Fukiko Mitsui

Common variation of IL28 affects gamma-GTP levels and inflammation of the liver in chronically infected hepatitis C virus patients

BACKGROUND & AIMS A common genetic variation at the IL28 locus has been found to affect the response of peg-interferon and ribavirin combination therapy against chronic hepatitis C virus (HCV) infection. An allele associated with a favorable response (rs8099917 T), which is the major allele in the majority of Asian, American, and European populations, has also been found to be associated with spontaneous eradication of the virus. METHODS As no studies have yet analyzed the effect of the polymorphism on biochemical and inflammatory changes in chronic infection, we analyzed a cohort of patients with chronic hepatitis C (n=364) for the effect of the IL28 polymorphism on viral, biochemical, and histological findings. RESULTS We found that the proportion of HCV wild type core amino acids 70 and 91 was significantly greater (p=1.21 x 10(-4) and 0.034) and levels of gamma-GTP significantly lower (p=0.001) in patients homozygous for the IL28 major allele. We also found that inflammation activity and fibrosis of the liver were significantly more severe in patients homozygous for the IL28 major allele (p=0.025 and 0.036, respectively). Although the higher gamma-GTP levels were also associated with higher inflammatory activity and fibrosis, multivariate analysis showed that only the IL28 allele polymorphism, sex, alcohol consumption, and liver fibrosis were independently associated with gamma-GTP levels (p=0.001, 0.0003, 0.0013, and 0.0348, respectively). CONCLUSIONS These results suggest that different cytokine profiles induced by the IL28 polymorphism resulted in different biochemical and inflammatory conditions during chronic HCV infection and contribute to the progression of liver diseases.

Predictive value of the IL28B polymorphism on the effect of interferon therapy in chronic hepatitis C patients with genotypes 2a and 2b

BACKGROUND & AIMS Common IL28B locus polymorphisms (SNPs rs8099917 and rs12979860) have been reported to affect peg-interferon plus ribavirin combination therapy (PEG-RBV) for hepatitis C virus (HCV) genotype 1b, but few reports have examined their effect on other two common genotypes, 2a and 2b. METHODS We analyzed predictive factors for sustained virological response (SVR) in a retrospective study of 719 patients with either genotype 2a (530) or 2b (189). Of these patients, 160 were treated with PEG-RBV and 559 were treated with interferon monotherapy. We evaluated predictive factors including HCV RNA, histological findings, IL28B SNP genotypes (rs8099917, rs12979860, and rs12980275), and the effect of treatment regimen and prior treatment history. RESULTS HCV RNA viral load, treatment regimen, and rs8099917 genotypes independently contributed to the effect of the therapy. For patients treated with PEG-RBV, rs8099917 and viral load were independent predictive factors for SVR in genotype 2b but not in genotype 2a. Conversely, in patients treated with interferon monotherapy, viral load and rs8099917 were independent predictive factors for SVR in genotype 2a but not in genotype 2b. The favorable rs8099917 genotype is also associated with a steep decline in viral load by the second week of treatment. CONCLUSIONS Initial viral load and rs8099917 genotype are significant independent predictors of SVR in genotype 2 patients.

HBx protein is indispensable for development of viraemia in human hepatocyte chimeric mice.

The non-structural X protein, HBx, of hepatitis B virus (HBV) is assumed to play an important role in HBV replication. Woodchuck hepatitis virus X protein is indispensable for virus replication, but the duck hepatitis B virus X protein is not. In this study, we investigated whether the HBx protein is indispensable for HBV replication in vivo using human hepatocyte chimeric mice. HBx-deficient (HBx-def) HBV was generated in HepG2 cells by transfection with an overlength HBV genome. Human hepatocyte chimeric mice were infected with HBx-def HBV with or without hepatic HBx expression by hydrodynamic injection of HBx expression plasmids. Serum virus levels and HBV sequences were determined with mice sera. The generated HBx-def HBV peaked in the sucrose density gradient at points equivalent to the generated HBV wild type and the virus in a patients serum. HBx-def HBV-injected mice developed measurable viraemia only in continuously HBx-expressed liver. HBV DNA in the mouse serum increased up to 9 log(10) copies ml(-1) and the viraemia persisted for more than 2 months. Strikingly, all revertant viruses had nucleotide substitutions that enabled the virus to produce the HBx protein. It was concluded that the HBx protein is indispensable for HBV replication and could be a target for antiviral therapy.

Elimination of hepatitis C virus by short term NS3-4A and NS5B inhibitor combination therapy in human hepatocyte chimeric mice

BACKGROUND & AIMS The current treatment regimen for chronic hepatitis C virus (HCV) infection is peg-interferon plus ribavirin combination therapy. The majority of developing therapeutic strategies also contain peg-interferon with or without ribavirin. However, interferon is expensive and sometimes intolerable for some patients because of severe side effects. METHODS Using human hepatocyte chimeric mice, we examined whether a short term combination therapy with the HCV NS3-4A protease inhibitor telaprevir and the RNA polymerase inhibitor MK-0608 with or without interferon eradicates the HCV from infected mice. The effect of telaprevir and MK-0608 combination therapy was examined using subgenomic HCV replicon cells. RESULTS Combination therapy with the two drugs enhanced inhibition of HCV replication compared with either drug alone. In in vivo experiments, early emergence of drug resistance was seen in mice treated with either telaprevir or MK-0608 alone. However, emergence was prevented by the combination of these drugs. Mice treated with a triple combination therapy of telaprevir, MK-0608, and interferon became negative for HCV RNA soon after commencement of the therapy, and HCV RNA was not detected in serum of these mice 12 weeks after cessation of the therapy. Furthermore, all mice treated with a high dose telaprevir and MK-0608 combination therapy for 4 weeks became negative for HCV RNA 1 week after the beginning of the therapy and remained negative after 18 weeks. CONCLUSIONS Eradication of HCV from mice with only 4 weeks of therapy without interferon points the way to future combination therapies for chronic hepatitis C patients.

Absence of viral interference and different susceptibility to interferon between hepatitis B virus and hepatitis C virus in human hepatocyte chimeric mice

BACKGROUND/AIMS Both hepatitis B virus (HBV) and hepatitis C virus (HCV) replicate in the liver and show resistance against innate immunity and interferon (IFN) treatment. Whether there is interference between these two viruses is still controversial. We investigated the interference between these two viruses and the mode of resistance against IFN. METHODS We performed infection experiments with either or both of the two hepatitis viruses in human hepatocyte chimeric mice. Huh7 cell lines with stable production of HBV were also established and transfected with HCV JFH1 clone. Mice and cell lines were treated with IFN. The viral levels in mice sera and culture supernatants and messenger RNA levels of IFN-stimulated genes were measured. RESULTS No apparent interference between the two viruses was seen in vivo. Only a small (0.3 log) reduction in serum HBV and a rapid reduction in HCV were observed after IFN treatment, regardless of infection with the other virus. In in vitro studies, no interference between the two viruses was observed. The effect of IFN on each virus was not affected by the presence of the other virus. IFN-induced reductions of viruses in culture supernatants were similar to those in in vivo study. CONCLUSIONS No interference between the two hepatitis viruses exists in the liver in the absence of hepatitis. The mechanisms of IFN resistance of the two viruses target different areas of the IFN system.

G to A hypermutation of TT virus

APOBEC3 proteins function as part of innate antiviral immunity and induce G to A hypermutation in retroviruses and hepatitis B virus (HBV) genomes. Whether APOBEC3 proteins affect viruses that replicate without a reverse transcription step is unknown. TT virus (TTV), known to present in serum of healthy individuals and HBV carriers, has a single-stranded circular DNA genome and replicates without reverse transcription. In this study, we examined 67 blood samples obtained from healthy individuals and HBV carriers and observed G to A hypermutation of genomes of TTV in both healthy individuals and HBV carriers. During ALT flare-up in HBV carriers, G to A hypermutation of HBV increased, but TTV genomes significantly decreased in number and hypermutated TTV genomes became undetectable. Our results show that hypermutated TTV exist in healthy individuals and HBV carriers and that TTV genomes were susceptible to immune reaction directed to HBV by interacting with APOBEC3 proteins.

Effects of structural variations of APOBEC3A and APOBEC3B genes in chronic hepatitis B virus infection.

Aim:  Human APOBEC3 deaminases induce G to A hypermutation in nascent DNA strand of hepatitis B virus (HBV) genomes and seem to operate as part of the innate antiviral immune system. We analyzed the importance of APOBEC3A (A3A) and APOBEC3B (A3B) proteins, which are potent inhibitors of adeno‐associated‐virus and long terminal repeat (LTR)‐retrotransposons, in chronic HBV infection.

Effects of Hepatitis B Virus Infection on the Interferon Response in Immunodeficient Human Hepatocyte Chimeric Mice

Complementary DNA microarray analysis of human livers cannot exclude the influence of the immunological response. In this study, complementary DNA microarray analysis was performed under immunodeficient conditions with human hepatocyte chimeric mice, and gene expression profiles were analyzed by hepatitis B virus (HBV) infection and/or interferon treatment. The expression levels of 183 of 525 genes upregulated by interferon treatment were significantly suppressed in response to HBV infection. Suppressed genes were statistically significantly associated with the interferon signaling pathway and pattern recognition receptors in the bacteria/virus recognition pathway (P = 1.0 × 10(-8) and P = 1.2 × 10(-8), respectively). HBV infection attenuated virus recognition and interferon response in hepatocytes, which facilitated HBV escape from innate immunity.

Amino acid substitutions in core and NS5A regions of the HCV genome can predict virological decrease with pegylated interferon plus ribavirin therapy.

BACKGROUND The current standard therapy for chronic hepatitis C is pegylated interferon (PEG-IFN) plus ribavirin (RBV) combination therapy. Recently, it has been reported that amino acid (aa) substitutions in the core region, as well as the IFN-sensitivity-determining region (ISDR), were predictive of non-virological response (NVR), sustained virological response (SVR) and early virological response. Despite the importance of these two predictive factors for combination therapy, their interaction is poorly understood. METHODS A total of 117 patients who were treated with PEG-IFN-α2b plus RBV combination therapy were selected for participation in this study. We determined the aa sequences in the core region and ISDR by direct sequencing and analysed them along with clinical data to identify predictive factors for therapeutic response. RESULTS The aa sequences in the core region and γ-glutamyl transpeptidase (GTP) levels were associated with SVR and NVR, but aa sequences in the ISDR were not. However, substitutions at both aa 70 and aa 91 in the core region without substitutions in the ISDR and higher levels of γ-GTP were independent predictive factors for NVR. Wild-type aa 70 and aa 91 in the core region, higher platelet counts and lower levels of γ-GTP were independent predictive factors for SVR. CONCLUSIONS These results indicate that analyses of aa substitutions in both the core region and the ISDR are useful for predicting the effectiveness of combination therapy, and could help to avoid therapy exposure for patients who have a low probability of SVR.

Prediction of response to peginterferon‐alfa‐2b plus ribavirin therapy in Japanese patients infected with hepatitis C virus genotype 1b

Variation at the IL‐28B locus was recently reported to be a significant predictive factor of viral response to pegylated‐interferon plus ribavirin combination therapy against chronic hepatitis C. Predictive factors for the effect of therapy, including IL‐28B polymorphism rs8099917 and viral and clinical factors were investigated. A total of 288 patients were enrolled who were chronically infected with hepatitis C virus (HCV) genotype 1b and treated with combination therapy. Among them, 87 patients completed 48 weeks of therapy without dose reduction or discontinuation. In multivariate regression analysis, the rs8099917 TT genotype was the only independent factor significantly associated with sustained viral response (P = 0.016, OR 61.5), whereas substitutions at amino acid 70 (aa 70) of the HCV core protein (P = 0.038, OR 5.9) and non‐TT genotypes (P = 0.002, OR 17.2) were associated with nonvirological response. Both factors were also associated with viral dynamics during the initial stage of the therapy. Correlation analysis revealed that rs8099917 genotype was correlated with γ‐glutamyl transpeptidase, hyaluronic acid, and HCV core aa 70. In conclusion, host (IL‐28B polymorphism) and viral (aa 70) factors independently affect response to combination therapy. J. Med. Virol. 83:981–988, 2011.