CA16感染恒河猴模型以及相关病原和免疫学特性研究

发布时间：2018-11-21 16:12

【摘要】：Coxsackievirus A16(CA16)做为人类手足口病(HFMD)的重要病原体之一,以其不断在亚洲-太平洋地区的儿童群体中所引起的HFMD大规模流行已经成为一个重要的公众关注对象。尽管CA16感染所导致的HFMD较为温和,但已有的流行病学报道证明,CA16仍然可能在感染人群中引起少数的重症病例,以及可能引起重复感染,因此,深入对该病原的相关研究,已经成为一个重要的公共卫生问题。本论文的研究工作主要针对CA16在恒河猴感染模型中的病理学特征及免疫学反应表现,以及相关的CA16灭活疫苗技术原理等做了初步的探索,所得到的结果为该病毒的感染机理分析及疫苗的研究提供了初步的资料。本课题的研究工作主要分为三个部分,首先,我们在前期建立EV71感染恒河猴婴猴模型研究的基础上,在恒河猴成年猴(4～5岁)幼猴(8～月10龄)和婴猴(1～3月龄)个体内建立了 CA16感染动物模型,并观察到该病毒可通过鼻腔粘膜引起恒河猴幼猴和婴猴出现与人类手足口病相似的口腔粘膜疱疹、手足部疱疹以及发热等临床症状,还可出现典型的病毒血症和各组织器官病毒载量的轻微上升,其中以淋巴器官和CNS中病毒载量的上升最为明显,同时,可见到外周血中一过性IL-6和TNF-α的升高,这与我们在建立EV71恒河猴婴猴模型时所表现出来的症状极其相似。另外,动物器官的组织病理观察表明,CA16的感染仅导致恒河猴出现肺脏、淋巴结等器官较轻的病理学改变,同时在皮肤疱疹病理组织中观察到部分嗜酸性细胞的出现。根据CA16在临床观察中可能出现反复感染的报道,我们对已感染的恒河猴婴猴、幼猴进行了第二、三次的重复感染试验,在此过程中,上述感染过程的表现均再次出现在相同动物的身上,但这些感染均不能明显诱导机体产生中和抗体反应,这似乎提示CA16病毒感染恒河猴、即使在重复感染的情况下,也未能诱导高滴度的中和抗体反应。其次,我们的工作探讨了以0、28天两剂免疫程序的实验性CA16灭活病毒疫苗免疫恒河猴后的免疫反应特征,实验结果表明,CA16灭活疫苗免疫恒河猴后可刺激其机体产生特异性细胞免疫反应,并且亦表现了相应的血清中和抗体上升,但在系统分析其病毒攻击后的免疫保护性和安全性的研究结果并不理想,免疫动物在受到病毒攻击后,85%的实验动物均出现了典型的疱疹、病毒血症以及相关临床症状,病毒攻击并未刺激机体血清中和抗体的四倍上升。在第三部分工作中,我们进一步观察表明,CA16可以直接感染DC细胞,并在其中增殖的过程中,刺激细胞上调包括IL6、IL5等细胞因子的表达。同时感染的DC细胞与T细胞孵育后,再次回输T细胞至供体猴体内后,仍可观察到猴体出现典型的口腔及手足口部疱疹。这一结果提示CA16感染所引起的粘膜及皮肤疱疹病变,很有可能是免疫反应的相关病理性反应。另外,对前期人群临床实验中CA16感染阳性的血清样品中细胞因子、趋化因子和中和抗体的分析进一步提示了 CA16感染人体后血清中部分细胞因子及趋化因子的特征性变化。尽管本研究尚未能对CA16感染恒河猴及其免疫病理机制有全面的认识,但通过不同层次的分析所获得的一系列研究资料,将为CA16在非人灵长类的感染及免疫保护应用的研究提供相应的理论依据,也为CA16疫苗的研发提供了参考资料。
[Abstract]:Coxsackievirus A16 (CA16) is one of the important pathogens of human hand-foot-mouth disease (HFMD), which has become an important public concern for the large-scale development of HFMD in the children's population in the Asia-Pacific region. Although the HFMD caused by the CA16 infection is mild, epidemiological studies have shown that the CA16 may still cause a small number of severe cases in the infected population, and may cause repeated infections, and therefore, in-depth study of the pathogen, It has become an important public health problem. The research work of this paper mainly focuses on the pathological characteristics and immunological reaction of CA16 in rhesus monkey infection model, and the related CA16 inactivated vaccine technical principle and so on. The results obtained provide a preliminary data for the analysis of the infection mechanism of the virus and the research of the vaccine. The research work of the subject was divided into three parts: first, we established the animal model of CA16 infection in the adult monkey (4-5 years old) and the baby (1-3 months) in the rhesus monkey (4-5 years old) and the infant (1-3 months), based on the study of the model of the EV71-infected rhesus monkey in the early stage. and observed that the virus can cause the clinical symptoms of oral mucosa herpes, hand-foot-foot-part herpes, fever and the like of the rhesus monkey and the baby monkey which can cause the rhesus monkey and the baby monkey to be caused by the nasal mucosa of the nasal cavity, and also can develop a typical viremia and a slight increase of the viral load of the tissues and organs, The most significant increase in viral load in the lymphoid organs and the CNS, while the increase in one of the peripheral blood mononuclear cells, IL-6 and TNF-1, is very similar to the symptoms we have shown in the establishment of the EV71 rhesus monkey model. In addition, the histopathological observation of animal organs showed that the infection of CA16 only resulted in the pathological changes of the lung, lymph nodes and other organs of the rhesus monkey, and the appearance of some of the eosinophils was observed in the pathological tissue of the skin. According to the report of the possible repeated infection of the CA16 in clinical observation, we conducted a second and a third repeated infection test on the infected rhesus monkey and the young monkey, in which the expression of the above-mentioned infection process again appeared on the same animal, These infections, however, do not significantly induce a neutralizing antibody reaction in the body, which appears to suggest that the CA16 virus-infected rhesus monkey, even in the case of repeated infection, fails to induce a neutralizing antibody reaction with high drop. The results show that the immune response of the CA16 inactivated vaccine to the rhesus monkey can be stimulated by the CA16 inactivated vaccine, and the immune response of the rhesus monkey can be stimulated by the CA16 inactivated vaccine. and the immune protection and the safety of the immune animals after the system analysis of the virus attack are not ideal, and after the immune animals are subjected to the virus attack, 85% of the experimental animals have typical herpes, Viremia and related clinical symptoms, viral attack did not stimulate a four-fold increase in serum and antibody levels in the body. In the third part, we have further observed that the CA16 can directly infect the DC cells and, in the course of the proliferation, stimulate the expression of cytokines including IL6, IL5, and the like. When the infected DC cells were incubated with T cells, the typical oral and hand-foot and hand-foot-mouth herpes of the monkey body could still be observed after the T-cells were returned to the donor monkey again. This result suggests that the mucosal and skin-herpes lesions caused by the infection of the CA16 are likely to be related to the pathological reaction of the immune response. In addition, the analysis of cytokines, chemokines and neutralizing antibodies in serum samples infected with CA16 in the clinical trials of the early population further showed the characteristic changes of some cytokines and chemokines in the serum of CA16 infected with the human body. Although this study has not been able to have a comprehensive understanding of the CA16 infected rhesus monkey and its immune and pathological mechanism, a series of research data obtained through different levels of analysis will provide a corresponding theoretical basis for the study of the application of CA16 in non-human primate infection and immune protection application. Reference is also made to the development of the CA16 vaccine.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R392

【相似文献】