The role of innate immune response in HBV infection and persistence

 描述（由申请方提供）：尽管有效疫苗可用，但目前仍有超过4亿人慢性感染B型肝炎病毒（HBV），其发展为肝癌的可能性高100倍。获批的抗病毒药可抑制病毒复制，但由于核共价闭合环状DNA（cccDNA）的持续存在，不能消除病毒。cccDNA的形成是生产性HBV感染的标志，并且其在感染细胞的细胞核中长时间维持是慢性的先决条件。这个过程的机制还没有很好的定义，但假设细胞和病毒因素都可能参与其中。先天免疫系统是第一个遇到入侵病毒的系统，并且在病毒感染后几乎普遍检测到其激活。对病毒感染的免疫应答的主要组分是I型和III型干扰素（IFN），其导致称为干扰素刺激基因（ISG）的细胞抗病毒效应物的合成。现有HBV感染系统的局限性使得研究这些过程变得困难。然而，Rice实验室先前的工作已经使得研究HBV感染和在与微图案形式（MPCCs）的基质细胞一起培养的原代肝细胞中诱导先天性免疫应答以及在诱导的多能干细胞衍生的肝细胞样细胞（iHep）中成为可能。该提案的主要目标是研究先天免疫应答在HBV感染和cccDNA维持过程中的作用，并确定干扰或促进HBV感染的相关途径和特定基因或宿主因子。在目的1中，我将在来自不同供体的MPCCs中进行肝细胞转录组学分析，以研究HBV感染过程中基因表达的变化。单细胞应用的发展，如激光捕获显微切割与微阵列分析相结合，将允许区分受感染的旁观者细胞的转录组。此外，CRISPR成像方法的建立将使我们能够识别受感染的细胞并跟踪活细胞中的cccDNA。在目标2中，我将进行高通量筛选，从实验室现有的cDNA文库中鉴定HBV前病毒和抗病毒ISG。Aim1中鉴定的其他基因和宿主因子也将纳入研究。最有效的抗病毒基因将进一步研究其作用机制。我的假设是先天免疫应答在限制HBV感染中起主要作用。重现体内HBV感染的创新体外系统以及新的分子工具和应用为研究HBV感染中的先天免疫应答提供了巨大的希望。这些方法将能够识别参与建立HBV感染和持续性的宿主因素。更好地了解HBV感染的先天免疫可能会导致开发新的治疗策略来消除cccDNA，并最终治愈慢性HBV感染。

 DESCRIPTION (provided by applicant): Despite the availability of an effective vaccine there are currently over 400 million people chronically infected with hepatitis B virus (HBV) that are 100 times more likely to develop liver cancer. Approved antivirals can suppress viral replication but do not eliminate the virus due to persistence of a nuclear covalently closed circular DNA (cccDNA). Formation of the cccDNA is the hallmark of productive HBV infection and its maintenance in the nucleus of infected cells over a long period of time is a prerequisite for chronicity. The mechanism of this process is not well defined but it is hypothesized that both cellular and viral factors may be involved. The innate immune system is the first to encounter an invading virus, and its activation is almost universally detected upon viral infection. Major components of the immune response to viral infections are types I and III interferons (IFN) which lead to synthesis of cellular antiviral effectors called interferon stimulated genes (ISGs). Limitations in the available HBV infection systems have made it difficult to study these processes. However, previous work in the Rice lab has made it possible to study HBV infections and the induction of innate immune responses in primary hepatocytes cultured with stromal cells in a micropatterned format (MPCCs) as well as in induced pluripotent stem cell-derived hepatocyte like cells (iHeps). The main goal of this proposal is to investigate the role of the innate immune response during HBV infection and cccDNA maintenance, and to identify relevant pathways and specific genes or host factors that either interfere with or promote HBV infection. In Aim 1, I will conduct a hepatocyte transcriptomic analysis in MPCCs derived from different donors to study changes in gene expression during the course of HBV infection. Development of single-cell applications such as laser capture microdissection coupled with microarray analysis will allow distinguishing the transcriptome of infected from bystander cells. In addition, establishment of a CRISPR-imaging method will allow us to identify infected cells and to follow the cccDNA in live cells. In Aim 2, I will perform a high-throughput screening to identify HBV proviral and antiviral ISGs from an existing cDNA library in the lab. Other genes and host factors identified in Aim1 will also be included in the study. The most potent antiviral genes will be further studied for their mechanism of action. My hypothesis is that the innate immune response plays a major role in limiting HBV infection. Innovative in vitro systems that recapitulate in vivo HBV infections together with novel molecular tools and applications offer a great promise to study the innate immune responses in HBV infection. These approaches will enable the identification of host factors that are involved in establishment of HBV infection and persistence. A better understanding of innate immunity to HBV infection may lead to the development of novel therapeutic strategies to eliminate cccDNA and ultimately a cure for chronic HBV infection.