ABSTRACT
Three decades after the discovery, hepatitis C virus (HCV) is still the leading cause of liver transplantation and poses a major threat to global health. In spite of recent advances in the development of direct acting antivirals, there is still a need for a prophylactic vaccine to limit the virus spread and protect at-risk populations, especially in developing countries, where the cost of the new treatments may severely limit access. The use of recombinant HCV glycoproteins E1E2 (rE1E2) in combination with the MF59, an oil-in-water emulsion-based adjuvant, has previously been shown to reduce the rate of chronicity in chimpanzees and to induce production of cross-neutralizing antibodies and cellular immune responses in human volunteers. To further improve neutralizing antibody responses in recipients along with robust T cell responses, we have explored the immunogenicity of different adjuvants when formulated with the HCV rE1E2 vaccine in mice. Our data show that cyclic di-adenosine monophosphate (c-di-AMP) and archaeosomes elicit strong neutralizing antibodies similar to those elicited using aluminum hydroxide/monophosphoryl lipid A (Alum/monophos. /MPLA) and MF59. However, both c-di-AMP and archaeosomes induced a more robust cellular immune response, which was confirmed by the detection of vaccine-specific poly-functional CD4+ T cells. We conclude that these adjuvants may substantially boost the immunogenicity of our E1E2 vaccine. In addition, our data also indicates that use of a partial or exclusive intranasal immunization regimen may also be feasible using c-di-AMP as adjuvant.
Subject(s)
Adjuvants, Immunologic/administration & dosage , Archaea/immunology , CD4-Positive T-Lymphocytes/immunology , Dinucleoside Phosphates/administration & dosage , Hepacivirus/immunology , Hepatitis C Antibodies/blood , Viral Envelope Proteins/immunology , Viral Vaccines/immunology , Administration, Intranasal , Antibodies, Neutralizing/blood , Humans , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , Viral Envelope Proteins/administration & dosage , Viral Envelope Proteins/genetics , Viral Vaccines/administration & dosage , Viral Vaccines/geneticsABSTRACT
The anti-hepatitis C virus (HCV) activity of a novel monoclonal antibody (mAb; AR4A) and epigallocatechin gallate (EGCG) were studied in vitro using a HCV cell culture system and in vivo using a humanized liver mouse model capable of supporting HCV replication. Alone, both exhibit reliable cross-genotype HCV inhibition in vitro, and combination therapy completely prevented HCV infection. In vitro AR4A mAb (alone and combined with EGCG) robustly protects against the establishment of HCV genotype 1a infection. EGCG alone fails to reliably protect against an HCV challenge. In conclusion, AR4A mAb represents a safe and efficacious broadly neutralizing antibody against HCV applicable to strategies to safely prevent HCV reinfection following liver transplantation, and it lends further support to the concept of HCV vaccine development. The poor bioavailability of EGCG limits HCV antiviral activity in vitro.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/pharmacology , Antiviral Agents/pharmacology , Catechin/analogs & derivatives , Hepatitis C/prevention & control , Hepatitis Viruses/drug effects , Liver/drug effects , Animals , Broadly Neutralizing Antibodies , Catechin/pharmacology , Cell Line , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Therapy, Combination , Genotype , Hepatitis C/diagnosis , Hepatitis C/immunology , Hepatitis Viruses/genetics , Hepatitis Viruses/immunology , Humans , Liver/immunology , Liver/virology , Mice, SCID , Time FactorsABSTRACT
Cytomegalovirus (CMV) encodes multiple microRNAs. While these have been partially characterized in vitro, their relevance to clinical CMV infection has not been evaluated. We analyzed samples from a cohort of solid organ transplant patients with CMV disease (n = 245) for viral microRNA expression. Several CMV microRNAs were readily detectable in patients with CMV disease in variable relative abundance. Expression level generally correlated with DNA viral load and the absence of viral microRNA was associated with faster viral clearance. Detection of hcmv-miR-UL22A-5p at baseline independently predicted the recurrence of CMV viremia upon discontinuation of antiviral therapy (OR 3.024, 95% CI: 1.35-6.8; p = 0.007). A combination of direct mRNA targeting by the microRNA and indirect modulation of gene expression involving isoforms of the transcriptional regulator C-MYC may be responsible for the broad effects seen in the association of gene transcripts with the RNA-induced silencing complex and in global protein expression upon hcmv-miR-UL22A-5p transfection. This novel study of in vivo viral microRNA expression profiles provides unique insight into the complexity of clinical CMV infection following transplantation. We provide evidence that viral microRNAs may have complex effects on gene expression and be associated with specific virologic and clinical outcomes, and thus could be further evaluated as biomarkers.
Subject(s)
Cytomegalovirus Infections/genetics , Cytomegalovirus/genetics , Gene Expression Profiling , Gene Expression Regulation, Viral , Host-Pathogen Interactions/genetics , MicroRNAs/genetics , Organ Transplantation , Biomarkers , Blotting, Western , Cohort Studies , Computational Biology , Cytomegalovirus Infections/blood , Cytomegalovirus Infections/virology , Follow-Up Studies , Graft Rejection , Graft Survival , Host-Pathogen Interactions/immunology , Humans , Immunoprecipitation , MicroRNAs/blood , Prognosis , RNA, Viral/blood , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction , Recurrence , Risk Factors , Virus ReplicationABSTRACT
Programmed death ligand-1 (PD-L1) plays an important role in the attenuation of adaptive immune responses in higher vertebrates. Here, we describe the identification of the Pekin duck PD-L1 orthologue (duPD-L1) and its gene structure. The duPD-L1 cDNA encodes a 311-amino acid protein that has an amino acid identity of 78% and 42% with chicken and human PD-L1, respectively. Mapping of the duPD-L1 cDNA with duck genomic sequences revealed an exonic structure of its coding sequence similar to those of other vertebrates but lacked a noncoding exon 1. Homology modelling of the duPD-L1 extracellular domain was compatible with the tandem IgV-like and IgC-like IgSF domain structure of human PD-L1 (PDB ID: 3BIS). Residues known to be important for receptor binding of human PD-L1 were mostly conserved in duPD-L1 within the N-terminus and the G sheet, and partially conserved within the F sheet but not within sheets C and C'. DuPD-L1 mRNA was constitutively expressed in all tissues examined with highest expression levels in lung and spleen and very low levels of expression in muscle, kidney and brain. Mitogen stimulation of duck peripheral blood mononuclear cells transiently increased duPD-L1 mRNA expression. Our observations demonstrate evolutionary conservation of the exonic structure of its coding sequence, the extracellular domain structure and residues implicated in receptor binding, but the role of the longer cytoplasmic tail in avian PD-L1 proteins remains to be determined.
Subject(s)
B7-H1 Antigen/genetics , Ducks/genetics , Amino Acid Sequence , Animals , B7-H1 Antigen/chemistry , Chromosome Mapping , Cloning, Molecular , DNA, Complementary , Gene Expression , Gene Order , Leukocytes, Mononuclear/metabolism , Models, Molecular , Molecular Sequence Data , Phylogeny , Protein Conformation , Protein Interaction Domains and Motifs , RNA, Messenger/chemistry , RNA, Messenger/genetics , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
Interleukin-10 (IL-10) mediates its broad anti-inflammatory and immunoregulatory effects through two cell surface receptors by which binding to the IL-10 receptor 1 (IL-10R1) is the initial step that leads to recruitment of IL-10R2 and initiation of the ternary complex signal transduction cascade. The duck IL-10R1 (duIL-10R1) cDNA was obtained by using RT-PCR and 5'RACE. The deduced 574 amino acid protein has an amino acid identity of 62%, 27% and 28% with chicken, mouse and human IL-10R1, respectively. Comparison of the duIL-10R1 cDNA with duck genomic sequences revealed a seven exon-six intron structure of the duck IL-10R1 gene that shares a similar size with the respective exons 1-7 of the chicken and human IL-10R1 genes, but the avian genes are more compact. Promoter analysis identified putative binding sites for regulatory elements such as CCAAT enhancer binding protein-α, specificity protein 1 (Sp1), nuclear factor 1 (NF1), transcriptional regulatory protein Oct-1, nuclear factor (NF) κB and interferon-stimulated gene factor-3 (ISGF-3). A canonical TATA box was absent in proximity of the transcription initiation site, but a CpG island was present. Sequence analysis of the predicted duIL-10R1 protein revealed characteristic features of class-II cytokine receptors (CFR2) family members and a considerable degree of conservation of residues implicated in ligand binding across higher vertebrates. The predicted secondary structure of the duIL-10R1 extracellular domain is compatible with the two-subdomain structure of the human IL-10R1 protein established by its crystal structure. The 3D model structure shows conservation of the positions of conserved contact residues within four of the five ligand-binding loops. Within the cytoplasmic domain, residues implicated in signal transduction were conserved including two redundant peptide motifs GYXXQ essential for recruitment and activation of STAT3. DuIL-10R1 mRNA expression was most abundant in spleen, thymus, peripheral blood mononuclear cells (PBMCs) and lung. Mitogen stimulation of PBMCs transiently increased duIL-10R1 mRNA expression. Our observations suggest significant evolutionary conservation of the IL-10R1 genomic organization, protein structure and receptor function through the JAK/STAT signalling pathway across higher vertebrates.
Subject(s)
Ducks/genetics , Interleukin-10 Receptor alpha Subunit/genetics , Amino Acid Sequence , Animals , Binding Sites , Chromosome Mapping , Cloning, Molecular , Computational Biology , Conserved Sequence , CpG Islands , DNA, Complementary/genetics , Ducks/classification , Ducks/immunology , Ducks/metabolism , Exons , Humans , Interferon-Stimulated Gene Factor 3/genetics , Interferon-Stimulated Gene Factor 3/metabolism , Interleukin-10 Receptor alpha Subunit/immunology , Interleukin-10 Receptor alpha Subunit/metabolism , Ionomycin/pharmacology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Mice , Molecular Sequence Data , NF-kappa B/genetics , NF-kappa B/metabolism , Phorbol Esters/pharmacology , Phylogeny , Promoter Regions, Genetic , Protein Structure, Secondary , Protein Structure, Tertiary , RNA, Messenger/genetics , Sequence Alignment , Signal Transduction , Sp1 Transcription Factor/genetics , Sp1 Transcription Factor/metabolism , Transcription, GeneticABSTRACT
N-Nonyl-deoxy-galactonojirimycin (N-nonyl-DGJ) has been shown to reduce the amount of hepatitis B virus (HBV) produced by tissue cultures under conditions where cell viability is not affected. We show here that the compound N-nonyl-DGJ was effective against lamivudine-resistant HBV mutants bearing the YMDD motif in the polymerase gene, consistent with the compound's activity being distinct from those of nucleoside inhibitors. To better understand the chemical structures that influence its antiviral activity, a series of imino sugar derivatives were made and tested for their antiviral activity against HBV. This work suggests that the antiviral activity of the alkovirs requires an alkyl chain length of at least eight carbons but that the galactose-based head group can be modified with little or no loss in activity.
Subject(s)
Antiviral Agents/pharmacology , Galactose/analogs & derivatives , Hepatitis B virus/drug effects , Antiviral Agents/chemistry , Structure-Activity RelationshipABSTRACT
We present the discovery of a novel category of 5-substituted acyclic pyrimidine nucleosides as potent antiviral agents. A series of 1-[(2-hydroxyethoxy)methyl] (5-7), 1-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl] (8-10), and 1-[4-hydroxy-3-(hydroxymethyl)-1-butyl] (11-13) derivatives of 5-(1-azido-2-haloethyl)uracil were synthesized and evaluated for their biological activity in cell culture. 1-[4-Hydroxy-3-(hydroxymethyl)-1-butyl]-5-(1-azido-2-chloroethyl)uracil (12) was the most effective antiviral agent in the in vitro assays against DHBV (EC(50) = 0.31-1.55 microM) and HCMV (EC(50) = 3.1 microM). None of the compounds investigated showed any detectable toxicity to several stationary and proliferating host cells.
Subject(s)
Antiviral Agents/chemical synthesis , Azides/chemical synthesis , Pyrimidine Nucleosides/chemical synthesis , Uracil/analogs & derivatives , Uracil/chemical synthesis , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Azides/chemistry , Azides/pharmacology , Cell Division/drug effects , Cells, Cultured , Chlorocebus aethiops , Cytopathogenic Effect, Viral , DNA/biosynthesis , Ducks , Hepatitis B virus/drug effects , Hepatocytes/drug effects , Hepatocytes/virology , Humans , Magnetic Resonance Spectroscopy , Pyrimidine Nucleosides/chemistry , Pyrimidine Nucleosides/pharmacology , Structure-Activity Relationship , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , Uracil/chemistry , Uracil/pharmacology , Virus ReplicationABSTRACT
Synthesis and antiviral activities of novel N-1 alkyl substituted pyrimidines, 1-[(2-hydroxyethoxy)methyl]-5-vinyluracil (5), 1-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl]-5-vinyluracil (6), and 1-[4-hydroxy-3-(hydroxymethyl)-1-butyl]-5-vinyluracil (7) are reported. Compounds 6 and 7 were potent inhibitors of DHBV in cell culture, in contrast, all of the compounds described were devoid of activity against TK(+) HSV-1 and TK(-) HSV-1.
Subject(s)
Antiviral Agents/pharmacology , Hepatitis B virus/drug effects , Pyrimidine Nucleosides/pharmacology , Uracil/chemical synthesis , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Humans , Indicators and Reagents , Molecular Conformation , Molecular Structure , Pyrimidine Nucleosides/chemical synthesis , Pyrimidine Nucleosides/chemistry , Structure-Activity Relationship , Uracil/analogs & derivatives , Uracil/pharmacologyABSTRACT
A new class of 5-(1-cyanamido-2-haloethyl)-2'-deoxyuridines (4-6) and arabinouridines (7, 8) were synthesized by the regiospecific addition of halogenocyanamides (X-NHCN) to the 5-vinyl substituent of the respective 5-vinyl-2'-deoxyuridine (2) and 2'-arabinouridine (3). Reaction of 2 with sodium azide, ceric ammonium nitrate, and acetonitrile-methanol or water afforded the 5-(1-hydroxy-2-azidoethyl)-(10) and 5-(1-methoxy-2-azidoethyl)-2'-deoxyuridines (11). In vitro antiviral activities against HSV-1-TK(+) (KOS and E-377), HSV-1-TK(-), HSV-2, VZV, HCMV, and DHBV were determined. Of the newly synthesized compounds, 5-(1-cyanamido-2-iodoethyl)-2'-deoxyuridine (6) exhibited the most potent anti-HSV-1 activity, which was equipotent to acyclovir and superior to 5-ethyl-2'-deoxyuridine (EDU). In addition, it was significantly inhibitory for thymidine kinase deficient strain of HSV-1 (EC(50) = 2.3-15.3 microM). The 5-(1-cyanamido-2-haloethyl)-2'-deoxyuridines (4-6) all were approximately equipotent against HSV-2 and were approximately 1.5- and 15-fold less inhibitory for HSV-2 than EDU and acyclovir, respectively. Compounds 4-6 were all inactive against HCMV but exhibited appreciable antiviral activity against VZV. Their anti-VZV activity was similar or higher to that of EDU and approximately 5-12-fold lower than that of acyclovir. The 5-(1-cyanamido-2-haloethyl)-(7,8) analogues of arabinouridine were moderately inhibitory for VZV and HSV-1 (strain KOS), whereas compounds 10 and 11 were inactive against herpes viruses. Compounds 5 and 6 also demonstrated modest anti-hepatitis B virus activity against DHBV (EC(50) = 19.9-23.6 microM). Interestingly, the related 5-(1-azido-2-bromoethyl)-2'-deoxyuridine (1n) analogue proved to be markedly inhibitory to DHBV replication (EC(50) = 2.6-6.6 microM). All compounds investigated exhibited low host cell toxicity to several stationary and proliferating host cell lines as well as mitogen-stimulated proliferating human T lymphocytes.
Subject(s)
Antiviral Agents/chemical synthesis , Azides/chemical synthesis , Uracil Nucleotides/chemical synthesis , Animals , Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/chemistry , Anti-HIV Agents/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Azides/chemistry , Azides/pharmacology , Cell Line , Chlorocebus aethiops , Cytomegalovirus/drug effects , Deoxyuridine/analogs & derivatives , Deoxyuridine/chemical synthesis , Deoxyuridine/chemistry , Deoxyuridine/pharmacology , Drug Screening Assays, Antitumor , Hepatitis B Virus, Duck/drug effects , Herpesvirus 3, Human/drug effects , Humans , Simplexvirus/drug effects , Structure-Activity Relationship , Tumor Cells, Cultured , Uracil Nucleotides/chemistry , Uracil Nucleotides/pharmacology , Virus Replication/drug effectsABSTRACT
Lamivudine (beta-L-(-)-2',3'-dideoxy-3'-thiacytidine) has been a major breakthrough in the care of patients with hepatitis B. With prolonged monotherapy the development of resistance is an increasingly recognized problem that limits the long term efficacy of this nucleoside analogue. The most common mutations associated with lamivudine resistance occur within the highly conserved YMDD motif in the C domain of the viral polymerase and are often associated with a compensatory mutation in the proximal B domain. The structural and functional relationship of resistance mutations is reflected in different in vitro sensitivities to lamivudine and changes in replication capacities. During prolonged lamivudine treatment there can be successive changes of different resistant mutants (genotypic succession) or a single mutant can remain the dominant viral species. In patients treated for chronic hepatitis B infection the cumulative incidence of viral resistance reaches over 50% after 3 years. Most patients will have lower serum HBV DNA levels after the emergence of resistance which is ascribed to the decreased replication capacity of these mutants. Although severe flares and ongoing HBe antigen seroconversion can occur in these patients with lamivudine-resistant HBV, the impact of continued therapy on the long-term outcome is still insufficiently studied. In the setting of liver transplantation for HBV-associated disease the clinical course after the emergence of viral resistance is variable but still may lead to disease progression and graft failure. Analogous to the success of combination therapies to delay the emergence of antiviral-resistant HIV, it will be important to combine anti-HBV agents with additive or synergistic antiviral properties and different resistance profiles for future de novo combination therapies for hepatitis B infection.
Subject(s)
Drug Resistance, Viral , Hepatitis B virus/drug effects , Hepatitis B, Chronic/drug therapy , Lamivudine/pharmacology , Lamivudine/therapeutic use , Reverse Transcriptase Inhibitors/pharmacology , Reverse Transcriptase Inhibitors/therapeutic use , Animals , Hepatitis B virus/enzymology , Hepatitis B, Chronic/enzymology , Humans , Lamivudine/chemistry , Reverse Transcriptase Inhibitors/chemistryABSTRACT
Lack of a small animal model of the human hepatitis C virus (HCV) has impeded development of antiviral therapies against this epidemic infection. By transplanting normal human hepatocytes into SCID mice carrying a plasminogen activator transgene (Alb-uPA), we generated mice with chimeric human livers. Homozygosity of Alb-uPA was associated with significantly higher levels of human hepatocyte engraftment, and these mice developed prolonged HCV infections with high viral titers after inoculation with infected human serum. Initial increases in total viral load were up to 1950-fold, with replication confirmed by detection of negative-strand viral RNA in transplanted livers. HCV viral proteins were localized to human hepatocyte nodules, and infection was serially passaged through three generations of mice confirming both synthesis and release of infectious viral particles. These chimeric mice represent the first murine model suitable for studying the human hepatitis C virus in vivo.
Subject(s)
Chimera , Hepacivirus/physiology , Liver/virology , Virus Replication , Animals , Cell Transplantation , Hepacivirus/genetics , Homozygote , Humans , Mice , Mice, SCID , RNA, Viral/isolation & purification , TransgenesABSTRACT
Effective antiviral agents are thought to inhibit hepatitis B virus (HBV) DNA synthesis irreversibly by chain termination because reverse transcriptases (RT) lack an exonucleolytic activity that can remove incorporated nucleotides. However, since the parameters governing this inhibition are poorly defined, fully delineating the catalytic mechanism of the HBV-RT promises to facilitate the development of antiviral drugs for treating chronic HBV infection. To this end, pyrophosphorolysis and pyrophosphate exchange, two nonhydrolytic RT activities that result in the removal of newly incorporated nucleotides, were characterized by using endogenous avian HBV replication complexes assembled in vivo. Although these activities are presumed to be physiologically irrelevant for every polymerase examined, the efficiency with which they are catalyzed by the avian HBV-RT strongly suggests that it is the first known polymerase to catalyze these reactions under replicative conditions. The ability to remove newly incorporated nucleotides during replication has important biological and clinical implications: these activities may serve a primer-unblocking function in vivo. Analysis of pyrophosphorolysis on chain-terminated DNA revealed that the potent anti-HBV drug beta-l-(-)-2',3'-dideoxy-3'-thiacytidine (3TC) was difficult to remove by pyrophosphorolysis, in contrast to ineffective chain terminators such as ddC. This disparity may account for the strong antiviral efficacy of 3TC versus that of ddC. The HBV-RT pyrophosphorolytic activity may therefore be a novel determinant of antiviral drug efficacy, and could serve as a target for future antiviral drug therapy. The strong inhibitory effect of cytoplasmic pyrophosphate concentrations on viral DNA synthesis may also partly account for the apparent slow rate of HBV genome replication.
Subject(s)
Diphosphates/metabolism , Ducks/virology , Hepatitis B Virus, Duck/enzymology , RNA-Directed DNA Polymerase/metabolism , Animals , Antiviral Agents/pharmacology , Catalysis/drug effects , DNA Replication/drug effects , DNA, Viral/biosynthesis , Diphosphates/pharmacology , Genome, Viral , Hepatitis B Virus, Duck/drug effects , Hepatitis B Virus, Duck/genetics , Hydrolysis/drug effects , Kinetics , Lamivudine/pharmacology , Liver/virology , Reverse Transcriptase Inhibitors/pharmacology , Virion/drug effects , Virion/enzymology , Virion/genetics , Zalcitabine/pharmacologyABSTRACT
A crucial step in the establishment and maintenance of a hepadnavirus infection is the formation of a pool of covalently closed circular viral genomes in the nucleus. Changes in the size of this pool occur when an infection is established, when acute infections are resolved, and when chronic infections are treated with antiviral drugs. However, the lack of a quantitative assay for the cccDNA form of the virus has hampered study of the biology of this replication intermediate. In response to this need we have devised a sensitive and accurate competitive PCR assay that is highly selective for the cccDNA form of the duck hepatitis B virus. Since only small amounts of DNA are needed for the assay, cccDNA pool sizes can be monitored in live animals using DNA derived from needle biopsies of infected liver.
Subject(s)
DNA, Circular/analysis , DNA, Viral/analysis , Hepadnaviridae Infections/virology , Hepatitis B Virus, Duck/isolation & purification , Polymerase Chain Reaction/methods , Animals , Biopsy , Blotting, Southern , Ducks , Hepatitis B Virus, Duck/genetics , Liver/virology , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
A detailed analysis of the hepatitis B virus (HBV) replication reaction is important both in understanding viral biology and in developing effective antiviral drugs. This can best be achieved by studying the viral reverse transcriptase (RT) in its natural context, encapsidated within viral core particles in a multiprotein complex, rather than as an isolated enzyme. In order to facilitate a precise enzymological analysis of the avian HBV-RT reaction and its inhibition within replicating cores, a scheme for the purification and analysis of intracellular core particles derived from infected liver tissue has been devised, optimized and evaluated. The purification scheme itself is simple and rapid, and results in preparations with a 25-fold increase in endogenous polymerase activity that persists for over 5 h under assay conditions. In order to assess the suitability of these preparations for mechanistic studies, a thorough evaluation of purity was undertaken, revealing predominantly pure viral protein and nucleic acid, free of contaminating cellular polymerases and phosphatase activities that potently degrade nucleotides and antiviral drugs. Parameters governing optimal polymerase activity have been determined, and an assay for DHBV-RT activity has been developed which offers the highest purity and specific polymerase activity currently available to study hepadnaviral replication and inhibition.
Subject(s)
Hepatitis B Virus, Duck/chemistry , RNA-Directed DNA Polymerase/chemistry , Viral Core Proteins/chemistry , Animals , Chromatography, Agarose , Chromatography, Gel , Detergents , Ducks , Hepatitis B Virus, Duck/enzymology , Hepatitis B Virus, Duck/isolation & purification , RNA-Directed DNA Polymerase/metabolism , Time Factors , Viral Core Proteins/isolation & purification , Virus ReplicationABSTRACT
A rapid on-column DNA labeling technique is used to detect viral restriction DNA fragments by capillary electrophoresis-laser induced fluorescence detection. Intercalating dyes such as POPO3 or ethidium homodimer-2 are incorporated into the detection buffer. The cationic dyes migrate into the capillary during electrophoresis and bind to the oppositely migrating DNA fragments. A post-column sheath-flow fluorescence detector is used in the experiment. Excellent labeling efficiency is achieved at minimal background fluorescence by diluting the dyes to between 1 x 10(-7) M and 5 x 10(-7) M in a buffer with low ionic strength relative to the running buffer within the capillary. This dilute sheath-flow buffer allows stacking of dye molecules inside the capillary when an electric field is applied. Calibration curves using a series of DNA size markers (between 72 and 1353 base pairs) were linear over an order of magnitude in DNA concentration. Sensitivity also increased linearly with fragment length, and detection limits ranged from 4 x 10(-14) M to 5 x 10(-13) M for the size-standards. Analysis of cloned viral DNA using duck hepatitis B virus demonstrated a concentration detection limit of 3.9 x 10(-16) M. Last, the technique produced very high separation efficiency, 14 x 10(6) theoretical plates which is greater than 47 x 10(6) plates m-1, for the duck hepatitis B viral genome.
Subject(s)
DNA, Viral/analysis , Electrophoresis, Capillary/methods , Fluorescent Dyes/chemistry , Hepatitis B Virus, Duck/genetics , Intercalating Agents/chemistry , Calibration , Cloning, Molecular , DNA, Viral/chemistry , Fluorescence , LasersABSTRACT
Genome replication of hepadnavirus proceeds by reverse transcription from a viral pregenomic RNA template by a virally encoded polymerase that possesses protein-priming, reverse transcriptase, DNA polymerase, and RNase H activities. Characterization of this enzyme has been hampered by failure to purify an active enzyme from virions and difficulties in expressing an active polymerase in heterologous systems. In this study, we constructed human hepatitis B virus polymerase cDNA under the control of a phage T7 promoter and expressed it in a rabbit reticulocyte lysate-coupled transcription-translation system. In vitro site-directed mutagenesis confirmed that the recombinant polymerase cDNA produced three products: a full-length protein (approximately 94 kDa), an internally initiated protein (approximately 81 kDa), and an N-terminal protein (approximately 40 kDa). The in vitro expressed polymerase possessed protein priming activity, as demonstrated by 32P-dGTP-labeling of the full size polymerase and the N-terminal portion of the molecule in an in vitro priming assay. The polymerase also exhibited polymerization activity, as detected in an in vitro polymerase assay by incorporation of radionucleotides into acid-precipitable polynucleotides and by synthesis of human hepatitis B virus (HBV) specific DNA with product lengths between 100 and 500 nucleotides. In addition, the polymerase was found to use an RNA sequence bearing HBV DR1/epsilon stem-loop motif as a template for DNA synthesis. Both the protein-priming and the reverse transcription activities of this recombinant polymerase are dependent on the RNA fragment containing the HBV DR1/epsilon stem-loop sequence known to be required for the polymerase activities. The in vitro systems used in this study will be applicable to further functional and biochemical studies of this enzyme.
Subject(s)
DNA-Directed DNA Polymerase/biosynthesis , Hepatitis B virus/enzymology , Protein Biosynthesis , Transcription, Genetic , Animals , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism , Deoxyguanine Nucleotides , Humans , Phosphorus Radioisotopes , RNA-Directed DNA Polymerase/biosynthesis , RNA-Directed DNA Polymerase/genetics , RNA-Directed DNA Polymerase/metabolism , Rabbits , Recombination, Genetic , Ribonuclease H/biosynthesis , Ribonuclease H/genetics , Ribonuclease H/metabolism , Templates, GeneticABSTRACT
Lamivudine, a novel cytosine analogue, exhibits potent antiviral activity against hepatitis B virus (HBV) in vitro and in vivo. The standard HBV DNA hybridization assay used in phase II clinical studies has a low sensitivity, the detection limit of HBV DNA levels being approximately 10(7) genome equivalents per ml (geq ml-1). In this work we used a semiquantitative polymerase chain reaction (PCR) assay (detection limit approximately 10(3) geq ml-1) to determine HBV DNA levels during a 24-week study of lamivudine in 51 stable chronic hepatitis B patients who were positive for hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). Patients were randomly allocated to receive oral doses of 25, 100 or 300 mg lamivudine once daily. At week 24 the median serum concentration of HBV DNA had fallen from 10(8) to 10(4) geq ml-1, a 4-log median reduction. A trend towards more profound suppression of viral replication with an increased dose of lamivudine was observed. After 12 weeks of therapy, 12% of patients had an HBV DNA level that was undetectable in the PCR assay; this increased to 26% after 24 weeks, while in an additional 20% of patients, HBV DNA decreased to the level of detection of the PCR assay. We conclude that a 24-week course of lamivudine decreases serum HBV DNA to the level of PCR detection in 46% of patients. Such additional viral suppressive activity with higher doses and more protracted lamivudine may be of clinical utility prior to liver transplantation. Further studies are needed to define the degree of virus suppression required in clinical practice, and methods are required to increase the efficacy of virus suppression.
Subject(s)
DNA, Viral/drug effects , Hepatitis B virus/drug effects , Hepatitis B, Chronic/drug therapy , Lamivudine/administration & dosage , Reverse Transcriptase Inhibitors/administration & dosage , Administration, Oral , Adolescent , Adult , Aged , DNA, Viral/blood , Female , Follow-Up Studies , Hepatitis B virus/genetics , Hepatitis B, Chronic/virology , Humans , Male , Middle Aged , Polymerase Chain Reaction/methods , Single-Blind Method , Time FactorsABSTRACT
Cirrhosis and hepatocellular carcinoma occur as long-term complications of chronic hepatitis B virus (HBV) infection. Antiviral therapy is potentially a successful approach for the treatment of patients with HBV infection, which includes the nucleoside analog, lamivudine [(-)2'-deoxy-3'-thiacytidine, 3TC]. Although resistance to lamivudine therapy has been reported in several HBV-infected patients, the pattern of resistance-associated mutations in HBV has not been fully characterized. We report a DNA sequence database that includes a 500-base pair region of the HBV polymerase gene from 20 patients with clinical manifestations of lamivudine resistance. Analysis of the database reveals two patterns of amino acid substitutions in the tyrosine, methionine, aspartate, aspartate (YMDD) nucleotide-binding locus of the HBV polymerase. HBV DNA from the sera of patients in Group I exhibits a substitution of valine for methionine at residue 552, accompanied by a substitution of methionine for leucine at residue 528. Patients in Group II had only an isoleucine-for-methionine substitution at position 552. Reconstruction of these mutations in an HBV replication-competent plasmid was performed in a transient transfection cell assay to determine the function/relevance of these mutations to lamivudine resistance. Both Group I and Group II mutations resulted in a substantial decrease in sensitivity to lamivudine treatment (> 10,000-fold shift in IC50 over wild-type [wt] IC50), strongly indicating that these mutations were involved in resistance to lamivudine. A hypothetical model of the HBV reverse transcriptase has been generated for further study of the role of these mutations in lamivudine resistance.
Subject(s)
Drug Resistance, Microbial/genetics , Hepatitis B virus/drug effects , Hepatitis B virus/genetics , Hepatitis B/virology , Lamivudine/pharmacology , Mutation , Reverse Transcriptase Inhibitors/pharmacology , Amino Acid Sequence , Amino Acid Substitution , Genes, Viral , Hepatitis B/drug therapy , Humans , Lamivudine/therapeutic use , Molecular Sequence Data , Reverse Transcriptase Inhibitors/therapeutic useABSTRACT
Lamivudine is a nucleoside analogue with efficacy in the suppression of hepatitis B viral (HBV) replication. In a previously reported study, lamivudine was administered to patients with chronic, actively replicating HBV infection who subsequently underwent liver transplantation. Patients became serum HBV DNA-negative in response to lamivudine before transplantation, which was continued in the post-transplant period. Two of four patients surviving the immediate postoperative period developed allograft reinfection 240 and 409 days post-transplant. The strain of the reinfecting virus was analyzed, and a mutation in the YMDD region of the viral polymerase conferring resistance to lamivudine was discovered. The long term follow-up of these two patients is reported. The first patient developed ascites 16.5 months after allograft reinfection. A transjugular liver biopsy performed 18 months after the emergence of the lamivudine-resistant strain revealed cirrhosis and lobular hepatitis without rejection. The gradient between hepatic vein wedged and free pressures was 13 mmHg, consistent with portal hypertension. The second patient, 16 months after allograft reinfection with the lamivudine-resistant strain, is without clinical evidence of portal hypertension, although liver enzymes remain elevated. Both patients were given a trial of famciclovir, which did not significantly suppress HBV viremia. In conclusion, lamivudine-resistant HBV strains with the YMDD mutation may have an aggressive clinical course with rapid progression to cirrhosis. Famciclovir did not appear to be an effective rescue agent in these two patients.
Subject(s)
Antiviral Agents/therapeutic use , Hepatitis B virus/drug effects , Hepatitis B/prevention & control , Lamivudine/therapeutic use , Liver Transplantation , Postoperative Complications/virology , 2-Aminopurine/analogs & derivatives , 2-Aminopurine/therapeutic use , Adult , Drug Resistance, Microbial , Famciclovir , Female , Follow-Up Studies , Hepatitis B/surgery , Hepatitis B virus/genetics , Humans , Liver/pathology , Liver Transplantation/pathology , Male , Middle Aged , Postoperative Complications/prevention & control , Recurrence , Time FactorsABSTRACT
PURPOSE: The objective of this study was to evaluate a dual action prodrug concept wherein an unnatural myristic acid analogue is coupled via an ester moiety to the 5'-position of FLT or AZT. Subsequent intracellular cleavage of the prodrug ester would simultaneously release FLT or AZT that could inhibit reverse transcriptase (RT), and the myristic acid analogue that could inhibit myristoyl-CoA:protein N-myristoyltransferase (NMT). METHODS: Cytotoxicity (2.2.15 cell culture), and anti-hepatitis B activity of 5'-O-myristoyl analogue prodrug derivatives of FLT and AZT (2-8) were evaluated in vitro using human liver hepatitis B virus (HBV) producing 2.2.15 cell lines. RESULTS: The 5'-O-(12-methoxydodecanoyl) ester derivatives of AZT (2, EC(50) = 2. 7 +/- 0.3 microM; CC(50)= 727 +/- 19 microM) and FLT (4, EC(50)= 2.8 +/- 0.3 microM; CC(50)= 186 +/- 20 microM) were the most effective anti-hepatitis B virus (anti-HBV) compounds of this series in a replication assay. In the series of 5'-O-myristic acid analogue ester prodrug derivatives of FLT, the relative anti-HBV potency order was MeO(CH(2))(11)CO(2)- > N(3)(CH(2))(11)CO(2)- and Br(CH(2))(11)CO(2)- > EtS(CH(2))(n)CO(2)- (n = 10 or 11) > Me(CH(2))(12)CO(2)- (myristoyl). CONCLUSIONS: The in vitro data suggest that the 5'-O-myristoyl analogue prodrug concept offers a potential drug design approach to design dual acting antiviral agents, with superior pharmacokinetic, biodistribution, reduced cytotoxicity and/or increased efficacy. In this regard, the 5'-O-(12-methoxydodecanoyl) prodrug ester of 3'-thia-3'-deoxythymidine (3TC) may offer the greatest potential for the treatment of HBV infection.