of cross-reactive antibodies to plasminogen during the immune response to dengue virus infection. J Infect Dis.1991; 164:294-301.
[37]Chungue, E., L. Poli, C. Roche, et al. Correlation between detection of plasminogen cross-reactive antibodies and hemorrhage in dengue virus infection. J Infect Dis.1994; 170:1304-1307.
[38]Huang, Y. H., B. I. Chang, H. Y. Lei, et al. Antibodies against dengue virus E protein peptide bind to human plasminogen and inhibit plasmin activity. Clin Exp Immunol.1997; 110:35-40.
[39]Monroy, V., and B. H. Ruiz. Participation of the Dengue virus in the fibrinolytic process. Virus Genes.2000; 21:197-208.
[2]梁文佳,何剑峰,罗会明等广东省2001-2006年登革热流行病学分析.华南预防医学2007,5：4-7.
[3]黄春文,陈敏红,陈艳,等.福州市2004～2008年登革热疫情监测分析.中国卫生检验杂志.2009,19(8)：1870-1871.
[4]George R, Lum L.C.S.1997. Clinical spectrum of dengue infection,in:Gubler, D.J., Kuno, G. (ed.), Dengue and dengue hemorrhagic fever. CAB International, London, United Kingdom,pp.89-113.89-114
[5]Kanakaratne N, Wahala WM, Messer WB, et al. Severe dengue epidemics in Sri Lanka,2003-2006. Emerg Infect Dis 2009;15:192-199
[6]Kurane I, Ennis FE. Immunity and immunopathology in dengue virus infections. Semin Immunol 1992;4:121-127
[7]Gubler DJ. Dengue and dengue hemorrhagic fever. Clin Microbiol Rev 1998; 11:480-496
[8]WHO. Dengue and dengue haemorrhagic fever.2009
[9]De Paula SO, Fonseca BA. Dengue:a review of the laboratory tests a clinician must know to achieve a correct diagnosis. Braz J Infect Dis 2004;8:390-398
[10]WHO. Dengue:Guidelines for diagnosis, treatment, prevention and control. New Edition 2009
[11]WHO. Dengue haemorrhagic fever:diagnosis, treatment and control. Handbook of the World Health Organization.Geneva:WHO,2000, pp.1-84.
[12]WHO Report of the Scientific Working Group meeting on Dengue Report on Dengue. Report on dengue.Geneva:WHO,2007, pp.1-150.
[13]Qiu LW, Di B, Wen K, et al. Development of an antigen capture immunoassay based on monoclonal antibodies specific for dengue virus serotype 2 nonstructural protein 1 for early and rapid identification of dengue virus serotype 2 infections. Clin Vaccine Immunol 2009; 16:88-95
[14]Lindenbach BD, and C. M. Rice. Flaviviridae:the viruses and their replication, p.991-1041. In D. M. Knipe, P. M. Howley, and D. E. Griffin (ed.), Fields virology. Lippincott William & Wilkins, Philadelphia, PA.2001.
[15]Costa SM, Azevedo AS, Paes MV, et al. DNA vaccines against dengue virus based on the nsl gene:the influence of different signal sequences on the protein expression and its correlation to the immune response elicited in mice. Virology 2007;358:413-423
[16]Costa SM, Freire MS, Alves AM, Alves AM. DNA vaccine against the non-structural 1 protein (NS1) of dengue 2 virus. Vaccine 2006;24:4562-4564
[17]Chung KM, Nybakken GE, Thompson BS, et al. Antibodies against West Nile Virus nonstructural protein NS1 prevent lethal infection through Fc gamma receptor-dependent and-independent mechanisms. J Virol 2006;80:1340-1351
[18]Falgout B, Bray M, Schlesinger JJ, Lai CJ. Immunization of mice with recombinant vaccinia virus expressing authentic dengue virus nonstructural protein NS1 protects against lethal dengue virus encephalitis. J Virol 1990; 64:4356-4363
[19]Henchal EA, Henchal LS, Schlesinger JJ. Synergistic interactions of anti-NS1 monoclonal antibodies protect passively immunized mice from lethal challengewith dengue 2 virus. J Gen Virol 1988;69 (Pt:8):2101-2107
[20]Schlesinger JJ, Brandriss MW, Walsh EE. Protection of mice against dengue 2 virus encephalitis by immunization with the dengue 2 virus non-structural glycoprotein NS1. J Gen Virol 1987;68 (Pt 3):853-857
[21]Chang HH, Shyu HF, Wang YM, et al. Facilitation of cell adhesion by immobilized dengue viral nonstructural protein 1 (NS1):arginine-glycine-aspartic acid structural mimicry within the dengue viral NS1 antigen. J Infect Dis 2002; 186:743-751
[22]Chen MC, Lin CF, Lei HY, et al. Deletion of the C-terminal region of dengue virus nonstructural protein 1 (NS1) abolishes anti-NS1-mediated platelet dysfunction and bleeding tendency. J Immunol 2009; 183:1797-1803
[23]Falconar AK. The dengue virus nonstructural-1 protein (NS1) generates antibodies to common epitopes on human blood clotting, integrin/adhesin proteins and binds to human endothelial cells:potential implications in haemorrhagic fever pathogenesis. Arch Virol 1997;142:897-916
[24]Sun DS, King CC, Huang HS, et al. Antiplatelet autoantibodies elicited by dengue virus non-structural protein 1 cause thrombocytopenia and mortality in mice. J Thromb Haemost 2007;5:2291-2299
[25]Alcon-LePoder S, Drouet MT, Roux P, et al. The secreted form of dengue virus nonstructural protein NS1 is endocytosed by hepatocytes and accumulates in late endosomes:implications for viral infectivity. J Virol 2005;79:11403-11411
[26]Kurosu T, Chaichana P, Yamate M, Anantapreecha S, Ikuta K. Secreted complement regulatory protein clusterin interacts with dengue virus nonstructural protein 1. Biochem Biophys Res Commun 2007;362:1051-1056
[27]Lin CF, Chiu SC, Hsiao YL, et al. Expression of cytokine, chemokine, and adhesion molecules during endothelial cell activation induced by antibodies against dengue virus nonstructural protein 1. J Immunol 2005; 174:395-403
[28]Lin CF, Lei HY, Shiau AL, et al. Antibodies from dengue patient sera cross-react with endothelial cells and induce damage. J Med Virol 2003;69: 82-90
[29]Lin CF, Lei HY, Shiau AL, et al. Endothelial cell apoptosis induced by antibodies against dengue virus nonstructural protein 1 via production of nitric oxide. J Immunol 2002; 169:657-664
[30]Lin CF, Wan SW, Chen MC, et al. Liver injury caused by antibodies against dengue virus nonstructural protein 1 in a murine model. Lab Invest 2008;88:1079-1089
[31]Avirutnan P, Zhang L, Punyadee N, et al. Secreted NS1 of dengue virus attaches to the surface of cells via interactions with heparan sulfate and chondroitin sulfate E. PLoS Pathog 2007;3:e183
[32]Falconar AK, Young PR. Production of dimer-specific and dengue virus group cross-reactive mouse monoclonal antibodies to the dengue 2 virus non-structural glycoprotein NS1. J Gen Virol 1991;72 (Pt 4):961-965
[33]Henchal EA, Henchal LS, Thaisomboonsuk BK. Topological mapping of unique epitopes on the dengue-2 virus NS1 protein using monoclonal antibodies. J Gen Virol 1987;68 (Pt 3):845-851
[34]Shu PY, Chen LK, Chang SF, et al. Dengue NS1-specific antibody responses: isotype distribution and serotyping in patients with Dengue fever and Dengue hemorrhagic fever. J Med Virol 2000;62:224-232
[35]Shu PY, Chen LK, Chang SF, et al. Potential application of nonstructural protein NS1 serotype-specific immunoglobulin G enzyme-linked immunosorbent assay in the seroepidemiologic study of dengue virus infection:correlation of results with those of the plaque reduction neutralization test. J Clin Microbiol 2002;40:1840-1844
[36]Xu H, Di B, Pan YX, et al. Serotype 1-specific monoclonal antibody-based antigen capture immunoassay for detection of circulating nonstructural protein NS1:Implications for early diagnosis and serotyping of dengue virus infections. J Clin Microbiol 2006;44:2872-2878
[37]Young PR, Hilditch PA, Bletchly C, Halloran W. An antigen capture enzyme-linked immunosorbent assay reveals high levels of the dengue virus protein NS1 in the-sera-of infected patients. J Clin Microbiol 2000;38:1053-1057
[38]Lai CY, Tsai WY, Lin SR, et al. Antibodies to envelope glycoprotein of dengue virus during the natural course of infection are predominantly cross-reactive and recognize epitopes containing highly conserved residues at the fusion loop of domain Ⅱ. J Virol 2008;82:6631-6643
[39]Mason PW, Dalrymple JM, Gentry MK, et al. Molecular characterization of a neutralizing domain of the Japanese encephalitis virus structural glycoprotein. J Gen Virol 1989;70 (Pt 8):2037-2049
[40]Kuhn RJ, Zhang W, Rossmann MG, et al. Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell 2002; 108:717-725
[41]Lindenbach BD, Thiel HJ, Rice CM. Flaviviridae:the viruses and their replication. In:Knipe DM, Howley PM eds, Fields Virology.5th Edition ed. Philadelphia:Lippincott Williams & Wilkins; 2007:1103-1113
[42]Crill WD, Roehrig JT. Monoclonal antibodies that bind to domain III of dengue virus E glycoprotein are the most efficient blockers of virus adsorption to Vero cells. J Virol 2001;75:7769-7773
[43]Falconar AK, Young PR, Miles MA. Precise location of sequential dengue virus subcomplex and complex B cell epitopes on the nonstructural-1 glycoprotein. Arch Virol 1994;137:315-326
[44]Wu HC, Huang YL, Chao TT, et al. Identification of B-cell epitope of dengue virus type 1 and its application in diagnosis of patients. J Clin Microbiol 2001;39:977-982
[45]Wu HC, Jung MY, Chiu CY, et al. Identification of a dengue virus type 2 (DEN-2) serotype-specific B-cell epitope and detection of DEN-2-immunized animal serum samples using an epitope-based peptide antigen. J Gen Virol 2003;84:2771-2779
[46]Thullier P, Demangel C, Bedouelle H, et al. Mapping of a dengue virus neutralizing epitope critical for the infectivity of all serotypes:insight into the neutralization mechanism. J Gen Virol 2001;82:.1885-1892
[47]Beasley DW, Aaskov JG. Epitopes on the dengue 1 virus envelope protein recognized by neutralizing IgM monoclonal antibodies. Virology 2001;279: 447-458
[48]da Silva AN, Nascimento EJ, Cordeiro MT, et al. Identification of continuous human B-cell epitopes in the envelope glycoprotein of dengue virus type 3 (DENV-3). PLoS One 2009;4:e7425
[49]Roehrig JT, Bolin RA, Kelly RG Monoclonal antibody mapping of the envelope glycoprotein of the dengue 2 virus, Jamaica. Virology 1998;246: 317-328
[50]晋晶,潘玉先,丁细霞等。 Ⅲ型登革病毒NS1单克隆抗体的制备及鉴定.广东医学2007,1726-1729
[1]Xu H, Di B, Pan YX, et al. Serotype 1-specific monoclonal antibody-based antigen capture immunoassay for detection of circulating nonstructural protein NS1:Implications for early diagnosis and serotyping of dengue virus infections. J Clin Microbiol 2006;44:2872-2878
[2]Qiu LW, Di B, Wen K, et al. Development of an antigen capture immunoassay based on monoclonal antibodies specific for dengue virus serotype 2 nonstructural protein 1 for early and rapid identification of dengue virus serotype 2 infections. Clin Vaccine Immunol 2009;16:88-95.
[3]晋晶,潘玉先,丁细霞等。 Ⅲ型登革病毒NS1单克隆抗体的制备及鉴定.广东医学2007,1726-1729
[4]温坤。Ⅰ型登革病毒E蛋白Ⅲ区抗体的制备、鉴定及禽流感H5N1病毒血凝素基因在昆虫细胞中的表达。南方医科大学硕士论文2009
[1]Falconar AK, Young PR, Miles MA. Precise location of sequential dengue virus subcomplex and complex B cell epitopes on the nonstructural-1 glycoprotein. Arch Virol 1994; 137:315-326
[2]Green S, Kurane I, Pincus S, Paoletti E, Ennis FA. Recognition of dengue virus NS1-NS2a proteins by human CD4+ cytotoxic T lymphocyte clones. Virology 1997;234:383-386
[3]Mathew A, Kurane I, Green S, et al. Predominance of HLA-restricted cytotoxic T-lymphocyte responses to serotype-cross-reactive epitopes on nonstructural proteins following natural secondary dengue virus infection. J Virol 1998;72:3999-4004
[4]Carter JM, Loomis-Price L. B cell epitope mapping using synthetic peptides. Curr Protoc Immunol 2004;Chapter 9:Unit 9 4
[5]Huang JH, Wey JJ, Sun YC, et al. Antibody responses to an immunodominant nonstructural 1 synthetic peptide in patients with dengue fever and dengue hemorrhagic fever. J Med Virol 1999;57:1-8
[6]Wu HC, Huang YL, Chao TT, et al. Identification of B-cell epitope of dengue virus type 1 and its application in diagnosis of patients. J Clin Microbiol 2001;39:977-982
[7]Westaway EG, Blok,.J..,1997.Taxonomy and evolutionary relationships of flaviviruses, in:Gubler, D.J., Kuno, G (Eds),.Dengue and dengue hemorrhagic fever.CAB International, London, United Kingdom,pp.147-173.
[8]Xu H, Di B, Pan YX, et al. Serotype 1-specific monoclonal antibody-based antigen capture immunoassay for detection of circulating nonstructural protein NS1:Implications for early diagnosis and serotyping of dengue virus infections. J Clin Microbiol 2006;44:2872-2878
[9]Qiu LW, Di B, Wen K, et al. Development of an antigen capture immunoassay based on monoclonal antibodies specific for dengue virus serotype 2 nonstructural protein 1 for early and rapid identification of dengue virus serotype 2 infections. Clin Vaccine Immunol 2009; 16:88-95
[10]Chen MC, Lin CF, Lei HY, et al. Deletion of the C-terminal region of dengue virus nonstructural protein 1 (NS1) abolishes anti-NS1-mediated platelet dysfunction and bleeding tendency. J Immunol 2009;183:1797-1803
[11]Lin CF, Chiu SC, Hsiao YL, et al. Expression of cytokine, chemokine, and adhesion molecules during endothelial cell activation induced by antibodies against dengue virus nonstructural protein 1. J Immunol 2005;174:395-403
[12]Sun DS, King CC, Huang HS, et al. Antiplatelet autoantibodies elicited by dengue virus non-structural protein 1 cause thrombocytopenia and mortality in mice. J Thromb Haemost 2007;5:2291-2299
[1]Kuhn RJ, Zhang W, Rossmann MG, et al. Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell 2002; 108: 717-725
[2]Jaiswal S, Khanna N, Swaminathan S. High-level expression and one-step purification of recombinant dengue virus type 2 envelope domain Ⅲ protein in Escherichia coli. Protein Expr Purif 2004;33:80-91
[3]Chin JF, Chu JJ, Ng ML. The envelope glycoprotein domain Ⅲ of dengue virus serotypes 1 and 2 inhibit virus entry. Microbes Infect 2007;9:1-6
[4]Thullier P, Demangel C, Bedouelle H, et al. Mapping of a dengue virus neutralizing epitope critical for the infectivity of all serotypes:insight into the neutralization mechanism. J Gen Virol 2001;82:1885-1892
[5]Lin B, Parrish CR, Murray JM, Wright PJ. Localization of a neutralizing epitope on the envelope protein of dengue virus type 2. Virology 1994;202: 885-890
[6]Roehrig JT, Bolin RA, Kelly RG Monoclonal antibody mapping of the envelope glycoprotein of the dengue 2 virus, Jamaica. Virology 1998;246:317-328
[7]Beasley DW, Aaskov JG Epitopes on the dengue 1 virus envelope protein recognized by neutralizing IgM monoclonal antibodies. Virology 2001;279: 447-458
[8]Crill WD, Roehrig JT. Monoclonal antibodies that bind to domain III of dengue virus E glycoprotein are the most efficient blockers of virus adsorption to Vero cells. J Virol 2001;75:7769-7773
[9]Roehrig JT. Antigenic structure of flavivirus proteins. Adv Virus Res 2003;59:141-175
[10]Thullier P, Lafaye P, Megret F, et al. A recombinant Fab neutralizes dengue virus in vitro. J Biotechnol 1999;69:183-190
[11]Lisova O, Hardy F, Petit V, Bedouelle H. Mapping to completeness and transplantation of a group-specific, discontinuous, neutralizing epitope in the envelope protein of dengue virus. J Gen Virol 2007;88:2387-2397
[12]Hung JJ, Hsieh MT, Young MJ, et al. An external loop region of domain III of dengue virus type 2 envelope protein is involved in serotype-specific binding
[1]Wallis TP, Huang CY, Nimkar SB, et al. Determination of the disulfide bond arrangement of dengue virus NS1 protein. The Journal of biological chemistry 2004;279:20729-20741
[2]Takada A, Kawaoka Y. Antibody-dependent enhancement of viral infection: molecular mechanisms and in vivo implications. Reviews in medical virology 2003;13:387-398
[3]Halstead SB. Dengue. Lancet 2007;370:1644-1652
[4]Young PR, Hilditch PA, Bletchly C, et al. An antigen capture enzyme-linked immunosorbent assay reveals high levels of the dengue virus protein NS1 in the sera of infected patients. Journal of clinical microbiology 2000;38:1053-1057
[5]Libraty DH, Young PR, Pickering D, et al. High circulating levels of the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever. The Journal of infectious diseases 2002; 186:1165-1168
[6]Kuno G, Vorndam AV, Gubler DJ, Gomez I. Study of anti-dengue NS1 antibody by western blot. Journal of medical virology 1990;32:102-108
[7]Falkler WA, Jr., Diwan AR, Halstead SB. Human antibody to dengue soluble complement-fixing (SCF) antigens. J Immunol 1973;111:1804-1809
[8]Chuansumrit A, Chaiyaratana W, Pongthanapisith V, et al. The use of dengue nonstructural protein 1 antigen for the early diagnosis during the febrile stage in patients with dengue infection. The Pediatric infectious disease journal 2008;27:43-48
[9]Xu H, Di B, Pan YX, et al. Serotype 1-specific monoclonal antibody-based antigen capture immunoassay for detection of circulating nonstructural protein NS1:Implications for early diagnosis and serotyping of dengue virus infections. Journal of clinical microbiology 2006;44:2872-2878
[10]Falconar AK. The dengue virus nonstructural-1 protein (NS1) generates antibodies to common epitopes on human blood clotting, integrin/adhesin proteins and binds to human endothelial cells:potential implications in haemorrhagic fever pathogenesis. Archives of virology 1997; 142:897-916
[11]Falconar—AK. Antibody responses are generated to immunodominant ELK/KLE-type motifs on the nonstructural-1 glycoprotein during live dengue virus infections in mice and humans:implications for diagnosis, pathogenesis, and vaccine design. Clin Vaccine Immunol 2007;14:493-504
[12]Lin CF, Lei HY, Shiau AL, et al. Endothelial cell apoptosis induced by antibodies against dengue virus nonstructural protein 1 via production of nitric oxide. J Immunol 2002; 169:657-664
[13]Lin CF, Lei HY, Shiau AL, et al. Antibodies from dengue patient sera cross-react with endothelial cells and induce damage. Journal of medical virology 2003;69:82-90
[14]Alcon-LePoder S, Drouet MT, Roux P, et al. The secreted form of dengue virus nonstructural protein NS1 is endocytosed by hepatocytes and accumulates in late endosomes:implications for viral infectivity. Journal of virology 2005;79:11403-11411
[15]Avirutnan P, Zhang L, Punyadee N, 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:e183
[16]Avirutnan P, Malasit P, Seliger B, Bhakdi S, Husmann M. Dengue virus infection of human endothelial cells leads to chemokine production, complement activation, and apoptosis. J Immunol 1998;161:6338-6346
[17]Winkler G, Maxwell SE, Ruemmler C, Stollar V. Newly synthesized dengue-2 virus nonstructural protein NS1 is a soluble protein but becomes partially hydrophobic and membrane-associated after dimerization. Virology 1989; 171: 302-305
[18]Avirutnan P, Punyadee N, Noisakran S, et al. Vascular leakage in severe dengue virus infections:a potential role for the nonstructural viral protein NS1 and complement. The Journal of infectious diseases 2006; 193:1078-1088
[19]Kurosu T, Chaichana P, Yamate M, Anantapreecha S, Ikuta K. Secreted complement regulatory protein clusterin interacts with dengue virus nonstructural protein 1. Biochemical and biophysical research communications 2007;362:1051-1056
[1]WHO.2009. Dengue and dengue haemorrhagic fever.
[2]Rey, F. A., F. X. Heinz, C. Mandl, et al. The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution. Nature 1995; 375:291-298.
[3]Modis, Y., S. Ogata, D. Clements et al.. A ligand-binding pocket in the dengue virus envelope glycoprotein. Proc Natl Acad Sci USA.2003; 100:6986-6991.
[4]Rey, F. A. Dengue virus envelope glycoprotein structure:new insight into its interactions during viral entry. Proc Natl Acad Sci U S A.2003; 100:6899-6901.
[5]AbuBakar, S., A. Azmi, N. Mohamed-Saad et al. Antibody responses of dengue fever patients to dengue 2 (New Guinea C strain) viral proteins. Malays JPathol.1997; 19:41-51.
[6]Crill, W. D., H. R. Hughes, M. J. Delorey, et al. Humoral immune responses of dengue fever patients using epitope-specific serotype-2 virus-like particle antigens. PLoS One.2009; 4:e4991.
[7]Lai, C. Y., W. Y. Tsai, S. R. Lin, et al. Antibodies to envelope glycoprotein of dengue virus during the natural course of infection are predominantly cross-reactive and recognize epitopes containing highly conserved residues at the fusion loop of domain II. J Virol.2008; 82:6631-6643.
[8]Churdboonchart, V., N. Bhamarapravati, S. Peampramprecha, et al. Antibodies against dengue viral proteins in primary and secondary dengue hemorrhagic fever. Am J Trop Med Hyg.1991; 44:481-493.
[9]Deubel, V., M. Bordier, F. Megret, et al. Processing, secretion, and immunoreactivity of carboxy terminally truncated dengue-2 virus envelope proteins expressed in insect cells by recombinant baculoviruses. Virology. 1991; 180:442-447.
[10]Aaskov, J. G, H. M. Geysen, and T. J. Mason. Serologically defined linear epitopes in the envelope protein of dengue 2 (Jamaica strain 1409). Arch Virol. 1989; 105:209-221.
[11]Innis, B. L., V. Thirawuth, and C. Hemachudha. Identification of continuous-epitopes of the envelope glycoprotein of dengue type 2 virus. Am J Trop Med Hyg.1989; 40:676-687.
[12]Henchal, E. A., J. M. McCown, D. S. Burke, et al. Epitopic analysis of antigenic determinants on the surface of dengue-2 virions using monoclonal antibodies. Am J Trop Med Hyg.1985; 34:162-169.
[13]Simantini, E., and K. Banerjee. Epitope mapping of dengue 1 virus E glycoprotein using monoclonal antibodies. Arch Viro.l 1995; 140:1257-1273.
[14]Mason, P. W., M. U. Zugel, A. R. Semproni, et al. The antigenic structure of dengue type 1 virus envelope and NS1 proteins expressed in Escherichia coli. J Gen Virol.1990; 71 (Pt9):2107-2114.
[15]Roehrig, J. T., A. J. Johnson, A. R. Hunt, et al. Antibodies to dengue 2 virus E-glycoprotein synthetic peptides identify antigenic conformation. Virology. 1990; 177:668-675.
[16]Megret, F., J. P. Hugnot, A. Falconar, et al. Use of recombinant fusion proteins and monoclonal antibodies to define linear and discontinuous antigenic sites on the dengue virus envelope glycoprotein. Virology.1992; 187:480-491.
[17]Lin, B., C. R. Parrish, J. M. Murray, et al. Localization of a neutralizing epitope on the envelope protein of dengue virus type 2. Virology.1994; 202: 885-890.
[18]Trirawatanapong, T., B. Chandran, R. Putnak, et al. Mapping of a region of dengue virus type-2 glycoprotein required for binding by a neutralizing monoclonal antibody. Gene 1992; 116:139-150.
[19]Hiramatsu, K., M. Tadano, R. Men, et al. Mutational analysis of a neutralization epitope on the dengue type 2 virus (DEN2) envelope protein: monoclonal antibody resistant DEN2/DEN4 chimeras exhibit reduced mouse neurovirulence. Virology.1996; 224:437-445.
[20]Roehrig, J. T., R. A. Bolin, and R. G. Kelly. Monoclonal antibody mapping of the envelope glycoprotein of the dengue 2 virus, Jamaica. Virology.1998; 246:317-328.
[21]Lai, C. J., A. P. Goncalvez, R. Men, et al. Epitope determinants of a chimpanzee dengue virus type 4 (DENV-4)-neutralizing antibody and protection against DENV-4 challenge in mice and rhesus monkeys by passively transferred humanized antibody. J Virol.2007; 81:12766-12774.
[22]Stiasny, K., S. Kiermayr, H. Holzmann, et al. Cryptic properties of a cluster of dominant flavivirus cross-reactive antigenic sites. J Virol.2006; 80:9557-9568.
[23]Goncalvez, A. P., R. H. Purcell, and C. J. Lai. Epitope determinants of a chimpanzee Fab antibody that efficiently cross-neutralizes dengue type 1 and type 2 viruses map to inside and in close proximity to fusion loop of the dengue type 2 virus envelope glycoprotein. J Virol.2004; 78:12919-12928.
[24]Goncalvez, A. P., R. Men, C. Wernly, et al. Chimpanzee Fab fragments and a derived humanized immunoglobulin G1 antibody that efficiently cross-neutralize dengue type 1 and type 2 viruses. J Virol.2004; 78:12910-12918.
[25]Goncalvez, A. P., R. E. Engle, M. St Claire, et al. Monoclonal antibody-mediated enhancement of dengue virus infection in vitro and in vivo and strategies for prevention. Proc Natl Acad Sci USA.2007; 104:9422-9427.
[26]Hung, J. J., M. T. Hsieh, M. J. Young, et al. An external loop region of domain III of dengue virus type 2 envelope protein is involved in serotype-specific binding to mosquito but not mammalian cells. J Virol.2004; 78:378-388.
[27]Crill, W. D., and J. T. Roehrig. Monoclonal antibodies that bind to domain III of dengue virus E glycoprotein are the most efficient blockers of virus adsorption to Vero cells. J Virol.2001; 75:7769-7773.
[28]Thullier, P., C. Demangel, H. Bedouelle, et al. Mapping of a dengue virus neutralizing epitope critical for the infectivity of all serotypes:insight into the neutralization mechanism. J Gen Virol.2001; 82:1885-1892.
[29]Lisova, O., F. Hardy, V. Petit, et al. Mapping to completeness and transplantation of a group-specific, discontinuous, neutralizing epitope in the envelope protein of dengue virus. JGen Virol. 2007; 88:2387-2397.
[30]Rajamanonmani, R., C. Nkenfou, P. Clancy, et al. On a mouse monoclonal antibody that neutralizes all four dengue virus serotypes. J Gen Virol.2009; 90:799-809
[31]Falconar, A. K.1999. Identification of an epitope on the dengue virus membrane (M) protein defined by cross-protective monoclonal antibodies: design of an improved epitope sequence based on common determinants present in both envelope (E and M) proteins. Arch Virol 144:2313-2330.
[32]Gromowski, G D., and A. D. Barrett. Characterization of an antigenic site that contains a dominant, type-specific neutralization determinant on the envelope protein domain Ⅲ (ED3) of dengue 2 virus. Virology.2007; 366:349-360.
[33]Gromowski, G. D., N. D. Barrett, and A. D. Barrett. Characterization of dengue virus complex-specific neutralizing epitopes on envelope protein domain III of dengue 2 virus. J Virol.2008; 82:8828-8837.
[34]Matsui, K., G. D. Gromowski, L. Li, et al. Characterization of dengue complex-reactive epitopes on dengue 3 virus envelope protein domain III. Virology.2009; 384:16-20.
[35]Serafin, I. L., and J. G Aaskov. Identification of epitopes on the envelope (E) protein of dengue 2 and dengue 3 viruses using monoclonal antibodies. Arch Virol.2001; 146:2469-2479.
[36]Markoff, L. J., B. L. Innis, R. Houghten, et al. Development of cross-reactive antibodies to plasminogen during the immune response to dengue virus infection. J Infect Dis.1991; 164:294-301.
[37]Chungue, E., L. Poli, C. Roche, et al. Correlation between detection of plasminogen cross-reactive antibodies and hemorrhage in dengue virus infection. J Infect Dis.1994; 170:1304-1307.
[38]Huang, Y. H., B. I. Chang, H. Y. Lei, et al. Antibodies against dengue virus E protein peptide bind to human plasminogen and inhibit plasmin activity. Clin Exp Immunol.1997; 110:35-40.
[39]Monroy, V., and B. H. Ruiz. Participation of the Dengue virus in the fibrinolytic process. Virus Genes.2000; 21:197-208.