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.

Elderly age is not a negative predictive factor for virological response to therapy with pegylated interferon-α and ribavirin in chronic hepatitis C virus patients.

BACKGROUND & AIMS: Age is frequently discussed as negative host factor to achieve a sustained virological response (SVR) to antiviral therapy of chronic hepatitis C. However, elderly patients often show advanced fibrosis/cirrhosis as known negative predictive factor. The aim of this study was to assess age as an independent predictive factor during antiviral therapy. METHODS: Overall, 516 hepatitis C patients were treated with pegylated interferon-α and ribavirin, thereof 66 patients ≥60 years. We analysed the impact of host factors (age, gender, fibrosis, haemoglobin, previous hepatitis C treatment) and viral factors (genotype, viral load) on SVR per therapy course by performing a generalized estimating equations (GEE) regression modelling, a matched pair analysis and a classification tree analysis. RESULTS: Overall, SVR per therapy course was 42.9 and 26.1%, respectively, in young and elderly patients with hepatitis C virus (HCV) genotypes 1/4/6. The corresponding figures for HCV genotypes 2/3 were 74.4 and 84%. In the GEE model, age had no significant influence on achieving SVR. In matched pair analysis, SVR was not different in young and elderly patients (54.2 and 55.9% respectively; P = 0.795 in binominal test). In classification tree analysis, age was not a relevant splitting variable. CONCLUSIONS: Age is not a significant predictive factor for achieving SVR, when relevant confounders are taken into account. As life expectancy in Western Europe at age 60 is more than 20 years, it is reasonable to treat chronic hepatitis C in selected elderly patients with relevant fibrosis or cirrhosis but without major concomitant diseases, as SVR improves survival and reduces carcinogenesis.

Interferon-γ-stimulated genes, but not USP18, are expressed in livers of patients with acute hepatitis C.

BACKGROUND & AIMS: Approximately 50% of patients with chronic hepatitis C (CHC) have a sustained virologic response to treatment with pegylated interferon (pegIFN)-α and ribavirin. Nonresponse to treatment is associated with constitutively increased expression of IFN-stimulated genes (ISGs) in the liver. Treatment of patients with acute hepatitis C (AHC) is more effective, with sustained virologic response rates greater than 90%. We investigated mechanisms of the different responses of patients with CHC and AHC to pegIFN-α therapy. METHODS: We analyzed IFN signaling and ISG expression in liver samples from patients with AHC, patients with CHC, and individuals without hepatitis C (controls) using microarray, immunohistochemical, and protein analyses. Findings were compared with those from primary human hepatocytes stimulated with IFN-α or IFN-γ, as reference sets. RESULTS: Expression levels of hundreds of genes, primarily those regulated by IFN-γ, were altered in liver samples from patients with AHC compared with controls. Expression of IFN-γ-stimulated genes was induced in liver samples from patients with AHC, whereas expression of IFN-α-stimulated genes was induced in samples from patients with CHC. In an expression analysis of negative regulators of IFN-α signaling, we did not observe differences in expression of suppresor of cytokine signaling 1 or SOCS3 between liver samples from patients with AHC and those with CHC. However, USP18 (another negative regulator of IFN-α signaling), was up-regulated in liver samples of patients with CHC that did not respond to therapy, but not in AHC. CONCLUSIONS: Differences in expression of ISGs might account for the greater response of patients with AHC, compared with those with CHC, to treatment with pegIFN-α and ribavirin. Specifically, USP18 is up-regulated in liver samples of patients with CHC that did not respond to therapy, but not in patients with AHC.

Serum ferritin levels are associated with a distinct phenotype of chronic hepatitis C poorly responding to pegylated interferon-alpha and ribavirin therapy.

UNLABELLED: Elevated serum ferritin levels may reflect a systemic inflammatory state as well as increased iron storage, both of which may contribute to an unfavorable outcome of chronic hepatitis C (CHC). We therefore performed a comprehensive analysis of the role of serum ferritin and its genetic determinants in the pathogenesis and treatment of CHC. To this end, serum ferritin levels at baseline of therapy with pegylated interferon-alpha and ribavirin or before biopsy were correlated with clinical and histological features of chronic hepatitis C virus (HCV) infection, including necroinflammatory activity (N = 970), fibrosis (N = 980), steatosis (N = 886), and response to treatment (N = 876). The association between high serum ferritin levels (> median) and the endpoints was assessed by logistic regression. Moreover, a candidate gene as well as a genome-wide association study of serum ferritin were performed. We found that serum ferritin ≥ the sex-specific median was one of the strongest pretreatment predictors of treatment failure (univariate P < 0.0001, odds ratio [OR] = 0.45, 95% confidence interval [CI] = 0.34-0.60). This association remained highly significant in a multivariate analysis (P = 0.0002, OR = 0.35, 95% CI = 0.20-0.61), with an OR comparable to that of interleukin (IL)28B genotype. When patients with the unfavorable IL28B genotypes were stratified according to high versus low ferritin levels, SVR rates differed by > 30% in both HCV genotype 1- and genotype 3-infected patients (P < 0.001). Serum ferritin levels were also independently associated with severe liver fibrosis (P < 0.0001, OR = 2.67, 95% CI = 1.68-4.25) and steatosis (P = 0.002, OR = 2.29, 95% CI = 1.35-3.91), but not with necroinflammatory activity (P = 0.3). Genetic variations had only a limited impact on serum ferritin levels. CONCLUSION: In patients with CHC, elevated serum ferritin levels are independently associated with advanced liver fibrosis, hepatic steatosis, and poor response to interferon-alpha-based therapy.

Lipid-specific ß-sheet formation in a mussel byssus protein domain.

Intrinsically disordered proteins (IDP) or regions (IDR) can adopt multiple conformational states, depending on the interaction partners they encounter. This enables proteins or individual domains to fulfill multiple functions. Here, we analyzed the flank sequences of preCol-NG, one of three collagenous proteins of a mussel byssus thread governing its mechanical performance. preCol-NG comprises a collagen domain and nonrepetitive termini enclosing specific flank regions characterized by tandem repeats known from silk proteins, protein elastomers, and plant cell wall-associated proteins. We recombinantly produced a protein mimicking the M. galloprovincialis preCol-NG C-terminal flank region. The protein was intrinsically unfolded in solution, even at elevated temperatures. However, upon contact with small unilamellar vesicles (SUVs) reversible ß-structure formation occurred, reminiscent of partitioning-folding coupling. This behavior of preCol-NG flank domains likely impacts byssogenesis and sheds new light on a distinct mechanism of how fibrous protein materials might be achieved by lipid-induced self-assembly in nature.

Rapid and strong antiviral activity of the non-nucleosidic NS5B polymerase inhibitor BI 207127 in combination with peginterferon alfa 2a and ribavirin.

BACKGROUND & AIMS: BI 207127 is a potent non-nucleoside hepatitis C virus (HCV) NS5B polymerase inhibitor in vitro. METHODS: In this double-blind, placebo-controlled study, 57 HCV genotype (GT)-1 patients (n=27 treatment-naïve [TN]; n=30 treatment-experienced [TE]) with compensated liver disease were randomised for 28-day treatment with 400, 600, or 800 mg BI 207127 three times daily (TID) or placebo (only TN) in combination with peginterferon alfa 2a and ribavirin (PegIFN/RBV). Plasma HCV RNA was measured by Roche COBAS TaqMan assay. RESULTS: HCV RNA decreased in a dose-dependent manner with little difference between 600 mg (TN 5.6 log(10), TE 4.2 log(10)) and 800 mg (TN 5.4 log(10), TE 4.5 log(10)). Rapid virological response (RVR; HCV RNA <15 IU/ml) at day 28 occurred in 11/19 TN and 4/30 TE patients treated with BI 207127. GT-1b patients had stronger reductions in HCV RNA than GT-1a (RVR: TN 64% vs. 43%; TE 33% vs. 5%). There were no breakthroughs (HCV RNA rebound >1 log(10) from nadir) in the TN groups, whereas 3/30 TE patients experienced breakthrough due to P495-mutations. Gastrointestinal adverse events (AEs) and rash were the major AEs and most frequent at higher doses. One and four patients discontinued due to AEs in the 600 and 800 mg groups, respectively. Overall, tolerability was good and better at 600 mg than 800 mg. CONCLUSIONS: BI 207127 in combination with PegIFN/RBV demonstrated strong antiviral activity with a favourable safety and tolerability profile. The best benefit/risk ratio was observed at 600 mg.

Interferon-induced gene expression is a stronger predictor of treatment response than IL28B genotype in patients with hepatitis C.

BACKGROUND & AIMS: The host immune response during the chronic phase of hepatitis C virus infection varies among individuals; some patients have a no interferon (IFN) response in the liver, whereas others have full activation of IFN-stimulated genes (ISGs). Preactivation of this endogenous IFN system is associated with nonresponse to pegylated IFN-α (pegIFN-α) and ribavirin. Genome-wide association studies have associated allelic variants near the IL28B (IFNλ3) gene with treatment response. We investigated whether IL28B genotype determines the constitutive expression of ISGs in the liver and compared the abilities of ISG levels and IL28B genotype to predict treatment outcome. METHODS: We genotyped 109 patients with chronic hepatitis C for IL28B allelic variants and quantified the hepatic expression of ISGs and of IL28B. Decision tree ensembles, in the form of a random forest classifier, were used to calculate the relative predictive power of these different variables in a multivariate analysis. RESULTS: The minor IL28B allele was significantly associated with increased expression of ISG. However, stratification of the patients according to treatment response revealed increased ISG expression in nonresponders, irrespective of IL28B genotype. Multivariate analysis of ISG expression, IL28B genotype, and several other factors associated with response to therapy identified ISG expression as the best predictor of treatment response. CONCLUSIONS: IL28B genotype and hepatic expression of ISGs are independent predictors of response to treatment with pegIFN-α and ribavirin in patients with chronic hepatitis C. The most accurate prediction of response was obtained with a 4-gene classifier comprising IFI27, ISG15, RSAD2, and HTATIP2.

Genetic variation in IL28B is associated with chronic hepatitis C and treatment failure: a genome-wide association study.

BACKGROUND & AIMS: Hepatitis C virus (HCV) induces chronic infection in 50% to 80% of infected persons; approximately 50% of these do not respond to therapy. We performed a genome-wide association study to screen for host genetic determinants of HCV persistence and response to therapy. METHODS: The analysis included 1362 individuals: 1015 with chronic hepatitis C and 347 who spontaneously cleared the virus (448 were coinfected with human immunodeficiency virus [HIV]). Responses to pegylated interferon alfa and ribavirin were assessed in 465 individuals. Associations between more than 500,000 single nucleotide polymorphisms (SNPs) and outcomes were assessed by multivariate logistic regression. RESULTS: Chronic hepatitis C was associated with SNPs in the IL28B locus, which encodes the antiviral cytokine interferon lambda. The rs8099917 minor allele was associated with progression to chronic HCV infection (odds ratio [OR], 2.31; 95% confidence interval [CI], 1.74-3.06; P = 6.07 x 10(-9)). The association was observed in HCV mono-infected (OR, 2.49; 95% CI, 1.64-3.79; P = 1.96 x 10(-5)) and HCV/HIV coinfected individuals (OR, 2.16; 95% CI, 1.47-3.18; P = 8.24 x 10(-5)). rs8099917 was also associated with failure to respond to therapy (OR, 5.19; 95% CI, 2.90-9.30; P = 3.11 x 10(-8)), with the strongest effects in patients with HCV genotype 1 or 4. This risk allele was identified in 24% of individuals with spontaneous HCV clearance, 32% of chronically infected patients who responded to therapy, and 58% who did not respond (P = 3.2 x 10(-10)). Resequencing of IL28B identified distinct haplotypes that were associated with the clinical phenotype. CONCLUSIONS: The association of the IL28B locus with natural and treatment-associated control of HCV indicates the importance of innate immunity and interferon lambda in the pathogenesis of HCV infection.

Multicompartment micelle-structured peptide nanoparticles: a new biocompatible gene- and drug-delivery tool.

Self-assembled, biodegradable materials that embed fragile, soluble, or insoluble compounds of therapeutic interest have potential use as drug delivery systems. The bead-forming peptide Ac-X3-gT can embed hydrophobic and hydrophilic payloads. Loaded peptide beads were internalized by human acute monocytic leukemia cell line (THP-1) macrophages, THP-1 monocytes, and hepatocellular carcinoma cells (Huh7). Furthermore, paclitaxel and doxorubicin coencapsulated in the peptide beads were delivered to THP-1 monocytes, causing a decrease in cell viability due to the activity of the anticancer drugs. In addition to the bead-forming peptide Ac-X3-gT, the use of a positively charged peptide analogue increased the RNA/DNA embedding efficiency to 99% by charge compensation and micellar complexation. Internalization of the resulting gene delivery systems by Huh7 cells led to specific gene silencing either by embedded small interfering RNA or by plasmid-encoding small hairpin RNA delivered in cells. The new class of purely peptidic material caused no measurable toxicity during in vitro experiments, thereby indicating potential use as a drug delivery system for multidrug delivery and gene therapy.

Pegylated IFN-α regulates hepatic gene expression through transient Jak/STAT activation.

The use of pegylated interferon-α (pegIFN-α) has replaced unmodified recombinant IFN-α for the treatment of chronic viral hepatitis. While the superior antiviral efficacy of pegIFN-α is generally attributed to improved pharmacokinetic properties, the pharmacodynamic effects of pegIFN-α in the liver have not been studied. Here, we analyzed pegIFN-α-induced signaling and gene regulation in paired liver biopsies obtained prior to treatment and during the first week following pegIFN-α injection in 18 patients with chronic hepatitis C. Despite sustained high concentrations of pegIFN-α in serum, the Jak/STAT pathway was activated in hepatocytes only on the first day after pegIFN-α administration. Evaluation of liver biopsies revealed that pegIFN-α induces hundreds of genes that can be classified into four clusters based on different temporal expression profiles. In all clusters, gene transcription was mainly driven by IFN-stimulated gene factor 3 (ISGF3). Compared with conventional IFN-α therapy, pegIFN-α induced a broader spectrum of gene expression, including many genes involved in cellular immunity. IFN-induced secondary transcription factors did not result in additional waves of gene expression. Our data indicate that the superior antiviral efficacy of pegIFN-α is not the result of prolonged Jak/STAT pathway activation in hepatocytes, but rather is due to induction of additional genes that are involved in cellular immune responses.

Faldaprevir (BI 201335), deleobuvir (BI 207127) and ribavirin oral therapy for treatment-naive HCV genotype 1: SOUND-C1 final results.

BACKGROUND: Faldaprevir (BI 201335) and deleobuvir (BI 207127) are direct-acting antiviral agents under development for the treatment of chronic HCV infection. This article describes the final results of the Phase Ib SOUND-C1 study that evaluated the interferon-free oral combination of faldaprevir, deleobuvir and ribavirin in 32 treatment-naive patients infected with HCV genotype 1. METHODS: Patients were randomized to receive deleobuvir 400 mg (n=15) or 600 mg (n=17) three times daily plus faldaprevir 120 mg once daily and weight-based ribavirin for 4 weeks. Interferon-free therapy was followed by response-guided faldaprevir plus pegylated interferon-α2a/ribavirin to week 24 or 48. RESULTS: At week 4, 73% (11/15) and 100% (17/17) of patients in the deleobuvir 400 mg and 600 mg groups achieved HCV RNA<25 IU/ml, respectively. During interferon-free treatment, virological breakthrough was reported in one patient and re-increase of HCV RNA in one patient. Both patients were successfully treated with interferon-containing therapy. The rate of sustained virological response 24 weeks after completion of treatment was 73% (11/15) in the deleobuvir 400 mg group and 94% (16/17) in the 600 mg group. During faldaprevir plus pegylated interferon-α2a/ribavirin treatment, the most common adverse events were pruritus (38% of patients), rash (31%) and asthenia (31%); these were severe in approximately 3% of patients. CONCLUSIONS: Potent antiviral activity and favourable safety of the treatment regimen were demonstrated. Furthermore, the results suggest that patients with breakthrough at week 4 may be rescued with an interferon-containing regimen. Clinical trials.gov number NCT01132313.

S-adenosyl-methionine and betaine improve early virological response in chronic hepatitis C patients with previous nonresponse.

BACKGROUND/AIMS: Treatment of chronic hepatitis C (CHC) with pegylated interferon α (pegIFNα) and ribavirin results in a sustained response in approximately half of patients. Viral interference with IFNα signal transduction through the Jak-STAT pathway might be an important factor underlying treatment failure. S-adenosyl-L-methionine (SAMe) and betaine potentiate IFNα signaling in cultured cells that express hepatitis C virus (HCV) proteins, and enhance the inhibitory effect of IFNα on HCV replicons. We have performed a clinical study with the aim to evaluate efficacy and safety of the addition of SAMe and betaine to treatment of CHC with pegIFNα/ribavirin. METHODS: In this open-label pilot study, 29 patients with CHC who failed previous therapy with (peg)IFNα/ribavirin were treated with SAMe, betaine, pegIFNα2b and ribavirin. Treatment duration was 6 or 12 months, depending on genotype, and the protocol comprised a stopping rule at week 12 if early virological response (EVR) was not achieved. Virological and biochemical response and safety were assessed throughout the treatment. RESULTS: 29 patients were enrolled and treated according to the study protocol. 79% of the patients were infected with genotype 1, 72% had advanced fibrosis, 76% had previously received pegIFNα/ribavirin, and only 14% achieved EVR to the previous treatment. When treated with the study medications, 17 patients (59%) showed an EVR, only 3 (10%) however achieved a sustained virological response (SVR). SAMe and betaine were found to be safe when used with pegIFNα/ribavirin. CONCLUSION: The addition of SAMe and betaine to pegIFNα/ribavirin improves early virological response in CHC. TRIAL REGISTRATION: ClinicalTrials.gov NCT00310336.

Treatment of hepatitis C in HCV mono-infected and in HIV-HCV co-infected patients: an open-labelled comparison study.

BACKGROUND/AIMS: Treatment of chronic HCV infection has become a priority in HIV+ patients, given the faster progression to end-stage liver disease. The primary endpoint of this study was to evaluate and compare antiviral efficacy of Peginterferon alpha 2a plus ribavirin in HIV-HCV co-infected and HCV mono-infected patients, and to examine whether 6 months of therapy would have the same efficacy in HIV patients with favourable genotypes 2 and 3 as in mono-infected patients, to minimise HCV-therapy-related toxicities. Secondary endpoints were to evaluate predictors of sustained virological response (SVR) and frequency of side-effects. METHODS: Patients with genotypes 1 and 4 were treated for 48 weeks with Pegasys 180 microg/week plus Copegus 1000-1200 mg/day according to body weight; patients with genotypes 2 and 3 for 24 weeks with Pegasys 180 microg/week plus Copegus 800 mg/day. RESULTS: 132 patients were enrolled in the study: 85 HCV mono-infected (38: genotypes 1 and 4; 47: genotypes 2 and 3), 47 HIV-HCV co-infected patients (23: genotypes 1 and 4; 24: genotypes 2 and 3). In an intention-to-treat analysis, SVR for genotypes 1 and 4 was observed in 58% of HCV mono-infected and in 13% of HIV-HCV co-infected patients (P = 0.001). For genotypes 2 and 3, SVR was observed in 70% of HCV mono-infected and in 67% of HIV-HCV co-infected patients (P = 0.973). Undetectable HCV-RNA at week 4 had a positive predictive value for SVR for mono-infected patients with genotypes 1 and 4 of 0.78 (95% CI: 0.54-0.93) and of 0.81 (95% CI: 0.64-0.92) for genotypes 2 and 3. For co-infected patients with genotypes 2 and 3, the positive predictive value of SVR of undetectable HCV-RNA at week 4 was 0.76 (95%CI, 0.50-0.93). Study not completed by 22 patients (36%): genotypes 1 and 4 and by 12 patients (17%): genotypes 2 and 3. CONCLUSION: Genotypes 2 or 3 predict the likelihood of SVR in HCV mono-infected and in HIV-HCV co-infected patients. A 6-month treatment with Peginterferon alpha 2a plus ribavirin has the same efficacy in HIV-HCV co-infected patients with genotypes 2 and 3 as in mono-infected patients. HCV-RNA negativity at 4 weeks has a positive predictive value for SVR. Aggressive treatment of adverse effects to avoid dose reduction, consent withdrawal or drop-out is crucial to increase the rate of SVR, especially when duration of treatment is 48 weeks. Sixty-one percent of HIV-HCV co-infected patients with genotypes 1 and 4 did not complete the study against 4% with genotypes 2 and 3.