Little is known about the functional significance of hepatitis B virus (HBV) sequence heterogeneity. Here we analyzed the type, frequency, and function of mutations in the core promoter/enhancer II region of HBV in immunosuppressed patients. The major HBV population in immunosuppressed patients with severe liver disease had deletions, insertions, and/or base changes in this region. Such mutations were not found in immunosuppressed patients with mild disease. Except for two mutations, all created a hepatocyte nuclear factor 1 (HNF1) binding site or a potential HNF3 binding site. Occasionally, known binding sites for C/EBP and HNF4 were additionally duplicated. Eleven mutated core promoter prototype sequences were functionally tested in the context of a wild-type genome by transfection in Huh7 cells. Despite the diversity of mutations tested, all decreased steady-state levels of pre-C mRNA drastically and increased those of the C mRNA/ pregenomic RNA. This correlated with reduced levels of secreted hepatitis B e antigen and increased intracellular levels of core and Pol proteins and replicative HBV DNA intermediates. The levels of secreted HBV DNA-containing particles were also increased although most of the mutations reduced the levels of pre-S/S mRNA and pre-S1, and pre-S2 proteins as well as secretion of hepatitis B surface antigen. These data reveal a novel class of HBV variants with HNF1 binding sites in the core promoter which are characterized by a defect in hepatitis B e antigen expression, enhanced replication, and altered protein levels, all probably mediated by altered transcription factor binding. The phenotype of these variants and their prevalence only in immunosuppressed patients with severe liver disease may indicate that they play a role in pathogenesis.

Hepatitis B immunoglobulin is used for prophylaxis against hepatitis B virus (HBV) and is thought to act by neutralization of virions and hepatitis B virus surface antigen (HBsAgHepatitis B immunoglobulin (HBIG) is used clinically as passive immunoprophylaxis for accidental exposure to hepatitis B virus (HBV) and long term to prevent HBV recurrence in the graft after liver transplantation (24). It contains high-titer antibodies against HBV surface antigen (HBsAg), which is the major component of the outer envelope of the 42-nm-diameter hepatitis B virion, as well as the 22-nm-diameter subviral particles. The therapeutic effect of HBIG is believed to be due to high-affinity binding with HBs-containing particles and neutralization of HBV in the circulation.Despite HBIG prophylaxis, HBV infection recurs in 30% of patients who receive transplants for HBsAg-positive cirrhosis (24). The failure of immunoprophylaxis is due either to a high HBV load and inadequate neutralization by HBIG or to the emergence of antibody-induced escape HBV mutants (3, 6, 25). These mutant HBV strains contain amino acid substitutions within the conserved a-determinant (a group-reactive region between amino acids 124 and 149 of HBsAg), which abrogate the binding affinity of anti-HBs (4, 5, 22, 27). The most frequent mutation occurs at codon 145 of the surface open reading frame, leading to a glycine (G)-to-arginine (R) substitution-G145R-which has been shown to emerge both in liver transplant patients receiving HBIG prophylaxis and in HBV vaccine recipients (2,6,22). The mechanism for the emergence of HBsAg mutations that escape antibody recognition has not been defined.Earlier studies have demonstrated the presence of membrane-bound and/or nuclear localization of immunoglobulin G (IgG) in hepatocytes of patients with chronic HBV infection, who express HBV core antigen or hepatitis delta virus antigen in the liver (17,19,23). Recently, a novel Fc receptor for IgG (FcRn) which mediates the transcytosis of IgG from serum to bile and protects the internalized IgG from catabolism has been identified on the plasma membranes of adult rat hepatocytes (1, 9). FcRn is a heterodimer of ␤ 2 -microglobulin light chain and a major histocompatibility complex class I-like heavy chain that binds IgG via Fc residues in a pH-dependent manner. IgG binding to FcRn is followed by endocytosis of the complex in the acidic endosome environment, trafficking through cellular conduits to bypass lysosomal activities and finally releasing IgG in the extracellular fluids (7). Whether hepatitis B immunoglobulin enters HBV-infected hepatocytes and whether an interaction with HBsAg occurs within cells, in addition to the interaction in serum, have not been investigated.In the present study, we investigated the hypothesis that HBIG is able to bind to hepatocytes and affect the secretion of HBsAg and HBV virions from the cells. For this purpose, we used a panel of human hepatocyte-derived cell lines cultured together with monoclonal and polyclonal HBs-specific antibodies. The...

The factors determining the responsiveness of different hepatitis B virus (HBV) genotypes to interferon treatment are not fully understood. We investigated the relationship between HBV genetic characteristics and the outcome of short (16 weeks) or prolonged (32 weeks) treatment with standard interferon-alpha in a prospectively followed cohort of 103 patients across Europe with HBeAg positive chronic hepatitis B. INNO-LiPA assays and HBV DNA sequencing were used to determine HBV genotypes, mutations in the core promoter and precore/core regions. After 16-weeks interferon-alpha treatment, the rate of HBeAg clearance was higher in genotype A versus all other genotypes (P = 0.014), or genotype D alone (P = 0.05). The HBV genome analysis revealed that: (i) after 16-weeks treatment, an HBV subpopulation with core promoter mutations emerged or increased (P < 0.001) only in genotype A; (ii) the core gene of genotype A has the lowest number of amino acid variations in comparison with genotypes B, C, or D. Logistic regression analysis identified genotype A as a positive predictor of short (16 weeks) treatment response (P = 0.001; odds ratio 6.19, 95 confidence interval 1.94-19.8), having a greater impact than baseline HBV DNA or alanine aminotransferase (ALT) levels. In contrast, the response to prolonged interferon-alpha treatment was not different between HBV genotypes. These results suggest that HBV genotype A responds earlier to interferon treatment than other genotypes, which is associated with its molecular characteristics. The optimal duration of interferon-based therapies in chronic hepatitis B may vary between different HBV genotypes.

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