Gladis Bortoletto

Large‐scale survey of naturally occurring HBV polymerase mutations associated with anti‐HBV drug resistance in untreated patients with chronic hepatitis B

Summary.  Drug resistance is a major limitation for the long‐term efficacy of antiviral therapy with nucleos(t)ide analogues (NAs) in chronic hepatitis B (CHB). Antiviral resistance mutations may pre‐exist in the overall viral population of untreated patients. We aimed to assess the prevalence of such hepatitis B virus (HBV) variants in a large cohort of NAs‐naïve patients with CHB and to explore possible association with viral and host variables. Serum samples from 286 NAs‐naïve consecutive patients with CHB were tested for serum HBV‐DNA, and 255 of them having HBV‐DNA > 1000 IU/mL were further analysed for drug resistance mutations by INNO‐LiPA HBV DRv2/v3. NAs‐naïve patients analysed were mainly men (73%), Caucasians (85%), hepatitis B e Antigen (HBeAg) negative (79%) and genotype D (69%), with a mean age of 43.2 ± 13.4 years. HBV mutations associated with antiviral drug resistance were detected in 13 (5%) patients: three patients infected with HBV genotype C had the rtM204V + rtL180M mutations associated with lamivudine (LMV) resistance. Four patients had the rtI233V mutation that may reduce sensitivity to adefovir, and three patients had the rtM250L/V mutation typical of entecavir resistance. LMV compensatory mutations rtL80V and rtV173L were seen in two and one patients, respectively. No relationship was seen between presence of resistant or compensatory mutations and HBV‐DNA levels, HBeAg/anti‐HBe status or previous IFN therapy. These results confirm that HBV mutations, which confer resistance against currently available anti‐HBV NAs, may already exist in patients who have never received the drug.

Hyperinsulinaemia reduces the 24-h virological response to PEG-interferon therapy in patients with chronic hepatitis C and insulin resistance

Summary.  Insulin resistance (IR) reduces response to pegylated‐interferon (PEG‐IFN)/ribavirin in chronic hepatitis C (CHC), but the mechanisms are still undefined. We examined the relationship between baseline insulin levels, the main component affecting homeostasis model of assessment – insulin resistance (HOMA‐IR) for assessment of IR in non‐diabetic patients, and the ‘acute’ virological response to PEG‐IFN measured 24 h after the first injection and taken as correlate of intracellular interferon signalling. In 62 patients treated with PEG‐IFN/Ribavirin, serum insulin and HOMA‐IR were assessed at baseline, while hepatitis C virus (HCV)‐RNA was measured at baseline and 24 h, 1, 2, 4 and 12 weeks after treatment initiation. Sustained virological response was examined 24 weeks after therapy discontinuation. Mean baseline insulin was 11.52 ± 8.51 U/L and mean HOMA‐IR was 2.65 ± 2.01 both being significantly higher with advanced liver fibrosis. Hepatitis C virus‐RNA decay observed 24 h after the first injection of PEG‐IFN was significantly lower (0.7 ± 0.8 log) in patients with HOMA ≥3 compared with those with HOMA <3 (1.7 ± 0.8, P = 0.001). A highly significant (r = −0.42) inverse correlation was observed between baseline insulin levels and the 24‐h HCV‐RNA decay. The difference in early viral kinetics between patients with HOMA ≥3 or <3 resulted in a significant difference in the percentage of patients achieving rapid (week 4) and sustained virological response. Multivariate analysis, inclusive of patient age, HCV genotype and fibrosis stage, identified baseline insulin levels as the main independent variable affecting the 24‐h response to PEG‐IFN. Hyperinsulinaemia reduces the cellular response to Pegylated‐interferon in CHC with IR. Strategies to reduce insulin levels before initiation of treatment should be pursued to improve efficacy of anti‐viral treatment.

Impact of NS5A Sequences of Hepatitis C Virus Genotype 1a on Early Viral Kinetics during Treatment with Peginterferon-α2a plus Ribavirin

UNLABELLED BACKGROUND. Hepatitis C virus (HCV) genotype 1 is the most prevalent genotype in Western countries, and treatment with pegylated interferon (pegIFN) plus ribavirin fails in 50%-60% of patients. Genetic variability within the NS5A dsRNA-dependent protein kinase binding domain (PKRbd) of HCV-1b has been associated with responsiveness to IFN- alpha . Little is known about NS5A sequences of HCV-1a. We investigated whether genetic variability of HCV-1a NS5A correlates with the early HCV kinetics during treatment. METHODS Twenty-four treatment-naive, HCV-1a-infected patients were treated with standard doses of pegIFN- alpha 2a plus ribavirin. HCV viremia was quantitated at days 0, 1, 2, and 3 and weeks 1, 2, 4, 8, and 12 of treatment. According to HCV kinetics, patients were classified as early rapid responders, early moderate responders, or early slow responders. The full-length HCV NS5A was sequenced at baseline and at week 1. RESULTS At baseline, variability of the NS5A C terminus (concentrated in the PKRbd) is associated with interferon efficacy but not with the second phase of the early viral decline that has been associated with a sustained virologic response. Comparisons between baseline and week-1 full-length sequences did not show significant increases in mutations. CONCLUSIONS Genetic variability of HCV-1a NS5A does not predict responsiveness to IFN- alpha . Differences in early kinetics during combination therapy are not due to selection of IFN-resistant HCV strains.

Reciprocal interference between insulin and interferon‐alpha signaling in hepatic cells: A vicious circle of clinical significance?

Insulin resistance (IR) is common in chronic hepatitis C (CHC) and associates with reduced virological response to pegylated‐interferon (PEG‐IFN)/ribavirin therapy, but the underlying mechanisms are still unclear. We have previously shown that, in CHC patients, insulin plasma levels are inversely related to antiviral effect induced by PEG‐IFN. Therefore, we investigated the in vitro effect of insulin on interferon alpha (IFN‐α) intracellular signaling as well as that of IFN‐α on insulin signaling. HepG2 cells, preincubated with or without insulin, were stimulated with IFN‐α2b and messenger RNA (mRNA) and protein expression of IFN‐stimulated genes (ISGs) were measured at different timepoints. The role of intracellular suppressors of cytokine signaling 3 (SOCS3) was evaluated with the small interfering RNA (siRNA) strategy. To assess the effect of IFN‐α on insulin signaling, HepG2 were preincubated with or without IFN before addition of insulin and cells were then analyzed for IRS‐1 and for Akt/PKB Ser473 phosphorylation. Insulin (100 and 1000 nM) significantly reduced in a dose‐dependent fashion IFN‐induced gene expression of PKR (P = 0.017 and P = 0.0017, respectively), MxA (P = 0.0103 and P = 0.00186), and 2′‐5′ oligoadenylatesynthetase 1 (OAS‐1) (P = 0.002 and P = 0.006). Insulin also reduced IFN‐α‐induced PKR protein expression. Although insulin was confirmed to increase SOCS3 expression, siRNA SOCS3 did not restore ISG expression after insulin treatment. IFN‐α was found to reduce, in a dose‐dependent fashion, IRS‐1 gene expression as well as Akt/PKB Ser473 phosphorylation induced by insulin. Conclusion: These results provide evidence of reciprocal interference between insulin and IFN‐α signaling in liver cells. These findings may contribute to understand the role of insulin in CHC: IR might be favored by endogenous cytokines including IFN‐α, and the resulting hyperinsulinemia then reduces the antiviral response to exogenous IFN in a vicious circle of clinical significance. (HEPATOLOGY 2011;)

Detailed analysis of the E2–IgM complex in hepatitis C-related type II mixed cryoglobulinaemia

Summary.  Hepatitis C virus (HCV) plays a major role in the induction of type II mixed cryoglobulinaemia (MCII). The role of HCV proteins and virus–host interaction in the pathogenesis of MC remains to be defined. To address this issue, we have characterized, in detail, the monoclonal IgM and the viral component of circulating immune complexes in eight patients with HCV‐associated MCII. The proportion of HCV‐RNA compartmentalized in the cryoprecipitate (CP) varied greatly (10–80% of total HCV‐RNA). The complementary determining region (CDR)3 sequences of monoclonal immunoglobulin M (IgM) VH and VK genes were highly homologous to rheumatoid factor and to antibodies against HCV‐E2. Furthermore, the CDR3 sequences in some of our MCII patients were highly similar to those described in HCV‐positive patients with non‐Hodgkins lymphoma (NHL). From these results, it appears that, as in the case of NHL, the IgM‐rheumatoid factor (RF) production in MCII patients is antigen driven, namely by E2. However, the limited number of mutations in VH and VK genes with respect to the germline and their distribution showed that the B‐cell response in these cases was prevented from undergoing affinity maturation. Furthermore, in patients with monoclonal IgM and definite compartmentalization of HCV in either CP or supernatant, a highly homogeneous E2‐hypervariable region (HVR)1 sequence distribution was found (90–100% identical clones), a feature of the quasispecies frequently associated with an impaired humoral immune response to HCV. These findings suggest that in patients with HCV‐associated MCII, maturation of monoclonal B lymphocytes may be blocked in a primitive stage preventing serious damaging effects because of the auto‐reactivity of their secreted immunoglobulins.

Comparable performance of TMA and Real-Time PCR in detecting minimal residual hepatitis C viraemia at the end of antiviral therapy.

BACKGROUND Antiviral therapy for chronic hepatitis C may cause transient on-treatment response followed by post-treatment relapse. OBJECTIVES We have compared the prognostic value for post-treatment relapse of minimal hepatitis C residual viraemia detected at end-of-therapy by transcription mediated assay (TMA) and by Abbott RealTime PCR HCV assay. STUDY DESIGN Minimal residual viraemia was investigated in 202 HCV patients who had completed a full course of Pegylated Interferon (PEG-IFN) plus ribavirin and were HCV-RNA negative by conventional PCR in two separate serum samples obtained during the last week of therapy and the results were then correlated with post-treatment outcome. RESULTS Minimal residual viraemia was detected in 22/202 (11%, 95% CI: 7-16%) and in 28/202 (13.8%, 95% CI 10-19%) patients by TMA and by Abbott RealTime HCV assay, respectively, with 92% concordant results. Post-treatment relapse was seen in 81.8% (95% CI: 60-93%) of TMA positive and in 82.1% (95% CI: 64-92%) of Abbott RealTime HCV assay positive cases compared to 16.6% (95% CI: 12-23%) of TMA negative and 17.2% (95% CI: 12-23%) of Abbott RealTime HCV assay negative patients. CONCLUSIONS These results indicate that TMA and the Abbott RealTime HCV assay have comparable sensitivity and specificity in identifying minimal residual viraemia at the end of antiviral therapy, with excellent predictive value for post-treatment relapse.

647 INSULIN DIRECTLY INHIBITS IFN-α SIGNALING IN HEPATIC CELLS THROUGH A SOCS3 INDEPENDENT PATHWAY

When HCVpp lacking the HVR were used (or when SRB1 was inhibited by BLT-4) serum enhancement was lost, particularly in HIV coinfected patients. In HIV patients, neutralization was positively and independently related to CD4 count (p < 0.01) and to antiretroviral therapy (p < 0.001). Selective pressure on HVR was higher in immunocompetent subjects and was related to the neutralization observed with serum immunoglobulin fraction. Conclusion: The sera of patients coinfected by HIV or liver transplanted are poorly neutralizing and facilitate the entry of HCVpp mainly through HVR and SRB1. Our findings are the first to show that low neutralization/high facilitation is related to the immune status and could be involved in the severity of HCV in immunocompromized subjects.