手足口病（EV71和CVA16）病毒样颗粒疫苗免疫原性及保护性研究
发布时间：2018-08-11 18:49
【摘要】：手足口病(Hand,Foot and Mouth Disease,HFMD)是一种全球范围内的传染性疾病,流行区域主要集中在发展中国家(特别是亚太地区)。中国流行病学调查结果显示:手足口病位居丙类传染病之首,其主要致病病原体是Enterovirus 71(EV71)型和Coxsackievirus A16(CVA16)型,两种病毒的流行率在80%以上。目前临床上还没有有效的药物治疗手足口病,疫苗的研发与接种是控制手足口病流行的重要手段。根据中国大陆临床试验结果证实,EV71灭活疫苗具有良好的保护效果,可以预防EV71所引起手足口病的保护率在90%以上,预防EV71相关疾病的保护率在80%以上,其诱导的中和抗体具有良好的广谱交叉活性,但其血清不能中和CVA16以及其它引起手足口病的病毒,所以包含EV71和CVA16的联合疫苗的研制显得尤其迫切。目前CVA16灭活疫苗仍处于临床前研究阶段,限制了二者联合疫苗在临床上的应用。EV71和CVA16分别只有一个血清型,但每种病毒含有多个基因亚型,而各基因亚型之间的中和抗体交叉活性有所不同。因此筛选出中和抗体覆盖率高、免疫原性好的疫苗株,制备成联合疫苗预防由EV71和CVA16引起的手足口病是疫苗研发的首选。目前研究只检测了单价疫苗的广谱性中和抗体交叉活性,而关于联合疫苗的中和抗体交叉活性是否有差别尚未有任何报道,联合疫苗同时接种时两种疫苗之间是否产生免疫抑制和免疫干扰,联合疫苗接种时是否可以预防两种病毒的入侵等方面亦没有详细、全面的研究。HFMD疫苗的研究形式以灭活疫苗和病毒样颗粒(VLPs)疫苗为主,随着基因工程技术的不断发展VLPs疫苗以其独有的天然优势而被广泛应用。本论文针对以上问题进行更为深入、具体、全面的研究,提供真实、可靠的动物试验数据,期望解决目前联合疫苗存在的问题,开发出有效、安全、交叉活性好的EV71 VLPs和CVA16 VLPs联合疫苗。另一方面,疫苗的抗原剂量效应关系(ED_(50))直接反映出疫苗的效力及最低免疫剂量,对疫苗的临床前评价和疫苗的临床免疫剂量具有指导意义。EV71灭活疫苗在临床前评价了抗原的ED_(50),但对于EV71 VLPs疫苗的ED_(50)还没有详细、可靠的研究数据。本论文就这一问题也进行了研究:应用层析纯化之后的EV71 VLPs作为免疫原,选择不同免疫剂量接种小鼠,不仅对EV71 VLPs抗原的ED_(50)进行了测定,而且测定了其抗体的ED_(50)。抗原和抗体ED_(50)的测定对EV71 VLPs疫苗的后续研究具有重要的指导意义。第一部分:单价疫苗和联合疫苗免疫原性及保护性研究一、本课题根据中国大陆流行病学调查,选用EV71流行株C4a(EU703814.1)及CVA16流行株B1a(AF177911.1),应用昆虫细胞杆状病毒表达系统生产EV71病毒样颗粒(VLPs)、CVA16病毒样颗粒(VLPs),阳性对照疫苗为EV71灭活疫苗和CVA16灭活疫苗,分别制备出单价疫苗和联合疫苗。二、依托本实验室的假病毒检测平台,评价了单价疫苗和联合疫苗的免疫原性,结果显示不论是单价疫苗还是联合疫苗都可以诱导出很强的中和抗体滴度;VLPs单价疫苗与灭活单价疫苗相比中和抗体滴度无显著性差异;联合疫苗同时接种时与单价疫苗相比中和抗体滴度亦没有显著性差异;联合免疫后两种疫苗之间不存在免疫抑制和免疫干扰;在此首次测定了联合疫苗的广谱性中和抗体。三、保护力试验是评价疫苗有效性的一个重要指标。本研究选用1日龄ICR乳鼠,分别建立出EV71乳鼠动物模型和CVA16乳鼠动物模型,并评价了单价疫苗和联合疫苗的保护力水平。结果显示无论是单价疫苗还是联合疫苗对新生乳鼠都可以提供很好的保护力水平,联合疫苗可以预防两种病毒的侵入。第二部分:EV71 VLPs抗原和抗体ED_(50)的研究本部分研究与中国食品药品检定研究院(NIFDC)合作,评价层析纯化之后的EV71 VLPs抗原和抗体的ED_(50)。EV71 VLPs不同剂量分别与铝佐剂吸附后免疫接种小鼠,中和抗体呈现出明显的剂量效应关系。通过乳鼠保护力实验结果显示根据不同免疫剂量新生乳鼠脑内攻毒后的存活率,计算出EV71 VLPs抗原ED_(50)为0.20μg/dose,根据攻毒时间点不同免疫剂量组的中和抗体滴度,计算出在此剂量下诱导出的中和抗体滴度大约为50。应用两种乳鼠保护力评定方式分别初步确定了抗体的ED_(50)大约为54和24。综上所述,本研究利用昆虫细胞杆状病毒系统表达出EV71 VLPs、CVA16VLPs,并应用假病毒中和抗体检测体系评价单价疫苗和联合疫苗的中和抗体滴度趋势;首次应用了所有基因亚型的假病毒评价了单价疫苗和联合疫苗的广谱性中和抗体滴度,证明联合疫苗中和抗体滴度与单价疫苗相比在相同基因亚型的假病毒中无显著性差异;并且联合疫苗同时接种不存在免疫抑制及免疫干扰。应用乳鼠动物模型体内评价了单价疫苗及联合疫苗的有效性,两种保护力评价方式结果证明联合疫苗可以同时预防两种病毒的攻击。广谱性中和抗体测定及乳鼠保护力的评价为联合疫苗的后续研究和临床应用提供了真实、可靠的数据。另外,本研究还首次研究了层析纯化后的EV71 VLPs抗原和抗体的ED_(50),为EV71 VLPs疫苗的进一步研究提供了试验基础。
[Abstract]:Hand, Foot and Mouth Disease (HFMD) is a worldwide infectious disease, and the epidemic areas are mainly concentrated in developing countries (especially in the Asia-Pacific region). The results of epidemiological survey in China show that HFMD is the most common type of infectious diseases, and the main pathogens are Enterovirus 71 (EV71) and Coxsackievirus. At present, there is no effective drug to treat HFMD. The development and vaccination of vaccine is an important means to control the epidemic of HFMD. According to the results of clinical trials in mainland China, the inactivated vaccine of EV71 has a good protective effect and can prevent the HFMD caused by EV71. The protective rate against EV71-related diseases is above 90%, and the protective rate against EV71-related diseases is above 80%. The induced neutralizing antibodies have good broad-spectrum cross-reactivity, but their serum can not neutralize CVA 16 and other viruses causing HFMD. Therefore, the development of a combined vaccine containing EV71 and CVA 16 is particularly urgent. EV71 and CV16 have only one serotype, but each virus contains multiple genotypes, and the neutralizing antibody cross-reactivity among the genotypes is different. Therefore, the vaccine strains with high coverage and good immunogenicity were screened and prepared to be linked. Preventing hand-foot-mouth disease (HFMD) caused by EV71 and CV16 with a combined vaccine is the first choice for vaccine development. Current studies have only examined the broad-spectrum neutralizing antibody cross-activity of monovalent vaccines, but there is no report on the difference in neutralizing antibody cross-activity between the combined vaccines. Whether there is immunity between the two vaccines when the combined vaccines are vaccinated simultaneously has not been reported. There is no detailed and comprehensive study on inhibition and immune interference, and whether combined vaccination can prevent the invasion of two viruses. HFMD vaccines mainly consist of inactivated vaccines and virus-like particles (VLPs) vaccines. With the development of genetic engineering technology, VLPs vaccines have been widely used for their unique natural advantages. Aiming at the above problems, this paper carries out a more in-depth, specific and comprehensive study to provide real and reliable animal test data, expecting to solve the existing problems of the combined vaccine and develop an effective, safe and cross-active EV71 VLPs and CVA16 VLPs combined vaccine. On the other hand, the antigen dose-response relationship (ED_ (50)) of the vaccine directly reflects. EV71 inactivated vaccine has been evaluated for ED_ (50) of antigen before clinic, but there is no detailed and reliable data for ED_ (50) of EV71 VLPs vaccine. This paper also studies this problem: application chromatography purity. The ED_ (50) of EV71 VLPs antigen and the ED_ (50) of its antibody were determined. The determination of antigen and antibody ED_ (50) is of great significance for the follow-up study of EV71 VLPs vaccine. Part I: Monovalent vaccine and combined vaccine immunization. First, according to the epidemiological investigation in mainland China, we selected EV71 strain C4a (EU703814.1) and CVA16 strain B1a (AF177911.1) to produce EV71 virus-like particles (VLPs), CVA16 virus-like particles (VLPs) by using insect cell baculovirus expression system. The positive control vaccine was EV71 inactivated vaccine and CVA16 inactivated vaccine. The results showed that both monovalent and combined vaccines could induce strong neutralizing antibody titers, and VLPs monovalent vaccines could neutralize antibody titers compared with inactivated monovalent vaccines. There was no significant difference in neutralizing antibody titer between the combined vaccine and the monovalent vaccine; there was no immunosuppression and immune interference between the two vaccines after combined immunization; the broad-spectrum neutralizing antibody of the combined vaccine was determined for the first time. Indicators. In this study, one-day-old ICR suckling mice were selected to establish EV71 suckling mice model and CVA16 suckling mice model, and the protective level of monovalent vaccine and combined vaccine was evaluated. The results showed that both monovalent vaccine and combined vaccine could provide a good protective level for neonatal suckling mice, and combined vaccine could prevent two kinds of vaccine. Invasion of the virus. Part II: Study on the antigen and antibody ED_ (50) of EV71 VLPs. This part, in cooperation with the China Institute of Food and Drug Control (NIFDC), evaluates the ED_ (50) of the purified EV71 VLPs antigen and antibody. Different doses of EV71 VLPs were immunized with aluminum adjuvant respectively, and the neutralizing antibody showed a significant dose. According to the protective effect of suckling mice, according to the survival rate of neonatal mice after intracerebral injection of different immune doses, the ED_ (50) of EV71 VLPs antigen was calculated to be 0.20 ug/dose. According to the titers of neutralizing antibodies at different immune doses at different time points, the titers of neutralizing antibodies induced at this dose were calculated to be about 50. In summary, the expression of EV71 VLPs and CVA116 VLPs by baculovirus system in insect cells and the neutralizing antibody titer trend of monovalent vaccine and combined vaccine were evaluated by using pseudovirus neutralizing antibody detection system. The broad-spectrum neutralizing antibody titers of the monovalent vaccine and the combined vaccine were evaluated by the pseudovirus genotype, which showed that there was no significant difference between the neutralizing antibody titers of the combined vaccine and the monovalent vaccine in the same genotype of pseudovirus, and there was no immunosuppression and immune interference in the combined vaccine. The validity of monovalent vaccine and combined vaccine was evaluated internally. The results showed that the combined vaccine could prevent the attack of both viruses simultaneously. The determination of broad-spectrum neutralizing antibody and the evaluation of suckling mice'protective power provided real and reliable data for the follow-up study and clinical application of the combined vaccine. The ED_ (50) of the purified antigen and antibody of EV71 VLPs was studied, which provided experimental basis for further study of EV71 VLPs vaccine.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R392
本文编号：2177899
[Abstract]:Hand, Foot and Mouth Disease (HFMD) is a worldwide infectious disease, and the epidemic areas are mainly concentrated in developing countries (especially in the Asia-Pacific region). The results of epidemiological survey in China show that HFMD is the most common type of infectious diseases, and the main pathogens are Enterovirus 71 (EV71) and Coxsackievirus. At present, there is no effective drug to treat HFMD. The development and vaccination of vaccine is an important means to control the epidemic of HFMD. According to the results of clinical trials in mainland China, the inactivated vaccine of EV71 has a good protective effect and can prevent the HFMD caused by EV71. The protective rate against EV71-related diseases is above 90%, and the protective rate against EV71-related diseases is above 80%. The induced neutralizing antibodies have good broad-spectrum cross-reactivity, but their serum can not neutralize CVA 16 and other viruses causing HFMD. Therefore, the development of a combined vaccine containing EV71 and CVA 16 is particularly urgent. EV71 and CV16 have only one serotype, but each virus contains multiple genotypes, and the neutralizing antibody cross-reactivity among the genotypes is different. Therefore, the vaccine strains with high coverage and good immunogenicity were screened and prepared to be linked. Preventing hand-foot-mouth disease (HFMD) caused by EV71 and CV16 with a combined vaccine is the first choice for vaccine development. Current studies have only examined the broad-spectrum neutralizing antibody cross-activity of monovalent vaccines, but there is no report on the difference in neutralizing antibody cross-activity between the combined vaccines. Whether there is immunity between the two vaccines when the combined vaccines are vaccinated simultaneously has not been reported. There is no detailed and comprehensive study on inhibition and immune interference, and whether combined vaccination can prevent the invasion of two viruses. HFMD vaccines mainly consist of inactivated vaccines and virus-like particles (VLPs) vaccines. With the development of genetic engineering technology, VLPs vaccines have been widely used for their unique natural advantages. Aiming at the above problems, this paper carries out a more in-depth, specific and comprehensive study to provide real and reliable animal test data, expecting to solve the existing problems of the combined vaccine and develop an effective, safe and cross-active EV71 VLPs and CVA16 VLPs combined vaccine. On the other hand, the antigen dose-response relationship (ED_ (50)) of the vaccine directly reflects. EV71 inactivated vaccine has been evaluated for ED_ (50) of antigen before clinic, but there is no detailed and reliable data for ED_ (50) of EV71 VLPs vaccine. This paper also studies this problem: application chromatography purity. The ED_ (50) of EV71 VLPs antigen and the ED_ (50) of its antibody were determined. The determination of antigen and antibody ED_ (50) is of great significance for the follow-up study of EV71 VLPs vaccine. Part I: Monovalent vaccine and combined vaccine immunization. First, according to the epidemiological investigation in mainland China, we selected EV71 strain C4a (EU703814.1) and CVA16 strain B1a (AF177911.1) to produce EV71 virus-like particles (VLPs), CVA16 virus-like particles (VLPs) by using insect cell baculovirus expression system. The positive control vaccine was EV71 inactivated vaccine and CVA16 inactivated vaccine. The results showed that both monovalent and combined vaccines could induce strong neutralizing antibody titers, and VLPs monovalent vaccines could neutralize antibody titers compared with inactivated monovalent vaccines. There was no significant difference in neutralizing antibody titer between the combined vaccine and the monovalent vaccine; there was no immunosuppression and immune interference between the two vaccines after combined immunization; the broad-spectrum neutralizing antibody of the combined vaccine was determined for the first time. Indicators. In this study, one-day-old ICR suckling mice were selected to establish EV71 suckling mice model and CVA16 suckling mice model, and the protective level of monovalent vaccine and combined vaccine was evaluated. The results showed that both monovalent vaccine and combined vaccine could provide a good protective level for neonatal suckling mice, and combined vaccine could prevent two kinds of vaccine. Invasion of the virus. Part II: Study on the antigen and antibody ED_ (50) of EV71 VLPs. This part, in cooperation with the China Institute of Food and Drug Control (NIFDC), evaluates the ED_ (50) of the purified EV71 VLPs antigen and antibody. Different doses of EV71 VLPs were immunized with aluminum adjuvant respectively, and the neutralizing antibody showed a significant dose. According to the protective effect of suckling mice, according to the survival rate of neonatal mice after intracerebral injection of different immune doses, the ED_ (50) of EV71 VLPs antigen was calculated to be 0.20 ug/dose. According to the titers of neutralizing antibodies at different immune doses at different time points, the titers of neutralizing antibodies induced at this dose were calculated to be about 50. In summary, the expression of EV71 VLPs and CVA116 VLPs by baculovirus system in insect cells and the neutralizing antibody titer trend of monovalent vaccine and combined vaccine were evaluated by using pseudovirus neutralizing antibody detection system. The broad-spectrum neutralizing antibody titers of the monovalent vaccine and the combined vaccine were evaluated by the pseudovirus genotype, which showed that there was no significant difference between the neutralizing antibody titers of the combined vaccine and the monovalent vaccine in the same genotype of pseudovirus, and there was no immunosuppression and immune interference in the combined vaccine. The validity of monovalent vaccine and combined vaccine was evaluated internally. The results showed that the combined vaccine could prevent the attack of both viruses simultaneously. The determination of broad-spectrum neutralizing antibody and the evaluation of suckling mice'protective power provided real and reliable data for the follow-up study and clinical application of the combined vaccine. The ED_ (50) of the purified antigen and antibody of EV71 VLPs was studied, which provided experimental basis for further study of EV71 VLPs vaccine.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R392
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