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Background & Aims: Bulevirtide (BLV) is a first-in-class entry inhibitor and the only approved treatment for patients chronically infected with HDV in Europe. We aimed to investigate the efficacy of BLV treatment in paired liver biopsies obtained at baseline and after 24 or 48 weeks of treatment., Methods: We performed a combined analysis of 126 paired liver biopsies derived from three clinical trials. In the phase II clinical trial MYR202, patients with chronic hepatitis D were randomised to receive 24 weeks of BLV at 2 mg, 5 mg or 10 mg/day. Patients in MYR203 (phase II) and MYR301 (phase III) received 48 weeks of BLV at 2 mg or 10 mg/day. Tenofovir disoproxil fumarate monotherapy or delayed treatment served as comparators. Virological parameters and infection-related host genes were assessed by qPCR and immunohistochemistry., Results: At week 24, median intrahepatic HDV RNA decline from baseline was 0.9Log 10 with 2 mg (n = 7), 1.1Log 10 with 5 mg (n = 5) and 1.4 Log 10 with 10 mg (n = 7) of BLV. At week 48, median reductions were 2.2Log 10 with 2 mg (n = 27) and 2.7Log 10 with 10 mg (n = 37) of BLV, while HDV RNA levels did not change in the comparator arms. Notably, a drastic decline in the number of hepatitis delta antigen-positive hepatocytes and a concomitant decrease in transcriptional levels of inflammatory chemokines and interferon-stimulated genes was determined in all BLV-treatment arms. Despite the abundance of HBsAg-positive hepatocytes, replication and covalently closed circular DNA levels of the helper virus HBV were low and remained unaffected by BLV treatment., Conclusion: Blocking viral entry diminishes signs of liver inflammation and promotes a strong reduction of HDV infection within the liver, thus suggesting that some patients may achieve HDV cure with long-term treatment., Impact and Implications: Chronic infection with HDV causes the most severe form of viral hepatitis, affecting approximately 12 million people worldwide. The entry inhibitor bulevirtide (BLV) is the only recently approved anti-HDV drug, which has proven efficacious and safe in clinical trials and real-word data. Here, we investigated paired liver biopsies at baseline and after 24 or 48 weeks of treatment from three clinical trials to understand the effect of the drug on viral and host parameters in the liver, the site of viral replication. We found that BLV treatment strongly reduces the number of HDV-infected cells and signs of liver inflammation. This data implies that blocking viral entry ameliorates liver inflammation and that prolonged treatment regimens might lead to HDV cure in some patients. This concept will guide the further development of therapeutic strategies and combination treatments for patients with CHD., Clinical Trial Numbers: NCT03546621, NCT02888106, NCT03852719., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background and Aims: To date, conflicting data exist as to whether hepatitis B virus (HBV) has the ability to induce innate immune responses. Here, we investigated cellular changes after the first contact between HBV and primary human hepatocytes (PHH) in vitro and in vivo., Approach and Results: The exposure of PHH to HBV particles resulted in nuclear translocation of NFκB, followed by the expression and secretion of inflammatory cytokines (IL [interleukin] 1B, IL6, and TNF [tumor necrosis factor]). Ultraviolet irradiation of viral particles suppressed HBV infectivity but not the induction of cytokines in PHH, suggesting that the inoculum contains the immune-inducing agent. Purified HBV particles on the whole, which were prepared from HBV DNA-positive and protein-rich fractions after heparin column separation, still had immune-inducing capacity in PHH. The HBV-induced gene expression profile was similar to that induced by toll-like receptor 2 (TLR2) ligand Pam3Cys, but different from those induced by the viral sensors TLR3 or TLR7-9. Treatment of PHH with both HBV particles and Pam3Cys led to phosphorylation of ERK (extracellular signal-regulated kinase), JNK, and p38 mitogen-activated protein kinases as well as NFκB (nuclear factor kappa B). Finally, HBV-induced gene expression could be neutralized by TLR2-specific antibodies. Of note, pretreatment with an HBV entry inhibitor attenuated the TLR2-mediated response to HBV, suggesting a receptor binding-related mechanism. In liver-humanized uPA/severe combined immunodeficient (SCID)/beige mice challenged with HBV in vivo, immune induction could only marginally be seen., Conclusions: PHHs are able to sense HBV particles through TLR2, leading to an activation of anti-HBV immune responses in vitro. These findings challenge the previously described stealth properties of HBV., (© 2020 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of American Association for the Study of Liver Diseases.)

Hepatocyte proliferation could result in the loss of covalently closed circular DNA (cccDNA) and the emergence of cccDNA-cleared nascent hepatocytes, which appear refractory to hepatitis B virus (HBV) reinfection with unknown mechanism(s). Sodium taurocholate cotransporting polypeptide (NTCP) is the functional receptor for HBV entry. In this study, down-regulation of cell membrane localized NTCP expression in proliferating hepatocytes was found to prevent HBV infection in HepG2-NTCP-tet cells and in liver-humanized mice. In patients, lower NTCP protein expression was correlated well with higher levels of hepatocyte proliferation and less HBsAg expression in HBV-related focal nodular hyperplasia (FNH) tissues. Clinically, significantly lower NTCP protein expression was correlated with more active hepatocyte proliferation in CHB patients with severe active necroinflammation and better antiviral treatment outcome. Mechanistically, the activation of cell cycle regulatory genes p53, S-phase kinase-associated protein 2 (SKP2) and cyclin D1 during cell proliferation, as well as proliferative and inflammatory cytokine Interleukin-6 (IL-6) could transcriptionally down-regulate NTCP expression. From these aspects, we conclude that within the milieu of hepatocyte proliferation, down-regulation of cell membrane localized NTCP expression level renders nascent hepatocytes resistant to HBV reinfection. This may accelerate virus clearance during immune-mediated cell death and compensatory proliferation of survival hepatocytes.

Objective: The stability of the covalently closed circular DNA (cccDNA) in nuclei of non-dividing hepatocytes represents a key determinant of HBV persistence. Contrarily, studies with animal hepadnaviruses indicated that hepatocyte turnover can reduce cccDNA loads but knowledge on the proliferative capacity of HBV-infected primary human hepatocytes (PHHs) in vivo and the fate of cccDNA in dividing PHHs is still lacking. This study aimed to determine the impact of human hepatocyte division on cccDNA stability in vivo., Methods: PHH proliferation was triggered by serially transplanting hepatocytes from HBV-infected humanised mice into naïve recipients. Cell proliferation and virological changes were assessed by quantitative PCR, immunofluorescence and RNA in situ hybridisation. Viral integrations were analysed by gel separation and deep sequencing., Results: PHH proliferation strongly reduced all infection markers, including cccDNA (median 2.4 log/PHH). Remarkably, cell division appeared to cause cccDNA dilution among daughter cells and intrahepatic cccDNA loss. Nevertheless, HBV survived in sporadic non-proliferating human hepatocytes, so that virological markers rebounded as hepatocyte expansion relented. This was due to reinfection of quiescent PHHs since treatment with the entry inhibitor myrcludex-B or nucleoside analogues blocked viral spread and intrahepatic cccDNA accumulation. Viral integrations were detected both in donors and recipient mice but did not appear to contribute to antigen production., Conclusions: We demonstrate that human hepatocyte division even without involvement of cytolytic mechanisms triggers substantial cccDNA loss. This process may be fundamental to resolve self-limiting acute infection and should be considered in future therapeutic interventions along with entry inhibition strategies., Competing Interests: Competing interests: SU is co-applicant and inventor of patents protecting myrcludex-B., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Background & Aims: NVR3-778 is a capsid assembly modulator in clinical development. We determined the in vivo antiviral efficacy and effects on innate and endoplasmic reticulum (ER) stress responses of NVR3-778 alone or in combination with pegylated interferon alpha (peg-IFN) and compared with entecavir., Methods: We performed 2 studies, with a total of 61 uPA/SCID mice with humanized livers. Mice were infected with a hepatitis B virus (HBV) genotype C preparation; we waited 8 weeks for persistent infection of the human hepatocytes in livers of mice. Mice were then randomly assigned to groups (5 or 6 per group) given vehicle (control), NVR3-778, entecavir, peg-IFN, NVR3-778 + entecavir, or NVR3-778 + peg-IFN for 6 weeks. We measured levels of HB surface antigen, HB e antigen, HBV RNA, alanine aminotransferase, and human serum albumin at different time points. Livers were collected and analyzed by immunohistochemistry; levels of HBV DNA, covalently closed circular DNA, and HBV RNA, along with markers of ER stress and IFN response, were quantified., Results: Mice given NVR3-778 or entecavir alone for 6 weeks had reduced serum levels of HBV DNA compared with controls or mice given peg-IFN. The largest reduction was observed in mice given NVR3-778 + peg-IFN; in all mice in this group, the serum level of HBV DNA was below the limit of quantification. NVR3-778 and peg-IFN, but not entecavir, also reduced serum level of HBV RNA. The largest effect was obtained in the NVR3-778 + peg-IFN group, in which serum level of HBV RNA was below the limit of quantification. Levels of HB surface antigen and HB e antigen were reduced significantly in only the groups that received peg-IFN. Levels of covalently closed circular DNA did not differ significantly among groups. NVR3-778 was not associated with any significant changes in level of alanine aminotransferase, the ER stress response, or IFN-stimulated genes., Conclusions: NVR3-778 has high antiviral activity in mice with humanized livers and stable HBV infection, reducing levels of serum HBV DNA and HBV RNA. Entecavir reduced levels of serum HBV DNA, but had no effect on HBV RNA. The combination of NVR3-778 and peg-IFN prevented viral replication and HBV RNA particle production to a greater extent than each compound alone or entecavir., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Cell-based therapies represent a promising alternative to orthotopic liver transplantation. However, therapeutic effects are limited by low cell engraftment rates. We recently introduced a technique creating human hepatocyte spheroids for potential therapeutic application. The aim of this study was to evaluate whether these spheroids are suitable for engraftment in diseased liver tissues. Intrasplenic spheroid transplantation into immunodeficient uPA/SCID/beige mice was performed. Hepatocyte transduction ability prior to transplantation was tested by lentiviral labeling using red-green-blue (RGB) marking. Eight weeks after transplantation, animals were sacrificed and livers were analyzed by immunohistochemistry and immunofluorescence. To investigate human hepatocyte-specific gene expression profiles in mice, quantitative real-time-PCR was applied. Human albumin and alpha-1-antitrypsin concentrations in mouse serum were quantified to assess the levels of human chimerism. Precultured human hepatocytes reestablished their physiological liver tissue architecture and function upon transplantation in mice. Positive immunohistochemical labeling of the proliferating cell nuclear antigen revealed that human hepatocytes retained their in vivo proliferation capacity. Expression profiles of human genes analyzed in chimeric mouse livers resembled levels determined in native human tissue. Extensive vascularization of human cell clusters was detected by demonstration of von Willebrand factor activity. To model gene therapy approaches, lentiviral transduction was performed ex vivo and fluorescent microscopic imaging revealed maintenance of RGB marking in vivo. Altogether, this is the first report demonstrating that cultured and retroviral transduced human hepatocyte spheroids are able to engraft and maintain their regenerative potential in vivo.

Unlabelled: Chronic hepatitis B virus (HBV) infection has been associated with alterations in lipid metabolism. Moreover, the Na+-taurocholate cotransporting polypeptide (NTCP), responsible for bile acid (BA) uptake into hepatocytes, was identified as the functional cellular receptor mediating HBV entry. The aim of the study was to determine whether HBV alters the liver metabolic profile by employing HBV-infected and uninfected human liver chimeric mice. Humanized urokinase plasminogen activator/severe combined immunodeficiency mice were used to establish chronic HBV infection. Gene expression profiles were determined by real-time polymerase chain reaction using primers specifically recognizing transcripts of either human or murine origin. Liver biopsy samples obtained from HBV-chronic individuals were used to validate changes determined in mice. Besides modest changes in lipid metabolism, HBV-infected mice displayed a significant enhancement of human cholesterol 7α-hydroxylase (human [h]CYP7A1; median 12-fold induction; P<0.0001), the rate-limiting enzyme promoting the conversion of cholesterol to BAs, and of genes involved in transcriptional regulation, biosynthesis, and uptake of cholesterol (human sterol-regulatory element-binding protein 2, human 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and human low-density lipoprotein receptor), compared to uninfected controls. Significant hCYP7A1 induction and reduction of human small heterodimer partner, the corepressor of hCYP7A1 transcription, was also confirmed in liver biopsies from HBV-infected patients. Notably, administration of Myrcludex-B, an entry inhibitor derived from the pre-S1 domain of the HBV envelope, provoked a comparable murine CYP7A1 induction in uninfected mice, thus designating the pre-S1 domain as the viral component triggering such metabolic alterations., Conclusion: Binding of HBV to NTCP limits its function, thus promoting compensatory BA synthesis and cholesterol provision. The intimate link determined between HBV and liver metabolism underlines the importance to exploit further metabolic pathways, as well as possible NTCP-related viral-drug interactions., (© 2014 by the American Association for the Study of Liver Diseases.)

Background & Aims: Interferon (IFN)-α therapy is not effective for most patients with chronic hepatitis B virus (HBV) infection for reasons that are not clear. We investigated whether HBV infection reduced IFN-α-mediated induction of antiviral defense mechanisms in human hepatocytes., Methods: Human hepatocytes were injected into severe combined immune-deficient mice (SCID/beige) that expressed transgenic urokinase plasminogen activator under control of the albumin promoter. Some mice were infected with HBV; infected and uninfected mice were given injections of human IFN-α. Changes in viral DNA and expression of human interferon-stimulated genes (ISGs) were measured by real-time polymerase chain reaction, using human-specific primers, and by immunohistochemistry., Results: Median HBV viremia (0.8log) and intrahepatic loads of HBV RNA decreased 3-fold by 8 or 12 hours after each injection of IFN-α, but increased within 24 hours. IFN-α activated expression of human ISGs and nuclear translocation of signal transducers and activators of transcription-1 (STAT1) in human hepatocytes that repopulated the livers of uninfected mice. Although baseline levels of human ISGs were slightly increased in HBV-infected mice, compared with uninfected mice, IFN-α failed to increase expression of the ISGs OAS-1, MxA, MyD88, and TAP-1 (which regulates antigen presentation) in HBV-infected mice. IFN-α did not induce nuclear translocation of STAT1 in HBV-infected human hepatocytes. Administration of the nucleoside analogue entecavir (for 20 days) suppressed HBV replication but did not restore responsiveness to IFN-α., Conclusions: HBV prevents induction of IFN-α signaling by inhibiting nuclear translocation of STAT1; this can interfere with transcription of ISGs in human hepatocytes. These effects of HBV might contribute to the limited effectiveness of endogenous and therapeutic IFN-α in patients and promote viral persistence., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)