RESUMEN
CD8+ T cells are key antiviral effectors against hepatitis B virus (HBV), yet their number and function can be compromised in chronic infections. Preclinical HBV models displaying CD8+ T cell dysfunction showed that interleukin-2 (IL-2)-based treatment, unlike programmed cell death ligand 1 (PD-L1) checkpoint blockade, could reverse this defect, suggesting its therapeutic potential against HBV. However, IL-2's effectiveness is hindered by its pleiotropic nature, because its receptor is found on various immune cells, including regulatory T (Treg) cells and natural killer (NK) cells, which can counteract antiviral responses or contribute to toxicity, respectively. To address this, we developed a cis-targeted CD8-IL2 fusion protein, aiming to selectively stimulate dysfunctional CD8+ T cells in chronic HBV. In a mouse model, CD8-IL2 boosted the number of HBV-reactive CD8+ T cells in the liver without substantially altering Treg or NK cell counts. These expanded CD8+ T cells exhibited increased interferon-γ and granzyme B production, demonstrating enhanced functionality. CD8-IL2 treatment resulted in substantial antiviral effects, evidenced by marked reductions in viremia and antigenemia and HBV core antigen-positive hepatocytes. In contrast, an untargeted CTRL-IL2 led to predominant NK cell expansion, minimal CD8+ T cell expansion, negligible changes in effector molecules, and minimal antiviral activity. Human CD8-IL2 trials in cynomolgus monkeys mirrored these results, achieving a roughly 20-fold increase in peripheral blood CD8+ T cells without affecting NK or Treg cell numbers. These data support the development of CD8-IL2 as a therapy for chronic HBV infection.
Asunto(s)
Hepatitis B Crónica , Interleucina-2 , Humanos , Animales , Ratones , Virus de la Hepatitis B , Linfocitos T CD8-positivos , Hepatitis B Crónica/tratamiento farmacológico , Antivirales/farmacología , Antivirales/uso terapéuticoRESUMEN
Bevirimat (BVM) is the first of a new class of anti-HIV drugs with a novel mode of action known as maturation inhibitors. BVM inhibits the last cleavage of the Gag polyprotein by HIV-1 protease, leading to the accumulation of the p25 capsid-small peptide 1 (SP1) intermediate and resulting in noninfectious HIV-1 virions. Early clinical studies of BVM showed that over 50% of the patients treated with BVM did not respond to treatment. We investigated the impact of prior antiretroviral (ARV) treatment and/or natural genetic diversity on BVM susceptibility by conducting in vitro phenotypic analyses of viruses made from patient samples. We generated 31 recombinant viruses containing the entire gag and protease genes from 31 plasma samples from HIV-1-infected patients with (n = 21) or without (n = 10) prior ARV experience. We found that 58% of the patient isolates tested had a >10-fold reduced susceptibility to BVM, regardless of the patient's ARV experience or the level of isolate resistance to protease inhibitors. Analysis of mutants with site-directed mutations confirmed the role of the V370A SP1 polymorphism (SP1-V7A) in resistance to BVM. Furthermore, we demonstrated for the first time that a capsid polymorphism, V362I (CA protein-V230I), is also a major mutation conferring resistance to BVM. In contrast, none of the previously defined resistance-conferring mutations in Gag selected in vitro (H358Y, L363M, L363F, A364V, A366V, or A366T) were found to occur among the viruses that we analyzed. Our results should be helpful in the design of diagnostics for prediction of the potential benefit of BVM treatment in HIV-1-infected patients.
Asunto(s)
Fármacos Anti-VIH/farmacología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Succinatos/farmacología , Triterpenos/farmacología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , ADN Viral/genética , Farmacorresistencia Viral/genética , Genes Virales , Genes gag , Proteasa del VIH/genética , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Técnicas In Vitro , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fenotipo , Proteínas Recombinantes/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Tenofovir, also known as PMPA, R-9-(2-(phosphonomethoxypropyl)adenine, is a nucleotide reverse transcriptase (RT) inhibitor. We have determined the crystal structures of two related complexes of HIV-1 RT with template primer and tenofovir: (i) a ternary complex at a resolution of 3.0 A of RT crosslinked to a dideoxy-terminated DNA with tenofovir-diphosphate bound as the incoming substrate; and (ii) a RT-DNA complex at a resolution of 3.1 A with tenofovir at the 3' primer terminus. The tenofovir nucleotide in the tenofovir-terminated structure seems to adopt multiple conformations. Some nucleoside reverse transcriptase inhibitors, including 3TC and AZT, have elements ('handles') that project beyond the corresponding elements on normal dNTPs (the 'substrate envelope'). HIV-1 RT resistance mechanisms to AZT and 3TC take advantage of these handles; tenofovir's structure lacks handles that could protrude through the substrate envelope to cause resistance.
Asunto(s)
Adenina/análogos & derivados , Adenina/química , ADN Viral/química , Transcriptasa Inversa del VIH/química , Organofosfonatos , Compuestos Organofosforados/química , Inhibidores de la Transcriptasa Inversa/química , Secuencia de Bases , Cartilla de ADN , Modelos Moleculares , TenofovirRESUMEN
Human rhinoviruses (HRV) represent the single most important causative agent of the common cold. The HRV genome encodes an RNA-dependent RNA polymerase (RdRp) designated 3D polymerase that is required for replication of the HRV RNA genome. We have expressed and purified recombinant HRV-16 3D polymerase to near homogeneity from Escherichia coli transformed with an expression plasmid containing the full-length 460 amino acid HRV-16 3D sequence with a methionine at the N-terminus and a glycine-serine linker followed by a 6-histidine affinity tag at the C-terminus. The purified recombinant protein has rifampicin-resistant activity in a poly(A)-dependent poly(U) polymerase assay while corresponding fractions similarly purified from E. coli transformed with an expression plasmid without the HRV-16 3D sequence showed no activity. The optimal conditions for temperature, pH, divalent cations Mg(2+) and Mn(2+), and KCl were determined. The recombinant protein has RNA polymerase activity on homopolymeric templates poly(A) and poly(C) and heteropolymeric RNA templates primed with either RNA or DNA oligonucleotide primers or self-primed by a copy-back mechanism. A unique, secondary structureless heteropolymeric RNA template that is an efficient substrate was developed to facilitate kinetic characterizations of the enzyme. In the presence of Mg(2+), the enzyme displayed strong base and sugar specificity. However, when Mg(2+) was replaced by Mn(2+) specificity for ribonucleotides was lost, utilization of deoxynucleotides became possible and primer-independent activity was observed on the poly(C) template. Zn(2+) was found to inhibit HRV-16 3D polymerase with an IC(50) as low as 0.6 microM by a mechanism distinct from the magnesium ion stimulation. The activity of this 6His-tagged HRV-16 3D polymerase was compared with that of a recombinant HRV-16 3D polymerase expressed without the 6His-tag and was found to be identical. The availability of recombinant rhinovirus RdRp in a purified form will facilitate the structure-function analysis of this enzyme as well as the identification of specific inhibitors to the rhinovirus 3D polymerase that have therapeutic value in the treatment of the common cold.
Asunto(s)
ARN Polimerasa Dependiente del ARN/metabolismo , Rhinovirus/enzimología , Cationes Bivalentes , Escherichia coli , Expresión Génica , Células HeLa , Histidina , Humanos , Cinética , ARN/biosíntesis , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/genética , Especificidad por Sustrato , Moldes Genéticos , ZincRESUMEN
CyaA is crucial for colonization by Bordetella pertussis, the etiologic agent of whooping cough. Here we report crystal structures of the adenylyl cyclase domain (ACD) of CyaA with the C-terminal domain of calmodulin. Four discrete regions of CyaA bind calcium-loaded calmodulin with a large buried contact surface. Of those, a tryptophan residue (W242) at an alpha-helix of CyaA makes extensive contacts with the calcium-induced, hydrophobic pocket of calmodulin. Mutagenic analyses show that all four regions of CyaA contribute to calmodulin binding and the calmodulin-induced conformational change of CyaA is crucial for catalytic activation. A crystal structure of CyaA-calmodulin with adefovir diphosphate, the metabolite of an approved antiviral drug, reveals the location of catalytic site of CyaA and how adefovir diphosphate tightly binds CyaA. The ACD of CyaA shares a similar structure and mechanism of activation with anthrax edema factor (EF). However, the interactions of CyaA with calmodulin completely diverge from those of EF. This provides molecular details of how two structurally homologous bacterial toxins evolved divergently to bind calmodulin, an evolutionarily conserved calcium sensor.
Asunto(s)
Toxina de Adenilato Ciclasa/química , Toxina de Adenilato Ciclasa/metabolismo , Bordetella pertussis/enzimología , Calmodulina/química , Calmodulina/metabolismo , Toxina de Adenilato Ciclasa/genética , Secuencia de Aminoácidos , Bordetella pertussis/genética , Dominio Catalítico , Cristalografía por Rayos X , Datos de Secuencia Molecular , Mutación/genética , Nucleótidos/química , Nucleótidos/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Alineación de Secuencia , Homología Estructural de Proteína , Venenos de Víboras/química , Venenos de Víboras/metabolismoRESUMEN
Thrombin is the ultimate protease of the blood clotting cascade and plays a major role in its own regulation. The ability of thrombin to exhibit both pro- and anti-coagulant properties has spawned efforts to turn thrombin into an anticoagulant for therapeutic purposes. This quest culminated in the identification of the E217K variant through scanning and saturation mutagenesis. The antithrombotic properties of E217K thrombin are derived from its inability to convert fibrinogen to a fibrin clot while maintaining its thrombomodulin-dependent ability to activate the anticoagulant protein C pathway. Here we describe the 2.5-A crystal structure of human E217K thrombin, which displays a dramatic restructuring of the geometry of the active site. Of particular interest is the repositioning of Glu-192, which hydrogen bonds to the catalytic Ser-195 and which results in the complete occlusion of the active site and the destruction of the oxyanion hole. Substrate binding pockets are further blocked by residues previously implicated in thrombin allostery. We have concluded that the E217K mutation causes the allosteric inactivation of thrombin by destabilizing the Na(+) binding site and that the structure thus may represent the Na(+)-free, catalytically inert "slow" form.
Asunto(s)
Trombina/química , Sitio Alostérico , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Proteica , Conformación Proteica , Sodio/química , Sodio/metabolismo , Trombina/genéticaRESUMEN
Combination therapies may be required for long-term management of some patients chronically infected with hepatitis B virus (HBV). Adefovir is a nucleotide analog that has similar activity against wild-type and lamivudine-resistant HBV. In contrast to lamivudine, clinical resistance to the prodrug adefovir dipivoxil emerges infrequently. Based on its clinical efficacy and low frequency of resistance, adefovir dipivoxil may form an important component of combination regimens. We therefore investigated the in vitro antiviral efficacy of combinations of adefovir with other nucleoside analogs (lamivudine, entecavir, emtricitabine [FTC],and telbivudine [L-dT]) and the nucleotide analog tenofovir. Using a novel stable cell line that expresses high levels of wild-type HBV, we assayed the antiviral activity of each drug alone and in combination with adefovir. All two-drug combinations resulted in greater antiviral effects than treatments with single agents and could be characterized as additive by the Bliss independence model. Analysis using the Loewe additivity model indicated that adefovir exerted additive antiviral effects when combined with lamivudine, FTC, or L-dT and moderately synergistic effects when combined with entecavir or tenofovir. There was no evidence of cytotoxicity with any of the drugs when used alone or in combination at the tested doses.
Asunto(s)
Adenina/análogos & derivados , Adenina/farmacología , Antivirales/farmacología , Virus de la Hepatitis B/efectos de los fármacos , Nucleósidos/farmacología , Organofosfonatos/farmacología , Southern Blotting , Línea Celular , Supervivencia Celular/efectos de los fármacos , Combinación de Medicamentos , Sinergismo Farmacológico , HumanosRESUMEN
BACKGROUND/AIMS: In contrast to lamivudine, adefovir dipivoxil (ADV) therapy is associated with delayed and infrequent selection of drug resistant hepatitis B virus (HBV). METHODS: A 52 year-old man was treated with lamivudine for an HBV recurrence on his liver graft. A viral breakthrough was observed and the patient received ADV. Serum HBV DNA decreased rapidly and lamivudine was discontinued while ADV monotherapy was maintained. Serum HBV DNA levels remained suppressed until a second breakthrough was observed. Lamivudine was then reintroduced together with ADV, and serum HBV DNA became undetectable by polymerase chain reaction. RESULTS: Sequence analyses of the HBV polymerase gene revealed a sequential selection of lamivudine resistance mutations L180M+M204V, followed by a reversion to wild-type, and subsequently the selection of a novel adefovir resistance mutation N236T. Phenotypic analyses in cell culture assays demonstrated that the HBV isolates at the time of ADV breakthrough had reduced susceptibility to ADV. This mutant remained sensitive to lamivudine, entecavir and emtricitabine in vitro. CONCLUSIONS: We describe the first case of sequential selection of lamivudine and adefovir resistant strains of HBV in a liver transplantation patient. The selection of the N236T polymerase mutant was associated with resistance to ADV but remained sensitive to lamivudine in vitro and in vivo.
Asunto(s)
Adenina/análogos & derivados , Adenina/administración & dosificación , Virus de la Hepatitis B/efectos de los fármacos , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/cirugía , Trasplante de Hígado , Organofosfonatos , Inhibidores de la Transcriptasa Inversa/administración & dosificación , Secuencia de Aminoácidos , Terapia Combinada , Farmacorresistencia Viral/genética , Virus de la Hepatitis B/genética , Hepatitis B Crónica/virología , Humanos , Lamivudine/administración & dosificación , Masculino , Persona de Mediana Edad , Datos de Secuencia MolecularRESUMEN
Seven hundred nucleoside treatment-naive patients were enrolled in two phase 3 trials of adefovir dipivoxil (ADV) for the treatment of chronic hepatitis B. To monitor for the emergence of potential adefovir resistance mutations over the first 48 weeks, all intent-to-treat patients (467 ADV-treated and 228 placebo patients) were included in a prospectively defined, treatment-blinded, virology substudy. The study protocol mandated genotypic analysis for all patients with detectable hepatitis B virus (HBV) DNA by Roche Amplicor polymerase chain reaction (PCR) at baseline and week 48, and in vitro phenotypic analyses for patients with conserved site substitutions in HBV polymerase or 1.0 log(10) or greater increase in HBV DNA from nadir. Paired sequences of the entire HBV reverse transcriptase were obtained for 271 ADV-treated and 227 placebo patients by using a sequencing method that detects down to 30% of minor species present within mixtures. Four substitutions (rtS119A, rtH133L, rtV214A, and rtH234Q) developed once each at conserved sites in HBV polymerase in 4 ADV-treated patients. Seven conserved site substitutions developed in 6 placebo patients. HBV mutants encoding the 4 substitutions that emerged in ADV-treated patients remained fully susceptible to adefovir in vitro. Furthermore, these 4 ADV-treated patients had HBV-DNA reductions of 3.3 to 5.9 log(10) copies/mL by week 48 with no rebound. All other substitutions occurred at very low frequencies (<1.6%) at polymorphic sites and were not associated with HBV-DNA increases in patients or adefovir resistance in vitro. In conclusion, no adefovir resistance mutations were identified in a large group of chronic hepatitis B patients treated with ADV for 48 weeks.
Asunto(s)
Adenina/análogos & derivados , Adenina/administración & dosificación , Antivirales/administración & dosificación , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/genética , Hepatitis B Crónica/tratamiento farmacológico , Organofosfonatos , Adenina/química , Adolescente , Adulto , Anciano , Sustitución de Aminoácidos , Antivirales/química , Secuencia de Bases , Secuencia Conservada , ADN Viral/análisis , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Estudios ProspectivosRESUMEN
Therapy of chronic hepatitis B virus (HBV) infection with the polymerase inhibitor lamivudine frequently is associated with the emergence of viral resistance. Genotypic changes in the YMDD motif (reverse transcriptase [rt] mutations rtM204V/I) conferred resistance to lamivudine as well as reducing the in vitro replication efficiency of HBV. A second mutation, rtL180M, was previously reported to partially restore replication fitness as well as to augment drug resistance in vitro. Here we report the functional characterization of a third polymerase mutation (rtV173L) associated with resistance to lamivudine and famciclovir. rtV173L was observed at baseline in 9 to 22% of patients who entered clinical trials of adefovir dipivoxil for the treatment of lamivudine-resistant HBV. In these patients, rtV173L was invariably found as a third mutation in conjunction with rtL180M and rtM204V. In vitro analyses indicated that rtV173L did not alter the sensitivity of wild-type or lamivudine-resistant HBV to lamivudine, penciclovir, or adefovir but instead enhanced viral replication efficiency. A molecular model of HBV polymerase indicated that residue rtV173 is located beneath the template strand of HBV nucleic acid near the active site of the reverse transcriptase. Substitution of leucine for valine at this residue may enhance polymerization either by repositioning the template strand of nucleic acid or by affecting other residues involved in the polymerization reaction. Together, these results suggest that rtV173L is a compensatory mutation that is selected in lamivudine-resistant patients due to an enhanced replication phenotype.
Asunto(s)
2-Aminopurina/análogos & derivados , Virus de la Hepatitis B/enzimología , Lamivudine/uso terapéutico , Mutación , ADN Polimerasa Dirigida por ARN/genética , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Selección Genética , 2-Aminopurina/farmacología , 2-Aminopurina/uso terapéutico , Secuencia de Aminoácidos , Antivirales/farmacología , Antivirales/uso terapéutico , Farmacorresistencia Viral , Famciclovir , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/fisiología , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/virología , Humanos , Lamivudine/farmacología , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Datos de Secuencia Molecular , Inhibidores de la Transcriptasa Inversa/farmacología , Replicación ViralRESUMEN
Edema factor (EF), a key virulence factor in anthrax pathogenesis, has calmodulin (CaM)-activated adenylyl cyclase activity. We have found that adefovir dipivoxil, a drug approved to treat chronic infection of hepatitis B virus, effectively inhibits EF-induced cAMP accumulation and changes in cytokine production in mouse primary macrophages. Adefovir diphosphate (PMEApp), the active cellular metabolite of adefovir dipivoxil, inhibits the adenylyl cyclase activity of EF in vitro with high affinity (K(i) = 27 nM). A crystal structure of EF-CaM-PMEApp reveals that the catalytic site of EF forms better van der Waals contacts and more hydrogen bonds with PMEApp than with its endogenous substrate, ATP, providing an explanation for the approximately 10,000-fold higher affinity EF-CaM has for PMEApp versus ATP. Adefovir dipivoxil is a clinically approved drug that can block the action of an anthrax toxin. It can be used to address the role of EF in anthrax pathogenesis.
Asunto(s)
Adenina/análogos & derivados , Adenina/farmacología , Inhibidores de Adenilato Ciclasa , Antivirales/farmacología , Hepatitis B Crónica/tratamiento farmacológico , Organofosfonatos , Adenina/química , Adenilil Ciclasas/química , Animales , Antígenos Bacterianos , Antivirales/química , Toxinas Bacterianas , Sitios de Unión , Células CHO , Línea Celular , Cricetinae , AMP Cíclico/metabolismo , Exotoxinas/antagonistas & inhibidores , Cinética , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/antagonistas & inhibidores , Spodoptera , TransfecciónRESUMEN
Current therapies for chronic hepatitis B virus (HBV) infection do not provide adequate long-term control of viral replication in the majority of patients. Monotherapy with nucleoside analogs, such as lamivudine and famciclovir, is effective for short periods but results in the emergence of drug-resistant HBV in a substantial number of patients within 1 year of therapy. Adefovir dipivoxil (ADV) has demonstrated clinical activity against wild-type and lamivudine-resistant HBV, but it is unclear whether resistance mutations will emerge after long-term therapy with this drug. To determine whether extended treatment with ADV led to the emergence of drug-resistant populations of HBV, we analyzed virus isolated from patients currently enrolled in a long-term open-label study. The reverse transcriptase domain of HBV polymerase was amplified and sequenced from patients that had received a cumulative exposure of up to 60 weeks of ADV. During our analyses, several previously unreported amino acid substitutions were observed in the reverse transcriptase domain of HBV. Importantly, none of the observed mutations occurred in more than 1 patient, nor were they associated with an adefovir-resistant phenotype in vitro. Furthermore, none of the patients from whom these mutant viruses were isolated had evidence of virologic rebound. In conclusion, these results, although based on a limited number of patients, suggest that treatment with ADV does not lead to the emergence of resistant virus after up to 60 weeks of therapy.
Asunto(s)
Adenina/análogos & derivados , Adenina/uso terapéutico , Antivirales/uso terapéutico , Virus de la Hepatitis B/efectos de los fármacos , Hepatitis B Crónica/tratamiento farmacológico , Organofosfonatos , Adulto , Secuencia de Bases , Carcinoma Hepatocelular , Secuencia Conservada , ADN Viral/análisis , ADN Polimerasa Dirigida por ADN/genética , Farmacorresistencia Viral , Femenino , Estudios de Seguimiento , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/crecimiento & desarrollo , Hepatitis B Crónica/virología , Humanos , Neoplasias Hepáticas , Masculino , Persona de Mediana Edad , Mutagénesis , Fenotipo , Análisis de Secuencia de ADN , Células Tumorales Cultivadas , Carga Viral , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND/AIMS: Combination of antiviral drugs with immunotherapeutic approaches may be a promising approach for the treatment of chronic hepatitis B. We used the duck HBV (DHBV) infection model to evaluate the efficacy of the combination of adefovir with DNA-immunization by comparison with the respective monotherapies. METHODS: Pekin ducks chronically infected with DHBV received adefovir treatment alone or in association with intramuscular immunization with a plasmid (pCI-preS/S) expressing the DHBV large envelope protein. Ducks immunized with pCI-preS/S plasmid alone and two control groups receiving empty plasmid injections or no treatment were followed in parallel. RESULTS: All animals treated with adefovir showed a marked drop in viremia titers during drug administration, followed by a rebound of viral replication after drug withdrawal. Eight weeks after the third DNA boost, the median of viremia within the duck group receiving the combination therapy tended to be lower compared to that of the other groups. In addition, our results suggest a trend to an additive effect of adefovir and DNA vaccine since a 51% decrease in DHBV DNA was observed in autopsy liver samples from combination therapy group, whereas pCI-preS/S or adefovir monotherapies decreased intrahepatic viral DNA by 38 and 14%, respectively. This effect was sustained since it was observed 12 weeks after the end of therapy. CONCLUSIONS: Our results suggest that combination of adefovir with DNA-vaccine may be able to induce a sustained antiviral effect in vivo.
Asunto(s)
Adenina/uso terapéutico , Antivirales/uso terapéutico , Infecciones por Hepadnaviridae/tratamiento farmacológico , Virus de la Hepatitis B del Pato , Hepatitis Viral Animal/tratamiento farmacológico , Inmunización , Organofosfonatos , Vacunas de ADN/uso terapéutico , Adenina/análogos & derivados , Animales , Formación de Anticuerpos , Patos , Infecciones por Hepadnaviridae/inmunología , Virus de la Hepatitis B del Pato/efectos de los fármacos , Virus de la Hepatitis B del Pato/inmunología , Hepatitis Viral Animal/inmunología , Hígado/virología , Viremia/tratamiento farmacológico , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND & AIMS: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is a unique episomal replicative intermediate responsible for persistent infection of hepatocytes. Technical constraints have hampered the direct study of cccDNA maintenance and clearance mechanisms in patients. The aim of this study was to develop a sensitive and specific assay for quantifying cccDNA in biopsy samples from chronic hepatitis B patients during different natural history phases and in patients undergoing antiviral therapy. METHODS: Intrahepatic cccDNA levels were quantified by a specific real-time PCR assay. Ninety-eight liver biopsy samples from patients in the major phases of the natural history of chronic hepatitis B and 32 pairs of samples from patients receiving adefovir dipivoxil (ADV) therapy were assessed. RESULTS: cccDNA was detected, at levels ranging over 3 orders of magnitude, in patients in different phases of the natural history of chronic hepatitis B. cccDNA levels were strongly correlated with levels of total intracellular HBV DNA and serum HBV DNA. Forty-eight weeks of ADV therapy resulted in a significant 0.8 log decrease in cccDNA copies/cell. Changes in cccDNA were correlated with a similar reduction in serum HBsAg titer but not with a decrease in the number of HBV antigen-positive cells during ADV treatment. CONCLUSIONS: cccDNA persists throughout the natural history of chronic hepatitis B, even in patients with serologic evidence of viral clearance. Long-term ADV therapy significantly decreased cccDNA levels by a primarily noncytolytic mechanism.