Prevalence and significance of naturally occurring mutations in the surface and polymerase genes of hepatitis B virus.
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The prevalence and clinical significance of naturally occurring mutations in the full-length surface and overlapping polymerase genes of hepatitis B virus (HBV) were analyzed in 42 patients with chronic hepatitis. Mutations were observed in 10 patients (24%) in the a determinant region, which is the neutralizing epitope within the major hydrophilic region of the surface gene. A high proportion of these mutations (17/18; 94%) occurred in the first loop, unlike mutations induced by immunization. The presence of serum antibody to hepatitis B surface antigen was significantly associated with these mutations. No other region of the surface gene contained any cluster of mutations. These results suggest that escape mutations commonly contribute to persistency in the natural course of HBV infection. In contrast, mutations affecting the major catalytic domains of the polymerase gene, which could alter susceptibility to antiviral nucleoside analogues, were not detected at all. (+info)
Lamivudine as initial treatment for chronic hepatitis B in the United States.
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BACKGROUND AND METHODS: Although the nucleoside analogue lamivudine has shown promise in patients with chronic hepatitis B, long-term data on patients from the United States are lacking. We randomly assigned previously untreated patients with chronic hepatitis B to receive either 100 mg of oral lamivudine or placebo daily for 52 weeks. We then followed them for an additional 16 weeks to evaluate post-treatment safety and the durability of responses. The primary end point with respect to efficacy was a reduction of at least 2 points in the score on the Histologic Activity Index. On this scale, scores can range from 0 (normal) to 22 (most severe abnormalities). RESULTS: Of the 143 randomized patients, 137 were included in the efficacy analysis: 66 in the lamivudine group and 71 in the placebo group. The other six patients were excluded at the base-line visit because of the absence of a documented history of hepatitis B surface antigen for at least six months. After 52 weeks of treatment, lamivudine recipients were more likely than placebo recipients to have a histologic response (52 percent vs. 23 percent, P<0.001), loss of hepatitis B e antigen (HBeAg) in serum (32 percent vs. 11 percent, P=0.003), sustained suppression of serum hepatitis B virus (HBV) DNA to undetectable levels (44 percent vs. 16 percent, P<0.001), and sustained normalization of serum alanine aminotransferase levels (41 percent vs. 7 percent, P<0.001), and they were less likely to have increased hepatic fibrosis (5 percent vs. 20 percent, P=0.01). Lamivudine recipients were also more likely to undergo HBeAg seroconversion, defined as the loss of HBeAg, undetectable levels of serum HBV DNA, and the appearance of antibodies against HBeAg (17 percent vs. 6 percent, P=0.04). HBeAg responses persisted in most patients for 16 weeks after the discontinuation of treatment. Lamivudine was well tolerated. Self-limited post-treatment elevations in serum alanine aminotransferase were more common in lamivudine recipients: 25 percent had serum alanine aminotransferase levels that were at least three times base-line levels, as compared with 8 percent of placebo recipients (P=0.01). The clinical condition of all patients remained stable during the study. CONCLUSIONS: In U.S. patients with previously untreated chronic hepatitis B, one year of lamivudine therapy had favorable effects on histologic, virologic, and biochemical features of the disease and was well tolerated. HBeAg responses were generally sustained after treatment. (+info)
Expression of hepatitis B virus polymerase in Ty1-his3AI retroelement of Saccharomyces cerevisiae.
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Hepatitis B virus (HBV), although a DNA virus, replicates using reverse transcriptase encoded by the HBV polymerase (pol) gene. The biochemical dissection of HBV pol has been hampered by failure to liberate enzymatically active protein from nucleocapsids. Here, we have employed a yeast-based genetic approach to express the HBV reverse transcriptase. In this strategy, the reverse transcriptase of yeast retrotransposon Ty1 element is replaced with the HBV pol gene to produce the hybrid Ty1/HBV element. Additionally, the indicator gene his3AI is combined in an antisense orientation to the transcripts of the hybrid Ty1/HBVRT element. The splicing of his3AI, cDNA synthesis of the Ty1/HBVRT RNA and subsequent integration relies on the reverse transcriptase activity. The production of histidine prototrophs results from the successful reverse transcription of Ty1/HBVRThis3AI transcripts followed by either homologous recombination or integrase-mediated insertion and subsequent expression of HIS3 gene. Using this approach we successfully detected the reverse transcriptase activity of HBV in yeast strains defective in endogenous Ty1 expression. Consistent with the unique priming activity associated with HBV pol, the minus strand DNA synthesis was protein-primed. Deletion of HBV reverse transcriptase (RT) or RNase H domains resulted in a dramatic drop in histidine prototrophs. The addition of HBV encoded HBx protein in virus-like particles during in vitro RT reaction stimulated the RT reaction by severalfold. Furthermore, in the presence of 3TC, a known inhibitor of HBV reverse transcriptase, yeast His(+) growth of His protrophs was not observed. Thus, this approach, which is based on genetic selection in yeast, is safe, economic, and a reliable strategy with a potential for large scale screening of cofactors and inhibitors of HBV polymerase functions. (+info)
Human transporters associated with antigen processing (TAPs) select epitope precursor peptides for processing in the endoplasmic reticulum and presentation to T cells.
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Antigen presentation by major histocompatibility complex (MHC) class I molecules requires peptide supply by the transporters associated with antigen processing (TAPs), which select substrates in a species- and, in the rat, allele-specific manner. Conflicts between TAPs and MHC preferences for COOH-terminal peptide residues in rodent cells strongly reduce the efficiency of MHC class I antigen presentation. Although human TAP is relatively permissive, some peptide ligands for human histocompatibility leukocyte antigen class I molecules are known to possess very low TAP affinities; the significance of these in vitro findings for cellular antigen presentation is not known. We studied two naturally immunodominant viral epitopes presented by HLA-A2 that display very low affinities for human TAP. Low TAP affinities preclude minimal epitope access to the endoplasmic reticulum (ER) and assembly with HLA-A2 in vitro, as well as presentation by minigene-expressing cells to cytotoxic T lymphocytes. However, NH(2)-terminally but not COOH-terminally extended epitope variants with higher TAP affinities assemble in vitro and are presented to cytotoxic T lymphocytes with high efficiency. Thus, human TAP can influence epitope selection and restrict access to the ER to epitope precursors. Analysis of TAP affinities of a panel of viral epitopes suggests that TAP selection of precursors may be a common phenomenon for HLA-A2-presented epitopes. We also analyzed HLA-A2-eluted peptides from minigene-expressing cells and show that an NH(2)-terminally extended variant with low A2 binding affinity undergoes ER processing, whereas another with high affinity is presented unmodified. Therefore, the previously reported aminopeptidase activity in the ER can also act on TAP-translocated peptides. (+info)
Quantitation of hepatitis B viremia and emergence of YMDD variants in patients with chronic hepatitis B treated with lamivudine.
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Hepatitis B viremia and emergence of hepatitis B virus (HBV) YMDD variants with reduced susceptibility to lamivudine were analyzed in patient sera from a phase II study of extended lamivudine therapy. Within 12 weeks, all patients exhibited a marked virologic response to lamivudine: >99% reduction (median 5 log decrease) in serum HBV DNA levels. Virus remained at >104 genomes/mL in 11 patients and decreased to <104 genomes/mL in the remaining 12 patients. In 10 patients, detectable YMDD variants emerged during the course of treatment. Six patients, including 3 with YMDD variants, experienced hepatitis B e antigen seroconversion while on lamivudine therapy or soon after its discontinuation. No patients with HBV DNA levels >104 genomes/mL seroconverted. Thus, patients who respond to lamivudine therapy with dramatic reductions in viral DNA level (to <104 genomes/mL) appear more likely to seroconvert than patients who do not achieve this level of HBV clearance. (+info)
Interference of antibody production to hepatitis B surface antigen in a combination hepatitis A/hepatitis B vaccine.
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A randomized trial comparing 3 manufacturing consistency lots of a combination hepatitis A/hepatitis B vaccine to each other and to hepatitis A vaccine and hepatitis B vaccine given separately and concurrently was done to evaluate safety, tolerability, and immunogenicity. Healthy volunteers >/=11 years of age were divided into 4 groups. Each of 3 groups received a separate consistency lot of the combination vaccine, and 1 group received separate but concurrent injections of hepatitis A and hepatitis B vaccines. Injections were given at weeks 0 and 24. The combination vaccine was generally well tolerated. The hepatitis A portion of the combination vaccine produced clinically acceptable high seropositivity rates 4 and 52 weeks after the first injection. The hepatitis B portion of the vaccine did not produce clinically acceptable seropositivity rates 4 weeks after the second injection. Lack of antibody production may be attributed, at least in part, to immunologic interference. (+info)
Subtype-independent immature secretion and subtype-dependent replication deficiency of a highly frequent, naturally occurring mutation of human hepatitis B virus core antigen.
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The most frequent mutation of the human hepatitis B virus (HBV) core antigen occurs at amino acid 97. Recently, a phenylalanine (F)-to-leucine (L) mutation at this position (mutant F97L) in HBV surface antigen subtype ayw has been shown to result in an immature secretion phenotype, which is characterized by the nonselective export of an excessive amount of virions containing minus-strand, single-stranded HBV DNA. While subtype ayw mutant F97L has been found in Europe, the major reservoir of HBV resides in Asia and Africa. We report here that the immature secretion phenotype indeed can be found in an HBV strain (subtype adr) prevalent in Asia, changing from an isoleucine (I) to a leucine (mutant I97L). Despite its immature secretion phenotype, the adr variant I97L replicates as well as its parental adr wild-type I97I, supporting the conclusion that the extracellular phenotype of immature secretion is not a consequence of the intracellular HBV DNA replication defect. Further studies demonstrated that it is the acquisition of a leucine, rather than the loss of a wild-type amino acid at codon 97, that is important for immature secretion. We conclude that immature secretion is a subtype-independent phenotype and deficiency in intracellular DNA synthesis is a subtype-dependent phenotype. The former is caused by the trans-acting effect of a mutant core protein, while the latter by a cis-acting effect of a mutated nucleotide on the ayw genome. These immature secretion variants provide an important tool for studying the regulation of HBV virion assembly and secretion. (+info)
In vivo regulation of hepatitis B virus replication by peroxisome proliferators.
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The role of the peroxisome proliferator-activated receptor alpha (PPARalpha) in regulating hepatitis B virus (HBV) transcription and replication in vivo was investigated in an HBV transgenic mouse model. Treatment of HBV transgenic mice with the peroxisome proliferators Wy-14,643 and clofibric acid resulted in a less than twofold increase in HBV transcription rates and steady-state levels of HBV RNAs in the livers of these mice. In male mice, this increase in transcription was associated with a 2- to 3-fold increase in replication intermediates, whereas in female mice it was associated with a 7- to 14-fold increase in replication intermediates. The observed increases in transcription and replication were dependent on PPARalpha. HBV transgenic mice lacking this nuclear hormone receptor showed similar levels of HBV transcripts and replication intermediates as untreated HBV transgenic mice expressing PPARalpha but failed to demonstrate alterations in either RNA or DNA synthesis in response to peroxisome proliferators. Therefore, it appears that very modest alterations in transcription can, under certain circumstances, result in relatively large increases in HBV replication in HBV transgenic mice. (+info)