Joerg Petersen

Peginterferon alpha-2b plus adefovir induce strong cccDNA decline and HBsAg reduction in patients with chronic hepatitis B

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is responsible for persistent infection of hepatocytes. The aim of this study was to determine changes in intrahepatic cccDNA in patients with chronic hepatitis B (CH‐B) during 48 weeks of antiviral therapy and its correlation to virological, biochemical, and histological parameters. Twenty‐six HBsAg‐positive CH‐B patients received combination treatment with pegylated interferon alpha‐2b (peg‐IFN) and adefovir dipivoxil (ADV) for 48 weeks. Paired liver biopsies from before and at the end of treatment were analyzed for intrahepatic HBV‐DNA. Median serum HBV‐DNA had decreased by −4.9 log10 copies/mL at the end of treatment and was undetectable in 13 individuals (54%). Median intrahepatic total HBV‐DNA and cccDNA had decreased by −2.2 and −2.4 log10, respectively. Changes in intracellular HBV‐DNA positively correlated with HBsAg serum reduction and were accompanied by a high number of serological responders. Eight of 15 HBeAg‐positive patients lost HBeAg, and five developed anti‐HBe antibodies during treatment. These eight patients exhibited lower cccDNA levels before and at the end of therapy than did patients without HBeAg loss. Four patients developed anti‐HBs antibodies. ALT normalized in 11 patients. The number of HBs‐antigen‐ and HBc‐antigen‐positive hepatocytes was significantly lower after treatment, suggesting the involvement of cytolytic mechanisms. In conclusion, combination therapy with peg‐IFN and ADV led to marked decreases in serum HBV‐DNA and intrahepatic cccDNA, which was significantly correlated with reduced HBsAg. (HEPATOLOGY 2006;44:675–684.)

IFN-α inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome

HBV infection remains a leading cause of death worldwide. IFN-α inhibits viral replication in vitro and in vivo, and pegylated IFN-α is a commonly administered treatment for individuals infected with HBV. The HBV genome contains a typical IFN-stimulated response element (ISRE), but the molecular mechanisms by which IFN-α suppresses HBV replication have not been established in relevant experimental systems. Here, we show that IFN-α inhibits HBV replication by decreasing the transcription of pregenomic RNA (pgRNA) and subgenomic RNA from the HBV covalently closed circular DNA (cccDNA) minichromosome, both in cultured cells in which HBV is replicating and in mice whose livers have been repopulated with human hepatocytes and infected with HBV. Administration of IFN-α resulted in cccDNA-bound histone hypoacetylation as well as active recruitment to the cccDNA of transcriptional corepressors. IFN-α treatment also reduced binding of the STAT1 and STAT2 transcription factors to active cccDNA. The inhibitory activity of IFN-α was linked to the IRSE, as IRSE-mutant HBV transcribed less pgRNA and could not be repressed by IFN-α treatment. Our results identify a molecular mechanism whereby IFN-α mediates epigenetic repression of HBV cccDNA transcriptional activity, which may assist in the development of novel effective therapeutics.

Virological response to entecavir is associated with a better clinical outcome in chronic hepatitis B patients with cirrhosis

Objective Entecavir (ETV) is a potent inhibitor of viral replication in chronic hepatitis B and prolonged treatment may result in regression of fibrosis. The aim of this study was to investigate the effect of ETV on disease progression. Design In a multicentre cohort study, 372 ETV-treated patients were investigated. Clinical events were defined as development of hepatocellular carcinoma (HCC), hepatic decompensation or death. Virological response (VR) was defined as HBV DNA <80 IU/ml. Results Patients were classified as having chronic hepatitis B without cirrhosis (n=274), compensated cirrhosis (n=89) and decompensated cirrhosis (n=9). The probability of VR was not influenced by severity of liver disease (p=0.62). During a median follow-up of 20 months (IQR 11–32), the probability of developing clinical events was higher for patients with cirrhosis (HR 15.41 (95% CI 3.42 to 69.54), p<0.001). VR was associated with a lower probability of disease progression (HR 0.29 (95% CI 0.08 to 1.00), p=0.05) which remained after correction for established risk factors such as age. The benefit of VR was only significant in patients with cirrhosis (HR 0.22 (95% CI 0.05 to 0.99), p=0.04) and remained after excluding decompensated patients (HR 0.15 (95% CI 0.03 to 0.81), p=0.03). A higher HBV DNA threshold of 2000 IU/ml was not associated with the probability of disease progression (HR 0.20 (95% CI 0.03 to 1.10), p=0.10). Conclusion VR to ETV is associated with a lower probability of disease progression in patients with cirrhosis, even after correction for possible baseline confounders. When using a threshold of 2000 IU/ml, the association between viral replication and disease progression was reduced, suggesting that complete viral suppression is essential for nucleoside/nucleotide analogue treatment, especially in patients with cirrhosis.

Hepatitis B Virus Limits Response of Human Hepatocytes to Interferon-α in Chimeric Mice

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.

The replication cycle of hepatitis B virus.

Liver unit, IFI Institute for InterdisciplinaryMedicine, Asklepios Clinics St. Georg, Hamburg, Germany(1) Reversible and non-cell-type specific attachment to cell-associated heparan sulfate proteoglycans.(2) Specific and probably irreversible binding to an unknownhepatocyte-specific preS1-receptor. This step presumablyrequires activation of the virus resulting in exposure ofthe myristoylated N-terminus of the L-protein [1].(3) Two different entry pathways have been proposed: (3A)endocytosis followed by release of nucleocapsids fromendocytic vesicles; (3B) fusion of the viral envelope atthe plasma membrane.(4) Cytoplasmic release of the viral nucleocapsid containingthe relaxed circular partially double stranded DNA (rcDNA)with its covalently linked polymerase.(5) Transport of the nucleocapsid along microtubules. Accu-mulation of the capsids at the nuclear envelope facilitatesinteractions with adaptor proteins of the nuclear porecomplex.(6) Possible trapping of the nucleocapsid in the nuclear basketand release of rcDNA into the nucleoplasm. The mecha-nisms determining the breakdown of the capsid and therelease of the viral DNA genome are unsolved [2].(7) ‘‘Repair” of the incoming rcDNA: Completion of the plusstrand of the rcDNA by the viral polymerase. Removal ofthe polymerase from the 5

Binding of hepatitis B virus to its cellular receptor alters the expression profile of genes of bile acid metabolism

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. (Hepatology 2014;60:1483–1493)

In vivo proliferation of hepadnavirus‐infected hepatocytes induces loss of covalently closed circular DNA in mice

Chronic hepatitis B virus (HBV) infection is maintained by the presence of covalently closed circular DNA (cccDNA), the template of viral transcription and replication. In quiescent hepatocytes, cccDNA is a stable molecule that can persist throughout the hepatocyte lifespan. However, in chronic HBV infection, immunomediated cell injury and compensatory hepatocyte proliferation may favor cccDNA decline and selection of cccDNA‐free cells. To investigate the impact of liver regeneration on cccDNA stability and activity in vivo, we used the urokinase‐type plasminogen activator (uPA)/severe combined immunodeficiency (SCID) mouse model. Primary tupaia hepatocytes (PTHs) chronically infected with woolly monkey HBV (WM‐HBV) were isolated from one highly viremic uPA/SCID chimeric mouse and transplanted into 20 uPA recipients. Expansion of transplanted PTHs and viral load changes were determined by real‐time polymerase chain reaction and immunohistochemistry. Transplantation of WM‐HBV infected hepatocytes led to an average of 3.8 PTH doublings within 80 days, 75% reduction of virion productivity (relaxed circular DNA/cccDNA), and lower expression levels of pregenomic RNA and hepatitis B core antigen. Remarkably, a median 2‐log decline of cccDNA per cell determined during PTH proliferation was due to both dilution of the cccDNA pool among daughter cells and a 0.5‐log loss of intrahepatic cccDNA loads (P = 0.02). Intrahepatic viral DNA sequences persisting at the end of the study were mostly present as replicative intermediates and not as integrated virus. Conclusion: Cell division in the setting of liver regeneration and without administration of antiviral drugs induced strong destabilization of the cccDNA reservoir, resulting in cccDNA clearance in the great majority of chronically infected hepatocytes. (Hepatology 2010)

Immune cell responses are not required to induce substantial hepatitis B virus antigen decline during pegylated interferon-alpha administration

BACKGROUND & AIMS Pegylated interferon-alpha (PegIFNα) remains an attractive treatment option for chronic hepatitis B virus (HBV) infection because it induces higher rates of antigen loss and seroconversion than treatment with polymerase inhibitors. Although early HBsAg decline is recognised as the best predictor of sustained response to IFN-based therapy, it is unclear whether immune cell functions are required to induce significant antigenemia reduction in the first weeks of treatment. Aim of the study was to investigate whether PegIFNα can induce sustained human hepatocyte responsiveness and substantial loss of circulating and intrahepatic viral antigen loads in a system lacking immune cell functions. METHODS HBV-infected humanized uPA/SCID mice received either PegIFNα, entecavir (ETV), or both agents in combination. Serological and intrahepatic changes were determined by qRT-PCR and immunohistochemistry and compared to untreated mice. RESULTS After 4 weeks of treatment, median viremia reduction was greater in mice treated with ETV (either with or without PegIFNα) than with PegIFNα. In contrast, levels of circulating HBeAg, HBsAg, and intrahepatic HBcAg were significantly reduced (p = 0.03) only in mice receiving PegIFNα alone or in combination, as compared to mice receiving ETV monotherapy. Progressive antigen reduction was also demonstrated in mice receiving PegIFNα for 12 weeks (HBeAg = Δ1log; HBsAg = Δ1.4log; p < 0.0001). Notably, repeated administrations of the longer-active PegIFNα could breach the impairment of HBV-infected hepatocyte responsiveness and induce sustained enhancement of human interferon stimulated genes (ISG). CONCLUSIONS The antiviral effects of PegIFNα exerted on the human hepatocytes can induce sustained responsiveness and trigger substantial HBV antigen decline without claiming the involvement of immune cell responses.

Optimization of hepatocyte spheroid formation for hepatic tissue engineering on three-dimensional biodegradable polymer within a flow bioreactor prior to implantation

We hypothesize that in vitro conditioning of hepatocytes within biodegradable poly-L-lactic acid (PLLA) polymer matrices prior to implantation may increase hepatocyte survival and function after transplantation. The purpose of this study was to optimize the culture conditions of hepatocytes in a pulsatile flow bioreactor. PLLA discs were seeded with rat hepatocytes in a concentration of 2.5, 5, 10, 20 and 40 × 106 cells/ml. Seeded discs were exposed to recirculated perpendicular flow of 0, 7, 15, 24, 32, 52 ml/min of supplemented Williams’ Medium E and harvested after 6 days in flow culture. Only under flow conditions the hepatocytes formed spheroidal aggregates (SphA) of 50–260 µm in diameter with a liver-like morphology and active metabolic function. The number of SphA was examined by phase contrast microscopy and the reductive enzyme function of the hepatocytes was tested using MTT. Hematoxylin and eosin histology showed vital hepatocytes within the SphA less than 200 µm in diameter but central necrosis in the SphA exceeding this size. Immunohistochemical staining confirmed albumin production of hepatocytes within the SphA. The optimal cell seeding concentration was 10 × 106 cells/ml with a flow speed of 24 ml/min. SphA of hepatocytes cultured with this flow bioreactor method may prove useful as a functional unit for tissue engineering of an in vivo liver substitute.

Virion half-life in chronic hepatitis B infection is strongly correlated with levels of viremia†

Analysis of hepatitis B virus (HBV) kinetics with mathematical models may disclose new aspects of HBV infection and host response mechanisms. To determine the kinetics of virion decay from the blood of patients in different phases of chronic infection, we applied mathematical modeling to real‐time polymerase chain reaction assays, which enable quantification of viremia and intrahepatic HBV productivity by measuring both copy number and activity of covalently closed circular DNA (relaxed circular DNA/covalently closed circular DNA) in the liver of 80 untreated chronically active HBV carriers (38 hepatitis B e antigen [HBeAg]‐positive and 42 HBeAg‐negative individuals). We found that the half‐life of circulating virions is very fast (median 46 and 2.5 minutes in HBeAg‐positive and HBeAg‐negative individuals, respectively) and strongly related to viremia, with clearance rates significantly accelerating as viral loads decrease. To investigate whether immune components can influence the kinetics of virion decay, we analyzed viral dynamics in immunodeficient urokinase‐type plasminogen activator chimera mice. Virion half‐life in mice (range, 44 minutes to >4 hours) was comparable to estimates determined in high viremic carriers, implying that clearance rates in these patients are mostly determined by common nonspecific mechanisms. Notably, the lack of correlation between virion half‐life and viremia in mice indicated that immune components significantly accelerate virion clearance rates in individuals with low titers. Conclusion: Our analyses suggest that both host defense mechanisms and levels of circulating virions affect the kinetics of HBV decay assessed in the serum of chronic carriers. Identification of the factors affecting clearance rates will be important for future antiviral drug developments and it may give insights into the mechanisms involved in clearance of other chronic infections, such as human immunodeficiency virus and hepatitis C virus. (HEPATOLOGY 2008.)