重组柯萨奇A16病毒致病机理及候选疫苗对动物模型致死性保护机制
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摘要
由肠道病毒引起的手足口病(Hand, Foot and Mouth Disease, HFMD)是全球范围内爆发流行的传染病。多发生于5岁以下婴幼儿，多数临床症状较轻微，表现为发热和手足口疱疹，一般2周后自愈。少数患儿可引起迟缓性瘫痪、无菌性脑膜脑炎、肺水肿、心肌炎等并发症。个别重症患儿死亡。近年来，手足口病疫情在亚太地区不断暴发，在我国出现多次大范围爆发流行,对我国儿童的健康造成了严重威胁。2008年卫生部将手足口病列入传染病防治法规定的丙类传染病进行管理，卫生部官方数据显示2008年以来手足口病发病数一直位居丙类传染病之首。引发手足口病的肠道病毒多达20多种，包括肠道病毒71型，柯萨奇病毒A组的16、4、5、9、10型，B组的2、5型以及埃可病毒，其中以肠道病毒71型(enterovirus71, EV71)和柯萨奇病毒A16型(Coxsachievirus A16,CA16)最常见。目前手足口病尚无特异的治疗方法，也无特异的疫苗和抗病毒药物。因此，有关手足口病的临床特征及最常见的病原体EV71和CA16的病毒生物学特性、致病机理及疫苗开发等的研究日益受到人们的重视。
引起婴幼儿手足口病的人肠道病毒在东南亚国家和太平洋地区广泛流行传播，其中EV71和CA16是两个主要的病原体。CA16和EV71属于小核糖核酸(Ribonucleic Acid, RNA)病毒科(Picornaviridae)，肠道病毒属(Enterovirus)，是人类多种疾病的致病病原体,可引起结膜炎、发热性皮疹、HFMD、疱疹性咽峡炎、心肌炎、心肌病及无菌性脑膜炎、脑炎、肺炎和急性弛缓性麻痹等疾病，严重影响婴幼儿的身体健康。EV71与CA16感染交替或同时出现，成为引起手足口病流行的主要病原体。
CA16和EV71病毒基因组是由大约7410个核苷酸组成的单股正链RNA，两端为5′端和3′端非编码区(Untranslated Region, UTR)。基因组中间是单一的开放读码框架(Open Reading Frame, ORF)，编码一个较大的多聚蛋白质前体，最终形成11个终末产物。CA16和EV71以非帽依赖形式(cap-independent)由内部核糖体进入位点(internalr ibosome entry site, IRES)直接将核糖体小亚基结合到病毒RNA的5′端而开始翻译过程。在病毒感染周期中，病毒与宿主细胞受体的结合起着重要的作用。目前认为与柯萨奇A16病毒结合的细胞受体主要有：脊髓灰质炎病毒受体(CD155)，三个整合蛋白(α2β3, αvβ3和αvβ6)，衰减加速因子(CD55)，柯萨奇-腺病毒受体(coxsackievirus adenovirus receptor, CAR)和细胞内粘附分子。同时，细胞受体及其体内其他辅助因子也CA16和EV71病毒组织趋向性和致病机理的主要决定因素。
因此本课题是从手足口病患者咽拭子中分离出CA16和EV71病毒，对其分子生物学特性、生物学特性、细胞适应性、致病机理、体外中和抗体及动物模型的保护性等方面进行了系统分析研究。通过基因组全长测序和与其他已知病毒基因进行对比分析，鉴定为多种病毒形式的重组体，并发现在基因遗传形式上与CA16病毒的原型株G10截然不同，特别是病毒的5′端和P2、P3区基因，当前的流行的CA16病毒是重组形式的A型人肠道病毒,包括CA4、CA16和可能的EV71、CA2、CA3、CA6、CA10和CA12重组体。
体外中和试验，免疫CA16和EV71病毒的小鼠血清体外中和试验，发现CA16鼠血清能中和多株异源性CA16病毒，且有较高的中和抗体效价，但与EV71病毒进行交叉中和试验时，发现两者之间无交叉中和作用。通过乳鼠体内保护性试验，进一步验证了CA16和EV71之间无相互保护作用。
在东南亚地区，流行传播的重组形式的CA16病毒是手足口疾病的主要病原体。目前，还没有有效的针对CA16病毒的疫苗。对重组CA16病毒敏感的致病性动物模型是疫苗发展和质量评价的关键因素。在这项研究中，我们从手足口病患者咽拭子中分离并鉴定为重组形式的CA16病毒。这些在人群中流行的CA16病毒对新发展的乳鼠动物模型有较高的致病性。同时，我们也观察到并分析了具有代表性的重组CA16病毒的发病机理。
在该动物模型试验中，我们研究发现临床分离株重组CA16病毒对新生乳鼠的致病性强于原型株G10病毒和EV71病毒。虽然这些病毒对乳鼠组织器官的特异趋向性存在相同之处，即都对肌肉组织有较强的亲嗜性，表明肌肉组织是引起手足口病毒的主要复制场所，除此之外，对其他组织的趋向性并不相同。分别通过小鼠组织器官的病理分析、免疫组织化学以及实时荧光定量PCR检测不同时间点的病毒增殖情况，证明了重组CA16对新生乳鼠的致病机理，第一次发现了CA16病毒对鼠类动物的肺部易感性。
该研究中，制备了一种CA16灭活疫苗，包含有氢氧化铝佐剂和微克级的病毒蛋白的试验性灭活疫苗，免疫母鼠，用多株致死性剂量病毒攻击其出生的新生乳鼠，发现疫苗能对乳鼠起到很好的保护性作用，我们又深入分析了被免疫母鼠和乳鼠的体液免疫，通过体外中和试验证明了母鼠和乳鼠血清抗体都能够中和多株异源CA16病毒。而且，在免疫组被致死性剂量攻击的乳鼠的组织病毒滴度和载量明显小于对照组，并且多数组织器官中未能检测到病毒。这种应用致病性的重组CA16病毒建立的致死性动物模型和保护该动物模型的候选疫苗株对未来疫苗的发展和CA16疫苗的评价提供了有利的条件。体外中和抗体试验证明了候选病毒疫苗株具有较强的广谱交叉保护性，小鼠体内攻毒保护性试验证明了免疫了候选株疫苗的乳鼠能很好的保护其他异源CA16病毒的感染。
为能更好的掌握手足口疾病的预防措施。我们对CA16和EV71病毒进行交叉中和抗体反应及体内动物模型保护性试验，证明两种病毒间无交叉中和及动物保护性。将灭活CA16和EV71制备成双价联合疫苗免疫母鼠，致死性攻毒感染所出生的乳鼠，我们观察到免疫联合疫苗组的体外中和相对单价苗有更高的抗体效价，体内保护性也同时证明了能够较好的保护EV71或CA16病毒致死性攻毒。为未来CA16和EV71联合疫苗的发展奠定了良好的理论基础。
Hand, foot and mouth disease (HFMD) is a global infectious disease caused bymultiple enterovirus and commonly affects children who are younger than5years old.Most patients have mild symptoms and recover spontaneously within2weeks, but asmall proportion of them may suffer from severe complications such as asepticmeningitis, encephalitis, acute flaccid paralysis, pulmonary edema, myocarditis andeven death. In recent years, the prevalence of HFMD in the Asia-Pacific region hasincreased greatly and there were several epidemics in mainland China in the past fewyears. It is a serious threat to health of infants and young children. As HFMD is lifethreatening for children, the disease was listed as one of the category “C” notifiablediseases in China since2nd May2008. The epidemic may further expand, and how toprevent the disease is still very important in future. At present, there is no specifictreatment, specific vaccines and antiviral drugs. Therefore, the clinical characteristicsof HFMD and the research of biology, pathogenesis and prevention of enterovirus areextremely necessary.
The enterovirus which cause hand, foot and mouth disease spread popular insoutheast Asia and the Pacific. EV71and CA16are the two main pathogens. EV71and CA16belong to Ribonucleic Acid (RNA), Picornaviridae and Enterovirus, whichcan cause a variety of human disease, including conjunctivitis, febrile, rash, HFMDand herpangina, myocarditis, cardiomyopathy and aseptic meningitis, encephalitis,diseases such as pneumonia and acute flaccid paralysis, seriously affects infants andchildren's health. EV71and CA16infection or alternately appear at the same time,become the main pathogens causing, foot and mouth disease epidemic.
The RNA genome of EV71and CA16consists of7400nucleotides and iscomposed of a5′-untranslated region (UTR) and a3′-UTR, and an open reading frame (ORF), encoding a polyprotein precurosor that is proteolytically cleaved to11endproducts. The translation of CA RNAs generally proceeds by a cap-independentmechanism, in which ribosomes bind directly to an internal ribosome entry site (IRES)in the5′-UTR of the RNA. It might play an important role in viruses binding cellularreceptor in the infectious cycle. Various cellular receptors were demonstratedincluding coxsackievirus-adenovirus receptor (CAR), the complement system (DAF),Polio virus receptors (CD155), α2β3, αvβ3and αvβ6and decay accelerating factorand so on. Meanwhile, the interactions between cellular receptor and some accessoryfactors are thought to be crucial factors in tissue tropism.
In the study， we have investigated the molecular biology, the biologicalcharacteristic, adaptation to cell culture, neutralization of antibody in vitro andanimal model of CA16which was isolated from FHMD patients. The CA16strainswere identified recombinant form by genome sequencing and compared with otherknown virus gene. Moreover, circulating CA16viruses from HFMD patients aregenetically distinct from the prototype CA16(G10), especially in5′UTR, P2and P3region. The currently circulating CA16viruses are actually complex recombinantviruses involving multiple types A HEV, including CA4, CA16, and possiblyEV71/CA2/CA3/CA6/CA10/CA12. The enterovirus can form different genotypesbecause it mutates extremely easily.
In neutralization test in vitro, we found the antibody of CA16can neutralizemultiple strains of heterologous CA16virus, even including G10virus, and theneutralizing antibody titer is relatively high. However, the antibody of CA16can notneutralize EV71and the antibody of EV71can not neutralize CA16, these resultswere demonstrated by lethal challenge neonatal mouse model.
Circulating recombinant forms of CA16(CA16) is a major cause of HFMD inSoutheast Asia. At present, there is no vaccine against CA16viruses. Pathogenicanimal models that are sensitive to diverse circulating CA16viruses would bedesirable for vaccine development and evaluation. In this study, we isolated andcharacterized several circulating CA16viruses from recent HFMD patients. TheseCA16viruses currently circulating in humans were highly pathogenic in a newly developed neonatal mouse model; we also observed and analyzed the pathogenesis ofrepresentative circulating recombinant form (CRF) CA16viruses.
In this animal model, the pathogenicity of the circulating recombinant formCA16viruses to neonatal mice was stronger than that of the prototype CA16(G10)and EV71virus. Although all the viruses were susceptible to the same organ andtissue including muscle and so on, the mice muscle was the main room where theCA16and EV71viruses replicated. These results were demonstrated throughpathological analysis, immunohistochemistry and viral load. We found in the firsttims that the recombinant form CA16viruses were very susceptible to lung inneonatal mouse model.
An inactivated CA16vaccine candidate, formulated with alum adjuvant andcontaining sub-microgram quantities of viral proteins, well protected neonatal miceborn to immunized female mice from lethal-dose challenge with a series of CA16viruses. Further analysis of humoral immunity showed that antibody elicited fromboth the immunized dams and their pups can neutralize various lethal viruses bycytopathic effect (CPE) in vitro. Moreover, viral titers and loads in the tissues ofchallenged pups in the vaccine group were far lower than those in the control group,and some were even undetectable. This lethal challenge model using pathogenicCA16viruses and the vaccine candidates that mediated protection in this model couldbe useful tools for the future development and evaluation of CA16vaccine.
引起婴幼儿手足口病的人肠道病毒在东南亚国家和太平洋地区广泛流行传播，其中EV71和CA16是两个主要的病原体。CA16和EV71属于小核糖核酸(Ribonucleic Acid, RNA)病毒科(Picornaviridae)，肠道病毒属(Enterovirus)，是人类多种疾病的致病病原体,可引起结膜炎、发热性皮疹、HFMD、疱疹性咽峡炎、心肌炎、心肌病及无菌性脑膜炎、脑炎、肺炎和急性弛缓性麻痹等疾病，严重影响婴幼儿的身体健康。EV71与CA16感染交替或同时出现，成为引起手足口病流行的主要病原体。
CA16和EV71病毒基因组是由大约7410个核苷酸组成的单股正链RNA，两端为5′端和3′端非编码区(Untranslated Region, UTR)。基因组中间是单一的开放读码框架(Open Reading Frame, ORF)，编码一个较大的多聚蛋白质前体，最终形成11个终末产物。CA16和EV71以非帽依赖形式(cap-independent)由内部核糖体进入位点(internalr ibosome entry site, IRES)直接将核糖体小亚基结合到病毒RNA的5′端而开始翻译过程。在病毒感染周期中，病毒与宿主细胞受体的结合起着重要的作用。目前认为与柯萨奇A16病毒结合的细胞受体主要有：脊髓灰质炎病毒受体(CD155)，三个整合蛋白(α2β3, αvβ3和αvβ6)，衰减加速因子(CD55)，柯萨奇-腺病毒受体(coxsackievirus adenovirus receptor, CAR)和细胞内粘附分子。同时，细胞受体及其体内其他辅助因子也CA16和EV71病毒组织趋向性和致病机理的主要决定因素。
因此本课题是从手足口病患者咽拭子中分离出CA16和EV71病毒，对其分子生物学特性、生物学特性、细胞适应性、致病机理、体外中和抗体及动物模型的保护性等方面进行了系统分析研究。通过基因组全长测序和与其他已知病毒基因进行对比分析，鉴定为多种病毒形式的重组体，并发现在基因遗传形式上与CA16病毒的原型株G10截然不同，特别是病毒的5′端和P2、P3区基因，当前的流行的CA16病毒是重组形式的A型人肠道病毒,包括CA4、CA16和可能的EV71、CA2、CA3、CA6、CA10和CA12重组体。
体外中和试验，免疫CA16和EV71病毒的小鼠血清体外中和试验，发现CA16鼠血清能中和多株异源性CA16病毒，且有较高的中和抗体效价，但与EV71病毒进行交叉中和试验时，发现两者之间无交叉中和作用。通过乳鼠体内保护性试验，进一步验证了CA16和EV71之间无相互保护作用。
在东南亚地区，流行传播的重组形式的CA16病毒是手足口疾病的主要病原体。目前，还没有有效的针对CA16病毒的疫苗。对重组CA16病毒敏感的致病性动物模型是疫苗发展和质量评价的关键因素。在这项研究中，我们从手足口病患者咽拭子中分离并鉴定为重组形式的CA16病毒。这些在人群中流行的CA16病毒对新发展的乳鼠动物模型有较高的致病性。同时，我们也观察到并分析了具有代表性的重组CA16病毒的发病机理。
在该动物模型试验中，我们研究发现临床分离株重组CA16病毒对新生乳鼠的致病性强于原型株G10病毒和EV71病毒。虽然这些病毒对乳鼠组织器官的特异趋向性存在相同之处，即都对肌肉组织有较强的亲嗜性，表明肌肉组织是引起手足口病毒的主要复制场所，除此之外，对其他组织的趋向性并不相同。分别通过小鼠组织器官的病理分析、免疫组织化学以及实时荧光定量PCR检测不同时间点的病毒增殖情况，证明了重组CA16对新生乳鼠的致病机理，第一次发现了CA16病毒对鼠类动物的肺部易感性。
该研究中，制备了一种CA16灭活疫苗，包含有氢氧化铝佐剂和微克级的病毒蛋白的试验性灭活疫苗，免疫母鼠，用多株致死性剂量病毒攻击其出生的新生乳鼠，发现疫苗能对乳鼠起到很好的保护性作用，我们又深入分析了被免疫母鼠和乳鼠的体液免疫，通过体外中和试验证明了母鼠和乳鼠血清抗体都能够中和多株异源CA16病毒。而且，在免疫组被致死性剂量攻击的乳鼠的组织病毒滴度和载量明显小于对照组，并且多数组织器官中未能检测到病毒。这种应用致病性的重组CA16病毒建立的致死性动物模型和保护该动物模型的候选疫苗株对未来疫苗的发展和CA16疫苗的评价提供了有利的条件。体外中和抗体试验证明了候选病毒疫苗株具有较强的广谱交叉保护性，小鼠体内攻毒保护性试验证明了免疫了候选株疫苗的乳鼠能很好的保护其他异源CA16病毒的感染。
为能更好的掌握手足口疾病的预防措施。我们对CA16和EV71病毒进行交叉中和抗体反应及体内动物模型保护性试验，证明两种病毒间无交叉中和及动物保护性。将灭活CA16和EV71制备成双价联合疫苗免疫母鼠，致死性攻毒感染所出生的乳鼠，我们观察到免疫联合疫苗组的体外中和相对单价苗有更高的抗体效价，体内保护性也同时证明了能够较好的保护EV71或CA16病毒致死性攻毒。为未来CA16和EV71联合疫苗的发展奠定了良好的理论基础。
Hand, foot and mouth disease (HFMD) is a global infectious disease caused bymultiple enterovirus and commonly affects children who are younger than5years old.Most patients have mild symptoms and recover spontaneously within2weeks, but asmall proportion of them may suffer from severe complications such as asepticmeningitis, encephalitis, acute flaccid paralysis, pulmonary edema, myocarditis andeven death. In recent years, the prevalence of HFMD in the Asia-Pacific region hasincreased greatly and there were several epidemics in mainland China in the past fewyears. It is a serious threat to health of infants and young children. As HFMD is lifethreatening for children, the disease was listed as one of the category “C” notifiablediseases in China since2nd May2008. The epidemic may further expand, and how toprevent the disease is still very important in future. At present, there is no specifictreatment, specific vaccines and antiviral drugs. Therefore, the clinical characteristicsof HFMD and the research of biology, pathogenesis and prevention of enterovirus areextremely necessary.
The enterovirus which cause hand, foot and mouth disease spread popular insoutheast Asia and the Pacific. EV71and CA16are the two main pathogens. EV71and CA16belong to Ribonucleic Acid (RNA), Picornaviridae and Enterovirus, whichcan cause a variety of human disease, including conjunctivitis, febrile, rash, HFMDand herpangina, myocarditis, cardiomyopathy and aseptic meningitis, encephalitis,diseases such as pneumonia and acute flaccid paralysis, seriously affects infants andchildren's health. EV71and CA16infection or alternately appear at the same time,become the main pathogens causing, foot and mouth disease epidemic.
The RNA genome of EV71and CA16consists of7400nucleotides and iscomposed of a5′-untranslated region (UTR) and a3′-UTR, and an open reading frame (ORF), encoding a polyprotein precurosor that is proteolytically cleaved to11endproducts. The translation of CA RNAs generally proceeds by a cap-independentmechanism, in which ribosomes bind directly to an internal ribosome entry site (IRES)in the5′-UTR of the RNA. It might play an important role in viruses binding cellularreceptor in the infectious cycle. Various cellular receptors were demonstratedincluding coxsackievirus-adenovirus receptor (CAR), the complement system (DAF),Polio virus receptors (CD155), α2β3, αvβ3and αvβ6and decay accelerating factorand so on. Meanwhile, the interactions between cellular receptor and some accessoryfactors are thought to be crucial factors in tissue tropism.
In the study， we have investigated the molecular biology, the biologicalcharacteristic, adaptation to cell culture, neutralization of antibody in vitro andanimal model of CA16which was isolated from FHMD patients. The CA16strainswere identified recombinant form by genome sequencing and compared with otherknown virus gene. Moreover, circulating CA16viruses from HFMD patients aregenetically distinct from the prototype CA16(G10), especially in5′UTR, P2and P3region. The currently circulating CA16viruses are actually complex recombinantviruses involving multiple types A HEV, including CA4, CA16, and possiblyEV71/CA2/CA3/CA6/CA10/CA12. The enterovirus can form different genotypesbecause it mutates extremely easily.
In neutralization test in vitro, we found the antibody of CA16can neutralizemultiple strains of heterologous CA16virus, even including G10virus, and theneutralizing antibody titer is relatively high. However, the antibody of CA16can notneutralize EV71and the antibody of EV71can not neutralize CA16, these resultswere demonstrated by lethal challenge neonatal mouse model.
Circulating recombinant forms of CA16(CA16) is a major cause of HFMD inSoutheast Asia. At present, there is no vaccine against CA16viruses. Pathogenicanimal models that are sensitive to diverse circulating CA16viruses would bedesirable for vaccine development and evaluation. In this study, we isolated andcharacterized several circulating CA16viruses from recent HFMD patients. TheseCA16viruses currently circulating in humans were highly pathogenic in a newly developed neonatal mouse model; we also observed and analyzed the pathogenesis ofrepresentative circulating recombinant form (CRF) CA16viruses.
In this animal model, the pathogenicity of the circulating recombinant formCA16viruses to neonatal mice was stronger than that of the prototype CA16(G10)and EV71virus. Although all the viruses were susceptible to the same organ andtissue including muscle and so on, the mice muscle was the main room where theCA16and EV71viruses replicated. These results were demonstrated throughpathological analysis, immunohistochemistry and viral load. We found in the firsttims that the recombinant form CA16viruses were very susceptible to lung inneonatal mouse model.
An inactivated CA16vaccine candidate, formulated with alum adjuvant andcontaining sub-microgram quantities of viral proteins, well protected neonatal miceborn to immunized female mice from lethal-dose challenge with a series of CA16viruses. Further analysis of humoral immunity showed that antibody elicited fromboth the immunized dams and their pups can neutralize various lethal viruses bycytopathic effect (CPE) in vitro. Moreover, viral titers and loads in the tissues ofchallenged pups in the vaccine group were far lower than those in the control group,and some were even undetectable. This lethal challenge model using pathogenicCA16viruses and the vaccine candidates that mediated protection in this model couldbe useful tools for the future development and evaluation of CA16vaccine.
引文
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