A novel inactivated enterovirus 71 vaccine can elicit cross-protective immunity against coxsackievirus A16 in mice

Hand, foot, and mouth disease (HFMD) is a highly contagious disease that mainly affects infants and children. Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the major pathogens of HFMD. Two EV71 vaccines were recently licensed in China and the administration of the EV71 vaccines is believed to significantly reduce the number of HFMD-related severe or fatal cases. However, a monovalent EV71 vaccine cannot cross-protect against CA16 infection, this may result in that it cannot effectively control the overall HFMD epidemic. In this study, a chimeric EV71, whose VP1/210–225 epitope was replaced by that of CA16, was constructed using a reverse genetics technique to produce a candidate EV71/CA16 bivalent vaccine strain. The chimeric EV71 was infectious and showed similar growth characteristics as its parental strain. The replacement of the VP1/210–225 epitope did not significantly affect the antigenicity and immunogenicity of EV71. More importantly, the chimeric EV71 could induce protective immunity against both EV71 and CA16, and protect neonatal mice against either EV71 or CA16 lethal infections, the chimeric EV71 constructed in this study was shown to be a feasible and promising candidate bivalent vaccine against both EV71 and CA16. The construction of a chimeric enterovirus also provides an alternative platform for broad-spectrum HFMD vaccines development.     

Hepatitis B virus core particles containing multiple epitopes confer protection against enterovirus 71 and coxsackievirus A16 infection in mice

Human enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two major causative agents of hand-foot-and-mouth disease (HFMD). To investigate novel combined vaccines to prevent EV71 and CA16 infection, we constructed chimeric virus-like particles (tHBc/SPA or tHBc/SP VLPs) displaying conserved epitopes of EV71 (aa 208–222 of VP1 and aa 248–263 of VP2) and CA16 (aa271-285 of VP1) using a truncated hepatitis B virus core carrier (tHBc). Immunization with the chimeric VLPs induced epitope- or virus-specific IgG and neutralization antibodies against EV71 and CA16 in the mice. Compared with inactivated EV71, the chimeric VLPs induced significantly increased Th1 cytokine (IFN-γ, IL-2) production and decreased Th2 cytokine (IL-4, IL-10) responses. Neonatal mice born to dams immunized with the recombinant particles were completely protected from lethal EV71 and partially protected from CA16 infection. Co-expression of the conserved human MHC class I CD4+ T cell epitope (aa248-263 of VP2) did not improve the antiviral immunity of the chimeric VLP vaccine in mice. Our results demonstrate that experimental combination vaccines comprised of EV71 and CA16 epitopes induce both humoral and cellular immune responses and therefore support further preclinical and clinical development of a bivalent VLP vaccine targeting both CA16 and EV71.     

A combination vaccine comprising of inactivated enterovirus 71 and coxsackievirus A16 elicits balanced protective immunity against both viruses

Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two major causative agents of hand, foot and mouth disease (HFMD), which is an infectious disease frequently occurring in children. A bivalent vaccine against both EV71 and CA16 is highly desirable. In the present study, we compare monovalent inactivated EV71, monovalent inactivated CA16, and a combination vaccine candidate comprising of both inactivated EV71 and CA16, for their immunogenicity and in vivo protective efficacy. The two monovalent vaccines were found to elicit serum antibodies that potently neutralized the homologous virus but had no or weak neutralization activity against the heterologous one; in contrast, the bivalent vaccine immunized sera efficiently neutralized both EV71 and CA16. More importantly, passive immunization with the bivalent vaccine protected mice against either EV71 or CA16 lethal infections, whereas the monovalent vaccines only prevented the homologous but not the heterologous challenges. Together, our results demonstrate that the experimental bivalent vaccine comprising of inactivated EV71 and CA16 induces a balanced protective immunity against both EV71 and CA16, and thus provide proof-of-concept for further development of multivalent vaccines for broad protection against HFMD.     

Chimeric enterovirus 71 virus-like particle displaying conserved coxsackievirus A16 epitopes elicits potent immune responses and protects mice against lethal EV71 and CA16 infection

Hand-foot-and-mouth disease (HFMD) is an infectious disease of infants and young children frequently caused by the enterovirus A species, mainly enterovirus 71 (EV71) and coxsackievirus A16 (CA16). In this study, we prepared the EV71 virus-like particle (EV71-VLP) and its chimeras using recombinant baculovirus (Bac-P1-3CD) co-expressing EV71 P1 (under polyhedrin promoter) and 3CD (under CMV-IE promoter) proteins in Sf9 cells. EV71-VLP chimera ChiEV71(1E)-VLP or ChiEV71(4E)-VLP displayed single CA16 PEP71 epitope in VP1 or four conserved CA16 neutralizing epitopes (PEP71 in VP1, aa136-150 in VP2, aa176-190 in VP3 and aa48-62 in VP4) by substitution of the corresponding regions of EV71 structure proteins, respectively. In mice, EV71-VLP and its chimeras elicited similar EV71-specific IgG and neutralizing antibody (NAb) titers compared to inactivated EV71. Expectedly, vaccination of ChiEV71(1E)-VLP or ChiEV71(4E)-VLP resulted in significantly increased CA16-specific IgG and NAb production and improved cross-protection against CA16 infection compared to EV71-VLP. Interestingly, the VLPs induced potent cellular immune responses and significantly decreased Th2 type (IL-4 and IL-10) cytokines secretion in the splenocytes of immunized mice compared to inactivated EV71 or inactivated CA16. Neonatal mice born to dams immunized with the chimeric VLPs or neonatal mice passively transferred with sera of immunized mice were completely protected from lethal EV71 challenge and partially protected from lethal CA16 infection. Our study provides a novel bivalent or multivalent vaccine strategy to prevent EV71 and related-enterovirus infections.     

Efficacy of an inactivated bivalent vaccine for enterovirus 71 and coxsackievirus A16 in mice immunized intradermally

Human hand, foot, and mouth disease (HFMD), an important infectious disease in children, is caused mainly by enterovirus 71 (EV71) and coxsackievirus A16 (CA16). In this study, a bivalent inactivated EV71/CA16 vaccine is developed and evaluated in immunized BALB/c mice injected through the intradermal route. Q-RT-PCR detection of the mRNA of immune signal molecules in local epithelial tissues inoculated with the vaccine indicates activation of innate immunity, which includes upregulation of immune-related chemokines, interferons and CD molecules. Further, the finding that neutralizing antibodies and specific T cellular responses were elicited in adult mice after two immunizations with the vaccine at a 28-day interval, which endowed offspring mice to defend a viral challenge, suggests the successful induction of specific protective antiviral immunity. All these data suggest that immunization with this bivalent EV71/CA16 vaccine via the intradermal route elicits effective immunity against EV71 and CA16 infection.     

A virus-like particle based bivalent vaccine confers dual protection against enterovirus 71 and coxsackievirus A16 infections in mice

Enterovirus 71(EV71) and coxsackievirus A16 (CA16) are responsible for hand, foot and mouth disease which has been prevalent in Asia-Pacific regions, causing significant morbidity and mortality in young children. Co-circulation of and co-infection by both viruses underscores the importance and urgency of developing vaccines against both viruses simultaneously. Here we report the immunogenicity and protective efficacy of a bivalent combination vaccine comprised of EV71 and CA16 virus-like particles (VLPs). We show that monovalent EV71- or CA16-VLPs-elicited serum antibodies exhibited potent neutralization effect on the homotypic virus but little or no effect on the heterotypic one, whereas the antisera against the bivalent vaccine formulation were able to efficiently neutralize both EV71 and CA16, indicating there is no immunological interference between the two antigens with respect to their ability to induce virus-specific neutralizing antibodies. Passive immunization with monovalent VLP vaccines protected mice against a homotypic virus challenge but not heterotypic infection. Surprisingly, antibody-dependent enhancement (ADE) of disease was observed in mice passively transferred with mono-specific anti-CA16 VLP sera and subsequently challenged with EV71. In contrast, the bivalent VLP vaccine conferred full protection against lethal challenge by either EV71 or CA16, thus eliminating the potential of ADE. Taken together, our results demonstrate for the first time that the bivalent VLP approach represents a safe and efficacious vaccine strategy for both EV71 and CA16.     

A new EV71 VP3 epitope in norovirus P particle vector displays neutralizing activity and protection in vivo in mice

Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16), as the main agents causing hand, foot and mouth disease (HFMD), have become a serious public health concern in the Asia-Pacific region. Recently, various neutralizing B cell epitopes of EV71 were identified as targets for promising vaccine candidates. Structural studies of Picornaviridae indicated that potent immunodominant epitopes typically lie in the hypervariable loop of capsid surfaces. However, cross-neutralizing antibodies and cross-protection between EV71 and CVA16 have not been observed. Therefore, we speculated that divergent sequences of the two viruses are key epitopes for inducing protective neutralizing responses. In this study, we selected 10 divergent epitope candidates based on alignment of the EV71 and CVA16 P1 amino acid sequences using the Multalin interface page, and these epitopes are conserved among all subgenotypes of EV71. Simultaneously, by utilizing the norovirus P particle as a novel vaccine delivery carrier, we identified the 71-6 epitope (amino acid 176–190 of VP3) as a conformational neutralizing epitope against EV71 in an in vitro micro-neutralization assay as well as an in vivo protection assay in mice. Altogether, these results indicated that the incorporation of the 71-6 epitope into the norovirus P domain can provide a promising candidate for an effective synthetic peptide-based vaccine against EV71.     

Study of integrated protective immunity induced in rhesus macaques by the intradermal administration of a bivalent EV71-CA16 inactivated vaccine

Enterovirus type 71 (EV71) and coxsackievirus A 16 (CA16) are recognized as the major pathogens responsible for human hand-foot-mouth disease. To develop a bivalent EV71-CA16 vaccine, rhesus macaques immunized with two doses of this vaccine via the intradermal route were challenged with EV71 or CA16, and their clinical symptoms, viral shedding, neutralizing antibodies, IFN-γ-specific ELISpots, and tissue viral load were examined longitudinally. Specific immunity against EV71 and CA16 was observed in the macaques, which exhibited controlled proliferation of the EV71 and CA16 viruses and upregulated expression of immune-related genes compared with the controls. Furthermore, broad protection against EV71 and CA16 challenge without immunopathological effects was observed in all the immunized macaques. These studies suggest that the bivalent EV71-CA16 inactivated vaccine was effective against wild-type EV71 or CA16 viral challenge in rhesus macaques.     

Intradermal injection of a fractional dose of an inactivated HFMD vaccine elicits similar protective immunity to intramuscular inoculation of a full dose of an Al(OH)3-adjuvanted vaccine

Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) are the two major causative agents of hand, foot and mouth disease (HFMD), which erupts in the Asia-Pacific regions. A bivalent vaccine against both EV71 and CVA16 is highly desirable. In the present study, on the bases that an experimental bivalent vaccine comprising of inactivated EV71 and CVA16 induces a balanced protective immunity against both EV71 and CVA16, we compare the immunogenicity and reactogenicity of one fourth of a full dose of an intradermal vaccine administered by needle-free liquid jet injector with a full dose of an intramuscular vaccine administered by needle-syringe in monkeys. The results suggest that intradermal injection of a fractional dose of an inactivated HFMD vaccine elicits similar immunogenicity and reactogenicity to intramuscular inoculation of a full dose of an Al(OH)₃-adjuvanted vaccine, regardless of whether monovalent or bivalent vaccines were used. Our results support the use of an intradermal bivalent vaccine strategy for HFMD vaccination in order to satisfy the requirements and reduce the costs.     

Identification and characterization of a cross-neutralization epitope of Enterovirus 71

Enterovirus 71 (EV71) infections in children manifest as exanthema and are most commonly known as hand-foot-and-mouth disease (HFMD). Because it can cause severe neurological complications like poliomyelitis, EV71 has now emerged as an important neurotropic virus in Asia. EV71 virus has been shown to consist of 3 (A, B and C) genotypes and many subgenotypes. Although EV71 vaccine development has recently yielded promising preclinical results, yet the correlation between the content of antigen(s) in vaccine candidates and the level of protective antibody responses is not established. The neutralization epitope(s) of EV71 antigens could be used as the surrogate biomarker of vaccine potency. Using peptide ELISA, antisera generated from animals immunized with formalin-inactivated EV71 virion vaccine formulated in alum, EV71-specific neutralizing monoclonal antibody (nMAb) and a panel of 153 overlapping synthetic peptides covering the entire sequences of VP1, VP2 and VP3 of EV71, we screened for immunodominant linear neutralization epitope(s). Synthetic peptide VP2-28, corresponding to residues 136-150 of VP2, was found to bind to and inhibit the binding to EV71 of nMAb MAB979 that was found to have cross-neutralizing activity against different genotypes of EV71 virus. In addition, VP2-28 was found to be recognized only by neutralizing antisera generated from rabbits immunized with the formalin-inactivated whole EV71 virion vaccine but not by antisera from immunized mice and rats. During the epitope mapping, a murine EV71 genotype- and strain-specific linear neutralization epitope VP1-43 was identified within residues 211-220 of VP1. Furthermore, based on sequence alignment and structure prediction analysis using poliovirus as the template for molecular modeling, the VP1-43 and VP2-28 epitopes were shown to run in parallel within 0.1 nm and form a rim of the canyon at the junction site of VP1 and VP2 in the viral capsid. In mouse, rat and rabbit immunogenicity studies, a dose-dependent relationship between the number of VP2-28 epitope units measured by a quantitative assay in vaccine preparations and the magnitude of neutralizing titers was demonstrated. VP2-28 has amino acid sequences that are highly conserved among EV71 genotypes, is not affected by formalin-treatment and long-term storage. Thus, VP2-28 could be used as the surrogate biomarker in the potency testing of candidate EV71 vaccines.     

2010年合肥儿童手足口病病例肠道病毒血清基因型分析

目的:对合肥地区2010年5~7月儿童手足口病流行期间的手足口病患儿进行病原学调查。方法:采集手足口病患儿的疱疹液、粪便、咽拭子进行病毒分离;分离的病毒接种非洲绿猴肾细胞(Vero)后待细胞病变(CPE)达到++++以上后提取RNA。分别用EV71和CA16的VP1基因的特异性引物进行RT-PCR鉴定,并根据血清型不同比较其临床特征。结果:77例患儿采集的标本中65例分离到病毒;经RT-PCR检测发现其中47例CA16阳性,18例为EV71,比例为2.6∶1。CA16感染和EV71感染在患者的年龄大小、症状轻重等方面无显著统计学差异。结论:手足口病患儿检测到的主要病原是CA16和EV71,两者感染对临床的影响无显著差异。     

环介导等温扩增法快速检测手足口病病原体

目的：建立逆转录（RT）-环介导等温扩增方法（LAMP）,以快速检测手足口病最重要的两种病原体：肠道病毒71型（EV71）和柯萨奇病毒A组16型（CA16）。方法：收集手足口病患儿咽拭子标本93份,针对EV71和CA16病毒的VP1基因特异性序列8个区域各设计6条LAMP引物,分别于63℃（EV71）、65℃（CA16A）扩增1 h,日光下观察结果,与实时荧光定量PCR比较检测特异性和敏感性。结果：EV71、CA16的LAMP最低检测限均为500拷贝/管,与对照病毒无交叉反应。93份标本中,RT-PCR方法检测显示EV71阳性44例,CA16阳性36例;RT-LAMP方法检测显示EV71阳性46例,CA16阳性36例,两种方法间比较差异均无统计学意义（P〉0.05）。结论：应用RT-LAMP检测手足口病病原体EV71、CA16快速、灵敏、经济、特异性高,适合在基层医疗机构推广应用。     

Expression of VP1 protein in the milk of transgenic mice: a potential oral vaccine protects against enterovirus 71 infection

Enterovirus 71 (EV71) is the most common etiological agent detected in cases of hand-foot-and-mouth disease (HFMD) resulting in incidences of neurological complications and fatality in recent years. The clinical data have already shown the significant increase in recent EV71 epidemic activity throughout the Asia-Pacific region. Due to the lack of an effective antiviral agent, primary prevention of the disease, including the development of an effective vaccine, has been the top priority in terms of control strategies. In this study, we first generated a transgenic animal system to produce the EV71 VP1 capsid protein under the control of alpha-lactalbumin promoter and alpha-casein leader sequences. A high level of recombinant VP1 protein (2.51 mg/ml) was expressed and secreted into the milk of transgenic mice. Mouse pups that received VP1-transgenic milk orally demonstrated relatively better health conditions after challenge with the respective virus as compared with the non-transgenic milk fed group; moreover, the mice fed with the VP1-milk had body weights similar to those of the PBS placebo control groups. According to the serum-neutralization assay and serum antibody detection, the littermates suckling VP1-milk generated antibodies specific to EV71. Our data suggest that EV71 VP1-containing milk is suitable for development as a potential oral vaccine.     

北京及其周边地区儿童手足口病病原学监测及流行病学分析(2017-2018年)

目的了解北京及其周边地区儿童手足口病(HFMD)的流行特点及病原学特征。方法采集北京及其周边地区2017—2018年儿童HFMD病例咽拭子样本,使用肠道病毒(EV)通用、EV71及柯萨奇病毒A16型(CA16)三通道Real-time PCR的方法进行肠道病毒核酸检测,对非EV71非CA16的EV阳性样本采用RT-PCR法扩增VP1基因片段,通过BLAST比对进一步进行型别鉴定。结果2017—2018年共纳入HFMD患者939例,包括重症病例2例;男女性别比为1.54∶1;3岁以下儿童占46.96%,5岁以下儿童占75.19%。EV总阳性检出率为89.24%(838/939),其中2017年、2018年分别为87.44%(181/207)和89.75%(657/732);2017年主要为CA6(47.51%)、EV71(16.02%)、CA16(7.73%)和未分型EV(27.62%);2018年包括CA6(46.88%)、CA16(14.31%)和未分型EV(36.99%)。结论2017—2018年,引起北京及其周边地区儿童HFMD的主要病原体是CA16,EV71的检出率显著下降,因此需要加强HFMD病原体中CA6等非EV71非CA16肠道病毒的监测工作。     

河北省2009年手足口病病原学研究

目的了解2009年引起河北省手足口病的病原谱及分布特征,为制定预防控制措施提供实验依据。方法通过流行病学调查,采集手足口病临床诊断病例的粪便、肛拭子、咽拭子、疱疹液和脑脊液标本,针对肠道病毒(EV)5′端非编码区保守基因序列以及EVVP1至2A区基因序列设计EV通用引物和型特异性引物,通过RT-PCR扩增检测HFMD临床诊断病例的标本,并对病原进行分型。结果采用RT-PCR法共检测HFMD临床诊断病例1990份标本,1296份标本EV阳性,阳性率为65.13%,其中752份标本EV71阳性,452份CA16阳性,1份EV71+CA16阳性,91份其他EV阳性。随机抽取55份其他EV阳性标本经RT-PCR和测序进一步分型,除24份未定型外,共鉴定出31份,其中CA10占18.18%(10/55),CA6占14.55%(8/55),ECO30占9.09%(5/55),ECO6、ECO9各占3.64%(2/55),CA5、CA14、CB5、ECO14各占1.82%(1/55),未定型占43.64%(24/55)。结论 2009年引起河北省手足口病流行的病原体主要为EV71和CA16,同时还有部分CA5、CA6、CA10、CA14、CB5、ECO6、ECO9、ECO14、ECO30等其他型别,引起河北省重症病例的EV主要型别为EV71。     

CA6阳性与EV71、CA16阳性手足口病患儿临床特征分析

目的 分析柯萨奇病毒A6型(CA6)阳性与肠道病毒71型(EV71)、柯萨奇病毒A16型(CA16)阳性手足口病患儿的临床特征.方法 选取商洛市中心医院2014年1月至2016年12月收治的1528例手足口病患儿,通过实时荧光定量聚合酶链反应法检测患儿肠道病毒核酸,以分析CA6、EV71、CA16三种病毒引起的手足口病患儿的临床特征.结果 在1528例手足口病患儿中,经病原学确诊出374例CA6阳性患儿、673例CA16阳性患儿、481例EV71阳性患儿.CA6阳性患儿发病平均年龄(t值分别为6.34、5.85)、齿龈部疱疹分布(χ2值分别为5.65、5.23)均低于CA16、EV71阳性患儿(均P<0.05),且发热分布均高于CA16、EV71阳性患儿(χ2值分别为8.56、8.23,均P<0.05).CA6阳性患儿C反应蛋白和WBC升高分布均高于CA16、EV71阳性患儿(χ2值分别为5.34、5.89;6.34、6.83,均P<0.05).结论 CA6已发展成一种手足口病患儿新型的流行病原,且其临床特征与因EV71及CA16引起的手足口病有所不同,可作为临床诊断的依据.     

2013-2014年泉州地区手足口病病原学监测结果分析

摘要:目的　了解2013-2014年福建泉州地区手足口病（HFMD）的病原学特征,为手足口病防治提供科学依据。方法　采集2013-2014年泉州市妇幼保健院·儿童医院收治的3839例手足口病患儿空腹静脉血标本,采用捕获法酶联免疫吸附技术检测肠道病毒71型（EV71-IgM）和柯萨奇病毒A组16型（CA16-IgM）。结果　3839例被检标本共检测出阳性标本2176例,阳性率为56.68%;其中32.87%（1262/3839）为EV71阳性,23.81%（914/3839）为CA16阳性,两者间阳性率差异有统计学意义（P＜0.05）。5岁以下HFMD患者3679例,占95.83%,其中1～2岁组患儿最多,占68.56%。3～4岁组患儿EV71阳性率（48.28%）和CA16阳性率（35.09%）最高。不同年龄组,EV71阳性率和CA16阳性率均存在统计学差异（P＜0.05）。手足口病发病随季节而发生变化,不同月份EV71和CA16阳性率均存在统计学差异（P＜0.05）。发病以4-7月份为主,占56.37%,2014年9-10月份发病率出现一定程度反弹。结论　2013-2014年泉州地区手足口病肠道病毒EV71型的阳性检出率高于CA16型,4-7月份为发病高峰期,5岁以下儿童为高发人群,其中1～2岁组手足口患儿居多,而3～4岁组病毒阳性率最高,需引起重视。     

2013 - 2017年无锡市手足口病流行病和病原学特征分析

目的 了解无锡市2013 - 2017年手足口病（HFMD）流行病学及病原学变化特征,为本地区HFMD防控提供科学依据。方法 利用2013 - 2017年疾病监测信息系统报告的HFMD病例进行描述性流行病学分析。同时收集无锡市哨点医院HFMD病例的粪便样本,采用肠道病毒通用（EV）、肠道病毒71型（EV71）和柯萨基病毒A组16型（CA16）荧光定量检测试剂盒进行核酸检测和测序分析。结果 无锡市2013 - 2017年共报告HFMD 82 132例,年均发病率249.32/10万。病例报告以1～3岁发病率最高,且以散童（53.72%）为主。全年有5 - 7月和11 - 12月2个发病高峰,重症发生集中在7 - 8月。2013 - 2017年病原学监测结果显示,普通病例以EV71（46.08%）和CA16（35.78%）为主,重症病例以EV71（86.45%）为主（P＜0.001）。2012 - 2016年其他肠道病毒以CB5为主,占72.50%;2017年其它肠道病毒以CA6为主,占90.00%。结论 无锡市HFMD的发生存在周期性和季节性,呈现每隔1～2年出现1次流行高峰的特点;居住在城乡结合部地区的1～3岁儿童是HFMD的重点防控人群;2017年其他肠道病毒优势株由CB5转为CA6,CA6可能成为继CA16、EV71的另一个其他肠道病毒优势株。     

北京市2010年手足口病死亡危险因素分析

目的探讨北京市手足口病死亡的危险因素,为防制手足口病、减少手足口病死亡提供科学依据。方法收集2010年北京市手足口病重症和死亡病例资料,采用多因素Logistic回归分析对手足口病死亡的危险因素进行分析。结果 2010年北京市报告重症手足口病610例,其中死亡病例18例,重症病例病死率为2.95%。结果显示:患者年龄、户口类型为流动人口、诊断重症病例的医院类型为县医院、到村级(个体)医疗机构就诊和病原检测结果为肠道病毒71型(enterovirus 71,EV71)是重症手足口病死亡的影响因素。结论为减少手足口病死亡病例的发生,应重视1岁以下婴幼儿,流动人口和EV71感染儿童的防控工作,手足口病患者应到正规医院就诊,发现重症早期症状者应立即转诊到具有诊治重症病例能力的医疗机构进行科学规范救治。