H. Wedemeyer

Treating viral hepatitis C: efficacy, side effects, and complications

The treatment of hepatitis C has dramatically improved over the past decade. Unlike any other chronic viral infection, a significant proportion of patients with chronic hepatitis C can be cured. However, the current standard therapy--pegylated interferon alpha and ribavirin--has its limitations. Limited efficacy in patients with hepatitis C virus (HCV) genotype 1 and the side effect profile will necessitate the development of new therapeutic approaches. This review describes the efficacy and optimisation of the current standard therapy of hepatitis C and its problems in special patient populations. New treatment directions beyond interferon alpha based therapies are on the horizon.

Hepatitis E in HIV‐positive patients in a low‐endemic country

To the Editor: Hepatitis E virus (HEV) infection has emerged as a special topic of interest in recent years as cases of chronic hepatitis E have been described in organ transplant recipients [1,2]. We therefore read with great interest the recent article by Madejon et al. [3] published in the April 2009 issue of the Journal of Viral Hepatitis describing a lack of evidence for chronic hepatitis E in HIV-infected Spanish patients. The authors tested a cohort of 93 HIV-positive patients for the presence of HEV RNA, and none were found to be positive for this marker in serum. However, the study was not able to answer the important question if HEV infection may take more frequently a chronic course in HIV-infected patients, as IgG anti-HEV antibodies were not tested for and no information was given on the overall seroprevalence of anti-HEV in Spanish HIV-infected patients. We therefore first determined the prevalence of HEV infection in German HIVinfected patients and secondly attempted to establish whether HIV-infected patients who acquire HEV are able to clear the virus or have a higher risk of developing chronic infection. We tested 123 HIV-positive patients [30 men; mean age 44 years (range 19–75)] recruited at the HIV-outpatient clinic of Hannover Medical School for anti-HEV IgG by a commercial ELISA kit (Abbott laboratories, Chicago, IL, USA). HIV viral load was determined by COBAS Taqman (Roche Diagnostics, Grenzach-Wyhlen, Germany) which has a lower limit of quantification of 47 copies per mL. Liver enzymes were normal in 83 patients whilst 40 patients had elevated ALT levels including 10 individuals who also had elevated bilirubin levels. The mean CD4+ T cell count was 392/lL (range 2–1602); 17% of patients had a CD4+ T cell count below 200/ll. HIV-RNA was not detectable by COBAS Taqman (<47 copies per mL) in 85 patients. The mean viral load for HIV in the remaining 38 patients was 94.594 copies per mL (50–26 200 000 copies per mL). The majority of patients were born in Germany (n = 82) whilst 31 had migrated to Germany from various other countries from Asia, Africa and Eastern Europe. Six patients had chronic hepatitis B virus (HBV) and 11 had chronic hepatitis C virus (HCV) infection, whilst one patient was co-infected with HBV and HCV. Anti-HEV-IgG-positive samples were tested for HEV RNA by RT-PCR [4]. IgG-antibodies against HEV were detected by the Abbott-ELISA kit in six HIV-infected patients (5%). All six patients were Germans, but one was born in Africa (three men, three women), and none of these patients was HBV-DNA or HCV-RNA positive. Two of the female patients acquired HIV infection by heterosexual contact, and the third through drug abuse. The three male patients were homosexual. The age of the anti-HEV-positive patients ranged between 39 and 51 years and CD4+ counts between 417 and 923 cells per lL. HIV-RNA was negative in five patients. Importantly, all six anti-HEV-positive patients tested negative for HEV RNA by nested RT-PCR. Five of the six patients had normal ALT levels, and one patient had a moderately elevated ALT level of 94 U/L. Previous studies from Russia and Belarus examining the frequency of anti-HEV IgG in HIV-infected patients demonstrated a significantly increased prevalence of anti-HEV IgG (11%) when compared to the general population (<2%). The anti-HEV prevalence among AIDS patients was even higher at 40% [5]. In addition, a study from Argentina also showed a raised HEV-IgG-seroprevalence in HIV-patients (6.6%) when compared to healthy controls (1.8%) [6]. In this study, we found a seroprevalence of 5% which seemed to be slightly higher than our experience with healthy employees and blood donors where anti-HEV IgG antibodies were found in one of 108 subjects studied [7]. The reason for the slightly higher anti-HEV seroprevalence in HIV-infected patients is unknown. HEV is usually transmitted by the faecal–oral route. However, there have been isolated reports on HEV transmission by blood transfusions [8,9]. The risk of HEV transmission by sexual exposure or by IV drug abuse is still not known. Importantly, HEV infection can also be acquired as a zoonosis because HEV genotype 3 is present in various animal species including swine, rats and cats [10,11]. We recently described two cases of chronic hepatitis E in liver transplant recipients; both patients were infected with HEV genotype 3 [7]. One may speculate that viral factors could contribute to the course of HEV infection, i.e. if an infection becomes chronic. However, no serotyping assays are available yet distinguishing between past HEV genotype 1 infections and exposure to genotype 3. Thus, we do not know if our six HIV-infected patients who were anti-HEV positive were actually infected with HEV genotype 1 or 3. In conclusion, HEV infection is a rather rare event in HIVinfected German patients. In line with the Madrid experience, we did not find evidence for chronic hepatitis E in our cohort. Unfortunately, we are not able to determine the exact time point when the six patients had been infected with HEV. It is indeed possible that the individuals may have been already anti-HEV positive when they acquired HIV infection and thus, our data do not exclude the possibility of chronic HEV infections in HIV-positive patients. Nevertheless, general screening for anti-HEV may not be necessary in countries of low endemicity. However, HEV infection should be considered in the differential diagnosis of otherwise unexplained hepatitis particularly in immunocompromised patients. Journal of Viral Hepatitis, 2010, 17, 598–599 doi:10.1111/j.1365-2893.2009.01219.x

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 B Surface Antigen Concentrations in Patients with HIV/HBV Co-Infection

HBsAg clearance is associated with clinical cure of chronic hepatitis B virus (HBV) infection. Quantification of HBsAg may help to predict HBsAg clearance during the natural course of HBV infection and during antiviral therapy. Most studies investigating quantitative HBsAg were performed in HBV mono-infected patients. However, the immune status is considered to be important for HBsAg decline and subsequent HBsAg loss. HIV co-infection unfavorably influences the course of chronic hepatitis B. In this cross-sectional study we investigated quantitative HBsAg in 173 HBV/HIV co-infected patients from 6 centers and evaluated the importance of immunodeficiency and antiretroviral therapy. We also compared 46 untreated HIV/HBV infected patients with 46 well-matched HBV mono-infected patients. HBsAg levels correlated with CD4 T-cell count and were higher in patients with more advanced HIV CDC stage. Patients on combination antiretroviral therapy (cART) including nucleos(t)ide analogues active against HBV demonstrated significant lower HBsAg levels compared to untreated patients. Importantly, HBsAg levels were significantly lower in patients who had a stronger increase between nadir CD4 and current CD4 T-cell count during cART. Untreated HIV/HBV patients demonstrated higher HBsAg levels than HBV mono-infected patients despite similar HBV DNA levels. In conclusion, HBsAg decline is dependent on an effective immune status. Restoration of CD4 T-cells during treatment with cART including nucleos(t)ide analogues seems to be important for HBsAg decrease and subsequent HBsAg loss.

Non-invasive fibrosis score for hepatitis delta.

Identifying advanced fibrosis in chronic hepatitis delta patients and thus in need of urgent treatment is crucial. To avoid liver biopsy, non‐invasive fibrosis scores may be helpful but have not been evaluated for chronic hepatitis delta yet.

HBV-specific T-cell responses in healthy seronegative sexual partners of patients with chronic HBV infection.

Summary.  The hepatitis B virus (HBV) is frequently transmitted by sexual intercourse. Thus, HBV‐guidelines recommend vaccination. However, we have identified healthy hepatitis B surface antigen and anti‐HBc‐negative unvaccinated sexual partners of patients with chronic hepatitis B. We investigated whether HBV‐specific cellular immune responses were present that could explain the apparent protection against HBV infection. In six anti‐HBc‐negative HBV‐exposed sexual partners, HBV‐specific T‐cell responses were studied by proliferation assay and cytometric bead array after stimulation with 74 overlapping peptides spanning the HBV core, pre‐S and S‐encoding regions. Eleven HBV‐unexposed individuals served as negative controls. HBV‐DNA was undetectable in serum and peripheral blood mononuclear cells in all cases. HBV‐specific cytokine secretion was observed in 4/6 seronegative partners, but only in 1/11 controls. Proliferative responses were detectable in 5/6 partners and 0/11 controls. HBV‐specific cytokine secretion exists in healthy seronegative virus‐exposed individuals. HBV core‐directed immune responses indicate past, but controlled viral replication. T‐cell immunity may prevent clinical manifestation of HBV infection in the absence of humoral immunity.

Viral dominance patterns in chronic hepatitis delta determine early response to interferon alpha therapy

Chronic hepatitis D is caused by coinfection of hepatitis B and hepatitis D virus. While HDV is the dominant virus over HBV in the majority of cases, mechanisms and consequences of viral dominance are largely unknown. We aimed to investigate associations between viral dominance patterns and patients’ characteristics and inflammatory features; 109 HDV‐infected patients treated with PEG‐IFNa‐2α within the international multicentre, prospective HIDIT‐2 trial were studied. Patients were classified as D‐ or B‐dominant if the viral load of one virus exceeded that of the other virus by more than 1log10. Otherwise, no viral dominance (ND) was described. We used Luminex‐based multiplex technology to study 50 soluble immune mediators (SIM) in pretreatment samples of 105 HDV RNA‐positive patients. Dominance of HDV was evident in the majority (75%) of cases. While only 7% displayed B‐dominance, 17% showed nondominance. D‐dominance was associated with downregulation of 4 interleukins (IL‐2ra, IL‐13, IL‐16 and IL‐18) and 5 chemokines/cytokines (CTACK (CCL27), MCP‐1 (CCL2), M‐CSF, TRAIL and ICAM‐1) while no analyte was increased. In addition, D‐dominance could be linked to a delayed HDV RNA response to pegylated interferon as patients with B‐dominance or nondominance showed higher early HDV RNA responses (61% at week 12) than D‐dominant patients (11%; P < .001). In conclusion, this study revealed unexpected effects of viral dominance on clinical and immunological features in chronic hepatitis delta patients. Individualizing PEG‐IFNa‐2α treatment duration should consider viral dominance. Overall, our findings suggest an activated but exhausted IFN system in D‐dominant patients.

438 BASELINE AND WEEK 24 HBeAg LEVELS ARE ASSOCIATED WITH TELBIVUDINE TREATMENT RESPONSE IN THE 2410 ROADMAP STUDY IN CHRONIC HEPATITIS B

Results: The slope of changes in LSMs over time in years is shown in Figure 1. LSMs changes over time (expressed in DkPa/month) showed a slight faster pattern of fibrosis regression at the beginning (first two years) and a slow steady pattern later during a follow-up period of four years. Improvement in fibrosis was seen in 33/55 patients (60%) (drop of −2.06±1.6 in kPa). Importantly, regression of cirrhosis (initial LSM >12kPa) to less than 6.1 kPa was observed in 3/55 (5.4%) patients. Worsening of LSMwas observed in 3 cases due to other liver disease cofactors. The baseline median LSM was 9.2±3.1 kPa. LSMs significantly decreased during the follow-up period after the start of NA treatment (8.4±0.9, 7.8±1.3, 7.3±1.9 and 6.8±1.6 kPa at years 1, 2, 3 and 4, respectively). Conclusions: LSMs changes annually showed an early faster and a later steady pattern of fibrosis regression over time as measured by transient elastography for chronic HBeAg (−) hepatitis treated with NA. Also LSMs showed a slight improvement of fibrosis in the first 4 years. Longer follow-up of these patients by TE is necessary to assess fibrosis regression in chronic hepatitis B patients under antiviral therapy.

433 QUANTIFICATION OF LARGE, MIDDLE AND SMALL HEPATITIS B VIRUS (HBV) SURFACE PROTEIN (HBsAg) FRACTIONS IN PATIENTS WITH ACUTE HEPATITIS B

Methods: 315 HBsAg positive mothers and their offspring received combined immunoprophylaxis (hepatitis B immune globulin and hepatitis B vaccine) were recruited in this study. The venous blood of 315 mothers and infants (315 at birth, 262 at 1-month-old and 251 at 7-month-old) was collected for tests of HBVDNA load and HBV markers titer. Result: The positive rates of HBVDNA, HBsAg and HBeAg in the newborns at birth were 17.8% (56/315), 25.1% (79/315) and 25.4% (80/315), respectively. Maternal HBVDNA load, HBsAg and HBeAg titer were associated with neonatal HBVDNA load, HBsAg and HBeAg titer at birth respectively. In the neonatus born to the pregnant mothers with HBVDNA>10 IU/ml, HBsAg>10 IU/ml and HBeAg>1 s/co, the probability of HBVDNA-, HBsAgand HBeAgpositive at birth significantly increased (areas under ORC curve were the largest, 0.770, 0.697 and 0.944, respectively). During the follow-up, the positive rates of HBVDNA, HBsAg and HBeAg in infants reduced gradually and HBVDNA (83.9%), HBsAg (87.3%) and HBeAg (87.5%) in the majority of infants turned to be negative at 7 months after birth, while 3.6% (9/251) infants became chronic HBV carriers (HBVDNAand HBsAg-positive from birth to 7-monthold) and 8.6% (18/242) were non-responders (anti-HBs <10mIU/ml at 7-month-old). High levels of HBsAg titer and HBVDNA load of newborns at birth were associated with chronic HBV infection, while the relevance between HBsAg, HBeAg and HBVDNA from mother to infant and the non-response to HB vaccination was uncertain. Conclusion: HBsAg and HBVDNA, as HBeAg, could cross the human placenta from mother to infant and lead to short-term antigenemia or virusemia in newborns, while HBsAgand HBVDNA-positive at birth may not be indication for HBV infection of infants. HBVDNA, HBsAg and HBeAg from mother to infant did not contribute to the non-response to HB vaccination, while further follow-up was still in need.

287 COMPREHENSIVE PHENOTYPING OF REGULATORY T CELLS FROM PATIENTS AFTER LIVER TRANSPLANTATION REVEALS DISTINCT T-REG PROPERTIES ASSOCIATED WITH GRAFT HEPATITIS AND REJECTION

infection and especially cccDNA elimination cytolytic mechanisms are crucial. It was the aim of this study to analyze HBV-specific CD8+ T cell effector functions in a human cell culture model. Methods: For this analysis HBV producing human hepatoma cell lines (HepG2.117) were utilized. These cells express significant amounts of HLA-A2 and allow antigen processing and presentation to HBV-specific CD8+ T cells. Co culture experiments were performed with T cell receptor transduced effector cells and HBV specific T cell lines. The effect on viral replication was measured by quantitative PCR and verified by southern blot. Results: Our results can be summarized as follows: i. The co culture of HBV producing hepatoma cells with HBVspecific CD8+ T cells led to upregulation of IFN-gamma and CD107a showing that HepG2.117 endogeneously process and present HBV-specific epitopes. ii. In co culture experiments, a significant more than 1 log suppression of HBVDNA was observed. iii. The decline in HBVDNA occured in concert with a increase in AST levels. iv. The antiviral effect was lost in transwell experiments, showing that direct cell-cell contact is required for the antiviral effect. v. The addition of recombinant IFN-gamma and/or TNF-alpha to HepG2.117 cells led to no significant reduction of HBVDNA. Conclusions: Taken together, these results show the dominant role of cytolysis in this model system of HBV infection. This new model will be useful to determine the cytolytic effector functions of HBV specific CD8+ T cells and other immune cells in HBV infection.