Phenotypic evaluation of resistance profiles of HBV polymerase - PowerPoint PPT Presentation

“AREVIR-GenaFor-Meeting”, Köln, 11.04.2013 Phenotypic evaluation of resistance profiles of HBV polymerase against Lamivudine reveals an unexpected resistance against Adefovir and Entecavir Dieter Glebe Institute for Medical Virology National Reference Centre for Hepatitis B and D Viruses Justus-Liebig-Universität Gießen, Germany

Available Nucleos(t)id-Analoga (HBV) Nucleosid-Analoga Resistance Lamivudin 100 mg/d 80 % after 5 years 1.2 % after 5 years 1 Entecavir 0,5 mg/d 22 % after 2 years 2 Telbivudin 1,0 mg/d Nucleotid-Analoga Adefovir dipivoxil 10 mg/d 30 % after 5 years Tenofovir disoproxil 245 mg/d 0 % after 2 years 1 treatment-naive patients 2 HBeAg positive patients aus: Schaefer, Glebe, Gerlich. Hepatitis B Virus ; in: Doerr & Gerlich, Medizinische Virologie, 2010

HBV Polymerase Zoulim & Locarnini, HBV resistance to nucleos(t)ide analogues . Gastroenterology, 2009, modified

HBV Polymerase Secondary- Primary Resistance- resistance mutations mutations Zoulim & Locarnini, HBV resistance to nucleos(t)ide analogues . Gastroenterology, 2009, modified

HBV Polymerase Secondary- Primary Resistance- resistance mutations mutations Zoulim & Locarnini, HBV resistance to nucleos(t)ide analogues . Gastroenterology, 2009, modified

HBV Polymerase Secondary- Primary Resistance- resistance mutations mutations Zoulim & Locarnini, HBV resistance to nucleos(t)ide analogues . Gastroenterology, 2009, modified

HBV genotypic assay Adapt treatment based on genotypic information Zoulim & Locarnini, HBV resistance to nucleos(t)ide analogues . Gastroenterology, 2009; 137:1593-1608

HBV resistance: from genotype to phenotype BMBF project (2009-2012): H epatitis B therapies o ptimized by p henotypic e valuation (HOPE) Goals: – Validate resistance profile of HBV polymerase as a biomarker – Rapid rating of HBV (antiviral)-mutants ( Geno2pheno ) – improved clinical outcome of antiviral therapy • Involved groups: • Helmholtz Zentrum München (HMGU) (Protzer), • Justus-Liebig-Universität-Giessen (JLU) (Glebe, Gerlich), • Universität Duisburg-Essen University (Roggendorf, Lu) • Universität zu Köln (Kaiser), • Humboldt Universität Berlin/ Charite (van Boemmel, Berg), • Medizinische Hochschule Hannover (MHH) (Manns, Wursthorn), • Max-Planck-Institut Saarbrücken (MPI) (Lengauer, Beggel) • Dade Behring/Siemens Medical Solutions Diagnostics GmbH Deutschland Leverkusen,

Replication cycle of hepatitis B virus

Replication cycle of hepatitis B virus

Replication cycle of hepatitis B virus

Replication cycle of hepatitis B virus Formation Resistant of cccDNA Virus MVB Secretion Nukleos(t)id-Analoga of virions

HBV resistance: from genotype to phenotype Phenotypic in vi vitr tro o resistance assay Expression plasmid with HBV-Insert Pol-frame amplicon from patients serum Wildtype pCH9-3091 pCH9-3091

HBV resistance: from genotype to phenotype - 72 datapoints for every clinical isolate

HBV resistance: from genotype to phenotype 1.day Cloning Wildtype Wildtype Wildtype pCH9-3091 pCH9-3091 pCH9-3091 pCH9-3091 pCH9-3091 pCH9-3091 pCH9-3091 3.day - Transfection of cells 4.day - 96-well-format - Incubation for 3 day - Determination of transfection-efficiency - Automated purification of DNA from 7.day cell culture supernatant - Quantification of viral HBV-DNA 8.day using discriminative quantitative PCR

HBV resistance: from genotype to phenotype WT-Adefovir WT-Entecavir 1,4 1,2 1,2 1 relative replication relative replication 1 0,8 0,8 0,6 0,6 0,4 0,4 0,2 0,2 0 0 0,01 0,1 1 10 100 1000 10000 100000 0,01 0,1 1 10 100 1000 Adefovir [nM] Entecavir [nM] WT-Lamivudin WT-Tenofovir 1,2 1,4 1,2 1 relative replication relative replication 1 0,8 0,8 0,6 0,6 0,4 0,4 0,2 0,2 0 0 0,01 0,1 1 10 100 1000 10000 100000 0,01 0,1 1 10 100 1000 10000 100000 Lamivudin [nM] Tenofovir [nM]

HBV resistance: from genotype to phenotype Regression model for the calculation of the IC 50

HBV resistance: from genotype to phenotype Adefovir Entecavir Lamivudine Tenofovir R 2 R 2 R 2 R 2 RF 50 RF 50 RF 50 RF 50 sign. sign. sign. sign. 1 1,9 ns 0,91 1,1 ns 0,76 0,7 ns 0,83 0,5 ns 0,65 2 1,1 ns 0,88 2,3 ns 0,76 0,9 ns 0,73 0,9 ns 0,82 3 0,9 ns 0,97 0,5 ns 0,81 0,5 ns 0,73 1,4 ns 0,93 replicates of WT 4 1,6 ns 0,87 0,7 ns 1,00 1,3 ns 0,86 1,7 ns 0,94 5 0,8 ns 0,97 1,1 ns 0,81 0,9 ns 0,83 0,7 ns 0,95 6 0,9 ns 0,97 1,1 ns 0,93 0,9 ns 0,99 0,9 ns 0,96 7 1,0 ns 0,83 1,9 ns 0,93 1,7 ns 0,94 1,4 ns 0,86 8 0,6 ns 0,83 0,7 ns 0,79 1,2 ns 0,98 0,5 ns 0,97 9 1,4 ns 0,88 0,6 ns 0,92 1,7 ns 0,84 1,8 ns 0,81 10 0,8 ns 0,98 0,6 ns 0,97 0,9 ns 0,96 1,0 ns 0,96

HBV resistance: the phenotype for Lamivudine Adefovir Entecavir L180M M204V R 2 R 2 RF 50 RF 50 sign. sign. Patient Mutations GT Fitness 1 180M,204V 2,07 ns 0,86 32,88 * 0,91 B 2,9 clinical resistance 2 180M,204V 0,75 ns 0,99 48,05 * 0,77 A 92,7 ADV ETV LMV TDF 3 180M,204V 1,67 ns 0,87 40,74 * 0,70 A 88,6 none low high none 4 180M,204V 0,65 ns 0,98 9,45 * 0,78 A 45,2 5 180M,204V 4,42 * 0,93 43,21 * 0,71 A 24,1 6 180M,204V 3,32 * 0,88 12,09 * 0,91 A 10,3 7 180M,204V 5,11 * 0,93 4,65 * 0,73 A 6,8 8 204I 0,98 ns 0,94 9,34 * 0,83 A 5,7 L80I/V M204I 9 80I,204I 0,86 ns 0,95 20,04 * 0,93 D 79,8 10 80I,204I 1,03 ns 0,97 3,52 * 0,84 D 50,9 Clinical resistance 11 80V,204I 1,52 ns 0,82 15,73 * 0,73 D 59,6 ADV ETV LMV TDF 12 80V,204I 2,37 ns 0,95 75,23 * 0,91 D 47,6 none low high none 13 80V,204I 0,91 ns 0,78 5,24 * 0,71 A 4,5 14 181V 3,19 * 0,90 1,03 ns 0,99 D 143,4 15 181V 4,46 * 1,00 1,30 ns 0,99 A 35,4

HBV resistance: the phenotype for Lamivudine Adefovir Entecavir L180M M204V R 2 R 2 RF 50 RF 50 sign. sign. Patient Mutations GT Fitness 1 180M,204V 2,07 ns 0,86 32,88 * 0,91 B 2,9 clinical resistance 2 180M,204V 0,75 ns 0,99 48,05 * 0,77 A 92,7 ADV ETV LMV TDF 3 180M,204V 1,67 ns 0,87 40,74 * 0,70 A 88,6 none low high none 4 180M,204V 0,65 ns 0,98 9,45 * 0,78 A 45,2 5 180M,204V 4,42 * 0,93 43,21 * 0,71 A 24,1 6 180M,204V 3,32 * 0,88 12,09 * 0,91 A 10,3 7 180M,204V 5,11 * 0,93 4,65 * 0,73 A 6,8 8 204I 0,98 ns 0,94 9,34 * 0,83 A 5,7 L80I/V M204I 9 80I,204I 0,86 ns 0,95 20,04 * 0,93 D 79,8 10 80I,204I 1,03 ns 0,97 3,52 * 0,84 D 50,9 Clinical resistance 11 80V,204I 1,52 ns 0,82 15,73 * 0,73 D 59,6 ADV ETV LMV TDF 12 80V,204I 2,37 ns 0,95 75,23 * 0,91 D 47,6 none low high none 13 80V,204I 0,91 ns 0,78 5,24 * 0,71 A 4,5 14 181V 3,19 * 0,90 1,03 ns 0,99 D 143,4 15 181V 4,46 * 1,00 1,30 ns 0,99 A 35,4

Acknowledgments BMBF-HOPE-GROUP Institute of Medical Virology Justus-Liebig-University Institute of Virology Giessen, Germany University Hospital, Cologne, Germany Andreas Geipel Pia Seiz Maria Neumann-Fraune Hauke Niekamp Jens Verheyen Corinna M. Bremer Rolf Kaiser Wolfram H. Gerlich John Ziebuhr Max-Planck Institute for Informatics University of Saarland, Germany Clinic for Gastroenterology Bastian Beggel and Rheumatology Thomas Lengauer University Hospital Leipzig,Germany Institute of Virology Helmholtz Center Munich , Germany Florian van Bömmel Thomas Berg Christian Bach Ke Zhang Ulrike Protzer

“AREVIR-GenaFor-Meeting”, Köln, 11.04.2013 Thank you for your attention ! Dieter Glebe Institute for Medical Virology National Reference Centre for Hepatitis B and D Viruses Justus-Liebig-Universität Gießen, Germany