M. Lütgehetmann

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.)

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)

The liver stage of Plasmodium berghei inhibits host cell apoptosis

Plasmodium berghei is the causative agent of rodent malaria and is widely used as a model system to study the liver stage of Plasmodium parasites. The entry of P. berghei sporozoites into hepatocytes has extensively been studied, but little is known about parasite–host interaction during later developmental stages of the intracellular parasite. Growth of the parasite far beyond the normal size of the host cell is an important stress factor for the infected cell. Cell stress is known to trigger programmed cell death (apoptosis) and we examined several apoptotic markers in P. berghei‐infected cells and compared their level of expression and their distribution to that of non‐infected cells. As none of the apoptotic markers investigated were found altered in infected cells, we hypothesized that parasite infection might confer resistance to apoptosis of the host cell. Treatment with peroxide or serum deprivation induced apoptosis in non‐infected HepG2 cells, whereas P. berghei‐infected cells appeared protected, indicating that the parasite interferes indeed with the apoptotic machinery of the host cell. To prove the physiological relevance of these results, mice were infected with high numbers of P. berghei sporozoites and treated with tumour necrosis factor (TNF)‐α/d‐galactosamine to induce massive liver apoptosis. Liver sections of these mice, stained for degraded DNA, confirmed that infected cells containing viable parasites were protected from programmed cell death. However, in non‐treated control mice as well as in TNF‐α‐treated mice a small proportion of dead intracellular parasites with degraded DNA were detected. Most hepatocytes containing dead parasites provoked an infiltration of immunocompetent cells, indicating that these cells are no longer protected from cell death.

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.

Entecavir plus tenofovir combination as rescue therapy in pre-treated chronic hepatitis B patients: An international multicenter cohort study

BACKGROUND & AIMS Long-term viral suppression is a major goal to prevent disease progression in patients with HBV. Aim of this study was to investigate the efficacy and safety of entecavir plus tenofovir combination in 57 CHB partial responders or multidrug resistant patients. METHODS Investigator-initiated open-label cohort study. Quantitative HBV-DNA measurement and resistance testing (line-probe-assays and direct-sequencing) at baseline and every 3 months. RESULTS Fifty seven patients (37 HBeAg+), median age 45 years, previously treated with a median of three lines of antiviral therapy (range 1-6), 24/57 with advanced liver disease, were included. Median ALT at baseline was 1.0 ULN (range 0.3-22) and HBV-DNA 1.5 × 10(4)IU/ml (range 500-1 × 10(11)IU/ml). Median treatment duration of combination therapy was 21 months. HBV-DNA level dropped 3 logs (median, range 0-8 log; p<0.0001), 51/57 patients became HBV-DNA undetectable, median after 6 months (95% CI, 4.6-7). The probability for HBV DNA suppression was not reduced in patients with adefovir or entecavir resistance or in patients with advanced liver disease. Viral suppression led to decline in ALT (median 0.7 ULN; range 0.2-2.4; p=0.001). Five patients lost HBeAg (after 15, 18, 20, 21, and 27 months, respectively), one patient showed HBs-seroconversion. Patients with advanced disease did not show clinical decompensation, two patients with cirrhosis and undetectable HBV DNA developed HCCs. No death, newly induced renal impairment or lactic acidosis were reported. CONCLUSIONS Rescue therapy with entecavir and tenofovir in CHB patients harboring viral resistance patterns or showing only partial antiviral responses to preceding therapies was efficient, safe, and well tolerated in patients with and without advanced liver disease (249).

RGB marking facilitates multicolor clonal cell tracking

We simultaneously transduced cells with three lentiviral gene ontology (LeGO) vectors encoding red, green or blue fluorescent proteins. Individual cells were thereby marked by different combinations of inserted vectors, resulting in the generation of numerous mixed colors, a principle we named red-green-blue (RGB) marking. We show that lentiviral vector–mediated RGB marking remained stable after cell division, thus facilitating the analysis of clonal cell fates in vitro and in vivo. Particularly, we provide evidence that RGB marking allows assessment of clonality after regeneration of injured livers by transplanted primary hepatocytes. We also used RGB vectors to mark hematopoietic stem/progenitor cells that generated colored spleen colonies. Finally, based on limiting-dilution and serial transplantation assays with tumor cells, we found that clonal tumor cells retained their specific color-code over extensive periods of time. We conclude that RGB marking represents a useful tool for cell clonality studies in tissue regeneration and pathology.

Clearance of persistent hepatitis C virus infection in humanized mice using a claudin-1-targeting monoclonal antibody

Hepatitis C virus (HCV) infection is a leading cause of liver cirrhosis and cancer. Cell entry of HCV and other pathogens is mediated by tight junction (TJ) proteins, but successful therapeutic targeting of TJ proteins has not been reported yet. Using a human liver–chimeric mouse model, we show that a monoclonal antibody specific for the TJ protein claudin-1 (ref. 7) eliminates chronic HCV infection without detectable toxicity. This antibody inhibits HCV entry, cell-cell transmission and virus-induced signaling events. Antibody treatment reduces the number of HCV-infected hepatocytes in vivo, highlighting the need for de novo infection by means of host entry factors to maintain chronic infection. In summary, we demonstrate that an antibody targeting a virus receptor can cure chronic viral infection and uncover TJ proteins as targets for antiviral therapy.

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.