Cross-genotype-specific T-cell responses in acute hepatitis E virus (HEV) infection.

Hepatitis E is an inflammatory liver disease caused by infection with the hepatitis E virus (HEV). In tropical regions, HEV is highly endemic and predominantly mediated by HEV genotypes 1 and 2 with >3 million symptomatic cases per year and around 70 000 deaths. In Europe and America, the zoonotic HEV genotypes 3 and 4 have been reported with continues increasing new infections per year. So far, little is known about T-cell responses during acute HEV genotype 3 infection. Therefore, we did a comprehensive study investigating HEV-specific T-cell responses using genotypes 3- and 1-specific overlapping peptides. Additional cytokines and chemokines were measured in the plasma. In four patients, longitudinal studies were performed. Broad functional HEV-specific CD4(+) and CD8(+) T-cell responses were detectable in patients acutely infected with HEV genotype 3. Elevated of pro- and anti-inflammatory cytokine levels during acute HEV infection correlated with ALT levels. Memory HEV-specific T-cell responses were detectable up to >1.5 years upon infection. Importantly, cross-genotype HEV-specific T-cell responses (between genotypes 1 and 3) were measurable in all investigated patients. In conclusion, we could show for the first time HEV-specific T-cell responses during and after acute HEV genotype 3 infection. Our data of cross-genotype HEV-specific T-cell responses might suggest a potential role in cross-genotype-specific protection between HEV genotypes 1 and 3.

Relevance of chronic hepatitis E in liver transplant recipients: a real-life setting.

The chronic course of hepatitis E virus (HEV) infections in orthotopic liver transplant (OLT) recipients has been described previously, but prospectively collected data are rare. We aimed to study the role of chronic hepatitis E in OLT in a real-life setting. Therefore, 287 adult OLT recipients (169 male [59%], median age 56 years) were prospectively tested by HEV polymerase chain reaction assay (lower level of detection = 10 IU/mL), irrespective of their level of liver enzymes. In 4 patients (1.4%), chronic HEV infection was diagnosed. All 4 patients were male, and their age (median 48.5 years), the time since transplantation (median 45.5 months), and bilirubin level (median 0.6 mg/dL) did not differ significantly from the total cohort. However, alanine transaminase and aspartame transaminase levels were significantly higher in HEV-infected patients (75-646 U/L, median 216 U/L and 68-317 U/L, median 108 U/L) than in non-infected patients (6-617 U/L, median 41 and 6-355 U/L, median 36; P = 0.004 and 0.040, Mann-Whitney test). In 3 patients, liver biopsy was performed and revealed signs of inflammation and chronic liver disease, as enlarged densely infiltrated portal tracts with mild-to-moderate interface hepatitis. All infected patients were treated with ribavirin with the starting dose adjusted to renal function (400-800 mg/day). In 2 patients, dose reduction was necessary. Transaminases normalized in all 4 patients, and all patients cleared their infection within 3 months of ribavirin treatment. However, 1 patient experienced viral relapse 12 weeks after discontinuation. Ribavirin medication was re-started and viral clearance occurred within 8 weeks and persisted. Sequence analysis of the HEV genome of this patient revealed that he was infected with an HEV variant, which recently has been shown to have a reduced response to ribavirin in cell culture. The risk of chronic HEV infections in OLT recipients in low-endemic countries should not be overestimated. No case of chronic hepatitis E was observed in patients with normal liver enzymes, indicating that general screening of all OLT recipients is not necessary. However, if chronic hepatitis E develops, it can be treated efficiently with ribavirin.

Chronic hepatitis e in heart transplant recipients.

Chronic courses of hepatitis E virus (HEV) infections have been described in immunosuppressed patients. We aimed to study the role of HEV infections in heart transplant recipients (HTR). 274 HTR were prospectively screened for HEV infection using an anti-HEV-IgG ELISA and HEV-PCR. In addition, 137 patients undergoing cardiac surgery (non-HTR) and 537 healthy subjects were studied cross-sectionally. The anti-HEV-IgG seroprevalence was 11% in HTR, 7% in non-HTR and 2% in healthy controls (HTR vs. healthy controls p<0.0001; non-HTR vs. healthy controls p<0.01). Anti-HEV tested positive in 4.0% in control cohorts of other immunocompromised patients (n = 474). Four HTR (1.5%) were chronically infected with HEV as shown by HEV-PCR and all four patients had liver transaminases of >200 IU/L and histological or clinical evidence of advanced liver disease. In three patients ribavirin treatment was successful with a sustained biochemical and virological response while treatment failed in one cirrhotic patient after ribavirin dose reduction. Heart transplant recipients and patients undergoing cardiac surgery have an increased risk for HEV infections. Chronic hepatitis E may explain elevated liver enzymes in heart transplant recipients. Treatment of HEV infection with ribavirin is effective but the optimal dose and duration of ribavirin therapy remains to be determined.

Intrahepatic long-term persistence of parvovirus B19 and its role in chronic viral hepatitis.

Parvovirus B19 (B19V) has been detected in the liver of Asian patients infected with HBV and may contribute to acute and chronic liver disease. This study aimed to investigate the impact of B19V infection in European patients with viral hepatitis. B19V DNA was detected in 1/91 and 0/50 serum samples from patients with chronic hepatitis C and B, respectively. In contrast, B19V DNA was amplified frequently from explanted end-stage liver tissues (37/50, 74%) and from routine biopsy samples (14/32, 44%) (P < 0.05). However, there was no significant difference in B19V copy number per cell between these two groups. B19V-specific CD4(+) T-cell responses to two dominant MHC-class-restricted epitopes were detected in a similar frequency in healthy anti-B19V-positive individuals (3/19; 16%) and patients with chronic hepatitis C (3/13; 23%). These results indicate that B19V can persist in the liver. However, there is no evidence that B19V is a "hepatitis virus" worsening liver disease in European patients with chronic hepatitis C.

Natural history of hepatic fibrosis progression in chronic hepatitis C virus infection in India.

BACKGROUND AND AIM: The rate of fibrosis progression per year can predict the time for the development of cirrhosis in chronic hepatitis C (CHC). We assessed the rate of fibrosis progression and the predictors of disease severity in Indian CHC patients. METHODS: Of the 355 treatment-naïve, histologically-proven CHC patients, the precise duration of infection (from the time of exposure to HCV until liver biopsy) could be determined in 213 patients (age = 41.6 +/- 14.7 years, male : female = 139 : 74, genotype 3 = 75%). The rate of fibrosis progression per year was calculated. The correlation of the advanced degree of fibrosis and age, duration of infection, age at the onset of infection, sex, mode of infection, hepatitis C virus (HCV) genotype, histological activity index (HAI), and the presence of diabetes mellitus were studied. RESULTS: The median rate of fibrosis progression per year was 0.25 (0.0-1.5) fibrosis units. The fibrosis progression rate was higher in patients who acquired infection at > 30 years of age, those < 30 years (0.33 vs 0.15; P < 0.001), and those who acquired HCV infection with a history of blood transfusion than with other modes of transmission (0.25 vs 0.19; P = 0.04). The median time to progress to cirrhosis was 16 years. The multivariate analysis found that the HAI score (odds ratio [OR]= 14.03; P < 0.001) and the duration of infection > 10 years (OR = 4.83; P < 0.001) correlated with severe liver disease (fibrosis > or = 3). CONCLUSION: The median rate of fibrosis progression per year in Indian CHC patients is 0.25 fibrosis units. A higher HAI and longer duration of infection are associated with a significant risk of advanced liver disease, and merit early therapeutic interventions.

CCR5Delta32 mutation does not influence the susceptibility to HCV infection, severity of liver disease and response to therapy in patients with chronic hepatitis C.

AIM: To study whether CCR5Delta32 mutation was associated with viral infection and severity of liver disease. METHODS: Two hundred and fifty two histologically proven, chronic HCV patients (mean age: 41 +/- 14 years; M/F: 164/88) were genotyped. PCR based genotyping of 32 bp deletion at the CCR5 locus was done. Four-hundred and eight matched healthy controls were studied to assess susceptibility to HCV infection. To assess correlation of immune gene polymorphism with severity of HCV related liver disease, patients with chronic HCV infection were divided into those with a fibrosis score of <= 2 (mild) or > 2 (severe) and histological activity index (HAI) of <= 5 or > 5. For correlation between CCR5Delta32 mutations and response to therapy, 129 patients who completed therapy were evaluated. RESULTS: The majority (89.4%) of the patients were infected with genotype 3. The frequency of homozygous CCR5Delta32 mutants was comparable to HCV patients as compared to the healthy controls (0.7% vs 0%, P = 0.1). Further more, the frequency of CCR5Delta32 mutation was comparable in patients with mild or severe liver disease. (P = NS). There was also no association observed with response to therapy and CCR5Delta32 mutation. CONCLUSION: CCR5Delta32 mutation does not have a role in disease susceptibility, severity or response to therapy in patients with chronic hepatitis C infection.

Association of IL-18 promoter polymorphism with liver disease severity in HCV-infected patients.

INTRODUCTION: Interleukin (IL)-18 plays an important dual role in Th1 polarization and viral clearance, as well as in the development of liver fibrosis. Single-nucleotide promoter polymorphisms influence the transcription of IL-18 mRNA. Promoter polymorphisms are linked to delayed virus clearance and disease susceptibility in many diseases. However, there is no information about their role in hepatitis C virus (HCV) infection. AIM: To investigate the association between -607 or -137 polymorphism with susceptibility and severity of HCV infection. PATIENTS AND METHODS: Two hundred and four serologically proven patients with chronic HCV infection and 350 matched healthy controls were included in this study. Patients were segregated in 2 groups: group A with mild liver disease and group B with severe liver disease on the basis of histological activity index (HAI 5) and hepatic fibrosis score (2). IL-18 promoter genotyping was performed with sequence-specific primers. RESULTS: There was no significant difference in the frequencies of -607 and -137 allelic distribution in patients and controls. The -607 A/A allele was more common in group A patients with mild liver disease than in patients with severe liver disease on the basis of HAI (38.6% vs. 21%, P = 0.05; odds ratio [OR] = 0.424, confidence interval [CI] = 0.233-0.773; R (2) = 0.631) and stage of fibrosis (38.7% vs. 16.7%, P = 0.008; OR = 0282, CI = 0.134-0.596; R (2) = 0.434). CONCLUSIONS: IL-18 promoter polymorphism at -607 position with A/A allele is a potential protective marker, as it is associated with milder liver disease in patients with chronic HCV infection.

Cross-genotype-reactivity of the immunodominant HCV CD8 T-cell epitope NS3-1073.

The HCV-specific HLA-A2-restricted NS3(1073) epitope is one of the most frequently recognized epitopes in hepatitis C. NS3(1073)-specific T-cell responses are associated with clearance of acute HCV-infection. Therefore this epitope is an interesting candidate for a HCV-peptide vaccine. However, heterogeneity between genotypes and mutations in the epitope has to be considered as an obstacle. We here identified 34 naturally occurring NS3(1073)-variants, as compared with the wild type genotype-1 variants (CVNGVCWTV/CINGVCWTV) by sequencing sera of 251 Greek and German patients and searching for published HCV-genomes. The frequency of variants among genotype-1 patients was 10%. Importantly, HLA-A2 binding was reduced only in 3 genotype 1 mutants while all non-genotype 1 variants showed strong HLA-A2-binding. By screening 28 variants in ELISPOT assays from T cell lines we could demonstrate that HCV-NS3(1073)-wild-type-specific T-cells displayed cross-genotype-reactivity, in particular against genotypes 4-6 variants. However, single aa changes within the TCR-binding domain completely abolished recognition even in case of conservative aa exchanges within genotype-1. NS3(1073)-specific T-cell lines from recovered, chronically infected, and HCV-negative individuals showed no major difference in the pattern of cross-recognition although the proliferation of NS3(1073)-specific T-cells differed significantly between the groups. Importantly, the recognition pattern against the 28 variants was also identical directly ex vivo in a patient with acute HCV infection and a healthy volunteer vaccinated with the peptide vaccine IC41 containing the NS3(1073)-wild-type peptide. Thus, partial cross-genotype recognition of HCV NS3(1073)-specific CD8 T cells is possible; however, even single aa exchanges can significantly limit the potential efficacy of vaccines containing the NS3(1073)-wild-type peptide.

Studies on TAQ1 polymorphism in the 3'untranslated region of IL-12P40 gene in HCV patients infected predominantly with genotype 3.

Host immunity plays an important role in viral persistence and progression of liver disease in HCV infected patients. IL-12 induces production of IFN-gamma, a potent antiviral agent. IL-12 comprises two subunits; IL-p35 and IL-12p40, which are encoded by two different genes located on chromosome 3 and 5, respectively. Single nucleotide polymorphism at A1188C in the 3'UTR of IL-12p40 gene is associated with immune mediated diseases. Association of IL-12p40 A1188C polymorphism with the outcome of HCV infection was investigated in this study. Two hundred and fifty three histologically proven chronic hepatitis C patients (43 +/- 13 years, male:female: 185:68) and 380 matched controls were included. Genotyping was performed by RFLP and confirmed by direct sequencing. To assess correlation of immune gene polymorphism with severity of HCV-related liver disease, patients were divided into those with fibrosis score of < or = 2 (mild) or > 2 (severe), and histological activity index (HAI) of = 5 (mild) or > 5 (severe). The distribution of A/A, A/C or C/C alleles in the controls was comparable to the patients. The distribution of C/C allele was significantly more common in patients with mild as compared to severe fibrosis (23.7% vs. 6.25%, P = 0.004). No significant difference was observed for any of the genetic markers with HAI or with normal or raised alanine aminotransferase (ALT). These results show that the C/C allele of IL-12p40 gene could render genetic protection against development of severe liver disease in patients infected with HCV.

Hepatitis C virus genotype 3 predominates in North and Central India and is associated with significant histopathologic liver disease.

Hepatitis C virus (HCV) genotypes help to tailor the treatment response, but their influence on the disease severity and association with hepatic steatosis is not well understood. The prevalence of HCV genotypes and their correlation with the histopathological severity of liver disease and steatosis in Indian patients were studied. HCV-RNA and genotyping was carried out in 398 patients with chronic hepatitis C. Liver biopsy was available in 292 (73.4%) patients. The severity of liver disease was graded on the basis of the histological activity index and the stage of hepatic fibrosis. The patients were categorized as having mild (histological activity index < or =5 and/or fibrosis < or =2) or severe (histological activity index > or =6 and/or fibrosis > or =3) liver disease. Steatosis was graded in 106 patients as 0 (no steatosis), 1 (<33% of hepatocytes affected), 2 (33%-66% of hepatocytes affected), or 3 (>66% of hepatocytes affected). HCV genotype 3 was detected in 80.2% patients (3a:24.4%, 3b:3.3%, 3c:0.5%, 3a/3b:36.7%, and un-subtypable 3:15.3%), genotype 1 in 13.1% (1a:3%, 1b:5.5%, 1a/1b:0.3%, and un-subtypable 1:4.3%), genotype 4 in 3% patients (4a:1.5%, 4b:0.3%, 4a/4c:0.5%, and un-subtypable 4:0.8%), 2 in 2.5% and mixed genotypes (more than one genotype) in 1.3% of patients. The median histological activity index and fibrosis scores were: 5 and 2 in genotype 1; 4 and 2 in genotype 2; 5 and 2 in genotype 3; 7 and 3 in genotype 4; and 5 and 2 in mixed genotypes, respectively. Severe liver disease was present in 17 of 38 (45%) with genotype 1; in 1 of 3 (33%) with genotype 2; in 128 of 236 (54%) with genotype 3; 7 of 10 (70%) with genotype 4; and in 1 of 4 (25%) with mixed genotype. Hepatic steatosis grade > or =2 was found in 28.1% of genotype 3; 23.5% of genotype 1; 20% of genotype 4; and in none of genotype 2 and mixed genotypes. In conclusion, genotype 3 is the most prevalent genotype in patients with chronic hepatitis C in North and Central India and this is associated with significant hepatic steatosis and fibrosis.