HSV-1突变毒株的构建及其生物学特性和免疫原性分析

发布时间：2018-04-16 17:06

本文选题：单纯疱疹病毒Ⅰ型 + ul7　；参考：《北京协和医学院》2017年博士论文

【摘要】：人类疱疹病毒作为一个由8个成员组成的DNA病毒家族,在全球不同人群中均具有较高的感染率,尤其是其中的HSV-1和HSV-2等病原所引起的包括口唇疱疹、生殖器官疱疹以及疱疹性脑炎等常见感染,已成为青少年人群中的重要传染性疾病,并受到公共卫生领域的广泛关注。因此,针对这一病原的相关临床治疗和预防性制品的研究已经得到了多方面的推进。迄今为止,尽管阿昔洛韦类药物已经在临床上得到了广泛的应用,但有关HSV-1和HSV-2的多个疫苗临床研究均未提供具有应用意义的结果。基于HSV-1和HSV-2感染病理的特征探索合适的疫苗形式以诱导有效的临床保护效率显然是目前一个重要的研究方向。我们前期关于HSV-1与宿主相互作用的分子相关研究以及HSV-1相关突变株的构建技术的研究,为进一步针对HSV-1衣被蛋白在病毒生物学特性及其毒力表型变化中的作用分析提供了初步的资料。在此基础上,本文的主要工作描述了一个具有转录调控作用的HSV-1衣被蛋白UL7突变后对HSV-1毒力表型的影响,及其可能机理的研究过程。基于这一突变毒株的生物学特性,我们在其基础上完成了 HSV-1复制过程中的重要调控分子Vhs编码基因u141和与潜伏感染的建立和维持密切相关的LAT基因的突变,构建了突变毒株M2和M3,并对三株毒株的生物学特性及致病性进行了分析。最后,我们以突变毒株M3为研究重点,评价了其在小鼠模型上的免疫原性,为HSV-1减毒病毒的研究提供了初步的基础。在第一部分工作中,针对UL7突变的毒株M1的分析表明,突变毒株M1表现出细胞水平的低增殖能力以及动物水平的急性期感染能力的下降,进一步的体外实验还表明UL7蛋白可能通过参与染色质化α-4基因而起到调控其转录激活的作用。在第二部分工作中,突变毒株M1、M2和M3的生物学特性分析结果表明,与野生型毒株相比,突变毒株M3在细胞中的增殖能力显著降低,在小鼠体内的致病能力和潜伏能力均显著降低,提示了 M3毒株的减毒特性。基于前两部分的研究资料,我们将突变毒株M3作为减毒活疫苗候选株免疫小鼠,并在免疫后1个月和2个月时分别予以野生型毒株滴鼻感染。随后的临床症状观察、免疫学、组织病理学和病毒载量测定结果显示,突变毒株M3免疫后诱导的以中和抗体和CD8+T细胞免疫反应为特征的免疫反应在针对野生型毒株攻击情况下,能够有效控制野生型毒株感染引起的多种临床症状,抑制野生型毒株在小鼠体内的增殖,显著减轻野生型毒株感染引起的病理损伤,限制野生型毒株在神经系统的潜伏感染过程。这些结果均表明了突变毒株M3可作为减毒活疫苗候选株的潜在可能。
[Abstract]:As a family of eight members of DNA virus, human herpesvirus has a high infection rate in different populations around the world, especially HSV-1, HSV-2 and other pathogens, including oral herpes.Herpes genitalis and herpes encephalitis and other common infections have become an important infectious disease in the adolescent population and have received extensive attention in the field of public health.Therefore, the research on clinical treatment and prophylaxis of this pathogen has been advanced in many aspects.So far, although acyclovir drugs have been widely used in clinical practice, many clinical studies on HSV-1 and HSV-2 have not provided significant results.Based on the pathological features of HSV-1 and HSV-2 infection, it is an important research direction to explore the appropriate vaccine form to induce effective clinical protection efficiency.We have studied the molecular correlation of HSV-1 interaction with host and the construction of HSV-1 related mutants.It provides preliminary data for the analysis of the role of HSV-1 coat protein in the changes of virus biological characteristics and virulence phenotypes.On this basis, the main work described a transcriptional regulation of HSV-1 coat protein UL7 mutation after the impact of HSV-1 virulence phenotype, and the possible mechanism of the study process.Based on the biological characteristics of the mutant strain, we have completed the mutation of the Vhs encoding gene U141, an important regulatory molecule in the process of HSV-1 replication, and the LAT gene closely related to the establishment and maintenance of latent infection.Mutants M2 and M3 were constructed and their biological characteristics and pathogenicity were analyzed.Finally, we evaluated the immunogenicity of mutant strain M3 in mouse model and provided a preliminary basis for the study of HSV-1 attenuated virus.In the first part of the work, the analysis of M1 strain with UL7 mutation showed that mutant M1 showed low proliferation ability at cell level and decreased infection ability at animal level in acute stage.Further in vitro experiments indicated that UL7 protein may regulate the transcriptional activation by participating in chromatin 伪 -4 gene.In the second part of the work, the biological characteristics of mutant strain M1M2 and M3 were analyzed. The results showed that the proliferation of mutant strain M3 was significantly lower than that of wild-type strain.The pathogenicity and latent ability in mice were significantly decreased, indicating the detoxification characteristics of M _ 3 strain.Based on the first two parts of the study, we immunized mice with mutant strain M3 as a candidate strain of attenuated live vaccine, and were inoculated with wild-type virus strain drip nasal infection at 1 month and 2 months after immunization, respectively.Subsequent clinical observation, immunology, histopathology, and viral load measurements showed that,The immune response induced by mutant strain M3, characterized by neutralizing antibody and CD8 T cell immune response, can effectively control the clinical symptoms caused by wild-type virus infection under the attack of wild-type strain.Inhibiting the proliferation of wild type virus strain in mice, reducing the pathological damage caused by wild type virus strain infection, limiting the latent infection process of wild type virus strain in nervous system.These results suggest that mutant strain M _ 3 is a potential candidate for attenuated live 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

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