A Prospective Five-Year Follow-up After peg-Interferon Plus Nucleotide Analogue Treatment or no Treatment in HBeAg Negative Chronic Hepatitis B Patients.

Background: Currently available treatment options for chronic hepatitis B (CHB) are not recommended for HBeAg-negative patients with a low viral load. These patients may however benefit from treatment by achieving a functional cure, defined by HBsAg-loss and undetectable HBV DNA. This study evaluated the long-term effect of combination treatment with peg-interferon-alpha-2a (peg-IFN) and adefovir or tenofovir compared to no treatment in these patients. Methods: HBeAg-negative CHB patients with HBV-DNA levels < 20,000 IU/mL (n = 151) were previously randomised 1:1:1 for peg-IFN 180 µg/week plus either adefovir 10 mg/day or tenofovir 245 mg/day, or no treatment and treated for 48 weeks in an open-label study. In this prospective long-term follow-up study, patients were monitored yearly up to five years after end of treatment (week 308). The primary outcome was sustained HBsAg-loss and secondary outcome the dynamics of HBsAg and HBV-DNA levels over time. Results: Of the 131 followed patients, the HBsAg-status was known for 118 patients after five-year follow-up. HBsAg-loss occurred similarly (P = 0.703) in all arms: 8/43 (18.6%) peg-IFN + adefovir, 4/34 (11.7%) peg-IFN + tenofovir, and 6/41 (14.6%) among the untreated patients. The time to HBsAg-loss did not differ between groups (P = 0.641). Low baseline HBsAg levels and genotype A were independently associated with HBsAg-loss irrespective of allocation. HBsAg and HBV-DNA levels declined similarly during follow-up in all patient groups. Conclusions: This prospective randomised controlled study showed that HBsAg-loss overtime was not influenced by treatment with a combination of nucleotide analogue and Peg-IFN. Low baseline HBsAg levels can predict HBsAg-loss irrespective of treatment allocation.

Deep sequencing identifies hepatitis B virus core protein signatures in chronic hepatitis B patients.

BACKGROUND: We aimed to identify HBc amino acid differences between subgroups of chronic hepatitis B (CHB) patients. METHODS: Deep sequencing of HBc was performed in samples of 89 CHB patients (42 HBeAg positive, 47 HBeAg negative). Amino acid types were compared using Sequence Harmony to identify subgroup specific sites between HBeAg-positive and -negative patients, and between patients with combined response and non-response to peginterferon/adefovir combination therapy. RESULTS: We identified 54 positions in HBc where the frequency of appearing amino acids was significantly different between HBeAg-positive and -negative patients. In HBeAg negative patients, 22 positions in HBc were identified which differed between patients with treatment response and those with non-response. The fraction non-consensus sequence on selected positions was significantly higher in HBeAg-negative patients, and was negatively correlated with HBV DNA and HBsAg levels. CONCLUSIONS: Sequence Harmony identified a number of amino acid changes associated with HBeAg-status and response to peginterferon/adefovir combination therapy.

Plasma MicroRNA Levels Are Associated With Hepatitis B e Antigen Status and Treatment Response in Chronic Hepatitis B Patients.

Background: Hepatitis B virus (HBV) modulates microRNA (miRNA) expression to support viral replication. The aim of this study was to identify miRNAs associated with hepatitis B e antigen (HBeAg) status and response to antiviral therapy in patients with chronic hepatitis B (CHB) , and to assess if these miRNAs are actively secreted by hepatoma cells. Methods: Plasma miRNA levels were measured by reverse-transcription quantitative polymerase chain reaction in healthy controls (n = 10) and pretreatment samples of an identification cohort (n = 24) and a confirmation cohort (n = 64) of CHB patients treated with peginterferon/nucleotide analogue combination therapy. Levels of HBV-associated miRNAs were measured in cells, extracellular vesicles, and hepatitis B surface antigen (HBsAg) particles of hepatoma cell lines. Results: HBeAg-positive patients had higher plasma levels of miR-122-5p, miR-125b-5p, miR-192-5p, miR-193b-3p, and miR-194-5p compared to HBeAg-negative patients, and levels of these miRNAs were associated with HBV DNA and HBsAg levels. Pretreatment plasma levels of miR-301a-3p and miR-145-5p were higher in responders (combined response or HBsAg loss) compared to nonresponders. miR-192-5p, miR-193b-3p, and miR-194-5p were present in extracellular vesicles and HBsAg particles derived from hepatoma cells. Conclusions: We identified miRNAs that are associated with HBeAg status, levels of HBV DNA and HBsAg, and treatment response in CHB patients. We demonstrated that several of these miRNAs are present in extracellular vesicles and HBsAg particles secreted by hepatoma cells.

Safety, tolerability, and antiviral effect of RG-101 in patients with chronic hepatitis C: a phase 1B, double-blind, randomised controlled trial.

BACKGROUND: miR-122 is an important host factor for hepatitis C virus (HCV) replication. The aim of this study was to assess the safety and tolerability, pharmacokinetics, and antiviral effect of a single dose of RG-101, a hepatocyte targeted N-acetylgalactosamine conjugated oligonucleotide that antagonises miR-122, in patients with chronic HCV infection with various genotypes. METHODS: In this randomised, double-blind, placebo-controlled, multicentre, phase 1B study, patients were randomly assigned to RG-101 or placebo (7:1). We enrolled men and postmenopausal or hysterectomised women (aged 18-65 years) with chronic HCV genotype 1, 3, or 4 infection diagnosed at least 24 weeks before screening who were either treatment naive to or relapsed after interferon-α based therapy. Patients with co-infection (hepatitis B virus or HIV infection), evidence of decompensated liver disease, or a history of hepatocellular carcinoma were excluded. Randomisation was done by an independent, unblinded, statistician using the SAS procedure Proc Plan. The first cohort received one subcutaneous injection of 2 mg/kg RG-101 or placebo; the second cohort received one subcutaneous injection of 4 mg/kg or placebo. Patients were followed up for 8 weeks (all patients) and up to 76 weeks (patients with no viral rebound and excluding those who were randomised to the placebo group) after randomisation. The primary objective was safety and tolerability of RG-101. This trial was registered with EudraCT, number 2013-002978-49. FINDINGS: Between June 4, 2014, and Oct 27, 2014, we enrolled 32 patients with chronic HCV genotype 1 (n=16), 3 (n=10), or 4 (n=6) infections. In the first cohort, 14 patients were randomly assigned to receive 2 mg/kg RG-101 and two patients were randomly assigned to receive placebo, and in the second cohort, 14 patients were randomly assigned to receive 4 mg/kg RG-101 and two patients were randomly assigned to receive placebo. Overall, 26 of the 28 patients dosed with RG-101 reported at least one treatment-related adverse event. At week 4, the median viral load reduction from baseline was 4·42 (IQR 3·23-5·00) and 5·07 (4·19-5·35) log10 IU/mL in patients dosed with 2 mg/kg RG-101 or 4 mg/kg RG-101. Three patients had undetectable HCV RNA levels 76 weeks after a single dose of RG-101. Viral rebound at or before week 12 was associated with the appearance of resistance associated substitutions in miR-122 binding regions in the 5' UTR of the HCV genome. INTERPRETATION: This study showed that one administration of 2 mg/kg or 4 mg/kg RG-101, a hepatocyte targeted N-acetylgalactosamine conjugated anti-miR-122 oligonucleotide, was well tolerated and resulted in substantial viral load reduction in all treated patients within 4 weeks, and sustained virological response in three patients for 76 weeks. FUNDING: Regulus Therapeutics, Inc.

Hepatitis B Virus Pregenomic RNA Is Present in Virions in Plasma and Is Associated With a Response to Pegylated Interferon Alfa-2a and Nucleos(t)ide Analogues.

BACKGROUND: Treatment of patients with chronic hepatitis B (CHB) with nucleos(t)ide analogues (NAs) suppresses hepatitis B virus (HBV) DNA production but does not affect the synthesis of the RNA pregenome or HBV messenger RNA. Whether HBV RNA-containing particles continue to be secreted into the bloodstream remains controversial. METHODS: We developed a sensitive polymerase chain reaction (PCR) assay to quantify the HBV RNA load in a supernatant of NA-treated HepG2-2.2.15 cells and in plasma specimens from 20 patients with CHB who were receiving NA therapy and 86 patients treated with pegylated interferon alfa (Peg-IFN) and adefovir. RESULTS: Treatment of HepG2-2.2.15 cells with NAs for 9 days reduced HBV DNA levels (by 1.98 log10 copies/mL), whereas HBV RNA levels increased (by 0.47 log10 copies/mL; P < .05). During long-term NA treatment of patients with CHB, HBV RNA levels remained higher than HBV DNA levels. Peg-IFN-based treatment induced a stronger decrease in the HBV RNA load than NA monotherapy, and this decline was more pronounced in responders than in nonresponders. In HBV e antigen-negative patients, a lower baseline plasma HBV RNA level was independently associated with response to Peg-IFN and adefovir (odds ratio, 0.44; P = .019). Immunoprecipitation with HBV core antigen-specific antibodies after removal of the HBV surface antigen envelope demonstrated the association of plasma HBV RNA with virions. CONCLUSIONS: HBV RNA is present in virions in plasma specimens from patients with CHB. HBV RNA levels vary significantly from those of established viral markers during antiviral treatment, which highlights its potential as an independent marker in the evaluation of patients with CHB.

Restoration of T cell function in chronic hepatitis B patients upon treatment with interferon based combination therapy.

BACKGROUND & AIMS: Chronic hepatitis B virus (HBV) infection is characterized by functional impairment of HBV-specific T cells. Understanding the mechanisms behind T cell dysfunction and restoration is important for the development of optimal treatment strategies. METHODS: In this study we have first analysed the phenotype and function of HBV-specific T cells in patients with low viral load (HBV DNA <20,000IU/ml) and spontaneous control over the virus. Subsequently, we assessed HBV-specific T cells in patients with high viral load (HBV DNA >17,182IU/ml) treated with peginterferon/adefovir combination therapy who had various treatment outcomes. RESULTS: HBV-specific T cells could be detected directly ex vivo in 7/22 patients with low viral load. These showed an early differentiated memory phenotype with reduced ability to produce IL-2 and cytotoxic molecules such as granzyme B and perforin, but with strong proliferative potential. In a cohort of 28 chronic hepatitis B patients with high viral load treated with peginterferon and adefovir, HBV-specific T cells could not be detected directly ex vivo. However, HBV-specific T cells could be selectively expanded in vitro in patients with therapy-induced HBsAg clearance (HBsAg loss n=7), but not in patients without HBsAg clearance (n=21). Further analysis of HBV-specific T cell function with peptide pools showed broad and efficient antiviral responses after therapy. CONCLUSIONS: Our results show that peginterferon based combination therapy can induce HBV-specific T cell restoration. These findings may help to develop novel therapeutic strategies to reconstitute antiviral functions and enhance viral clearance.

An intrahepatic transcriptional signature of enhanced immune activity predicts response to peginterferon in chronic hepatitis B.

BACKGROUND & AIMS: Differences in intrahepatic gene expression patterns may be associated with therapy response in peginterferon-treated chronic hepatitis B (CHB) patients. METHODS: We employed gene expression profiling in baseline liver biopsies of 40 CHB patients (19 HBeAg-positive; 21 HBeAg-negative) treated with peginterferon and adefovir for 48 weeks, and compared expression patterns of combined responders (HBeAg loss, HBV-DNA <2000 IU/ml, alanine aminotransferase normalization after 1 year of treatment-free follow-up) with non-responders. Genes identified by transcriptome analysis in 15 biopsies were confirmed in 25 additional biopsies by RT-qPCR. RESULTS: Transcriptome analysis demonstrated significant differences in expression of 41 genes between responders and non-responders. In responders, pathway analysis showed specific upregulation of genes related to the immune response, including chemotaxis and antigen processing and presentation. Genes upregulated in responders exhibited strongest similarity with a set of genes induced in livers of chimpanzees with acute Hepatitis B infection. Differential expression was confirmed for eight selected genes. A 2-gene subset (HLA-DPB1, SERPIN-E1) was found to predict response most accurately. Incorporation of these genes in a multivariable model with HBeAg status, HBV genotype and baseline HBsAg level correctly classified 90% of all patients, in which HLA-DPB1 and SERPIN-E1 were independent predictors of response. CONCLUSION: We identified an intrahepatic transcriptional signature associated with enhanced immune activation which predicts therapy response. These novel associations could lead to better understanding of responsiveness to peginterferon in CHB patients, and may assist in selecting possible responders to interferon-based treatment.

Long-term safety and efficacy of microRNA-targeted therapy in chronic hepatitis C patients.

BACKGROUND AND AIMS: MicroRNA-122 (miR-122) is an important host factor for hepatitis C virus (HCV) and promotes HCV RNA accumulation. Decreased intra-hepatic levels of miR-122 were observed in patients with hepatocellular carcinoma, suggesting a potential role of miR-122 in the development of HCC. Miravirsen targets miR-122 and resulted in a dose dependent and prolonged decrease of HCV RNA levels in chronic hepatitis C patients. The aim of this study was to establish the sustained virological response rate to peginterferon (P) and ribavirin (R) following miravirsen dosing and to assess long-term safety in patients treated with miravirsen. METHODS: In this multicenter, retrospective follow-up study we included 36 treatment naïve patients with chronic hepatitis C genotype 1 who received five weekly subcutaneous injections with miravirsen or placebo over a 29-day period in a phase 2a study. Patients were offered PR therapy 3weeks (3mg/kg group) or 6weeks (5 or 7mg/kg group) after completion of miravirsen or placebo dosing. RESULTS: PR therapy was started in 14/36 patients of whom 12 had received miravirsen. SVR was achieved in 7/12 patients previously dosed with miravirsen. All patients dosed with 7mg/kg miravirsen who were subsequently treated with PR achieved SVR. One patient had a prolonged undetectable HCV RNA period from week 14 to week 29 after baseline without subsequent antiviral therapy and relapsed thereafter. None of the patients treated with anti-miR-122 developed HCC or other liver-related complications. CONCLUSION: No long-term safety issues were observed among 27 miravirsen-treated patients. Targeting miR-122 may be an effective and safe treatment strategy for HCV infection and should be investigated in larger clinical trials.

MicroRNAs: role and therapeutic targets in viral hepatitis.

MicroRNAs regulate gene expression by binding to the 3'-untranslated region (UTR) of target messenger RNAs (mRNAs). The importance of microRNAs has been shown for several liver diseases, for example, viral hepatitis. MicroRNA-122 is highly abundant in the liver and is involved in the regulation of lipid metabolism. MicroRNA-122 is also an important host factor for the HCV and promotes HCV replication. In contrast to HCV, microRNA-122 inhibits replication of the HBV. MicroRNA-122 acts as a tumour suppressor and reduced levels of microRNA-122 are associated with hepatocellular carcinoma. MicroRNAs other than microRNA-122 have been linked to viral hepatitis, fibrosis and inflammation. In this review, we discuss function and clinical implications of microRNA-122 and other microRNAs in liver diseases, especially viral hepatitis.

Baseline hepatitis B surface antigen (HBsAg) as predictor of sustained HBsAg loss in chronic hepatitis B patients treated with pegylated interferon-α2a and adefovir.

BACKGROUND: In this study, we aimed to identify baseline predictors of response in chronic hepatitis B patients treated with a combination of pegylated interferon (PEG-IFN)-α2a and adefovir. METHODS: We treated 92 chronic hepatitis B patients (44 hepatitis B e antigen [HBeAg]-positive and 48 HBeAg-negative) with HBV DNA > 100,000 copies/ml (> 17,182 IU/ml) with PEG-IFN and adefovir for 48 weeks and followed them up for 2 years. Baseline markers for HBeAg loss, combined response (HBeAg negativity, HBV DNA levels ≤ 2,000 IU/ml and alanine aminotransferase [ALT] normalization) and hepatitis B surface antigen (HBsAg) loss were evaluated. RESULTS: Two years after the end of treatment, rates of HBeAg loss and HBsAg loss in HBeAg-positive patients were 18/44 (41%) and 5/44 (11%), respectively. In HBeAg-negative patients, rates of combined response and HBsAg loss were 12/48 (25%) and 8/48 (17%), respectively. HBeAg-negative patients with HBsAg loss had lower baseline HBsAg levels than those without HBsAg loss (mean HBsAg 2.35 versus 3.55 log10 IU/ml; P < 0.001). They also had lower HBV DNA levels and were more often (PEG-)IFN experienced. Baseline HBsAg was the only independent predictor of HBsAg loss (OR 0.02; P = 0.01). CONCLUSIONS: With combination therapy of PEG-IFN and adefovir for 48 weeks, a high rate of HBsAg loss was observed in both HBeAg-positive (11%) and HBeAg-negative (17%) patients 2 years after treatment ended. In HBeAg-negative patients, a low baseline HBsAg level was a strong predictor for HBsAg loss.