Our scientific interest is to better understand the interaction of hepatitis B (HBV) and C (HCV) virus with their host and to develop new (gene) therapeutic strategies to treat chronic viral hepatitis and hepatocellular carcinoma.
The majority of the team is currently concentrating on the question, how HBV is controlled by cellular defense mechanisms and by the systemic immune response, and how HBV manages to escape this immune control. To be able to study this, we have developed new animal and cell culture models of HBV infection, have established cellular infection models for HBV and HCV and use molecular and immunological diagnostic assays to follow the human situation. We currently use HepaRG and HuH7.5 cells as well as primary human hepatocytes as cell culture infection models for HBV and HCV, respectively, have established two HBV-transgenic mouse lines as models for vertical transmission of HBV, and have developed a mouse model of self-limited hepatitis B.
1. Control of HBV infection at the cellular level
(Silke Arzberger, Mathias Broxtermann, Theresa Asen, Xioaming Cheng, Gergor Ebert, Knud Esser, Julie Lucifora)
HBV replication is strictly non-cytopathic and is hardly recognized by the infected cell. Thus, hepatitis B viruses avoid harming their hosts. Since they are optimized to persist life long, these viruses established a well-controlled replication strategy and an intimate cross talk with their host cells. Therefore, genome replication and gene expression of HBV greatly vary in response to extra cellular stimuli such as cytokines or hormones, and in response to the state of the host cell, e.g. cellular differentiation.
To study this, we established preparation and culture of primary hepatocytes from animal and human liver tissue (Protzer 1999, Schulze-Bergkamen 2003, Untergasser 2004, Untergasser 2006) as well as the infectable cell line HepaRG and the novel cell line HepG2-H1.3, which allow us to study the effect of antivirals and interferons on the persistence form of HBV, the nuclear covalently closed circular (ccc) DNA (Jost 2007, Protzer 2007, Quasdorff 2008).
The HBx protein proofed essential to establish and maintain HBV infection (Lucifora, submitted). Since HBx hereby controls transcription from HBV cccDNA we investigate the molecular mechanisms of this control.
We recently showed that HBV is recognized by liver macrophages (Kupffer cells) leading to the secretion of proinflammatory cytokines such as e.g. IL-6. IL-6 was able to block HBV replication at the level of transcription (Hösel 2009) by depleting HNF1a and HNF4a, two essential transcription factors (Quasdorff 2008, Quasdorff 2010). IL6 in addition helps to prevent apoptosis of the infected hepatocyte – which is deadly for the virus (Arzberger 2010).
Interferon treatment and expression very efficiently block HBV replication (Klöcker 2000, Dumortier 2005), but rarely eliminate the virus. An efficient way to induce interferon is triggering nucleic acid recognition receptors such as the cytosolic helicase Rig I. We have shown that siRNAs, which are bi-functional and besides gene silencing trigger Rig I, control HBV very efficiently and long-lasting (Ebert 2010 in revision).
2. Control of HBV infection by the systemic immune response
(Leon Stross, Claudia Dembek, Tanja Bauer)
HBV only infects humans and chimpanzees. Systemic studies on the immune control of infection using HBV in a convenient, well characterized animal model are therefore limited. To overcome the species barrier, we have established adenoviral vectors that transfer replication competent HBV genomes (AdHBV) into a broad range of cultured cells and in vivo into animals (Sprinzl 2001, Sprinzl 2004). Following infection of mice with AdHBV, the mice replicate HBV for 2-3 weeks and develop a T-cell immune response against HBV as well as neutralizing antibodies (Isogawa 2005, John von Freyend 2010). This system now allows for the first time to follow onset and clearance of HBV infection and will help to dissect cellular and molecular mechanisms essential for HBV clearance. Close immune monitoring of patient samples shall confirm and extend the findings in the human situation.
3. Hepatitis B virus based vectors: gene therapy vectors and molecular tools
(Gregor Ebert, Ke Zhang)
Our group developed HBV-based vectors for a liver directed gene transfer (Protzer 1999) and we still hold the patent on these vectors. HBV-based vectors are promising because they specifically target and infect quiescent hepatocytes, express genes in a hepatocyte specific fashion and are non-cytopathic with a favorable ratio of infectious to defective particles.
We established packaging cell lines, which avoid recombination and accidental co-production of wild-type HBV (Klöcker 2003). We completely eliminated HBV gene expression, improved transgene expression levels (Untergasser 2004) and maximize transgene capacity (Ebert submitted). In addition, we successfully used HBV based vectors in collaboration with scientists from the NIH in a preclinical study in chimpanzees and proofed that they are safe and target the liver after intravenous application (Shin 2005). HBV-based vectors, however, also proofed to be a very useful tool for experimental purposes since they allow studying the early steps of HBV infection, namely uptake and release of the viral genome (Klöcker 2000, Untergasser 2006).
4. Interference of HCV with insulin signaling pathways
(Julia Graf, Katrin Singethan)
HCV infected patients have a more than 60% increased risk to develop insulin resistance and type 2 diabetes mellitus if compared to healthy controls and also if compared to patients with HBV infection. Insulin resistance in HCV infected patients is associated with increased liver fibrosis and reduced responsiveness to antiviral therapy. How HCV infection causes insulin resistance is so far unknown.
Hepatic endoplasmic reticulum (ER)-stress is known to induce IR via the activation of JNK. We recently showed that counteracting ER stress in hepatocytes is sufficient to normalize blood glucose levels (Bailly-Maitre 2010). HCV translates a large polyprotein into the ER membrane and several groups describe ER-stress in models with subgenomic HCV replicons. Our aim is to investigate whether ER-stress can explain for IR in HCV infection using the model of full-length HCV replicons and HCV infection of Huh7.5 cells.
5. Testing of novel anti-HBV therapies and HBV drug resistance
(Ke Zhang, Andrea Weicht, Julie Lucifora, Thomas Michler, Yuchen Xia, Christian Bach)
To be able to study anti-HBV therapies, we use HBV replicating cell lines after transient or stable transfection of replication competent HBV genomes, the cell line HepaRG, which can be infected with HBV and supports the complete replication cycle, and HBV transgenic mouse models for in vivo testing.
We established a transfection-based HBV replication assay that allows phenotypic testing of HBV variants isolated from patient material in a 96-well format. The assay determines viral fitness as well as sensitivity or resistance to a given drug. By site directed mutagenesis we generated a series of HBV variants with all known resistance mutations and tested them alone or in combination. Within the BMBF-funded, German-wide consortium HOPE, we join our efforts to phenotype HBV and establish a reliable prediction tool for drug resistance. Together with several external collaborators we test antivirally active peptides, cytokines and shRNAs.
6. Therapeutic vaccination for chronic hepatitis B
(Simone Backes, Clemens Jäger, Raindy Tedjokusumo, Tanja Bauer)
Since we are convinced that immune tolerance needs to be overcome to cure viral hepatitis, we follow strategies of therapeutic vaccination and redirect T cells to infected hepatocytes for T cell-based therapies.
To break tolerance in chronic HBV infection, CD4+ as well as CD8+ effector T cells need to be induced. To block further virus spread and tolerizing effects of HBsAg, neutralizing anti-HBs antibody responses are also desired. HBV transgenic mice proofed to be a suitable model to study this, since they only start to express HBV antigens after birth and therefore allow breaking tolerance against HBV envelop and core proteins (Quasdorff unpublished). In close collaboration with Rheinbiothech/Dynavax Europe, we recently showed that a properly formulated, particulate HBV vaccine can induce anti-HBs seroconversion in HBVtg mice as well as a CD8+ effector T cell response (Buchmann unpublished). In addition, we exploit MVA-based vaccination schemes.
7. Development of T cell based therapeutic strategies for viral hepatitis and hepatocellular carcinoma
(Karin Krebs, Nina Böttinger, Sarah Kutscher, Martin Sprinzl)
An efficient T cell response is essential to control and finally clear HBV infection. We therefore follow two different strategies to induce an efficient HBV specific immune response. On the one hand, we have an industrial collaboration to develop and test suitable adjuvant compositions to break tolerance in chronic HBV infection.
On the other hand, we graft primary T cells with T cell receptors (TCR) to retarget them against HBV infected cells. Therefore, TCR are currently cloned from patients with resolved HBV infection. Alternatively, we use chimeric antigen receptors (CAR), which are composed of intracellular TCR signaling domains and „single chain” antibody fragments (scFv) directed against HBV envelope proteins recognizing these HBV proteins on infected cells independent of peptide presentation on MHC. We recently showed that this allows the generation of primary human T cells, which are activated by and kill HBV infected hepatocytes of the same donor (Bohne et al. 2008).