表达免疫刺激因子的重组狂犬疫苗对狂犬病的暴露前及暴露后预防研究

发布时间：2018-01-03 10:16

本文关键词：表达免疫刺激因子的重组狂犬疫苗对狂犬病的暴露前及暴露后预防研究　出处：《吉林大学》2010年博士论文　论文类型：学位论文

【摘要】： 狂犬病(Rabies)是由RNA病毒引起、通过患狂犬病动物咬、抓伤传播的严重威胁人类健康的人兽共患传染病。每年,全世界大约有55000人死于狂犬病感染,且多于1000万的人接受狂犬病暴露后免疫治疗。大多数人类狂犬病主要发生在亚洲、非洲等发展中国家。在发达国家,由于对宠物的常规免疫,已基本消除了人类狂犬病。当前用于人狂犬病免疫的疫苗主要为灭活疫苗,此种疫苗具有安全、有效等优点,但狂犬病暴露人群通常需要在较长时间(14天)内进行多次免疫注射(至少4次)。此外,此种疫苗价格昂贵,进一步阻止了其在发展中国家的使用。弱毒疫苗或重组活疫苗也被批准用于动物,尤其是野生动物的免疫。这些疫苗已在欧洲及北美洲很大范围内消除了野生动物的狂犬病,但通常会引起副反应,且对犬及臭鼬没有良好的保护效果。因此,狂犬病的控制需要更加廉价、有效的疫苗,尤其是在广大发展中国家。目前,狂犬病仍没有有效的治疗手段,一旦出现临床症状,几乎所有的方法都无能无力。截止今天,全世界共有9人在出现狂犬病症状后依然存活,然而他们中的大多数或产生严重的神经系统后遗症或死于持续的疾病。因此,探讨新的狂犬病治疗方法对于控制狂犬病具有重要意义。近来研究表明,先天性免疫应答的激活,尤其是趋化因子及干扰素,是狂犬病致弱的重要分子机制。为进一步研究趋化因子在狂犬病感染中的作用,通过反向遗传技术,将巨噬细胞炎性蛋白(Macrophage inflammatory protein 1α, MIP-1α)克隆入HEP-Flury致弱狂犬病毒株,拯救获得重组病毒并命名为rHEP-MIP1α。rHEP-MIP1α与母本病毒在NA细胞的生长特性相似,这表明重组病毒的体外增殖不受外源基因表达的影响。ELISA检测病毒感染细胞后MIP-1α的表达,发现rHEP-MIP1α以剂量依赖形式表达MIP-1α。将重组病毒脑内注射小鼠,检测MIP-1α表达对重组病毒致病性的影响。与母本病毒相比,rHEP-MIP1α可在中枢神经系统(Central nervous system, CNS)诱导短暂的趋化因子表达及炎性细胞浸润,且其对小鼠的致病性降低。结果说明rHEP-MIP1α可通过诱导短暂的CNS先天性免疫应答进一步降低病毒的致病性。为进一步研究rHEP-MIP1α的免疫原性,将重组病毒肌肉免疫小鼠后,以快速荧光灶抑制试验(RFFIT)测定病毒诱导机体产生的中和抗体水平。与母本病毒相比,rHEP-MIP1α肌肉注射后可诱导机体产生显著高的狂犬病中和抗体。以CVS-24强毒进行攻击后,显著多的rHEP-MIP1α免疫小鼠可抵抗强病毒感染。通过荧光定量PCR分析免疫局部的病毒增殖、趋化因子表达及先天性免疫应答分子聚集,结果发现不同病毒在免疫局部的增殖相似,这说明rHEP-MIP1α诱导的适应性免疫应答与病毒在免疫局部的增殖无关。与母本病毒相比,rHEP-MIP1α可在免疫局部诱导表达更高水平的MIP-1α、IL-4、CD19及CD11c。此外,流式细胞术分析发现rHEP-MIP1α可在淋巴结及外周血聚集并激活更多的树突状细胞(Dendritic cells, DCs)及B淋巴细胞。这些数据说明,rHEP-MIP1α可在免疫局部表达高水平的MIP-1α,进而在淋巴结及外周血聚集并激活更多的DCs及B细胞,从而产生高水平的中和抗体。因此,DCs的聚集及激活在增强病毒的保护性免疫应答过程中起重要作用。进一步分析了表达粒细胞-巨噬细胞集落刺激因子(Granulocyte-macrophage colony-stimulating factor, GMCSF )、巨噬细胞来源趋化因子( Macrophage derived chemokines, MDC)及MIP-1α等DCs刺激分子的重组病毒的免疫原性。结果表明,重组病毒表达这些分子后可显著增强DCs的聚集及激活,进而增强病毒的保护性免疫应答。为研究表达免疫刺激因子的重组狂犬病毒对狂犬病的暴露后预防作用,在小鼠肌肉感染高致病性狂犬病街毒株后不同时间,用不同重组病毒以脑内(i.c.)、肌注(i.m.)、皮下(i.d.)及滴鼻(i.n.)等途径进行治疗。结果发现,即使在街毒感染后5天开始治疗,表达MIP-1α、GM-CSF、MDC及IP-10的重组病毒仍具有明显的保护作用。尽管紫外线灭活的重组病毒诱导机体产生了较高的中和抗体,但对街毒感染没有保护作用。测定荧光素钠(Sodium fluorescein, NaF)由血液循环进入CNS的量,发现表达免疫刺激因子的重组病毒可显著提高大脑及小脑的血脑屏障(Blood brain barrier, BBB)通透性。通过荧光定量PCR、多重ELISA(Multiplex ELISA)及流式细胞术分析CNS的趋化因子、细胞因子表达及炎性细胞浸润。结果表明,与紫外线灭活病毒相比,表达免疫刺激因子的重组病毒脑内注射后在CNS及外周血引起更高水平的趋化因子及细胞因子表达,炎性细胞浸润。更为重要的是,可增强血脑屏障通透性的趋化因子MCP-1(Chemoattractant protein 1, MCP-1)显著增加了灭活病毒的保护效果。这些数据说明,表达免疫刺激因子的重组病毒可通过在CNS诱导炎性因子表达,增强炎性细胞浸润及提高血脑屏障的通透性,允许更多的炎性细胞或免疫效应因子进入CNS,进而加速病毒的清除及阻止狂犬病感染的发生。本研究成功构建了表达免疫刺激因子的重组狂犬病毒,证实了外源基因在病毒基因组的表达对病毒的体外生长没有影响,且重组病毒可通过诱导短暂的先天性免疫应答分子表达进一步降低病毒的致病性,DCs的聚集及激活在增强病毒的保护性免疫应答中具有重要作用,筛选获得的重组疫苗不仅可用于狂犬病的暴露前及暴露后预防免疫,而且还可用于狂犬病的临床治疗。此研究结果为研究狂犬病的致病机理及筛选新型、低毒、高效狂犬病疫苗提供了重要理论依据,并为狂犬病的治疗提供了新的手段及工具。
[Abstract]:Rabies virus (Rabies) is caused by the RNA virus, rabies through animal bites, scratches the spread of a serious threat to human health zoonotic disease. Every year, there are about 55000 people died of rabies infection all over the world, and more than 10 million people received post exposure rabies immune therapy. Most human rabies occurred mainly in Asia, Africa etc. developing countries in developed countries, due to routine immunization of pets, has been basically eliminated. The current human rabies rabies vaccines used for human mainly for the inactivated vaccine, the vaccine is safe, effective and other advantages, but the population exposed to rabies usually in the long time (14 days) in the number of immunization (at least 4 time). In addition, the vaccine is expensive, further to prevent its use in developing countries. The attenuated vaccine or recombinant live vaccine has been approved for animal, especially wild Animal immune system. These vaccines have been eliminated in the wild animal rabies in Europe and North America in a large range, but often cause side effects, and there is no good protective effect on canine and skunk rabies control. Therefore, the need for more cheap and effective vaccine, especially in developing countries. At present, rabies is still not the effective treatment method, once the clinical symptoms, almost all methods are powerless. As of today, 9 people throughout the world are still alive in rabies, but most of them or cause serious neurological sequelae or died of persistent disease. Therefore, to explore the methods of treatment for rabies is new the important significance of rabies control.
Recent studies have shown that activation of the innate immune response, particularly chemokines and interferons, is an important mechanism of rabies virus attenuation. Chemokines in rabies infection. For further research, by reverse genetic technique, the macrophage inflammatory protein (Macrophage inflammatory protein 1 alpha, alpha MIP-1) was cloned into HEP-Flury attenuated rabies virus strains, save the recombinant virus named rHEP-MIP1 alpha.RHEP-MIP1 alpha and female parent virus were similar in the growth characteristics of NA cells, suggesting that the detection of.ELISA virus infected cells MIP-1 expression on proliferation of recombinant virus in vitro by exogenous gene expression, rHEP-MIP1 expression of MIP-1 alpha in dose-dependent manners. The recombinant virus was injected into the brain of mice, to detect MIP-1 expression effects on pathogenicity of the recombinant virus. Compared with the parent virus, rHEP-MIP1 alpha in the central nervous system (Cen Tral nervous system (CNS) induced transient chemokine expression and inflammatory cell infiltration, and reduced its pathogenicity to mice. Results showed that rHEP-MIP1 alpha could further reduce the pathogenicity of virus by inducing transient CNS immune response.
For further study of immunogenicity of rHEP-MIP1 alpha, the recombinant virus muscle of mice immunized with the rapid fluorescent focus inhibition test (RFFIT) neutralizing antibody level determination of virus induced. Compared with the parent virus, rHEP-MIP1 alpha after intramuscular injection can induce significantly higher rabies neutralizing antibody attack to CVS-24. After inoculation, rHEP-MIP1 alpha mice significantly more resistant to strong virus infection. By fluorescence quantitative PCR analysis of virus proliferation of local immune, chemokine expression and innate immune response of molecular aggregation, found with the virus in the local immune proliferation is similar, which shows that the adaptive immune response and virus induced by rHEP-MIP1 in the independent the proliferation of local immune. Compared with the parent virus, alpha rHEP-MIP1 can induce local higher expression levels of MIP-1, CD19 and CD11c. in the immune IL-4, in addition, flow cytometry Analysis found that rHEP-MIP1 alpha in lymph nodes and peripheral blood aggregation and activation of dendritic cells (Dendritic more cells, DCs) and B lymphocytes. These data suggest that rHEP-MIP1 alpha can be local expression of high levels of MIP-1 in the immune system, and in the lymph node and peripheral blood aggregation and activating DCs and B cells. In order to produce high levels of neutralizing antibodies. Therefore, recruitment and activation of DCs play an important role in the enhancement of virus protective immune response. Further analysis of the expression of granulocyte macrophage colony stimulating factor (Granulocyte-macrophage colony-stimulating, factor, GMCSF), macrophage derived chemokine (Macrophage derived, chemokines, MDC) immunogenicity DCs and MIP-1 alpha stimulation recombinant virus molecule. The results showed that the recombinant virus expression of these molecules can significantly enhance aggregation and induced DCs activities, thus enhancing the virus Protective immune response.
Immune stimulating factor recombinant rabies virus for prevention of rabies exposure on expression in muscle of mice infected with highly pathogenic strains of rabies street after different time, with different recombinant virus in the brain (i.c.), (i.m.), subcutaneous injection (i.d.) and intranasal (i.n.) and other means of treatment. Found that, even in the street virus infection 5 days after the start of treatment, the expression of MIP-1 alpha, GM-CSF, recombinant virus MDC and IP-10 still has obvious protective effect. Although recombinant virus UV inactivated induce neutralizing antibodies is higher, but no protective effect on street virus infection. The determination of fluorescein sodium (Sodium fluorescein. NaF) from the circulation into the amount of CNS, found that the expression of the recombinant virus immune stimulating factor can significantly improve the blood brain barrier in brain and cerebellum (Blood brain, barrier, BBB). The permeability by fluorescence quantitative PCR, multiplex ELISA (Multiplex ELISA) and flow cytometry analysis of CNS chemokines, cytokines and inflammatory cell infiltration. The results showed that compared with UV inactivated virus, expression of recombinant virus immune stimulating factor in brain after injection of CNS in peripheral blood and induced higher levels of chemokines and cytokines, inflammation infiltration of inflammatory cells. More importantly, can enhance the permeability of blood brain barrier chemokine MCP-1 (Chemoattractant protein 1, MCP-1) significantly increased the protective effect of inactivated virus. These data suggest that expression of recombinant virus immune stimulating factors can be induced by inflammatory cytokine expression in CNS enhanced inflammatory cells infiltration and increase the permeability of the blood-brain barrier, allowing inflammatory cells or immune effectors more into CNS, thereby accelerating the clearance and prevents the occurrence of rabies virus.
This study successfully constructed recombinant rabies virus expressing immunostimulatory factors, confirmed the expression of exogenous gene in the viral genome of the virus in vitro growth was not affected, and the recombinant virus can induce transient innate immune response molecule expression further reduced the pathogenicity of the virus, recruitment and activation of DCs play an important role in the enhancement of protection immune responses to the virus, the screening of recombinant vaccine can be used not only for rabies pre exposure and post exposure immunization, but also can be used for clinical treatment of rabies. The results of this research and study the pathogenic mechanism of rabies and screening model, low toxicity, provides an important basis for effective rabies vaccine, and provides new means and tools for the treatment of rabies.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

【引证文献】