Phenotypic assays for HBV resistance tests by Maria Fraune - PowerPoint PPT Presentation

University of Cologne Institute of Virology Phenotypic assays for HBV resistance tests by Maria Fraune AREVIR-GenaFor-Meeting 10.-11.04.2008 | Phenotypic assays for HBV resistance tests

The HBV Genome University of Cologne Institute of Virology AREVIR-GenaFor-Meeting 10.-11.04.2008 | Phenotypic assays for HBV resistance tests

The Replication-Cycle University of Cologne Institute of Virology Gerlich and Kann in Topley & Wilson AREVIR-GenaFor-Meeting 10.-11.04.2008 | Phenotypic assays for HBV resistance tests

Drug Targets University of Cologne Institute of Virology 3TC, ADF, TDF, ETV (LdT) 3TC, ADF, TDF, ETV (LdT) Gerlich and Kann in Topley & Wilson AREVIR-GenaFor-Meeting 10.-11.04.2008 | Phenotypic assays for HBV resistance tests

Resistance Mutations University of Cologne Institute of Virology Terminal Protein Angus, Gastroenterology 2003; Delaney ,J Virol 2003; Allen, Hepatology 1998; Colonno, Hepatology 2004 Schilden, NEJM 2006 AREVIR-GenaFor-Meeting 10.-11.04.2008 | Phenotypic assays for HBV resistance tests

Experimental Setup University of Cologne Institute of Virology Huh7 Insert nt384-nt3065 of the HBV Genome Transfection direct cloning of patient material side directed mutagenesis or AREVIR-GenaFor-Meeting 10.-11.04.2008 | Phenotypic assays for HBV resistance tests

Experimental Setup University of Cologne Institute of Virology d4: medium change d-1: plate cells d0: transfection of d1: medium followed by (12,27x10 5 c/ml) plasmid change drugtreatment in different concentrations medium tested on HBsAg/HBeAg d6/7: • harvest cells, extract DNA • southern blot, PCR, medium tested on HBsAg/HBeAg AREVIR-GenaFor-Meeting 10.-11.04.2008 | Phenotypic assays for HBV resistance tests

Example of possible result University of Cologne Institute of Virology Southern Blot Drug -Treatment wildtype 10% 50% 100% 500% 0% Working Concentration HBV Resistance AREVIR-GenaFor-Meeting 10.-11.04.2008 | Phenotypic assays for HBV resistance tests

Example of possible result University of Cologne Institute of Virology Southern Blot Drug -Treatment wildtype 10% 50% 100% 500% Working Concentration 0% HBV Resistance Durantel et al., 2004 AREVIR-GenaFor-Meeting 10.-11.04.2008 | Phenotypic assays for HBV resistance tests

...to be done in the future... University of Cologne Institute of Virology • Replace Southern blot method by real-time PCR method • Southern blot: long and fastidous • Real-time PCR: scale down culture volume to 96-well plates • Make the whole assay less expensive more straight forward AREVIR-GenaFor-Meeting 10.-11.04.2008 | Phenotypic assays for HBV resistance tests

Acknowledgments University of Cologne Institute of Virology Thank you... AG Ulrike Protzer Jens Verheyen Ke Zhang Andrea Klöckner Rolf Kaiser Herbert Pfister AREVIR-GenaFor-Meeting 10.-11.04.2008 | Phenotypic assays for HBV resistance tests

University of Cologne Institute of Virology Thank you for your Attention!!! AREVIR-GenaFor-Meeting 10.-11.04.2008 | Phenotypic assays for HBV resistance tests