15th International HIV Drug Resistance Workshop 13-17 June 2006, Sitges, Spain

DEVELOPMENT OF A NEW HIGH THROUGHPUT PHENOTYPING TEST TO EVALUATE THE DRUG SUSCEPTIBILITY OF HBV STRAINS ISOLATED FROM PATIENTS: PHENOSCRIPT-HBV®

Antivir Ther. 2006, 11:S9 (abstract no. 7)

L Barraud¹, S Durantel¹, A Ollivet¹, D Durantel², S Lebel-Binay¹, K Skrabal¹, JL Faudon³, G Avenard¹ and F Zoulim^2
1Bioalliance Pharma, Paris, France; ²INSERM U271, Lyon, France; ³Eurofins VIRAlliance Inc., Paris, France

BACKGROUND: During chronic hepatitis B virus (HBV) infection, as for HIV, the efficiency of antiviral treatments can be compromised due to the emergence of HBV drug-resistant mutants. Thus, the development of HBV drug resistance assays may become essential for the management of infected patients. There are currently three nucleos(t)ides analogues approved for the treatment of chronic hepatitis B: lamivudine, adefovir, and entecavir. Three others are actually in clinical trial phase III: tenofovir, emtricitabine and telbivudine. We developed and validated a high throughput in vitro phenotyping assay to evaluate in vitro HBV drug resistance in clinical isolates.

METHODS: We modified our described cloning based phenotypic assay (Durantel et al, Hepatology. 2004 Oct;40(4):855-64) using a high throughput cell culture format and a highly sensitive HBV quantification by qPCR developed in the same format. In addition, we developed a more rapid one-step HBV PCR amplification which can replace the cloning strategy for a high throughput and rapid phenotyping test in clinical isolates: Phenoscript-HBV®

RESULTS: We demonstrate that this new technique is suitable for all HBV genotypes (A to H) obtained from patients since our qPCR is adapted to amplify all HBV genomes. This test has been validated on wild type laboratory strains, on plasma virus obtained from drug naïve patients and on mutant strains obtained from patients with clinical breakthrough. IC₅₀s and fold changes in IC₅₀ were calculated for all current anti-HBV drugs. For wild type viruses, the IC₅₀ obtained for the tested drugs are in accordance with published data. For mutant viruses, the fold change in IC₅₀ is well correlated with clinical observations and mutations identified during treatment with lamivudine (rtM204I and rtL180M-rtM204V), adefovir (rtN236T and rtV173L-rtL180M-rtA181V-rtN236T) and entecavir (rtL180M-rtA181V-rtS202G-rtM204V and rtL180M-rtS202G-rtM204V).

CONCLUSIONS: We have developed a high throughput technique using a new whole HBV genome amplification method that allows phenotyping of the overall population found in patients, in a quick and sensitive way. This method should allow a better monitoring of patients receiving antiviral therapy, as well as the screening and the optimization of new drugs against HBV.

2006-06-13
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