Human IgG Fc promotes expression, secretion and immunogenicity of enterovirus 71 VP1 protein

Enterovirus （EV71） can cause severe neurological diseases, but the underlying pathogenesis remains unclear. The capsid protein, viral protein 1 （VP1）, plays a critical role in the pathogenicity of EVT1. High level expression and secretion ofVP 1 protein are necessary for structure, function and immunogenicity in its natural conformation. In our previous studies, 5 codon-optimized VP 1 DNA vaccines, including wt-VP 1, tPA-VP 1, VP l-d, VP 1-hFc and VP 1 - mFc, were constructed and analyzed. They expressed VP1 protein, but the levels of secretion and immunogenicity of these VP1 constructs were significantly different （P〈0.05）. In this study, we further investigated the protein lev- els of these constructs and determined that all of these constructs expressed VP1 protein. The secretion level was increased by including a tPA leader sequence, which was further increased by fusing human IgG Fc （hFc） to VP1. VP 1-hFc demonstrated the most potent immunogenicity in mice. Furthermore, hFc domain could be used to purify VPI-hFc protein for additional studies.

Therapeutic and prevention strategies against human enterovirus 71 infection

Human enterovirus 71(HEV71) is the cause of hand,foot and mouth disease and associated neurological complications in children under five years of age.There has been an increase in HEV71 epidemic activity throughout the Asia-Pacific region in the past decade,and it is predicted to replace poliovirus as the extant neurotropic enterovirus of highest global public health significance. To date there is no effective antiviral treatment and no vaccine is available to prevent HEV71 infection. The increase in prevalence, virulence and geographic spread of HEV71 infection over the past decade provides increasing incentive for the development of new therapeutic and prevention strategies against this emerging viral infection. The current review focuses on the potential, advantages and disadvantages of these strategies. Since the explosion of outbreaks leading to large epidemics in China, research in natural therapeutic products has identified several groups of compounds with anti-HEV71 activities. Concurrently, the search for effective synthetic antivirals has produced promising results. Other therapeutic strategies including immunotherapy and the use of oligonucleotides have also been explored. A sound prevention strategy is crucial in order to control the spread of HEV71. To this end the ultimate goal is the rapid development, regulatory approval and widespread implementation of a safe and effective vaccine. The various forms of HEV71 vaccine designs are highlighted in this review. Given the rapid progress of research in this area, eradication of the virus is likely to be achieved.

Positive Selection Analysis of VP1 Genes of Worldwide Human Enterovirus 71 Viruses

Human enterovirus 71 viruses have been long circulating throughout the world. In this study, we performed a positive selection analysis of the VP1 genes of capsid proteins from Enterovirus 71 viruses. Our results showed that although most sites were under negative or neutral evolution, four positions of the VP1 genes were under positive selection pressure. This might account for the spread and frequent outbreaks of the viruses and the enhanced neurovirulence. In particular, position 98 might be involved in neutralizing antibodies, modulating the virus-receptor interaction and enhancing the virulence of the viruses. Moreover, both positions 145 and 241 might correlate to determine the receptor specificity. However, these positions did not display much difference in amino acid polymorphism. In addition, no position in the VP1 genes of viruses isolated from China was under positive selection.

Evaluation of Reverse Transcription Loop-Mediated Isothermal Amplification assays for Rapid Detection of Human Enterovirus 71 and Coxsackievirus A16 in Clinical Samples

A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for human enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) infection was further evaluated. The one step reaction was performed in a single tube at 65?C for 45 min for EV71 and 35 min for CVA16. The detection limits of RT-LAMP assays for both EV71 and CVA16 were 0.1 of a 50% tissue culture infective dose (TCID50) per reaction, based on 10—Fold dilutions of a titrated EV71 or CVA16 strain. The specific assay showed there were no cross-reactions with Coxsackievirus A (CVA) viruses (CVA 2, 4, 5, 7, 9, 10, 14, and 25), Coxsackievirus B (CVB) viruses (CVB 1, 2, 3, 4, and 5) or ECHO viruses (ECHO 3, 6, 11, and 19). In parallel with commercial quantitative real-time polymerase chain reaction (qRT-PCR) diagnostic kits for EV71 and CVA16, the RT-LAMP assay was evaluated with 515 clinical specimens, the results showed the RT-LAMP assay and the qRT-PCR assay were in complete agreement for 513/515 (99.6%) of the specimens. Two samples with discrepant results from two methods were further verified by nested reverse transcription polymerase chain reaction (nRT-PCR) assay and sequencing to be true positives for CVA16. In conclusion, RT-LAMP assay is demonstrated to be a sensitive and specific assay and have a great potential for the rapid and visual screening of EV71 and CVA16 in China, especially in those resource-limited hospitals and rural clinics of provincial and municipal regions.

Human Enterovirus 71 DNA Vaccine Constructs Containing 5’UTR with Complete Internal Ribosome Entry Site Sequence Stimulated Improved Anti-Human Enterovirus 71 Neutralizing Immune Responses

Recent improvement in the technologies for efficient delivery of DNA vaccines has renewed interest in the DNA-based vaccines. Several DNA-based vaccines against human enterovirus 71 (EV71), the causative agent for hand, foot and mouth disease (HFMD) have been developed. Here we examined the potential of improving the vaccines by inserting the EV71 5’ untranslated region (5’ UTR) containing the full length internal ribosome entry site (IRES) sequence to the EV71 VP1-based DNA vaccine constructs. Four vaccine constructs designated as 5’ UTR-VP1/EGFP, VP1/EGFP, 5’ UTR-VP1/pVAX and VP1/pVAX, were designed using the pEGFP-N1 and pVAX-1 expression vectors, respectively. Transfection of Vero cells with the vaccine constructs with the 5’-UTR (5’-UTR-VP1/EGFP and 5’ UTR-VP1/pVAX) resulted in higher percentages of cells expressing the recombinant protein in comparison to cells transfected with vectors without the 5’-UTR (67% and 57%, respectively). Higher IgG responses (29%) were obtained from mice immunized with the DNA vaccine construct with the full length 5’ UTR. The same group of mice when challenged with life EV71 produced significantly higher neutralizing antibody (NAb) titers (>5-fold). These results suggest that insertion of the EV71 5’ UTR sequence consisting of the full length IRES to the EV71 DNA vaccine constructs improved the efficacy of the constructs with enhanced elicitation of the neutralizing antibody responses.

轻或重症手足口病患儿HEV71分离鉴定及其VP1基因型分析

目的从不同病情手足口病（HFMD）患儿中分离鉴定人肠道病毒71型（HEV71）并分析其VP1基因型及药物结合位点突变情况。方法从2014年浙江省绍兴地区轻、重症各4例HFMD患儿标本中分离HEV71并用RT-PCR进行鉴定。采用RT-PCR扩增病毒分离株全长VP1基因并测序。采用专业生物信息学软件,对HEV71分离株进行VP1基因分型、确定VP1基因中药物结合位点突变情况及构建HEV71分离株系统进化树。结果8例HFMD患儿标本中均分离出HEV71。HEV71分离株与文献报道的VP1-C4a基因亚型的核苷酸和氨基酸序列相似性分别高达97.2%～98.8%和99.3%～99.7%。2例重症患儿HEV71分离株分别在VP1基因序列第179位药物结合位点发生V179L点突变。5株HEV71与安徽和湖南分离株、3株与上海和山东分离株亲缘关系较近。结论本地区不同病情HFMD患儿分离的HEV71均为VP1-C4a基因亚型,重症患儿HEV71分离株VP1基因第179位药物结合位点突变可能与病情严重程度有关。

Rational design of a DNA-launched live attenuated vaccine against human enterovirus 71

Human Enterovirus 71(EV71)has emerged as one of the predominant causative agents of hand,foot and mouth disease(HFMD)with global impact.Despite the inactivated vaccine being licensed,other vaccine candidates based on advanced technology platforms are under development.In this report,we rationally designed and constructed two DNA-launched live attenuated vaccine candidates(pDL-EV71)under the control of specific promoters.In vitro and in vivo transfection with pDL-EV71 driven by the CMV promoter successfully yielded fully infectious EV71.More importantly,the administration of pDL-EV71 did not cause clinical symptoms following intracranial or intramuscular inoculation in neonatal and IFNα/βR
/
mice,demonstrating its safety profile.Moreover,a single-dose or two-dose immunization with pDL-EV71 elicited robust neutralizing antibodies against EV71 as well as an antigen-specific cellular response in mice.A single-dose immunization with 10μg of pDL-EV71 conferred complete protection against lethal EV71 infection in neonates born to immunized maternal mice.Overall,our present results demonstrate that pDL-EV71 is a safe and effective vaccine candidate against EV71 for further development.

内标多重荧光RT-PCR同时检测肠道病毒71型和柯萨奇病毒A16型

[目的]为对当前爆发的手足口病进行快速准确的检测。[方法]本研究建立了含内标的同时检测EV71和CA16的多重荧光RT-PCR方法，对该方法的特异性、灵敏度等进行评估，并对400多份临床样品进行了检测。[结果]实验结果表明，该检测方法特异性强，对10株EV71病毒、8株CA16病毒和25株其他人类病毒进行了检测，特异性为100%；该检测方法对EV71和CA16的检测灵敏度分别达到0.1 TCID50和1 TCID50；将0.1-104TCID50/mL EV71和CA16样本进行重复性实验，其变异系数分别为0.9%-2.0%和0.9%-2.3%。对400多份临床样品分别进行荧光RT-PCR检测和传统方法检测，结果显示，荧光RT-PCR对EV71和CA16的阳性检出率平均为46.1%和14.2%，比传统方法（34.5%和12.8%）的阳性检测率高。另外，实验数据显示，在粪便、直肠拭子、咽喉拭子样本中，PCR抑制物存在的比例为1.8%-3.4%，表明内标对监控PCR抑制物的存在具有重要作用。[结论]本方法能同时对EV71和CA16进行快速检测，并且灵敏度高，特异性好，由于加入了内标，能有效地监控假阴性的出现，适合于手足口病的临床检测。

重症EV71型手足口病炎症因子的临床意义

目的了解5种炎症因子MCP-1、IL-1β、IL-18、HMGB1、IL-10在重症EV71型手足口病(HFMD)中的临床意义。方法选取某院2014年3—8月确诊为重症EV71型HFMD的住院患儿为HFMD组,同期本院门诊体检的健康儿童为对照组,动态观察两组外周血MCP-1、IL-1β、IL-18、HMGB1、IL-10表达水平的变化,并收集HFMD组临床资料。结果 HFMD组共102例患儿,平均年龄为(2.18±0.91)岁,其中主要以≤3岁为主,占80.39%;HFMD组患儿入院时为第2期77例,第3期16例,第4期9例。经过第2期共77例,第3期共52例,第4期共21例,第5期共88例。HFMD组第2、3、4期分别与对照组的5种炎症因子表达水平比较,差异均有统计学意义(均P<0.05);HFMD组第3期与第4期的IL-10表达水平比较,差异无统计学意义(P>0.05)。HFMD组第2、3期进展组HMGB1表达水平均高于痊愈组,差异均有统计学意义(均P<0.05)。死亡组与存活组在入院时5种炎症因子表达水平的比较,差异均有统计学意义(均P<0.05)。结论 MCP-1、HMGB1、IL-1β、IL-10、IL-18的表达水平与HFMD病情轻重相关,对患儿预后的预测有一定临床意义。HMBG1在HFMD中对病情转归有一定预测价值。

肠道病毒71型感染人脑微血管内皮细胞的初步研究

目的 初步探讨肠道病毒71型（enterovirus 71,EV71）穿血脑屏障（blood-brain barrier,BBB）的相关机制.方法 用人脑微血管内皮细胞（human brain microvascular endothelial cells,HBMECs）建立体外BBB模型,采用EV71毒株感染HBMECs,并设置阴性对照.通过形态学观察、免疫荧光、透射电镜及实时荧光定量PCR方法探讨EV71能否感染HBMECs,激光共聚焦方法观察EV71能否诱导HBMECs微丝骨架的重排.结果 EV71感染后能诱导HBMECs的细胞病变,细胞内可见EV71抗原红色荧光表达及直径为20～30 nm的EV71颗粒存在,实时荧光定量PCR方法证实EV71能在HBMECs内复制,不同时间水平差异有统计学意义（P＜0.01）.细胞感染EV71后失去正常形态,变得极不规则,胞内微丝排列紊乱,极性消失,微丝聚集在细胞边缘.结论 本研究首次证明EV71可以感染HBMECs并复制,并且可以诱导部分HBMECs的骨架重排.

人肠道病毒71型感染免疫抑制恒河猴的分析

目的:通过建立人肠道病毒71型(EV71)感染在免疫抑制恒河猴的动物模型,进一步分析人EV71在免疫抑制动物体内病毒增殖、病理变化以及病毒抗原在不同组织的表达情况。方法:按照每天15 mg/kg的剂量给8只恒河猴口服环孢菌素A连续7 d后造成其免疫抑制状态,再经呼吸道和消化道感染EV71病毒FY23株,对其进行14 d的临床观察后处死,采集各器官组织进行病毒载量检测,同时对不同的组织进行病理学分析和免疫组化分析。结果:感染EV71病毒后,免疫抑制组的临床症状、病毒载量、病理分析和免疫组化结果都明显比非免疫抑制组严重,从病毒对机体造成的影响来看呼吸系统感染较消化系统感染明显严重。结论:本实验分析了人EV71在免疫抑制动物体内病毒增殖的情况,为建立用于EV71疫苗评价的恒河猴动物模型提供了依据,并对未来的EV71疫苗的使用人群范围做出了新的限定。

肠道病毒71型灭活疫苗上市后安全性监测与评价

目的 评价不同细胞基质制备的肠道病毒71型（EV71）灭活疫苗上市后常规免疫安全性。 方法 EV71灭活疫苗疑似预防接种异常反应（AEFI）数据来源于中国免疫规划信息管理系统，为2017年接种EV71 灭活疫苗后江苏省报告的监测数据；受种儿童人口学信息和接种剂次数来源于江苏省预防接种信息管理系统。 结果 2017年江苏省共接种EV71灭活疫苗316 889剂，报告AEFI 208例，报告发生率为65.64/10万剂（95% CI ：56.72/10万剂～74.56/10万剂），其中一般反应55.54/10万剂（95% CI ：47.34/10万剂～63.74/10万剂），异常反应 7.89/10万剂（95% CI ：4.80/10万剂～10.98/10万剂）；不同细胞基质的疫苗AEFI发生率（χ 2 =35.61， P ＜0.01）和一般反应发生率（χ 2 =34.09， P ＜0.01）差异有统计学意义，异常反应发生率差异无统计学意义（χ 2 =1.88， P =0.17），均未见严重异常反应，208例AEFI均未住院且均痊愈。EV71灭活疫苗（Vero细胞）首剂接种一般反应发生率高于第二剂（χ 2 =6.94， P ＜0.01），不同季度接种EV71灭活疫苗（人二倍体细胞）一般反应发生率差异有统计学意义（χ 2 =18.86， P ＜0.01）。 结论 不同细胞基质的EV71灭活疫苗在常规免疫接种中AEFI发生率均不高，有良好的安全性；一般反应以发热为主，异常反应以过敏性-荨麻疹、过敏性皮疹和过敏性斑丘疹等过敏性反应为主。

陕西省手足口病重症和死亡病例的流行特征及肠道病毒71型VP1区基因特征分析

目的了解陕西省手足口病(HFMD)重症和死亡病例的流行特征及肠道病毒71型(EV-A71)基因特征,为制定防控措施提供参考依据。方法对2010-2015年陕西省HFMD监测数据和病原学资料进行分析,选择重症和死亡病例的EV-A71分离株用RT-PCR方法扩增VP1基因片段,MEGA5.0软件分析同源性并构建系统进化树。结果 2010-2015年陕西省共报告HFMD重症病例5 054例,死亡病例118例,年均报告死亡率0.052 09/10万,重症病死率2.32%。5-7月为重症和死亡病例发病高峰,西安、渭南、咸阳为高发地区。病例集中于3岁以下儿童,分别占重症和死亡病例总数的90.84%和93.22%,男性高于女性,男女性别比1.67∶1。病原学监测重症病例中EV-A71、CV-A16和其他肠道病毒的构成比分别为66.39%、6.26%和27.35%,不同年份的各病原构成有统计学差异(χ~2=307.827,P<0.001)。不同年龄组的EV-A71检出率最高,且随着年龄的增加不断增高,差异有统计学意义(χ~2=50.688 18,P<0.001)。死亡病例中EV-A71检出率达96.83%。29株EV-A71分离株的核苷酸和氨基酸同源性分别为94.2%～99.7%和98.0%～100%,与我国C4亚型代表株之间具有高度同源性,属于EV-A71病毒的C4基因亚型C4a进化分支。结论应做好重点地区、高发季节和≤3岁儿童的HFMD防控工作,密切关注EV-A71病原流行变化趋势,采取有效措施降低重症和死亡病例的发生。

检测EV71和EV-U的TaqMan-LNA探针多重荧光RT-PCR的建立

目的建立一种针对手足口病病原体的快速准确检测方法。方法利用TaqMan-LNA探针建立多重荧光定量RT-PCR方法,同时检测肠道病毒71型(EV71)和肠道病毒通用型(EV-U),用含有目的序列RNA的标准品制备标准曲线,对病毒进行准确定量,同时对该方法的特异性、灵敏度、重复性进行评估。结果用TaqMan-LNA探针多重荧光RT-PCR法检测CoxA16、CoxB2、EV71病毒和其他人类病毒RNA,证实其特异性为100%;对EV71和EV-U的检测灵敏度均达到102拷贝/μl;将EV71和EV-U的RNA标准品进行重复性实验,其批内、批间变异系数分别为0.97%～2.02%和0.89%～2.13%。结论本方法灵敏度较高,特异性强,重复性好,适用于手足口病的临床检测。

人免疫球蛋白中肠道病毒71型中和抗体效价的测定

采用经典微量细胞病变法,应用近两年分离自中国手足口病（HFMD）高发区的3株肠道病毒71型（EV71）病毒株,对中国不同血液制品厂家生产的35批人免疫球蛋白制品进行抗-EV71中和效价检测。结果显示,3株不同EV71病毒株间的抗-EV71中和效价差异均在4倍以内,差异无显著的统计学意义（F=2.323,P〉0.05）。根据这3株毒株检测结果判定,35批人免疫球蛋白的抗-EV71均为阳性,肌肉注射用免疫球蛋白（简称肌丙）的抗-EV71-GMTs（525.9）显著高于静脉注射用免疫球蛋白（简称静丙）的GMTs（252.3,F=66.518,P〈0.01）。30批静丙的抗-EV71-GMTs中和效价分布在128.0-384.0之间。应开展原料血浆中抗-EV71中和效价的筛选,研制高效价的EV71特异性免疫球蛋白制品,用于HFMD的治疗和预防。

赣榆县儿童肠道病毒71型血清流行病学调查

目的:了解赣榆县儿童肠道病毒71型(HEV71)抗体水平及自然感染状况,为预防控制儿童手足口病传播与流行提供依据。方法:2010年8月采集赣榆县2个乡镇400名1～15岁儿童、20名新生儿与其母血清标本,采用中和试验方法检测血清的HEV71中和抗体。结果:420名儿童中HEV71中和抗体阳性178人,阳性率为42.4%。阳性率与年龄有关,5月龄的儿童阳性率最低,随着年龄的增长,抗体阳性率及几何平均滴度先下降后上升,HEV71中和抗体几何平均滴度水平为1∶15.05,各年龄组间抗体阳性率、几何抗体平均滴度差异均有统计学意义;不同性别间儿童、高低发乡镇间儿童、新生儿与母亲间抗体阳性率、几何抗体平均滴度差异均无统计学意义。结论:赣榆县<3岁儿童是目前手足口病防控的重点人群。儿童在4～5月龄时是接种EV71疫苗的最佳时机。

重症肠道病毒71型感染早期误诊的临床分析

目的提高对重症肠道病毒71型(EV71)感染的认识,分析总结救治的经验教训,减少误诊,提高抢救成功率,及早做好消毒隔离,防止疾病扩散流行。方法选择2011—2012年县级医院及我院收治的6例重症EV71感染患儿,均早期误诊为重症肺炎并发中毒性脑病及感染性休克,回顾性分析其临床表现、实验室检查结果、影像学结果、抢救经过及治疗转归。结果 6例患儿均有较明显的临床特征,如高热不退、心动过速、血压过高、高血糖、无典型的皮疹,咽拭子EV71病毒特异性核酸检测阳性。颅脑及颈椎MRI检查,延髓区,颈段及上胸段脊髓内可见长T1、T2高信号影。6例患儿均给予大剂量丙种球蛋白等治疗,其中4例痊愈,2例死亡。结论对临床出现高热不退、心动过速、血压过高、高血糖等表现的危重患儿,应及早做咽拭子EV71病毒抗体检测,减少误诊、漏诊;早诊断,尽早使用机械通气;早隔离,防止EV71感染传播流行。

肠道病毒71型诱导人脑微血管内皮细胞的凋亡

目的探讨肠道病毒71型(enterovirus 71,EV71)诱导人脑微血管内皮细胞(human brain microvascular endothelial cells,HBMECs)的凋亡及其相关机制。方法流式细胞仪检测EV71能否诱导HBMECs的凋亡;免疫荧光方法观察EV71诱导HBMECs线粒体膜电位的改变情况;免疫印迹检测DJ-1蛋白在EV71感染HBMECs不同时间点的表达;酶联免疫方法检测EV71感染HBMECs不同时间点TNF-α、IL-10、IL-4的分泌情况。结果 EV71感染HBMECs不同时间点的HBMECs的早期凋亡数与晚期凋亡、坏死细胞数具有显著差异(P<0.01);随着感染时间延长,HBMECs早期凋亡数与晚期凋亡、坏死细胞数均有所增加,但以晚期凋亡、坏死细胞数增加更明显(P<0.01)。与对照组相比,EV71感染HBMECs8 h线粒体膜电位明显降低(P<0.01);EV71感染HBMECs 16、24 h,DJ-1表达增强(P<0.05)。EV71感染HBMECs不同时间点TNF-α、IL-10、IL-4的浓度具有明显差异(P<0.01)。结论 EV71能诱导HBMECs的凋亡以及改变线粒体膜通透性,EV71还能诱导HBMECs中DJ-1的表达改变和TNF-α、IL-10、IL-4的分泌改变。

人肠道病毒71型动物模型研究进展

人肠道病毒71型是婴幼儿手足口病的致病原之一,其严重的并发症可导致神经系统疾病,甚至死亡,是近期威胁中国儿童健康的因素之一。目前尚无临床疫苗可以预防该病毒感染,而EV71的动物模型是进行致病机理、疫苗评价和药物等研究的基础。本文对EV71的两种常用动物模型:小鼠和猕猴(Macaca mulatta)模型进行了描述,并对其在研究中的应用给与概括,为研究者选择合适的动物模型提供了依据。

EV71-VP1抗原在双歧杆菌中表达的免疫效果检测

目的检测表达肠道病毒71型(enterovirus 71,EV71)VP1基因的重组双歧杆菌免疫后小鼠Ig G、Ig A表达变化,并对免疫的孕鼠产下的新生小鼠注射EV71病毒,以检测免疫保护作用可否从母代传给子代。方法 ELISA法检测免疫小鼠血清Ig G、Ig A抗体效价;用EV71毒株对免疫孕鼠所产下的新生小鼠攻毒。结果两种抗体效价都有明显升高(P<0.01);实验组受病毒攻击新生小鼠存活率明显高于对照组。结论口服表达VP1蛋白的双歧杆菌能够激活体内免疫系统产生特异性抗体,重组双歧杆菌可通过免疫孕鼠而使新生幼鼠获得保护性抗体。为研制EV71亚单位口服活疫苗奠定了研究基础。