Nov. 2 Abstracts       

 

Disease Progression – Role of HBeAg Status

 

861. Hepatitis B Viral Factors Influencing the Histological Changes Of HBeAg-negative Chronic HBV Infected Patients With Persistently Normal Serum Alanine Aminotransferase Levels

Y. Yang; Q. Xie; H. Zhou; H. Wang; H. Gui; W. Cai; L. Lin; G. Zhao; C. Shi; H. Yu; Q. Guo

 

Background and Aims:

Several viral factors have been identified to be associated with a higher risk of HCC and cirrhosis development, while studies in HBeAg-negative chronic HBV infected patients with persistently normal serum ALT levels (PNAL) are limited. We thus investigated the correlation between viral factors and liver histological changes of such patients.

 

Methods:

HBV DNA, HBV genotypes, BCP and precore mutations were determined in 52 HBeAg-negative chronic HBV infected patients with PNAL (defined as normal ALT measured on at least 3 occasions in the intervals of about two months over a period of 12 or more months apart prior to biopsy) who underwent percutaneous liver biopsy(Ishak scoring system).

 

Results:

Of 52 subjects, 67.3% and 59.6% had BCP and precore mutations respectively. Those with both mutations had significantly higher HBV DNA levels than those without (4.9 vs. 4.1 log10copies/mL,P=0.025). Results of both genotypes and mutations were successfully obtained in 40 samples with HBV DNA≥10^4copies/mL. Subjects with genotype B had a higher chance of harboring precore mutations compared to those with genotype C, but there was no significantly statistical difference (75.0% vs. 50.0%,P=0.102). The higher viral load was observed in the patients with genotype B than genotype C (5.1 vs. 4.3log10copies/mL,P=0.046). Moreover, in patients infected with genotype C virus, HBV DNA levels were significantly higher in those with precore mutations compared to those without (4.9 vs. 3.7log10 copies/mL, P=0.004). A higher proportion of HAI≥4 was found in patients with precore mutations (29.1% vs. 19.1%) and those with both mutations (32.1% vs. 16.7%) as well, but there was no statistical difference (P=0.415, 0.119); in terms of the histological finding of F≥3, no difference was found between patients with and without both mutations (4/28 vs. 3/24, P=1.000). In those with precore or BCP wild type infection, there was a strong positive correlation between viral load and liver inflammation and fibrosis (precore:r=0.626,P=0.002;r=0.592,P=0.005;BCP:r=0.730,P=0.001;r=0.641,P=0.007).

 

Conclusions:

Patients of HBeAg negative chronic HBV infection with PNAL have a higher prevalence of BCP or precore mutations. Virus harboring both BCP and precore mutant sequences has the highest replication level and precore mutants may play a more important role in that, especially in genotype C virus. There was a strong positive correlation between viral load and liver histological changes including inflammation and fibrosis in patients with precore or BCP wild type infection. Quite of few subjects with both mutations have moderate/severe inflammation and need antiviral therapy.

 

862. Hepatitis B Virus Basal Core Promoter Mutation and DNA Load Correlate with Expression of Hepatitis B Core Antigen

C. Liu; P. Chen; Y. Jeng; J. Kao; D. Chen

 

Background & Aims:

Expression of intrahepatic hepatitis B core antigen (HBcAg) and mutations of hepatitis B virus (HBV) genome are closely related to the immunopathogenesis and disease activity of chronic HBV infection. The role of HBV genotype and basal core promoter (BCP) mutation in subcellular expression of HBcAg was investigated. Methods: A cohort of 70 HBeAg-positive patients with chronic active hepatitis was consecutively enrolled in a medical center. Their clinical, virologic and histologic features were compared with regard to subcellular localization and expression of intrahepatic HBcAg. The effects of HBV genotype and BCP T1762/A1764 mutation on the expression of HBcAg were further evaluated by in vitro mutagenesis and transfection assays.

 

Results:

Fifty-two (74.3%) patients were infected with HBV genotype B and 18 (25.7%) with genotype C. Virus nucleotide sequences displayed BCP T1762/A1764 mutation in 16 (22.9%). Predominant cytoplasmic, mixed cytoplasmic/nuclear, and nuclear localization of intrahepatic HBcAg were found in 38 (56.7%), 25 (37.3%) and 4 (6.0%), respectively. Fifty-eight (80.6%) of these patients expressed a high level of HBcAg. In multivariate analysis, cytoplasmic localization of HBcAg correlated with low serum viral load (P=0.045) and BCP mutation (P=0.04), but not genotype. High expression level of HBcAg also correlated with high serum viral load (P=0.015) and BCP wild-type sequence (P=0.037). In vitro assays supported that HBV BCP mutant had lower subcellular expression of HBcAg compared with BCP wild-type strain.

 

Conclusions:

HBV BCP mutation and viral load contribute to the expression and localization of intrahepatic HBcAg in HBeAg-positive patients with chronic active hepatitis.

 

838. Histological Findings Of HBeAg-negative Chronic HBV -Infected Patients With Persistently Normal Serum Alanine Aminotransferase Levels

Q. Xie; Y. Yang; H. Zhou; H. Wang; H. Gui; L. Lin; W. Cai; W. Zhu; X. Xiang; B. Gong; Q. Guo; H. Yu

 

Background and Aims:

As the increasing prevalence, poor clinical prognosis and lower response of anti-viral therapy, HBeAg-negative chronic hepatitis has attracted the attention of experts in liver disease. However, research on HBeAg-negative carriers with persistently normal ALT levels (PNAL) are limited. Our study aims to find out the histological features of them and factors influencing their disease progression.

 

Methods:

154 HBeAg-negative subjects underwent percutaneous liver biopsy were enrolled, 56 with abnormal ALT and 98 with PNAL (defined as normal ALT for at least 1 year in periodic biochemical examinations before biopsy). Liver pathology was evaluated by Ishak scoring system. A comparison of histological features between the two groups was performed and the correlations between histological changes and host as well as viral factors in PNAL group were analyzed.

 

Results:

Compared with abnormal ALT group, PNAL group had lower histological activity index(HAI) and fibrosis(F) score (5.5 vs. 0,P=0.000; 3 vs. 0,P=0.000). However, 22.4% and 17.3% PNAL subjects still had HAI≥4 and F≥3 respectively. Subgroup analysis showed that subjects with ALT>0.50×ULN had a remarkably higher rate of histological findings above-mentioned than those with ALT<0.50×ULN (36.4% vs. 11.1%,P=0.003; 27.3% vs. 9.3%,P=0.019), and older subjects (more than 45yrs) had a higher proportion of F≥3 than the younger (33.3% vs. 13.4%,P=0.027). Multivariate logistic regression analysis revealed that fibrosis and an increase in age by a decade were independent predictors of HAI≥4 (OR=2.584, P=0.000; OR=2.410, P=0.023), while histological grade was the only independent predictor of F≥3 (OR=2.179, P=0.000). HBV DNA was detectable (≥10^3copies/mL) in 91.3% PNAL subjects, and 27.2% of whom had HBV DNA≥105copies/mL.

 

Significant histological findings may be seen in various HBV DNA levels. We also found that even in the patients with HBV DNA<10^4copies/mL the percentage of HAI≥4 and F≥3 were 14.9% and 12.8% respectively. 63.1% of HBeAg-negative patients were infected with genotype C virus, the percentage was higher in abnormal ALT group compared with PNAL group (87% vs. 50%,P=0.003), while there was no difference between all PNAL subgroups.

 

Conclusions:

HBeAg-negative chronic HBV infected patients with PNAL are not a homogenous group. Those with age>45yrs, ALT>0.50×ULN share some characteristics that have been associated with significant histological findings. Inactive carriers can’t be distinguished from HBeAg-negative patients with severe liver damage by viral load.Liver biopsy is still a golden standard for recognizing the candidates of antiviral treatment within this population.

 

815. Age-specific Prognosis Following HBeAg Seroconversion in Chronic Hepatitis B

Y. Chen; C. Chu; C. Yeh; Y. Liaw

 

Background and Objectives:

HBeAg seroconversion confers favorable prognosis but untoward outcomes may develop thereafter. Since age is an important factor in the events of chronic HBV infection, we conducted a cohort study to elucidate the prognosis following spontaneous HBeAg seroconversion in different age groups in chronic hepatitis B infection.

 

Patients and Methods:

Five hundred and 51 patients with biopsy-proved chronic hepatitis B virus (HBV) monoinfection had been followed up in our liver unit for at least 1 year since 1977. Eight-three patients with persistent HBeAg > 3 years and 441 patients with HBeAg seroconversion and then followed > 1 year were included in our analysis. Follow-up studies included liver biochemistry, alfa-fetoprotein and ultrasonography every 3-6 months or more frequently if clinically indicated. Occurrence of hepatitis relapse, development of cirrhosis and hepatocellular carcinoma (HCC) and HBsAg seroclearance were compared between different age groups by chi-square test, student t test and Kaplan-Meier survival analysis.

 

Results:

Of 441patients with spontaneous HBeAg seroconversion, 207 were seroconverted before age 30 (group A), 181 between age 31 and 40 (group B) and 53 after age 40 (group C). The calculated annual incidence of hepatitis relapse, cirrhosis, HCC and HBsAg seroclearance in these 441 patients were 1.9%, 0.7%, 0.2% and 0.6% respectively. The cumulative incidence of hepatitis relapse, development of cirrhosis and HCC increased with increasing age of HBeAg seroconversion, being significantly higher in group C than group A and B (p=0.005, p<0.0001 and p=0.024, respectively). The cumulative incidence of HBsAg seroclearance tended to be higher in patients seroconverted at younger age but the differences were nonsignificant statistically (p=0.530).

 

Conclusion:

Untoward outcomes can develop after spontaneous HBeAg seroconversion, especially in patients HBeAg seroconverted after age 40.

 

829. Evolution of HBV Quasipecies Generates New CpG Dinucleotides Prior to HBeAg Seroconversion

Y. Cheng; M. Aung; S. Lim

 

We recently showed that HBV quasispecies diversity was significantly higher in HBeAg seroconverters compared to non-seroconverters (Lim et al, Gastroenterology 2007; 133:951-8). We postulate that this increase in viral diversity may result in formation of new CpG motifs that may stimulate toll receptor 9 receptors. In this study, we analyzed sequences of HBV quasispecies in patients before seroconversion compared to non-seroconverters to determine if significant new CpG motifs had developed.

 

Methods:

Eight chronic hepatitis B HBeAg seroconverters, with long term stored serum and clinical follow-up of at least 6-15 years were studied. Two chronic hepatitis B patients without HBeAg seroconversion with matching length of follow-up were included as controls. Serum samples from 6-10 time points per patients were involved in this study. PCR, cloning were carried out followed by sequencing at least 20 clones per sample. CpG motifs from all sequences were then identified. Results: 19 new CpG dinucleotides developed in the ten patients and occurred predominantly in the HBV precore/core gene. Eleven out of 19 CpG sites were at conserved sites, while 8 of 19 CpG dinucleotides transformed to non-CpG nucleotides. Fourteen HBV CpG sites in the precore/core gene occurred at different time points in HBeAg seroconverters only, and were absent in the non-seroconverters. In addition, 7 out of 8 HBeAg seroconverter patients showed development of new HBV CpG dinucleotides before seroconversion, but control patients showed no such new CpG sites and no change in the previously identified common CpG sites. New CpG sites were also found before seroconversion in HBeAg seroconverters beyond the precore/core gene. Moreover, seven of the new HBV CpG dinucleotides observed in HBeAg seroconverters were similar to previously reported immuno-stimulatory CpGs.

 

Conclusion:

New CpG dinucleotides occurred along during course of HBeAg seroconversion and may play an important role in HBeAg seroconversion. Functional studies of these new CpG dinucleotides are needed.

 


Disease Progression:  Viral Replication and Integration

 

858. High HBV DNA Load, HBeAg Positivity and Considerable Hepatic Necro Inflammation in Patients with Incidentally Detected, Asymptomatic Chronic HBV-infected Individuals from Bangladesh

A. Mamun; R. Salimur; S. Akbar; K. Mobin; K. Md. Fazal; K. Md.

 

Introduction:

Inactive hepatitis B virus (HBV) carriers constitute one of the major reservoirs of the HBV, especially in the developing countries of the world. But, present management guidelines provide inadequate treatment modalities for these patients. Generally, these patients are recommended for regular check up, and treatment is only recommended when patients exhibit evidences of liver damages. The condition can mainly be attributable to lack of information about extent of liver damages of inactive HBV carriers in developing countries. The purpose of this study was to assess the extent of liver damages in patients who were completely unaware of their HBV infection at Bangladesh, an Asian country that harbor about 6-10 million chronic HBV-infected persons.

 

Methods:

Three hundred ten HBV-infected subjects were enrolled in this study. HBV infection was detected in these subjects incidentally either during (1) health check up, or (2) pre-vaccination screening, or (3) assessment before blood donation or (4) screening due to presence of HBsAg-positive patients among family members. They did not know that they were infected with the HBV, and none of them had any symptoms related to liver damages. HBV infection was detected from hepatitis B surface antigen (HBsAg) positivity in these subjects. Subsequently, all patients were tested for serum alanine aminotransferase (ALT), hepatitis B e antigen (HBeAg), and HBV DNA. All of them underwent ultrasonographic examination of hepato-biliary system and spleen. Per-cutaneous liver biopsies were done to assess the extent of liver damages in all subjects. Results: Almost half of the patients were positive for HBeAg in the sera (48.7%). The mean level of ALT was 82 U/L (range 12-948 U/L) in HBeAg +ve patients and 64 U/L (range 12- 388 U/L) in HBeAg -ve patients. High levels of HBV DNA (more than 1 million copies/ml) were detected in 96% and 54.1% HBeAg +ve and HBeAg –ve patients, respectively. Significant necro-inflammation of the liver was seen in 90.7% and 45.3% of HBeAg +ve and HBeAg -ve patients, respectively. Liver histology revealed that moderate degrees of hepatic fibrosis in about one-fourth of these patients.

 

Conclusion:

This study indicates that machinery should be developed to characterize undetected HBV carriers in developing countries by conducting multicenter clinical studies. We have shown that considerable numbers of patients those are unaware of their HBV infection suffer from progressive liver damages. The overall strategy of management of chronic HBV infection in developing countries should be revisited.

 

859. Lack of Correlation Between Viral Load and Extent of Liver Damage in Patients with Chronic HBV Infection in Bangladesh

A. Mamun; R. Salimur; S. Akbar; K. Mobin; K. Md. Fazal; K. Md.

 

Introduction:

In general, it is assumed that patients with chronic hepatitis B virus (HBV) infection with high viral load exhibit increased liver damages. Accordingly, the treatment guidelines emphasize on reducing viral load in chronic HBV carriers. The ethical and scientific basis of these observations was mainly accumulated from investigations from developed countries of the world. However, more than 80% chronic HBV carriers live in the developing nations of the world, but little is known about relationship between HBV viral load and extent of liver damages in these countries. In this study, we addressed this issue to provide insights about this so that better management guidelines can be developed for chronic HBV carriers of developing nations.

 

Methods:

A total of 402 Bangladeshi patients with chronic hepatitis B (CHB) were enrolled in this study, and were tested for serologic markers of HBV and serum alaninetransaminase (ALT) level. All patients also underwent per-cutaneous liver biopsy. HBV genotyping was done in 45 patients.

 

Results:

High HBV DNA load (more than 100,000 copies/ml) was detected in 64.4% patients. 43.5% patients were HBeAg +ve, whereas, the rest 56.5% patients were HBeAg -ve. HAI-NI was observed in 62.9% HBeAg +ve and 49.8% HBeAg -ve patients, respectively. In those with high HBV DNA, 59.8% had significantly higher HAI-NI, as opposed to 45.5% with low HBV DNA. On the contrary, much higher percentage of patients with low HBV DNA had considerable levels of hepatic fibrosis compared to patients with high HBV DNA levels. However, there was no significant difference about hepatic fibrosis (HAI-F) score between these two groups. HBV genotyping was done in 45 patients. Genotype C (38%) and D (49%) were two prominent genotypes. However, a comparison between HBV genotype with HBV load and extent of liver damages could not be done because genotypin was done in only 45 patients.

 

Conclusion:

This study shows that a correlation could not be established between viral load and liver damage in patients with CHB at Bangladesh. Further study may be needed to find out the influence of other factors on liver damages in CHB patients in developing nations like Bangladesh where about 6-10 million chronic HBV carriers are living. Most of these patients have not been characterized and treatment modalities could not be defined for these patients.

 

860. Modulation of ERK, AKT, apoptosis and cell cycle in primary mouse hepatocytes and Huh7 cells by wild-type and rtM204I HBV.

R. Chin; U. Nachbur; C. Bock; J. Silke; J. Torresi

 

Introduction:

Lamivudine resistant hepatitis B viruses (HBV) are not infrequent in patients on antiviral therapy and who develop hepatocellular carcinoma (HCC). We have investigated the alterations in MAPK signalling, apoptosis and cell cycle induced by infection with wild-type (wt) and rtM204I HBV.

 

Methods:

Recombinant adenoviruses expressing a full-length infectious genome of wt and rtM204I HBV together with an internal GFP reporter or a control adeno-GFP virus were used to infect primary mouse hepatocytes (PMH) and Huh7 cells. Intracellular HBV cccDNA was analysed by real time PCR. Western immunoblot analyses were performed for MAPK intermediates, cIAP, procaspase 3, cleaved caspase 3 and cell cycle regulatory proteins. Cell viability was analyzed after incubation of cells with TNF-alpha, FasL and TRAIL. Cell cycle was examined by flow cytometry.

 

Results:

Infection of PMH and Huh7 cells with rAd-HBV-wt and rAd-HBV-M204I resulted in up-regulation of ERK phosphorylation when compared to mock infected or control adeno-GFP-infected cells. In contrast to mock-infected cells infection of cells with rAd-HBV-M204I resulted in a 150% increase in pERK, a 160% increase pAkt, and a 30% increase in p-cMyc. In both rAd-HBV-wt and rAd-HBV-M204I infected cells ERK phosphorylation was also strongly increased following PDGF stimulation. The level of the inactivated p-Ser9-GSK3-beta was increased by 140%. The p53 and p21cip1 proteins were both increased by 40% and 35% respectively. Relative to mock-infected cells cyclin A was increased by 95% in rAd-HBV-wt and 140% in rAd-HBV-M204I infected cells. Cyclin B1 was increased by 225% in rAd-HBV-wt and 210% in rAd-HBV-M204I infected cells. Both rAd-HBV-wt and rAd-HBV-M204I infected cells demonstrated increased levels of p-cdc2 (158% wild type and 128% rtM204I). Cell cycle analysis showed a greater proportion of the rAd-M204I HBV infected cells (16% ±SD 0.6) were in G2 phase compared to mock infected cells (7% ±SD 3.3). Cell viability of HBV infected hepatocytes was significantly reduced after treatment with TNF-alpha, FasL and TRAIL. cIAP was increased in the first 24 hours post-infection but this was followed by a reduction in cIAP and a rise in cleaved caspase 3, consistent with increased apoptosis in HBV infected hepatocytes.

 

Conclusion:

In both PMH and Huh7 cells wild-type and rtM204I HBV strongly upregulate the pERK, pAkt and cMyc. These changes were accompanied by increased levels of p53, p21, cyclin B1 and pcdc2 and in Huh7 cells and resulted in G2/M cell cycle arrest. Apoptosis was increased in both HBV infected PMH and Huh7 cells. The combination of these effects may contribute to HCC development.

 

854. Plasmacytoid Dendritic Cell Function Is Impaired in Chronic Hepatitis B Infection

D. Ratnam; K. Visvanathan; P. U. Cameron; W. Sievert

 

Background:

Dendritic cells (DC) are potent antigen presenting cells involved in the induction of adaptive immune responses and may also play a role in immunological tolerance. The two major subtypes found in human peripheral blood are myeloid (mDC) and plasmacytoid (pDC) and much of their function is mediated through pattern recognition receptors known as Toll like receptors (TLR). pDCs play an important role in host defences against viruses, with the ability to secrete large amounts of type 1 interferons. Previous studies of DC frequency and function in chronic HBV have yielded conflicting results. The aim of this study was do determine whether a TLR mediated impairment of DC function contributes to the suboptimal immune responses seen in chronic HBV(CHB) infection.

 

Methods:

Peripheral blood was obtained from patients in immunoactive (both HBeAg+ and HBeAg-), immunotolerant (IT) and inactive stages of chronic HBV infection and from healthy volunteers. All patients in the immunoactive and IT phase had viral loads above four log IU/ml compared to undetectable viral loads in patients in the inactive stage. PBMCs were isolated using Ficoll Density Centrifugation and then cultured with purified lipopolysaccharide (pLPS), or Influenza virus (H1 strain) in order to stimulate TLR4 on mDCs, and TLR9 on pDCs respectively. Cells were then stained with combinations of flurochrome conjugated antibodies to identify the individual DC subsets as well as intracellular TNF-α and IFN-α. Flow cytometry was then used to determine the frequency of the DC subsets and their levels of intracellular cytokine production (expressed as the percentage of the DC subset expressing either cytokine).

 

Results:

pDCs from individuals in the immunoactive and immunotolerant phases of infection demonstrated an impaired capacity to secrete IFN-α in response to Influenza virus compared to healthy controls (p<0.02). There was no difference in pDC IFN-α production from patients in the inactive phase compared to controls. mDCs from chronic HBV individuals did not demonstrate a difference in capacity to secrete TNF-α in response to pLPS stimulation compared to controls. No difference was detected in the frequency of circulating mDCs or pDCs in CHB individuals compared to uninfected controls.

 

Conclusion:

An impaired capacity of pDCs to produce IFN-α in CHB patients with active viral replication may contribute to the inability of such individuals to achieve viral clearance. Future studies are required to determine whether TLR mediated functional deficits contribute to the impaired CD4 and CD8 T-Cell responses associated with CHB.

 

855. Assessment of Delta and Hepatitis B Viremia in Patients with Chronic Hepatitis Delta. Influence of HIV Infection and Antiviral Therapy.

A. Madejón; G. Gaeta; M. Bottecchia; J. García-Samaniego; M. Romero; V. Soriano

 

Background:

Complex interactions between hepatitis B (HBV) and delta (HDV) viruses, occur in chronic HDV carriers.

 

Methods:

A total of 226 sera from 42 patients with chronic HDV infection were examined (mean follow-up: 26.1+16.4 months). Twenty-four (57%) were anti-HIV-neg, 36(85%) anti-HBe+ and 39 (93%) anti-HCV-neg. Six HIV-neg patients (25%) were treated with interferon (IFN), and 18 HIV+ patients received HAART with anti-HBV drugs (tenofovir and/or lamivudine/emtricitabine). Serum HDV-RNA was quantified with a real time PCR (detection limit, 10 copies/ml). Serum HBV-DNA was quantified whith a commercial assay and genotyped with LiPA (Innogenetics).

 

Results:

Baseline mean serum HDV-RNA tended to be higher in HIV-pos than in HIV-neg patients (4x107 vs 6.8x106 copies/ml; p=0.073). Different HDV replication patterns were observed in HIV-neg untreated patients; serum HDV-RNA was detected during the entire follow-up in 11/18 (61%) patients, intermittently in 3 (17%), and was always negative in the remaining 3 (17%) patients. In contrast, almost all HIV patients were delta viremic. Serum HBV-DNA was intermittently positive in the majority of these patients, with low viral titers. A significant reduction in serum HDV-RNA was observed in 5/6 (83%) HIV-neg treated patients, and in 10 (62%) of HIV+ patients treated with tenofovir+/-3TC/FTC. Serum HBV-DNA declined in all HIV-neg treated patients. However, in all viremic HIV-neg cases, both HBV and HDV reactivation occurred upon IFN discontinuation. Serum HBV-DNA was positive in 11 (46%) HIV-neg patients,with low titers (range: 1x102 - 105 IU/ml). HBV genotype could be determined in 10 of these patients: 8D, 1A and 1E. In HIV-pos patients, serum HBV-DNA was positive in 10/18 (56%) cases (range: 2x103 - 107 IU/ml) and HBV genotype distribution was 6D and 4A.Serum HDV-RNA levels tended to be higher in HIV-neg patients with undetectable serum HBV-DNA than in HBV viremic patients (1x107 vs 9.1x105; p=0.132). No correlation was found between serum HDV-RNA titers and ALT levels.

 

Conclusions:

Most patients with chronic HDV infection show an inhibition of HBV replication. Serum HDV-RNA titers are generally greater than HBV-DNA titers regardless HIV status. Serum HDV-RNA is persistently or intermittently recognized in most patients, even among HIV-neg patients. Patients with HIV infection show higher viral load levels for both HBV and HDV than HIV-neg counterparts. In HIV patients, long-lasting treatment with active anti-HBV antiretroviral regimens might be beneficial, indirectly inhibiting HDV replication. IFN therapy may suppress HDV replication but HDV and HBV relapse is upon treatment discontinuation.

 

856. Viral Hepatitis B Virus (HBV) Promotes Extracellular Matrix Disruption Promoted by Viral Transactovatpr X (HBx) and Mediated by Gelatinase A.

Y. Rodriguez-Muñoz; S. Martin-Vilchez; P. Sanz-Cameno; M. Lopez-Cabrera; R. Moreno-Otero; E. Lara-Pezzi

VHB chronic infection in liver fibrosis may lead to cirrhosis and higher risk of hepatocellular carcinoma, due to the host immune response and to the expression of the viral transactivator HBx, which exhibits pleiotropic biological effects, being able to modify different cell functions. HSC activation is a key step in liver fibrogenesis, due to the synthesis of profibrotic factors and their ability to regulate extracellular matrix (ECM) homeostasis. Gelatinase A (MMP-2),increased in liver fibrosis, is a key molecule in matrix remodelling. HBx is a HBV protein that has been widely studied in hepatocellular carcinoma but its role in liver fibrosis has not been investigated yet.

 

Aim:

Our aim is to analyze HBx contribution to liver fibrosis through its effects over ECM homeostasis, MMP-2 expression and HSCs activation.

 

Methods:

Primary human HSCs were incubated with conditioned media from hepatic cell lines: CMX which expresses HBx in a dexamethasone (DX)-inducible manner; 2.2.15, which bears two copies of the HBV genome. Parental cell lines CHL and HepG2 were used as controls. HSCs were incubated during 24 hours with different conditioned media. Activation marker α-SMA, and MMP2 activity, was analyzed by immunofluorescence and zymography, respectively. HSC proliferation was spectrophotometric analyzed using a cell growth determination kit.

 

Results:

HSCs activation was demonstrated by the “de novo” expression of α-SMA as showed in immunofluorescence. Zymography from HSCs exposed to conditioned media from HBx expressing cells displayed an increased expression of MMP-2 as compared to HSCs incubated with control media. In the same cases HSCs showed an enhanced proliferation rate too in comparison with those exposed to control media. This increased proliferation was aborted when we blockade MMP-2 activity with TIMP2.

 

Conclusion:

HBx contribute to the fibrotic process inducing HSCs activation and proliferation. Moreover, HBx protein also mediates in ECM integrity, through its capacity to induce MMP-2 in HSCs. Interestingly, MMP-2 neutralization with TIMP-2 prevented HSCs proliferation, suggesting the role of this metalloproteinase in the enhacement of HSCs proliferation by HBx. Taken together our results demonstrate that HBx could be a promoting agent of hepatic fibrosis through its ability to modulate key features, like HSCs activation and ECM integrity modulated at least by MMP-2 expression.

 

857. Modulation of Human Hepatic Stellate Cells (HSCS) Behavior by Hepatitis B Virus (HBV) X Protein (HBX) Through Transforming Growth Factor Beta Signalling Pathway

S. Martin-Vilchez; Y. Rodriguez-Muñoz; P. Sanz-Cameno; M. Lopez-Cabrera; R. Moreno-Otero; E. Lara-Pezzi

 

Introduction:

Hepatic fibrosis is a scarring process that leads to extracellular matrix (ECM) accumulation and whose central mediators are HSCs. HSCs activation, is characterized by increased expression of matrix components and growth factors such collagen-I (COL-I), connective tissue growth factor (CTGF) and TGF-beta, as well as their enhanced proliferation. HBx protein is a promoting agent of HCC, an end-stage of liver injury; however its possible role in hepatic fibrosis remains unclear.

 

Aim:

Our aimis to study HBx contribution to the fibrotic process through the analysis of its effects on human HSCs behaviour.

 

Methods:

Primary human HSCs were incubated with conditioned media from the following hepatic cell lines; CMX, which expresses HBx in a dexamethasone inducible manner; 2.2.15, which bears two head to tail copies of the HBV genome. Parental cell lines CHL and HepG2 were used as controls. Activation markers α-SMA, COL-I, and CTGF were analyzed by FACS and Western Blot, respectively. Proliferation assays were performed using trypan blue exclusion under a phase-contrast microscope. To discriminate the possible signalling pathways implicated, neutralization assays against the main growth factors involved in fibrosis such as VEGF, TNF-α, PDGF and TGF-β were carried out.

 

Results:

α-SMA expression was detected in HSCs exposed to CMX and 2.2.15 media indicating an activated state that was not detected in HSCs exposed to control media. COL-I and CTGF synthesis, were enhanced in the same cases. Similar results were obtained in proliferation assays. Related with neutralization assays only TGF-β blockade produced a downregulation of the fibrosis markers studied. None of the other neutralized factor had effect related with HSCs activation. CONCLUSIONS: HBx protein has a remarkable influence in human HSCs behaviour, demonstrated by the increased expression of the fibrotic markers α-SMA, COL-I and CTGF and the enhanced proliferation in HSC exposed to the viral protein HBx. Neutralization assays showed that HBx influence is mediated at least by TGF-β signalling pathway. These results strongly suggest that HBx could be an inducing agent of liver fibrosis, being capable to induce the secretion of soluble mediators that result in the paracrine activation of HSCs, at least in a TGF-β manner.

 

849. Control of HBV Replication by HepaRG cells Innate Response

J. Lucifora; D. Durantel; B. Testoni; O. Hantz; M. Levrero; F. Zoulim

 

Background and Aims:

To gain more insight on the HBV/host-cell interactions, one of the major problems is to improve the existing cellular models for the study of HBV replication. As human primary hepatocytes are difficult to obtain, we have improved the system based on the transduction of HepG2 cells with a recombinant baculovirus thanks to the construction of a new HBV recombinant baculovirus (Bac-HBV-1.1-WT).Then, we asked whether a persistent HBV infection may be obtained after transduction of the differentiated HepaRG cells which are susceptible to viral infection.

 

Methods:

HepaRG cells were transduced by Bac-HBV-1.1-WT and kinetics of synthesis of HBV replication intermediates were analyzed. Cellular response to infection via the IFN pathway was studied by qRT-PCR. Cells transduced with baculovirus vectors harbouring the beta-galactosidase gene served as controls.

 

Results:

Following transduction by Bac-HBV-1.1-WT, HepaRG cells initiate an HBV replication cycle with viral DNA synthesis and viral protein expression. Interestingly, a rapid arrest of HBV replication was observed several hours post-transduction. Challenge experiments showed that transduction with Bac-HBV-1.1-WT (but not with the control baculovirus) lead to an antiviral state in HepaRG cells. Cellular gene expression analyses showed that IFN-β and other genes involved in the innate response were up-regulated in HBV-transduced HepaRG cells but not in control HepaRG cells. This demonstrates that the antiviral state is mediated by HBV and not by the baculovirus vector. To inhibit the cell response, a new HepaRG cell line down regulated for the IFN pathway by a short hairpin RNA strategy was designed. Transductions experiments demonstrated that HBV replication is improved in these new cells compared to the WT cells.

 

Conclusions:

Our results suggest that the innate response may play a critical role in the control of HBV infection. The magnitude of the innate response was dependent on the cell type and may therefore play a role in the clearance or control of HBV infection. The modulation of the IFN-β pathway may be a prerequisite to establish persistent hepatocyte infection by HBV.

 

843. Genetic Variability on the cccDNA Regulatory Regions Changes Significantly in the Different Phases of Chronic HBV Infection

A. Koepke; T. Volz; M. Lutgehetmann; A. W. Lohse; M. Dandri; J. Petersen

 

Knowledge of factors regulating the template of HBV replication, the covalently closed circular DNA (cccDNA), is essential to elucidate mechanism determining viral persistence. By applying molecular assays which permit assessment of viral loads and transcriptional activity of the cccDNA in human liver biopsies, we found that the HBV replication pathway was significantly reduced in most treatment naïve HBeAg-negative individuals and that impairment of virion production was not linked to the emergence of the common HBeAg variants (Gastroenterology 2007). Here we investigated whether genetic variability within the regulatory regions (Enhancer I and II) and the X ORF may account for the lower replicative activity observed in the HBeAg-negative phase.

 

Methods:

The cccDNA of treatment naïve 21 HBeAg-positive and 22 HBeAg-negative, anti-HBe positive individuals was compared by direct sequencing of cccDNA-specific PCR products (nt. 968 to 1974). Results: 104 nucleotide mutations were detected in 17/21 HBeAg-positive individuals (median 4; range: 0-13 mutations/patient) with active viral replication (median 43 rcDNA/cccDNA), while only 27 mutations (range: 0-5 mutations/patient) were identified in 11/15 HBeAg-negative patients displaying low replicative activity (median 6 rcDNA/cccDNA) and without detectable HBeAg variants. Of note, 7/22 HBeAg-negative individuals harboured only cccDNA with precore and basal core promoter (BCP) mutations and displayed significantly higher replicative activity (median 150 rcDNA/cccDNA) and mutation frequency (0.46% vs. 0.18%; p=0.001; range: 2-8 additional mutations/patient) compared to HBeAg-negative patients without detectable PC/BCP mutations. Aminoacid substitutions in the X protein were mostly due to the occurrence of BCP mutations.

 

Conclusions:

The significantly lower mutation rates determined in the low replicative phase of HBV chronic infection reveals that impairment of virion productivity is not due to the emergence of specific mutation patterns within the regulatory regions, suggesting that other host-mediated molecular mechanisms control HBV replicative activity, while the selection of BCP mutations re-established high virion productivity and accumulation of mutations in HBeAg-negative patients.

 

844. Single nucleotide polymorphisms and functional analysis of Class II transactivator (CIITA) coding region in chronic HBV infection

X. Zhang

 

Aims:

To investigate the association of the polymorphisms in CIITA coding region with the outcome of chronic HBV infection.

 

Methods:

The two SNPs at C30799G (Ala500Gly) and C19170G (Leu45Val) sites of CIITA gene coding region were genotyped using ARMS-PCR among 627 patients with chronic HBV infection and 101 healthy non-HBV infected blood donors. The cDNA fragments of three different CIITA haplotypes were prepared by inducing one or two single nucleotide mutation of wild type recombinant plasmid EBS-NPL-CIITA cDNA using overlap extension PCR site-directed mutagenesis technology. The eukaryotic expression vectors were constructed for three different haplotypes cDNA of human CIITA gene, and transfected into Hela cells respectively. CIITA mRNA and HLA class II antigen were respectively detected by RT-PCR and indirect cell immunofluorescence technique and flow cytometry in the untransfected and transfected Hela cells.

 

Results:

1. The frequencies of C allele and CC genetype at 30799 site were significantly higher among patients with hepatocellular carcinoma than those among patients with liver cirrhosis(χ2=4.861,4.993;P=0.027,0.025). Logistic regression analysis indicated that there were significantly different with the genotype frequencies at 30799 site in the patients between liver cirrhosis and hepatocellular carcinoma(OR 0.557;95%CI,0.334-0.930;P=0.025).

2. The frequencies of G allele and genetypes carring G allele (GG and GC) at 19170 site were significantly higher among patients with liver cirrhosis than those among nonprogressed liver diseases including asymptomatic carriers and patients with chronic hepatitis B χ2=7.128,6.464;P=0.008,0.011). Logistic regression analysis indicated that there were significantly different with the allele frequencies at 19170 site in the patients between liver cirrhosis and nonprogressed liver diseases (OR 0.742;95%CI,0.552-0.998;P=0.048).3.There were not significantly different with the levels of HLA class II antigen (HLA-DR,DP,DQ) expression among Hela cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA (P>0.05).

 

Conclusions:

1. The polymorphism at 30799 site of CIITA gene was associated with hepatocellular carcinoma in chronic HBV infection. The polymorphism at 19170 site of CIITA was associated with cirrhosis in chronic HBV infection.

2. The two SNP at the sites C19170G(Leu45Val) and C30799G(Ala500Gly) in the coding site of CIITA gene did not influence capability of CIITA trans-activating HLA class II gene expression.

 

 

837. Intrahepatic Analysis of Cytokine and Toll-like Receptor Signaling Expression in Chronic Hepatitis B Patients with Different HBV Replicative Activity

J. Bockmann; M. Lutgehetmann; T. Volz; A. W. Lohse; M. Dandri; J. Petersen

 

Accumulating evidence suggests that HBV may prevent activation of the Toll-like receptor signaling, while antiviral noncytolytic mechanisms can inhibit virion productivity.

 

Aim:

The aim of the study was to investigate whether intrahepatic changes in the expression of innate immunity genes correlate with changes in viral activity in chronic HBV infection (CH-B).

 

Methods:

DNA and RNA isolated from liver biopsies obtained from 2 uninfected and 17 treatment naïve CH-B patients (9 HBeAg+ and 8 HBeAg-) with significant different viremia (>4log) and replicative activity (median 42 vs. 3 rcDNA/cccDNA) were analysed. SuperArray assays which combine RT-PCR with microarray technology were used to determine the expression profiles of TLR-signalling pathways.

 

Results:

In general, moderate changes in cytokine levels (i.e. TGF-beta, IFN-alpha/beta) were found in different patient cohorts and only TNF alpha, IFN gamma and IL10 levels significantly differed in CH-B livers compared to uninfected controls, but did not vary between HBeAg+ and HBeAg- patients. Intrahepatic expression of TLR3 and TLR4 did not differ between groups, but levels of TLR4 and down-stream effector HMGB1 inversely correlated with ALT levels (p=0.02; r=0.6), indicating that TLR4 signalling is negatively regulated in the context of CH-B inflammation. Of note, expression of MD1 effector molecule, which was shown to inhibit TLR4 signalling, was significantly up-regulated in CH-B patients and levels correlated with intrahepatic viral loads, suggesting that HBV components may suppress some TLR-mediated immune mechanisms. Furthermore, TLR2 and MyD88 levels were only slightly increased in the liver of HBeAg- individuals, though changes became significant when expression levels of macrophages specific makers were taken into account.

 

Conclusions:

This analysis indicates that interactions between HBV and components of the innate immunity occur in the clinical setting and that intrahepatic HBV levels may favour suppression of the immune surveillance.

 

832. RIG-I signaling Expression in Monocyte-derived Dendritic Cells in Patients with Hepatitis B virus infection

G. Zhao; Q. Xie; H. Wang; L. Lin; H. Zhou; N. Jia; Y. Yang; C. Shi; B. Gong; X. Xiang; Q. Huang; Q. Guo; H. Yu

 

Background and Aims:

The retinoic acid-inducible gene I (RIG-I) can recognize viral RNA and result in beta interferon (IFN-β) induction. Dendritic Cells (DC) function as the most important part of the native immunity against virus. This study aims to investigate the function of RIG-I in Monocyte-derived Dendritic Cells(MoDC) at various stages of HBV infection, to explore the role of RIG-I in the disease progression after HBV infection.

 

Methods:

Peripheral blood was collected from 28 hepatitis B (HB) virus-infected persons, including 21 cases of chronic hepatitis B (CHB),7 of acute hepatitis B (AHB). 18 healthy subjects are as controls.Purified Monocytes were isolated by combination of HISTOPAQUE-1.077 and CD14 Microbeads.MoDCs were induced with GM-CSF and IL-4 for 7 days from CD14+ monocytes,then infected with vesicular stomatitis virus (VSV) to stimulate RIG-I expression. The mRNA expression level of RIG-I,IPS-1(IFN-promoter stimulating factor-1) and IFN-β after infection with VSV at 16h and 24h were measured by Real-time PCR.

 

Results:

The expression of RIG-I in MoDC of CHB were significantly lower than in AHB and healthy at 16 hours (2.44±2.03,19.54±3.15,21.48±8.39; P=0.002)and 24 hours(2.68±2.93, 10.31±3.88, 14.01±5.04;P=0.001) following VSV stimulation. There was a significant difference in RIG-I expression at 16 hours between AHB and healthy, while there was no difference at 24 hours. The mRNA level of IPS-1 in CHB and AHB was higher than healthy at 16 hours(2.05±1.08, 1.99±1.56, 0.60±0.31;P=0.024) and 24 hours(2.27±2.16, 3.24±1.21, 1.08±0.73;P=0.018). However, there was no significant difference in mRNA level of IPS-1 at 16 hours and 24 hours between CHB and AHB. Furthermore it was found that the IFN-β expression was lower in CHB than healthy at 16 hours(5.76vs1.4,P=0.021) and 24 hours(5.68vs1.23,P=0.011).

 

Conclusions:

Our results showed that the expression of RIG-I and IPS-1 in MoDC were abnormal in HBV infection.Chronic HBV infection may down-regulate RIG-I and IPS-1 expression in MoDC,which could decreased the IFN production. Our results suggest that RIG-I signaling pathway might be blocked by HBV and the impaired function of MoDC may play a role in HBV infection and chronicity.

 

833. Hepatitis B virus-replication could induce sHSP60 and enhance regulatory T cell activity via toll like receptor 2

Y. Kondo; Y. Ueno; S. Kon, J. Inoue; M. Ninomiya; E. Kakazu; M. Shiina; K. Tamai; Y. Wakui; K. Kobayashi; K. Fukushima; T. Kogure; H. Niitsuma; Y. Tanaka; M. Mizokami; T. Shimosegawa

 

Background:

We previously reported that HBcAg specific regulatory T cells (Tregs) play an important role in the pathogenesis of chronic hepatitis B virus. Some groups reported that heat shock protein 60 (HSP60) could affect the regulatory T cell function via Toll like receptor 2 (TLR2). Recently, HBV replication systems that replicate in-vitro are used for the analysis of the direct effects of HBV replication in hepatocytes.

 

Aim:

The aim of this study is to analyze whether soluble HSP60 (sHSP60) produced by HBV-infected-hepatocytes may affect the functions of Tregs functions.

 

Methods:

Chronic Hepatitis B (CH-B) HBeAg+ (n=24), CH-B HBeAb+ (n=24) were studied. As control subjects, CH-C (n=24) and Healthy subjects (n=10) were simultaneously studied.

 

Ex-vivo study. The amounts of sHSP60 in serum were quantified by ELISA. PBMCs of selected CH-B subjects who had received Entercavir (ETV, 5mg/day) were used for the analysis of Tregs. The study protocol comprised 6 months of patient monitoring before the start of treatment, followed by 6 months of ETV therapy. The frequency of Tregs and the expression level of TLR2 during ETV treatment were quantified sequentially up to 6 months during treatment by flow cytometry analysis. Tregs were isolated from PBMCs by magnetic beads methods. IL-10 secretion assay and cell suppression assay were carried out using isolated Tregs with or without recombinant HSP60 (rHSP60).

 

In-vitro study. Two kinds of plasmids carrying a 1.3-fold of the HBV genome that could replicate in HepG2 cells were used to analyze whether HBV replication may affect the production of sHSP60 in culture medium.

 

Results:

The serum levels of sHSP60 in CHB HBeAg+ patients, CHB HBeAg-patients, CHC patients and Healthy subjects were 5.77±1.19, 4.12±1.37, 2.11±0.96 and 0.54±0.46 ng/ml (Mean±SD). The frequency of IL-7R-CD4+CD25+ cells and TLR2 expression had gradually declined during-ETV treatment. Pre-incubation in vitro of CD4+ cells from CHB patients with rHSP60 (0.01 -1 ng/ml) dose-dependently increased the frequnecy of HBcAg specific IL-10 secreting Tregs. These effects of rHSP60 on Tregs were canceled with Anti-TLR2 antibody. The amounts of sHSP60 in HBV replicating HepG2 cells were significantly higher than those of Mock transfected HepG2 cells. [pBFH(29.56±0.47), pBAH(24.77±1.17), Mock(13.07±2.03) ng/ml.

 

Conclusion:

 sHSP60 produced from HBV infected hepatocytes could enhance the HBcAg specific function of regulatory T cells. The suppression of HBV replication by ETV could suppress the frequency and function of Tregs, which might contribute to the persistence of HBV infection.

 

834. T-1993C Polymorphism in The Promoter of TBX21 Gene Is Associated With Susceptibility to Persistent Hepatitis B Virus Infection

S. Chen; W. Zhao; G. Deng

 

Transcription factor T-bet is responsible for the differentiation of naive T lymphocytes, and its expression level is linked with different responses to some viral infections, including hepatitis B virus (HBV) infection. In this report we examine whether promoter polymorphisms of TBX21 gene (encoding T-bet) are associated with persistent HBV infection. Three previously reported promoter polymorphisms, the T-1993C, T-1514C, and G-1499A , were analyzed by PCR restriction fragment length polymorphism analysis.

 

Two common polymorphisms, the T-1993C and T-1514C were selected for genotyping in 436 persistent HBV-infected cases, 310 spontaneously recovered controls, and 302 HBV naïve controls. Haplotypes were constructed for each subject and association with the susceptibility to persistent HBV infection was estimated by logistic regression. Case-control association study showed that the -1993C and -1514C allele were associated with persistent HBV infection (for T-1993C, χ2 = 20.4, P = 0.0000063; for T-1514C, χ2 = 5.6, P = 0.018). Similarly, haplotype -1993T-1514T was associated with decreased susceptibility to the persistence of HBV infection (P = 0.000017, OR = 0.46, 95% CI 0.32-0.65). Our results suggest that it is the common regulatory T-1993C polymorphism, not the uncommon G-1499A polymorphism in TBX21 promoter, influences susceptibility to persistent HBV infection in a Chinese population.

 

Table 1.TBX21 genotype distributions in naïve controls, persistent HBV infected group and spontaneously recovered controls.

 

Naïve controls

(n = 302)

Spontaneously recovered individuals

(n = 310)

Persistent HBV

infected group

(n = 436)

Allele T versus allele C†

 

χ2 P Value

T-1993C

 

 

 

 

TT, no. (%)

231 ( 76.5 )

262 ( 84.5 )

310 ( 71.1 )

 

TC, no. (%)

66 ( 21.8 )

45 ( 14.5 )

111 ( 25.5

 

CC, no. (%)

5 ( 1.7 )

3 ( 1.0 )

15 ( 3.4 )

 

T allele, no. (%)

528 ( 87.4 )

569 (91.8 )

731 (83.8 )

20.4 0.0000063

C allele, no. (%)

76 (12.6 )

51 (8.2 )

141 (16.2 )

 

T-1514C

 

 

 

 

TT, no. (%)

265 ( 87.8 )

281 ( 90.7 )

366 ( 84.0 )

 

TC, no. (%)

36 ( 11.9 )

27 ( 8.7 )

69 ( 15.8 )

 

69 ( 15.8 )

1 ( 0.3 )

2 ( 0.6 )

1 ( 0.2 )

 

T allele, no. (%)

566 ( 93.7 )

589 (95.0 )

801 (91.9 )

5.6 0.018

C allele, no. (%)

38 (6.3 )

31 (5.0 )

71 (8.1 )

 

 

†Comparison is between persistent HBV infected group and spontaneously recovered individuals

 

 

 

835. A previously unidentified subset of CD4-CD8-T cells contributes to the persistence of chronic viral hepatitis via virus specific killing of CD8+ T cells

W. Yan; X. Wang; Q. Xiaoming; J. Zhang; T. Chen; H. Wang; X. Luo; Q. Ning

 

Objective:

To study the contribution of previously unidentified subset of double negative T cells (DNT cells) in MHV-3 induced chronic viral hepatitis in C3H/HeJ mouse and patients with chronic hepatitis B(CHB).

 

Methods:

Murine hepatitis virus stain 3(MHV-3) infected C3H/HeJ displayed as chronic hepatitis and Balb/cJ mice displayed as fulminant hepatitis were used in this study. Splenocytes from MHV-3 infected C3H/HeJ mice were adaptively transferred into Balb/cJ mice, the surviral rate and pathohistology of Balb/cJ mice were examined. The proportions of various T cell subsets in CD3+ T cells in C3H/HeJ mice were examined post infection. Magnetic bead sorting was applied for the purification of T cell subsets. The cytotoxic effects, Transwell experiment and Perforin/granzyme/Fas-FasL pathways identification were performed. Cell surface markers and cytokines of the DNT cells were identified by cytofluorometric analysis. The frequencies of DNT cells from healthy and patients with CHB were also observed.

 

Results:

About 63% of MHV-3 infected C3H/HeJ mice developed a chronic course of virus infection and all of the Balb/cJ mice receiving MHV-3 died within 3 to 5 days. After adaptive transfer of splenocytes from MHV-3 infected C3H/HeJ mice, the survival rate(~30%)of Balb/cJ mice post MHV-3 infection increased significantly to. The DNT cell and CD4+CD25+T cell proportions raised Significantly since 2 days post MHV-3 infection in C3H/HeJ mice. DNT cells showed significant and specific cytotoxic effects on CD8+T cells. More than 14% of infected DNT cells expressing IL-2, and about 8% expressing IFN-γ. None has shown the expression of IL-4,IL-10, TNF-γ or Foxp3. The cell surface markers of the DNT cells is TCRαβ+ CD4-CD8-CD25-CD28-CD30-CD44+. Cytotoxicity of DNT cells is only about 20% when cultured with CD8+T cells in separate chambers of Transwell. Fas-FasL but not Perforin/granzyme pathway was involved in the virus specific cytotoxicity of DNT cells.

 

Patients with CHB also showed a higher rcentage of DNT cells within their population of CD4+T cells in peripheral blood compared with healthy controls. conclusions: Here we first report that a unique subset of DNT cells with production of IL-2 and IFN-γ have profound immunomodulatory effects on CD8+T cells in MHV-3 induced chronic viral hepatitis in C3H/HeJ mice via a Fas/FasL dependent pathway, suggesting their contribution to viral persistence. Primary studies performed on CHB patients also reveal the correlation of increased DNT cells and persistence of viral hepatitis.

 

828. Cellular Immune Response to Recombinant Surface and Core HBV Antigens Is Not Impaired in Blood Donors with Occult Hepatitis B Infection

M. Bes; S. Sauleda; N. Casamitjana; A. Esplugas; J. I. Esteban; M. Pirón; L. Puig; J. Guardia; V. Vargas

 

Introduction:

Since implementation of highly sensitive HBV DNA screening, occult hepatitis B infection (OBI) has become a finding among blood donors from an area of medium prevalence of HBV markers.

 

Aim:

To assess cellular immune response to HBV recombinant proteins in blood donors with occult hepatitis B infection.

 

Subjects and Methods:

Nineteen blood donors with OBI (HBsAg negative, HBV DNA positive, total anti-HBc positive, anti-HBc-IgM negative) identified during screening were included in the study. PBMCs were obtained and challenged in vitro with HBV recombinant proteins from the surface, core and HBe antigens. HBV cellular immune response was assessed by IFN-gamma ELISPOT assay. Results were compared to those obtained from 27 inactive HBV carriers. Eleven donors negative to all HBV markers were included as negative controls for the assay and ten HBV vaccinated donors as positive surface (HBsAg) responses. Results were expressed as median of IFN-gamma spot forming units per 106 PBMCs and range. Groups were compared with the Mann-Whitney test.

 

Results:

From January 2006 to April 2008, 647.953 blood donations were routinely screened for HBV DNA and 41 blood donors were identified to carry OBI; of them, nineteen were included in this study. Mean age of the OBIs was 56.9±7.2, 17 (88%) were male and 71% presented genotype D. Mean age in the inactive HBV carrier group was 39±11 (p<0.001), 15 (56%) were male (p=0.022). OBI donors had a significantly higher cellular immune response to IFN-gamma production than inactive HBV carriers. For HBsAg protein, median values were 300 SFU (30-2030) in the OBI group versus 130 SFU(10-1130) in the inactive HBV carriers (p=0.031). For core protein; median values were 450 SFU (30-1900) in the OBI group compared to 180 SFU(20-1150) in the inactive HBV carriers (p=0.004). Finally, HBeAg response was also significantly higher (370 SFU vs. 100 SFU, respectively, p=0.004). Median IFN-gamma production in the negative (non-exposed) controls was 160 SFU for surface antigen, 190 SFU for core antigen and 180 SFU for HBeAg. These values were lower than the OBI group (p<0.05) and similar to the inactive HBV carriers. IFN-gamma production in HBV vaccinated controls was similar to OBI donors for HBsAg (415 SFU vs. 300, p=n.s), but different for core (115 vs. 450 SFU respectively, p= 0.012).

 

Conclusions:

Cellular immune response to HBV recombinant proteins in OBI donors is multispecific (surface, core and HBeAg), shows a Th1 profile (IFN-gamma production) and is significantly higher than in inactive HBV carriers. In OBI donors, low level HBV DNA replication persists in spite of conserved anti-HBV cellular immune response.

 

822. Cytopathic Effect of Hepatitis B Virus Obtained from Fulminant Hepatitis Patients

J. Inoue; Y. Ueno; Y. Wakui; K. Fukushima; T. Kogure; Y. Kondo; E. Kakazu; H. Niitsuma; T. Shimosegawa

 

Background and Aims:

It is generally thought that hepatitis B virus (HBV) has no direct cytopathic effect and that the host immune response to infected hepatocytes leads to liver injury. However, the presence of a direct effect on hepatocytes was suggested based on the HBV-induced histological changes of a uPA-SCID mouse model with the liver replaced by human liver. We reported in the 58th annual meeting of AASLD that genotype B2 HBV obtained from patients of epidemic fulminant hepatitis accumulates its replicative intermediates in cells, and mutations in the core promoter region (A1762T/G1764A) and precore region (G1862T and G1896A) are related to its effects. We aimed to analyze further the functions of these mutations in the view of the cytopathic effects. Methods: A plasmid carrying 1.3-fold the HBV genome obtained from a fulminant hepatitis patient (pBFH2) was constructed, and the mutations at nt 1762/1764, 1862 and 1896 in pBFH2 were converted to the wild type nucleotides using site-directed mutagenesis. Another plasmid was constructed from an acute hepatitis B patient (pBAH2) in the same way. These plasmids were transfected into HepG2 cells, and cell viability was measured by MTS assay four days after transfection. Cytotoxicity was analyzed by LDH assay, and participation of apoptosis was evaluated by measurement of caspase-3/7 activity and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay.

 

Results:

The cell viability of pBFH2-transfected HepG2 was significantly lower than pBAH2, and the release of LDH was increased in pBFH2. In the analysis of mutations in pBFH2, the cell viability was most greatly recovered when nt 1862 mutation was converted to the wild type. Nucleotide conversions in combination revealed that the presence of both nt 1762/1764 and 1862 mutations decreased the cell viability, whereas single mutations showed no effect. Based on these results, these mutations seemed to be associated with the cytopathic effects of this HBV strain. Because caspase-3/7 and TUNEL-positive cells were not increased in pBFH2-transfected cells, necrosis but not apoptosis was thought to lead to the cytopathy. Conclusions: All of the five patients with fulminant hepatitis caused by this HBV strain were more than 60 years old, in which the immune reaction seemed to be relatively low. This fact prompted us to investigate the direct effect of this strain on hepatocytes. In our study, it was suggested that the presence of both core promoter mutations (A1762T/G1764A) and a precore mutation (G1862T) might be associated with the cytopathic effects of this HBV strain, which might lead to the progression of fulminant hepatitis.

 

816. Cleavage of Duck Hepatitis B Virus Envelope Protein by Endosomal Proprotein Convertase Promotes Viral cccDNA Formation in Non-susceptible LMH Cells

Y. Tong; R. Machida; S. Tong; J. R. Wands; J. Li

 

Background and Aim:

The mechanism of hepadnavirus entry into hepatocytes is not well defined. Duck carboxypeptidase (DCPD) has been identified as a virus docking receptor. In addition, glycine decarboxylase (DGD), a liver specific protein capable of binding to truncated viral pre-S protein with C-terminal dibasic residues (Arg-Arg), is required for a post-binding step. Processing of substrate proteins via dibasic residues such as Arg-Arg is a characteristic of proprotein convertases such as furin and PC7. Therefore, we investigated whether viral envelope proteins can be cleaved by endoproteinases, and whether this cleavage triggers viral entry.

 

Methods:

Endosome was isolated from duck liver by sucrose gradient ultracentrifugation. Co-localization of DCPD, DGD and viral pre-S protein in endosome was documented from DHBV infected liver by Western blot. Cleavage of viral envelope proteins was examined by Western blot after incubation of viral particles with the endosomal fraction. The effect of the envelope cleavage on viral infection was determined in LMH cells, a chicken hepatoma cell line resistant to viral infection but supportive of viral replication. Role of furin and PC7 in DHBV infection was investigated using specific inhibitors. Results: Both DCPD and DGD were found to be co-localized with the viral pre-S protein in the fraction enriched for EEA1, an early endosome marker. Interestingly, incubation of virus particles with the endosomal fractions isolated from Peking duck liver resulted in cleavage of the viral envelope protein, similar to that observed in vivo in DHBV infected duck liver. In contrast, no such cleavage could be demonstrated for endosomes isolated from Muscovy ducks, a duck species resistant to DHBV infection. Moreover, the cleavage was dependent on the amount of endosomes applied and was optimal at pH 5.5 in the presence of Mg++ or Ca++. Interestingly, such pretreated virus particles showed infectivity in LMH cells as evidenced by increased viral DNA titer according to PCR analysis, and cccDNA formation/amplification as revealed by rolling circle amplification and Southern blot. Such infection could be efficiently blocked by either polyclonal antibody against DGD or by a PC7 inhibitor. In addition, a furin inhibitor partially blocked DHBV infection of primary duck hepatocytes when added immediately after virus infection.

 

Conclusion:

Cleavage of viral envelope protein by Furin/PC7 like-convertases may be critical for initiation of productive viral infection.

 

817. HBx Protein Is Necessary for HBV Replication in Human Hepatocytes

M. Tsuge; N. Hiraga; M. Imamura; S. Takahashi; K. Chayama

 

Background & Aims:

The HBx protein is one of the hepatitis B virus (HBV) proteins that is considered to play an important role in replication of the hepatitis B virus. Previous reports have shown that the X protein is important in the replication of the woodchuck hepatitis virus, but not in the duck hepatitis B virus. Recently, we developed reverse genetics of the HBV using human hepatocyte chimeric mice. In this study, we examined whether or not the X protein of the HBV is dispensable in viral replication in vivo.

 

Methods:

For expression of the HBx protein, we constructed plasmids containing the entire HBx protein in pcDNA3 and pcDNA3-HA plasmid vectors. A stop codon was introduced to the HBx region of the plasmid containing the entire HBV genome with C1395T nucleotide substitution (CAA to TAA). Wild and HBx-deficient HBV particles were produced from HepG2 cells after transfection of 1.4 genome length plasmids with the calcium phosphate method. Produced HBV particles were inoculated into the human hepatocyte chimeric mice from the tail vein, with or without a hydrodynamic injection of HBx expression plasmids. We extracted the serum samples from the mice every two weeks and analyzed the HBV titers by real time PCR.

 

Results:

The HBx protein expression by hydrodynamic injection of the HBx protein expressing plasmid was confirmed by the Western Blot analysis, using antibody directed to HA tag. Quantitatively measurable viremia developed in six of seven HBx injected mice. In contrast, none of the 16 mice without the expression of the HBx developed the measurable viremia (P= 0.000016). The HBV DNA levels in serum increased up to 10e9 copies/ml and the viremia continued for more than 2 months. Nucleotide sequence analysis showed that revertant viruses with nucleotide substitutions revert the stop codon to amino acids. Interestingly, not only an original wild type revertant (TAA to CAA), but a novel virus with a nucleotide sequence of AAA instead of CAA also emerged.

 

Conclusions:

Complementation of the HBx protein with hydrodynamic injection rescued the HBx deficient virus infection to the human hepatocyte chimeric mice, and resulted in the emergence of the revertant virus. Our results suggest that the HBx protein is necessary in active replication of the virus in vivo.

 

819. Mechanism of Hepatitis B Virus Persistence Following an Acute Infection – A study using duck hepatitis B virus of the roles of virus replication and cell turnover.

G. Reaiche; M. Le Mire; W. Mason; A. R. Jilbert

 

Traces of hepatitis B virus persist in the liver after clearance of acute infections, but the extent to which persistence depends upon ongoing viral DNA synthesis is unknown. To address this question, we determined the effect on residual DNA levels of treatment with the nucleoside anaolog, entecavir (ETV), an inhibitor of viral DNA synthesis, using ducks that had recently recovered from duck hepatitis B virus infection (DHBV).

 

Six week-old ducks were infected with DHBV and monitored until recovery. Following recovery, the ducks were treated continuously with ETV. Levels of DHBV total and cccDNA in the liver were measured by qPCR in treated ducks and untreated controls before and after 3 and 6 months of treatment. Changes in DHBV DNA levels were also correlated with hepatocyte turnover using proliferating cell nuclear antigen (PCNA) as a marker of cell replication.

 

At the time of initiation of ETV therapy and at later times, no infected cells were detectable in the liver by immunostaining of liver biopsies for viral protein expression. Most of the residual DHBV DNA in the recovered ducks was found to be cccDNA, which declined slowly over the next six months. Levels of cccDNA were 0.05-0.12 copies/cell at commencement of ETV treatment and declined to 0.0003-0.04 copies/cell after 6 months of treatment. The rate of decline was not affected by entecavir treatment.

 

Hepatocyte proliferation correlated strongly with loss of residual DHBV cccDNA. Assuming loss of cccDNA with cell death, but not with mitosis a mathematical model was used to predict the required turnover of hepatocytes, which would explain the observed half-life of cccDNA. In 5 out of the 6 ducks studied, estimated cell turnover matched that predicted using the model.

 

Conclusion:

In conclusion, residual DHBV DNA declined over the duration of the study. Inhibition of viral replication did not affect the level of residual DHBV DNA, suggesting that replication is not required for persistence of DHBV DNA. The study suggests that DHBV DNA declines as cells containing viral DNA are lost and replaced. These findings have implications for development of strategies for curing chronic hepatitis B infection.

 

825. Increased Innate and Adaptive Immune Pressure Is Responsible for the Reduced Transcriptional Activity of Intrahepatic Covalently-Closed Circular (ccc) DNA That Occurs in HBeAg-negative Chronic Hepatitis B (CHB)

J. Thompson; J. Chang; N. A. Skinner; S. Lewin; K. Visvanathan; P. V. Desmond; S. Locarnini

 

 

Introduction:

The factors regulating the transcriptional activity (TA) of cccDNA remain unclear. The G1896A (PC) and A1762T/G1764A (BCP) mutations, associated with HBeAg(–) CHB, increase TA in cell culture models. However, studies of the molecular biology of HBV in vivo show that TA is reduced in patients with HBeAg(–) versus HBeAg(+) CHB (1,2). This paradox may be due to host immunological factors.

 

Aim:

To investigate the relationship between intrahepatic markers of TA, and host innate and adaptive immune reactivity, comparing patients with HBeAg(+) versus HBeAg(–) CHB.

 

Methods:

Serum and liver tissue were collected from 44 treatment naïve patients with CHB and raised ALT (17 HBeAg(+) 27 HBeAg(–)). Detailed virological characterization included genotype, BCP/PC sequence, and measurement of intrahepatic cccDNA, total HBV-DNA (rcDNA) and TA. TA was defined as the ratio of rcDNA: cccDNA (rc DNA was corrected for cccDNA) (2). The expression of TLR2 and TLR4 on hepatocytes, Kupffer cells and peripheral CD14+ monocytes was compared using flow cytometry. Whole blood, cultured PBMCs, and cultured liver-infiltrating lymphocytes were stimulated with an overlapping peptide library (15mer) to the whole HBV genome. The expression of Th1 cytokines (IFN-γ, TNF-α, and IL-2) and IL-10 was analysed by intracellular cytokine staining and flow cytometry.

 

Results:

HBeAg(–) CHB was associated with lower median levels of serum HBV DNA (-4 log), cccDNA (-1 log) and TA (-2 log) compared to HBeAg(+) CHB (p<0.05). The PC mutation was associated with reduced TA (p<0.05). CD14+ monocyte, hepatocyte and Kupffer cell TLR2 expression were increased in the HBeAg(–) cohort (p<0.05). Intrahepatic IL-10+ HBV-specific CD8+ T cells were reduced in HBeAg(–) CHB (p<0.05). This difference was not apparent in the peripheral CD8+ T cells. No difference in HBV-specific T cells was noted for any cytokine in ex vivo whole blood or PBMCs

 

Conclusion:

This is the first study to directly examine the relationship between markers of HBV transcriptional activity and host immunity within the liver of patients with CHB. The lower viral load in patients with HBeAg(–) CHB was due to both lower levels of cccDNA and lower TA. TLR2 expression was upregulated in the liver compartment of patients with HBeAg-negative CHB, consistent with heightened innate immune control. HBV-specific regulatory IL-10+ CD8+ T cells were less common in the liver of patients with HBeAg(–) CHB, consistent with a more active adaptive immune response. The data suggests an important role for both innate and adaptive immunity in regulating the TA of HBV.

(1) Laras,Hepatology,2006,44:694

(2) Volz,Gastroenterology,2007,133:843

 

831. High Prevalence of Hepatic Steatosis and its Impact on Fibrosis in Chronic Hepatitis B

W. Bleibel; G. Hussain; M. Lai; C. Hayne; N. H. Afdhal; D. Lau

 

 

Background and Aim:

There is strong evidence that nonalcoholic fatty liver disease (NAFLD) is associated with increased disease progression in hepatitis C. The impact of hepatic steatosis and metabolic syndrome on chronic hepatitis B (CHB) is not well understood. The aim of this study is to examine the prevalence and effects of hepatic steatosis in CHB and to evaluate the accuracy of abdominal ultrasound in assessing hepatic steatosis and fibrosis.

 

Methods:

All adult patients with treatment naïve CHB who had liver biopsy at our Liver Center between 2000 and 2007 were included in this retrospective study. Patients with history of other coexisting liver disease or excessive alcohol intake (>50g daily) were excluded. The analysis was performed for the entire cohort; and for the HBeAg positive and negative CHB patients separately.

 

Results:

A total of 220 patients met the inclusion criteria and they were predominantly male (63%) and Asian American (68%). HBeAg positive CHB patients accounted for 51% (N=113) and were younger compared to HBeAg negative ones (34.6 vs. 43.5 years, p<0.0001). Hepatic steatosis was present in 73 (33.9%) patients. There was a higher proportion of males than females with steatosis (40% vs. 24%, p=0.034). Those with steatosis were older (43.5 vs. 36.4 years, p<0.0001) and had higher BMI (26.6 vs. 23.6, p=0.00057). Patients with steatosis were more likely to have increased hepatic fibrosis (70% vs. 56%, p=0.05). More importantly, presence of advanced fibrosis, such as bridging fibrosis and cirrhosis, was significantly higher among those with steatosis (29% vs.16%, p=0.022). The two groups had similar rates of inflammation, hepatic iron deposition, elevated ALT (>40IU/L) and HBV DNA (>105 copies/ml) levels. Similar trends were observed for both HBeAg positive and negative CHB; namely, hepatic steatosis was associated with older age, higher BMI and advanced fibrosis. Hepatic ultrasound, both models used prior to and after 2002, was only able to detect 30% of patients with hepatic steatosis and 40% with biopsy proven cirrhosis.

 

Conclusion:

There is a high prevalence of hepatic steatosis in this Asian American predominant CHB population. Those with steatosis have increased hepatic fibrosis and higher BMI. However, their BMI was lower than the conventional cut-off for obesity suggesting that a lower BMI for overweight and obesity should be used for Asians. Abdominal ultrasound has low sensitivity in detecting steatosis and early cirrhosis in CHB. These findings underscore the importance of NAFLD in contributing to HBV disease progression and the prognostic value of liver biopsy in assessing steatosis and fibrosis among patients with CHB.

 

 


Disease Progression – Role of Genotypes and Mutations

 

989. Precore (PC) and Basic Core Promoter (BCP) Mutations and Genotypes in Relation to Viral Replication in Asymptomatic HBsAg-Positive Blood Donors in Greece

D. Zoulas; M. J. Schina; A. Kostourou; H. Theochari; G. Mousoulis; M. Parara

 

Introduction:

Virological characteristics of HBV such as efficiency of viral replication and viral genome variability may explain the possible pathogenic and therapeutic differences among chronic hepatitis B (CHB) patients. HBsAg positive, first time volunteers blood donors, consist a representative sample of general population, in order to study the virologic characteristics of HBV in Greece with special interest in genotypes and pre core mutations.

 

Methods:

321 out of 41,181 blood donors found to be positive for HBsAg between 2004 and 2007, prevalence 0,78%. 119 from them came for a second test and only 80 subjects with a positive HBV viral load were finally enrolled in the study. Age, sex and nationality were recorded. HBsAg, HBeAg and anti-HBe were assayed using standard EIAs (AXSYM, Abbott) while HBV viral load was determined by real-time PCR with artus HBV PCR kit, Qiagen (detection limit 25 copies/mL). HBV viral characteristics such as genotype, PC and BCP Mut were detected using line-probe assays (INNO-LiPA, immunogenetics). Relation of PC and BCP Mut with demographics and viral replication were studied.

 

Results:

All but six were Greek. 79% male, mean age 33,96 ±10,83 years. Median HBV DNA=444 copies/mL (IQR 80-3155 copies/mL). 9% were eAg+, 91% eAg-. Genotype D was the dominant with an incidence of 77/80 (96%). The overall prevalence of PC/BCP Mut was 93,75%. The prevalence of PC Mut was 87,5% with predominance of G1896A (64%) and mixed (24%), while the prevalence of BPC Mut was 54% with predominance of mixed population (36,2%) rather than single Mut. Comparison between eAg+ and eAg- subjects did not reveal any significant difference. In eAg- group only the presence of PC Mut was significantly correlated with lower HBV DNA levels (p=0,01) and there was no correlation with gender or age.

 

Conclusions:

In general, Greek population genotype D is the predominant and eAg- CHB is very common (91%). The overall prevalence of PC/BCP Mut is 94%. PC Mut has a high prevalence among patients with eAg- CHB (70%) and is the only virologic characteristic that is significantly correlated with a lower HVB DNA in this group. The possible implication of PC/BCP Mut in the natural history of CHB and therapeutic decision process remains to be assessed.

 

868. Increasing Frequency of Acute Hepatitis B with Imported Strain Genotype A in Japan

Y. Tamada; K. Yano; A. Komori; S. Abiru; K. Migita; M. Nakamura; H. Fujioka; H. Yatsuhashi; H. Ishibashi

 

Background and Aim:

An increasing trend of acute hepatitis B (AH-B) in Japan has been suggested. This may due to an increase of imported strains such as genotype (gt) A and Ba, which were not originally indigenous to Japan. The aim of this study is to reveal the incidence and trend of AH-B in Japan and to clarify the role of imported strains on the increasing trend.

 

Methods:

A total of 457 cases of AH-B were enrolled in a nationwide prospective survey on acute hepatitis, in which 28 national hospitals participated, between 1991 and 2007. The diagnosis of AH-B was made by positivity for IgM-HBc and HBsAg with an elevation of ALT. Sera drawn on admission were collected and stored at −20°C until being examined. HBV-DNA was extracted from serum, amplified, and analyzed for the gt based on the nucleotide sequences from preS1 to S region.

 

Results:

Of 457 cases, gt A, B, and C accounted for 93 (20.4%), 40 (8.8%), and 320 (70.0%), respectively. Other gts, such as gt D, E, G and H, were detected in 1 case (0.2%) each. Gt A accounted for 6.4%, 9.2%, 23.1%, 38.5% in 1991-1994, 1995-1999, 2000-2004, and 2005-2007, respectively. The prevalence in the recent 3 years was significantly higher than those in the first and second period each (p<0.0001, p<0.01, respectively). Particularly in 2007, gt A accounted for as much as 52.0% of AH-B. In Kanto region, where the capital locates and the population flux is highest in Japan, gt A became present in the late 1990s and showed steady increase in 2000s, and then reached up to 76% in 2007. In other regions, gt A has been increasing since around 2000. Of 457 cases, 191 (41.8%), 14 (3.1%), 14 (3.1%), 5 (0.9%), 4 (0.9%), 2 (0.4%), and 227 (49.7%) seemed to be attributed to sexual contact (either with Japanese or non-Japanese), medical accident, trip to foreign countries, intrafamilial horizontal infection, intravenous drug use or tattooing, horizontal infection in a rehabilitation facility, and unknown etiology as modes of transmission.

 

Discussion and Conclusion:

Gt A infection, which was considered rare in Japan, now accounts for over 50% of the AH-B. Regional analysis revealed that the silent epidemic initiated in urban areas and gradually spread over rural areas. Both numbers of people coming from foreign countries and going abroad from Japan have been increasing. Most HBV infection, however, occurred in Japan in our study, indicating that gt A was imported from other countries at first where gt A is prevalent, and consequently indigenized to Japan. Our data indicate that gt A has been spreading over Japan explosively. An immediate action, such as implementation of universal vaccination, is needed.

 

869. Non-A Hepatitis B Genotypes are Associated with More Liver Fibrosis in HIV/HBV Patients

D. Y. Dao; H. Yuan; R. Joshi; N. Attar; W. M. Lee; M. K. Jain

 

Background:

Approximately 10% of HIV-infected patients are co-infected with hepatitis B virus (HBV). Few studies have evaluated the prevalence of HBV genotypes and its impact on fibrosis. Evidence exists that genotypes are important in the progression of chronic liver disease in HBV mono-infected patients. The purpose of this study was to determine the prevalence of HBV genotypes and their association with advanced liver fibrosis in co-infected patients. Method: We examined a cohort of 141 HIV and HBV surface antigen (HBsAg) positive patients, for whom sera were available from a large inner city HIV clinic. HBV genotyping was done by direct sequencing. Baseline demographics and current liver function tests were obtained by chart review. Non-invasive serum biomarkers (APRI and Fib-4) were used to assess fibrosis stage at the last clinical follow-up. APRI and Fib-4 scores were dichotomized to examine scores indicating stage 1 fibrosis or ≥ stage 2 fibrosis.

 

Indeterminate values were not assessed. Categorical variables were analyzed by chi-square and continuous by Wilcoxon Rank Sum test. Results: HBV genotype A was the most frequent genotype (n=100; 75%) while non-A genotypes were present in 25% (n=41). The frequency of the non-A genotypes were the following: 13% (n=17) genotype G, 3% (n=4) genotype A/G mix, 6% genotype D (n=8) and 1.5% (n=2) 2 each were identified for genotypes F and H. Seven patients could not be genotyped. African Americans (AA) composed 47% (n=66) and Caucasians 37% (n=51) of the study population, and the median time of follow-up was 37 months. African Americans were more likely to be genotype A than non-A compared to Caucasians (AA 83% genotype A vs. 67% in CA, p=0.06).

 

In the non-A genotypes 7/17 (41%) had advanced fibrosis compared to 6/64 (9%) in genotype A, p=0.0015. Similarly, Fib-4 showed 8/24 (33%) of non-A genotypes with advanced fibrosis compared to 9/68 (13%), p=0.03. Median ALTs were higher in non-A genotypes [65 (7-193)] compared to genotype A [30 (8-159)], p<0.0001. In addition, HBeAg was positive in 18/20 (90%) of non-A genotypes compared to 44/62 (71%) of genotype A, p=0.08).

 

Conclusion:

This cross-sectional analysis reveals that HBV genotype appears to play a role in the natural history of the HIV/HBV co-infected patients. Genotype A is the most prevalent but there is a high prevalence of genotype G in HIV/HBV co-infected population. Non-A genotypes are associated with more liver fibrosis on follow-up evaluation. HBV genotypes may be an important tool to predict those who may be at greatest risk for liver disease progression in the HIV/HBV co-infected population.

 

870. Higher Levels of HBV-DNA in Genotypes B and C Compared to Genotypes A and D

H. Krarup; P. Madsen; A. Bentzen-Petersen; J. M. Møller; N. Weis

 

Hepatitis B viral factors such as viral load and HBV genotype have been suggested to influence progression of chronic hepatitis B.

 

Aim:

To explore relations between 1) genotype and viral load, 2) gender and viral load, 3) gender and genotype and 4) genotype and country of origin.

 

Material:

Samples from 802 consecutive HBV-DNA positive patients in Denmark were genotyped if possible. In house real-time PCR based methods were used. HBV-DNA levels were in the range of 10(2) – 10(10) IU/mL. Genotypes were determined using genotype specific primer pairs selected from the pre-S region of the genome.

 

Results:

Male/female ratio 427 to 375, age given as median (25;75 percentiles) was for males 37 (27;46) and females 30 (24;38) years. In total 195 samples could not be genotyped; 106 due to lack of sufficient amount of material and 89 due to various reasons, mostly too low viral load. Of 607 samples 11.2% were genotype A, 14.3% B, 19.4% C, 50.3% D, 2.3% E, 0.5% F and 1.5% genotype G, 0.8% had more than one genotype.

 

1) A significant difference was observed in viral load in genotype A compared to B and C (p values <0.008) and genotype D compared to B and C (p values <0.007), but no difference was observed in viral load between A and D or B and C. The viral loads in genotypes A to D were 1.2 x 10(5) (4.4 x 10(3); 3.8 x 10(7)) IU/mL, 1.4 x 10(7) (2.9 x 10(4); 1.4 x 10(8)) IU/mL, 1.4 x 10(7) (8.5 x 10(4); 1.4 x 10(8)) IU/mL and 1.0 x 10(5) (4.0 x 10(3); 5.9 x 10(7)) IU/mL respectively.

 

2) No difference in viral load among gender was observed either overall or in different genotypes.

 

3) A significant difference in distribution of gender over the 4 dominant genotypes was observed: more women had genotypes B or C while more men had genotypes A or D (p values <0.001). Women were significantly younger than men, 30 years versus 41 years (p value <0.001) and 30 years versus 35 years (p value <0.004) in genotypes B and D respectively, whereas no significant age difference was observed in genotypes A or C, men 32 years versus 35 years and 29 years versus 31 years respectively.

 

4) More than 65% of patients with genotype A originated from Africa.73% and 95% of patients with genotypes B and C originated from South East Asia, while 75% of patients with genotype D originated from the Middle East, Afghanistan or India.

 

Conclusion:

We found the median of serum HBV-DNA levels 100 times higher in genotypes B and C compared to genotypes A and D. More women, than men, had genotypes B or C, as opposed to genotypes A or D. The distribution of genotypes was as expected from ethnic origin of the patients.

 

871. The Frequency of T126 in Surface Antigen Increase with Disease Progression in Patients with Chronic Hepatitis B Virus (Genotype C) Infection

C. Kim; S. Yoon; C. You; J. Jang; S. Bae; J. Choi; J. Yang; J. Han; S. Choi; N. Han; C. Lee; Y. Lee

 

 

Background:

'a' determinant, which is in major hydrophilic region (MHR) of surface antigen (HBsAg) of hepatitis B virus (HBV), is neutralizing epitope located between amino acid (aa) 124 and 147 of HBsAg. Amino acid substitutions in MHR of HBsAg can be appeared inside or outside 'a' determinant, which make HBsAg variants. 126 aa of HBsAg has two kinds as wild type, isoleucine (I126) and threonine (T126). In HBV genotype C, 126 aa is usually I126 instead of T126. It is well known that the association between aa substitutions in MHR of HBsAg and HBV subtype or escape mutant but, there is a few data about the relationship between variants in MHR of HBsAg and disease progression from chronic hepatitis via cirrhosis to hepatocellular carcinoma (HCC).

 

Methods:

Serums from total 122 patients with chronic HBV infection were used for HBV DNA extraction. By nested PCR, MHR in HBV surface gene was amplified and analyzed for DNA sequence. Amino acid substitutions in MHR of HBsAg were detected by DNA sequence analysis of MHR. For HBV genotyping, HBV DNA was also amplified with genotype specific primers. Clinical status of chronic HBV infection divided into 3 groups as chronic hepatitis, cirrhosis and HCC. The associations were looked for between variants in MHR of HBsAg and clinical status or clinical variables.

 

Results:

A total of 122 patients, infected all with HBV genotype C (100%), were composed of 78 chronic hepatitis B, 24 cirrhosis and 20 HCC patients. HBV serotype showed adr (93.4%), adw (4.9%) and ayr (1.6%). Total number of patients who showed variants in MHR was 22 (18.0%). Total number of variants in MHR was 33. 19 (57.6%) of 33 were detected inside 'a' determinant on 11 aa positions. 14 (42.4%) of 33 were detected outside 'a' determinant on 10 aa positions. All kinds of 126 aa were isoleucine (77%), threonine (18.8%) and serine (S, 4.1%).

 

The frequency of T126 as another wild type instead of I126 showed significant relationship with disease progression from chronic hepatitis via cirrhosis to HCC (10.3% vs. 20.8% vs. 50.0% respectively, P < 0.001, Linear-by-Linear association). The most frequent kind of variants in MHR of HBsAg was I126S. Variants status in MHR of HBsAg was not differ according to sex, age, serum ALT level, HBeAg status and serum HBV DNA level.

 

Conclusions:

126 aa of HBsAg is important position in variations of HBsAg. The frequency of T126 showed significant relationship with disease progression associated with HBV (genotype C) from chronic hepatitis via cirrhosis to HCC. The role of 126 aa and T126 as a biomarker of chronic liver disease including HCC could be investigated in the future study.

 

 

872. Mechanism for the Dependence of Hepatitis B Virus Genotypes G on Coinfection with Other Genotypes for Viral Replication

T. Sakamoto; Y. Tanaka; M. Sugiyama; F. Kurbanov; K. Matsuura; A. Kusakabe; N. Shinkai; F. Sugauchi; M. Mizokami

 

Background:

Hepatitis B virus genotype G (HBV/G) has stop codons in the precore region and is usually being detected in coinfection with the other genotypes, typically HBV/A2/Ae (Europe/USA). HBV/G reportedly accelerates liver fibrosis in patients with HIV infection (Lacombe K, et al. AIDS 2006). As recently reported, HBV/G monoinfection in chmeric mice resulted in viral replication at very low levels, but the replication increased markedly when animals coinfected with HBV/A2 and induces fibrosis, that concurs with findings among immunosuppressive patients. However, it is still unclear how the interaction with other genotypes enhances the replication of HBV/G.

 

Aim:

To investigate which viral proteins of HBV/A2 contribute in the replication of HBV/G in course of their coinfection. Methods: Four HBV/A2 recombinant plasmids were constructed (HBV/A2/S, HBV/A2/C, HBV/A2/pol and HBV/A2/X) in which only one of the four viral proteins (large surface, precore/core, polymerase or X, respectively) was selectively translated whereas translation of the other three was prevented by introduction of the corresponding stop codons. Huh7 cells were co-transfected with HBV plasmids encoding replication-competent 1.24-fold HBV genome of HBV/G and the recombinant HBV/A2 plasmids in following five protocols: (1) HBV/G alone; (2) HBV/G and HBV/A2/S; (3) HBV/G and HBV/A2/C; (4) HBV/G and HBV/A2/pol; and (5) HBV/G and HBV/A2/X. HBsAg and HBeAg in culture supernatant and core-related antigens (HBcrAg) in cell lysates were determined by CLEIA. Viral replication was estimated by Southern blot hybridization.

 

Results:

Comparing with HBV/A2, HBV/G produced barely detectable intracellular HBV DNA in cell lysates, and secreted HBsAg (307.2 vs. 28.1 IU/mL) and HBcrAg (247.4 vs. 20.8 kU/mL) in lower levels and no HBeAg into culture media. Co-transfection with HBV/G and recombinant HBV/A2/C plasmid greatly enhanced viral replication of HBV/G (around 2.5 times), as compared to mono-transfection of HBV/G, although there was no influence on HBV/G replication by the other proteins of HBV/A2. Additionally, transfection of a chimeric HBV/G plasmid with rearrangement of HBV/A2 within core promoter/precore/core significantly enhanced viral replication, as compared to wild HBV/G or a chimeric HBV/G with precore/core genome of HBV/A2.

 

Conclusion:

Low replication of HBV/G would be contributed by low synthesis or dysfunction of HBV/G core protein due to weak core promoter activity. HBV/G might take advantage of core protein of the other genotypes to replicate efficiently on coinfection with them, and the enhanced replication might result in disease progression under immunocompromised states.

 

864. Large Deletion Mutants of Hepatitis B Virus with Amino Acid Substitutions in the Core Region Emerges and Dominates Around the Onset of Active Hepatitis in Persistent HBV Carriers

N. Yamada; H. Yotsuyanagi; H. Miyoshi; T. Tsutsumi; H. Fujie; Y. Shintani; K. Moriya; C. Okuse; M. Suzuki; F. Itoh; K. Yasuda; S. Iino; K. Koike

 

Background:

Most HBe antigen (HBeAg)-positive asymptomatic carriers (AsC) suffer active liver injury in their adolescence, the mechanism of which is not clear yet. To elucidate contributing viral factors, we analyzed the structure and full nucleic/amino acid sequences of cloned HBV recovered from the sera of HBeAg-positive AsC and patients with active hepatitis.

 

Methods:

Nine HBeAg-positive HBV carriers persistently infected with HBV of genotype C (4 AsC, 5 chronic hepatitis (CH) patients with an acute exacerbation) were studied. The full-length genome of HBV was amplified using a single-step PCR and subcloned into the vector. Subsequently, nucleic/amino acid sequences of full-length genome of HBV were determined. Serum samples, serially collected from a patient who was followed-up from HBeAg-positive to HBeAg-negative AsC phases with an intervening acute exacerbation of hepatitis, were analyzed by the same methods.

 

Results:

1)     Surprisingly, HBV clones with large deletions at from the end of core to the middle of preS-S region or and with those in the core region were detected.

2)     2) In AsC, the HBV deletion mutants circulated as minor clones (median 7.0%), whereas those circulated as major clones (median 67.6%), in CH patients.

3)     3) In CH patients, amino acid substitutions in the core region at AA13, 87 and 97 were detected more frequently than in AsC (AA13: 14.7% vs. 0.0%, AA87: 55.6% vs. 0.0%, AA97: 55.6% vs. 0.0%).

4)     4) Double mutations in the basal core promoter (BCP) region (T1762/A1764) and precore mutations (A1896) were not detected in any clones from AsC group, but were very frequently found in CH group (88.6/92.6%, 37.5%).

5)     5) Time-course analysis of the sera showed that the HBV deletion mutants emerged on the exacerbation and dominated along with seroconversion to anti-HBe.

 

Conclusions:

The clinical course of HBeAg-positive carriers with genotype C HBV is accompanied by the appearance of large deletions in the core and preS-S regions and amino acid substitutions in the core region in addition to nucleic acid substitutions in the BCP and precore regions. The emergence of such mutants may be associated with viral vigorousness and define the fate of HBV carriers.

 

863. Drifting of HBV-specific CTL Epitope HBcAg18-27V to HBcAg18-27I in Most HBV Genotype B and C Virus Strains in China

S. Ma; M. Liang; X. Hu; Y. Yu; J. Chen; L. Yang; C. Li; X. Xu; Z. Wang; J. Hou

 

Background:

HLA-A2 restricted HBcAg18-27V (C18-27V) specific cytotoxicity T lymphocyte (CTL) epitope, derived from hepatitis B virus (HBV) genotype D and A strains, has been widely used to monitor specific CTL response and design HBV vaccine. However, C18-27I motif is present naturally in most of genotype B and C strains, and is not derived from mutation of prototype C18-27V under short-term immune selection. Moreover, very little is known about the difference of immunogenicity between these two mutants in prototype C18-27I background.

 

Aims:

The aims of this study are to profile the difference between C18-27V/I mutants in the prevalence and immunogenicity in China. Methods: A total of 230 serum samples from study cohort of HBV genotype distribution in China were screened for C18-27V/I mutants by PCR-sequencing and PCR-RFLP. Moreover, peripheral blood mononuclear cells were isolated from 27 chronic hepatitis B patients (HLA-A2+,C18-27I+ and ALT≥2ULN). The frequency and magnitude of the immune responses by C18-27V/I specific CTL in periphery blood were detected by tetramer staining and IFN-γ ELISPOT assay.

 

Results:

We found that C18-27I motif was the predominant virological background in 80 genotype B strains, 41 genotype C1, 78 genotype C2 and 26 CD recombinants (97.5%, 82.9%, 78.1% and 100%), whereas total 5 genotype D strains showed C18-27V. The proportion of C18-27I specific CTL was significantly higher than C18-27V specific CTL in periphery blood(0.335±0.092% vs. 0.221±0.075%, P=0.012). Using short-term CTL lines generated by peptide-C18-27V/I, we found peptide-C18-27V showed cross reactivity to C18-27I specific CTL, but peptide-C18-27I showed stronger proliferation capacity to prototype C18-27I specific CTL. Additionally, both peptide-C18-27V and peptide-C18-27I stimulated weak IFN-γ secretion of C18-27I specific CTL in chronic hepatitis B patients, and there was no significant difference between two peptides stimulus (8.6±4.2 SFCs/million cells vs. 7.3±3.1 SFCs/million cells, P=0.837).

 

Conclusions:

It suggests that C18-27I is an immunodominant peptide than C18-27V under prototype C18-27I virological background. It will be further exploited in the development of the diagnostic and vaccine in the control of HBV infection.

 

851. Influences of Hepatitis B Virus Genotype and Precore Mutations on Reactivation of Occult Hepatitis B in Patients with Hematological Malignancy

F. Sugauchi; Y. Tanaka; T. Sakamoto; A. Kusakabe; K. Mtsuura; M. Sugiyama; S. Nojiri; J. Takashi; M. Mizokami

 

Background and Aim:

Virologic characteristics of hepatitis B virus (HBV) on the reactivation of occult hepatitis B in patients who received hematopoietic stem cell transplantation (HSCT) or cytotoxic chemotherapy for the hematological malignancy are not well defined. Influences of HBV genotype and viral mutations on reactivation of occult hepatitis B in patients with hematological malignancy were investigated.

 

Methods:

Twenty eight HBsAg-negative patients who received HSCT for the hematological malignancy and 138 HBsAg-negative patients treated for malignant lymphoma (ML) with cytotoxic chemotherapy containing rituximab at the Nagoya City University Hospital from January 2005 to December 2007 were included. De novo HBV hepatitis was diagnosed when a patient’s HBsAg status turned from negative to positive after the initiation of therapy. HBV genotype and full-genome sequences were determined in patients of the de novo HBV hepatitis. HBV clones encoding a replication competent 1.24-fold the HBV genomes obtained from patients of the de novo HBV hepatitis were constructed, and intracellular HBV DNA levels isolated from Huh7 cells were analyzed by Southern blotting.

 

Results:

Three of the 28 patients (10.7%) received HSCT and 1 of the 138 (0.72%) patients treated for ML with cytotoxic chemotherapy developed de novo HBV hepatitis. The four patients with de novo hepatitis B had the mean age of 49 + 10 years including 3 men, and anti-HBc was detected in 4 and anti-HBs in 2 patients before commencement of HSCT or chemotherapy. HBV genotype Bj was detected in 2 (50%) and C in 2 (50%).They all possessed wild type sequences (A1762/G1764) in core promoter region, and the precore stop mutation (G1896A) was detected only in a patient with genotype Bj who developed fulminant hepatic failure.

 

HBV DNA was detected on nested PCR in pretreatment HBsAg-negative serum samples in 2 of the 4 patients with de novo hepatitis B, and their sequence homology encompassing core promoter to precore region (481 bp) between sera commencement of chemotherapy and at time of de novo HBV hepatitis showed 100%. In in vitro replication model, southern blot analysis showed that the replicative activity of the genotype Bj with G1896A clone obtained from a fulminant case was much higher than those of the clones obtained from the de novo HBV hepatitis patients with genotype Bj with G1896 and C with G1896.

 

Conclusions:

Fulminant outcome on reactivation of occult hepatitis B in patients received HSCT or cytotoxic chemotherapy for the hematological malignancy is associated with genotype Bj accompanied by high replication due to the G1896A mutation, which is supported by in vitro replication model.

 

818. Reactive Oxygen Species Induce Liver Fibrosis in uPA/SCID Mice with Human Hepatocytes Infected with Specific Hepatitis B Virus Genotypes

M. Sugiyama; Y. Tanaka; I. Maruyama; T. Shimada; S. Takahashi; T. Shirai; K. Hino; I. Sakaida; M. Mizokami

 

Background:

Hepatitis B virus (HBV) genotypes/subgenotypes indicate different clinical features, i.e. genotype C is associated with hepatocellular carcinoma, and Bj with precore mutation (PCm) is associated with fulminant hepatitis. We previously showed the difference of each genotype in vitro and partially in vivo.

 

Aim:

Our purpose of this study is to investigate the difference of viral replication, protein production and liver damage among HBV genotypes in vivo.

 

Methods:

Sera of six HBV carriers [three each for Bj_wild-type (from acute HB patients) and Bj_PCm (fulminant HB patients)] were prepared, and the other sera were obtained from pre-infected mice which were inoculated with culture medium produced by transfection with plasmids (3 strains each for Ae, Ce, Bj_wild-type and Bj_PCm) carrying HBV genome in Huh7 cells. These 18 sera were inoculated into uPA/SCID mice harboring human hepatocytes (chimeric mice). HBV DNA in mouse serum was measured by real-time detection PCR weekly, and alpha-smooth muscle actin (α-SMA)and 8-OHdG were detected by immunostaining 6 month post-infection. Reactive oxygen species (ROS) was detected by in situ hybridization with dihydroethidium.

 

Results:

Chimeric mice inoculated with HBV/C or Bj_PCm for 6 months showed that human hepatocytes had somewhat ground-glass appearance and fibrosis, while chimeric mice inoculated with HBV/Ae or Bj_wild-type were not induced. Immunostaing analysis showed strong staining of α-SMA around fibrosis. ROS and 8-OHdG were highly expressed in HBV/C and Bj_PC mice, and ALT and TGF-beta1 production of fibrosis group were higher than that of non-fibrosis group (P<.01). The HBV-DNA level in sera was the highest in mice infected with HBV/C compared to those with HBV/A and HBV/B (109, 107 and 104 log copies/ml levels, respectively, P<.05) during 6–8 weeks post-inoculation. HB core-related antigen excretion had a similar trend to replication, whereas secretion of HBsAg was more pronounced for HBV/A followed by HBV/C and much lesser for HBV/B. Introduction of precore stop-codon mutation in the HBV/B caused significant increase in viral replication, antigens expression, and the histopatological picture similar to HBV/C.

 

Conclusion:

Using humanized in vivo model, we demonstrate that different HBV genotypes and even particular mutation resulted in different virological and histopathological outcomes of infection, indicating that distinct HBV variants may be directly cytopathic in immunosuppressive conditions. This would represent a novel mouse model for human liver fibrosis associated with ROS production leading to the activation of hepatic stellate cells by infection.

 

Disease Progression – Development of Cirrhosis and Liver Cancer

 

867. Accumulation of Hepatitis B Virus DNA in Hepatocellular Carcinoma of Patients with Sustained Virological Response for Hepatitis C Virus

A. Tamori; T. Hayashi; M. Shinzaki; S. Kobayshi; S. Iwai; H. Morikawa; M. Enomoto; H. Sakaguchi; S. Shiomi; S. Kubo; N. Kawada

 

Background and Aim:

Hepatocellular carcinoma (HCC) has been reported in patients in whom hepatitis C virus (HCV) was eliminated by interferon (IFN) therapy. HBV genome is frequently detectable in liver tumors in HBsAg-negative patients with HCV. To elucidate the role of occult HBV infection on HCC in patients with sustained viral response (SVR HCC), we compared the clinical factors of SVR HCC and those of HCV HCC.

 

Patients and Methods:

Operable HCC developed in fifteen of 342 patients cured of HCV infection by IFN monotherapy. We examined 15 patients with SVR HCC and 50 patients with HCV. All patients were male. No patient abused alcohol or autoimmune hepatitis or primary biliary cirrhosis. Mean age was 66 years in patients with SVR HCC and 65 years in patients with HCV HCC. Anti-HBc and anti-HBs in serum were examined with enzyme-linked immunoassay. HBV-DNA in serum and in liver was examined by nested polymerase chain reaction (PCR) using primers specific for HBx, HBs, HBc, cccHBV. In seven samples from SVR HCC, HBV integration sites in human genome were identified by cassette-ligation-mediated PCR.

 

Results:

Anti-HBc was positive in 10 (66.7%) of 15 patients with SVR HCC and in 25 (50%) of 50 patients with HCV HCC. Anti-HBs was positive in six (40%) of 15 SVR HCC and nine (18%) of 50 HCV HCC. HBV DNA was not amplified in serum samples from SVR HCC and HCV HCC. Occult HBV infections were twelve (80%) of 15 in SVR HCC and 20 (40%) of 50 in HCV HCC (p = 0.015). HBV DNA integration was detected in four of 7 SVR HCC. The liver of SVR HCC with HBV DNA integration had not progressed to cirrhosis. Three tumors with HBV integration had one integration site each, located at chromosomes 11q12, 11q22-23, and 22q11, respectively. The other tumor had two integration sites, situated at chromosomes 11q13 and 14q32. At chromosome 11q12, HBV DNA was integrated into protein-coding genome, the function of which remains unclear.

 

Conclusion:

Occult HBV infection might be one of the most important factors in SVR HCC in Japan.

 

847. Future Hepatitis Activation and Hepatocarcinogenesis in HBeAg-Negative HBV Carriers with Persistently Normal Alanine Aminotransferase, a Prospective Cohort Study

T. Nakazawa; Y. Shibata; A. Takeuchi; Y. Okuwaki; K. Ono; Y. Miura; H. Hidaka; Y. Tanaka; J. Takada; M. Watanabe; A. Shibuya

 

Background/Aims:

To elucidate the incidence of future hepatitis activation and hepatocarcinogenesis in HBeAg-negative HBV carriers with persistently normal alanine aminotransferase (PNALT), long-term observation was performed in a prospective cohort.

 

Methods:

Ninety-nine consecutive untreated HBeAg-negative carriers with PNALT (40IU/L) were prospectively included in the study. The physical examinations, laboratory tests, and abdominal ultrasound sonography were performed every 6 months after the enrollment. The incidence of hepatitis activation (ALT levels of more than 1.5x upper limit of normal) and hepatocarcinogenesis were examined. In addition, we statistically investigated the contributed factors for hepatitis activation and hepatocarcinogenesis among baseline clinical and virological features at the enrollment. ALT level was classified into two groups of low-normal ALT (30IU/L) and high-normal ALT (30-40IU/L).

 

Results:

The mean follow-up period was 65.3±36.8 months. Thirteen carriers (13%) revealed hepatitis activation, and hepatocarcinogenesis was observed in 4 (3.7%). The cumulative rate of hepatitis activation was 10.5% and 14.9% at 2 and 5 years, while the cumulative rate of hepatocarcinogenesis was 2.9% and 12.3% at 5 and 8 years. In statistical analyses for future hepatitis activation, the univariate analyses revealed high-normal ALT and serum HBV DNA level 105 (copies/ml), and high-normal ALT was an independent contributed factor in a Cox model [odds ratio, 13.45; 95% confidence interval, 4.34-41.64, P<0.0001]. Future hepatocarcinogenesis was significantly associated with HBV carriers with older age (>50 years), high-normal ALT, the incidence of hepatitis activation, and serum HBV DNA level 105 (copies/ml) (P = 0.0045, 0.0017, <0.0001, and <0.0001, respectively).

 

Conclusions:

HBeAg-negative HBV carriers with high-normal ALT and high viral load should be carefully followed up to monitor future hepatitis activation and hepatocarcinogenesis, and antiviral treatment is necessary.

 

845. Clinical Significance of Low Viral Load in Antiviral Treatment-naive HBsAg Positive Patients

H. Woo; J. Choi; A. Min; J. Kwon; J. Kim; J. Jang; S. Bae; S. Yoon; S. Cho; J. Yang; Y. Lee

 

Background/Aim:

The low limit of HBV DNA associated with progressive liver disease is still controversial. Even patients with low viral load (HBV DNA <5 log copies(C)/mL) have been shown to be at risk for progression to cirrhosis or hepatocellular carcinoma. The aim of this study is to know the clinical significance of low viral load among HBsAg positive patients without antiviral treatment in high endemic area of HBV infection.

 

Methods:

All HBsAg-positive subjects with HBV DNA <5 log copies/ml by quantitative real-time-PCR (detection limit 28 C/mL) were recruited in our liver clinic from May 2006 to Dec 2007. The amplitude and pattern of past HBV DNA and ALT changes (cut off 40 U/L, pattern; persistent low fluctuation/persistent high) were retrospectively analyzed.

 

The following patients were excluded; those with prior antiviral treatment, with prior or concomitant chemotherapy or immunosuppressive treatment, with other acute or chronic viral infection and with diagnosis of intrahepatic or extrahepatic malignancy. Liver cirrhosis was defined when pathology was confirmed or two of 3 following criteria are met; Platelet <100,000/mm3, definitive cirrhotic figure by ultrasonography, and variceal change by endoscopy.

 

Results:

Of the 1025 samples, 241 could not be genotyped:  137 due to lack of material and 104 for other reasons, mostly very low viral load.

 

We found no gender difference in viral load, neither overall nor in genotype A, B, C and E.  A slight difference was observed in genotype D (p=0.048)

 

Distribution of gender over the 4 dominant genotypes differed markedly (p<0.001) while more men had genotypes A (p=0.015) or D (p<0.001). 

 

Women were younger than men, 28 years vs. 39 years (p=0.001) and 30 years vs. 33 years (p=0.026) in genotypes B and D respectively.  No age difference was observed in genotypes A or C, men 32 years vas. 35 years and 29 years vs. 31 respectively.

·        62% o patients with genotype A originated from Africa,

·        82% with genotype B and 95% with genotype C from East Asia,

·        62% with genotype D from  the Middle East and

·        75% with genotype E from Africa

 

Conclusion

The median of serum HBV-NDA levels was 100 times higher in genotypes B, C and E compared to genotypes A and D.

·        Men had a slightly higher viral load in genotype D compared to women.  A similar difference is not seen in the other genotypes.

·        More women than men had genotypes B or C, as opposed to genotypes A or D.

·        The distribution of genotypes was in keeping with the origin of the patients. 

 

820. Fusion HBx from HBV integrant in human hepatoma cell line is implicated in the development and progression of hepatocellular carcinoma

R. Muroyama; N. Kato; M. Otsuka; J. Chang; M. Omata

 

Background/Aim:

Hepatitis B virus (HBV) is a major risk factor for hepatocellular carcinoma (HCC), and HBx has been suggested to play an important role in hepatocarcinogenesis. However, the molecular mechanism of HBx in the pathogenesis of HCC remains unclear. In this study, we identified fusion HBx from HBV integrant in human hapatoma cell line, and investigated its role in the development and progression of HCC.

 

Methods:

1)     Using cassette-ligation-mediated PCR and 3’-/5’-RACE, we determined viral-host flanking sequences and full-length mRNA from HBV integrant in Hep3B cells, which had been established from HBV-related HCC.

2)     2) We silenced the expression of mRNA from HBV integrant with siRNA in Hep3B cells, and established stably knocked-down clones (KD cells). Using KD cells, we investigated the expression of fusion HBx by immunofluorescence staining.

3)     3) Using KD cells, we evaluated the effect of fusion HBx on cell growth and invasiveness by MTT/BrdU assay and matrigel invasion assay.

4)     4) We compared the transactivation activity and the ability to transform cells between wild HBx and fusion HBx by luciferase assay and soft agar assay.

 

Results:

1)     We identified full-length mRNA from HBV integrant with the length of 3725bp containing 1877bp human sequences at 3’ end, and fusion HBx (3’-truncated HBx + human peptides) was supposed to be translated.

2)     2) Nearly 90% reduction of fusion mRNA expression by siRNA was confirmed using Realtime PCR and Northern blot analysis, and fusion HBx was disappeared in KD cells by immunofluorescence staining.

3)     3) KD cells demonstrated significant reduction in cell proliferation and invasion ability compared to parental cells.

4)     4) Although fusion HBx had significantly decreased transactivation ability compared to wild HBx, fusion HBx had anchorage-independent growth ability in soft agar whereas wild HBx did not have.

 

Conclusion:

Fusion HBx from HBV integrant may play an important role in the development and progression of hepatocellular carcinoma.

 

841. Pattern of serial HBV DNA level and HCC development in Patients with Liver Cirrhosis

J. Kwon; J. Choi; H. Woo; J. Kim; C. You; S. Bae; S. Yoon; N. Han; C. Lee; Y. Lee

 

Aims:

Hepatitis B virus (HBV) DNA level was known to be a strong predictor of hepatocellular carcinoma (HCC) in REVEAL study. The majority of studies assessed the one time of HBV DNA level at the time of chronic hepatitis or/and HCC development. The aim of this study is to know whether the pattern of serial HBV DNA level is associated with the development of HCC.

 

Methods:

Among 352 patients who diagnosed a newly HBV related HCC between Jan 2005 and Dec 2007, 49 cirrhosis patients with serial HBV DNA level over 1 year before the HCC development was enrolled in the study. 98 consecutive cirrhosis patients with serial HBV DNA level during the same period were recruited as controls. 29 patients (59.2%) in HCC group and 52 patients (55.1%) in the control have been treated with antiviral agent. The changing patterns of serial HBV DNA were arbitrarily classified as follows; Type 1, persistently high pattern (≥104 copies/mL (C/mL))of DNA titer without interval change of 1-2 log C/mL, type 2, fluctuation pattern of DNA titer over the range of 2-3 log C/mL, type 3, decreasing pattern of DNA titer <104 C/mL, type 4, increasing pattern of DNA titer ≥104 C/mL, and type 5, persistently low pattern of DNA titer (<104 C/mL).

 

Results:

There was no difference of age, sex, HBe Ag status, antiviral therapy status and Child–Pugh class in HCC and control group. For a median 1,682 (365-2521) interval days, type 1 and type 2 were frequent in the HCC group compared with the control (28.6% vs 21.4%, 34.7% vs 32.7%, respectively). Frequency of type 3 and type 5 were lower in the HCC group than in the control (10.2% vs 13.3%, 14.3% vs 19.4%, respectively). With reference to the pattern of type 5, the patients of type 1 had the highest risk of HCC (HR=2.650; 95% CI, 1.061-6.617; P=0.037). On multivariate analysis adjusted by age, sex, HBe Ag status and antiviral therapy status, type 1 (HR 3.109; 95% CI, 1.161-8.327, P=0.024) and age (HR 1.042; 95% CI, 1.007-1.077, P=0.017) were independent risk factors of HCC. Among the patients with type 5, the number of patients with regenerating nodules on the ultrasonography was more in the HCC group than in the control (42.8% vs 27.7%).

 

Conclusions:

The cirrhosis patients with persistently high pattern (≥104 C/mL) of HBV DNA titer have more than three times of risk of HCC than those with persistently low pattern (<104 C/mL). Our results suggest that sequential analysis and persistent suppressing of HBV DNA are important in the monitoring of cirrhosis patients for the HCC development. Even in those with persistently low pattern of HBV DNA titer, the patients with regenerating nodules should be carefully observed for the HCC development.

 


Disease Progression : Reactivation of Infection with Chemotherapy

 

848. Rituximab Leads to Reactivation of Hepatitis B in Individuals with Resolved Infection

F. Metzler; I. Mederacke; M. P. Manns; H. Wedemeyer; K. Wursthorn

 

Background

Rituximab is a chimeric anti-CD20 antibody causing a massive reduction of B-cells without affecting their progenitors. It is indicated for the treatment of B-cell Non-Hodgkin Lymphoma (NHL) and Rheumatoid Arthritis (RA). There are reports that the administration of rituximab can lead to reactivation of hepatitis B virus (HBV) infection in HBV carriers resulting in hepatic failure and death. Current guidelines recommend HBsAg testing prior to initiation of chemotherapy and antiviral prophylaxis for hepatitis B carriers for a finite course.

 

Aim:

The aim of this study was to analyse the course of hepatitis B in patients with rituximab treatment.

 

Patients & Results

A total of 709 courses of rituximab (range 1-11) were given to 258 hospitalized patients at the Hannover Medical School in the years 2005 to 2007. Patients who received at least one course were included in this retrospective analysis. The median age at the time of the first dose was 46 years (range 2–87). 119 patients were female (46.1%), 139 were male (53.9%). For 78 patients there were no records of testing for hepatitis B (30.2%). In 6 patients only HBsAg has been checked (2.3%) and was negative. 70 patients have been tested negative for all relevant HBV serological markers with no further action taken (29.5%). 83 individuals had been vaccinated either before or during the course of rituximab treatment (32.2%). Titres of anti-HBs were found to drop over time. 21/258 patients were positive for anti-HBc (8.1%), 20 of those were also positive for anti-HBs antibody identifying them as individuals with a resolved hepatitis B infection.

 

One patient remained an inactive HBsAg carrier with detectable HBsAg, low HBV viremia at 1.1x103 IU/mL and normal ALT (0.4%). Median follow-up of anti-HBc+ patients was 88 days (range 0–1208) with no serological or virological data available for 7 patients after the last dose of rituximab. 3/20 patients with resolved HBV infection became HBV-DNA positive 92, 146 and 320 days after administration of 2 to 3 doses of rituximab. 2 of the 3 patients had protective anti-HBs antibodies prior to therapy and lost those to become HBsAg+. The third patient with HBV DNA recurrence remained anti-HBs and anti-HBc antibody positive. Two patients developed ALT flares of up to 5x ULN. There were no HBV associated deaths.

 

Conclusions

Our results show that reactivation of hepatitis B can occur. Decline and subsequent loss of neutralizing anti-HBs antibodies and reappearance of HBV DNA in patients with resolved hepatitis B must be considered after rituximab administration. Testing for HBV DNA and serology should be performed on a regular basis.

 

982. Oncologist Compliance with Recommendations for Hepatitis B Serostatus Testing: Experience from an academic hospital.

R. Lee; K. Vu; H. Shah; C. Bell; L. K. Hicks

 

Reactivation of hepatitis B (HBV) is a recognized complication of chemotherapy associated with substantial morbidity and mortality. Prophylaxis with lamivudine in chronic HBV carriers prior to chemotherapy can reduce the incidence and severity of reactivation hepatitis. Published guidelines recommend screening all patients receiving chemotherapy for HBV prior to initiation. There is currently no such policy at St. Michael’s Hospital in Toronto, Canada. St. Michael’s Hospital is a large teritiary referral centre with a high local prevalence of HBV infection.

 

Aim:

The objectives of this study are to 1) determine the frequency with which HBsAg is tested prior to intravenous (IV) chemotherapy at St. Michael’s Hospital, and 2) to compare the incidence of HBsAg testing pre-chemotherapy, with the incidence of cardiac function testing prior to potentially cardiotoxic chemotherapy.

 

Patients who were started on IV chemotherapy at St. Michael’s Hospital between March 2006 to March 2007 were retrospectively identified using existing pharmacy records. The frequency of HBsAg and LV function testing within 1 year prior to starting chemotherapy were determined via electronic chart review. Standard descriptive statistical modeling was used. Between March 2006 and March 2007, 208 patients were started on IV chemotherapy at St. Michael’s Hospital. 28 patients (13.5%) had HBsAg testing within 1 year prior to chemotherapy commencement. None of these patients were found to be HBsAg positive. In comparison, 138 patients were administered potentially cardiotoxic chemotherapy during the same time interval. All 138 (100%) patients had either a MUGA scan or an echocardiogram prior to chemotherapy; less than 1% had abnormal left ventricular function. There were no reports of HBV reactivation in any of the patients.

 

Results:

Despite the recognized importance of HBV testing prior to chemotherapy, the proportion of patients being tested for HBV status prior to chemotherapy at our academic institution is low. In contrast, cardiac function is routinely determined prior to cardiotoxic chemotherapy. The high rate of testing for cardiac function indicates that programs aimed at predicting and preventing side effects to chemotherapy can have a high compliance. Barriers to universal HBV testing likely include oncologists’ perception of the importance of testing as low and lack of a systematic requirement to test prior to chemotherapy. A quality assurance intervention will be performed in response to this data to determine whether the frequency of HBsAg testing can be increased to 90% or more at our academic institution.

 


Experimental Therapies

 

949. Serine Palmitoyltransferase Inhibitor Suppresses Hepatitis B Virus Replication

K. Tatematsu; Y. Tanaka; M. Sugiyama; M. Mizokami

 

Background:

Serine palmitoyltransferase (SPT) is a first-step enzyme in the sphingolipid biosynthetic pathway. Myriocin is an inhibitor of SPT and suppresses replication of the hepatitis C virus (HCV) replicon. However, it is still unknown whether this SPT inhibitor can suppress HBV replication.

 

Aim:

To investigate the anti-HBV effect of myriocin against intact HBV using Huh7 cells transfected with HBV (in vitro) and uPA/SCID mice with the liver replaced by human hepatocytes (chimeric mice) with HBV (in vivo).

 

Method:

Huh7 cells were transfected with plasmids carrying 1.24-fold the HBV genome of genotype C which is the most common in Asia. Myriocin was added in the medium of Huh7 cells at a final concentration of 0.1 to 20 μ M. After 72h incubation, culture supernatants were collected and HBV-DNA was quantified by real-time detection PCR to determine the 50% inhibitory concentration (IC50) of myriocin. These culture media were also injected into chimeric mice. Myriocin or pegylated interferon (PEG-IFN) and the combination were treated in chimeric mice (n=9) for a week, and HBV-DNA in mice serum was measured by real-time PCR.

PEG-IFN was subcutaneously injected at 30 μ g/kg (3 times injection per week). The amount of myriocin intraperitoneally injected was adjusted to be 1 mg/kg.

 

Result:

We succeed in reducing the HBV DNA levels in culture supernatants of Huh7 cells. As a result, IC50 value of myriocin was about 5 μ M. Although we administered myriocin into HBV-infected chimeric mice and it was not effective enough to reduce HBV-DNA levels, PEG-IFN reduced the HBV levels in serum to 1/10 of the levels prior to 8 days treatment. Interestingly, myriocin with PEG-IFN reduced the HBV levels to less than 1/1000 of the control levels. Concurrently, we monitored the concentration of human albumin and found slightly reductions only in the combined treatment group, but not reaching to significance.

 

Conclusion:

Myriocin could inhibit HBV replication in Huh7 cells. Although myriocin monotherapy was not effective, combined treatment with PEG-IFN cooperatively and synergistically inhibits the replication and proliferation of HBV in a chimeric mouse model of humanized liver.) Our results suggest that SPT may be an effective target of drugs designed to inhibit HBV replication, and that SPT inhibitors such as myriocin are good candidate on which to base the development of new anti-HBV drugs.

 

879. Efficacy of RNAi Constructs for Inhibiting Hepatitis B Virus Replication Has Implications for Antiviral Cell and Gene Therapy

M. Kumar; A. Follenzi; S. Gupta

 

Despite the success of interferon alfa (IFNα) and nucleoside/nucleotide-based antiviral therapies for HBV, treatment failures and emergence of drug-resistant HBV strains pose problems. Also, HBV infection of the graft after orthotopic liver transplantation may produce aggressive liver disease and transplant failure. Therefore, alternative approaches to control HBV replication are required.

 

Method:

To identify optimal strategies for suppressing HBV replication, in initial studies, we used two published shRNAs sequences inserted in a miR-30 backbone to target HBV. These sequences were cloned either individually or in combination in lentiviral vectors (LVs) under control of CMV promoter upstream of green fluorescent protein (GFP) to track gene expression. Anti-HBV LVs were designated LV-miK-GFP (HBVno.6 sequence from Kay et al. Nat Biotech. 2003;21:639), LV-miSH-GFP (pSUPERcore sequence from Shaul et al. Hepatology 2003;37:764) and LV-miK+SH for the combined sequence. Another LV was generated to express IFNα, which was PCR-cloned from human liver under the control of ECMV promoter. These LV constructs were efficiently expressed in liver cells, including HepG 2.2.15 cells capable of supporting HBV replication. Transduction of HepG2.2.15 cells with LV constructs either individually or in combination inhibited HBV replication as demonstrated by HBc protein and HBV DNA analyses. LV-miK-GFP was more effective than LV-miSH-GFP and both LVs were more effective than LV-IFNα-GFP in suppressing HBV replication. HBV suppression with the combined sequence LV-miK+SH was greater than either LV alone. We then studied HepG2 cells stably transduced with LV-miK-GFP, LV-miSH-GFP, or LV-miK+SH. These HepG2 clones were transduced with an HBV adenoviral vector (J. Liang, NIH) to establish productive HBV replication. Again, the combination LV-miK+SH most effectively decreased HBV replication. No cytotoxicity of change in proliferation of LV-transduced HepG2 was observed. Finally, we found that although hepatoblasts from fetal human liver were effectively transduced with HBV adenoviral vector, HBV replication was spontaneously prevented in these cells.

 

Conclusions:

Suitable antiviral shRNAs could be combined to effectively limit HBV replication in cells. The ability of fetal human hepatoblasts to spontaneously suppress HBV replication coupled with their known avidity for LV transduction and engraftment in the liver after LV transduction suggests that the RNAi approach will be practical for cell and gene therapy. Repopulation of virus-infected liver with disease-resistant cells should thus offer new opportunities for overcoming chronic HBV infection.

 

877. Drug-resistant and Wild-type Strains of HBV Are Inhibited Equally by Simvastatin

T. Bader; B. Korba; M. Bronze

 

The purpose of this report is to compare the in vitro inhibition of a panel of clinically relevant drug resistant viruses of HBV by simvastatin (SIM), lamivudine (3TC), and adefovir (ADV). We have previously reported significant in vitro activity of SIM against HBV. Inhibition occurs both for extracellular virion production and intracellular DNA intermediate forms (J Hepatol 2008;48:S207). Fifty percent reduction of extracellular and intracellular activity occurs at 3 μM and 9 μM, respectively. Others have shown in mammals that these concentrations of SIM should be easily achieved in humans with currently FDA-approved doses for cholesterol. This study was performed in collaboration with Georgetown University and Southern Research Institute under NIAID contracts, N01-AI-30046 and N01-AI-95364, respectively.

 

Methods:

Cultures of HBV-producing HepG2 2.15 cells were maintained as previously described (Antivir.Res.19:55-70, 1992). Confluent cultures of 2.2.15 cells were maintained on 96-well flat-bottomed tissue culture plates. Cultures were treated with nine consecutive daily doses of the test compounds. Activity against 3TC-resistant and ADV-resistant HBV mutants was tested in a 5-day assay using a tran¬sient transfection method. Antiviral activity was determined by quantitative Southern blot hybridization of intracellular HBV DNA replication intermediates.

 

Discussion:

The safety of SIM has recently been reported in a large RCT where SIM significantly reduced portal HTN and was tolerated better than placebo (J Hepatol 2008;48:S25). We have observed in vitro reduction of HCC proliferation in multiple cell lines by SIM (unpublished). Others have reported epidemiological data for the reduction of the prevalence of hepatocellular cancer and SIM use.

 

Conclusion:

Comparable efficacy against a panel of clinically relevant drug resistant viruses is yet another indicator of the potential usefulness of SIM for treatment of HBV. Studies of synergism, addition or antagonism of SIM with the nucleoside analogues 3TC, ADV, etc are being investigated now. Whether or not SIM is a new direction in combating the most common chronic infectious disease in the world remains to be determined.

 

Simvastatin (μM)

Lamivudine (μM)

Adefovir (μM)

HBV STRAIN

EC50

EC90

EC50

EC90

EC50

EC90

Wild-type

8.2

35

0.2

0.7

1.5

6.5

L180M

9.5

32

5.2

20

1.9

8.5

M204V

9

39

>100

>100

1.8

7

M204I

8.4

40

>100

>100

1.3

7.2

LM/MV

8.8

44

>100

>100

1.3

7.8

N236T

9.3

45

0.2

0.6

8.1

32

EC50, EC90 are concentrations for 50% and 90% viral inhibition, respectively.

 

875. Treatment of Chronic Viral Hepatitis in Woodchucks by Prolonged Intrahepatic Expression of Interleukin-12

I. Otano; J. Crettaz; L. Ochoa; A. Benito; A. Paneda; I. Aurrekoetxea; A. Astudillo; F. Kreppel; S. Kochanek; J. Ruiz; S. Menne; J. Prieto; G. Gonzalez-Aseguinolaza

 

Background and Aims:

Chronic hepatitis B is a major cause of liver-related death worldwide. Interleukin-2 (IL-12) induction accompanies viral clearance in chronic hepatitis B virus (HBV) infection. We tested the therapeutic potential of IL-12 gene therapy in woodchucks with chronic woodchuck hepatitis virus (WHV) infection, a condition that closely resembles chronic hepatitis B.

 

Methods:

Eight woodchucks treated by intrahepatic injection of a helper-dependent adenoviral vector encoding murine IL-12 under the control of a liver-specific RU486-responsive promoter. IL-12 expression was induced for 42 days. Five animals with viremias over 10e11 viral genomes/ml (vg/ml) received combination therapy with nucleos(t)ide analogues for four weeks and IL-12 gene therapy for 42 days.

 

Results:

All woodchucks with viral load below 10e10 vg/ml showed a marked and sustained reduction of viremia accompanied by loss of WHVe and WHV surface antigens and improved liver histology. In contrast, none of the woodchucks with viremia higher than 10e10 vg/ml responded to therapy. In the responder animals, the antiviral effect was associated with induction of T cell immunity against viral antigens and reduction of hepatic expression of Foxp3, the nuclear factor that characterizes regulatory T cells. The animals that received combination therapy experienced a transient decrease of viremia with relapse after cessation of therapy.

 

Conclusions:

IL-12-based gene therapy is an efficient approach to treat chronic hepadnavirus infection. This therapy is able to break immunotolerance and to control viral replication in all cases with viral load below 10e10 vg/ml, which is the situation of most patients with chronic hepatitis B.

 

821. Surface Antigen Seroconversion after Allogenic Hematopoietic Stem Cell Transplantation in Patient with Chronic Hepatitis B Virus Infection

S. Woo; C. Kim; S. Nam; J. Choi; S. Cho; J. Yang; J. Han; Y. Lee

 

Background:

In chronic hepatitis B virus (HBV) infection, antiviral drugs are the effective way of restraining disease progression, but small number of cases showed surface antigen (HBsAg) seroconversion by antiviral drugs. Though some reports showed the effect of immune cell therapy in which sensitized lymphocyte induced HBsAg seroconversion, the effect of immune cell therapy is still uncertain. The aim of this study was to investigate the effect of allogenic hematopoietic stem cell transplantation (allo-HSCT) on HBsAg seroconversion in patients with chronic HBV infection.

 

Methods:

HBsAg and antibody to HBsAg (anti-HBs) were surveyed retrospectively in total 537 patients who underwent allo-HSCT from January 2000 to June 2007 in single bone marrow transplantation center. Forty-five (8.4%) of 537 allo-HSCT patients showed HBsAg positive/Anti-HBs negative. HBV serologic markers and clinical variables were inspected in these 45 HBsAg positive allo-HSCT recipients and their donors before and after allo-HSCT. Median observational period was 36.6 months (range 3~80 months) after transplantation.

 

Results:

Among 45 HBsAg positive patients, 21 were transplanted with hematopoietic stem cells (HSC) from anti-HBs positive donors and 24 were transplanted with HSC from anti-HBs negative donor. Compared to anti-HBs negative donor group (0/24, 0%), anti-HBs positive donor group (4/21, 19.0%) showed frequent HBsAg seroconversion rate significantly (P=0.04, Fisher’s Exact test). Among these 21 HBsAg positive recipients who have anti-HBs positive donor, 13 were male and 8 were female, mean age was 46 years (range 22~60), and all were leukemia. Among them, 5 were chronic active hepatitis B, 15 were inactive carriers and 1 was under immune tolerance state. According to hepatitis e antigen (HBeAg) status, 5 were HBeAg positive and 16 were HBeAg negative before allo-HSCT. Four who showed HBsAg seroconversion after allo-HSCT were all HBeAg negative and inactive carriers before allo-HSCT.

 

Conclusion:

The rate (19.0%) of HBsAg seroconversion in chronic HBV infection patients who underwent allo-HSCT is so high compare to natural HBsAg seroconversion. This high rate of HBsAg seroconversion shows a possibility of immune cell therapy in chronic HBV infection.

 

823. Efficacy of Hepatitis B Vaccine Against Antiviral Drug-resistant Hepatitis B Virus Mutants in the Chimpanzee Model

S. Kamili; T. Sozzi; G. Thompson; K. Campbell; C. M. Walker; S. Locarnini; K. Krawczynski

 

Hepatitis B virus (HBV) mutants resistant to treatment with nucleoside or nucleotide analogues and those with the ability to escape from HBV neutralizing antibody have the potential to infect HBV-vaccinated individuals. To address this potential serious public health challenge, we tested the efficacy of immunity induced by a commercial hepatitis B vaccine against a tissue culture-derived, clonal HBV polymerase mutant in HBV seronegative chimpanzees. The polymerase gene mutant, generated in vitro by genetic engineering technology, contained a combination of three mutations (rtV173L+rtL1806M+rtM204V) two of which resulted in changes to the overlapping viral envelope of the hepatitis B surface antigen (sE164D+sI195M).

 

The in vitro generated polymerase gene mutant inoculum was proven infectious in three HBV seronegative chimpanzees. Cloning and sequencing experiments determined that the three mutations in the polymerase gene mutant remained stable. Two naïve chimpanzees were vaccinated with a pediatric dose of hepatitis B vaccine, which resulted in both humoral (steady levels of >75 mIU/L anti-HBs) and cellular immune responses against HBsAg in the vaccinated chimpanzees. The two vaccinated chimpanzees were challenged with infectious polymerase gene mutant inoculum; following challenge anti-HBs titers increased more than 100 fold, anti-HBc was detected on day 7 in one chimpanzee and on days 7, 10 and 17 in the second chimpanzee. Analysis of cellular immunity in one chimpanzee also provided evidence of productive infection with the polymerase gene mutant. Specifically, a transient T cell response targeting the polymerase protein was detected on day 28 post-infection by the interferon-γ ELISpot assay. T cell lines specific for the HBV polymerase protein derived from the peripheral blood of the chimpanzee on day 28 were CD4+, suggesting that the response was mediated in part by T helper cells.

 

Conclusion:

The data from this study showed vaccination of naïve chimpanzees with commercial hepatitis B vaccine did not confer sterilizing immunity against a clonal rtV173L+rtL1806M+rtM204V HBV polymerase gene mutant.

 

824. New Insights into HBV Pathogenesis from Kinetic Analysis of HBeAg Titre Decay During Potent Antiviral Therapy.

A. J. Thompson; E. Shudo; R. Ribeiro; K. Visvanathan; P. V. Desmond; G. K. Lau; A. S. Perelson; S. Locarnini

 

Background:

HBeAg seroconversion is one of the major therapeutic endpoints used when treating HBeAg-positive chronic hepatitis B (CHB). HBeAg titre has been proposed as a surrogate marker for infected liver cell mass in the setting of CHB (1). This assumption has not been tested in vivo. During antiviral therapy, it is not known whether HBeAg declines in a typical biphasic pattern, as is frequently seen for HBV DNA. The aim of this study was to define the kinetics of HBeAg decline in a cohort of patients treated with potent nucleos(t)ide analogue therapy.

 

Methods:

Mathematical modelling was used to analyze and compare the kinetics of the decline in serum HBV DNA and HBeAg titre in a cohort of 25 HBeAg-positive CHB patients treated with adefovir monotherapy (n=13) or adefovir/emtricitabine combination (n=12) for 48 weeks (2). During therapy, serum samples were collected every 2nd day to 2 weeks, weekly to 4 weeks and then every 4 weeks. HBeAg titre was measured using the Architect platform (Abbott, Ill.) and expressed in Paul-Ehrlich IU. Detailed virological characterization included genotype and PC/BCP sequence.

 

Results:

The pattern of HBV DNA decline was biphasic in all patients. A prominent lag phase prior to decline in HBeAg was noted. In all patients, the lag in HBeAg was longer than in HBV DNA (average 3.5 wks (0 – 16 wks) vs 0.01 weeks for HBV DNA, p<0.05). The duration of this lag phase was not correlated to baseline variables (including HBV DNA, HBeAg), or the slope of 1st / 2nd phase HBV DNA decay. In a number of patients the HBeAg lag phase continued into the 2nd phase decline of HBV DNA, suggesting a disconnect between infected cell number and HBeAg production. HBeAg seroconversion was not observed; even when the viral load fell below the detection limit (10/25) the serum concentration of HBeAg remained detectable. The baseline level of both HBV DNA and HBeAg were significantly higher (p<0.01) in patients infected with wild type HBV compared to PC/BCP variant HBV. However, the presence of PC/BCP variant HBV did not influence the kinetics of HBeAg decline in these patients.

 

Conclusion:

This is the first mathematical analysis of the kinetics of HBeAg titre during potent antiviral therapy. A lag phase was identified prior to HBeAg titre decline, consistent with a drug-induced reduction in cccDNA level through inhibition of the intracellular recycling pathway. Furthermore, the occurrence in a number of patients of a prolonged lag phase despite 2nd phase HBV DNA decline suggests an extrahepatic source of HBeAg production.

(1) Nowak M, PNAS, 1996, 93(9): 4398

(2) Lau G, AVT, 2007, 12(5): 705

 

826. CD4+LAP+ Regulatory T Cells Mediate the Enhancement of the Anti-HBV Immune Response: A novel method for an adjuvant-dependent augmentation of anti-viral immunity

M. Mizrahi; G. Lalazar; A. Ben Ya'akov; T. Adar; L. Zolotarov; Y. Ilan

 

Latency-associated peptide (LAP) is an amino-terminal domain of the TGF-beta precursor peptide and remains non-covalently associated with TGF-beta. after cleavage, forming the latent complex. The presence of membrane-bound LAP on the surface of regulatory T cells (Tregs) is associated with their regulatory function. Tregs responses may be either beneficial or detrimental to those infected with HBV, by either limiting liver immunopathology or suppressing protective T-cell responses.

 

Aim:

To determine the ability to enhance the anti-HBV immunity by promoting LAP+ Tregs.

 

Methods:

Thirteen groups of mice were vaccinated with HBV vaccine along with oral (PO), intraperitoneal (IP), intrarectal (IR),intranasal (IN) administration of different beta-glycosphingolipid adjuvants including beta-glucosylceramide (GC), beta-lactosylceramide (LC), or there combination (IGL. Mice were followed for anti HBV response and for the induction of Tregs.

 

Results:

A five fold increase in spleen-derived CD4+LAP+ lymphocytes was induced by oral administration of GC with the HBV vaccine (0.62 vs.0.12%, for GC vs. controls, respectively, p<0.001), and was associated with a three fold elevation of the anti-HBV antibody titer (945 vs. 312 mIU/ML, for GC vs. controls, respectively, p<0.02). Mode of adjuvant administration determined the increase in CD4+LAP+ and anti HBV antibody titer. Spleen levels of CD4+LAP+ increase with oral, IP, and IN administration of GC, but to a much lower extent following IR administration (0.62, 0.78, 0.64, and 0.14%, for GC, PO, IP, IN, and IR, respectively, p<0.001). These data correlated with the anti-HBV antibody titers (604, 945, 620, and 283 mIU/ML, for GC, PO, IP, IN, and IR, respectively, p<0.03). Interestingly, a negative correlation was found between antibody titer and CD4+CD25+ cells (0.5, 0.45, 0.92, vs. 1.51%, for IP, PO, IN, vs. control, respectively, p<0.001), and with NKT regulatory cells (6, 4.2, 1.03 vs. 9.22%, for IP, PO, IN, vs. control, respectively, p<0.001). Intrahepatic derived CD4+LAP+ increased mainly in IP GC administered mice (0.23, 0.62, 0.38, 0.13%, for PO, IP, IN, and IR, respectively, p<0.001). The use of LC or IGL as adjuvants showed a similar correlation between CD4+LAP+ and anti-HBV antibody titer (CD4+LAP+ levels were 0.65 and 1.26 vs. 0.14%, p<0.001; anti HBV titers were 763, 520.86, vs. 312 mIU/ML, for LC and IGL vs. controls, respectively, p<0.01). In summary, CD4 + LAP+ regulatory lymphocyte is a novel subset of Tregs which play a role in augmenting the anti-HBV immunity. The use of different adjuvants can promotes this subset of Tregs and serves as a tool for the design of anti-HBV immune-based therapies.

 


Diagnostic Tools

 

933. Hepascore: A Mon-invasive Marker for Advanced Fibrosis and Response to Antiviral Therapies in Chronic Hepatitis B Infection

S. C. Raftopoulos; E. Rossi; B. De Boer; G. P. Jeffrey; D. J. Speers; G. C. MacQuillan; L. A. Adams

 

Staging liver fibrosis in chronic hepatitis B (CHB) infection is important for prognostication and management, however few validated non-invasive markers of liver fibrosis are currently available, and it is unknown whether these are responsive to changes in fibrosis over time. We examined the accuracy of a non-invasive model developed for hepatitis C infection (Hepascore) in determining liver fibrosis in CHB infection, and secondly, the longitudinal response of Hepascore over time among patients with or without anti-viral therapy.

 

Methods:

Patients with CHB infection had serum taken within 6 months of a liver biopsy analysed for hyaluronic acid, alpha 2 macroglobulin, bilirubin, gamma glutamyltransferase and combined with age and gender to produce a Hepascore ranging from 0-1. A subset of patients with serum stored over time had serial Hepascore’s performed. Liver biopsies were interpreted by a single liver pathologist and scored according to METAVIR. Fibrosis was quantified as significant (F2-4), advanced (F3-4) or cirrhosis (F4). Accuracy of Hepascore was calculated by receiver operator characteristic (ROC) curves.

 

Results: 70 patients (73% male) with a mean ±SD age of 42.5 ±12.6 years underwent biopsy. The majority were Asian (59%) and eAg negative (60%). The median time between serum collection and biopsy was 0.7 months (range 0-6). Thirty two (46%) had significant fibrosis, 21 (30%) had advanced fibrosis and 12 (17%) were cirrhotic. Hepascore had an area under the ROC curve for significant fibrosis, advanced fibrosis and cirrhosis of 0.91 (95% CI 0.84-0.98), 0.94 (95% CI 0.89-0.99) and 0.92 (95% CI 0.85-0.99) respectively. A cutoff of 0.62 provided sensitivity, specificity, positive and negative predictive values (NPV) of 85%, 84%, 82% and 87% respectively for significant fibrosis. The same cutoff provided a 100% NPV for advanced fibrosis and cirrhosis. Among 18 patients who were monitored over a median of 1.7 (range 0.4-6.8) years without anti-viral treatment, mean Hepascore tended to increase over time (0.59 ±0.30 to 0.72 ±0.29, p=0.09). Among 21 patients who had anti-viral therapy over a median of 2.9 (0.8-7.0) years, mean Hepascore did not change significantly (0.65 ±0.29 to 0.61 ±0.35, p=0.56). The annual rate of change of Hepascore was significantly higher in those that did not receive anti-viral therapy compared to those who did (0.07 ±0.15 vs -0.04 ±0.13, p=0.04).

 

Conclusion:

Hepascore is an accurate predictor of liver fibrosis in patients with CHB infection with potential to avoid liver biopsy. The rate of change for hepascore varies with anti-viral therapy suggesting it is responsive to changes in fibrosis over time.

 

934. Quantitative Assessment of Serum HBsAg Level Using the Elecsys® HBsAg II Screening Assay: Results of a feasibility study

F. Bonino; B. Ofenloch; W. Melchior; B. Upmeier; D. Rössler; W. van der Helm

 

Background/Aims:

Recent data suggest that on-treatment monitoring of HBsAg during peginterferon alfa-2a therapy in patients with chronic hepatitis B may be useful for predicting response to therapy. We hypothesized that it may be possible to use an available qualitative HBsAg screening assay to determine HBsAg quantitatively. By selection of appropriate dilutions, limitations of the measuring range can be overcome, allowing quantification of HBsAg in a suitable linear range. We evaluated the feasibility of a simple algorithm for quantification of serum HBsAg based on the Elecsys® HBsAg II screening assay (Roche Diagnostics).

 

Methods:

A total of 29 patient serum samples were tested according to the following algorithm. Samples were tested at a dilution of 1:400 in serum negative for HBsAg and anti-HBs. If the cut-off index (c.o.i.) was ≥1, results were expressed as c.o.i x 400. If the c.o.i was <1, the sample was retested undiluted. Samples giving a value >300 c.o.i. were re-tested with 1:8000 dilution and results were calculated as c.o.i x 8000. Quantitative results (IU/mL) were determined by reading off resulting c.o.i. values from a master calibrator curve (target values assigned with WHO standard material).

 

Results:

There was a good correlation between the c.o.i and IU/mL and this was linear over the entire range tested (5–6 logs) [Figure]. The concentration of the samples was in the range <0.05–46382 IU/mL. 21/29 samples gave a value <1 c.o.i. after 1:400 dilution and were re-tested undiluted. 2/29 samples gave a value >300 c.o.i. and required retesting at 1:8000 dilution.

 

Conclusions:

This simple testing algorithm used in connection with the qualitative Elecsys®HBsAg II screening assay appears to be suitable for the effective quantification of serum HBsAg in clinical samples. This assay may provide useful information during on-treatment monitoring of HBsAg levels to guide patient management.

 

939. Changes in Liver Stiffness During Entecavir Therapy in Patients with Chronic Hepatitis B

S. Lim; J. Cheong; S. Cho

 

Background/Aims:

Liver stiffness (LS) measurement is a non-invasive method for assessment of fibrosis in patients with liver disease. The usefulness of LS measurement for the follow-up of fibrosis in hepatitis B patients receiving antiviral therapy is unknown. The aim of this study was to evaluate changes in LS and factors associated with LS changes in chronic hepatitis B (CHB) patients receiving entecavir.

 

Methods:

Study population included 24 CHB patients and 22 cirrhotic (LC) patients. All patients received entecavir more than 12 months. LS and HBV DNA levels were measured by Fibroscan and b-DNA assay, respectively. LS was measured at baseline and 48 weeks of therapy. Results: Among 46 patients treated, 42 patients (91%) achieved HBV DNA < 2×103 copies/mL, and 31 patients (67%) achieved ALT normalization at 48 weeks of therapy. Mean LS value at baseline was 28 KPa (13 KPa in CHB, 28KPa in LC). After 48 weeks of therapy, mean LS value decreased significantly to 14.5 KPa (7 KPa in CHB, 14 KPa in LC). High AST level at baseline was significantly associated with decrease in LS value during therapy (P=0.05), LS value tended not to decrease in patients with abnormal ALT level at 48 weeks of therapy. Decrease in LS value during therapy was significantly correlated with decrease in AST level (P=0.02), ALT level (P=0.01), and APRI score (P=0.04).

 

Conclusions:

LS is significantly reduced during entecavir therapy. Regression of hepatic inflammation and fibrosis during therapy may result in reducing LS.

 

964. Rapid, Reliable and Cost-effective Detection of Hepatitis B Virus in Human Plasma

C. M. Habibullah; Y. Naresh; C. Madhavi; H. Aejaz

 

A number of common mutations have been described in HBV strains and recombinant genomes have been reported. These include variability within the basic core promoter, precore/core and the pre-S1 gene, which shows great heterogeneity. Designing primer and probe sets to detect and quantify all HBV variants by real-time PCR is challenging at best. HBV viral load was the strongest predictor of progression to cirrhosis and directly related to necroinflammation and progression of fibrosis.

 

Materials and Methods:

A total of 250 samples consisting of HBV positive (n=100); HBV, HCV and HIV negative (n=100); HCV positive (n=25) and HIV positive (n=25) were collected from out patient unit, Centre for Liver research and diagnostics. Complete Human HBV DNA sequences (n=944) were selected from the National centre for biotechnology information (NCBI) nucleotide database, Primers and probes were designed and synthesized from core, surface and x region. Real-time based quantification was done using standard kit and in-house generated standards and RT-PCR protocols. Results and discussion: The standard calibration curve was generated by using the Smart Cycler II software and serial dilution 102 to 108. The calibration curve was linear in a range from 102 to108 copies/ml, with R2 value of 0.999. Reproducibility as measured by dual testing of triplicates of serum samples was acceptable, with coefficients of variation at 6.5%, 7.5% and 10.5%. Our results showed that amplification performance was good in case of primer and probe set designed from x region.

 

We constructed a plasmid with x region using p-GEMT easy vector for being used as a control for deriving standard curve. Out of 100 negative samples screened by ELISA and standard RT-PCR kit, one sample was detected as positive with the in-house developed RT-PCR assay, the positivity of the sample was confirmed by sequencing the amplified product, NCBI accession EU684022. To avoid the possibility of contamination, this sample was simultaneously processed twice using both standard and in-house assays. Out of four oligo sets designed for HBV detection by RT-PCR, X gene set showed highest specificity and sensitivity (98%) followed by surface (74%) and core (70%).

 

Conclusion:

This assay is reproducible showing limited inter and intra assay variability and good amplification efficacy in different HBV genotypes and serotypes. We demonstrate that the results of our assay correlated well with the standard kit for HBV viral load monitor. In this study we eliminated the drug inducible mutation and variable regions for selecting the conserved portion of viral genome for effective oligo designing and evaluated the same.

 

947. Development of Kinetic Amplification-Based Assay for Detection and Quantification of HBV DNA

C. L. Wong; L. Ku; A. Ying; A. Jacobson; S. K. Aung; Z. Li; D. G. Sherman; L. Shen

 

Background:

Hepatitis B virus (HBV) can cause lifelong infection, cirrhosis of the liver, liver cancer, liver failure and death. Worldwide, an estimated 400 million people are persistently infected with HBV. HBV quantification is useful for therapeutic monitoring of disease progression and detecting resistance to antiviral therapy. The authors are developing a highly sensitive, automated, kinetic amplification assay for the detection and quantification of HBV DNA levels in serum or plasma of HBV-infected individuals.

 

Method:

The kinetic amplification-based HBV assay was performed using the VERSANT kPCR Molecular System*, which is under development by Siemens Healthcare Diagnostics. The HBV assay includes HBV primers and probe sequences, a noncompetitive internal control (IC) to monitor the sample preparation process, and dUTP/UNG to eliminate chemical contamination during a heat-labile DNA glycosylase step. The assay was standardized with HBV recombinant DNA quantified by a phosphate assay traceable to the US-NIST phosphate standard. Assay sensitivity, linearity and precision were evaluated with an HBV viral panel with concentrations ranging from 25 copies/mL to 2.0E+9 copies/mL. Specificity of the assay was determined by testing 300 unique HBsAg-negative specimens representing 150 serum and 150 EDTA-plasma specimens from normal blood donors. Equivalent detection of the assay was demonstrated by using plasmid DNA samples representing multiple isolates from eight HBV genotypes (A–H).

 

Results:

The assay dynamic range was 28 copies/mL to 1.0E+09 copies/mL in plasma or serum of HBV-infected individuals, with a sensitivity of below 28 copies/mL of HBV DNA. All panel levels demonstrated good reproducibility, with total CVs of quantitation <25% over most of the range of the assay. The specificity was 100.00%, achieved by testing 300 negative EDTA-plasma and serum samples from normal blood donors. The assay detected eight HBV genotypes (A–H) equivalently, with ±0.5 log relative difference.

 

Conclusion:

The automated HBV kinetic amplification-based assay currently in development demonstrated a wide dynamic range of 28 copies/mL to 1.0E+09 copies/mL, with sensitivity of 28 copies/mL. In addition, the assay performed with excellent specificity and good precision. The incorporation of UNG eliminates the potential for contamination up to 1.0E+9 amplicons per reaction. A DNA molecule added during sample processing acts as a noncompetitive internal control.

* System and assay not commercially available

 

899. Utility of Liver Stiffness Measured by Transient Elastography for Determining Significant Liver Fibrosis in Patients with Chronic Hepatitis B

T. Tanwandee; P. Charatcharoenwitthaya; V. Viboolsirikul; W. Chotiyaputta; S. Chainuvati; M. Maneerattanaporn; V. Prachayakul; S. Pongprasobchai; S. Manatsathit; S. Leelakusolvong; N. Pausawasdi; W. Srikureja; U. Kachintorn

 

Background:

Transient Elastography (TE) is a reliable non-invasive predictor of hepatic fibrosis but data on TE in patients with chronic hepatitis B (CHB) is still limited.

 

Aim:

To prospectively evaluate the accuracy of TE for diagnosis of hepatic fibrosis in patients with CHB.

 

Methods:

One hundred and four consecutive patients with CHB who underwent liver biopsy prior to antiviral therapy at Siriraj Hospital between May 2007 and December 2007 were enrolled. Liver stiffness measurement with TE (Fibroscan®) was performed by an experienced operator who was unaware of the clinical, biochemical, and radiological data. Liver histology done on the same day was graded by pathologists using the METAVIR classification. Spearman rank correlation was used to evaluate the association between liver stiffness and liver histology. Area-under-receiver-operating-curves (AUROC) was used to evaluate the accuracy of TE in diagnosing significant fibrosis (F≥2) and advanced fibrosis (F≥3).

 

Results:

Patients had a mean age of 44 years and 63% were male. Mean body mass index at baseline (standard deviation) was 23.6 (4.2) kg/m2. The HBeAg was positive in 27 (26%) patients, and 77 (74%) patients were HBeAg-negative. Mean level of serum aspartate aminotransferase, albumin, and platelet count was 49 U/L, 4.4 g/dl, and 219,000/ml, respectively. The median liver stiffness was 6.9 kPa (range 3.3-46.4 kPa). The median liver stiffness measurement value correlated well with histological fibrosis grade (r=0.719, p<0.001) and good with necroinflammation activity (r=0.656, p<0.001). AUROC for diagnosis of significant fibrosis was 0.757 (95% CI: 0.66, 0.84) and advanced fibrosis was 0.793 (95% CI: 0.70, 0.87). Optimal liver stiffness value was 6.9 kPa for diagnosis of significant fibrosis, which yielded a sensitivity of 70%, specificity of 79%, positive predictive value (PPV) of 82%, and negative predictive value (NPV) of 66%. Optimal liver stiffness value was 7.3 kPa for diagnosis of cirrhosis, which provided a sensitivity of 93%, specificity of 61%, PPV of 31%, and NPV of 98%.

 

Conclusion:

Liver stiffness is a reliable predictor of hepatic fibrosis in patients with CHB. This non-invasive measurement should help clinicians in identification of significant fibrosis in this population.

 

988. Application of Magnetic Bead-based Proteomic Fingerprinting Technology to the Detection of Liver Fibrosis in Patients with Chronic Hepatitis B Infection

M. Wong; T. Poon; V. W. Wong; J. Yu; Y. Chan; K. Yu; J. Sung; H. Chan

 

Most noninvasive predictive models of liver fibrosis are complicated and have suboptimal sensitivity. Serum proteomic fingerprinting by SELDI ProteinChip technology is a potential approach for detecting liver fibrosis in patients with chronic hepatitis B virus (HBV) infection. Unfortunately, the diagnostic SELDI peak cannot be directly recovered for protein sequence analysis. To overcome this problem, a similar mass spectrometry-based technology which is based on chromatographic magnetic beads has been developed by our team. This technology allows simultaneous analytic profiling and obtaining preparative fractions for subsequent protein identification experiments. Serum samples were collected from 100 treatment-naïve chronic HBV infected patients [76 males and 24 females; mean (SD) age, 35.0 (10.3) years] who underwent liver biopsy as per protocol procedure for clinical trials. 57 patients had minimal fibrosis (Ishak score = 0 or 1); 22 had mild fibrosis (Ishak score = 2); 9 had moderate fibrosis (Ishak score = 3 or 4), 6 had severe fibrosis (Ishak score = 5); and 6 had probable/definite cirrhosis (Ishak score = 6). The mean alanine aminotransferase, HBV DNA and platelet count were 144 IU/L, 6.8 x 108 copy/mL and 186 x 109/L, respectively. Serum samples were subjected to quantitative proteomic profiling by using C18 hydrophobic magnetic bead and strong anion exchange bead.

 

Linear regression method (forward stepwise) was used to develop a diagnostic model for predicting the degrees of liver fibrosis from the serum proteomic profiles, clinical data, serological data, and biochemical data. Two proteomic features and platelet counts were included in the final diagnostic model. Other factors including liver biochemistries or HBV DNA were not included for predicting liver fibrosis. Ten-fold cross-validation showed that ROC curve area of the diagnostic model was 0.781 (p < 0.0005), and the overall accuracy was 75% (76% sensitivity, 72% specificity).

 

Conclusion:

At a specificity of 89%, the sensitivity was 67%, suggesting that combined use of serum proteomic fingerprinting and platelet count could avoid about 65% of liver biopsy for detection of liver fibrosis in the CHB patients. In conclusion, serum proteomic fingerprint is a good non-invasive method to supplement liver biopsy for assessment of liver fibrosis.

 

975. Do Serum ALT and Quantitative HBV DNA Levels Predict Histologic Activity in Patients with Decompensated Cirrhosis? Analysis in an explanted liver

J. Kim; J. Choi; J. Kwon; C. You; S. Bae; S. Yoon; S. Cho; J. Yang; J. Han; N. Han; S. Choi; Y. Lee; C. Lee; E. Jung

 

Previous studies of antiviral therapy in patients with decompensated cirrhosis showed that the nucleos(t)ide analog was well-tolerated and could improve clinical outcomes. The aim of this study was to assess the relationship between pretransplantational serum ALT and HBV DNA levels and the degree of histologic activities in explanted livers. Ninety consecutive patients undergoing liver transplantation between January 2000 and March 2008 for HBV-related decompensated cirrhosis were evaluated. The patients with fulminant or subfulminant diseases, and hepatocellular carcinoma were excluded. None of the patients had received nucleos(t)ide analogs for more than 2 weeks before transplantation. The extent of histologic activity was graded by assessing the severity of lobular and portoperiportal inflammations (absent or minimal, mild, moderate, severe).

 

Serum ALT and HBV DNA levels are compared with histologic activities. The mean ALT level was 42.7±36.8 U/L and the mean HBV DNA level was 5.18±1.81 log10 copies/mL. There were no differences in ALT levels according to lobular and portoperiportal activities (P=.105, P=.257, respectively), and there were no differences in HBV DNA levels according to histologic grades (P=.552, P=.117, respectively). Among 29 patients (32.2%) with HBV DNA level below 2000 copies/mL, 14 patients (48.3%) had moderate and severe portoperiportal activities and the median ALT level was 28 U/L (range: 12-135). There was no correlation between ALT and HBV DNA levels.

 

Conclusion:

In conclusion, although the patients with decompensated cirrhosis show normal ALT and low level of HBV DNA (<2000 copies/mL), they could have significant portoperiportal activities.

 

987. Clinical Usefulness of Liver Stiffness Measurement in HBeAg-positive Chronic Hepatitis B Patients with ALT Level < 2 Times Upper Limit of Normal

J. Choi; D. Kim; J. Park; S. Ahn; K. Yoon; J. Lee; J. Kim; Y. Paik; K. Lee; B. Moon; C. Chon; K. Han

 

Background/Aims:

In patients with HBeAg positive chronic hepatitis B (CHB) having alanine aminotransferase (ALT) levels less than two 2 times normal, antiviral treatment may be initiated if moderate or severe necroinflammation or significant fibrosis is seen on liver biopsy. We investigated the usefulness of liver stiffness measurement (LSM) using FibroScan® in patients with CHB who were HBeAg-positive and had ALT levels less than two times normal and asked whether we could determine the need for antiviral therapy in these patients using LSM instead of liver biopsy.

 

Subjects & Methods:

Liver biopsy and LSM were carried out in patients with CHB who were HBeAg-positive in Severance Hospital between December 2005 and May 2008. Patients with a serum HBV DNA ≥5log10copies/ml and an ALT level less than two times normal were enrolled. Patients suspected of having liver cirrhosis clinically were excluded.

 

Results:

Forty-eight patients underwent liver biopsy and LSM. Their mean age was 41.7±13.3 years and 28 (58.3%) were men. The mean body mass index (BMI) was 23.3±3.1 kg/m2 and the mean ALT level was 43.2±18.0 IU/L. The mean platelet count was 192,670±68,680 /mm3, the mean size of the spleen was 9.6±1.4cm, and the mean HBV DNA level was 8.0±8.3 log10copies/ml. LSM was correlated with hepatic inflammation and fibrosis (r=0.66, p<0.001 and r=0.308, p=0.039). The areas under the receiver operating characteristic curve (AUROC) of the LSM for ≥F2, ≥F3, and F4 were 0.88 (95% CI: 0.76-1.00), 0.86 (0.75-0.97), and 0.86 (0.76-0.97), respectively. The cutoff value of ≥F2 with the optimal diagnostic accuracy was 7.7 kPa (sensitivity 88%, specificity 88%). The cutoff value of liver cirrhosis was 10.4 kPa (sensitivity 79%, specificity 83%). The AUROC values for the diagnosis of hepatic inflammation ≥A2 or hepatic fibrosis ≥F2 was 0.88 (0.75-1.00). The cutoff value was 7.7 kPa (sensitivity 85%, specificity 86%).

 

Discussion:

LSM using FibroScan® was effective for predicting inflammatory activity and fibrosis in patients with CHB who were HBeAg-positive and had ALT levels less than two times the upper limit of normal (ULN). LSM offered the best diagnostic performance for significant fibrosis (F≥2). The cutoff value was 7.7 kPa. LSM has the potential to reduce the number of liver biopsies in patients with CHB who require antiviral therapy.

 

891. A Novel Three-Dimensional Culture System of Immortalized Hepatocytes; Assessment on HBV Vaccine Escape Mutant Strain Infectivity and on Ability of anti-PreS2 to Prevent the Infection

A. Kusakabe; Y. Tanaka; T. Yamaguchi; M. Segawa; F. Kurbanov; M. Sugiyama; F. Sugauchi; M. Hijikata; K. Shimotohno; M. Mizokami

 

Background:

We reported development of clinical hepatitis in a healthy individual with antibody to hepatitis B virus (HBV) surface antigen (HBsAg) and isolation of a strain with sG145R amino acid replacement (J Gastrol 2008), suggesting that mutations within the ‘a’ determinant may affect antigenicity and enable HBV to evade the neutralizing antibodies. Investigations on vaccine escape mutants (VSM) have been hampered by the absence of accessible in vitro infection modeling tools. Recently, we applied three-dimensional (3D) principle to culture immortalized human hepatocytes (HuS-E/2) and reported on ability of this system to maintain HBV infection and replication (AASLD 2007).

 

Aims:

To examine the infectivity of the sG145R mutant in comparison to the wild type of the clone in the 3D culture system, and to evaluate ability of PreS2 monoclonal antibody (anti-PreS2) (PNAS 1986) to prevent the infection. Methods: The 3D culture system was prepared by injecting HuS-E/2 cell suspension into hollow fiber bundles. The fibers were then cultured in serum-free medium. A point mutation leading to sG145R amino-acid substitution was engineered by site-directed mutagenesis on the basis of 1.24-fold HBV/Ce genomic (C-AT wild) replicative construct, and infectious virions were propagated in Huh7 cells transfected with the mutant or wild construct. The culture supernatant aliquots containing different amount of the virions (3, 2, 1 and 0 log copies) were inoculated into the 3D culture. In infection-block experiment, infectious aliquots (2 log copies) were incubated at 37 degrees C in presence of anti-PreS2 at different concentrations (1, 10, 20 and 40μg/mL) for 2 hours before inoculation into the 3D culture. Anti-PreS2 was added every 3 days when culture medium was changed. HBV DNA in culture supernatants was quantified by real-time PCR. Results: Both sG145R and C-AT wild strains successfully established infection of the 3D culture system when inoculated in quantity of 2 log copies, and intracellular HBV DNA as well as ccc DNA were detected in HuS-E/2. The levels of HBV DNA detected in culture supernatants of sG145R strain decreased dose-dependently in inverse proportion with applied concentration of anti-PreS2. Anti-PreS2 concentration of 20μg/mL completely inhibited the infection of both C-AT wild and sG145R clones.

 

Conclusion:

In the 3D culture system, we demonstrated that the infectivity of sG145R clone was comparable with that of C-AT wild and that anti-PreS2 might be protective against infections with VSM. The new modification of the 3D culture system using HuS-E/2 appears to be useful in examining of viral replication capacity and phylaxis ability of antibody.

 

850. Limited Value of Single ALT Determination for Assessing Chronic Hepatitis B

E. Heathcote; P. Marcellin; I. M. Jacobson; M. L. Shiffman; H. N. Trinh; K. J. Peschell; D. Goodwin; J. Sorbel; E. A. Fagan; F. Rousseau

 

Background/Aims:

Current practice guidelines for chronic hepatitis B (CHB) recommend monitoring patients with a normal range ALT (NRALT) at ≥ 3 - 6 month intervals (AASLD), but in clinical practice, patients often are monitored once per year, or even less frequently. We sought to determine: 1) the degree of concordance between ALT values on repeat testing within a shorter than recommended interval (i.e., at ≤ 60 days); 2) whether a single NRALT is predictive of a normal or benign liver histology in CHB; and 3) whether other predictors (e.g., age, HBV DNA level) could help identify patients with underlying liver disease despite a NRALT.

 

Methods:

Patients with ≥ 2 ALT tests measured within a 60-day interval prior to treatment (screening, baseline) who consented to participate in Gilead Sciences CHB registration studies of adefovir dipivoxil (studies 437, 438) and tenofovir disoproxil fumarate (studies 102, 103) were identified and stratified into 2 groups; Group 1: intermittently-elevated ALT, including ≥ 1 NRALT value (IE ALT); Group 2: persistently-elevated ALT (PE ALT) (ALT lab range: 6-43, males; 6-34, females). These groups were compared for age, gender, Asian/non-Asian ethnicity, HBV DNA level at last ALT (baseline) and liver biopsy findings (Knodell scores) near baseline.

 

Results:

Group 1 and 2 patients were similar in age (~ 40 yrs old) and Asian ethnicity (~ 40% Asian). Compared to Group 2, Group 1 patients were less likely to be male (63% vs. 76%), and had lower Baseline HBV DNA levels (mean ± SD of 6.3 ± 1.3 vs. 7.8 ± 1.2 log10 copies/mL) and Knodell scores. Sixty (60)/1334 patients (Group 1) had IE ALT values measured within a mean interval of 36 ± 11 days. Of patients with available liver biopsy, 31/57 (54%) in Group 1 had a Knodell necroinflammatory score ≥ 6, and 18/57 (32%) had a Knodell fibrosis score ≥ 3, indicating significant CHB disease, compared to 959/1199 (80%) in Group 2 with Knodell necroinflammatory score ≥ 6 and 554/1199 (46%) with Knodell fibrosis score ≥ 3.

 

Conclusion:

These results indicate that CHB patients with IE ALT values often have significant liver disease, which cannot be excluded by a single NRALT test. IE ALT patients are not distinguished from patients with PE ALT by demographics or HBV DNA levels. In CHB, early repeat testing of ALT (e.g., ≤ 2 months interval) may reveal an elevated ALT and identify CHB patients with underlying liver disease that should be considered for further evaluation and antiviral therapy. Further studies are needed in unselected patients to confirm these findings.

 

846. Usefulness of Serum Markers for Predicting Liver Cirrhosis in Patients with Chronic Hepatitis B

K. Lee; Y. Seo; H. An; E. Jung; B. Keum; Y. Kim; H. . Yim; Y. Jeen; H. Chun; C. Kim; H. Ryu; S. Um

 

Background and Aims:

Liver biopsy is still considered as the gold standard method for the evaluation of liver cirrhosis. However, there are several limitations. It is an invasive method that is associated with patient discomfort and in some case with serious complication of bleeding and infection. In addition, its accuracy is limited by sampling errors and inter- or intra-observer variability. Recently, various serum markers were proposed for non-invasive methods for predicting liver fibrosis stage. However, most of the studies were performed about chronic hepatitis C patients and the studies about chronic hepatits B patients are insufficient. This study was performed to evaluate the significance of various noninvasive markers for predicting cirrhosis in chronic hepatitis B patients.

 

Methods:

Liver biopsy was performed in 125 chronic hepatitis B patients. In this patients, WBC, platelet count, prothrombin time (PT), AST, ALT, GGT, ALP, total bilirubin, albumin, cholesterol, glucose, haptoglobin, apolipoprotein A-1 (apo-A1), α2-macroglobulin, MMP-2, TIMP-1, YKL-40, MMP-9, and procollagen III N-terminal peptide were measured on the biopsy-performed day. Fibrosis stage was assessed according to METAVIR system. All patients were divided into two groups: chronic hepatitis group, fibrosis stage F0-3; cirrhosis group, F4.

 

Results:

Among 125 patients with chronic hepatitis B, 34 patients (27.2%) were diagnosed as liver cirrhosis on liver biopsy. Age, platelet count, WBC count, AST, ALT, haptoglobin, apo-A1, MMP-2, and YKL-40 were significantly different between chronic hepatitis group and cirrhosis group by univariate analysis. By multivariate analysis, platelet count, AST, haptoglobin, and apo-A1 were independent predictive factors for liver cirrhosis. With these four factors, we made a new scoring system (PAHA index). PAHA indices were significantly different between chronic hepatitis group and liver cirrhosis group (3.6±2.3 and 6.8±1.9, respectively; P <0.001). Area under ROC curve (AUROC) of PAHA index for predicting liver cirrhosis was 0.85 (95% CI, 0.78-0.92), which was higher than the AUROCs of previously published fibrosis models including AAR, PGA index, PGAA index, AP index, FFI, APRI, and CDS. With cut-off value of 5.5, sensitivity, specificity, positive predictive value and negative predictive value were 75.8%, 81%, 60.9%, and 89.5%.

 

Conclusion:

The PAHA index including platelet count, AST, haptoglobin and apo-A1 was useful for predicting liver cirrhosis in patients with chronic hepatitis B. The PAHA index might be useful for planning treatment and reducing the need for liver biopsy.

 

973. Usefulness of Liver Biopsy in Chronic HBV with Normal Transaminases in an European Setting: High prevalence of advanced liver fibrosis and cirrhosis

T. Goebel; U. Heinzel-Pleines; C. Poremba; S. E. Baldus; M. Herwig; M. Molderings; D. Haussinger; A. Erhardt

 

 

Introduction:

In chronic HBV-infection indication for antiviral treatment depends on ALT-levels, liver histology and viral load. In general, antiviral treatment is initiated if transaminases are elevated and/or significant liver fibrosis or inflammation is detected in liver histology. The present study aimed at investigating the prevalence of advanced fibrosis in patients with normal ALT and near normal (<2x ULN) in a non-Asian setting.

 

Patients and Methods:

We retrospectively analysed consecutive 252 patients with chronic, replicative hepatitis B who underwent liver biopsy. Patients with HIV/HDV or HCV-coinfection were excluded. Normal transaminases were defined as ALT-levels within reference range (<23 U/l males, <19 U/l females). Stage of liver fibrosis was classified according to Desmet/Scheuer by two independent pathologists.

 

Results: Of the 252 patients, 214 had elevated and 38 normal ALT. Moderately elevated ALTs were found in 84 patients.

 

Discussion: The present study shows that in patients of a non-Asian origin with normal or slightly elevated transaminases, advanced liver fibrosis or even liver cirrhosis is frequently found. Therefore, liver biopsy should be considered even in patients with normal or near normal transaminases.

Parameter

Normal ALT (n=38)

ALT <2xULN

(n=84)

ALT >2ULN

(n=130)

Significant liver fibrosis [≥F3]

16/38 (42%)

45/84 (54%)

82/130 (63%)

Liver cirrhosis

9/38 (24%)

21/84 (25%)

28/130 (22%)

Significant inflammation [≥G2]

7/27 (26%)

34/66 (52%)

71/104 (68%)

HBeAg-positivity

17/38 (45%)

24/84 (29%)

61/130 (47%)

Male/Female [%]

53/47

73/27

79/21

HBV-DNA [mean IU/ml]

1782101

788698

1869311

 

836. Liver Stiffness Measurement and Serum Markers for Predicting Significant Fibrosis in Chronic Hepatitis B

Y. Seo; E. Jung; H. An; B. Keum; Y. Kim; H. Yim; Y. Jeen; H. Chun; C. Kim; H. Ryu; S. Um

 

Background and Aims:

Various non-invasive methods were reported to be very useful for predicting fibrosis stage in patients with chronic hepatitis. However, most published studies focused on the patients with chronic hepatitis C. Therefore, it is still uncertain which non-invasive method is most accurate for fibrosis assessment in patients with chronic hepatitis B (CHB). This study was performed to evaluate the non-invasive indicators for predicting significant fibrosis in patients with CHB.

 

Methods:

We enrolled patients with CHB who performed liver biopsy. Fibrosis stage on liver biopsy was determined according to METAVIR scoring system. Liver stiffness measurement (LSM) and laboratory tests (AST, ALT, prothrombin time [PT], gamma-GT, cholesterol, triglyceride, platelet, haptoglobin, collagen-IV, hyaluronic acid, apolipoprotein-A1, α2-macroglobulin, and procollagen III N-terminal peptide [PIIINP]) were performed on biopsy-performed day. Fibrosis models including AST/ALT ratio (AAR), AST-to-platelet ratio index (APRI), age-platelet index (API), Forns index, and PGA index were determined using the results of laboratory tests. Patients were divided into two groups according to the fibrosis stage: mild fibrosis group.

 

Results:

Eighty-five patients with CHB were enrolled (66 males and 19 females; age, 35±10 years). Significant fibrosis was noted in 58 patients (68.2%): F0-1, 27 patients (31.8%); F2, 25 (29.4%); F3, 23 (27.1%); F4, 10 (11.8%). Liver stiffness was 6.1±2.0 kPa, 11.5±6.6 kPa, 15.2±6.7 kPa, and 18.3±7.6 kPa in patients with F0-1, F2, F3, and F4, respectively. Liver stiffness, platelet, PT, AST, ALT, bilirubin, gamma-GT, triglyceride, haptoglobin, collagen-IV, hyaluronic acid, α2-macroglobulin, and PIIINP were significantly different between two groups. When binary logistic analysis was performed with the results of laboratory tests, bilirubin, PIIINP, and PT were the significant independent indicators for significant fibrosis. However, when liver stiffness was added into the variables, only liver stiffness (P=0.009) and PT (P=0.014) were independent predictive factors. In area under ROC curve analysis for significant fibrosis, LSM was superior to APRI, AAR, API, Forn’s index, and PGA index.

 

Conclusions:

LSM was useful method for predicting significant fibrosis in patients with CHB. PT might improve the diagnostic accuracy of LSM for predicting significant fibrosis.

 

840. Is Liver Biopsy Required for All Patients (pts) with HBeAg-negative (HBeAg-) Chronic HBV Infection and HBV DNA <20,000 IU/mL?

G. V. Papatheodoridis; E. K. Manesis; S. Manolakopoulos; I. Elefsiniotis; I. Giannousis; G. Kafiri; A. J. Archimandritis

 

Background/Aim:

The optimal HBV DNA level for differentiation between HBeAg- chronic hepatitis B pts and inactive HBV carriers remains unclarified. This HBV DNA threshold has been recently decreased from 20,000 to 2,000 IU/mL and liver biopsy is recommended for pts with HBV DNA≥2,000 IU/mL. We evaluated the severity of liver histology in HBeAg- chronic HBV pts with HBV DNA <20,000 IU/mL.

 

Methods:

From 9/2001-12/2007, liver biopsy was performed in 105 HBeAg- pts with increased ALT (>ULN on ≥2 monthly occasions) and HBV DNA 80-20,000 IU/mL (Group A, age:46±15 yrs, M=83) (Group-A1, n=63: HBV DNA 2,000-19,999; -A2, n=42: HBV DNA 80-1,999 IU/mL) and in 35 HBeAg- pts with persistently normal ALT (≤ULN on ≥4 occasions within 1 year) and HBV DNA 2,000-19,999 IU/mL (Group B, age:43±13 yrs, M=22). No biopsy was performed in pts with persistently normal ALT and HBV DNA<2,000 IU/mL. HBV DNA levels were determined by a quantitative PCR assay (Monitor, Roche; sens. 80 IU/mL). Histological lesions were evaluated by one liver pathologist (Ishak’s classification) and histological treatment indication was defined as grading score≥7 and/or stage≥2.

 

Results:

There was no significant difference in any characteristic between Group A1 and A2, except for a trend for worse fibrosis (stage:2.8±1.7 vs 2.2±1.7, P=0.087) or more frequent severe fibrosis/cirrhosis (stage 4-6:37% vs 19%, P=0.079) in group A1 than A2 pts. Group A compared to Group B pts did not differ in age, sex, BMI, alcohol abuse or HBV DNA levels, but they had higher ALT (by definition), worse grading score (6.1 vs 2.9, P<0.001) and worse stage (2.6 vs 1.0, P<0.001). Histological treatment indication was present in 73 (70%) Group A (A1:75% vs A2:62%, P=0.24) and in only 6 (17%) Group B pts. In Group B, histological treatment indication was diagnosed always due to stage 2 fibrosis without active necroinflammation (grading score 2-4 in all 6 pts).

 

Conclusions:

Our results demonstrate that a) there are no substantial differences between HBeAg- chronic HBV pts with elevated ALT and HBV DNA <2,000 or 2,000-20,000 IU/mL and b) liver biopsy is required for all HBeAg- pts with elevated ALT regardless of viremia, since histological lesions widely accepted as treatment indications are present in the majority of such pts even with HBV DNA <2,000 IU/mL. In contrast, immediate liver biopsy is not required for HBeAg- pts with persistently normal ALT and HBV DNA 2,000-20,000 IU/mL, as minimal lesions are found in the majority and just moderate fibrosis without active necroinflammation may be found in <20% of them.

 

958. Quantitative HBsAg Titer Correlates Well with HBV DNA Level in Chronic Hepatitis B Patients

T. Su; C. Hsu; C. Liu; Y. Huang; C. Liu; P. Chen; J. Kao; D. Chen

 

Background and Aims:

Serum HBV DNA level remains an important diagnostic and monitoring tool for patients with chronic HBV infection; however, this assay is expensive and cannot be widely used in clinical practice. We thus studied the correlation between quantitative HBsAg titer and HBV DNA level in chronic hepatitis B patients and the role of quantitative HBsAg in monitoring those with different anti-HBV treatments.

 

Methods:

A total of 209 chronic hepatitis B patients without previous anti-HBV treatment were enrolled as treatment-naïve group. Serum HBsAg was quantified by a commercial kit (ARCHITECT, Abbott laboratories, Illinois, USA), and HBV DNA by a real-time PCR method with the sensitivity of 20 IU/mL. Another cohort of 107 chronic hepatitis B patients who had received various antiviral treatments (lamivudine, adefovir, entecavir, pegylated-interferon immunotherapy or in combination) were enrolled as treatment group. HBsAg and HBV DNA levels were quantified at baseline and two occasions during anti-HBV therapy.

 

Results:

In treatment-naïve group, a Pearson correlation coefficient of 0.59 (p<0.01) was found between log (HBsAg) and log (HBV DNA), and a linear regression equation was obtained as follows: Log (HBV DNA) = 1.98 + 0.97 Log (HBsAg). Further stratification according to age, sex, HBeAg status and HBV genotype showed that patients < 50 years and genotype C infection had higher correlation coefficients of 0.60 and 0.62, respectively (p<0.01). A highest correlation coefficient of 0.64 (p<0.01) was found among genotype C patient < 50 years. Using ROC curve to determine the best cut-off value of HBsAg for the prediction of HBV DNA level, an HBsAg titer of 314 IU/mL had the sensitivity of 83% and specificity of 50% to predict HBV DNA level of 2,000 IU/mL. The HBsAg titer of 768 IU/mL had the sensitivity of 82% and specificity of 60% to predict HBV DNA level of 20,000 IU/mL By using the mixed model, data of treatment group showed a significant linear correlation between log (HBsAg) and log (HBV DNA), regardless of the types of anti-HBV treatment: Log (HBV DNA) = 0.49 + 1.25 Log (HBsAg) (p<0.0001).

 

Conclusions:

Quantitative HBsAg assay is a useful and economic tool to monitor serum HBV DNA level before and during anti-HBV treatment. A cut-off HBsAg titer of 314 IU/mL and 768 IU/mL may be used to predict serum HBV DNA level of 2,000 IU/mL and 20,000 IU/mL, respectively. Serial HBsAg measurements are also practicable in monitoring on-treatment responses.

Children

923. High Prevalence of HBV-DNA in Children with Chronic Hepatitis B after Seroconversion to Anti-Hbe

S. Gehring; T. Gumbrich; F. Zepp; U. Kullmer

 

Introduction & Aim:

The goal of therapy for chronic hepatitis B virus (HBV) infection in children is to eliminate HBV replication and prevent the progression of liver disease to cirrhosis. Interferon-alpha (IFN-α) and Lamivudine (LMV) have been approved for treatment in children, Adefovir is currently investigated in a multi-centre trial. In western countries, treatment with IFN-α has been found to lead in up to 58% of children to the loss of HBV-DNA and HBe antigen (Ag) seroconversion. After the introduction of a highly sensitive HBV-DNA real-time PCR system at our institution the overall prevalence of HBV-DNA in our pediatric population was re-evaluated.

 

Methods:

Forty pediatric patients ages 4 – 22 years (average 13,4) were evaluated. 24/40 children underwent a treatment with IFN-α. 8/40 children received treatment with LMV. Patients were monitored with liver function tests (LFT), complete blood cell count, auto-antibody screening (LKM, ANA, SMA), thyroid screening, hepatitis screens (HBe, HBs, anti-HBe, anti-HBs, anti-HBc), and HBV DNA by quantitative PCR at each visit. Quantitative real-time PCR for HBV-DNA was performed with the commercially available COBAS® test system.

 

Results:

Eighty percent of the children in our study group were infected by neonatal transmission. Ethnic background included 23 South/Eastern-European (Poland, Russia, Albania, and Turkey), 3 Asian, and 14 German children. 9/24 children (37%) that received IFN-α converted HBeAg negative and developed HBe antibodies. Only one child receiving LMV treatment became HBeAg negative during therapy. All children had copies > 2 million/ml prior to therapy. 10/40 children (25%) developed spontaneously HBe antibodies and converted HBeAg negative. Development of anti-HBe in association with treatment or spontaneously led to a significant reduction of LFT levels and a drop of HBV-DNA from a median of 447 million copies/ml to 2100 copies/ml. Importantly, only 2 out of 20 anti-HBe positive children developed HBV-DNA levels below detection level (< 100 copies/ml).

 

Conclusions:

Key elements of a successful treatment in HBV infected individuals (sustained response) are seroconversion from HBeAg to anti-HBe and / or loss of HBV-DNA. With the introduction of a highly sensitive quantitative HBV-DNA PCR testing system, we observed that the vast majority (90%) of our seroconverted children remained to be at a low level HBV-DNA positive. Subsequently, the risk of infectivity after seroconversion appears to be diminished but not eliminated, which remains to be a challenging burden particularly during adolescence.