有关B型肝炎的研究, 病毒学研究所，慕尼黑工业大学，慕尼

StephenW

有关B型肝炎的研究, 病毒学研究所，慕尼黑工业大学，慕尼黑，德国,的一些简介。

他们研究的一个新的B肝治疗:

http://www.ncbi.nlm.nih.gov/pubmed/21684282
Gastroenterology. 2011 Aug;141(2):696-706, 706.e1-3. Epub  2011 May 13.
5' Triphosphorylated small interfering RNAs control replication of hepatitis B virus and induce an interferon response in human liver cells and mice.
Ebert G, Poeck H, Lucifora J, Baschuk N, Esser K, Esposito I, Hartmann G, Protzer U.
SourceInstitute of Virology, Technische Universität München/Helmholtz Zentrum München, Munich, Germany.

Abstract
BACKGROUND & AIMS: Approved therapies for chronic hepatitis B include systemic administration of interferon (IFN)-alfa and inhibitors of hepatitis B virus (HBV) reverse-transcription. Systemic application of IFN-alfa is limited by side effects. Reverse-transcriptase inhibitors effectively control HBV replication, but rarely eliminate the virus and can select drug-resistant variants. We aimed to develop an alternative therapeutic approach that combines gene silencing with induction of IFN in the liver.
METHODS: To stimulate an immune response while inhibiting HBV activity, we designed 3 small interfering (si)RNAs that target highly conserved sequences and multiple HBV transcripts of all genotypes. A 5'-triphosphate (3p) was added to the siRNAs, turning them into a ligand for the cytosolic helicase retinoic acid-inducible protein I, which becomes activated and induces expression of type-I IFNs. Antiviral activity was investigated in cell lines that replicate HBV, in HBV-infected primary human hepatocytes, and in HBV transgenic mice.
RESULTS: 3p-double-stranded RNA (3p-RNA) activated retinoic acid-inducible protein I, induced a strong type I IFN response (expression of IFN-β) in liver cells and showed transient but strong antiviral activity. Bifunctional, HBV-specific, 3p-siRNAs controlled replication of HBV more efficiently and for longer periods of time than 3p-RNAs without silencing capacity or siRNAs that targeted identical sequences but did not contain 3p.
CONCLUSIONS: HBV-specific 3p-siRNAs are bifunctional antiviral molecules that induce production of type I IFNs in the liver and target HBV RNAs to inhibit viral replication.
Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

StephenW

消化科。 2011 08期141（2）:696 - 706，706.e1 - 3。 EPUB 2011 5月13日。
5'Triphosphorylated小分子干扰核糖核酸控制乙肝病毒的复制，在人类肝细胞和小鼠，诱导干扰素反应。
Baschuk ñ Poeck埃伯特G，H，Lucifora J，K ESSER，埃斯波西托我，哈特曼摹，Protzer U。
来源

病毒学研究所，慕尼黑工业大学/亥姆霍兹慕尼黑中心，慕尼黑，德国。
摘要
背景及目的：

批准治疗慢性乙型肝炎的治疗包括全身给药干扰素（IFN）-α和B型肝炎病毒（HBV）逆转录酶抑制剂。全身应用IFN -阿尔法是有限的副作用。逆转录酶抑制剂有效控制乙肝病毒复制，但很少消灭病毒，并可以选择抗药性的变种。我们的目的是开发一种替代治疗方法，它结合了在肝脏中干扰素诱导基因沉默。
方法：

为了刺激免疫反应，同时抑制乙肝病毒的活动，我们设计了3个小干扰（SI）RNA的目标高度保守的序列和多个乙肝病毒的所有基因型的成绩单。添加5' -三磷酸腺苷（3P）的siRNA，变成一个配体胞质的解旋酶维甲酸诱导蛋白，我被激活并诱导的I型干扰素的表达。复制乙肝病毒，HBV感染的主要人体肝细胞，并在HBV转基因小鼠的细胞株的抗病毒活性进行了调查。
结果：

3P -双链RNA（3P - RNA）激活维甲酸诱导蛋白我，我干扰素的反应（IFN​​ -β的表达）在肝细胞诱导强类型和显示瞬态但强大的抗病毒活性。双功能，HBV特异性，3P - siRNA的控制HBV的复制更有效率和更长的时间比没有沉默，有针对性的相同的序列，但不包含3P的能力或siRNA 3P - RNA的。
结论：

HBV特异性3P - siRNA的双功能抗病毒的分子，促使我在肝脏和目标乙肝病毒的RNA干扰素抑制病毒复制的类型生产。

StephenW

http://www.virologie.med.tu-muenchen.de/en/forschung-tum/ag-protzer/forschungsthemen/      
Research topics
                        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.

Research topics1.
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).
        

Date last modified: Wednesday August 18th, 2010

StephenW

研究课题

我们的科学兴趣，是为了更好地了解肝炎病毒（HBV）和C（HCV）病毒与其宿主的相互作用，并开发新的治疗策略（基因）治疗慢性病毒性肝炎和肝细胞癌。

大多数团队目前集中的问题，乙肝病毒是如何控制细胞的防御机制和全身免疫反应，以及如何乙肝设法逃脱这种免疫控制。为了能够研究，我们已经开发出新的乙肝病毒感染的动物和细胞培养模型，建立了乙型肝炎病毒和丙型肝炎病毒细胞感染模型，并利用分子生物学和免疫学诊断方法，按照人类的情况。我们目前使用HepaRG和HuH7.5细胞，以及细胞培养模型，分别为乙型肝炎和丙型肝炎感染的主要人肝细胞，建立了两个HBV基因乙肝病毒母婴垂直传播模型的鼠标线，并已制定的小鼠模型自有限的B型肝炎
研究课题
1。在细胞水平上乙肝病毒感染的控制

（西尔克Arzberger，恩威Broxtermann，特里萨阿森，Xioaming程，Gergor艾伯特，克努兹ESSER，朱莉Lucifora）

乙肝病毒复制的是严格的非细胞病变，是很难被感染的细胞确认。因此，乙肝病毒，避免伤害他们的主机。这些病毒，由于他们是优化要坚持终身，建立一个良好的控制复制策略与宿主细胞的亲密串扰。因此，乙肝病毒基因组复制和基因表达的差异很大反应，如细胞因子或激素的细胞外刺激，并在宿主细胞的状态，例如细胞分化。

要研究这个问题，我们建立了从动物和人体肝脏组织（，Untergasser 2006年，Untergasser 2004年，2003年Bergkamen舒尔茨，1999年Protzer）以及infectable细胞线HepaRG和新的细胞株HepG2 - H1，原代肝细胞的制备和文化。 3，这使我们能够研究HBV的核共价闭合的圆形（CCC）DNA（约斯特2007年，Protzer 2007年，Quasdorff 2008）的持久形式的抗病毒药物和干扰素的效果。

HBx蛋白隔音必须建立和维护乙肝病毒感染（Lucifora提交）。由于HBx的现控制乙肝病毒cccDNA的转录，我们调查这个控制的分子机制。

最近，我们发现，乙肝病毒是导致炎性细胞因子的分泌，比如：肝巨噬细胞（枯否细胞）的认可IL - 6。 IL - 6是能够阻止乙肝病毒复制，在转录水平（杆颈2009）消耗HNF1A HNF4a，两个重要的转录因子（Quasdorff 2008年，Quasdorff 2010）。除了IL - 6，有助于防止受感染的肝细胞凋亡的 - 这是致命的病毒（Arzberger 2010）的。

干扰素治疗和表达非常有效地阻止乙肝病毒复制（Klöcker 2000，Dumortier 2005年），但很少消除病毒。我们已经表明的一种有效的方式来诱导干扰素引发胞质解旋钻机一，如核酸识别受体的siRNAs，这是非常有效和持久的双功能除了基因沉默触发钻机我，控制乙肝（埃伯特2010在修订版）。
2。控制乙肝病毒感染的全身免疫反应

（莱昂斯特罗斯，克劳迪亚Dembek，鲍尔的Tanja）

乙肝病毒只感染人类和黑猩猩。因此，有限的方便，以及特点的动物模型中使用乙肝病毒感染的免疫控制的系统性研究。为了克服物种屏障，我们已经建立了腺病毒载体转移复制主管乙型肝炎病毒基因组（AdHBV）广泛的培养细胞到动物体内（Sprinzl 2001年，Sprinzl 2004年）。感染小鼠AdHBV，小鼠复制2-3周HBV和发展了T细胞对HBV的免疫反应，以及（Isogawa 2005年，2010年约翰冯Freyend）中和抗体。现在这个系统可以在第一时间跟进的清除乙肝病毒感染和发病的情况，将有助于解剖细胞和分子机制，对HBV的清除至关重要。关闭免疫监测患者的样本应确认和扩大在人权状况的调查结果。
3。基于B型肝炎病毒载体基因治疗载体和分子工具

（格里艾伯特，张克）

我们集团发展为肝定向基因转移（Protzer 1999年）HBV为基础的载体，我们仍然认为这些载体的专利。 HBV为基础的载体是有希望的，因为他们明确的目标和感染静态的肝，在肝细胞的特定的方式表达的基因，并非是一个有利的比率传染病缺陷颗粒细胞病变。

我们建立了包装细胞系，从而避免重组和意​​外野生型HBV（Klöcker 2003年）合作生产。我们完全消除乙肝病毒基因的表达，提高转基因表达水平（Untergasser 2004年），并最大限度地提高转基因的能力（艾伯特提交）。此外，我们成功地使用从美国国立卫生研究院的科学家合作，在临床前研究黑猩猩HBV基于向量和隔音，他们是安全和肝后静脉应用目标（新2005）。然而，乙肝病毒的载体，也隔音实验目的是一个非常有用的工具，因为它们允许研究乙肝病毒感染，即吸收和释放的病毒基因组（Klöcker 2000，Untergasser 2006年）的早期步骤。
4。丙型肝炎病毒的干扰胰岛素信号通路

（朱格拉夫，卡特琳Singethan）

丙型肝炎病毒感染者已超过60％的风险增加，开发胰岛素抵抗和2型糖尿病，如果健康对照相比，也比与乙肝病毒感染的病人。丙型肝炎病毒感染患者的胰岛素抵抗与肝纤维化的增加和减少抗病毒治疗的反应。如何丙型肝炎病毒感染引起的胰岛素抵抗是迄今未知的。

肝脏内质网（ER）应激诱导IR通过JNK活化。最近，我们发现，抵制在肝细胞内质网压力是足够的血糖水平（2010年贝利 - 贝耶）正常化。丙型肝炎转化成一个大聚在ER膜和几组描述与丙型肝炎病毒亚基因组复制子模型ER应力。我们的目的是探讨ER应力是否可以解释丙型肝炎病毒感染，使用红外全长的丙型肝炎病毒复制子和丙型肝炎病毒感染Huh7.5细胞模型。
5。新颖的抗乙肝病毒治疗和抗乙肝病毒药物测试

（张克，安德烈Weicht，朱莉Lucifora，托马斯米氏，雨辰夏，基督徒Bach）

为了能够研究抗乙肝病毒治疗中，我们使用后，主管乙型肝炎病毒基因组复制，细胞系HepaRG，可感染HBV和支持，完整的复制周期，和HBV转基因小鼠模型的瞬时或稳定转乙肝病毒复制的细胞株在体内试验。

我们建立了一个基于转染乙肝病毒复制检测，允许在96孔板格式的表型检测从病人物质中分离出的乙肝病毒的变种。检测确定病毒的健身以及一个给定的药物的敏感性或抵抗。由网站定向诱变，我们产生了一系列与所有已知的耐药突变的乙肝病毒的变种，他们单独或联合测试。在BMBF的资助，德国宽财团希望，我们加入我们的努力，乙肝病毒的表型和耐药性建立一个可靠的预测工具。加上一些外部合作者，我们测试antivirally活性肽，细胞因子的shRNA。
6。治疗慢性B型肝炎疫苗接种

（西蒙Backes，克莱门斯耶格尔，Raindy Tedjokusumo，鲍尔的Tanja）

由于我们深信需要克服免疫耐受治疗病毒性肝炎，我们遵循的治疗性疫苗接种战略和重定向T细胞T细胞为基础的治疗感染的肝细胞。

打破慢性乙肝病毒感染的宽容，需要的CD4 +以及CD8 +效应T细胞被诱导。要阻止进一步传播的病毒和乙肝表面抗原的tolerizing影响，中抗- HBs抗体反应也理想。 HBV转基因小鼠隔音是一个合适的模型来研究，因为它们只在出生后开始表达HBV抗原，因此允许对乙肝病毒包膜和核心蛋白​​（Quasdorff未出版）打破宽容。在与Rheinbiothech / Dynavax欧洲的密切合作，我们最近发现，正确制定，颗粒乙肝疫苗可以诱导小鼠HBVtg的抗- HBs血清转换，以及一个CD8 +效应T细胞的反应（Buchmann未出版）。此外，我们利用基于MVA的疫苗接种计划。
7。开发基于T细胞病毒肝炎和肝癌的治疗策略

（卡琳克雷布斯，Böttinger龚如心，Sprinzl马丁，莎拉Kutscher）

一个有效的T细胞的反应是必要的控制和最终明确HBV感染。因此，我们遵循两种不同的策略，以促使一个高效的HBV特异性免疫反应。一方面，我们的工业合作，以开发和测试合适的辅助成分，打破慢性乙肝病毒感染的宽容。

另一方面，我们与T细胞受体（TCR）的主要的T细胞移植重定向他们对乙肝病毒感染的细胞。因此，TCR目前克隆解决乙肝病毒感染患者。另外，我们使用嵌合抗原受体（CAR），它是细胞内的TCR信号域的“单链”抗体片段（scFv的）针对HBV的信封承认独立的感染细胞的MHC肽介绍这些乙肝病毒蛋白的蛋白质组成。最近，我们发现，这使得小学人类T细胞的生成，激活和杀死乙肝病毒感染的肝细胞相同的捐助（Bohne等人，2008年）。
最后修改日期：2010年8月18日，日（星期三）