The role of innate immune response in HBV infection and persistence

 描述(由申请人提供)：尽管有有效的疫苗可用，但目前仍有超过4亿慢性乙肝病毒(乙肝)感染者患肝癌的可能性是后者的100倍。批准的抗病毒药物可以抑制病毒复制，但由于核共价闭合环状DNA(CccDNA)的持续存在而不能消除病毒。CccDNA的形成是生产性乙肝病毒感染的标志，它在感染细胞的细胞核中长期维持是慢性感染的先决条件。这一过程的机制尚不清楚，但据推测，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，在病毒感染时，几乎所有人都能检测到它的激活。对病毒感染的免疫反应的主要成分是I型和III型干扰素(干扰素)，它们导致合成称为干扰素刺激基因(ISGs)的细胞抗病毒效应器。现有的乙肝病毒感染系统的局限性使得研究这些过程变得困难。然而，莱斯实验室之前的工作已经使研究乙肝病毒感染和在以微图案形式的基质细胞(MPCCs)培养的原代肝细胞以及在诱导的多能干细胞来源的肝细胞样细胞(IHEPS)中诱导先天免疫反应成为可能。本研究的主要目的是探讨先天免疫反应在乙肝病毒感染和cccDNA维持中的作用，并找出干扰或促进乙肝病毒感染的相关途径和特异性基因或宿主因子。在目标1中，我将对不同供体来源的MPCCs进行肝细胞转录分析，以研究乙肝病毒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，将使受感染的转录组与旁观者细胞区分开来。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文库中鉴定乙肝病毒前病毒和抗病毒ISG。在Aim1中确定的其他基因和宿主因子也将包括在研究中。最有效的抗病毒基因将进一步研究它们的作用机制。我的假设是，先天免疫反应在限制乙肝病毒感染方面起着主要作用。创新的体外系统，结合新的分子工具和应用，概括了体内的乙肝病毒感染，为研究乙肝病毒感染的先天免疫反应提供了巨大的希望。这些方法将能够确定与确定乙肝病毒感染和持久性有关的宿主因素。更好地了解对乙肝病毒感染的先天免疫可能导致新的治疗策略的发展，以消除cccDNA，并最终治愈慢性乙肝病毒感染。

 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.