Identification of Liver and Plasma microRNAs in Chronic Hepatitis B Virus infection.

Background and Aims: With current standard of care a functional cure for Chronic Hepatitis B (CHB) is only achieved in 1-3% of patients and therefore novel therapies are needed. Disease activity during CHB can be determined by a broad range of virological biomarkers, however these biomarkers are also targets for novel treatment strategies. The aim of this study was to identify novel miRNAs that are differentially expressed in plasma and liver in CHB, and determine whether these miRNAs may serve as biomarkers of disease stage or treatment outcome. Methods: miRNA Next-Generation-Sequencing of plasma and liver samples from CHB patient and controls was performed to identify differentially expressed miRNAs. The identified candidate miRNAs were validated by qPCR in additional plasma and liver samples from two CHB cohorts. Results: Several miRNAs in plasma and liver were found to be differentially expressed between CHB patients and controls. Of the identified miRNAs expression levels of miR-122-5p in plasma were associated with plasma HBsAg, and plasma and liver HBV-DNA levels. Expression levels of miR-223-3p, miR-144-5p and miR-133a-3p in liver were associated with plasma alanine aminotransferase levels. No correlation was observed between miRNA expression levels at baseline and treatment outcome. Conclusions: Limited overlap between plasma and liver miRNAs was found, indicating that plasma miRNAs could be useful as biomarkers for treatment outcome or viral activity during treatment. Whereas liver miRNAs are more likely to be regulated by HBV and could be potential therapeutic targets to control viral activity in liver.

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.

Hepatitis B core related antigen in relation to intrahepatic and circulating viral markers, before and after combination therapy.

INTRODUCTION AND OBJECTIVES: Covalently closed circular (ccc)DNA acts as a viral reservoir in the liver of patients with a chronic hepatitis B (CHB) infection and can only be quantified in liver biopsies. Hepatitis B core-related antigen (HBcrAg) levels in plasma/serum have been proposed to reflect intrahepatic cccDNA-levels and may therefore monitor treatment efficacy. This study aimed to validate the relationship between HBcrAg and other intrahepatic and circulating viral markers in CHB patients with high viral load, before and after combination treatment. MATERIALS AND METHODS: Plasma/serum levels of HBcrAg, HBsAg, HBV-DNA, and HBV pregenomic RNA (HBV-pgRNA), and intrahepatic cccDNA and HBV-DNA levels and fibrosis scores were measured in 89 CHB patients with HBV-DNA levels of >100,000 copies/mL (17,182 IU/mL). Measurements were done before and after a 48-week treatment with pegylated interferon alfa-2a and adefovir in a prospective study (ISRCTN77073364). RESULTS: Baseline HBcrAg-values correlated strongly with intrahepatic cccDNA (ρ 0.77, p < 0.001), intrahepatic HBV-DNA (ρ 0.73, p < 0.001) and plasma/serum HBV-DNA (ρ 0.80, p < 0.001), HBV-pgRNA (ρ 0.80, p < 0.001), and to lesser extend HBsAg (ρ 0.56, p < 0.001). Baseline HBcrAg-levels could not predict functional cure (FC) but HBcrAg-levels declined more strongly in patients who developed FC or HBeAg-loss. Furthermore, most correlations persisted at the end of treatment and follow-up. CONCLUSIONS: HBcrAg reflects cccDNA transcription activity more accurately than HBsAg and may replace HBV-DNA as a marker during future treatment regimens, especially when cccDNA transcription is targeted or nucleot(s)ide analogues are included in the treatment regime.

The Observed Effect of Gastric Bypass Surgery on Direct-acting Antiviral Treatment: a Case Report.

Chronic hepatitis C virus (HCV) infection can be cured with treatment using direct-acting antivirals (DAAs). Although these drugs have been widely studied, information about certain special populations is missing. In this case report we describe a treatment-experienced patient with chronic HCV infection genotype 1b, treated with 150 mg/day simeprevir, 400 mg/day sofosbuvir, and 1,000 mg/ day ribavirin for 24 weeks, after a Roux-and-Y gastric bypass. At steady-state a pharmacokinetic curve was recorded of sofosbuvir, GS-331007, and simeprevir. Ribavirin trough plasma concentration (Ctrough) was determined. The simeprevir area under the-concentration time curve (AUClast) and Ctrough were 9.42 h.mg/L and 0.046 mg/L, respectively. Compared to what was described in the literature, simeprevir exposure was low and therefore the simeprevir dose was increased to 300 mg/day. The increased dose of simeprevir was well tolerated and Ctrough was 0.532 mg/L. Sofosbuvir AUClast and Ctrough were 0.63 h.mg/L and 0.0013 mg/L. GS-331007 AUClast and Ctrough were 21.02 h.mg/L and 0.35 mg/L. Ribavirin Ctrough was 2.5 mg/L. Sofosbuvir, GS-331007, and ribavirin exposure were comparable with levels described in literature. The patient achieved a sustained virological response twelve weeks after the completion of treatment.

Immune responses in DAA treated chronic hepatitis C patients with and without prior RG-101 dosing.

BACKGROUND&AIMS: With the introduction of DAA's, the majority of treated chronic hepatitis C patients (CHC) achieve a viral cure. The exact mechanisms by which the virus is cleared after successful therapy, is still unknown. The aim was to assess the role of the immune system and miRNA levels in acquiring a sustained virological response after DAA treatment in CHC patients with and without prior RG-101 (anti-miR-122) dosing. METHODS: In this multicenter, investigator-initiated study, 29 patients with hepatitis C virus (HCV) genotype 1 (n = 11), 3 (n = 17), or 4 (n = 1) infection were treated with sofosbuvir and daclatasvir ± ribavirin. 18 patients were previously treated with RG-101. IP-10 levels were measured by ELISA. Ex vivo HCV-specific T cell responses were quantified in IFN-γ-ELISpot assays. Plasma levels of miR-122 were measured by qPCR. RESULTS: All patients had an SVR12. IP-10 levels rapidly declined during treatment, but were still elevated 24 weeks after treatment as compared to healthy controls (median 53.82 and 39.4 pg/mL, p = 0.02). Functional IFN-γ HCV-specific T cell responses did not change by week 12 of follow-up (77.5 versus 125 SFU/106 PBMC, p = 0.46). At follow-up week 12, there was no difference in plasma miR-122 levels between healthy controls and patients with and without prior RG-101 dosing. CONCLUSIONS: Our data shows that successful treatment of CHC patients with and without prior RG-101 dosing results in reduction of broad immune activation, and normalisation of miR-122 levels (EudraCT: 2014-002808-25). TRIAL REGISTRATION: EudraCT: 2014-002808-25.

Peg-interferon plus nucleotide analogue treatment versus no treatment in patients with chronic hepatitis B with a low viral load: a randomised controlled, open-label trial.

BACKGROUND: Antiviral treatment is currently not recommended for patients with chronic hepatitis B with a low viral load. However, they might benefit from acquiring a functional cure (hepatitis B surface antigen [HBsAg] loss with or without formation of antibodies against hepatitis B surface antigen [anti-HBs]). We assessed HBsAg loss during peg-interferon-alfa-2a (peg-IFN) and nucleotide analogue combination therapy in patients with chronic hepatitis B with a low viral load. METHODS: In this randomised controlled, open-label trial, patients were enrolled from the Academic Medical Center (AMC), Amsterdam, Netherlands. Eligible patients were HBsAg positive and hepatitis B e antigen (HBeAg) negative for more than 6 months, could be treatment naive or treatment experienced, and had alanine aminotransferase (ALT) concentrations less than 5 × upper limit of normal (ULN). Participants were randomly assigned (1:1:1) by a computerised randomisation programme (ALEA Randomisation Service) to receive peg-IFN 180 µg/week plus adefovir 10 mg/day, peg-IFN 180 µg/week plus tenofovir disoproxil fumarate 245 mg/day, or no treatment for 48 weeks. The primary endpoint was the proportion of patients with serum HBsAg loss among those who received at least one dose of study drug or had at least one study visit (modified intention-to-treat population [mITT]). All patients have finished the initial study of 72 weeks and will be observed for up to 5 years of follow-up. This study is registered with ClinicalTrials.gov, number NCT00973219. FINDINGS: Between Aug 4, 2009, and Oct 17, 2013, 167 patients were screened for enrolment, of whom 151 were randomly assigned (52 to peg-IFN plus adefovir, 51 to peg-IFN plus tenofovir, and 48 to no treatment). 46 participants in the peg-IFN plus adefovir group, 45 in the peg-IFN plus tenofovir group, and 43 in the no treatment group began treatment or observation and were included in the mITT population. At week 72, two (4%) patients in the peg-IFN plus adefovir group and two (4%) patients in the peg-IFN plus tenofovir group had achieved HBsAg loss, compared with none of the patients in the no treatment group (p=0·377). The most frequent adverse events (>30%) were fatigue, headache, fever, and myalgia, which were attributed to peg-IFN dosing. Two (4%) serious adverse events were reported in the peg-IFN plus adefovir group (admission to hospital for alcohol-related pancreatitis [week 6; n=1] and pregnancy, which was electively aborted [week 9; n=1]), three (7%) in the peg-IFN plus tenofovir group (admission to hospital after a suicide attempt during a severe depression [week 23; n=1], admission to hospital for abdominal pain [week 2; n=1], and an elective laminectomy [week 40; n=1]), and three (7%) in the no treatment group (admission to hospital for septic arthritis [week 72; n=1], endocarditis [week 5; n=1], and hyperthyroidism [week 20; n=1]). INTERPRETATION: In patients with chronic hepatitis B with a low viral load, combination treatment (peg-IFN plus adefovir and peg-IFN plus tenofovir) did not result in significant HBsAg loss compared with no treatment, which does not support the use of combination treatment in this population of patients. FUNDING: Roche, Fonds NutsOhra.

Immune phenotype and function of natural killer and T cells in chronic hepatitis C patients who received a single dose of anti-MicroRNA-122, RG-101.

MicroRNA-122 is an important host factor for the hepatitis C virus (HCV). Treatment with RG-101, an N-acetylgalactosamine-conjugated anti-microRNA-122 oligonucleotide, resulted in a significant viral load reduction in patients with chronic HCV infection. Here, we analyzed the effects of RG-101 therapy on antiviral immunity. Thirty-two chronic HCV patients infected with HCV genotypes 1, 3, and 4 received a single subcutaneous administration of RG-101 at 2 mg/kg (n = 14) or 4 mg/kg (n = 14) or received a placebo (n = 2/dosing group). Plasma and peripheral blood mononuclear cells were collected at multiple time points, and comprehensive immunological analyses were performed. Following RG-101 administration, HCV RNA declined in all patients (mean decline at week 2, 3.27 log10 IU/mL). At week 8 HCV RNA was undetectable in 15/28 patients. Plasma interferon-γ-induced protein 10 (IP-10) levels declined significantly upon dosing with RG-101. Furthermore, the frequency of natural killer (NK) cells increased, the proportion of NK cells expressing activating receptors normalized, and NK cell interferon-γ production decreased after RG-101 dosing. Functional HCV-specific interferon-γ T-cell responses did not significantly change in patients who had undetectable HCV RNA levels by week 8 post-RG-101 injection. No increase in the magnitude of HCV-specific T-cell responses was observed at later time points, including 3 patients who were HCV RNA-negative 76 weeks postdosing. CONCLUSION: Dosing with RG-101 is associated with a restoration of NK-cell proportions and a decrease of NK cells expressing activation receptors; however, the magnitude and functionality of ex vivo HCV-specific T-cell responses did not increase following RG-101 injection, suggesting that NK cells, but not HCV adaptive immunity, may contribute to HCV viral control following RG-101 therapy. (Hepatology 2017;66:57-68).

Dynamics of the Immune Response in Acute Hepatitis B Infection.

BACKGROUND: Acute hepatitis B virus infection in adults is generally self-limiting but may lead to chronicity in a minority of patients. METHODS: We included 9 patients with acute hepatitis B virus (HBV) infection and collected longitudinal follow-up samples. Natural killer (NK) cell characteristics were analyzed by flowcytometry. HBV-specific T-cell function was analyzed by in vitro stimulation with HBV peptide pools and intracellular cytokine staining. RESULTS: Median baseline HBV DNA load was 5.12 log IU/mL, and median ALT was 2652 U/mL. Of 9 patients, 8 cleared HBsAg within 6 months whereas 1 patient became chronically infected. Early time points after infection showed increased CD56bright NK cells and an increased proportion of cells expressing activation markers. Most of these had normalized at week 24, while the proportion of TRAIL-positive CD56bright NK cells remained high in the chronically infected patient. In patients who cleared HBV, functional HBV-specific CD8+ and CD4+ responses could be observed, whereas in the patient who developed chronic infection, only low HBV-specific T-cell responses were observed. CONCLUSIONS: NK cells are activated early in the course of acute HBV infection. Broad and multispecific T-cell responses are observed in patients who clear acute HBV infection, but not in a patient who became chronically infected.

Cost-Effectiveness of Hepatitis C Treatment for People Who Inject Drugs and the Impact of the Type of Epidemic; Extrapolating from Amsterdam, the Netherlands.

BACKGROUND: People who inject drugs (PWID) are disproportionally affected by the hepatitis C virus (HCV) infection. The efficacy of HCV treatment has significantly improved in recent years with the introduction of direct-acting antivirals (DAAs). However, DAAs are more costly than pegylated-interferon and ribavirin (PegIFN/RBV). We aimed to assess the cost-effectiveness of four HCV treatment strategies among PWID and treatment scale-up. METHODS: An individual-based model was used describing HIV and HCV transmission and disease progression among PWID. We considered two epidemiological situations. A declining epidemic, based on the situation in Amsterdam, the Netherlands, and a stable HCV epidemic, as observed in other settings. Data on HCV incidence, prevalence, treatment setting and uptake were derived from observed data among PWID in Amsterdam. We assessed the incremental cost-effectiveness ratio (ICER, costs in €/quality-adjusted life year (QALY)) of four treatment strategies: 1) PegIFN/RBV; 2) sofosbuvir/RBV for genotype 2-3 and dual DAA for genotype 1-4; 3) Dual DAA for all genotypes; 4) Dual DAA with 3x treatment uptake. RESULTS: In both types of epidemic, dual DAA therapy was most cost-effective strategy. In the declining epidemic, dual DAA yielded an ICER of 344 €/QALY while in the stable epidemic dual DAA led to cost-savings. Scaling-up treatment was also highly cost-effective. Our results were robust over a range of sensitivity analyses. CONCLUSION: HCV treatment with DAA-containing regimens is a highly cost-effective intervention among PWID. Based on the economic and population benefits of scaling-up treatment, stronger efforts are needed to achieve higher uptake rates among PWID.

Limited Generalizability of Registration Trials in Hepatitis C: A Nationwide Cohort Study.

BACKGROUND: Approval of drugs in chronic hepatitis C is supported by registration trials. These trials might have limited generalizability through use of strict eligibility criteria. We compared effectiveness and safety of real world hepatitis C patients eligible and ineligible for registration trials. METHODS: We performed a nationwide, multicenter, retrospective cohort study of chronic hepatitis C patients treated in the real world. We applied a combined set of inclusion and exclusion criteria of registration trials to our cohort to determine eligibility. We compared effectiveness and safety in eligible vs. ineligible patients, and performed sensitivity analyses with strict criteria. Further, we used log binomial regression to assess relative risks of criteria on outcomes. RESULTS: In this cohort (n = 467) 47% of patients would have been ineligible for registration trials. Main exclusion criteria were related to hepatic decompensation and co-morbidity (cardiac disease, anemia, malignancy and neutropenia), and were associated with an increased risk for serious adverse events (RR 1.45-2.31). Ineligible patients developed significantly more serious adverse events than eligible patients (27% vs. 11%, p< 0.001). Effectiveness was decreased if strict criteria were used. CONCLUSIONS: Nearly half of real world hepatitis C patients would have been excluded from registration trials, and these patients are at increased risk to develop serious adverse events. Hepatic decompensation and co-morbidity were important exclusion criteria, and were related to toxicity. Therefore, new drugs should also be studied in these patients, to genuinely assess benefits and risk of therapy in the real world population.