897. In Vitro Infection of Immortalized Human Hepatocytes by Hepatitis B Virus and Its Application to Anti-viral Treatment

Y. Tanaka; T. Yamaguchi; M. Segawa; M. Sugiyama; M. Hijikata; H. Aly; K. Shimotohno; M. Mizokami



We have developed a novel three-dimensional culture system of human hepatocytes in terms of ability to metabolite ammonia and CYP3A enzyme activity as well as hepatitis B virus (HBV) infection. Recently HPV18/E6E7-immortalized human hepatocytes (HuS-E/2 cells; primary hepatocytes isolated from a 9-year-old male patient with Primary Hyperoxaluria) which could be infected by hepatitis C virus were established.



To investigate the ability of HBV infection using the three-dimensional culture system of HuS-E/2 cells and to compare viral replication and sensitivity of anti-viral drugs (lamivudine, adefovir, entecavir, inferferon, etc) among HBV genotypes/subgenotypes. Methods: The three-dimensional culture system was made as follows: immortalized HuS-E/2 cells suspension (3x10^5cells) was injected into hollow fiber bundles after inoculation with HBV-infected serum samples or virions of HBV recovered from culture supernatants, that were produced by transfection with plasmids carrying 1.24-fold the HBV genome of different genotypes/subgenotypes (Ae, Ba, Bj, C) in Huh7 cells. The hollow fibers were placed in a 12-well plate and cultured in serum-free medium.



The hepatocytes aggregated in hollow fiber bundles formed a hepatocyte organoid and HBV DNA was detected in supernatants within 7 days. The HBV DNA increased in titer 10 days post-inoculation and continued to be detectable until 5–6 weeks. The extracellular HBV DNA levels were significantly higher in genotype C (>10^7copies/ml) than the others. Five weeks post-inoculation, intrahepatic total HBV DNA and covalently closed circular DNA were detected in hepatocytes by real-time detection PCR. Futhermore, HBV DNA levels of these genotypes were inhibited by several anti-viral drugs with different manners in this culture system. The combination of entecavir and interferon could inhibit HBV replication more efficiently.



This in vitro system using immortalized hepatocytes could provide a simple and cheap long-term culture of human hepatocytes infected with HBV. The infection model of HBV would allow us to examine differences of viral replication capacity, changes of host factors and anti-viral efficiency of new drugs on HBV genotypes/subgenotypes.


898. The Role of Serial Measurement of Serum HBV-DNA Levels in Patients with Chronic HBeAg(-) Hepatitis B Infection; Association with Liver Disease Progression: A Prospective Cohort Study

G. Zacharakis; J. Koskinas; S. Kotsiou; F. Tzara; N. Vafeiadis; M. Papoutselis; E. Maltezos; D. Kountouras; S. Eleftherios; A. Archimandritis; K. Papoutselis



The role of serum HBV-DNA levels in the natural history and management of chronic HBV infection is currently under investigation. We evaluated the fluctuating course of serum HBV-DNA levels during the natural history of chronic HBV infection in the general population of northeaster Greece, in association with liver disease progression.



We recruited and prospectively followed-up for a period up to 12 years (1992/94-2006) 257 adults (34±12.3yrs old) with chronic HBV infection. Viral markers/liver biochemistry/physical examination were performed every 6 months. Liver biopsy/abdominal ultrasound were performed at entry and every 2 to 4 years. Serum HBV-DNA levels measured by Amplicor HBV Monitor.



Fourteen of 257 (5.5%) patients were HBeAg(+) with mean HBV-DNA levels 2.5x10(7)copies/ml (6.2x10(6)-7.1x10(7)). At entry, 195/257 (76%) were HBeAg(-)/anti-HBe(+), with a history of normal ALT for at least 12 months: a) 97/195 (50%) individuals had undetectable serum HBV-DNA levels at entry although 31/97(32%) had detectable in at least one occasion, always below 10(4)copies/ml, had no liver disease at entry and at follow-up period by imaging evaluation, b) 94/195(48%) had HBV-DNA levels <10(4)copies/ml, normal liver histology (97%) at entry and at follow-up period and, c) 4/195(2%) had HBV-DNA >10(4)copies/ml, nearly normal liver histology of whom 2 exhibited abnormal ALT levels after 3 years. At entry, 48/257 (19%) patients were HBeAg(-)/anti-HBe(+), with a history of elevated ALT levels for >6m; a)12/48 (25%) had an erratic ALT pattern, mean HBV-DNA levels 3.5x10(5)copies/ml (2.1x10(3)-1.5x10(8)) of whom 5/12 (42%) had HBV-DNA levels <10(4)copies/ml for at least one occasion and, b) 36/48 (75%) had persistent elevated ALT(>1.5xUNL), mean HBV-DNA levels 1.2x10(6)copies/ml (6.7x10(3)-8.7x10(8)) of whom 8/36 (22%) had HBV-DNA <10(4)copies/ml for at least one occasion.


In HBeAg(-) patients with active disease, 22/48 (46%) had moderate/severe histology at entry and 5/48 (10%) developed liver disease progression during the follow-up period. The multivariate-adjusted OR (95% CI) of fibrosis was 604.865(67.233-999.999) for serum HBV-DNA levels >10(4) vs. <10(4)copies/ml; 0.002(><0.001-0.015) for serum ALT level at study entry >40IU vs <40IU; 0.057(0.006-0.519) for age >40 vs <40 years old.



Twenty-seven percent of patients with chronic HBeAg(-) hepatitis could be misclassified as inactive carriers if testing was performed on one occasion and not serially serum HBV DNA levels >10(4)copies/ml were significantly associated with liver disease activity and, close monitoring of serum HBV-DNA is a useful clinical tool in the management of chronic HBeAg(-) patients.


900. Large Scale Longitudinal Study of Chronic Hepatitis B Patients with Hepatitis B Surface Antigen Seroclearance

D. Wong; C. Lai; J. Fung; D. But; I. Hung; J. Yuen; F. Fung; J. Young; M. Yuen


Background and Aim

Seroclearance of hepatitis B surface antigen (HBsAg) is a rare event in chronic hepatitis B patients. We conducted a large-scale longitudinal study investigating the virological, histological and clinical aspects, including the risk of development of hepatocellular carcinoma (HCC) in patients with HBsAg seroclearance.


Patients and Methods

Two hundred and ninety-eight patients (211 male and 87 female; median age on presentation: 43.1 years) with HBsAg seroclearance were recruited and followed up every 3–6 months for clinical assessment. Intrahepatic HBV DNA and covalently closed circular DNA (cccDNA) were measured by real-time PCR. Serum HBV DNA was measured by the Artus HBV RG Test (QIAGEN, Germany). Liver stiffness was assessed by FibroScan (Echosens, France).



The median age of HBsAg seroclearance was 49.6 years. The median follow-up duration was 108.9 months and the median follow-up duration after HBsAg seroclearance was 36.4 months. Liver biopsies were performed on 29 patients (median time of biopsy: 48.6 months after HBsAg seroclearance). All have detectable intrahepatic HBV DNA (median: 1.68 copies/cell), and cccDNA were detectable in 23 patients (79.3%, median: 0.03 copies/cell). Of the 29 patients with liver biopsy, 9 and 16 patients had sera available within 1 year and between 5–10 years after HBsAg seroclearance, respectively, for HBV DNA analysis. All 9 patients had undetectable HBV DNA (<1.1 IU/mL) within 1 year of HBsAg seroclearance, and 4/16 patients had detectable HBV DNA levels between 5–10 years of HBsAg seroclearance (median: 2.37 IU/mL).


Of the 26 patients with adequate liver tissues for histological examination, 4 had mild fibrosis (F1) and 5 had minimal necroinflammation. FibroScan was performed on 76 and 78 patients who had HBsAg seroclearance at age <50 and ≥50 years respectively. Significant fibrosis (liver stiffness > 8.1 kPa) was observed only in 7.9% (6/76) patients with HBsAg seroclearance at age <50 compared to 29.5% (23/78) patients with HBsAg seroclearance at age ≥50 (p = 0.001). Seven patients developed HCC (median age: 69.3). Kaplan-Meier analysis showed that the chance of HCC development in patients with HBsAg seroclearance at age <50 was significantly less than those with HBsAg seroclearance at age ≥50 (p = 0.004).



Although serum HBV DNA was detectable in only a small proportion of patients with HBsAg seroclearance, intrahepatic HBV DNA was still present in all patients. Nevertheless, patients who cleared HBsAg at age <50 had significantly less fibrosis and lower chance of HCC development than those with HBsAg seroclearance at ≥50 years.


902. Reduced HBV Rreplication in HBeAg-negative Patients Is Due to Lower Covalently Closed Circular DNA Content and Impaired Virion Productivity

J. Petersen; T. Volz; M. Lutgehetmann; A. Quaas; J. W. Murray; M. Dandri


Background and Aims

Knowledge of factors regulating transcriptional activity of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) may help understanding mechanisms of viral decay and how these processes are thwarted in chronically HBV-infected patients. Methods: Liver biopsies from 119 treatment naïve chronically infected patients (42 HBeAg-positive and 77 HBeAg-negative) were determined for transcriptional and replicative activity of HBV.



Significantly lower median serum HBV-DNA (-4log), intrahepatic HBV-DNA (-2log) and cccDNA (-1log) amounts were measured in HBeAg-negative vs. HBeAg-positive patients. Despite a good correlation found between intrahepatic amounts of progeny virions and serum HBV-DNA titres in all patients, cccDNA levels did not correlate with serum titres in HBeAg-negative individuals. Analysis of HBV-RNA transcripts demonstrated that impaired virion productivity in HBeAg-negative individuals was due to lower steady-state levels of pregenomic RNA produced per cccDNA molecule. Interestingly, preS/S RNAs levels and serum HBsAg concentrations did not differ between HBeAg-positive and HBeAg-negative patients when normalized for cccDNA contents, demonstrating that subviral particle production was not impaired in HBeAg-negative patients and correlated with cccDNA levels.


Though the majority of HBeAg-negative individuals harbored cccDNA with common precore and/or basal core promoter (PC/BCP) mutations, occurrence of these variants was not responsible for reduced viral replication. Instead, replacement of wild type cccDNA with populations of BCP mutations re-established high virion productivity.



Lower viremia in HBeAg-negative individuals is not only due to lower cccDNA content but also to impaired virion productivity, which can arise without emergence of HBeAg variants and without affecting HBsAg production.


903. High Viral Load and Hepatitis B Virus (HBV) Subgenotype Ce Have Increased Risk of Hepatocellular Carcinoma (HCC) – A 7-year Prospective Follow-up Study

H. Chan; C. Tse; F. Mo; J. Koh; V. W. Wong; G. Wong; J. J. Sung; T. Mok



The determination of risk factors for HCC is important to design cancer surveillance strategy. Genotype C HBV is associated with a higher risk of HCC than genotype B HBV, but the effect of HBV subgenotypes on HCC is uncertain. We aimed to investigate the impact HBV DNA levels and HBV genotypes/subgenotypes on the risk of HCC.



A prospective cohort of chronic HBV infected patients recruited in a surveillance program for hepatocellular carcinoma (HCC) since 1997 was studied. Ultrasound and alfa-fetoprotein were regularly performed for HCC surveillance. HBV DNA at recruitment was measured by Taqman real-time polymerase chain reaction assay. HBV subgenotyping was determined by direct sequencing of the S gene.



Among 1006 patients (68% male) aged 48±7 years, 86 (9%) patients developed HCC at a median follow-up of 7.7 years. 377 (38%) patients had liver cirrhosis. 146 (15%) patients had low serum albumin. 851 (85%) patients had ALT levels below 1.5 x upper limit of normal and the mean log HBV DNA was 4.72±1.84 copies/ml. 152 (15%) patients had received anti-viral treatment during the follow-up period. The hazard ratio of each log step increase in HBV DNA was 1.38 (95% CI 1.23, 1.55; p<0.0001). With referent to the low HBV DNA stratum (log HBV DNA ≤ 4.5 copies/ml), the hazard ratio for HCC of the intermediate HBV DNA stratum (log HBV DNA > 4.5 – 6.5 copies/ml) was 1.62 (95% CI 1.05, 2.48; p=0.027) and that of the high HBV DNA stratum (log HBV DNA > 6.5 copies/ml) was 2.73 (95% CI 1.76, 4.25; p<0.001). Among 769 patients with genotyping results, 330 patients had genotype B HBV (all subgenotype Ba) and 439 patients had genotype C HBV (94 subgenotype Ce and 345 subgenotype Cs). With referent to genotype B HBV, HBV subgenotype Ce has the highest risk of HCC (hazard ratio 2.75; 95% CI 1.66, 4.56; p<0.001) and HBV subgenotype Cs has intermediate risk (hazard ratio 1.70; 95% CI 1.09, 2.64; p=0.02). On multivariate analysis, HBV DNA (hazard ratio 1.79; 95% CI 1.27, 2.52; p<0.001) and HBV subgenotypes (hazard ratio 1.53; 95% CI 1.17, 2.01; p=0.002) were independent risk factors of HCC. Liver cirrhosis, male gender, older age and low serum albumin were also risk factors of HCC whereas serum bilirubin, ALT level, ascites and use of anti-viral treatment were not. Using genotype B HBV and low HBV DNA stratum as referent, the hazard ratio of HCC for HBV subgenotype Cs was 2.85 (95% CI 1.68, 4.84; p<0.001) and that for HBV subgenotype Ce was 4.00 (95% CI 2.08, 7.69; p<0.001).



High HBV DNA level and HBV genotype C, particularly subgenotype Ce, are independent risk factors of HCC in chronic hepatitis B.

1480. Liver Disease Progression Among HIV-infected Patients with Viral Hepatitis

E. Seremba; R. K. Joshi; N. Attar; W. M. Lee; M. K. Jain



Co-infection with viral hepatitis B (HBV), C (HCV) or both is common in HIV-infected patients due to shared routes of transmission. Little is known about the risk of liver disease progression among those coinfected with HIV/HCV, HIV/HBV or triply infected with HIV/HBV/HCV.



Medical records of 151 HIV-infected patients including 67 HIV/HBV, 43 HIV/HCV, and 41 triply infected patients were reviewed for demographics, CD4 cell count, HIV viral load, liver function tests, hepatitis B, C, and hepatitis delta (HDV) serologies, and radiologic images. Charts were reviewed in 124 for evidence of progression to end stage liver disease (ESLD) and alcohol consumption. HBV DNA was quantified using VERSANT® HBV 3.0 (bDNA) and HCV RNA with VERSANT® HCV RNA 3.0 (Siemens Diagnostics, Tarrytown NY) from stored sera. Eighty samples were obtained prior to start on antiretroviral therapy (ART) and were thus considered baseline. ANOVA was used to compare continuous variables across the three groups and chi square for dichotomous variables.



ESLD and cirrhosis was seen more frequently in those with HIV/HCV/HBV compared to those with HIV/HCV or HIV/HBV (p=0.02 and 0.03, respectively). Log HCV load was similar in those with HIV/HCV (5.08) vs. triple (5.37). Log HBV DNA was lower in those with triple (2.58) compared to those with HIV/HBV (7.25), p=<0.001, however many samples in the triple group were obtained while patients were on ART, often using nucleosides with anti-HBV activity. Baseline samples [HIV/HBV (n=55); HIV/HCV(n=12); triple (n=13)] were examined and showed no difference in HCV viral load between HIV/HCV and triple infection, but log HBV viral load was slightly lower in those with triple infection compared to those with HIV/HBV ( 5.06 vs. 7.25, p=0.07). A history of alcohol abuse was more frequent in those with HIV/HCV than other groups (p=0.06).



HIV-infected patients with both HBV and HCV are more likely to progress to ESLD compared to those with HIV/HBV or HIV/HCV. Cirrhosis is also more likely to be seen in this population. Most HIV-infected patients will receive anti-HBV as part of ART. Triply infected patients should be considered additionally for HCV treatment, given the propensity for disease progression.