Premature stop codons in the hepatitis B virus (HBV) surface protein can be associated with nucleos(t)ide analogue resistance due to overlap of the HBV surface and polymerase genes. The aim of this study was to determine the effect of the replication of three common surface stop codon variants on the hepatocyte. Cell lines were transfected with infectious HBV clones encoding surface stop codons rtM204I/sW196*, rtA181T/sW172*, rtV191I/sW182*, and a panel of substitutions in the surface proteins. HBsAg was measured by Western blotting. Proliferation and apoptosis were measured using flow cytometry. All three surface stop codon variants were defective in HBsAg secretion. Cells transfected with these variants were less proliferative and had higher levels of apoptosis than those transfected with variants that did not encode surface stop codons. The most cytopathic variant was rtM204I/sW196*. Replication of HBV encoding surface stop codons was toxic to the cell and promoted apoptosis, exacerbating disease progression.

Introduction: HBsAg plays a critical role in mediating HBV-induced hepatocellular carcinoma (HCC).

ABSTRACT Chronic hepatitis B (CHB) is prevalent worldwide. The infectious agent, hepatitis B virus (HBV), replicates via an RNA intermediate and is error prone, leading to the rapid generation of closely related but not identical viral variants, including those that can escape host immune responses and antiviral treatments. The complexity of CHB can be further enhanced by the presence of HBV variants with large deletions in the genome generated via splicing (spHBV variants). Although spHBV variants are incapable of autonomous replication, their replication is rescued by wild-type HBV. spHBV variants have been shown to enhance wild-type virus replication, and their prevalence increases with liver disease progression. Single-molecule deep sequencing was performed on whole HBV genomes extracted from samples, including the liver explant, longitudinally collected from a subject with CHB over a 15-year period after liver transplantation. By employing novel bioinformatics methods, this analysis showed that the dynamics of the viral population across a period of changing treatment regimens was complex. The spHBV variants detected in the liver explant remained present posttransplantation, and a highly diverse novel spHBV population as well as variants with multiple deletions in the pre-S genes emerged. The identification of novel mutations outside the HBV reverse transcriptase gene that co-occurred with known drug resistance-associated mutations highlights the relevance of using full-genome deep sequencing and supports the hypothesis that drug resistance involves interactions across the full length of the HBV genome. IMPORTANCE Single-molecule sequencing allowed the characterization, in unprecedented detail, of the evolution of HBV populations and offered unique insights into the dynamics of defective and spHBV variants following liver transplantation and complex treatment regimens. This analysis also showed the rapid adaptation of HBV populations to treatment regimens with evolving drug resistance phenotypes and evidence of purifying selection across the whole genome. Finally, the new open-source bioinformatics tools with the capacity to easily identify potential spliced variants from deep sequencing data are freely available.

Hepatitis B virus (HBV) infection can result in a spectrum of outcomes from immune-mediated control to disease progression, cirrhosis, and liver cancer. The host molecular pathways that influence and contribute to these outcomes need to be defined. Using an immunocompetent mouse model of chronic HBV infection, we identified some of the host cellular and molecular factors that impact on infection outcomes. Here, we show that cellular inhibitor of apoptosis proteins (cIAPs) attenuate TNF signaling during hepatitis B infection, and they restrict the death of infected hepatocytes, thus allowing viral persistence. Animals with a liver-specific cIAP1 and total cIAP2 deficiency efficiently control HBV infection compared with WT mice. This phenotype was partly recapitulated in mice that were deficient in cIAP2 alone. These results indicate that antagonizing the function of cIAPs may promote the clearance of HBV infection.

SummaryPatients with hepatitis B e antigen (HBeAg)‐positive chronic hepatitis B (CHB) have suppressed TLR2 expression, function and cytokine production. The aim of this study was to explore the importance of hepatitis B virus (HBV) genotype in innate immune responses and investigate whether Toll‐like receptor (TLR) expression/function has potential roles as predictive biomarkers of successful therapy with pegylated interferon (Peg‐IFN) therapy of HBeAg seroconversion in HBeAg‐positive patients. We showed that as early as 4 weeks after initiation of Peg‐IFN, future HBeAg seroconverters had significantly elevated levels of TLR2 expression on monocytes. TLR2‐associated IL‐6 production at baseline and week 4 of therapy and TLR4 IL‐6 production at week 4 were also markedly elevated in HBeAg seroconverters. HBV genotype also influenced treatment response, with genotypes A and B more likely to seroconvert than D. We were able to demonstrate that these differences were due in part to the interaction of the specific HBeAg proteins with TLR pathway adaptor molecules, and these interactions were genotype dependent. HBeAg‐mediated modulation of TLR signalling was also observed in Huh7 cells, following stimulation with Pam3Cys. Importantly, the addition of IFN‐α to TLR2‐stimulated cells cotransfected with an HBeAg expression plasmid reversed HBeAg‐mediated suppression of hepatocytes. These findings demonstrate that patients with an activated inflammatory response are much more likely to respond to IFN therapy, with TLR responses showing promise as potential biomarkers of HBeAg seroconversion in this setting. Furthermore, our findings suggest there is differential genotype‐specific HBeAg suppression of innate signalling pathways which may account for some of the clinical differences observed across the CHB spectrum.

ABSTRACTThe mechanisms by which hepatitis B virus (HBV) establishes and maintains chronic hepatitis B infection (CHB) are poorly defined. Innate immune responses play an important role in reducing HBV replication and pathogenesis. HBV has developed numerous mechanisms to escape these responses, including the production of the secreted hepatitis B e antigen (HBeAg), which has been shown to regulate antiviral toll-like receptor (TLR) and interleukin-1 (IL-1) signaling. IL-18 is a related cytokine that inhibits HBV replication in hepatoma cell lines and in the liver through the induction of gamma interferon (IFN-γ) by NK cells and T cells. We hypothesized that HBV or HBV proteins inhibit IFN-γ expression by NK cells as an accessory immunomodulatory function. We show that HBeAg protein inhibits the NF-κB pathway and thereby downregulates NK cell IFN-γ expression. Additionally, IFN-γ expression was significantly inhibited by exposure to serum from individuals with HBeAg-positive but not HBeAg-negative chronic HBV infection. Further, we show that the HBeAg protein suppresses IL-18-mediated NF-κB signaling in NK and hepatoma cells via modulation of the NF-κB pathway. Together, these findings show that the HBeAg inhibits IL-18 signaling and IFN-γ expression, which may play an important role in the establishment and/or maintenance of persistent HBV infection.IMPORTANCEIt is becoming increasingly apparent that NK cells play a role in the establishment and/or maintenance of chronic hepatitis B infection. The secreted HBeAg is an important regulator of innate and adaptive immune responses. We now show that the HBeAg downregulates NK cell-mediated IFN-γ production and IL-18 signaling, which may contribute to the establishment of infection and/or viral persistence. Our findings build on previous studies showing that the HBeAg also suppresses the TLR and IL-1 signaling pathways, suggesting that this viral protein is a key regulator of antiviral innate immune responses.

A 37-year-old Asian woman was referred for assessment of abnormal liver function following a needle-stick injury. She was asymptomatic. Liver function testing revealed albumin 40 g/L, alkaline phosphatase 62 IU/L, alanine aminotransferase 43 IU/L and bilirubin 23 μmol/L. Serum hepatitis B surface antigen (HBsAg) testing was negative using the Roche Cobas assay (Roche Elecsys platform, Roche Diagnostics, IN, USA), and antiHBs titre was 616 IU/mL (Roche Elecsys). Standard serology for antibodies to hepatitis C virus and HIV was negative, as was testing for autoimmune and metabolic liver diseases. She was clinically well and taking no regular medication. The patient was a primary care doctor (general practitioner). The needle-stick injury occurred to the patient when vaccinating an infant. The patient was noted to be anti-HBs positive at the time of the needle-stick injury, and the infant (donor) was hepatitis B virus (HBV) negative. There were no prior recognised occupational exposures to HBV. The patient’s mother was born in Hong Kong and was confirmed to have chronic hepatitis B infection with detectable serum HBsAg and HBV DNA. The patient was born in the UK, but did not receive immunoprophylaxis at the time. She received a complete course of HBV vaccine as a teenager. In 1991, HBsAg testing was negative, with a low positive anti-HBs titre, and subsequently three booster doses of vaccine were administered. Anti-HBs levels were >100 IU/mL in 1994. She received a booster vaccine in 1999, and serum HBsAg was again negative in 2003. Further testing at presentation showed that serum was positive for antibodies directed against the core protein of HBV (anti-HBc). The HBV precore protein (or hepatitis B ‘e’ antigen) was also detectable in serum, at a level of 133 Paul-Ehrlich IU/mL (Roche Cobas Assay). Serum HBV DNA level was 134 488 IU/mL (Roche Cobas AmpliPrepTaqman HBV Test v2.0, Roche Diagnostics). Sequencing of the HBV genome was performed and identified genotype C HBV with a four amino acid (aa) repeat insertion at position 115 in the surface protein (Fig. 1). We present an unusual case of a HBV diagnostic escape variant, where the standard HBsAg immunoassay was negative despite persistent viraemia and active hepatitis. The standard diagnostic assay for HBV infection is an enzyme immunoassay for detection of HBsAg. The assay relies on antigenic interaction between HBsAg and antibodies directed against HBsAg (anti-HBs). Most commercial assays use a panel of monoclonal antibodies directed against antigenic determinants (epitopes) between aa99 and aa169. HBsAg is a transmembrane protein, and this region exists as a large and exposed hydrophilic segment containing multiple cysteine residues and likely stabilised by the formation of numerous disulphide bonds, with the antigenic determinants being highly conformation dependent. The major antigenic determinant lies between aa121 and 149 (the protective immunity conferred by HBsAg vaccination is associated with neutralising antibody against this ‘a’ determinant). Structural variations in this region (Fig. 1), including substitutions and insertions, may alter the conformation of the region, disrupt antibody binding and result in diagnostic escape and/or vaccine escape (e.g. G154R vaccine escape mutation). We believe that this is the first description of an insertion at aa115 leading to diagnostic failure. Routine testing for HBV infection should always include serology for HBsAg and anti-HBs, as well as antiHBc. Although HBsAg diagnostic escape is uncommon using modern immunoassays, this case demonstrates the importance of maintaining an index of suspicion in highrisk individuals, particularly with a family history of chronic hepatitis B. In such a setting, nucleic acid testing for serum HBV DNA is required.

Previous clinical studies have demonstrated an association between the hepatitis B e antigen and Toll-like receptor (TLR) expression and signalling. Therefore, the aim of this study was to develop an in vitro assay to measure the effect of hepatitis B virus proteins, including the precore protein, on signalling mediated by members of the Toll-like/interleukin 1 (TIR) superfamily, by measuring NF-kappa B promoter activity. The basal level of NF-kappa B reporter activity was measured in three hepatocyte cell lines (Huh7, HepG2 and PH5CH8) and one kidney cell line (HEK293) using a luciferase assay. All cell lines were virtually refractory to stimulation with lipopolysaccharide; however, PH5CH8 cells had a robust activation of NF-kappa B in response to IL-1 beta stimulation, with similar to 40-fold higher activation than the unstimulated control, a higher degree of activation than that observed in either Huh7 and HepG2, or HEK293 and HEK293-TLR2 cells. In PH5CH8 cells transfected with pCI expression constructs and stimulated with IL-1 beta, we showed that the precursor form of the precore protein, p25, inhibits NF-kappa B activation by up to 30% and the cytosolic form, p22, inhibits NF-kappa B activation by 70%. The core protein, p21, which shares significant homology with the precore protein except for a 10-amino acid extension at the N-terminus, had no effect on NF-kappa B activation. We hypothesize that the inhibition of IL-1 beta-mediated NF-kappa B activation by the precore protein may be a mechanism that allows the virus to persist, suggesting a role for the pool of precore protein that remains intracellular.

Nucleos(t)ide analogue antiviral therapy for chronic hepatitis B has proven to be effective in the short term but the frequent development of resistance limits its clinical utility. Agents targeting other stages of viral replication are needed in order to develop improved combination therapies. The phenylpropenamide derivatives AT-61 and AT-130 have been shown to inhibit HBV replication in vitro, but the mechanism of action of these compounds remains undefined. The aim of this study was to determine the mechanism of action of AT-130, a non-nucleoside inhibitor of HBV in several in vitro models of replication. These studies found that AT-130 inhibited HBV DNA replication in hepatoma cells but had no effect on viral DNA polymerase activity or core protein translation. Total HBV RNA production was also unaffected in the presence of the drug whilst the amount of encapsidated RNA was significantly reduced, thereby inhibiting subsequent viral reverse transcription. These studies have established that the inhibition of HBV genome replication by a non-nucleoside analogue acting at the level of viral encapsidation and packaging is a potentially useful strategy for future therapeutic drug development in the management of chronic hepatitis B.

05E. Viral Hepatitis (e) Hepatitis' B Clinical therapy S179Results: Median time of therapy was 18 months (12 24).No patient became HBsAg negative, lost HBsAg and/or seroconverted to anti-HBs.2/14 HBeAg-positive patients seroconverted to anti-HBe.Overall in group 1 and 2 a BR was observed in 93% and 91% and a VR in 55% and 83% of patients, respectively (p NS); but considering only patients with a basal HBV-DNA />5 Log a VR was achieved in 81% of group 2 and only in 40% of ADV mono-therapy group (p <0.05).On multivariate analysis VR was significantly associated with a basal viral load <5 Log (p <0.01, RR 3.83) and with HBeAg-negativity (p <0.03, RR 3.619).In 4/12 (33%) patients of group 1, all with a basal HBV DNA />5 Log, who did not achieve a VR, we observed a primary nonresponse (basal A181V) or a virological rebound (1 pt.N236T, 1 pt.A181V +N236T, 1 pt.wild-type).No serious side effects developed during ADV therapy.Conclusions: Overall in LAM-R patients ADV alone or combined with lamivudine was safe and effective.In highly viremic subjects combined therapy was more efficacious.Baseline HBeAg positivity and/or HBV DNA />5 Log were significantly associated with a lower virological response to monotherapy and with a higher rate of ADV resistance.

Background: The expression of the hepatitis Be antigen (HBeAg) is one of several strategies used by hepatitis B virus (HBV) to ensure persistence. The HBeAg may function as a toleragen in utero and has been shown to regulate the host's immune response.Aim: The aim of this study was to examine the effect of the HBV precore and core protein on cellular gene expression in the hepatoma cell line Huh-7.Study design: Huh-7 cells with tight regulated expression of the HBV core or precore protein were produced using the Tet-Off tetracycline gene expression system. Changes in cellular gene expression in response to core/precore expression compared to Huh-7 cells not expressing the proteins were determined using a commercial high-density oligonucleotide array (Affymetrix Hu95A GeneChip) containing probes for 12,626 full-length human genes.Results: Analysis of differential mRNA gene expression profiles at 7 days post precore and core expression revealed 45 and 5 genes, respectively. with mRNA changes greater than three-fold. The most striking feature was in Huh-7 cells expressing the precore protein in which 43/45 genes were downregulated 3-11-fold. These included genes that encoded products that regulate transcription/DNA binding proteins. cell surface receptors, cell-cycle/nucleic acid biosynthesis and intracellular signalling and trafficking. The only known gene, which was upregulated encoded a cytoskeletal protein. For the core cell line, 4/5 genes were downregulated 3-15-fold upon core induction and included genes that encoded products that affect intermediary metabolism, cell surface receptors and intracellular signalling. The one gene, which was upregulated was a cyrokine gene.Conclusion: The results of this study show that HBV precore protein has a much greater effect on cellular gene expression in comparison to the core protein, suggesting that core and precore proteins may have diverse effects on cellular functions and equally different roles in modulating HBV pathogenesis. (C) 2004 Elsevier B.V. All rights reserved.