http://www.virologie.med.tu-muen ... ung-tum/ag-protzer/ Prof. Ulrike Protzer, M.D. Institute of Virology TU Munich Trogerstr. 30 81675 München Germany . Our main scientific interests are to better understand the interaction of hepatitis B virus (HBV) with its 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 defence mechanisms and by the systemic immune response, and how HBV manages to escape this immune control. To be able to study these, we have developed new animal and cell culture models of HBV infection and new molecular diagnostic assays. We have established two HBV-transgenic mouse models as models for a vertical transmission, and have developed a mouse model of self-limited hepatitis B. Based on an improved basic understanding of the control of HBV infection we are developing new treatment strategies to treat chronic viral hepatitis. We for example study the molecular mechanisms of antiviral effects of cytokines and other immune mediators, and investigate the possibility of a therapeutic cytokine gene transfer. Since we are convinced that immune tolerance needs to be overcome to cure chronic infection, we also follow strategies of therapeutic vaccination and try to redirect T cells to infected cells by generating recombinant T cell receptors. By grafting them onto primary T cells we follow a novel strategy in the treatment of infectious diseases. A second focus of our group is on developing new vectors systems for a liver directed gene transfer. As the first group worldwide, we managed to generate HBV based viral vectors and hold the patent on these vectors. Recently, we successfully used HBV based vectors in collaboration with scientists from the NIH in a preclinical study in chimpanzees. Research topics 1. Anti-HBV activity of cytokines and other immune mediators A strict tissue tropism and a very narrow host range characterize all hepatitis B viruses. Viral 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 establish persistent, often life long infection, these viruses use a well-balanced replication strategy and an intimate cross talk with their hosts. 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. the cellular differentiation state. HBV as well as hepatitis C virus (HCV) replication have been shown to be extremely sensitive to antiviral cytokines e.g. interferon alpha and gamma. Using adenoviral vectors which allow a liver specific and regulated cytokine gene expression, we found that expression of interferon gamma very efficiently blocks HBV replication but at a posttranscriptional step (Dumortier 2005). This we had already observed in the duck model of HBV infection, where mediators secreted by liver macrophages upon stimulation with LPS inhibited viral replication (Klöcker 2000). A gene transfer of constitutively active STAT-1 did hardly affected HBV replication at all (Siebler 2006). These results indicate that HBV replication is non-cytopathically suppressed by interferons, but interferon induced pathways are not sufficient to eliminate the virus. At the moment, we are investigating whether the sensitivity towards interferons may be a viral escape mechanism allowing the viruses to hide from cytotoxic T cells rather than an efficient immune control mechanism. In addition, we developed a new cell line, which allows 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). 2. Control of HBV infection by the systemic immune response HBV only infects humans and chimpanzees. Systemic studies on the immune control of infection in a convenient, well characterized animal model were so far limited. To systematically study immune control of HBV infection, we promoted the development of a mouse model of self-limiting hepatitis B. 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 via the tail vein, the mice replicate HBV for several weeks and develop a T-cell immune response against HBV as well as neutralizing antibodies (Isogawa 2005, Svorcova in preperation ). This system now allows for the first time to follow onset and clearance of HBV infection and to test new prevention strategies in a small animal model. Using the system in cooperation with Prof. T.J. Liang, NIH, Bethesda, USA, we found that HBV X-protein interacts with the proteasome, and that this interaction is essential for the viral life cycle (Zhang 2004). In cooperation with Prof. G. Tiegs, University of Erlangen, we showed using the AdHBV system that heme oxygenase I reduces HBV associated liver damage and in addition is antivirally active in vivo (Protzer 2007). 3. Interaction of HBV with cell autonomous defence We have a broad experience with preparation and culture of primary hepatocytes from animal and human liver tissue (Protzer 1999, Schulze-Bergkamen 2003, Untergasser 2004, Untergasser 2006). HBV replication essentially depends on the differentiation state of the host cell. Hereby, a concerted action of transcription factors HNF1a and HNF4a is essential for efficient HBV replication (Quasdorff, Cellular Micobiology in revision). Together with the groups of Prof. P. Krammer and Dr. M. Müller-Schilling, Heideberg, we showed that primary human hepatocytes are not only suited to study HBV infection, but allow studying apoptosis signalling pathways better than hepatoma cells. In addition, we showed that HBV infection does not induce spontaneous apoptosis of hepatocytes (Schulze-Bergkamen 2003). Intact CD95 pathway, however, seem to be essential to allow immune control of HBV. Although HBV is thought to be a stealth virus, we have good evidence that it is recognized by pattern recognition receptors and activates NFkB dependent cellular defence pathways, which we currently analyse in detail. 4. Hepatitis B virus based vectors: gene therapy vectors and molecular tools Using vectors based on HBV is a promising new concept for liver directed gene therapy because HBV specifically targets and infects quiescent hepatocytes, expresses genes in a hepatocyte specific fashion and is a non-cytopathic virus with a favourable ratio of infectious to defective particles. As the first group worldwide, we succeeded to convert HBV into a viral vector allowing hepatocyte specific gene transfer. Expression of an interferon type I, a secreted protein of therapeutic use, by HBV based vectors interfered with the replication of resident virus. With this, we have established the potential use of local cytokine production as a new concept for the treatment of acquired liver diseases such as chronic viral hepatitis (Protzer 1999). Recently, we managed to establish packaging cell lines which allow to avoid recombination and accidental co-production of wild-type HBV (Klöcker 2003). In addition, we succeeded to completely eliminate HBV gene expression and to improve transgene expression levels (Untergasser 2004). We could perform a first preclinical evaluation of human interferon gamma expressing vectors in chimpanzees in collaboration with Barbara Rehermann’s group from the NIH (Shin 2005). HBV-based vectors, however, are not only candidate gene therapy vectors, but also proofed to be a very useful tool for experimental purposes since they allow to study the early steps of HBV infection, namely uptake and release of the viral genome (Klöcker 2000, Untergasser 2006). 5. Development of T cell based therapeutic strategies 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 adjuvans compositions to break tolerance in chronic HBV infection. On the other hand, we graft primary T cells with artificial T cell receptors (TCR) to retarget them against HBV infected cells. These TCR use „single chain” antibody fragments (scFv) directed against HBV envelope proteins to recognize these HBV proteins on infected cells independent of MHC dependent antigen presentation. We recently showed that this allows the generation of T cells which are activated by and kill HBV infected cells (Bohne et al. 2008). Selected Publications 1. Bohne F, Chmielewski M, Ebert G, Wiegmann K, Kürschner T, Schulze A, Urban S, Krönke M, Abken H, Protzer U. T cells Redirected Against Hepatitis B Virus Surface Proteins Eliminate Infected Hepatocytes. Gastroenterology 2008, 134: 239-47. 2. Jost S, Turelli P, Mangeat B, Protzer U, Trono D. Induction of antiviral cytidine deaminases does not explain the inhibition of hepatitis B virus replication by interferons. J Virology 2007; 81:10588-96. 3. Protzer U, Seyfried S, Quasdorff M, Sass G, Svorcova M, Webb D, Bohne F, Hösel M, Schirmacher P, Tiegs G. Antiviral activity and hepatoprotection by heme oxygenase-1 in hepatitis B virus infection Gastroenterology 2007, 133: 1156-65. 4. Untergasser A, Zedler U, Langenkamp A, Hösel M, Quasdorff M, Esser K, Tapperzhofen B, Kolanus W and Protzer U. Dendritic cells take up viral antigens but do not support the early steps of hepatitis B virus infection. Hepatology 2006, 43: 539-547. 5. Siebler J, Protzer U, Wirtz S, Schuchmann M, Hohler T, Galle PR, Neurath MF. Overexpression of STAT-1 by adenoviral gene transfer does not inhibit hepatitis B virus replication.Eur J Gastroenterol Hepatol. 2006,18:167-74. 6. Shin E-C, Protzer U, Untergasser A, Feinstone SM, Rice CM, Hasselschwert D, Rehermann B. Liver-Directed Interferon-? Gene Delivery in Chronic Hepatitis C. Journal of Virology 2005, 79:13412-13420. 7. Isogawa M, Kakimi K, Kamamoto H, Moriyasu F, Protzer U and Chisari FV. Differential Dynamics of the Peripheral and Intrahepatic Cytotoxic T Lymphocyte Response to a Secreted Viral Antigen Produced in the Liver. Virology 2005, 333: 293-300. 8. Dumortier J, Schönig K, Giese T, Bujard H, Protzer U. The Tet-system allows tight control and liver-specific gene expression in vivo following adenoviral gene transfer into mice. Gene Therapy 2005, 12:668-677. 9. Sprinzl M, Dumortier J, Protzer U. Construction of recombinant adenoviruses that produce infectious HBV. In: R. Hamatke, J. Lau (eds.). Methods in Molecular Medicine. „Hepatitis B Virus Protocols”. The Humana Press Inc., Totowa NJ, 2004, 96:203-218. 10. Zhang Z, Protzer U, Hu Z, Jacob J, Liang TJ. Inhibition of cellular proteasome activities enhances hepadnaviral replication in a HBx dependent manner. Journal of Virology 2004, 78: 4566-4572. 11. Untergasser A & Protzer U. Hepatitis B virus vectors allow elimination of viral gene expression and insertion of foreign promoters. Human Gene Therapy 2004, 15: 203-210. 12. Schulze-Bergkamen H, Untergasser A, Dax A, Vogel H, Büchler P, Klar E, Lehnert T, Friess H, Büchler M, Kirschfink M, Stremmel W, Krammer P, Müller M, Protzer U. Primary human hepatocytes - a valuable tool for investigation of apoptosis and hepatitis B virus infection Journal of Hepatology 2003, 38: 736-744. 13. Klöcker U, Kürschner T, Oberwinkler H, Protzer U. Presence of replicating virus in recombinant hepadnavirus stocks results from homologous recombination and can be eliminated be the use of a packaging cell line. Journal of Virology 2003, 77 (5): 2873-2881. 14. Sprinzl M, Oberwinkler H, Schaller H, Protzer U. Hepatitis B virus genome transfer with adenoviral vectors into cultured cells and mice: crossing the species barrier. Journal of Virology 2001, 75: 5108-5118. 15. Klöcker U, Schultz U, Schaller H, Protzer U. Liver macrophages release mediators after endotoxin stimulation that inhibit an early step in hepadnavirus replication. Journal of Virology 2000, 74 (12): 5525-5533. 16. Protzer U & Schaller H. Immune Escape of Hepatitis B Viruses. Virus Genes 2000, 21: 27-37. 17. Protzer U, Nassal M, Chiang PE, Kirschfink M, Schaller H. Interferon gene transfer by a novel hepatitis B virus vector efficiently suppresses wildtype-virus infection. PNAS 1999, 96: 10818-10823. 18. Protzer U, Naumann U, Bartenschlager R, Berg T, Hopf U, Meyer zum Büschenfelde KH, Neuhaus P, Gerken G. Hepatitis B virus with antigenically altered hepatitis B surface antigen is selected by high-dose hepatitis B immune globulin after liver transplantation. Hepatology 1998, 27(1): 254-263. Patents „Methods and Compositions for Expressing Heterologous Genes in Hepatocytes using Hepadnaviral Vectors”, Heinz Schaller, Ulrike Protzer, Michael Nassal European Patent No. 1108050 U.S. Application No. 60/098,173, 26.8.1998; PCT/US99/19452, 26.8.1999 (WO 00/12739) Bigben 发表于 2009-10-8 19:56 |
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