L. Allweiss

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.

Humanized chimeric uPA mouse model for the study of hepatitis B and D virus interactions and preclinical drug evaluation

No specific drugs are currently available against hepatitis delta virus (HDV), a defective virus leading to the most severe form of chronic viral hepatitis in man. The lack of convenient HDV infection models has hampered the development of effective therapeutics. In this study, naïve and hepatitis B virus (HBV) chronically infected humanized uPA/SCID mice were employed to establish a small animal model of HBV/HDV coinfection and superinfection. For preclinical antiviral drug evaluation, the GMP version of the myristoylated preS‐peptide (Myrcludex‐B), a lipopeptide derived from the pre‐S1 domain of the HBV envelope, was applied to prevent de novo HBV/HDV coinfection in vivo. Virological parameters were determined at serological and intrahepatic level both by real‐time polymerase chain reaction (PCR) and by immunohistochemistry. Establishment of HDV infection was highly efficient in both HBV‐infected and naïve chimeric mice with HDV titers rising up to 1 × 10E9 copies/mL. Notably, HDV superinfection led to a median 0.6log reduction of HBV viremia, which although not statistically significant suggests that HDV may hinder HBV replication. In the setting of HBV/HDV simultaneous infection, a majority of human hepatocytes stained HDAg‐positive long before HBV spreading was completed, confirming that HDV can replicate intrahepatically also in the absence of HBV infection. Furthermore, the increase of HBV viremia and intrahepatic cccDNA loads was significantly slower than in HBV mono‐infected mice. Treatment with the HBV entry inhibitor Myrcludex‐B, efficiently hindered the establishment of HDV infection in vivo. Conclusion: We established an efficient model of HBV/HDV infection to exploit mechanisms of viral interference in human hepatocytes and to test the efficacy of an HDV‐entry inhibitor in vivo. (HEPATOLOGY 2011)

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.

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)

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.

Experimental in vitro and in vivo models for the study of human hepatitis B virus infection

Chronic infection with the hepatitis B virus (HBV) affects an estimate of 240 million people worldwide despite the availability of a preventive vaccine. Medication to repress viral replication is available but a cure is rarely achieved. The narrow species and tissue tropism of the virus and the lack of reliable in vitro models and laboratory animals susceptible to HBV infection, have limited research progress in the past. As a result, several aspects of the HBV life cycle as well as the network of virus host interactions occurring during the infection are not yet understood. Only recently, the identification of the functional cellular receptor enabling HBV entry has opened new possibilities to establish innovative infection systems. Regarding the in vivo models of HBV infection, the classical reference was the chimpanzee. However, because of the strongly restricted use of great apes for HBV research, major efforts have focused on the development of mouse models of HBV replication and infection such as the generation of humanized mice. This review summarizes the animal and cell culture based models currently available for the study of HBV biology. We will discuss the benefits and caveats of each model and present a selection of the most important findings that have been retrieved from the respective systems.

Hepatitis Delta co-infection in humanized mice leads to pronounced induction of innate immune responses in comparison to HBV mono-infection

BACKGROUND & AIMS The limited availability of hepatitis Delta virus (HDV) infection models has hindered studies of interactions between HDV and infected hepatocytes. The aim was to investigate the antiviral state of HDV infected human hepatocytes in the setting of co-infection with hepatitis B virus (HBV) compared to HBV mono-infection using human liver chimeric mice. METHODS Viral loads, human interferon stimulated genes (hISGs) and cytokines were determined in humanized uPA/SCID/beige (USB) mice by qRT-PCR, ELISA and immunofluorescence. RESULTS Upon HBV/HDV inoculation, all mice developed viremia, which was accompanied by a significant induction of hISGs (i.e. hISG15, hSTATs, hHLA-E) compared to uninfected mice, while HBV mono-infection led to weaker hISG elevations. In the setting of chronic infection enhancement of innate defense mechanisms was significantly more prominent in HBV/HDV infected mice. Also the induction of human-specific cytokines (hIP10, hTGF-ß, hIFN-ß and hIFN-λ) was detected in HBV/HDV co-infected animals, while levels remained lower or below detection in uninfected and HBV mono-infected mice. Moreover, despite the average increase of hSTAT levels determined in HBV/HDV infected livers, we observed a weaker hSTAT accumulation in nuclei of hepatocytes displaying very high HDAg levels, suggesting that HDAg may in part limit hSTAT signaling. CONCLUSIONS Establishment of HDV infection provoked a clear enhancement of the antiviral state of the human hepatocytes in chimeric mice. Elevated pre-treatment ISG and interferon levels may directly contribute to inflammation and liver damage, providing a rationale for the more severe course of HDV-associated liver disease. Such antiviral state induction might also contribute to the lower levels of HBV activity frequently found in co-infected hepatocytes.

Human liver chimeric mice as a new model of chronic hepatitis E virus infection and preclinical drug evaluation

BACKGROUND & AIMS Hepatitis E virus (HEV) is a major cause of acute hepatitis as well as chronic infection in immunocompromised individuals; however, in vivo infection models are limited. The aim of this study was to establish a small animal model to improve our understanding of HEV replication mechanisms and permit the development of effective therapeutics. METHODS UPA/SCID/beige mice repopulated with primary human hepatocytes were used for infection experiments with HEV genotype (GT) 1 and 3. Virological parameters were determined at the serological and intrahepatic level by real time PCR, immunohistochemistry and RNA in situ hybridization. RESULTS Establishment of HEV infection was achieved after intravenous injection of stool-derived virions and following co-housing with HEV-infected animals but not via inoculation of serum-derived HEV. GT 1 infection resulted in a rapid rise of viremia and high stable titres in serum, liver, bile and faeces of infected mice for more than 25 weeks. In contrast, viremia in GT 3 infected mice developed more slowly and displayed lower titres in all analysed tissues as compared to GT 1. HEV-infected human hepatocytes could be visualized using HEV ORF2 and ORF3 specific antibodies and HEV RNA in situ hybridization probes. Finally, six-week administration of ribavirin led to a strong reduction of viral replication in the serum and liver of GT 1 infected mice. CONCLUSION We established an efficient model of HEV infection to test the efficacy of antiviral agents and to exploit mechanisms of HEV replication and interaction with human hepatocytes in vivo.

Persistent hepatitis D virus mono-infection in humanized mice is efficiently converted by hepatitis B virus to a productive co-infection

BACKGROUND & AIMS Clinical studies have shown that hepatitis delta virus (HDV) infection can persist for years and intrahepatic latency of the large delta antigen (HDAg) has been detected following liver transplantation. However, large HDAg arising via RNA-editing is associated with increasing amounts of non-infectious HDV quasi-species. This study investigated whether HDV could persist intrahepatically in the absence of HBV in vivo and whether infectious HDV could subsequently be released following HBV super-infection. METHODS Humanized mice were infected with HDV particles lacking HBV. To test for rescue of latent HDV infection 3 and 6 weeks HDV mono-infected mice were super-infected with HBV. Viral loads and cell toxicity were determined by qRT-PCR and immunohistochemistry. RESULTS The presence of HDAg-positive human hepatocytes determined after 2, 3, and 6 weeks of HDV inoculation demonstrated establishment and maintenance of intrahepatic HDV mono-infection. Although intrahepatic amounts of large HDAg and edited HDV RNA forms increased over time in HDV mono-infected livers, HBV super-infection led to prompt viremia development (up to 10(8) HDV RNA and 10(7) HBV-DNA copies/ml) even after 6 weeks of latent mono-infection. Concurrently, the number of HDAg-positive human hepatocytes increased, demonstrating intrahepatic HDV spreading. The infectivity of the rescued HDV virions was verified by serial passage in naive chimeric mice. CONCLUSIONS HDV mono-infection can persist intrahepatically for at least 6 weeks before being rescued by HBV. Conversion of a latent HDV infection to a productive HBV/HDV co-infection may contribute to HDV persistence even in patients with low HBV replication and in the setting of liver transplantation.

Proliferation of primary human hepatocytes and prevention of hepatitis B virus reinfection efficiently deplete nuclear cccDNA in vivo

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.