Publications by authors named "Daniela Stadler"

12 Publications

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T cell engager antibodies enable T cells to control HBV infection and to target HBsAg-positive hepatoma in mice.

J Hepatol 2021 Jun 23. Epub 2021 Jun 23.

Institute of Virology, School of Medicine, Technical University of Munich, Helmholtz Zentrum München, Munich, Germany; German Center for Infection Research (DZIF), Munich and Hamburg Partner sites, Germany. Electronic address:

Background & Aims: Current antiviral therapies control but rarely eliminate HBV, leaving chronic HBV carriers at risk of developing hepatocellular carcinoma (HCC). Lacking or dysfunctional virus-specific adaptive immunity prevents control of HBV and allows the virus to persist. Restoring antiviral T cell immunity could lead to HBV elimination and cure of chronically infected patients.

Methods: We constructed bispecific T cell engager antibodies that are designed to induce antiviral immunity through simultaneous binding of HBV envelope proteins (HBVenv) on infected hepatocytes and CD3 or CD28 on T cells. T cell engager antibodies were employed in co-cultures with healthy donor lymphocytes and HBV-infected target cells. Activation of the T cell response was determined by detection of pro-inflammatory cytokines, effector function (by cytotoxicity) and antiviral effects. To study in vivo efficacy, immune-deficient mice were transplanted with HBVenv-positive and -negative hepatoma cells.

Results: The 2 T cell engager antibodies synergistically activated T cells to become polyfunctional effectors that in turn elicited potent antiviral effects by killing infected cells and in addition controlled HBV via non-cytolytic, cytokine-mediated antiviral mechanisms. In vivo in mice, the antibodies attracted T cells specifically to the tumors expressing HBVenv resulting in T cell activation, tumor infiltration and reduction of tumor burden.

Conclusion: This study demonstrates that the administration of HBVenv-targeting T cell engager antibodies facilitates a robust T cell redirection towards HBV-positive target cells and provides a feasible and promising approach for the treatment of chronic viral hepatitis and HBV-associated HCC.

Lay Summary: T cell engager antibodies are an interesting, novel therapeutic tool to restore immunity in patients with chronic hepatitis B. As bispecific antibodies, they bind envelope proteins on the surface of the hepatitis B virus (HBV) and CD3 or CD28 on T cells. This way, they induce a potent antiviral and cytotoxic T cell response that leads to the elimination of HBV-positive cells. These bispecific T cell engager antibodies are exciting therapeutic candidates for chronic hepatitis B and HBV-associated hepatocellular carcinoma.
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http://dx.doi.org/10.1016/j.jhep.2021.06.022DOI Listing
June 2021

Interferon-induced degradation of the persistent hepatitis B virus cccDNA form depends on ISG20.

EMBO Rep 2021 06 9;22(6):e49568. Epub 2021 May 9.

Institute of Virology, School of Medicine, Technical University of Munich / Helmholtz Zentrum München, Munich, Germany.

Hepatitis B virus (HBV) persists by depositing a covalently closed circular DNA (cccDNA) in the nucleus of infected cells that cannot be targeted by available antivirals. Interferons can diminish HBV cccDNA via APOBEC3-mediated deamination. Here, we show that overexpression of APOBEC3A alone is not sufficient to reduce HBV cccDNA that requires additional treatment of cells with interferon indicating involvement of an interferon-stimulated gene (ISG) in cccDNA degradation. Transcriptome analyses identify ISG20 as the only type I and II interferon-induced, nuclear protein with annotated nuclease activity. ISG20 localizes to nucleoli of interferon-stimulated hepatocytes and is enriched on deoxyuridine-containing single-stranded DNA that mimics transcriptionally active, APOBEC3A-deaminated HBV DNA. ISG20 expression is detected in human livers in acute, self-limiting but not in chronic hepatitis B. ISG20 depletion mitigates the interferon-induced loss of cccDNA, and co-expression with APOBEC3A is sufficient to diminish cccDNA. In conclusion, non-cytolytic HBV cccDNA decline requires the concerted action of a deaminase and a nuclease. Our findings highlight that ISGs may cooperate in their antiviral activity that may be explored for therapeutic targeting.
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http://dx.doi.org/10.15252/embr.201949568DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8183418PMC
June 2021

Efficient and reproducible depletion of hepatitis B virus from plasma derived extracellular vesicles.

J Extracell Vesicles 2020 12 22;10(2):e12040. Epub 2020 Dec 22.

Institute of Virology School of Medicine Helmholtz Zentrum München/Technical University of Munich Garching Germany.

Extracellular vesicles (EVs) are emerging fundamental players in viral infections by shuttling viral components, mediating immune responses and likely the spread of the virus. However, the obstacles involved in purifying EVs and removing contaminating viral particles in a reliable and effective manner bottlenecks the full potential for the development of clinical and diagnostic treatment options targeting EV. Because of the similarities in size, density, membrane composition and mode of biogenesis of EVs and virions there are no standardized approaches for virus-removal from EV preparations yet. Functional EV studies also require EV samples that are devoid of antibody contaminants. Consequently, the study of EVs in virology needs reliable and effective protocols to purify EVs and remove contaminating antibodies and viral particles. Here, we established a protocol for EV purification from hepatitis B virus (HBV)-containing plasma by a combination of size-exclusion chromatography and affinity-based purification. After purification, EV samples were free of virus-sized particles, HBV surface antigen, HBV core antigen, antibodies or infectious material. Viral genomic contamination was also decreased following purification. By using appropriate antibodies and size parameters, this protocol could potentially be applied to purification of EVs from other viral samples. In summary, we established a fast, reproducible and robust approach for the removal of HBV from EV preparations. Looking forward to the point of purifying EVs from clinical samples, this method should enable studies shedding light on the underlying mechanisms of EVs in viral infections and their diagnostic and prognostic potential.
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http://dx.doi.org/10.1002/jev2.12040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7754750PMC
December 2020

Comparative Analysis of the Antiviral Effects Mediated by Type I and III Interferons in Hepatitis B Virus-Infected Hepatocytes.

J Infect Dis 2019 07;220(4):567-577

Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Munich.

Background: Type III interferons (IFNs) (λ1-3) activate similar signaling cascades as type I IFNs (α and β) via different receptors. Since IFN-α and lymphotoxin-β activate cytosine deamination and subsequent purging of nuclear hepatitis B virus (HBV) DNA, we investigated whether IFN-β and -λ may also induce these antiviral effects in differentiated HBV-infected hepatocytes.

Methods: After determining the biological activity of IFN-α2, -β1, -λ1, and -λ2 in differentiated hepatocytes, their antiviral effects were analyzed in HBV-infected primary human hepatocytes and HepaRG cells.

Results: Type I and III IFNs reduced nuclear open-circle DNA and covalently closed circular DNA (cccDNA) levels in HBV-infected cells. IFN-β and -λ were at least as efficient as IFN-α. Differential DNA-denaturing polymerase chain reaction and sequencing analysis revealed G-to-A sequence alterations of HBV cccDNA in IFN-α, -β, and -λ-treated liver cells indicating deamination. All IFNs induced apolipoprotein B messenger RNA-editing enzyme-catalytic polypeptide-like (APOBEC) deaminases 3A and 3G within 24 hours of treatment, but IFN-β and -λ induced longer-lasting expression of APOBEC deaminases in comparison to IFN-α.

Conclusions: IFN-β, IFN-λ1, and IFN-λ2 induce cccDNA deamination and degradation at least as efficiently as IFN-α, indicating that these antiviral cytokines are interesting candidates for the design of new therapeutic strategies aiming at cccDNA reduction and HBV cure.
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http://dx.doi.org/10.1093/infdis/jiz143DOI Listing
July 2019

PASylated interferon α efficiently suppresses hepatitis B virus and induces anti-HBs seroconversion in HBV-transgenic mice.

Antiviral Res 2019 01 12;161:134-143. Epub 2018 Nov 12.

Institute of Virology, Technische Universität München/Helmholtz Zentrum München, 81675, Munich, Germany; German Center for Infection Research (DZIF), Munich Partner Site, 81675, Munich, Germany. Electronic address:

Interferon α (IFNα) so far is the only therapeutic option for chronic hepatitis B virus (HBV) infection that can lead to virus clearance. Unfortunately, its application is limited by side effects and response rates are low. The aim of this study was to generate a novel long-acting IFNα with the help of PASylation technology that adds a polypeptide comprising Proline, Alanine and Serine (PAS) to increase plasma half-life. Following evaluation of four selected recombinant murine IFNα (mIFNα) subtypes in cell culture, the most active subtype, mIFNα11, was fused with a 600 amino acid PAS chain. The activity of PAS-mIFNα was assessed by interferon bioassay and further evaluated for induction of interferon-stimulated genes (ISG) and antiviral efficacy in cell culture as well as in HBV-transgenic mice. PAS-mIFNα induced expression of ISG comparable to unmodified mIFNα and, likewise, evoked dose-dependent reduction of HBV replication in vitro. In vivo, PAS-mIFNα led to pronounced suppression of HBV replication without detectable liver damage whereas conventional mIFNα treatment only had a modest antiviral effect. Importantly, all PAS-mIFNα treated mice showed an anti-HBs antibody response, lost HBsAg and achieved seroconversion after three weeks. PASylated IFNα showed a profoundly increased antiviral effect in vivo compared to the non-modified version without toxicity, providing proof-of-concept that an improved IFNα can achieve higher rates of HBV antiviral and immune control.
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http://dx.doi.org/10.1016/j.antiviral.2018.11.003DOI Listing
January 2019

Hepatitis B virus genome recycling and de novo secondary infection events maintain stable cccDNA levels.

J Hepatol 2018 12 22;69(6):1231-1241. Epub 2018 Aug 22.

Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Munich, Germany; Technische Universität München, Institute for Advanced Study, Munich, Germany; German Center for Infection Research (DZIF), Munich partner site, Munich, Germany. Electronic address:

Background & Aims: Several steps in the HBV life cycle remain obscure because of a lack of robust in vitro infection models. These steps include particle entry, formation and maintenance of covalently closed circular (ccc) DNA, kinetics of gene expression and viral transmission routes. This study aimed to investigate infection kinetics and cccDNA dynamics during long-term culture.

Methods: We selected a highly permissive HepG2-NTCP-K7 cell clone engineered to express sodium taurocholate co-transporting polypeptide (NTCP) that supports the full HBV life cycle. We characterized the replication kinetics and dynamics of HBV over six weeks of infection.

Results: HBV infection kinetics showed a slow infection process. Nuclear cccDNA was only detected 24 h post-infection and increased until 3 days post-infection (dpi). Viral RNAs increased from 3 dpi reaching a plateau at 6 dpi. HBV protein levels followed similar kinetics with HBx levels reaching a plateau first. cccDNA levels modestly increased throughout the 45-day study period with 5-12 copies per infected cell. Newly produced relaxed circular DNA within capsids was reimported into the nucleus and replenished the cccDNA pool. In addition to intracellular recycling of HBV genomes, secondary de novo infection events resulted in cccDNA formation. Inhibition of relaxed circular DNA formation by nucleoside analogue treatment of infected cells enabled us to measure cccDNA dynamics. HBV cccDNA decayed slowly with a half-life of about 40 days.

Conclusions: After a slow infection process, HBV maintains a stable cccDNA pool by intracellular recycling of HBV genomes and via secondary infection. Our results provide important insights into the dynamics of HBV infection and support the future design and evaluation of new antiviral agents.

Lay Summary: Using a unique hepatocellular model system designed to support viral growth, we demonstrate that hepatitis B virus (HBV) has remarkably slow infection kinetics. Establishment of the episomal transcription template and the persistent form of the virus, so called covalently closed circular DNA, as well as viral transcription and protein expression all take a long time. Once established, HBV maintains a stable pool of covalently closed circular DNA via intracellular recycling of HBV genomes and through infection of naïve cells by newly formed virions.
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http://dx.doi.org/10.1016/j.jhep.2018.08.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611400PMC
December 2018

Analyses of HBV cccDNA Quantification and Modification.

Methods Mol Biol 2017 ;1540:59-72

Institute of Virology, Technische Universität München/Helmholtz Zentrum, Munich, Germany.

Covalently closed circular DNA (cccDNA) serves as the transcriptional template of hepatitis B virus (HBV) replication in the nucleus of infected cells. It ensures the persistence of HBV even if replication is blocked. Immune-mediated killing of infected hepatocytes, cell division, or cytokine induced non-cytolytic degradation of cccDNA can induce the loss of cccDNA. For studies on HBV control, the analysis of cccDNA integrity and its exact quantification is very important. Here, we describe different methods for HBV cccDNA quantification and modification.
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http://dx.doi.org/10.1007/978-1-4939-6700-1_6DOI Listing
January 2018

Lack of immunological DNA sensing in hepatocytes facilitates hepatitis B virus infection.

Hepatology 2016 09 26;64(3):746-59. Epub 2016 Jul 26.

Department of Biomedicine and, University of Aarhus, Aarhus, Denmark.

Unlabelled: Hepatitis B virus (HBV) is a major human pathogen, and about one third of the global population will be exposed to the virus in their lifetime. HBV infects hepatocytes, where it replicates its DNA and infection can lead to acute and chronic hepatitis with a high risk of liver cirrhosis and hepatocellular carcinoma. Despite this, there is limited understanding of how HBV establishes chronic infections. In recent years it has emerged that foreign DNA potently stimulates the innate immune response, particularly type 1 interferon (IFN) production; and this occurs through a pathway dependent on the DNA sensor cyclic guanosine monophosphate-adenosine monophosphate synthase and the downstream adaptor protein stimulator of IFN genes (STING). In this work we describe that human and murine hepatocytes do not express STING. Consequently, hepatocytes do not produce type 1 IFN in response to foreign DNA or HBV infection and mice lacking STING or cyclic guanosine monophosphate-adenosine monophosphate synthase exhibit unaltered ability to control infection in an adenovirus-HBV model. Stimulation of IFN production in the murine liver by administration of synthetic RNA decreases virus infection, thus demonstrating that IFN possesses anti-HBV activity in the liver. Importantly, introduction of STING expression specifically in hepatocytes reconstitutes the DNA sensing pathway, which leads to improved control of HBV in vivo.

Conclusion: The lack of a functional innate DNA-sensing pathway in hepatocytes hampers efficient innate control of HBV infection; this may explain why HBV has adapted to specifically replicate in hepatocytes and could contribute to the weak capacity of this cell type to clear HBV infection. (Hepatology 2016;64:746-759).
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http://dx.doi.org/10.1002/hep.28685DOI Listing
September 2016

Interferon-γ and Tumor Necrosis Factor-α Produced by T Cells Reduce the HBV Persistence Form, cccDNA, Without Cytolysis.

Gastroenterology 2016 Jan 28;150(1):194-205. Epub 2015 Sep 28.

Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Munich, Germany; German Center for Infection Research, Munich and Hannover, Germany. Electronic address:

Background & Aims: Viral clearance involves immune cell cytolysis of infected cells. However, studies of hepatitis B virus (HBV) infection in chimpanzees have indicated that cytokines released by T cells also can promote viral clearance via noncytolytic processes. We investigated the noncytolytic mechanisms by which T cells eliminate HBV from infected hepatocytes.

Methods: We performed a cytokine enzyme-linked immunosorbent assay of serum samples from patients with acute and chronic hepatitis B. Liver biopsy specimens were analyzed by in situ hybridization. HepG2-H1.3 cells, HBV-infected HepaRG cells, and primary human hepatocytes were incubated with interferon-γ (IFNγ) or tumor necrosis factor-α (TNF-α), or co-cultured with T cells. We measured markers of HBV replication, including the covalently closed circular DNA (cccDNA).

Results: Levels of IFNγ and TNF-α were increased in serum samples from patients with acute vs chronic hepatitis B and controls. In human hepatocytes with stably replicating HBV, as well as in HBV-infected primary human hepatocytes or HepaRG cells, IFNγ and TNF-α each induced deamination of cccDNA and interfered with its stability; their effects were additive. HBV-specific T cells, through secretion of IFNγ and TNF-α, inhibited HBV replication and reduced cccDNA in infected cells without the direct contact required for cytolysis. Blocking IFNγ and TNF-α after T-cell stimulation prevented the loss of cccDNA. Deprivation of cccDNA required activation of nuclear APOBEC3 deaminases by the cytokines. In liver biopsy specimens from patients with acute hepatitis B, but not chronic hepatitis B or controls, hepatocytes expressed APOBEC3A and APOBEC3B.

Conclusions: IFNγ and TNF-α, produced by T cells, reduce levels of HBV cccDNA in hepatocytes by inducing deamination and subsequent cccDNA decay.
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http://dx.doi.org/10.1053/j.gastro.2015.09.026DOI Listing
January 2016

[Specific degradation of nuclear hepatitis B virus covalently closed circular DNA].

Med Sci (Paris) 2014 Aug-Sep;30(8-9):724-6. Epub 2014 Sep 1.

Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Munich, Allemagne - German Center for Infection Research (DZIF), Allemagne.

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http://dx.doi.org/10.1051/medsci/20143008003DOI Listing
November 2014

Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA.

Science 2014 Mar 20;343(6176):1221-8. Epub 2014 Feb 20.

Institute of Virology, Technische Universität München-Helmholtz Zentrum München, 81675 Munich, Germany.

Current antiviral agents can control but not eliminate hepatitis B virus (HBV), because HBV establishes a stable nuclear covalently closed circular DNA (cccDNA). Interferon-α treatment can clear HBV but is limited by systemic side effects. We describe how interferon-α can induce specific degradation of the nuclear viral DNA without hepatotoxicity and propose lymphotoxin-β receptor activation as a therapeutic alternative. Interferon-α and lymphotoxin-β receptor activation up-regulated APOBEC3A and APOBEC3B cytidine deaminases, respectively, in HBV-infected cells, primary hepatocytes, and human liver needle biopsies. HBV core protein mediated the interaction with nuclear cccDNA, resulting in cytidine deamination, apurinic/apyrimidinic site formation, and finally cccDNA degradation that prevented HBV reactivation. Genomic DNA was not affected. Thus, inducing nuclear deaminases-for example, by lymphotoxin-β receptor activation-allows the development of new therapeutics that, in combination with existing antivirals, may cure hepatitis B.
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http://dx.doi.org/10.1126/science.1243462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6309542PMC
March 2014

Structure of LdtMt2, an L,D-transpeptidase from Mycobacterium tuberculosis.

Acta Crystallogr D Biol Crystallogr 2013 Mar 16;69(Pt 3):432-41. Epub 2013 Feb 16.

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-17 177 Stockholm, Sweden.

The transpeptidase LtdMt2 catalyzes the formation of the (3-3) cross-links characteristic of the peptidoglycan layer in the Mycobacterium tuberculosis cell wall. Bioinformatics analysis suggests that the extramembrane part of the enzyme consists of three domains: two smaller domains (denoted as A and B domains) and a transpeptidase domain (the C domain) at the C-terminus. The crystal structures of two fragments comprising the AB domains and the BC domains have been determined. The structure of the BC module, which was determined to 1.86 Å resolution using Se-SAD phasing, consists of the B domain with an immunoglobulin-related fold and the catalytic domain belonging to the ErfK/YbiS/YbnG fold family. The structure of the AB-domain fragment, which was solved by molecular replacement to 1.45 Å resolution, reveals that despite a lack of overall sequence identity the A domain is structurally very similar to the B domain. Combining the structures of the two fragments provides a view of the complete three-domain extramembrane part of LdtMt2 and shows that the protein extends at least 80-100 Å from the plasma membrane into the peptidoglycan layer and thus defines the maximal distance at which cross-links are formed by this enzyme. The LdtMt-related transpeptidases contain one or two immunoglobulin domains, which suggests that these might serve as extender units to position the catalytic domain at an appropriate distance from the membrane in the peptidoglycan layer.
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http://dx.doi.org/10.1107/S0907444912049268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3605044PMC
March 2013
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