TP15: Viral determinants and innate immunity in persistent hepatitis B virus and hepatitis D virus infections

Human hepatitis B (HBV) and hepatitis D viruses (HDV) cause persistent infections in more than 240 million people worldwide. Maintenance of persistence requires viral strategies to counteract intrinsic host immune responses. In this project, we aim to understand such responses on the molecular level in order to develop novel strategies to improve therapeutic outcomes of infection (functional cure). Specifically we will (i) investigate the impact of HBV integration on preservation of HBV and HDV templates (cccDNA and HDV RNA); (ii) elucidate the mechanism of innate immune sensing of replicating HDV RNA; (iii) uncover how IFNs suppress HDV spread during cell division.

Prof. Dr. Dr. Stephan Urban

Allweiss L, Testoni B, Yu M, Lucifora J, Ko C, Qu B, Lütgehetmann M, Guo H, Urban S, Fletcher SP, Protzer U, Levrero M, Zoulim F, Dandri M. (2023) Quantification of the hepatitis B virus cccDNA: evidence-based guidelines for monitoring the key obstacle of HBV cure. Gut. 72(5):972-983. doi: 10.1136/gutjnl-2022-328380.

Wedemeyer H, Schöneweis K,Bogomolov P, Blank A, Voronkova N, Stepanova T, Sagalova O, Chulanov V, Osipenko M, Morozov V, Geyvandova N, Sleptsova S, Bakulin IG, Khaertynova I, Rusanova M, Pathil A, Merle U, Bremer B, Allweiss L, Lempp FA, Urban S. (2023) Safety and efficacy of bulevirtide in combination with tenofovir disoproxil fumarate in patients with hepatitis B virus and hepatitis D virus coinfection (MYR202): a multicentre, randomised, parallel-group, open-label, phase 2 trial. Lancet Infect Dis. 23(1):117-129. doi: 10.1016/S1473-3099(22)00318-8.

Gillich N, Zhang Z, Binder M, Urban S, Bartenschlager R. (2022) Effect of variants in LGP2 on MDA5-mediated activation of interferon response and suppression of hepatitis D virus replication. J Hepatol. 21:S0168-8278(22)03113-0. doi: 10.1016/j.jhep.2022.08.041.

Zhang Z, Ni Y, Lempp FA, Walter L, Mutz P, Bartenschlager R, Urban S. (2022) Hepatitis D virus-induced interferon response and administered interferons control cell division-mediated virus spread. J Hepatol. 77(4):957-966. doi: 10.1016/j.jhep.2022.05.023.

Qu B, Nebioglu F, Leuthold MM, Ni Y, Mutz P, Beneke J, Erfle H, Vondran FWR, Bartenschlager R, Urban S. (2022) Dual role of neddylation in transcription of hepatitis B virus RNAs from cccDNA and production of viral surface antigen. JHEP Rep. 100551, ISSN 2589-5559, https://doi.org/10.1016/j.jhepr.2022.100551.

Tu T, Zehnder B, Wettengel JM, Zhang H, Coulter S, Ho V, Douglas MW, Protzer U, George J, Urban S. (2022) Mitosis of hepatitis B virus-infected cells in vitro results in uninfected daughter cells. JHEP Rep. 15;4(9):100514. doi: 10.1016/j.jhepr.2022.100514.

Lempp FA, Roggenbach I, Nkongolo S, Sakin V, Schlund F, Schnitzler P, Wedemeyer H, Le Gal F, Gordien E, Yurdaydin C, Urban S. (2021) A rapid point-of-care test for the serodiagnosis of Hepatitis Delta virus infection viruses. 13(12): 2371. doi: 10.3390/v13122371.

Roggenbach I, Chi X, Lempp FA, Qu B, Walter L, Wu R, Gao X, Schnitzler P, Ding Y, Urban S, Niu J. (2021) HDV seroprevalence in HBsAg-positive patients in China occurs in hotspots and is not associated with HCV mono-infection. Viruses. 13(9):1799. doi: 10.3390/v13091799.

Oswald A, Chakraborty A, Ni Y, Wettengel JM, Urban S, Protzer U. (2021) Concentration of Na+-taurocholate-cotransporting polypeptide expressed after in vitro-transcribed mRNA transfection determines susceptibility of hepatoma cells for hepatitis B virus. Sci Rep. 5;11(1):19799. doi: 10.1038/s41598-021-99263-3.

Urban S, Neumann-Haefelin C, Lampertico P. (2021) Hepatitis D virus in 2021: virology, immunology and new treatment approaches for a difficult-to-treat disease. Gut. 70(9):1782-1794. doi: 10.1136/gutjnl-2020-323888.

Tu T, Zhang H, Urban S. (2021) Hepatitis B virus DNA integration: in vitro models for investigating viral pathogenesis and persistence. Viruses. 26;13(2):180. doi: 10.3390/v13020180.

Zhang Z, Urban S. (2021) New insights into HDV persistence: The role of interferon response and implications for upcoming novel therapies. J Hepatol. 74(3):686-699. doi: 10.1016/j.jhep.2020.11.032.

Tu T, Zehnder B, Qu B, Urban S. (2020) De novo synthesis of hepatitis B virus nucleocapsids is dispensable for the maintenance and transcriptional regulation of cccDNA. JHEP Rep. 14;3(1):100195. doi.org/10.1016/j.jhepr.2020.100195

Tu T, Zehnder B, Qu B, Ni Y, Main N, Allweiss L, Dandri M, Shackel N, George J, Urban S. A novel method to precisely quantify hepatitis B virus covalently closed circular (ccc)DNA formation and maintenance. Antiviral Res. 2020 Sep;181:104865. doi: 10.1016/j.antiviral.2020.104865. (TP15)

Dao Thi VL, Wu X, Belote RL, Andreo U, Takacs CN, Fernandez JP, Vale-Silva LA, Prallet S, Decker CC, Fu RM, Qu B, Uryu K, Molina H, Saeed M, Steinmann E, Urban S, Singaraja RR, Schneider WM, Simon SM, Rice CM. Stem cell-derived polarized hepatocytes. Nat Commun. 2020 Apr 3;11(1):1677. doi: 10.1038/s41467-020-15337-2. (TP22, TP15)

Lempp FA, Schlund F, Rieble L, Nussbaum L, Link C, Zhang Z, Ni Y, Urban S. 2019. Recapitulation of HDV infection in a fully permissive hepatoma cell line allows efficient drug evaluation. Nat Commun. 10(2):2265.

Giersch K, Bhadra OD, Volz T, Allweiss L, Riecken K, Fehse B, Lohse AW, Petersen J, Sureau C, Urban S, Dandri M, Lutgehetmann M. 2019. Hepatitis delta virus persists during liver regeneration and is ampli-fied through cell division both in vitro and in vivo. Gut. 68(1):150-157.

Ni Y, Zhang Z, Engelskircher L, Verch G, Tu T, Lempp FA, Urban S. 2019. Generation and characteriza-tion of a stable cell line persistently replicating and secreting the human hepatitis delta virus. Sci Rep. 9(1):10021.

Nkongolo S, Nußbaum L, Lempp FA, Wodrich H, Urban S, Ni Y. 2019. The retinoic acid receptor (RAR) α-specific agonist Am80 (tamibarotene) and other RAR agonists potently inhibit hepatitis B virus tran-scription from cccDNA. Antiviral Res. 168:146-155.

Zhang Z, Filzmayer C, Ni Y, Sultmann H, Mutz P, Hiet MS, Vondran FWR, Bartenschlager R, Urban S. 2018. Hepatitis D virus replication is sensed by MDA5 and induces IFN-β/λ responses in hepatocytes. J Hepatol. 69(1):25-35.

Qu B, Ni Y, Lempp FA, Vondran FWR, Urban S. 2018. T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR. J Virol. 92(23): pii: e01117-18.

Mutz P, Metz P, Lempp FA, Bender S, Qu B, Schoneweis K, Seitz S, Tu T, Restuccia A, Frankish J, Dachert C, Schusser B, Koschny R, Polychronidis G, Schemmer P, Hoffmann K, Baumert TF, Binder M, Urban S, Bartenschlager R. 2018. HBV Bypasses the Innate Immune Response and Does Not Protect HCV From Antiviral Activity of Interferon. Gastroenterology. 154(6):1791-1804.

Allweiss L, Volz T, Giersch K, Kah J, Raffa G, Petersen J, Lohse AW, Beninati C, Pollicino T, Urban S, Lutgehetmann M, Dandri M. 2018. Proliferation of primary human hepatocytes and prevention of hepati-tis B virus reinfection efficiently deplete nuclear cccDNA in vivo. Gut. 67(3):542-552.

Tu T, Budzinska MA, Vondran FWR, Shackel NA, Urban S. 2018. Hepatitis B virus DNA integration oc-curs early in the viral life cycle in an in vitro infection model via NTCP-dependent uptake of enveloped vi-rus particles. J Virol. 14;92(11). pii: e02007-17.

Lempp FA, Wiedtke E, Qu B, Roques P, Chemin I, Vondran FW, Le Grand R, Grimm D, Urban S. 2017. Sodium taurocholate cotransporting polypeptide is the limiting host factor of Hepatitis B Virus infection in macaque and pig hepatocytes. Hepatology. 66(3): 703-716.

Project in detail +

Hepatitis B virus (HBV) causes chronic infections of ~240 million people worldwide, which is a key risk fac-tor for the development of liver cirrhosis and hepatocellular carcinoma. Coinfection with the human hepatitis D virus (HDV), a satellite virus depending on HBV (and possibly other viruses) and affecting 15-25 million people, accelerates progression of the disease. Currently, there are no curative regimens for chronic HBV and no approved medication for HDV infections, however new drugs are in development and one of them (the entry inhibitor Myrcludex B (clinical name Bulevirtide) affecting both viruses) will be clinically accessible soon. For extracellular spreading of both viruses, blockade of entry with Myrcludex B is very efficient, lead-ing to continuous reduction of infected hepatocytes in the liver of HDV patients. However, due to its indirect mode of interfering with replication, 48 weeks of Myrcludex B mono-therapy cannot eliminate HBV/HDV infected hepatocytes. This and the peculiar modes of episomal replication indicate that additional persis-tence strategies exist. For HBV, persistence is mediated by the covalently closed circular (ccc)DNA, a nu-clear episome in hepatocytes serving as a viral reservoir. HDV, a continuously replicating RNA can also be propagated by cell division.
In the previous funding period, we investigated the Hepatitis B Virus X-protein mediated regulation of tran-scription from cccDNA and discovered a neddylation inhibitor (MLN4924) specifically blocking this mecha-nism and may become important to achieve “functional cure”. This translational aspect has therefore been implemented into a DZIF-project. In the next funding period of the TRR179, we will continue to characterize the amplification, turnover and maintenance of cccDNA in resting and dividing infected hepatocytes, and focus on the crucial role of the large viral surface protein (L-protein). In collaboration with Cerwenka and Thimme, we will evaluate new aspects of immune cells (NK, T, etc.) with respect to their possible role for clearance of cccDNA-containing hepatocytes.
During the first funding period, we implemented HDV as new project and established robust cell culture models (Lempp et al., Nat Commun 2019). Regarding persistence of HDV, our recent studies showed that HDV spread, in addition to propagation via progeny viruses, occurs by hepatocyte proliferation. Cell-division mediated spread cannot be blocked by Myrcludex B and represents an alternative strategy of the virus to persist (Giersch et al., Gut 2019). Moreover, HBV integrates, although not essential for HBV replica-tion may encode envelope proteins that can be used by HDV to assemble even in the absence of the cccDNA template. We found that HDV, in contrast to HBV, induces an MDA5-mediated IFN response in hepatocytes (Zhang et al. 2018). This endogenously induced IFN-response, but also exogenous IFN used as a therapeutic, only marginally affects an established HDV infection in resting hepatocytes. However, IFN severely impairs HDV spread during cell division. The importance of this IFN effect is highlighted by the clinical observations (phase II Myr-203 study) showing a strong synergy between Myrcludex B and PEG-IFNα. In the next funding period, we therefore plan to: (i) unveil the role of HBV integration on amplification, turnover and maintenance of cccDNA and HDV RNA in infected hepatocytes (with Klingmüller/Timmer); (ii) elucidate the mechanism for innate immune recognition of HDV RNA (with Bartenschlager, Lohmann); (iii) unveil the mechanism of IFN mediated suppression of HDV spread during cell division and develop effec-tive co-treatment targeting different HDV spread pathways. Finally, based on a recent report demonstrating HDV – HCV co-replication in vitro and in mouse models in the absence of HBV as helper virus, we will im-plement epidemiological studies on possible HDV/HCV co-infections using a newly developed point of care assay (with Neumann-Haefelin), thus studying a possible contribution of HCV to HDV spread (with Barten-schlager).

Milestones of the first funding period:
- Elucidation of the mechanism of DDB1/CRL5 mediated regulation of cccDNA-driven HBV transcription and its counteraction by HBx
- Molecular understanding of the dynamics of HBV cccDNA formation, maintenance and elimination in resting and dividing hepatocytes
- Identification of viral and cellular restriction factors preventing recognition of persistently infected hepatocytes by the immune system