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

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

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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

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University of Cologne Institute of Virology

AREVIR-GenaFor-Meeting 10.-11.04.2008 | Phenotypic assays for HBV resistance tests

Phenotypic assays for HBV resistance tests

by Maria Fraune

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University of Cologne Institute of Virology

AREVIR-GenaFor-Meeting 10.-11.04.2008 | Phenotypic assays for HBV resistance tests

The HBV Genome

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University of Cologne Institute of Virology

AREVIR-GenaFor-Meeting 10.-11.04.2008 | Phenotypic assays for HBV resistance tests

The Replication-Cycle

Gerlich and Kann in Topley & Wilson

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University of Cologne Institute of Virology

AREVIR-GenaFor-Meeting 10.-11.04.2008 | Phenotypic assays for HBV resistance tests

Gerlich and Kann in Topley & Wilson

Drug Targets

3TC, ADF, TDF, ETV (LdT) 3TC, ADF, TDF, ETV (LdT)

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University of Cologne Institute of Virology

AREVIR-GenaFor-Meeting 10.-11.04.2008 | Phenotypic assays for HBV resistance tests

Resistance Mutations

Terminal Protein

Angus, Gastroenterology 2003; Delaney ,J Virol 2003; Allen, Hepatology 1998; Colonno, Hepatology 2004 Schilden, NEJM 2006

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University of Cologne Institute of Virology

AREVIR-GenaFor-Meeting 10.-11.04.2008 | Phenotypic assays for HBV resistance tests

Experimental Setup

Insert nt384-nt3065

  • f the HBV Genome

Transfection

Huh7

side directed mutagenesis

  • r

direct cloning of patient material

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University of Cologne Institute of Virology

AREVIR-GenaFor-Meeting 10.-11.04.2008 | Phenotypic assays for HBV resistance tests

Experimental Setup

d-1: plate cells (12,27x105 c/ml) d0: transfection of plasmid d1: medium change d4: medium change followed by drugtreatment in different concentrations d6/7:

  • harvest cells, extract

DNA

  • southern blot, PCR,

medium tested on HBsAg/HBeAg medium tested on HBsAg/HBeAg

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University of Cologne Institute of Virology

AREVIR-GenaFor-Meeting 10.-11.04.2008 | Phenotypic assays for HBV resistance tests

Example of possible result

Drug -Treatment

Southern Blot

HBV Resistance wildtype

0% 10% 50% 100% 500% Working Concentration

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University of Cologne Institute of Virology

AREVIR-GenaFor-Meeting 10.-11.04.2008 | Phenotypic assays for HBV resistance tests

Example of possible result

Drug -Treatment

HBV Resistance wildtype

Southern Blot

0% 10% 50% 100% 500% Working Concentration

Durantel et al., 2004

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University of Cologne Institute of Virology

AREVIR-GenaFor-Meeting 10.-11.04.2008 | Phenotypic assays for HBV resistance tests

...to be done in the future...

  • 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
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University of Cologne Institute of Virology

AREVIR-GenaFor-Meeting 10.-11.04.2008 | Phenotypic assays for HBV resistance tests

Acknowledgments

Thank you...

AG Ulrike Protzer Jens Verheyen Ke Zhang Andrea Klöckner Rolf Kaiser Herbert Pfister

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University of Cologne Institute of Virology

AREVIR-GenaFor-Meeting 10.-11.04.2008 | Phenotypic assays for HBV resistance tests

Thank you for your Attention!!!