A sequence-independent approach for attenuating hepaciviruses

摘要 人肝病毒或丙型肝炎病毒(丙型肝炎病毒)在约70%的感染者中建立慢性嗜肝感染 个人。在这个项目中，中心问题是：我们能否突变一个肝炎病毒基因组来产生一个 将减毒病毒用作疫苗？致病病毒在体外或体内生物系统中的连续传代 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。丙型肝炎病毒的减毒仍然是不可想象的，因为有效的细胞培养系统不能用于 病毒发现几十年后，动物模型仍然难以捉摸。同样，对于许多其他慢性病患者来说 病毒的物种特异性限制了免疫活性动物模型的发展，这些动物模型对 对免疫和发病机制的有意义的研究。该项目将使用一种啮齿动物肝炎病毒(RHV)，该病毒与 人类感染丙型肝炎病毒的特征。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，以产生减弱的变异体。我们的初步结果提供了一个坚实的 为该项目提供了理由，并确保了其可行性。我们确定了RHV的高频变异株 UPA(尿嘧啶后腺嘌呤)未能在大鼠中形成慢性感染。我们提出了两个目标来完善 我们的减毒肝炎病毒的产生策略和天然免疫和获得性免疫的特征 由它们的感染引起的。目的-1是优化产生高滴度的肝炎病毒突变体的设计 病毒血症，不会发展为慢性感染。目的-2是为了描述先天免疫和获得性免疫 由减毒的肝炎病毒突变体诱导。由于我们将使用与序列无关的方法(仅 为了减弱RHV，研究结果可以为丙型肝炎疫苗的设计提供参考。另外， 慢性病毒在其自然宿主中的成功减毒将开辟新的研究途径并产生新的病毒 用于定义与病毒清除和持久性相关的免疫反应的变体以及与 对慢性病毒的保护。

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.