两种猪流感通用疫苗的构建和免疫效果的初步研究

发布时间：2018-04-03 05:31

本文选题：猪流感　切入点：通用疫苗　出处：《吉林大学》2017年博士论文

【摘要】：大约90年前,猪流感病毒首次被证实,尽管在过去的50年内,猪流感引起了广泛的关注,但它仍然是养猪业存在的隐患,同时也对人类健康带来威胁。在二十世纪人流感的4次大流行、大爆发过程中,有三次与猪流感相关,分别是1957、1968和2009流感的大流行。H5亚型高致病性禽流感自从1996年在广东的家鹅的体内发现后,2003年开始在中国大规模的流行。近些年来,在中国、越南和埃及的猪群的流行病学调查中,发现H5亚型高禽流感病毒的已经开始在猪群内适应,H5亚型高致病性禽流感病毒可能会通过在猪群的适应,获得人传人的能力,由于H5亚型高致病性流感病毒在人类感染后,具有高达60%的致死率,这将会给人类带来巨大的灾难。高致病性禽流感H5亚型的病毒经过十多年的遗传进化,已经演化出了10个亚类。其中,1、2和7亚类进一步分化出二级亚类、三级亚类等。如此高度分化的遗传学性质给疫苗研发带来了挑战和巨大的经济压力。在本次研究中我们选择了在遗传进化树与共有序列(Consensus Sequence)距离最近的病毒株A/Thailand/406H/04(H5N1)作为免疫原,以BALB/c小鼠为动物模型,用表达A/Thailand/1(KAN)-1/04(H5N1)HA蛋白的DNA质粒免疫两次,用含有A/Thailand/1(KAN)-1/04(H5N1)病毒株的表面膜蛋白HA和NA的病毒样颗粒(VLP)加强免疫一次(这种异源性免疫策略简称为TH DDV)。在最后一次免疫后14天分析免疫后小鼠的CD8 T细胞的激活程度,根据活化的T细胞的数量和分泌的细胞因子的水平来评价免疫小鼠体内细胞免疫的水平。用假病毒中和实验、血凝抑制实验、微孔中和实验和蚀斑减少实验等多种检测手段来确定免疫鼠体内中和性抗体对同亚型不同亚类的病毒株的中和效果。我们也检测了免疫血清对不同亚型病毒株HA蛋白的结合情况和对这些病毒株的中和效价。随后,用活病毒对免疫小鼠进行了攻毒实验来检测这种免疫策略的体内保护效果。用攻毒后免疫小鼠的体重和存活情况来评价疫苗所提供的保护程度,用肺脏内的病毒载量来评价免疫后小鼠对病毒感染后的清除水平。实验结果显示TH DDV免疫策略不仅可以诱导出较高的T细胞免疫水平,还可以诱导出广谱的中和性抗体,这些抗体不仅可以中和所有的H5亚型10个亚类的和亚亚类代表毒株的假病毒和一些亚类的真病毒,还可以中和来自不同亚型的流感病毒株。结合性抗体检测显示TH DDV免疫血清可以结合H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15和H16亚型的流感HA蛋白。THDDV免疫小鼠可以对同亚型的不同亚类病毒的主动免疫和被动免疫攻毒提供完全的保护,对不同亚型的流感病毒也有一定的保护效果。随后,我们发现这种免疫策略的主要中和性抗体的表位在HA蛋白的头部,也就是说HA蛋白的头部存在保守的中和表位。然后以猪为动物模型,用DNA质粒免疫两次,病毒样颗粒(VLP)加强免疫一次后采集免疫血清,用假病毒中和实验检测显示有较广谱的中和效果。由于上面的研究发现THDDV免疫血清主要的广谱性中和抗体是由HA头部区域的保守表位产生的,为了进一步提高HA头部区域的保守表位产生的中和性抗体的效价和广谱性,本实验设计了表位依赖的流感通用疫苗。TH DDV免疫血清所针对的HA头部蛋白的保守表位,由于它诱导的中和性抗体的滴度不高,为了提高HA头部蛋白的保守表位诱导的中和抗体的滴度,将HA头部蛋白的保守表位放在一个可以产生高中和性抗体滴度的骨架蛋白上,构建了嵌合体的免疫原,以BALB/c小鼠为动物模型,用表达嵌合体HA蛋白的DNA质粒免疫两次,用含有嵌合体HA蛋白的病毒样颗粒(VLP)加强免疫一次。免疫后分析免疫血清所识别的HA蛋白的表位和评价嵌合体的免疫原的免疫效果。结果显示:这种免疫策略产生了HA表位特异性的中和性抗体,而且这种抗体由于抗原表位的减少而显示更高的、更广谱的抗体反应。免疫后小鼠的攻毒实验显示:这种免疫策略不仅可以保护相同亚型流感的病毒的感染,也可以保护不同亚型流感的病毒的感染。同样在以猪为动物模型,检测免疫血清也有很好的广谱性。本次研究为猪流感通用疫苗的研究提供了新的思路:1)选择离共有序列较近的病毒株作为免疫原,用DNA初免和病毒样颗粒加强免疫的策略;2)利用抗原表位减少的方式去设计表位疫苗。
[Abstract]:About 90 years ago, the swine flu virus was confirmed for the first time, although in the past 50 years, the swine flu has aroused widespread concern, but it is still a pig industry existence hidden danger, also pose a threat to human health. In twentieth Century 4 pandemic influenza outbreak, in the process, with three the swine flu related, were 19571968 and 2009.H5 influenza pandemic avian influenza since 1996 found home in Guangdong goose, beginning in 2003 China large-scale epidemic. In recent years, in Chinese, epidemiological investigation of Vietnam and Egypt pigs in, found H5 subtype the bird flu virus has started to adapt in pigs, H5 subtype of highly pathogenic avian influenza virus in pigs may be through the adaptation, the ability to obtain human transmission, because the H5 subtype of highly pathogenic avian influenza virus infection in humans, with the death of up to 60% This rate, it will bring great disaster to human beings. The highly pathogenic avian influenza H5 virus through the evolution of more than 10 years, has evolved in 10 subgroups. Among them, 1,2 and 7 subtypes of further differentiation of level two sub categories, three sub categories. So highly differentiated Nature Genetics for vaccine development challenges and tremendous economic pressure. In this study we chose in the phylogenetic tree and sequence (Consensus Sequence) from the nearest virus strain A/Thailand/406H/04 (H5N1) as immunogen, using BALB/c mice as animal models, with the expression of A/Thailand/1 (KAN) -1/04 (H5N1) DNA plasmid immune HA protein two, containing A/Thailand/1 (KAN) -1/04 (H5N1) virus like particles of virus membrane protein HA and NA (VLP) a second immunization (referred to as the heterologous immunization strategy for TH DDV). In the last 14 days after immunization of free The degree of activation of mouse CD8 T cells after disease, immune cell immunity of mice in vivo evaluation according to the number of activated T cells and cytokines level level. With a fake virus neutralization test, hemagglutination inhibition test, micro neutralization assay and plaque reduction test and other testing means to determine the immune rats in vivo antibodies of the same subtype in different subgroups of virus neutralizing effect. We also detected the immune serum with HA protein in different subtypes of the virus strains and the virus neutralization titer. Subsequently, with live virus on the immune mice were challenged in vivo experiments to test the protective effect of the immunization strategy used to immunize mice after infection. The body weight and survival to evaluate the degree of protection provided by the vaccine, the virus load in the lung of immunized mice to evaluate the virus clearance levels in experiments. The results showed that T cell immunity level TH DDV immunization strategy can not only induce higher, neutralizing antibodies can also induce broad-spectrum, these antibodies can not only neutralize all H5 subtypes of 10 subtypes and strains of the virus like Yaya and some subtypes of true virus, influenza virus strains can also neutralize from different subtypes. The combination of antibody detection showed that TH immune serum DDV can be combined with H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, HA flu protein in mice immunized with.THDDV H15 and H16 subtypes of with different subtypes subtype virus active immunization and passive immunity to provide complete protection to different subtypes of influenza viruses also have a protective effect. Then, we found the head immune strategy main neutralizing antibody epitopes in HA protein, that is conserved in the presence of HA protein in the head And then the epitope. The pig animal model with DNA immunized two times, virus like particles (VLP) collected after a second immunization serum with pseudotyped virus neutralization assay showed a broad-spectrum neutralization effect. Due to the above findings THDDV the main immune serum neutralizing antibody by broad spectrum table HA conservative head region position, in order to further improve the HA head region of conserved epitopes of neutralizing antibody titer and broad spectrum, the experimental design of conservative table HA head protein dependent universal influenza vaccine.TH DDV immune serum against the titer of neutralizing antibodies, because it induces the high titer of HA, in order to improve the head protein conserved epitope induced neutralizing antibody, HA head protein conserved epitopes in a high school and can produce antibody skeleton protein, constructed the chimeric The immunogen, using BALB/c mice as animal models, chimeric HA protein expression by DNA plasmid immunized two times, virus like particles containing the chimeric HA protein (VLP) at a time. To strengthen the immune epitope identification and evaluation of immune serum HA protein immune analysis of chimerism after immunogen immune effect. The results showed that this immunization strategy produced neutralizing antibody HA epitope specific antibody, and this epitope due to reduced and showed higher, more broad-spectrum antibody response. According to experimental mice after immunization challenge: this immunization strategy can not only protect the same subtype of influenza virus infection, also can protect the different subtypes of influenza virus infection in pigs. The same as animal model, detection of immune serum also have broad spectrum very well. This study provides a new way for the study of porcine universal influenza vaccine: 1) to choose from The more closely related virus strains were used as immunogen, the strategy of enhancing immunity with DNA primer and virus like particles. 2) design epitope vaccine by reducing the antigen epitope.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S852.4

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