A sequence-independent approach for attenuating hepaciviruses

摘要 人肝炎病毒或丙型肝炎病毒（HCV）在约70%的感染者中建立慢性嗜肝性感染 个体在这个项目中，核心问题是：我们能否使肝炎病毒基因组突变， 减毒病毒来做疫苗吗致病性病毒在体外或体内生物系统中的连续传代 然后选择致病性较低的病毒株是产生减毒病毒的传统方法 疫苗HCV减毒仍然是不可想象的，因为没有有效的细胞培养系统可用于 病毒发现几十年后，动物模型仍然难以捉摸。同样，对于许多其他慢性人类 病毒，它们的物种特异性限制了免疫活性动物模型的发展， 免疫和发病机制的有意义的研究。该项目将使用啮齿动物肝炎病毒（RHV）， 丙型肝炎病毒感染的标志我们将使用生物信息学方法来改变二核苷酸 RHV基因组中的频率以产生减毒变体。我们的初步结果提供了一个坚实的 他对项目进行了论证，并保证了其可行性。我们确定了高频率的RHV变体 的UpA（尿嘧啶，然后腺嘌呤）未能在大鼠中发展成慢性感染。我们提出了两个目标， 我们的策略是产生减毒的肝炎病毒，并表征先天性和适应性免疫 由其感染引起的。目的-1是优化肝炎病毒高滴度突变株的设计 病毒血症和不发展成慢性感染。目的-2是描述先天免疫和获得性免疫 由减毒肝病毒突变体诱导。由于我们将使用序列独立的方法（仅 同义突变）来减弱RHV，结果可以为HCV疫苗的设计提供信息。此外，本发明还 慢性病毒在其天然宿主中成功减毒将开辟新的研究途径并产生新的病毒 定义与病毒清除和持续存在相关的免疫反应以及病毒的免疫相关性的变体 保护慢性病毒。

Abstract Human hepacivirus or hepatitis C virus (HCV) establishes chronic hepatotropic infection in ~70% of infected individuals. In this project, the central question is: Can we mutate a hepacivirus genome to generate an attenuated virus to use as a vaccine? Serial passaging of pathogenic viruses in vitro or in vivo biological systems followed by the selection of less-pathogenic viral strains is the traditional way to generate an attenuated virus vaccine. HCV attenuation remained inconceivable since an efficient cell culture system was unavailable for decades after virus discovery, and an animal model is still elusive. Similarly, for many other chronic human viruses, their species specificity restricts the development of immunocompetent animal models critical for meaningful studies of immunity and pathogenesis. This project will use a rodent hepacivirus (RHV) that shares the hallmarks of HCV infection in humans. We will use a bioinformatics approach to alter the dinucleotide frequencies in RHV genomes to generate attenuated variants. Our preliminary results provided a solid rationale for the project and assured its feasibility. We determined that the RHV variant with high frequencies of UpA (uracil followed by adenine) failed to develop a chronic infection in rats. We propose two aims to refine our strategy of generating attenuated hepaciviruses and characterize the innate and adaptive immunity induced by their infection. Aim-1 is to optimize the design of the hepacivirus mutants that produce high-titer viremia and fail to develop chronic infection. Aim-2 is to characterize the innate and adaptive immunity induced by attenuated hepacivirus mutants. Since we will use a sequence-independent approach (only synonymous mutations) to attenuate RHV, the results can inform the design of an HCV vaccine. Additionally, successful attenuation of a chronic virus in its natural host will open new research avenues and yield new viral variants to define immune responses associated with viral clearance and persistence and immune correlates of protection for chronic viruses.