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.
Hepatitis B virus (HBV) affects the lives of over 400 million chronically infected individuals. Current therapies do not eliminate the virus. Here we propose to investigate the role of the innate immune response against HBV infections that would lead to the development of novel antiviral strategies.
|Michailidis, Eleftherios; Pabon, Jonathan; Xiang, Kuanhui et al. (2017) A robust cell culture system supporting the complete life cycle of hepatitis B virus. Sci Rep 7:16616|
|Xiang, Kuan-Hui; Michailidis, Eleftherios; Ding, Hai et al. (2017) Effects of amino acid substitutions in hepatitis B virus surface protein on virion secretion, antigenicity, HBsAg and viral DNA. J Hepatol 66:288-296|