Showing papers by "Fabien Zoulim published in 2002"

Monitoring drug resistance in chronic hepatitis B virus (HBV)-infected patients during lamivudine therapy: evaluation of performance of INNO-LiPA HBV DR assay.

Abstract: Sensitive and early detection of emerging hepatitis B virus (HBV) drug resistance may not only help monitor the viral dynamics associated with lamivudine treatment but could also improve therapeutic decision making. This is especially important when new antivirals effective against lamivudine-resistant HBV become available. A total of 159 serum samples from 33 chronic HBV patients receiving lamivudine treatment were analyzed at four centers for the presence of lamivudine-resistant mutations at codons 528 [180] (proposed revised nomenclature according to Stuyver et al. [Hepatology 33:751-757, 2001] shown in brackets), 552 [204], and 555 [207] of the HBV polymerase. Sequencing data were compared with results generated by the INNO-LiPA HBV DR line probe assay (LiPA), an assay based on reverse hybridization of amplified HBV DNA fragments with specific nucleotide probes immobilized on nitrocellulose strips. LiPA provided at least the same information as sequencing for 97.5% of all codons analyzed for codon 528 [180], 95% for codon 552 [204], and 100% for codon 555 [207]. The most common reason for discrepant or indeterminate results (0.4% and 1.5%, respectively) in a small percentage of the population tested could be attributed to polymorphisms not yet covered by LiPA probes. In at least five patients, a mutant could be detected earlier by LiPA than by sequencing. In 15 patients, LiPA detected mixed wild-type and mutant virus populations before viral breakthrough. These results demonstrate that INNO-LiPA HBV DR is a highly sensitive and easily applicable assay for the detection and monitoring of lamivudine-resistant mutations in chronic hepatitis B patients and that the assay is more sensitive than sequencing in detecting mixed mutant and wild-type sequences.

Inhibitory Effect of Adefovir on Viral DNA Synthesis and Covalently Closed Circular DNA Formation in Duck Hepatitis B Virus-Infected Hepatocytes In Vivo and In Vitro

Abstract: The elimination of viral covalently closed circular DNA (CCC DNA) from the nucleus of infected hepatocytes is an obstacle to achieving sustained viral clearance during antiviral therapy of chronic hepatitis B virus (HBV) infection. The aim of our study was to determine whether treatment with adefovir, a new acyclic nucleoside phosphonate, the prodrug of which, adefovir dipivoxil, is in clinical evaluation, is able to suppress viral CCC DNA both in vitro and in vivo using the duck HBV (DHBV) model. First, the effect of adefovir on viral CCC DNA synthesis was examined with primary cultures of DHBV-infected fetal hepatocytes. Adefovir was administered for six consecutive days starting one day before or four days after DHBV inoculation. Dose-dependent inhibition of both virion release in culture supernatants and synthesis of intracellular viral DNA was observed. Although CCC DNA amplification was inhibited by adefovir, CCC DNA was not eliminated by antiviral treatment and the de novo formation of CCC DNA was not prevented by pretreatment of the cells. Next, preventive treatment of experimentally infected ducklings with lamivudine or adefovir revealed that both efficiently suppressed viremia and intrahepatic DNA. However, persistence of viral DNA even when detectable only by PCR was associated with a recurrence of viral replication following drug withdrawal. Taken together, our results demonstrate that adefovir is a potent inhibitor of DHBV replication that inhibits CCC DNA amplification but does not effectively prevent the formation of CCC DNA from incoming viral genomes.

Assessing hepatitis B virus resistance in vitro and molecular mechanisms of nucleoside resistance.

Abstract: The development of viral resistance to drugs can significantly reduce the efficacy of therapy for chronic hepatitis B virus (HBV) infection, slowing the rate of seroconversion, reversing previous gains in liver function and histology, and allowing disease progression The efficacy of the nucleoside analogue lamivudine has been compromised by the development of viral resistance Research into the mechanisms of nucleoside resistance has intensified and has included the development of new resistance assays Resistance to lamivudine appears to be caused by the compound's structural features Mutations are selected in HBV polymerase that can discriminate among the structural differences between lamivudine triphosphate and the natural substrate mimicked by lamivudine, dCTP, causing steric hindrance and reduced efficiency of incorporation of lamivudine but not the natural substrate Unlike lamivudine, adefovir dipivoxil, an oral anti-HBV agent in late-stage development, has not been associated with HBV resistance, either in vitro or in vivo One long-term clinical study has shown a lack of resistance to adefovir dipivoxil in patients treated for up to 136 weeks In addition, adefovir dipivoxil is a nucleotide (rather than a nucleoside) analogue that exhibits no cross-resistance with lamivudine and is effective against lamivudine-resistant HBV In one hypothesis, this lack of resistance has been attributed to the minimal flexible acyclic structure of adefovir that subverts resistance due to steric hindrance By reducing the rate and frequency of development of drug resistance, adefovir dipivoxil and other new oral antiviral agents maximize the clinical advantages of long-term sustained viral suppression (HBV DNA reduction) to achieve lasting therapeutic benefits

A preliminary benefit-risk assessment of lamivudine for the treatment of chronic hepatitis B virus infection.

Abstract: Chronic hepatitis B virus (HBV) infection remains a major public health problem worldwide. Until recently, interferon (IFN)-α was the only approved drug for the treatment of chronic HBV infection. The recent registration of lamivudine, a dideoxycytidine analogue that inhibits both the HIV and HBV reverse transcriptases, has provided new perspectives for the treatment of chronic HBV infection. Lamivudine treatment for 12 months leads to a control of viral replication during therapy in the majority of the patients and to sustained anti-hepatitis B e (anti-HBe) seroconversion in 16 to 22% of the patients, associated with a biochemical and histological response. Further studies showed that extended lamivudine therapy increases the rate of anti-HBe seroconversion. However, long-term therapy is associated with the progressive emergence of drug resistant mutants. In most cases these mutants are not associated with a deterioration of the liver disease within the available follow-up. In the remaining patients and in particular settings such as liver transplantation, a severe exacerbation of the liver disease is observed and that requires add-on therapy. Lamivudine treatment of patients infected with a pre-core mutant also showed beneficial effect with the control of viral replication, and a biochemical and histological response in approximately 60% of the patients at 1 year. These patients face the same problem of drug resistant mutants, and the optimal duration of lamivudine treatment still needs to be determined in this clinical situation. Moreover, lamivudine therapy is the only therapeutic option in decompensated cirrhotic patients to allow liver transplantation, and in liver transplant patients with HBV recurrence following transplantation. Adverse effects of lamivudine therapy are comparable to those observed in placebo-treated patients. ALT flares have been observed mainly in relation to the re-occurrence of viral replication due to the rebound of viral replication after therapy withdrawal, or to the emergence of drug resistance mutants. Therefore, lamivudine provides a new treatment alternative for patients with chronic HBV infection. For each patient, its indication has to be weighed against the risk of developing viral resistance but also against the risk of natural history of the disease.

Animal models for the study of HBV infection and the evaluation of new anti‐HBV strategies

Abstract: Backgroud: Our aim was to evaluate the anti-HBV activity of a novel L-nucleoside analog, 2',3'-dideoxy-2',3'-didehydro-β-L-5-fluorocytidine (β-L-Fd4C), in study models of HBV infection. Method: Its mechanism of action was evaluated on the in vitro expressed duck HBV (DHBV) reverse transcriptase and in primary hepatocyte cultures of duck and human origin. The capacity of antiviral therapy to clear viral infection was analyzed in vivo in the duck and woodchuck models. Results: β-L-Fd4C-TP exhibited a more potent inhibitory effect on the RT activity of the DHBV polymerase than other cytidine analogs (lamivudine-TP, ddC-TP, β-L-FddC-TP). In primary duck hepatocyte cultures, β-L-Fd4C exhibited a long-lasting inhibitory effect on viral DNA synthesis but could not clear viral cccDNA. In vivo treatment with β-L-Fd4C in infected ducklings and woodchucks, induced a greater suppression of viremia and intrahepatic viral DNA synthesis than with lamivudine. However, covalently closed circular DNA persistence explained the relapse of viral replication after treatment withdrawal. Viral spread was strongly reduced in the case of early therapeutical intervention, but the number of infected cells did not decline when therapy was started during chronic infection. Liver histology analysis showed a decrease in the inflammatory activity of chronic hepatitis while no ultrastructural modification of liver cells was observed in electron microscopy studies. Furthermore, in human primary hepatocyte cultures, β-L-Fd4C induced a significant inhibition of HBV DNA synthesis. Conclusion: β-L-Fd4C is a potent inhibitor of hepadnavirus RT and inhibits viral DNA synthesis in hepatocytes both in vitro and in vivo. These experimental studies allowed as to show that β-L-Fd4C is a promising anti-HBV agent. Combination therapy should be evaluated to eradicate viral infection.

Les hépatites chroniques B : histoire naturelle et traitements

Abstract: La prise en charge des infections chroniques virales B s’est considérablement modifiée au cours des dernières années en raison des progrès de la virologie qui ont permis une meilleure connaissance physiopathologique de l’infection et le développement d’outils diagnostiques plus performants, et en raison de l’apparition récente de nouveaux médicaments antiviraux. La meilleure connaissance des interactions entre l’hôte et le virus permet une analyse des situations clinico-biologiques, rencontrées aux différentes phases de l’infection au virus de l’hépatite B (VHB), indispensable pour poser une indication thérapeutique. Le développement des méthodes qualitatives et quantitatives de biologie moléculaire a considérablement amélioré la sensibilité de détection de l’ADN du VHB et a permis une surveillance précise des traitements antiviraux. Enfin, la meilleure connaissance du cycle de réplication du VHB et des mécanismes moléculaires qu’il implique a permis le développement de nouveaux antiviraux capables d’inhiber spécifiquement cette réplication virale. Nous aborderons successivement dans cette synthèse quelques bases virologiques, l’histoire naturelle de l’infection à VHB en évoquant le cas particulier des mutants pré-C et les traitements actuels des hépatites chroniques B.

Study of the antiviral mechanism of action of ribavirin in the bovine viral diarrhea virus model

Abstract: Buts - En l'absence de modele de culture cellulaire du virus de l'hepatite C (VHC), le but de notre etude etait d'evaluer l'hypothese du mecanisme d'action de mutagenese letale de la ribavirine, analogue de la guanosine, dans le modele du virus de la diarrhee bovine (BVDV). Methodes - Nous avons evalue la capacite d'inhibition de la replication du BVDV par la ribavirine en suivant l'inhibition de l'apparition de l'effet cytopathique de la souche NADL (National Animal Disease Laboratory) cytopathique du BVDV en culture de cellules BT (bovine turbinate, cellules intestinales bovines). Ensuite, nous avons amplifie par RT-PCR les regions 5'NTR et NS5B du genome du BVDV dans les surnageants provenant de cultures cellulaires infectees, traitees ou non par la ribavirine, puis sequence les produits amplifies afin de deceler d'eventuelles mutations. Resultats - Au niveau phenotypique, la ribavirine etait capable d'inhiber la replication du BVDV de facon d'autant plus puissante qu'elle etait ajoutee tot par rapport a l'inoculation virale. Ces resultats sont en faveur d'un effet antiviral direct de la ribavirine sur le BVDV a une etape precoce du cycle viral. Au niveau genotypique, la ribavirine a diminue le taux d'ARN viral. Conclusion - Nous avons montre que la ribavirine exerce un effet inhibiteur direct sur la replication du BVDV. Ces resultats pourraient expliquer l'effet synergique de la ribavirine et de l'interferon lors d'une bitherapie chez les malades porteurs chroniques du VHC. Cet effet demande a etre confirme avec les replicons sous genomiques du VHC recemment disponibles.