【作者】 王化磊；

【导师】 夏咸柱；

【作者基本信息】 吉林大学 ， 预防兽医学， 2010， 博士

【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。更多还原

【Abstract】 Rabies is a life-threatening disease caused by an RNA virus that is usually transmitted to human through bites from rabid animals. Each year, approximately 55000 individuals worldwide die of rabies and millions more undergo post-exposure prophylaxis (PEP). Most of the human cases occur in the developing nations of Asia and Africa where dog rabies remains to be the main source for human exposure. In the developed countries, human rabies has dramatically declined as a direct consequence of routine vaccination of pet animals. Current rabies vaccines used for human are inactivated virus vaccine. Although these vaccines are safe and efficacious, multiple doses (at least 4) must be administered over an extended period of time (14 days) to people who are exposed to rabies or suspected rabid animals. In addition, the high cost associated with these inactivated rabies virus (RABV) vaccines prevents their effective use in developing countries where the vaccines are needed most. Live attenuated RABV vaccines or recombinant live vaccines have been licensed, particularly for wild animals. These vaccines have been used for large scale elimination of rabies in Europe as well as in North America. However, they usually induce intensive skin inflammation and not worked well for dogs and skunks. Furthermore, those vaccines are not so effective in oral vaccination for dogs and skunks. Therefore, more efficacious and affordable RABV vaccines are needed, particularly in the developing nations. Once the clinic manifestations of rabies developed, however, treatment options for rabies are limited. To date, only nine individuals have survived rabies virus infection. Unfortunately, most of these patients died either after prolonged illness or developed severe neurogical sequel. Therefore, more effective therapeutics is needed in management of clinical rabies.It was found previously that induction of innate immunity, particularly chemokines and type I interferons, is an important mechanism of rabies virus attenuation. To evaluated the effect of overexpression of chemokines on RABV infection. Macrophage inflammatory protein 1α(MIP-1α, CCL3) was cloned into the genome of attenuated RABV strain HEP-Flury, the recombinant rabies virus (rRABV) was rescued and designated as rHEP-MIP1α. To characterize the rRABV in vitro, virus growth kinetics was examined in NA cells. No significant difference was found between the rRABV and parental virus, indicating that the viral growth was not affected by the insertion of MIP-1α. The ability of rHEP-MIP1αto produce MIP-1αwas determined by measuring MIP-1αin virus-infected cells with ELISA kits. It was found that MIP-1αwas expressed by rHEP-MIP1αin a dose-dependent manner. To determine the effect of chemokine expression on RABV infection, mice were infected with the rRABV by intracerebral route. A transient MIP-1αexpression and inflammatory cell infiltration was induced in the central nervous system (CNS) of mice infected with rHEP-MIP1α, compared with those infected with parental virus. The pathogenicity of rHEP-MIP1αwas more decreased than parental virus. It indicates that overexpression of MIP-1αfurther decreased RABV pathogenicity by inducing a transient innate immune response in the CNS.To investigate the immunogenicity of rHEP-MIP1α, mice were immunized with rRABV by the intramuscular (i.c.) route. The virus neutralizing antibody titer (VNA) in serum was measured by the rapid fluorescent focus inhibiton test (RFFIT). It was found that the level of VNA was significantly higher in mice immunized with rHEP-MIP1αthan that induced by immunization with parent virus. After challenge with virulent CVS-24, more mice immunized with rHEP-MIP1αsurvived than mice immunized with the parental virus. Virus replication, expression of chemokines, and/or recruitment of immune cells at local sites was determined by quantitative real-time PCR (qRT-PCR). There was no difference in virus replication in mice infected with each virus, indicating the induction of adaptive immunity by rHEP-MIP1αis not due to the rate of virus replication at the local site. However, significantly more MIP-1α, CD19, CD11c and IL-4 mRNA was detected in the muscle tissue of mice infected with rHEP-MIP1αthan in mice infected with the parent virus. CD11c dendritic cells (DCs) and B cells were analyzed by flow cytometry. It was found that the recruitment of DCs and B cells in lymph node and peripheral blood was significantly increased in mice immunized with rHEP-MIP1α, than those immunized with parent virus. All the data demonstrate that expression of MIP-1αenhances the immunogenicity by recruiting DCs and B cells to the site of immunization, lymph nodes, and the blood. Therefore, recruitment and/or activation of DCs play an important role in enhancing the protective immune response against RABV. To address the importance of DCs activation for RABV vaccine efficacy, several DCs stimulatory molecules, e.g. granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage derived chemokines (MDC), and MIP-1α, were cloned into the genome of RABV strain SAD L16. The rRABVs were rescued and designated as rLBNSE-GM-CSF, rLBNSE-MDC, and rLBNSE-MIP1α, respectively. It was found that the rRABVs expressed these molecules activate/recruit DCs and enhanced protective immune response.To investigate if the rRABVs expressing the immunostimulatory molecules have the ability to prevent animals from developing rabies, mice were infected with lethal doses of street RABV and then treated with rRABV at different time points after infection by intracerebral (i.c.), intramuscular (i.m.), intradermal (i.d.) or intranasal (i.n.) route. It was found that significantly more mice intracerebrally treated with rRABV expressing MIP-1α, GM-CSF, macrophage-derived chemokine (MDC), or IP-10 as late as day 5 after infection with virulent RABV were protected from developing rabies than sham-treated mice. On the other hand, treatment with UV-inactivated rRABV did not provide protection despite the fact that virus VNA were induced in the periphery. The leakage of fluorescein sodium (NaF) from the circulation into CNS tissues was measured in cerebrum, cerebellum or spinal cord. It was found that BBB permeability to NaF was significantly elevated in the cerebrum and cerebellum of mice treated with rRABVs than sham-treated mice. Expression of chemokines/cytokines, and infiltration of inflammatory cells were measured by qRT-PCR, multiplex ELISA and flow cytometry. Intracerebral treatment of mice with live rRABVs induced significantly higher levels of chemokine/cytokine expression in the CNS and in the periphery, and more infiltration of inflammatory and immune cells into the CNS, than sham-treated mice or mice treated with UV-inactivated rRABV. Most importantly, treatment with a chemokine (chemoattractant protein-1, MCP-1, also termed CCL2) with doses known to enhance BBB permeability increased the protective efficacy of UV-inactivated rRABV. In sum, these studies confirm that chemokines can enhance the BBB permeability, allow infiltration of inflammatory cells and other immune effectors enter into the CNS to clear the virus and to prevent the development of rabies.In the present study, rRABVs expressing the immunostimulatory molecules were constructed. It was found that the viral growth was not affected by the insertion of foreign genes. Overexpression of these molecules further decreased RABV pathogenicity by inducing a transient innate immune response. Recruitment and activation of DCs is important in enhancing the protective immune responses against rabies. These rRBAVs expressing immunostimulatory molecules could have the potential to be used not only for pre- and post-exposure immunization but also for therapy in clinical rabies. The data from this study provides an important theoretical basis for study on pathogenesis of RABV, and selection of more viable and affordable RABV vaccines. It may also provide a new therapeutic of management of clinical rabies.更多还原