Analysis of ribavirin mutagenicity in human hepatitis C virus infection

The addition of ribavirin to alpha interferon therapy significantly increases response rates for patients with chronic hepatitis C virus (HCV) infection, but ribavirin's antiviral mechanisms are unknown. Ribavirin has been suggested to have mutagenic potential in vitro that would lead to "error catastrophe," i.e., the generation of nonviable viral quasispecies due to the increment in the number of mutant genomes, which prevents the transmission of meaningful genetic information. We used extensive sequence-based analysis of two independent genomic regions in order to test in vivo the hypothesis that ribavirin administration accelerates the accumulation of mutations in the viral genome and that this acceleration occurs only when HCV replication is profoundly inhibited by coadministered alpha interferon. The rate of variation of the consensus sequence, the frequency of mutation, the error generation rate, and the between-sample genetic distance were measured for patients receiving ribavirin monotherapy, a combination of alpha interferon three times per week plus ribavirin, or a combination of alpha interferon daily plus ribavirin. Ribavirin monotherapy did not increase the rate of variation of the consensus sequence, the mutation frequency, the error generation rate, or the between-sample genetic distance. The accumulation of nucleotide substitutions did not accelerate, relative to the pretreatment period, during combination therapy with ribavirin and alpha interferon, even when viral replication was profoundly inhibited by alpha interferon. This study strongly undermines the hypothesis whereby ribavirin acts as an HCV mutagen in vivo.     

Chronic viral hepatitis C: management update

The management of chronic viral hepatitis C is evolving rapidly. Monotherapy with interferon, the accepted standard of treatment until recently, achieves only a modest sustained virological response rate of 15%. Combination treatment with alpha-2b interferon and ribavirin has been shown to increase sustained response rates to 40% in patients who have never been treated with interferon and to 50% in those who have relapsed following monotherapy with interferon. However, side effects, which have led to the discontinuation of combination treatment in a significant proportion of patients, must be carefully monitored. Treatment with interferon alpha-2b and ribavirin has now been approved in Canada, but the selection and monitoring of patients suitable for combination treatment requires special expertise. Although improvements in current therapeutic options may be possible with more frequent, higher doses or long-acting forms of interferon together with ribavirin, low sustained response rates (i.e., below 30%) for patients with hepatitis C virus genotype 1 emphasize the need for novel antiviral medications that will target the functional sites of the HCV genome.     

Treatment of genotype 1b HCV-related chronic hepatitis: efficacy and toxicity of three different interferon alfa-2b/ribavirin combined regimens in naive patients

This prospective open-label randomized trial of chronic hepatitis C genotype-1b patients compared compared the efficacy and safety of peg-interferon alfa-2b administered once-weekly versus interferon alfa-2b thrice-weekly or daily, both in combination with ribavirin. Seventy-eight previously untreated patients, with biopsy-documented genotype 1 chronic HCV and persistently elevated ALT levels and detectable HCV RNA, were randomized (26 subjects each) to receive: interferon alfa-2b at 6MIUs.c./three-times-weekly (group A) or interferon alfa-2b, 3MIUs.c./daily (group B) or peg-interferon alfa-2b 1.5mcg/Kg s.c./once-weekly (group C). All regimens included standard weight-based doses of ribavirin (800, 1,000 or 1,200 mg/day) administered for 52-weeks. Patients in the three groups were comparable for age, sex, viral load, ALT value and histological-activity-index (HAI). Therapy was completed by 22, 20 and 23 patients in groups A, B and C, respectively. At the end of treatment, a complete (biochemical and virological) response was observed in 50.0% patients of group A, 57.7% of group B and 65.4% of group C. After an additional 24-weeks of follow-up, a sustained response was observed in 26.9%, 46.1% and 50.0% of patients in groups A, B or C, respectively. Therapy was discontinued by 4, 6 and 2 patients because of adverse events in the above three groups. In naive patients with chronic genotype-lb hepatitis C, a 48 week therapy with peg-interferon or interferon at daily doses combined with ribavirin were both more effective than treatment with thrice-weekly interferon in inducing end of treatment and sustained response. Peg-interferon treatment was better tolerated and provoked significantly fewer therapy discontinuations.     

Changes in gene expression during pegylated interferon and ribavirin therapy of chronic hepatitis C virus distinguish responders from nonresponders to antiviral therapy

Treating chronic hepatitis C virus (HCV) infection using pegylated alpha interferon and ribavirin leads to sustained clearance of virus and clinical improvement in approximately 50% of patients. Response rates are lower among patients with genotype 1 than with genotypes 2 and 3 and among African-American (AA) patients compared to Caucasian (CA) patients. Using DNA microarrays, gene expression was assessed for a group of 33 African-American and 36 Caucasian American patients with chronic HCV genotype 1 infection during the first 28 days of treatment. Results were examined with respect to treatment responses and to race. Patients showed a response to treatment at the gene expression level in RNA isolated from peripheral blood mononuclear cells irrespective of degree of decrease in HCV RNA levels. However, gene expression responses were relatively blunted in patients with poor viral response (<1.5 log(10)-IU/ml decrease at 28 days) compared to those in patients with a marked (>3.5 log(10)-IU/ml decrease) or intermediate (1.5 to 3.5 log(10)-IU/ml decrease) response. The number of genes that were up- or down-regulated by pegylated interferon and ribavirin treatment was fewer in patients with a poor response than in those with an intermediate or marked viral response. However AA patients had a stronger interferon response than CA patients in general. The induced levels of known interferon-stimulated genes such as the 2'5'-oligoadenylate synthetase, MX1, IRF-7, and toll-like receptor TLR-7 genes was lower in poor-response patients than in marked- or intermediate-response patients. Thus, the relative lack of viral response to interferon therapy of hepatitis C virus infection is associated with blunted interferon cell signaling. No specific regulatory gene could be identified as responsible for this global blunting or the racial differences.     

New interferons in the treatment of chronic hepatitis C

The current standard therapy for chronic HCV infection is a combination of pegylated-interferon (PEG-IFN) and weight-based ribavirin, administered for 24-48 weeks, according to the viral genotype. Although the weekly administration of pegylated interferons provides superior antiviral efficacy over standard interferon alpha, the rate of sustained virological response rarely overpasses 50% in patients infected with HCV genotypes 1 and 4. Consequently, multiple clinical trials with congeners of interferon (consensus interferon, interferon lambda, albinterferon, and controlled-release interferons) are ongoing. Their main advantages consist in maintenance of viral suppression across a longer dosing interval, avoidance of interdose trough and reduced dosing frequencies (twice or even once per month compared to once per week for the actual PEG-IFNs). Along with these superior pharmacokinetic properties, new interferons are expected to have improved side-effect profiles and better tolerability compared with the currently available formulations, providing an option for otherwise difficult to treat, challenging populations. New interferon formulation can be incorporated into future combination with direct acting antivirals, in order to maintain viral suppression over longer periods and minimize the development of viral resistance.     

Evaluating treatment of hepatitis C for hemolytic anemia management

The combination therapy of antiviral peg-interferon and ribavirin has evolved as one of the better treatments for hepatitis C. In spite of its success in controlling hepatitis C infection, it has also been associated with treatment-related adverse side effects. The most common and life threatening among them is hemolytic anemia, necessitating dose reduction or therapy cessation. The presence of this side effect leads to a trade-off between continuing the treatment and exacerbating the side effects versus decreasing dosage to relieve severe side effects while allowing the disease to progress. The drug epoietin (epoetin) is often administered to stimulate the production of red blood cells (RBC) in the bone marrow, in order to allow treatment without anemia. This paper uses mathematical models to study the effect of combination therapy in light of anemia. In order to achieve this we introduce RBC concentration and amount of drug in the body as state variables in the usual immunological virus infection model. Analysis of this model provides a quantification of the amount of drug a body can tolerate without succumbing to hemolytic anemia. Indirect estimation of parameters allow us to calculate the necessary increment in RBC production to be ?2.3 times the patient’s original RBC production rate to sustain the entire course of treatment without encountering anemia in a sensitive patient.     

Replicative Homeostasis III: implications for antiviral therapy and mechanisms of response and non-response

While improved drug regimens have greatly enhanced outcomes for patients with chronic viral infection, antiviral therapy is still not ideal due to drug toxicities, treatment costs, primary drug failure and emergent resistance. New antiviral agents, alternative treatment strategies and a better understanding of viral pathobiology, host responses and drug action are desperately needed. Interferon (IFN) and ribavirin, are effective drugs used to treat hepatitis C (HCV), but the mechanism(s) of their action are uncertain. Error catastrophe (EC), or precipitous loss of replicative fitness caused by genomic mutation, is postulated to mediate ribavirin action, but is a deeply flawed hypothesis lacking empirical confirmation. Paradoxically ribavirin, a proven RNA mutagen, has no impact on HCV viraemia long term, suggesting real viruses, replicating in-vitro, as opposed to mathematical models, replicating in-silico, are likely to resist EC by highly selective replication of fit (~consensus sequence) genomes mediated, in part, by replicative homeostasis (RH), an epicyclic mechanism that dynamically links RNApol fidelity and processivity and other viral protein functions. Replicative homeostasis provides a rational explanation for the various responses seen during treatment of HCV, including genotype-specific and viral load-dependent differential response rates, as well as otherwise unexplained phenomena like the transient inhibition and rebound of HCV viraemia seen during ribavirin monotherapy. Replicative homeostasis also suggests a primarily non-immunological mechanism that mediates increased immune responsiveness during treatment with ribavirin (and other nucleos(t)ide analogues), explicating the enhanced second-phase clearance of HCV ribavirin promotes and, thus, the apparent immunomodulatory action of ribavirin. More importantly, RH suggests specific new antiviral therapeutic strategies.     

Assessment of Hepatitis C Virus Protein Sequences with Regard to Interferon/Ribavirin Combination Therapy Response in Patients with HCV Genotype 1b

Hepatitis C virus (HCV) infection is a major and rising global health problem, affecting about 170 million people worldwide. The current standard of care treatment with interferon alpha and ribavirin in patients with the genotype 1 infection, the most frequent genotype in the USA and Western Europe, leads to a successful outcome in only about 50% of individuals. Accurate prediction of hepatitis C treatment response is of great benefit to patients and clinicians. The informational spectrum method, a virtual spectroscopy method for structure/function analysis of nucleotide and protein sequences, is applied here for the identification of the conserved information of the HCV proteins that correlate with the combination therapy outcome. Among the HCV proteins that we have analyzed the informational property of the p7 of HCV genotype 1b was best related to the therapy outcome. On the basis of these results, a simple bioinformatics criterion that could be useful in assessment of the response of HCV-infected patients to the combination therapy has been proposed.     

Advances in therapy for hepatitis C infection

The first approved therapy for chronic hepatitis C virus (HCV) infection was recombinant interferon. Subsequently, controlled studies demonstrated that the combination of interferon-alpha and ribavirin leads to significantly higher virologic sustained responses in patients with chronic hepatitis C. A novel modification of the interferon molecule resulted in the formulation of pegylated interferons, which have a longer half-life than standard interferon. Two recent trials have established the superiority of pegylated interferons compared with interferon-alpha in inducing sustained virologic responses in patients with chronic HCV infection, with or without cirrhosis. Presumably, pegylated interferons will replace standard interferon in treating HCV infection. Phase 3 trials of pegylated interferons in combination with ribavirin are currently under way. Noninterferon-based therapies for the treatment of HCV infection are also in the developmental and experimental phases. Our aims in this review are to present the currently available therapeutic options for HCV infection and the evidence supporting their use in typical patients with chronic hepatitis C or in patients with special circumstances. We also briefly review novel therapeutic approaches, including noninterferon-based therapies.     

Mathematical analysis of multiscale models for hepatitis C virus dynamics under therapy with direct-acting antiviral agents

Chronic hepatitis C virus (HCV) infection remains a world-wide public health problem. Therapy with interferon and ribavirin leads to viral elimination in less than 50% of treated patients. New treatment options aiming at a higher cure rate are focused on direct-acting antiviral agents (DAAs), which directly interfere with different steps in the HCV life cycle. In this paper, we describe and analyze a recently developed multiscale model that predicts HCV dynamics under therapy with DAAs. The model includes both intracellular viral RNA replication and extracellular viral infection. We calculate the steady states of the model and perform a detailed stability analysis. With certain assumptions we obtain analytical approximations of the viral load decline after treatment initiation. One approximation agrees well with the prediction of the model, and can conveniently be used to fit patient data and estimate parameter values. We also discuss other possible ways to incorporate intracellular viral dynamics into the multiscale model.     

Quasispecies heterogeneity within the E1/E2 region as a pretreatment variable during pegylated interferon therapy of chronic hepatitis C virus infection

A series of 29 patients undergoing treatment for chronic hepatitis C virus (HCV) genotype 1 infection with pegylated alpha-2a interferon plus ribavirin were studied for patterns of response to antiviral therapy and viral quasispecies evolution. All patients were treatment naive and had chronic inflammation and fibrosis on biopsy. As part of an analysis of pretreatment variables that might affect the outcome of treatment, genetic heterogeneity within the viral E1-E2 glycoprotein region (nucleotides 851 to 2280) was assessed by sequencing 10 to 15 quasispecies clones per patient from serum-derived PCR products. Genetic parameters were examined with respect to response to therapy based on serum viral RNA loads at 12 weeks (early viral response) and at 24 weeks posttreatment (sustained viral response). Nucleotide and amino acid quasispecies complexities of the hypervariable region 1 (HVR-1) were less in the responder group in comparison to the nonresponder group at 12 weeks, and genetic diversity was also less both within and outside of the HVR-1, with the difference being most pronounced for the non-HVR-1 region of E2. However, these genetic parameters did not distinguish responders from nonresponders for sustained viral responses. Follow-up studies of genetic heterogeneity based on the HVR-1 in selected responders and nonresponders while on therapy revealed greater evolutionary drift in the responder subgroup. The pretreatment population sequences for the NS5A interferon sensitivity determinant region were also analyzed for all patients, but no correlations were found between treatment response and any distinct genetic markers. These findings support previous studies indicating a high level of genetic heterogeneity among chronically infected HCV patients. One interpretation of these data is that early viral responses are governed to some extent by viral factors, whereas sustained responses may be more influenced by host factors, in addition to effects of viral complexity and diversity.     

The role of HCV proteins on treatment outcomes

For many years, the standard of treatment for hepatitis C virus (HCV) infection was a combination of pegylated interferon alpha (Peg-IFN-α) and ribavirin for 24–48 weeks. This treatment regimen results in a sustained virologic response (SVR) rate in about 50 % of cases. The failure of IFN-α-based therapy to eliminate HCV is a result of multiple factors including a suboptimal treatment regimen, severity of HCV-related diseases, host factors and viral factors. In recent years, advances in HCV cell culture have contributed to a better understanding of the viral life cycle, which has led to the development of a number of direct-acting antiviral agents (DAAs) that target specific key components of viral replication, such as HCV NS3/4A, HCV NS5A, and HCV NS5B proteins. To date, several new drugs have been approved for the treatment of HCV infection. Application of DAAs with IFN-based or IFN-free regimens has increased the SVR rate up to >90 % and has allowed treatment duration to be shortened to 12–24 weeks. The impact of HCV proteins in response to IFN-based and IFN-free therapies has been described in many reports. This review summarizes and updates knowledge on molecular mechanisms of HCV proteins involved in anti-IFN activity as well as examining amino acid variations and mutations in several regions of HCV proteins associated with the response to IFN-based therapy and pattern of resistance associated amino acid variants (RAV) to antiviral agents.     

Pretreatment sequence diversity differences in the full-length hepatitis C virus open reading frame correlate with early response to therapy

Pegylated alpha interferon and ribavirin therapy for hepatitis C virus (HCV) genotype 1 infection fails for half of Caucasian American patients (CA) and more often for African Americans (AA). The reasons for these low response rates are unknown. HCV is highly genetically variable, but it is unknown how this variability affects response to therapy. To assess effects of viral diversity on response to therapy, the complete pretreatment genotype 1 HCV open reading frame was sequenced using samples from 94 participants in the Virahep-C study. Sequences from patients with >3.5 log declines in viral RNA levels by day 28 (marked responders) were more variable than those from patients with declines of <1.4 log (poor responders) in NS3 and NS5A for genotype 1a and in core and NS3 for genotype 1b. These correlations remained when all T-cell epitopes were excluded, indicating that these differences were not due to differential immune selection. When the sequences were compared by race of the patients, higher diversity in CA patients was found in E2 and NS2 but only for genotype 1b. Core, NS3, and NS5A can block the action of alpha interferon in vitro; hence, these genetic patterns are consistent with multiple amino acid variations independently impairing the function of HCV proteins that counteract interferon responses in humans, resulting in HCV strains with variable sensitivity to therapy. No evidence was found for novel HCV strains in the AA population, implying that AA patients may be infected with a higher proportion of the same resistant strains that are found in CA patients.     

Effects of added dietary taurine on erythrocyte lipids and oxidative stress in rabbits fed a high cholesterol diet

Lipid peroxidation leads to damage of polyunsaturated fatty acids of membrane phospholipids. The contribution of oxidative stress to hypercholesterolemia-induced hemolytic anemia and the effects of addition of taurine on erythrocyte lipid composition, oxidative stress, and hematological data were studied in rabbits fed on a high cholesterol (HC) diet (1%, w/w) for 2 months. The effects of taurine on erythrocyte hemolysis and H2O2-induced lipid peroxidation were investigated in normal rabbit erythrocytes in vitro. The HC diet resulted in increases in plasma lipids and lipid peroxide levels as well as increases in cholesterol levels and the cholesterol:phospholipid ratio in the erythrocytes. This diet caused a hemolytic anemia, but lipid peroxide levels remained unchanged in the erythrocytes of the rabbits. Taurine (2.5%, w/w) added to the food has an ameliorating effect on plasma lipids and lipid peroxide levels in rabbits fed on a HC diet. This treatment also caused decreases in elevated erythrocyte cholesterol levels and cholesterol:phospholipid ratio due to the HC diet, but it did not prevent the hemolytic anemia and did not change erythrocyte lipid peroxide levels. In addition, in an in vitro study, taurine did not protect erythrocytes against H2O2-induced hemolysis or lipid peroxidation. These results show that the HC diet causes hemolytic anemia without any changes in erythrocyte lipid peroxidation, and taurine treatment was not effective against hemolytic anemia caused by the HC diet.     

Interleukin-4 polymorphisms and response to combination therapy in Egyptian chronic hepatitis C patients

In hepatitis C infection, the production of inappropriate cytokines levels may contribute to viral persistence and may affect the response to antiviral therapy. We investigate the effect of IL4 C-590T and C-33T polymorphisms on the response to combination therapy with interferon and ribavirin in chronic HCV patients. These single nucleotide polymorphisms were determined by PCR-RFLP in 235 responder and 210 non-responder to combination therapy. The IL4-590 T/T and -33 T/T genotypes were associated with resistance to the therapy (p<0.001, p=0.001 respectively). Haplotypes T(-590) T(-33) and T(-590) C(-33) were associated with a higher risk in non-responder patients than the responders (p<0.001 for each) while frequency of haplotype C(-590) C(-33) (with all wild alleles) was significantly higher in responders as compared to non-responders (p<0.001). These results suggest that inheritance of the IL4 polymorphisms may be associated with resistance to combined antiviral therapy in Egyptian HCV patients.     

Predictive value of early virological response to treatment with different interferon-based regimens plus ribavirin in patients with chronic hepatitis C

Early virological response (EVR) to different interferon-based regimens plus ribavirin and its ability to predict the outcome of therapy in patients with chronic hepatitis C were investigated. The study design was as follows: 64 naive patients were considered, 32/64 received pegylated interferon alpha-2b (Peg-IFN-alpha2b) plus ribavirin and the remaining 32 received leucocyte interferon alpha (IFN-alpha) plus ribavirin. At week 4 of treatment, EVR was present in 68.7% and 37.5% of patients treated with Peg-IFN-alpha2b plus ribavirin, and with leucocyte interferon alpha (IFN-alpha) plus ribavirin, respectively (p = 0.024). At week 12, the cumulative EVR rates did not differ between the two groups (71.9% vs 56.2%, p >0.05) because a higher proportion of patients achieved EVR for the first time after more than 4 weeks of therapy in the standard IFN-alpha group. Sustained virological response (SVR) rates, however, resulted significantly higher in the Peg-IFN-alpha2b group (65.6% vs 37.5%; p = 0.045) since a higher proportion of patients who received standard IFN-alpha relapsed during the follow-up. In the standard IFN-alpha group, HCV genotype 1 (p = 0.035), high baseline viral load (p = 0.035) and the presence of bridging fibrosis/cirrhosis (p = 0.011) were closely associated with significantly lower SVR rates. In the Peg-IFN-alpha2b group, only bridging fibrosis/cirrhosis (p = 0.02) negatively influenced the outcome of treatment. Overall, 33/41 (80.5%) patients with EVR at week 12 were sustained responders, yielding a positive predictive value (PPV) of 0.80. However, when SVR was related to the time taken to reach EVR, 32/34 (94.1%) patients with EVR at week 4 of therapy (PPV = 0.94) versus 1/7 (14.3%) patients who had EVR after more than 4 weeks of therapy (PPV = 0.14) resulted sustained responders (p = 0.000057). In conclusion, EVR at week 4 of treatment is strongly associated with the likelihood of achieving SVR, regardless of the therapeutic regimen. However, when compared with standard IFN-alpha plus ribavirin, treatment with Peg-IFN-alpha2b plus ribavirin significantly increases the probability of viral clearance within the first 4 weeks of treatment. Finally, patients who do not clear the virus within the first 12 weeks of treatment have no chance of achieving SVR, justifying discontinuation of therapy in these patients.     

Effect of alpha interferon on the hepatitis C virus replicon

Chronic hepatitis C virus (HCV) infections can be cured only in a fraction of patients treated with alpha interferon (IFN-alpha) and ribavirin combination therapy. The mechanism of the IFN-alpha response against HCV is not understood, but evidence for a role for viral nonstructural protein 5A (NS5A) in IFN resistance has been provided. To elucidate the mechanism by which NS5A and possibly other viral proteins inhibit the cellular antiviral program, we have constructed a subgenomic replicon from a known infectious HCV clone and demonstrated that it has an approximately 1,000-fold-higher transduction efficiency than previously used subgenomes. We found that IFN-alpha reduced replication of HCV subgenomic replicons approximately 10-fold. The estimated half-life of viral RNA in the presence of the cytokine was about 12 h. HCV replication was sensitive to IFN-alpha independently of whether the replicon expressed an NS5A protein associated with sensitivity or resistance to the cytokine. Furthermore, our results indicated that HCV replicons can persist in Huh7 cells in the presence of high concentrations of IFN-alpha. Finally, under our conditions, selection for IFN-alpha-resistant variants did not occur.     

Pathogenesis of hepatitis C virus

Hepatitis C virus (HCV) infects approximately 170 million people worldwide including 2 million in Japan and induces serious chronic hepatitis that results in the development of steatosis, cirrhosis and ultimately hepatocellular carcinoma. The current combination therapy using pegylated interferon alpha and a nucleotide analogue ribavirin achieved a sustained virological response in about half population of individuals infected with HCV genotypes la and lb. More than two-thirds of the HCV-positive population has been chronically infected with genotype 1 in Western countries and Japan. Therefore, more effective therapeutics and preventative measures are needed for the treatment of hepatitis C patients who are not responsive to the current chemotherapy. HCV core protein is well known to be the viral capsid protein as well as the pathogenic factor that induces steatosis and hepatocellular carcinoma in the transgenic mice. In this review, we summarize the current status of our knowledge regarding the molecular mechanism by which HCV core protein induces liver steatosis and hepatocellular carcinoma and discuss on a future perspective for the development of novel therapeutics for chronic hepatitis C.     

Modeling hepatitis C virus dynamics: liver regeneration and critical drug efficacy

Mathematical models for hepatitis C viral (HCV) RNA kinetics have provided a means of evaluating the antiviral effectiveness of therapy, of estimating parameters such as the rate of HCV RNA clearance, and they have suggested mechanism of action against HCV for both interferon and ribavirin. Nevertheless, the model that was originally formulated by Neumann et al. [1998. Hepatitis C viral dynamics in vivo and the antiviral efficacy of interferon-alpha therapy. Science 282 (5386), 103-107] is unable to explain all of the observed HCV RNA profiles under treatment e.g., a triphasic viral decay and a viral rebound to baseline values after the cessation of therapy. Further, the half-life of productively HCV-infected cells, estimated from the second phase HCV RNA decline slope, is very variable and sometimes zero with no clear understanding of why. We show that extending the original model by including hepatocyte proliferation yields a more realistic model without any of these deficiencies. Further, we define and characterize a critical drug efficacy, such that for efficacies above the critical value HCV is ultimately cleared, while for efficacies below it, a new chronically infected viral steady-state level is reached.