Hepatitis E virus ORF 1 induces proliferative and functional T-cell responses in patients with ongoing and resolved hepatitis E.

BACKGROUND AND AIMS: Hepatitis E virus (HEV) is a major cause of acute viral hepatitis with >3 million symptomatic cases per year accounting for 70 000 HEV-related deaths. HEV-specific T-cell responses have been investigated against structural proteins expressed by open reading frames (ORF) 2 and 3. T-cell responses against non-structural HEV proteins encoded by ORF1 are hardly studied. The aim of this study was to determine HEV ORF1-specific T-cell responses in comparison to ORF2/3 in patients exposed to HEV. METHODS: HEV-specific CD4+ and CD8+ T-cell responses against HEV genotype 3 were investigated in patients with acute and chronic hepatitis E as well as in HEV seropositive and seronegative individuals. HEV-specific T-cell responses were determined by proliferation and intracellular cytokine assay upon stimulation of PBMCs with HEV-specific overlapping peptide pools spanning the entire HEV genome. HEV-antigen was measured using an anti-HEV antigen-specific ELISA. RESULTS: Broad HEV ORF1-specific T-cell responses were detected in patients with acute, resolved and chronic hepatitis E without distinct dominant regions. The magnitude and frequency in recognition of ORF1-specific T-cell responses were similar compared to responses against HEV ORF2/3. Longitudinal studies of HEV-specific T-cell responses displayed similar behaviour against structural and non-structural proteins. HEV-antigen levels were inversely correlated with HEV-specific T-cell responses. CONCLUSIONS: HEV-specific T-cell responses are detectable against the entire HEV genome including the non-structural proteins. HEV-specific T-cell responses are associated with control of HEV infection. These findings have implications for the design of HEV vaccines.

In vivo evidence for ribavirin-induced mutagenesis of the hepatitis E virus genome.

OBJECTIVE: Hepatitis E virus (HEV) infection can take chronic courses in immunocompromised patients potentially leading to liver cirrhosis and liver failure. Ribavirin (RBV) is currently the only treatment option for many patients, but treatment failure can occur which has been associated with the appearance of a distinct HEV polymerase mutant (G1634R). Here, we performed a detailed analysis of HEV viral intrahost evolution during chronic hepatitis E infections. DESIGN: Illumina deep sequencing was performed for the detection of intrahost variation in the HEV genome of chronically infected patients. Novel polymerase mutants were investigated in vitro using state-of-the-art HEV cell culture models. RESULTS: Together, these data revealed that (1) viral diversity differed markedly between patients but did not show major intraindividual short-term variations in untreated patients with chronic hepatitis E, (2) RBV therapy was associated with an increase in viral heterogeneity which was reversible when treatment was stopped, (3) the G1634R mutant was detectable as a minor population prior to therapy in patients who subsequently failed to achieve a sustained virological response to RBV therapy and (4) in addition to G1634R further dominant variants in the polymerase region emerged, impacting HEV replication efficiency in vitro. CONCLUSIONS: In summary, this first investigation of intrahost HEV population evolution indicates that RBV causes HEV mutagenesis in treated patients and that an emergence of distinct mutants within the viral population occurs during RBV therapy. We also suggest that next-generation sequencing could be useful to guide personalised antiviral strategies.

Hepatitis E Virus Seroprevalence Rate in HIV-Infected Patients in Germany: A Comparison of Two Commercial Assays.

OBJECTIVES: Cases of chronic hepatitis E virus (HEV) infection have been described in HIV-infected patients. There are several commercial anti-HEV assays, but anti-HEV seroprevalence rates differ largely depending on the assay used. The aim of this study was to (1) compare two commercial anti-HEV assays in a German cohort of HIV-positive individuals, and (2) determine whether HEV takes chronic courses in controlled HIV infection. METHODS: 246 HIV patients were tested for both HEV RNA and HEV antibodies. All patients received antiretroviral therapy, if this was indicated, according to European guidelines. All but 19 individuals had CD4+ counts above 200/µl. Anti-HEV IgG was determined by two independent commercial assays (Wantai and MP). RESULTS: None of the patients tested HEV RNA positive. Anti-HEV IgG was detected more frequently by the Wantai assay (26%) than the MP assay (1.6%, p < 0.001). Patients born in Europe tested more frequently positive for anti-HEV (p = 0.047) than individuals from other regions. Increasing age but not CD4 count correlated with a higher likelihood of anti-HEV positivity (R = 0.313, p < 0.001). CONCLUSIONS: About one quarter of HIV-infected patients show evidence of previous HEV contact. The risk of developing chronic HEV infection is very low in individuals receiving appropriate antiretroviral therapy. The large variability in HEV seroprevalence rates determined by different assays requires consideration for the diagnostic workup of HIV patients.

A mutation in the hepatitis E virus RNA polymerase promotes its replication and associates with ribavirin treatment failure in organ transplant recipients.

We analyzed blood samples collected from 15 patients with chronic hepatitis E who were recipients of solid-organ transplants. All patients cleared the hepatitis E virus (HEV) except for 2 (nonresponders); 1 patient died. A G1634R mutation in viral polymerase was detected in the HEV RNA of the nonresponders; this mutation did not provide the virus with resistance to ribavirin in vitro. However, the mutant form of a subgenomic replicon of genotype 3 HEV replicated more efficiently in vitro than HEV without this mutation, and the same was true for infectious virus, including in competition assays. Similar results were obtained for genotype 1 HEV. The G1634R mutation therefore appears to increase the replicative capacity of HEV in the human liver and hence reduce the efficacy of ribavirin.

Increased HEV seroprevalence in patients with autoimmune hepatitis.

BACKGROUND: Hepatitis E virus (HEV) infection takes a clinically silent, self-limited course in the far majority of cases. Chronic hepatitis E has been reported in some cohorts of immunocompromised individuals. The role of HEV infections in patients with autoimmune hepatitis (AIH) is unknown. METHODS: 969 individuals were tested for anti-HEV antibodies (MP-diagnostics) including 208 patients with AIH, 537 healthy controls, 114 patients with another autoimmune disease, rheumatoid arthritis (RA), and 109 patients with chronic HCV- or HBV-infection (HBV/HCV). Patients with AIH, RA and HBV/HCV were tested for HEV RNA. HEV-specific proliferative T cell responses were investigated using CFSE staining and in vitro stimulation of PBMC with overlapping HEV peptides. RESULTS: HEV-antibodies tested more frequently positive in patients with AIH (n = 16; 7.7%) than in healthy controls (n = 11; 2.0%; p = 0.0002), patients with RA (n = 4; 3.5%; p = 0.13) or patients with HBV/HCV infection (n = 2; 2.8%; p = 0.03). HEV-specific T cell responses could be detected in all anti-HEV-positive AIH patients. One AIH patient receiving immunosuppression with cyclosporin and prednisolone and elevated ALT levels had acute hepatitis E but HEV viremia resolved after reducing immunosuppressive medication. None of the RA or HBV/HCV patients tested HEV RNA positive. CONCLUSIONS: Patients with autoimmune hepatitis but not RA or HBV/HCV patients are more likely to test anti-HEV positive. HEV infection should been ruled out before the diagnosis of AIH is made. Testing for HEV RNA is also recommended in AIH patients not responding to immunosuppressive therapy.