肠道病毒71型灭活疫苗（人二倍体细胞）免疫后攻毒及未免疫攻毒恒河猴婴猴丘脑组织的比较蛋白质组学研究
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摘要
手足口病是由肠道病毒引起的一种急性传染病。近年来,国内报告的手足口病发病例数和死亡例数均大幅度上升。在导致手足口病的20多种病毒中,由肠道病毒71型作为主要病原引起的手足口病发病病例数在总病例数中的比例在不断上升。肠道病毒71型感染人体除了引起手足口病外,还可导致脑膜炎、脑炎、局部麻痹和急性迟缓性麻痹等神经系统疾病以及疱疹性咽冈峡炎等非神经系统疾病,严重者能导致患者中枢神经系统并发症或肺水肿等致死性的临床后果。目前关于肠道病毒71型病毒的研究主要集中在病原学、流行病学方面,其对人体的致病机理尚不是十分清楚。随着肠道病毒71型引起的手足口病发病态势不断扩大,而临床上尚无公认的特效治疗药物,因此研制一种安全有效的疫苗成为防治肠道病毒71型所致手足口病的最有效的预治措施。
本研究采用初步研制成功的肠道病毒71型灭活疫苗,利用恒河猴婴猴这一经典动物模型进行了该疫苗的保护性实验。用疫苗免疫动物后攻毒,疫苗免疫组恒河猴婴猴神经系统的各个部分均未检出超过可信限的病毒RNA阳性结果,而感染对照组动物各部位均检测到明显的病毒RNA阳性结果。灭活疫苗能够诱导免疫后的恒河猴婴猴产生中和抗体,免疫后病毒攻击后第7天恒河猴婴猴血清中和抗体水平达1：512,能够形成有效的抗病毒免疫保护效应。免疫组化结果显示,免疫动物所诱导产生的免疫保护效应能够抑制病毒在体内器官及组织中的增殖。结合攻毒后动物相应靶器官组织切片病理观察,血清中和抗体检测和免疫组化结果,分析表明：肠道病毒71型灭活疫苗免疫恒河猴婴猴后,继而以病毒攻击,将不会引起导致机体组织器官受损的病理学反应,疫苗具有保护性。
此外,取上述研究对象中,免疫后攻毒和未免疫攻毒的恒河猴婴猴中枢神经系统的丘脑组织进行了蛋白质组学研究。运用蛋白质组学相关技术：蛋白质双向电泳分离蛋白和专业软件Imagemaster 7.0进行图像分析,定量研究在病毒感染组及疫苗保护组中表达量有所差异的蛋白质点,通过基质辅助激光解析电离飞行时间质谱和蛋白信息数据库建立差异蛋白质谱。
本研究共发现48个表达量变化的蛋白,按功能分为7类：①、细胞骨架相关蛋白8个,其中CFL2表达量变化结合功能分析提示该蛋白是研究EV71感染所致肌张力变化、心肌原纤维退化、心肌细胞凋亡潜在的靶点。结合各组PFN1、PFN2表达量变化与Profilins功能分析,提示在感染过程中PFN1、PFN2与细胞膜完整性或细胞内吞作用相关,疫苗的保护性降低了感染对细胞形态完整性的影响。②、小热休克蛋白1个,CRYAB,结合该蛋白在感染高峰期表达上调而与其它组不同的表现及其在生理或者神经变性疾病的压力下能够抑制中间纤维的聚集的能力,提示这种小热休克蛋白与感染压力下的细胞骨架稳定性维持相关；③、生物大分子合成相关蛋白4个,其中,CyPA作为一种具有调控免疫相关的因子,可能通过某种未知的信号传导途径激活T细胞活化所需的细胞因子,而促进T细胞的增殖,从而显示感染高峰CyPA表达量与其它组的差异。NUTF2作为一种入核转运因子,其表达量变化提示其是感染状态下宿主实现关闭或下调相关因子以抵抗病毒感染的可能途径之一。④、代谢相关蛋白13个,这些蛋白含量变化提示感染状态下的神经细胞增殖周期的改变、神经元发育的阻滞、突触后神经元的保护性抑制状态改变等与EV71感染致病机理密切相关,对疫苗组这些蛋白表达量的分析进一步映证了疫苗的保护效应。⑤、能量代谢与生物氧化相关蛋白11个,提示病理状态下和疫苗保护效应下机体能量代谢系统和生物氧化途径发生了改变。⑥、泛素-蛋白酶体途径相关蛋白2个,UCHL1和UBE2N在病毒感染组和疫苗保护组中的表达量差异及功能分析也支持了灭活疫苗所诱导的免疫保护的有效性。⑦、信号传导相关蛋白9个,PARK7表达变化提示中枢神经系统对感染的具有保护性,且可能通过PARK7实现。蛋白质组学实验中共结合差异谱中表达量发生改变的蛋白的功能与免疫后攻毒和未免疫攻毒恒河猴婴猴丘脑蛋白表达量变化分析,进一步验证了灭活疫苗具有保护效应,发现了一些在感染中有重要意义的分子靶标,并为探讨肠道病毒71型感染在疫苗免疫和非疫苗免疫机体中的宿主反应的分子生物学特征,以及从这一水平上认识肠道病毒71型感染的致病机理和疫苗的免疫保护机理而提供资料。
由于本研究对象为猕猴属的恒河猴,现有蛋白质数据库中关于猕猴属蛋白的数据还不全面,结合直系同源序列聚类分析(COG)以及蛋白数据库中现有灵长目其它物种的蛋白信息,初步证实了13个尚未在氨基酸或蛋白水平报道的灵长目某已知蛋白在恒河猴中的同源蛋白。
Hand-foot-mouth disease (HFMD) is an acute infectious disease which caused by enterovirus. In recent years, the morbidity and mortality of HFMD are observably increased. There are more than 20 variety virus that cause HFMD and enterovirus 71 (EV71) is the major one. EV71 infection can cause diseases of the nervous system, such as meningitis, encephalitis, monoplegia, acute flaccid paralysis and so on. It also associated with non-nervous system such as HFMD, herpetic angina and pulmonary oedema. In severe patients, it can cause central nervous system complication and pulmonary oedema which are fatal diseases. At present, the research about EV71 mainly focuses on the etiology and epidemiology of the virus. The pathogenesis of the virus has not yet understood. Due to continuous expanding of HFMD caused by EV71 and absence of effective drugs, there is a compelling need to develop a safe and effective vaccine. An elementary inactivated vaccine which prepared from the diploid cell cultured virus is used in this study. In this research, the protective effects of this vaccine were tested in a classic animal model—rhesus monkey neonate.Animal immunized or non-immunized with inactived vaccine were inoculated with EV71 and examined. In vaccine immunized rhesus monkey neonate, viral RNA was detected from infected control each parts of animals the neural system tissue, but not form vaccinated one. The inactivated vaccine can induce the monkeys produce neutralizing antibody which could reach 1:512 in the serum at the 7th day after viral attack.It can form effective antivirus immune barrier. Immunohistochemistry researches show that the protection effects which inoculate with inactivated vaccine could induce virus multiplication.It can be sure that inoculate with this inactivated vaccine, there were no immunopathogenesis damage in animals.Further testing by pathological examination, comprehensive biopsy pathological observation and immunohistochemistry analysis were carried out and all results indicated that inactivated EV71 vaccine protected rhesus monkey from virus attack.
Protein differential expression profiling of infected, vaccinated and control monkey brain tissue was analyzed by two-dimensional-electrophoresis and matrix assisted laser-desorption ionization time of flight mass spectrometry.48 proteins were found expressed differently in these group, including 8 cytoskeleton related proteins (6 microfilament related and 2 microtube related) which relate to maintain integrality of cytoskeleton structure and cell membrane,a heat shock protein which,4 proteins related to macromolecular biosynthesis (2 related with protein translation and 2 related with protein translocation and metabolism,13 metabolism related proteins (2 protein related with carbohydrate metabolism,6 protein related with lipids metabolism,5 protein related with amino acid metabolism),11 protein related with biological oxidation and 9 protein related with signal transduction. These results further confirmed the protective effects of inactivated vaccine and set the foundation for the research on the biological characters of host reaction during EV71 infection and mechanisms that vaccine protected the host from EV71 infection.
本研究采用初步研制成功的肠道病毒71型灭活疫苗,利用恒河猴婴猴这一经典动物模型进行了该疫苗的保护性实验。用疫苗免疫动物后攻毒,疫苗免疫组恒河猴婴猴神经系统的各个部分均未检出超过可信限的病毒RNA阳性结果,而感染对照组动物各部位均检测到明显的病毒RNA阳性结果。灭活疫苗能够诱导免疫后的恒河猴婴猴产生中和抗体,免疫后病毒攻击后第7天恒河猴婴猴血清中和抗体水平达1：512,能够形成有效的抗病毒免疫保护效应。免疫组化结果显示,免疫动物所诱导产生的免疫保护效应能够抑制病毒在体内器官及组织中的增殖。结合攻毒后动物相应靶器官组织切片病理观察,血清中和抗体检测和免疫组化结果,分析表明：肠道病毒71型灭活疫苗免疫恒河猴婴猴后,继而以病毒攻击,将不会引起导致机体组织器官受损的病理学反应,疫苗具有保护性。
此外,取上述研究对象中,免疫后攻毒和未免疫攻毒的恒河猴婴猴中枢神经系统的丘脑组织进行了蛋白质组学研究。运用蛋白质组学相关技术：蛋白质双向电泳分离蛋白和专业软件Imagemaster 7.0进行图像分析,定量研究在病毒感染组及疫苗保护组中表达量有所差异的蛋白质点,通过基质辅助激光解析电离飞行时间质谱和蛋白信息数据库建立差异蛋白质谱。
本研究共发现48个表达量变化的蛋白,按功能分为7类：①、细胞骨架相关蛋白8个,其中CFL2表达量变化结合功能分析提示该蛋白是研究EV71感染所致肌张力变化、心肌原纤维退化、心肌细胞凋亡潜在的靶点。结合各组PFN1、PFN2表达量变化与Profilins功能分析,提示在感染过程中PFN1、PFN2与细胞膜完整性或细胞内吞作用相关,疫苗的保护性降低了感染对细胞形态完整性的影响。②、小热休克蛋白1个,CRYAB,结合该蛋白在感染高峰期表达上调而与其它组不同的表现及其在生理或者神经变性疾病的压力下能够抑制中间纤维的聚集的能力,提示这种小热休克蛋白与感染压力下的细胞骨架稳定性维持相关；③、生物大分子合成相关蛋白4个,其中,CyPA作为一种具有调控免疫相关的因子,可能通过某种未知的信号传导途径激活T细胞活化所需的细胞因子,而促进T细胞的增殖,从而显示感染高峰CyPA表达量与其它组的差异。NUTF2作为一种入核转运因子,其表达量变化提示其是感染状态下宿主实现关闭或下调相关因子以抵抗病毒感染的可能途径之一。④、代谢相关蛋白13个,这些蛋白含量变化提示感染状态下的神经细胞增殖周期的改变、神经元发育的阻滞、突触后神经元的保护性抑制状态改变等与EV71感染致病机理密切相关,对疫苗组这些蛋白表达量的分析进一步映证了疫苗的保护效应。⑤、能量代谢与生物氧化相关蛋白11个,提示病理状态下和疫苗保护效应下机体能量代谢系统和生物氧化途径发生了改变。⑥、泛素-蛋白酶体途径相关蛋白2个,UCHL1和UBE2N在病毒感染组和疫苗保护组中的表达量差异及功能分析也支持了灭活疫苗所诱导的免疫保护的有效性。⑦、信号传导相关蛋白9个,PARK7表达变化提示中枢神经系统对感染的具有保护性,且可能通过PARK7实现。蛋白质组学实验中共结合差异谱中表达量发生改变的蛋白的功能与免疫后攻毒和未免疫攻毒恒河猴婴猴丘脑蛋白表达量变化分析,进一步验证了灭活疫苗具有保护效应,发现了一些在感染中有重要意义的分子靶标,并为探讨肠道病毒71型感染在疫苗免疫和非疫苗免疫机体中的宿主反应的分子生物学特征,以及从这一水平上认识肠道病毒71型感染的致病机理和疫苗的免疫保护机理而提供资料。
由于本研究对象为猕猴属的恒河猴,现有蛋白质数据库中关于猕猴属蛋白的数据还不全面,结合直系同源序列聚类分析(COG)以及蛋白数据库中现有灵长目其它物种的蛋白信息,初步证实了13个尚未在氨基酸或蛋白水平报道的灵长目某已知蛋白在恒河猴中的同源蛋白。
Hand-foot-mouth disease (HFMD) is an acute infectious disease which caused by enterovirus. In recent years, the morbidity and mortality of HFMD are observably increased. There are more than 20 variety virus that cause HFMD and enterovirus 71 (EV71) is the major one. EV71 infection can cause diseases of the nervous system, such as meningitis, encephalitis, monoplegia, acute flaccid paralysis and so on. It also associated with non-nervous system such as HFMD, herpetic angina and pulmonary oedema. In severe patients, it can cause central nervous system complication and pulmonary oedema which are fatal diseases. At present, the research about EV71 mainly focuses on the etiology and epidemiology of the virus. The pathogenesis of the virus has not yet understood. Due to continuous expanding of HFMD caused by EV71 and absence of effective drugs, there is a compelling need to develop a safe and effective vaccine. An elementary inactivated vaccine which prepared from the diploid cell cultured virus is used in this study. In this research, the protective effects of this vaccine were tested in a classic animal model—rhesus monkey neonate.Animal immunized or non-immunized with inactived vaccine were inoculated with EV71 and examined. In vaccine immunized rhesus monkey neonate, viral RNA was detected from infected control each parts of animals the neural system tissue, but not form vaccinated one. The inactivated vaccine can induce the monkeys produce neutralizing antibody which could reach 1:512 in the serum at the 7th day after viral attack.It can form effective antivirus immune barrier. Immunohistochemistry researches show that the protection effects which inoculate with inactivated vaccine could induce virus multiplication.It can be sure that inoculate with this inactivated vaccine, there were no immunopathogenesis damage in animals.Further testing by pathological examination, comprehensive biopsy pathological observation and immunohistochemistry analysis were carried out and all results indicated that inactivated EV71 vaccine protected rhesus monkey from virus attack.
Protein differential expression profiling of infected, vaccinated and control monkey brain tissue was analyzed by two-dimensional-electrophoresis and matrix assisted laser-desorption ionization time of flight mass spectrometry.48 proteins were found expressed differently in these group, including 8 cytoskeleton related proteins (6 microfilament related and 2 microtube related) which relate to maintain integrality of cytoskeleton structure and cell membrane,a heat shock protein which,4 proteins related to macromolecular biosynthesis (2 related with protein translation and 2 related with protein translocation and metabolism,13 metabolism related proteins (2 protein related with carbohydrate metabolism,6 protein related with lipids metabolism,5 protein related with amino acid metabolism),11 protein related with biological oxidation and 9 protein related with signal transduction. These results further confirmed the protective effects of inactivated vaccine and set the foundation for the research on the biological characters of host reaction during EV71 infection and mechanisms that vaccine protected the host from EV71 infection.
引文
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[1]贺福初.蛋白质组(proteome)研究——后基因组时代的生力军[J].科学通报,1999，44： 113-122.
[2]Coll ins FS, Morgan M, Patrinos A. The Human Genome Project:Lessons from Large-Scale Biology [J]. Science,2003,300:285-290.
[3]Chumakow IM, Rigoult P, Le GI, et al. A YAC contig map of the human genome [J]. Nature,1995,377 (Suppl):175-297
[4]Chen Z, Peng B, Wang S, et al. Rapid screening of highly efficient vaccine candidates by immunoproteomics[J]. Proteomics.2004.4(10):3203-13.
[5]Abbott A. And now for the proteome [J]. Nature,2001,49:747.
[6]Fields S. Proteomics in genomeland [J]. Science,2001,291:1221.
[7]Fenselau C. A review of quantitative methods for proteomic studies [J]. Chromatogr B Analyt Technol Biomed Life Sci,2007,855:14-20.
[8]Takatalo M S, Kouvonen P, Corthals G, et al. Identification of new Golgi complex specific proteins by direct organelle proteomic analysis [J]. Proteomics,2006, 6 (12):3502-3508.
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[12]Jung E, Heller M, Sanchez J C, et al. Proteomics meets cell biology:the establishment of subcellular proteomes [J]. Electrophoresis,2000,21:3369-3377.
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[20]Hass G, Karaali C, Ebermayer K, et al. Immunoproteomics of Helicobacter pylori infection and relation to gastric disease [J]. Proteomics 2002,2 (3):3132324.
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