白介素-7促进狂犬病病毒免疫记忆的作用机制研究

发布时间：2018-01-07 06:04

本文关键词：白介素-7促进狂犬病病毒免疫记忆的作用机制研究　出处：《华中农业大学》2017年博士论文　论文类型：学位论文

【摘要】：狂犬病(Rabies)作为最古老的传染病之一,是由狂犬病病毒(Rabies Virus,RABV)引起的人兽共患中枢神经系统传染病,分布于世界大部分地区。我国每年死于狂犬病的人数居世界第二位及全国各类传染病死亡人数的前五位,其中95%以上的病例是因直接或间接接触患病或带毒的动物引发的。目前,狂犬病发病后的临床治疗仍是一个世界性难题,其致死率高达100%,尚无有效的治疗手段。数据显示,70%以上犬的免疫覆盖率可以有效控制人间狂犬病,因此,动物的免疫预防成为遏制人类狂犬病最为有效的措施。当前用于动物狂犬病免疫的疫苗主要为安全的灭活疫苗,但价格昂贵,并需多次免疫才能获得良好的长期保护,阻碍了其在发展中国家的广泛应用。因此,发展低毒、单剂量及长期有效的狂犬病疫苗对控制狂犬病具有重要意义。近来研究表明,过表达树突状细胞(Dendritic cells,DCs)刺激因子的重组狂犬病病毒可在淋巴结及外周血中招募并成熟DCs及B淋巴细胞,进而迅速产生显著高水平的抗病毒中和抗体(Virus-neutralizing antibodies,VNA),但其有效持续时间短。当前,尚未有研究通过应用免疫记忆的作用机制来延长狂犬病疫苗诱导的体液免疫。本研究通过反向遗传技术,在弱毒疫苗株r LBNSE的基础上过表达对免疫细胞的生长、存活及分化方面具有广泛效应的白介素-7(Interleukin-7,IL-7),并拯救获得重组狂犬病病毒(r LBNSE-IL7)。在确定r LBNSE-IL7的体外生长特性不因外源基因IL-7的插入而受到影响之后,通过ELISA检测重组病毒感染细胞上清中IL-7的表达,发现只有rLBNSE-IL7以剂量依赖形式表达IL-7,并对细胞无明显副作用。将r LBNSE-IL7在BSR细胞中连续传代10次,各代次均保持IL-7的稳定遗传。为鉴定IL-7的表达对狂犬病病毒致病性的影响,将rLBNSE、rLBNSE-IL7以及阴性对照重组病毒rLBNSE-IL7(-)脑内途径感染免疫功能完全和不完全的小鼠。我们发现脑内高表达的IL-7能够限制病毒在脑内的转录和复制,进一步减弱狂犬病病毒的致病力,而非IL-7的插入造成病毒基因组长度的增加所导致。为进一步分析IL-7对狂犬病病毒免疫原性的作用,将重组病毒后肢肌肉途径免疫小鼠,进行依赖T细胞的与B细胞相互作用的相关免疫机制研究。与亲本病毒r LBNSE相比,重组病毒表达的IL-7可以显著增加淋巴结中滤泡性T辅助细胞(T follicular helper cells,Tfh cells)、生发中心B细胞(Germinal center cells,GC B cells)、长寿命记忆性B细胞(Memory B cells,Bmem)的数量和骨髓中长寿命浆细胞(Plasma cells,PCs)的比例。由单剂量免疫rLBNSE-IL7所增殖的长寿命PCs可产生更加强大的免疫效力,维持显著高水平的VNA和相关抗体亚类长达360天,且能够长期有效保护绝大多数小鼠抵抗强毒株攻击。此外,IL-7所增加的长寿命Bmem能够于再次共同免疫r LBNSE后,分化成抗体分泌细胞,迅速产生更高水平的VNA及相应抗体亚类。上述结果表明IL-7促进了Tfh细胞的增殖,并在其协助下增强了GCs反应,进一步生成与维持免疫记忆相关的长寿命Bmem和PCs,从而增强了机体针对狂犬病病毒的特异性初次免疫反应和再次免疫应答。综上所述,本研究成功构建了表达IL-7的重组狂犬病病毒r LBNSE-IL7,鉴定了外源基因IL-7在病毒基因组中的表达不影响病毒的体外生长特性;且脑内高表达的IL-7可以降低狂犬病病毒的致病力;并通过促进GCs中的Tfh细胞与B细胞的相关免疫反应从而增殖长寿命Bmem和PCs以增强狂犬病病毒的免疫记忆,改善初次免疫保护性反应和再次免疫应答。此研究结果表明rLBNSE-IL7具有成为低毒、高效的动物狂犬病疫苗的潜能。
[Abstract]:Rabies virus (Rabies) infection as one of the oldest, is composed of rabies virus (Rabies Virus, RABV) zoonotic infectious disease caused by central nervous system, distributed in most parts of the world. China's annual number of deaths in the rabies ranked second in the world and all kinds of infectious diseases before the five, of which more than 95% the cases are caused by direct or indirect contact with diseased or poisonous animal caused. At present, the incidence of rabies after clinical treatment is still a difficult problem in the world, the mortality rate as high as 100%, there is no effective treatment. The data show that more than 70% of the coverage rate of immune dogs can effectively control rabies, therefore. Animal immune prevention is the most effective containment of human rabies. The measures currently used for animal rabies vaccines are inactivated vaccine is safe, but the price is expensive, and needs more time to be immune Have good long-term protection, has hindered its widespread use in the developing world. Therefore, the development of low toxicity, single dose and long-term effective rabies vaccine is important for rabies control. Recent research showed that over expression of dendritic cells (Dendritic, cells, DCs) stimulation of recombinant rabies virus factors in lymph nodes and peripheral blood recruit and mature DCs and B lymphocytes, and then quickly produced significantly higher levels of neutralizing antibody (Virus-neutralizing, antibodies, VNA), but the effective duration is short. At present, there has not been a mechanism through the application of immune memory to extend the humoral immunity induced by rabies vaccine. This study by reverse genetic technique, overexpression of immune cell growth based on attenuated vaccine strain R LBNSE, has a wide effect on survival and differentiation of interleukin -7 (Interleukin-7, IL-7), and. Save the recombinant rabies virus (R LBNSE-IL7) r LBNSE-IL7. After determining the in vitro growth characteristics due to the insertion of exogenous gene IL-7 affected, recombinant virus ELISA infection by detecting the expression of IL-7 in the supernatant, found that only rLBNSE-IL7 expression of IL-7 in a dose dependent form, and no obvious side effects on R LBNSE-IL7 cells. In BSR cells were passaged 10 times and each generation are to maintain the stability of the IL-7 genetic expression. To identify the effect of IL-7 on the pathogenicity of rabies virus, rLBNSE, rLBNSE-IL7 and negative control recombinant virus rLBNSE-IL7 (-) complete and incomplete the immune function of mice infected brain. We found high expression in the brain the IL-7 transcription and replication in the brain to limit the virus, further attenuated rabies virus pathogenicity, rather than the increase of IL-7 due to the insertion of the viral genome length caused by in. Further analysis of the effect of IL-7 on the immunogenicity of rabies virus, the recombinant virus immunized mice by way of hindlimb muscles, dependent T cells and B cells related to immune mechanism of the interaction of LBNSE and R. Compared with the parental virus, the recombinant virus IL-7 can significantly increase the expression of T in lymph node follicular helper cells (T follicular helper cells, Tfh cells), germinal center B cell (Germinal center cells GC, B cells), long-lived memory B cells (Memory B cells, Bmem) and the number of long-lived plasma cells in the bone marrow (Plasma, cells, PCs). The proportion of long-lived PCs proliferation by single dose immunization can be rLBNSE-IL7 have stronger immunity, maintain significantly higher levels of VNA and related antibody subclasses for up to 360 days, and can effectively protect the most mice against virulent attacks. In addition, long-life Bmem added to the IL-7 to another LBNSE R after immunization, differentiate into antibody secreting cells, quickly produce higher levels of VNA and the corresponding subunits. These results indicated that IL-7 promoted the proliferation of Tfh cells and enhanced GCs reaction in its assistance, further generation associated with maintaining immunological memory long life Bmem and PCs, so as to enhance the the specificity of the primary immune response to rabies virus and secondary immune response. In summary, this study successfully constructed IL-7 expressing recombinant rabies virus R LBNSE-IL7, identified the expression of IL-7 gene in the viral genome does not affect the virus in vitro growth characteristics; and in the brain of the high expression of IL-7 can reduce the rabies virus force; and through the immune response in GCs Tfh cells and promote B cells proliferation and long life Bmem and PCs to enhance the rabies virus immune memory, improve primary immune protection The results of this study suggest that rLBNSE-IL7 has the potential to become a low toxic and efficient animal rabies vaccine.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S852.65

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