Inhibition of hepatitis C virus (HCV)-RNA-dependent translation and replication of a chimeric HCV poliovirus using synthetic stabilized ribozymes

Ribozymes are catalytic RNA molecules that can be designed to cleave specific RNA sequences. To investigate the potential use of synthetic stabilized ribozymes for the treatment of chronic hepatitis C virus (HCV) infection, we designed and synthesized hammerhead ribozymes targeting 15 conserved sites in the 5' untranslated region (UTR) of HCV RNA. This region forms an internal ribosome entry site that allows for efficient translation of the HCV polyprotein. The 15 synthetic ribozymes contained modified nucleotides and linkages that stabilize the molecules against nuclease degradation. All 15 ribozymes were tested for their ability to reduce expression in an HCV 5' UTR/luciferase reporter system and for their ability to inhibit replication of an HCV-poliovirus (HCV-PV) chimera. Treatment with several ribozymes resulted in significant down-regulation of HCV 5' UTR/luciferase reporter expression (range 40% to 80% inhibition, P <.05). Moreover, several ribozymes showed significant inhibition (>90%, P <.001) of chimeric HCV-PV replication. We further show that the inhibitory activity of ribozymes targeting site 195 of HCV RNA exhibits a sequence-specific dose response, requires an active catalytic ribozyme core, and is dependent on the presence of the HCV 5' UTR. Treatment with synthetic stabilized anti-HCV ribozymes has the potential to aid patients who are infected with HCV by reducing the viral burden through specific targeting and cleavage of the viral genome.     

Development of a hepatitis C virus relapse model using genome-length hepatitis C virus ribonucleic acid-harboring cells possessing the interferon-α-resistance phenotype

Aim:  The cure rate of current interferon (IFN) therapy is limited to approximately 50% and most of the relapses after therapy are caused by genotype-1. To develop a relapse model in cell culture, we attempted to obtain genome-length hepatitis C virus ribonucleic acid (HCV RNA) harboring cells possessing the IFN-α-resistance phenotype from previously established OR6 cells, which enabled the luciferase reporter assay for monitoring of HCV RNA replication.
Methods:  The IFN-α-resistant HCV RNA-harboring cells and control cells were obtained by the treatment of OR6 cells with and without IFN-α, respectively. Then, we examined the relapse of HCV in IFN-α-resistant HCV RNA-harboring cells.
Results:  Only type I IFN (α and β) showed significantly different anti-HCV activity between IFN-α-resistant HCV RNA-harboring cells and control cells. There was no significant difference in the anti-HCV activity of IFN-γ, fluvastatin, or cyclosporine A between the two types of cells. Furthermore, we showed that fluvastatin or cyclosporine A in combination with IFN-α could prevent the relapse after therapy in the IFN-α-resistant HCV RNA-harboring cells.
Conclusion:  We developed a HCV relapse model in cell culture using IFN-α-resistant HCV RNA-harboring cells. Thus anti-HCV reagents, which have a mechanism different from IFN-α, were shown to be useful for preventing a relapse of IFN-α-resistant HCV.     

Suppression of hepatitis C virus replication by protein kinase C-related kinase 2 inhibitors that block phosphorylation of viral RNA polymerase

Summary.  Hepatitis C virus (HCV) infection is a serious threat to human health worldwide. In spite of the continued search for specific and effective anti-HCV therapies, the rapid emergence of drug-resistance variants has been hampering the development of anti-HCV drugs designed to target viral enzymes. Targeting host factors has therefore emerged as an alternative strategy offering the potential to circumvent the ever-present complication of drug resistance. We previously identified protein kinase C-related kinase 2 (PRK2) as a cellular kinase that phosphorylates the HCV RNA-dependent RNA polymerase (RdRp). Here, we report the anti-HCV activity of HA1077, also known as fasudil, and Y27632, which blocks HCV RdRp phosphorylation by suppressing PRK2 activation. Treatment of a Huh7 cell line, stably expressing a genotype 1b HCV subgenomic replicon RNA, with 20 μ m each of HA1077 and Y27632 reduced the HCV RNA level by 55% and 30%, respectively. A combination of the inhibitors with 100 IU/mL interferon α (IFN-α) significantly potentiated the anti-HCV drug activities resulting in approximately a 2-log₁₀ viral RNA reduction. We also found that IFN-α does not activate PRK2 as well as its upstream kinase PDK1 in HCV-replicating cells. Furthermore, treatment of HCV-infected cells with 20 μ m each of HA1077 and Y27632 reduced the levels of intracellular viral RNA by 70% and 92%, respectively. Taken together, the results identify PRK2 inhibitors as potential antiviral drugs that act by suppressing HCV replication via inhibition of viral RNA polymerase phosphorylation.     

Interferon-α-2B and ribavirin in combination for chronic hepatitis C patients not responding to interferon-α alone: an italian multicenter, randomized, controlled, clinical study

Objective: The aim of the study was to assess the efficacy of interferon (IFN)-α-2b and ribavirin in combination in the treatment of chronic hepatitis C (CHC) patients unresponsive to a previous treatment with IFN-α−2b alone.
Methods: We conducted a randomized study in 303 CHC patients. One hundred fifty-two patients received subcutaneous administration of recombinant IFN-α−2b (3 MU thrice weekly) and ribavirin (1000–1200 mg/daily per os ), whereas 151 received IFN-α−2b alone (6 MU thrice weekly). Both ribavirin and IFN-α-2b were given for 24 wk, regardless of treatment response. Alanine aminotransferase (ALT) levels and HCV RNA titer were checked during the treatment period and for a further 24 wk.
Results: Normal ALT levels were observed in 64.5% of the patients treated with IFN-α and ribavirin and in 22.6% of the patients treated with IFN-α alone. In the group of patients receiving IFN-α and ribavirin HCV RNA was not detectable in 40% of patients responders and remained undetectable in 44.2% of sustained responders. In the group of patients receiving IFN-α alone HCV RNA was not detectable in 24.2% of patients responders and remained not detectable in 33.3% of sustained responders.
Conclusion: A 24-wk treatment course with IFN-α and ribavirin given to patients with a previous lack of response to IFN-α alone offers a chance of a sustained biochemical and virological response, at least in a subset of such patients. The role of long-term therapy in inducing prolonged remission still remains to be explored.     

Early Loss of Serum Hepatitis C Virus RNA Can Predict a Sustained Response to Interferon Therapy in Patients with Chronic Hepatitis C

Objectives : We evaluated whether the loss of serum hepatitis C virus (HCV) RNA early in interferon (IFN) therapy would indicate a subsequent response to IFN therapy. Methods : One hundred fourteen patients with chronic hepatitis C were treated with IFN-α for 24 weeks. All patients were positive for anti-HCV antibodies and serum HCV RNA. Serum HCV RNA was measured by highly sensitive and specific RT-PCR (modified Amplicor HCV). Results : Of 114 patients who were treated with IFN-α for 24 weeks, 22 of 29 patients (75.9%) who lost HCV RNA at the first week of treatment, 5 of 14 patients (35.7%) who lost HCV RNA at the second week, and 2 of 16 patients (12.5%) who lost HCV RNA at fourth week were judged as sustained responder (SR). The SR rate was significantly higher in patients who lost HCV RNA at the first week of therapy ( p < 0.05). On the contrary, none of 55 patients who retained HCV RNA during the first 4 weeks of IFN therapy were judged as SR. Concerning the patients who lost HCV RNA at the first week of therapy, there were no significant differences in the SR rate in either HCV genotype (1b, 2a, and 2b). Conclusions : Our study confirms that the early response to IFN (loss of HCV RNA at the end of the first week of IFN therapy) can be a predictor of the subsequent sustained response to IFN therapy. Additionally, positivity of HCV RNA at the fourth week of IFN therapy can be a predictor of the subsequent non-sustained response to IFN therapy.     

Zinc supplementation enhances the response to interferon therapy in patients with chronic hepatitis C

We evaluated the synergistic effect of zinc supplementation on the response to interferon (IFN) therapy in patients with intractable chronic hepatitis C in a pilot study using natural IFN-α with or without zinc. No clinical differences were observed between patients treated with IFN alone ( n =40) and IFN with polaprezinc (IFN + Zn, n =35). All patients were positive for HCV genotype Ib and had more than 10⁵ copies of the virus/mL serum. Ten million units of natural IFN-α was administered daily for 4 weeks followed by the same dose every other day for 20 weeks. In the IFN + Zn group, patients received an additional dose of 150 mg/day polaprezinc orally throughout the 24-week IFN course. No additional side-effects of polaprezinc were noted but four out of 40 IFN alone treatment and three out of 35 IFN + Zn group withdrew because of side-effects. Complete response (CR) was defined as negative HCV RNA in the serum on PCR and normal aminotransferase level 6 months after therapy. Incomplete response (IR) was normal liver enzyme and positive serum HCV RNA. Both of them were evaluated at the 6 months after the completion of the treatment. Patients with higher levels of serum HCV (more than 5 × 10⁵ copies/mL) had little response in both treatment groups. Patients with moderate amount of HCV (10⁵ to 4.99 × 10⁵/mL) showed high response rates in combination group (CR: 11/27, 40.7%; CR + IR 15/27, 64.3%), better than IFN alone (CR: 2/15, 18.2%; CR + IR: 2/15, 18.2%). Serum zinc levels were higher in patients with IFN + Zn group than in the IFN group. Our results indicate that zinc supplementation enhances the response to interferon therapy in patients with intractable chronic hepatitis C.     

Histological and virological long-term outcome in patients treated with interferon-alpha2b and ribavirin for chronic hepatitis C

Long-term virological and histological outcome following interferon-α2b (IFN-α2b) and ribavirin treatment for 24 weeks was studied in 20 patients with chronic hepatitis C who were without a lasting response to IFN as monotherapy. Following combination therapy, sustained virological response (SR) was achieved in 12 patients (i.e. hepatitis C virus (HCV) RNA negative in serum 6months post-treatment). Eleven of these patients remained HCV RNA negative in serum 2 years post-treatment. A virological long-term response (LTR) was more frequent in patients with a previous end-of-treatment response to IFN monotherapy than in non-responders. Liver histology at follow-up, ≥24months post-treatment, showed substantial improvement in patients with a virological LTR to the combination treatment. In all nine patients biopsied at the 2-year follow-up, liver inflammation had disappeared totally (grade=0), and the stage (fibrosis) had improved. In contrast, no significant changes in grade or stage were noted in patients with a virological non-LTR to combination treatment. A significant improvement in inflammation was noted, in patients with a virological LTR, from 3.6 to 0.2 ( P <0.01) and in fibrosis from 2.0 to 1.4 ( P <0.05) whereas the corresponding scores for patients with a virological non-LTR did not change significantly, from 3.1 to 1.5 for inflammation and for fibrosis from 1.3 to 1.3. We conclude that patients with chronic hepatitis C who achieve a virological sustained response 6months post-treatment with IFN-α2b and ribavirin will remain virological responders for a follow-up period of least 24months, concomitant with a disappearance of inflammatory activity and a marked improvement of fibrosis in the liver.     

Immunological response to interferon-gamma priming prior to interferon-alpha treatment in refractory chronic hepatitis C in relation to viral clearance

The aim of this study was to clarify the immunological and virological responses to pre-administration of interferon-γ prior to initiation of interferon-α treatment in patients with refractory chronic hepatitis C. Twenty-two nonresponders to 6-months of IFN-α treatment were enrolled. The hepatitis C virus (HCV) genotype was Ib in all. Natural IFN-γ (1 MIU/day) was administered daily for 14 days followed by natural IFN-α (5 MIU/day) daily for 14 days and then three times weekly for 22 weeks. Serum immunological parameters (IL-10, neopterin, BMG, sCD8, sCD4, IL-6, IL-12) were measured as were the levels of several cytokines (IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-6, IL-10). Three patients dropped out; two because of the occurrence of other diseases and one because of an adverse effect. At the end of the period of IFN-α treatment, HCV-RNA had become negative in six of 19 patients (end-of treatment response; ETR). Six months after the completion of IFN administration, a virological sustained response (SR) was seen in two of 19 patients. The mean serum levels of IL-10 were significantly decreased 6 weeks after the start of treatment. Other immunological parameter levels increased significantly during the period of IFN-γ administration, and tended to return to the pretreatment level after the start of IFN-α administration. Univariate logistic regression analysis showed that the initial change in the levels of these parameters or the change in the ratios of Th1/Th2 parameter levels are useful factors indicative of the end of the treatment response. These findings suggest that priming with IFN-γ prior to the initiation of IFN-α treatment in patients with refractory chronic hepatitis C can modulate the host immune response and this might contribute to viral clearance.     

Interferon-α plus amantadine in chronic hepatitis C resistant to interferon alone: a pilot randomized study

The optimal therapy for patients with chronic hepatitis C who have not responded to interferon (IFN) is still an unsolved issue. The aim of this study was to evaluate the efficacy and tolerability of a high dose of IFN-α2a plus amantadine for chronic hepatitis C patients who were non-responders to a previous course of IFN.
Forty consecutive patients with chronic hepatitis C, genotype 1b, who had not responded to IFN-α, were randomized to receive: (i) IFN 4.5  M U daily plus amantadine 200 mg/day for 4 weeks and then IFN 6  M U thrice weekly plus amantadine 200 mg/day for an additional 5 months (group A) or (ii) IFN alone at the same dosage and duration (group B). After 1 month of therapy, normal alanine aminotransferase (ALT) values were observed in three of 21 (14.3%) patients in group A and in three of 19 (15.8%) in group B; serum hepatitis C virus (HCV)-RNA clearance was observed in one patient (4.8%) in group A and in six (31.6%) in group B. At the end of treatment, six patients (28.6%) in group A and three (15.8%) in group B had normal ALT levels; however, HCV-RNA in serum was detectable in all of them at levels comparable to the basal values; an ALT relapse occurred within 3 months of stopping therapy. The combination of daily IFN plus amantadine was ineffective in this setting.     

Prediction of relapse or sustained response in biochemical responders by serum hepatitis C virus RNA monitoring during interferon therapy

Normalization of serum aminotransferase levels is achieved in ≈ 50% of chronic hepatitis C patients treated with interferon (IFN); however, in about one-half of these patients the hepatitis relapses after therapy. In this study we investigated the efficacy of serum hepatitis C virus (HCV) RNA monitoring during IFN therapy to predict the outcome of a biochemical end-of-treatment (ETR) response. Eighty patients with chronic hepatitis C received leucocyte (natural) IFN-α (13 patients) or recombinant IFN-α2a (67 patients). Antiviral therapy was given for 12 months to 43 (53.7%) responders and this group was analysed further. During follow-up, 15 relapsed and 28 showed a sustained response (median follow-up 50 months, range 39–67 months). Viraemia was monitored at baseline, and at months 1, 3, 6, 9 and 12 of treatment, by nested polymerase chain reaction (PCR) (sensitivity 10–100 copies ml^–1). A combination of positive nested PCR and HCV RNA values at the 3rd and 6th months of treatment was 100% predictive of relapse (sensitivity, 66.6%; specificity, 100%). A combination of negative nested PCR and HCV RNA values at the 1st and 3rd months of treatment was 100% predictive of sustained response (sensitivity, 39.3%; specificity, 100%). In conclusion, serum HCV RNA monitoring is an appropriate and reliable tool for predicting early outcome of the biochemical ETR response after IFN discontinuation. This could be useful in the modulation of therapeutic management of chronic hepatitis C.     

Genotype and viral load as prognostic indicators in the treatment of hepatitis C

Interferon-α (IFN-α), either alone or in combination with ribavirin, is the standard treatment for patients with hepatitis C. However, most patients do not achieve a sustained remission with this treatment regimen. A number of studies have demonstrated that genotype, baseline viral load and/or a decrease in viral load early after treatment induction are the major predictive factors for response to treatment with IFN. Patients with hepatitis C virus (HCV) genotype 1 are more resistant to treatment with IFN, whereas low viral load at baseline and a marked decline in the HCV RNA level during the first 2–12 weeks of IFN therapy are associated with enhanced treatment efficacy. These variables could potentially be used to develop treatment algorithms that tailor therapies for specific clinical situations. Continued development and refinement of such algorithms would facilitate both the selection of patients who are most likely to benefit from therapy and the development of optimal treatment regimens for different patient groups. Predictive factors will also enable clinicians to identify subsets of patients who are not expected to respond well to current treatment. The development of new delivery methods for IFN that produce sustained antiviral pressure may provide a means of treating these previously difficult-to-treat patient groups.     

Pegylated interferon-alpha2a/ribavirin treatment of recurrent hepatitis C after liver transplantation

Abstract: Hepatitis C virus (HCV) infection invariably recurs after liver transplantation (LT), leading to significant morbidity and mortality. Although the combination of pegylated interferon-alpha (IFN-α)/ribavirin is the preferred treatment for these patients, the optimal schedule remains undetermined. In an uncontrolled trial, 19 patients with HCV infection recurring after LT received pegylated IFN-α_2a, 180 μg weekly, and ribavirin, 10 mg/kg body weight daily, for 48 weeks. The proportion of patients with undetectable HCV RNA in their serum after 12 weeks of treatment was 53%. Five patients (26%) dropped out of the study due to intolerance (in 2 cases), depression (in 1), or infectious complications (in 2). A sustained virological response (SVR), defined as undetectable serum HCV RNA 24 weeks after the end of treatment, was observed in 9/19 patients (47%). SVR was associated with an early virological response after 12 weeks of therapy ( P <0.001) and a treatment duration >80% ( P =0.02), but not with baseline HCV RNA level or a cumulative dose of pegylated IFN-α_2a or ribavirin >80% of the scheduled dose. All 4 patients with genotype 2 or 3 reached SVR, as compared with 33% of patients with genotype 1 or 4 ( P =0.03). A 48-week course of pegylated IFN-α_2a/ribavirin therapy is effective in patients with recurrent HCV infection after LT.     

Expression of interferon-alpha subtypes in peripheral mononuclear cells from patients with chronic hepatitis C: a role for interferon-alpha5

Interferon (IFN)-α is a family of antiviral proteins encoded by different genes. The biological significance of the existence of various IFN-α subtypes is not clear. We have investigated the interferon system in chronic hepatitis C virus (HCV) infection, a disease that responds to interferon-α2 therapy in only a limited proportion of cases. We analysed the expression of interferon regulatory factor (IRF)-1, IRF-2, and IFN-α subtypes in nonstimulated and Sendai virus-stimulated peripheral blood mononuclear cells (PBMC) from HCV infected patients and healthy controls. We observed that the IRF-1 mRNA and IRF-1/IRF-2 ratios were increased in PBMC from hepatitis C patients with respect to normal subjects. Sendai virus stimulation of PBMC led to a significant increase in the levels of IRF-1, IRF-2 and IFN-α mRNAs and in the production of IFN-α protein with respect to basal values in healthy controls as well as in patients with HCV infection. In addition, we found that while natural HCV infection induced increased IFN-α5 expression in PBMC, in vitro infection of these cells with Sendai virus caused a raise in the expression of IFN-α8 in both patients and normal controls. In summary, our results indicate that virus-induced activation of the IFN system in human PBMC is associated with selective expression of individual IFN-α subtypes, IFN-α5 being the specific subtype induced in PBMC from patients with chronic HCV infection.     

Virus-specific antibody titres in different phases of hepatitis C virus infection

summary . This study aimed to examine anti hepatitis C virus (HCV) antibody titres, their changes and differences in acute, chronic and past HCV infection and to examine them after IFN-α-therapy. Ninety five patients were studied in a cross-sectional investigation and 18 of them were followed long-term. Titres of IgM and IgG antibodies against core, NS3, NS4 (A + B), NS5A proteins were determined by the third generation enzyme immunoassays. Patients with acute hepatitis C developed IgG antibodies against core protein in titres 1/5–1/800 and against individual NS proteins at the same titres. During the first to second month of acute hepatitis C IgG antibody titres to HCV proteins were very low, but they had risen considerably by the fourth to sixth month. Anti-HCV IgM antibodies were found in half the acute hepatitis serum samples, titres were 1/5–1/40. Sixty individuals with chronic hepatitis C showed IgG antibodies against core in titres 1/800–1/40000 and against individual NS proteins in titres 1/5–1/20000. Eight patients with chronic hepatitis C had invariable anti-HCV IgG antibodies over 2–3 years. About 81.7% of chronically infected patients had anti-HCV IgM antibodies in titres 1/5–1/160. Patients with resolution of HCV infection showed only anti-core IgG antibodies (titres 1/5–1/200) or no virus-specific antibodies. Individuals with different response to IFN-α-therapy showed two distinct patterns of anti-HCV antibody titres. Acute and chronic HCV infection may be distinguished by anti-core titres.     

Does fluvastatin favour HCV replication in vivo? A pilot study on HIV–HCV coinfected patients

Summary.  Fluvastatin showed anti-hepatitis C virus (HCV) activity in vitro , through the inhibition of geranylgeranylation of cellular proteins, and a synergistic effect with interferon (IFN)-α. Nevertheless statins up-regulate low-density lipoprotein (LDL) receptor, required for HCV cell entry, and the closely related scavenger receptors SRBI and CD36; moreover they reduce class II major histocompatibility complex expression on antigen presenting cell, modulating T-cell activation. In vivo LDL levels have been identified as prognostic indicator of sustained viral response to IFN in patients with HCV infection, suggesting that lipid-lowering agents might conversely favour HCV entry into the hepatocytes and translate into higher viral replication. We evaluated the effect of fluvastatin on HCV-RNA levels, CD36 expression and T-cell homeostasis in HCV-RNA positive patients. HCV-RNA was measured at baseline and after 4 weeks in 42 HCV/HIV-1 co-infected patients, randomized to receive either fluvastatin 80 mg qd or no treatment. CD36 expression and markers of T-cell activation were evaluated by means of flow cytometry. Plasma interleukin (IL)-10, IFN-γ and IL-7 were measured by ELISA. Serum cholesterol and LDL decreased significantly in the treatment group ( P  = 0.0001 and 0.01, respectively). Surprisingly a significant increase of HCV-RNA levels was seen after 4 weeks of fluvastatin ( P =  0.03). The percentages of naive/activated/apoptotic cells and CD36 expression remained unchanged. Fluvastatin did not inhibit HCV-RNA replication in vivo ; conversely we observed a significant increase of HCV-RNA levels. CD36 expression on monocytes were not up-regulated by statins as previously reported in vitro . The correlation between HCV infectivity, oxidized-LDL receptor and statins in HCV infection need further evaluation.     

Randomized, placebo-controlled, double-blind trial with interferon-α with and without amantadine sulphate in primary interferon-α nonresponders with chronic hepatitis C

In primary interferon-α (IFN-α) nonresponders with chronic hepatitis C, retreatment with IFN-α has only limited efficacy with sustained response rates below 10%. Therefore, the aims of the present study were to compare the efficacy and safety of IFN-α alone or in combination with amantadine sulphate in nonresponders to previous IFN-α monotherapy. Fifty-five IFN-α nonresponders with chronic hepatitis C (mean age: 46.6 years) received IFN-α 6 MIU thrice weekly for 24 weeks followed by 3 MIU thrice weekly for additional 24 weeks. Amantadine sulphate ( n =26) or a matched placebo ( n =29) was given orally twice daily for 48 weeks. Because of a low initial response rate at week 12 (13/55 patients) and a high breakthrough rate (8/13 patients) after IFN-α dose reduction in week 24, a virological end-of-treatment response with undetectable serum HCV-RNA (< 1000 copies/mL) was achieved in only five patients (IFN-α/amantadine sulphate, one patient; IFN-α/placebo, four patients). After 24 weeks follow-up a sustained virological response was observed in only two patients receiving IFN-α and placebo. Health-related quality-of-life analysis showed a substantial improvement of the Profile of Mood States (POMS) scale concerning the subscales fatigue ( P  < 0.05) and vigor ( P  < 0.05) in patients receiving combined IFN-α/amantadine sulphate treatment compared with those treated with IFN-α alone. IFN-α/amantadine sulphate combination therapy was well tolerated without any serious adverse events. In conclusion, retreatment with IFN-α and amantadine sulphate does not increase the low sustained virological response rates of IFN-α therapy in primary IFN-α nonresponders with chronic hepatitis C, but may lead to a sustained improvement of health-related quality-of-life.     

Characterization of the immune response against hepatitis C infection in recovered,and chronically infected chimpanzees

summary . The immune response to hepatitis C virus (HCV) is believed to be critical in determining the outcome of the disease. In this study we have analysed epitope recognition, cytokine profile, and anti-HCV antibody responses in chronically HCV-infected and recovered chimpanzees. Quantitative measurement of anti-HCV antibody in HCV-infected chimpanzees revealed that the response in HCV- recovered chimpanzees peaked within 4–20 weeks. In contrast, the anti-HCV antibody responses in chronically HCV infected chimpanzees did not peak until 100–200 weeks after infection, and decreased gradually thereafter. T cell proliferation assays measuring responses to pooled HCV proteins revealed significant increases in the ³H-uptake during the early stages of infection in recovered chimpanzees in comparison to the chronically infected ones. Class I-restricted epitopes of the core, and NS3 proteins of HCV were analysed using 9–10 mer overlapping peptides covering the core and NS3 proteins, and IFN-γ ELISPOT technique. Our data indicated early and broad class-I restricted core, and NS3 protein epitope recognitions in HCV-recovered chimpanzees but not in chimpanzees that had been chronically infected. Additionally, dominant epitopes recognized early in infection (8 weeks) were no longer recognized later in infection (followed up to 64 weeks). Cytokines profiling revealed a 50-fold increase in TNF-α secretion in the supernatant of core-specific CD8 memory cells of the chronically infected chimpanzees in comparison to the recovered ones. In summary, multiple parameters correlate with HCV recovery in chimpanzees.     

Ribavirin treatment in dialysis patients with chronic hepatitis C virus infection – a pilot study

Standard treatment for chronic hepatitis C is with interferon (IFN)-α and ribavirin for 6–12 months. In dialysis patients only interferon therapy is currently used due to the lack of knowledge concerning ribavirin dosage and side-effects. The aim of this study was to investigate if ribavirin can be added to interferon when treating dialysis patients with hepatitis C.
Five patients on haemodialysis and one patient on peritoneal dialysis with chronic hepatitis C, five with genotype 1 and one with genotype 4, were given interferon-α2b 3  M U thrice weekly for 4 weeks, whereafter ribavirin 200–400 mg was added, for an intended total treatment period of 28 weeks. Ribavirin plasma concentrations were monitored, using HPLC.
Four patients completed the treatment. One patient suffered marked side-effects from interferon and therapy was terminated. One patient developed heart failure and died after 14 weeks of treatment but the death was not considered treatment related. Based on plasma concentrations, ribavirin doses were frequently adjusted initially. The target concentration (10–15 μmol/L) was reached with average daily doses of 170–300 mg ribavirin. Ribavirin induced anaemia was managed with high doses of erytropoietin (20 000–30 000 IU/week). Five of six patients became hepatitis C virus (HCV)-RNA negative during treatment, but four relapsed post-treatment; one is HCV-RNA negative.
Hence ribavirin, in combination with IFN-α, can be used to treat dialysis patients with HCV. However, this requires reduced ribavirin doses and close monitoring of ribavirin plasma concentrations and haemoglobin. Ribavirin-induced anaemia can be managed with high doses of erythropoeitin.