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

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phenotypic evaluation of resistance profiles of hbv

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

Mar 12, 2024 369 likes •595 views

AREVIR-GenaFor-Meeting, Kln, 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

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“AREVIR-GenaFor-Meeting”, Köln, 11.04.2013

Dieter Glebe

Institute for Medical Virology National Reference Centre for Hepatitis B and D Viruses Justus-Liebig-Universität Gießen, Germany

Phenotypic evaluation of resistance profiles

f HBV polymerase against Lamivudine

reveals an unexpected resistance against Adefovir and Entecavir

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Nucleosid-Analoga

Resistance

Lamivudin 100 mg/d 80 % after 5 years Entecavir 0,5 mg/d 1.2 % after 5 years1 Telbivudin 1,0 mg/d 22 % after 2 years2 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

Available Nucleos(t)id-Analoga (HBV)

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HBV Polymerase

Zoulim & Locarnini, HBV resistance to nucleos(t)ide

analogues. Gastroenterology, 2009, modified

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SLIDE 4

HBV Polymerase

Zoulim & Locarnini, HBV resistance to nucleos(t)ide

analogues. Gastroenterology, 2009, modified

Primary Resistance- mutations Secondary- resistance mutations

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SLIDE 5

HBV Polymerase

Zoulim & Locarnini, HBV resistance to nucleos(t)ide

analogues. Gastroenterology, 2009, modified

Primary Resistance- mutations Secondary- resistance mutations

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HBV Polymerase

Zoulim & Locarnini, HBV resistance to nucleos(t)ide

analogues. Gastroenterology, 2009, modified

Primary Resistance- mutations Secondary- resistance mutations

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SLIDE 7

Zoulim & Locarnini, HBV resistance to nucleos(t)ide

analogues. Gastroenterology, 2009; 137:1593-1608

Adapt treatment based on genotypic information

HBV genotypic assay

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Goals:

– Validate resistance profile of HBV polymerase as a biomarker – Rapid rating of HBV (antiviral)-mutants (Geno2pheno) – improved clinical outcome of antiviral therapy

BMBF project (2009-2012): Hepatitis B therapies optimized by phenotypic evaluation (HOPE)

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,

HBV resistance: from genotype to phenotype

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Replication cycle of hepatitis B virus

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Replication cycle of hepatitis B virus

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Replication cycle of hepatitis B virus

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MVB

Replication cycle of hepatitis B virus

Nukleos(t)id-Analoga

Formation

f cccDNA

Secretion

f virions

Resistant Virus

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pCH9-3091 pCH9-3091

Wildtype

Expression plasmid with HBV-Insert Pol-frame amplicon from patients serum

Phenotypic in vi vitr tro

resistance assay

HBV resistance: from genotype to phenotype

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72 datapoints for every clinical isolate

HBV resistance: from genotype to phenotype

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pCH9-3091 pCH9-3091 pCH9-3091 pCH9-3091

Wildtype

pCH9-3091

Wildtype

pCH9-3091 pCH9-3091

Wildtype

Transfection of cells
Incubation for 3 day
Determination of transfection-efficiency
Automated purification of DNA from

cell culture supernatant

96-well-format
Quantification of viral HBV-DNA

using discriminative quantitative PCR Cloning 1.day 3.day 4.day 7.day 8.day

HBV resistance: from genotype to phenotype

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WT-Adefovir

0,2 0,4 0,6 0,8 1 1,2 1,4 0,01 0,1 1 10 100 1000 10000 100000

Adefovir [nM] relative replication

WT-Lamivudin

0,2 0,4 0,6 0,8 1 1,2 0,01 0,1 1 10 100 1000 10000 100000

Lamivudin [nM] relative replication

WT-Entecavir

0,2 0,4 0,6 0,8 1 1,2 0,01 0,1 1 10 100 1000

Entecavir [nM] relative replication

WT-Tenofovir

0,2 0,4 0,6 0,8 1 1,2 1,4 0,01 0,1 1 10 100 1000 10000 100000

Tenofovir [nM] relative replication

HBV resistance: from genotype to phenotype

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HBV resistance: from genotype to phenotype

Regression model for the calculation of the IC50

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HBV resistance: from genotype to phenotype

RF50 sign. R2 RF50 sign. R2 RF50 sign. R2 RF50 sign. R2 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 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 Tenofovir replicates of WT Adefovir Entecavir Lamivudine

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HBV resistance: the phenotype for Lamivudine

M204V L180M

clinical resistance ADV ETV LMV TDF none low high none

RF50 sign. R2 RF50 sign. R2 1 180M,204V 2,07 ns 0,86 32,88 * 0,91 B 2,9 2 180M,204V 0,75 ns 0,99 48,05 * 0,77 A 92,7 3 180M,204V 1,67 ns 0,87 40,74 * 0,70 A 88,6 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 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 11 80V,204I 1,52 ns 0,82 15,73 * 0,73 D 59,6 12 80V,204I 2,37 ns 0,95 75,23 * 0,91 D 47,6 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 GT Fitness Adefovir Entecavir Patient Mutations

Clinical resistance ADV ETV LMV TDF none low high none

M204I L80I/V

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HBV resistance: the phenotype for Lamivudine

M204V L180M

clinical resistance ADV ETV LMV TDF none low high none

RF50 sign. R2 RF50 sign. R2 1 180M,204V 2,07 ns 0,86 32,88 * 0,91 B 2,9 2 180M,204V 0,75 ns 0,99 48,05 * 0,77 A 92,7 3 180M,204V 1,67 ns 0,87 40,74 * 0,70 A 88,6 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 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 11 80V,204I 1,52 ns 0,82 15,73 * 0,73 D 59,6 12 80V,204I 2,37 ns 0,95 75,23 * 0,91 D 47,6 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 GT Fitness Adefovir Entecavir Patient Mutations

Clinical resistance ADV ETV LMV TDF none low high none

M204I L80I/V

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Institute of Medical Virology Justus-Liebig-University Giessen, Germany Andreas Geipel Pia Seiz Hauke Niekamp Corinna M. Bremer Wolfram H. Gerlich John Ziebuhr

Institute of Virology Helmholtz Center Munich, Germany Christian Bach Ke Zhang Ulrike Protzer Max-Planck Institute for Informatics University of Saarland, Germany Bastian Beggel Thomas Lengauer

Clinic for Gastroenterology and Rheumatology

University Hospital Leipzig,Germany

Florian van Bömmel Thomas Berg

Acknowledgments

Institute of Virology

University Hospital, Cologne, Germany

Maria Neumann-Fraune Jens Verheyen Rolf Kaiser

BMBF-HOPE-GROUP

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“AREVIR-GenaFor-Meeting”, Köln, 11.04.2013

Dieter Glebe

Institute for Medical Virology National Reference Centre for Hepatitis B and D Viruses Justus-Liebig-Universität Gießen, Germany

Thank you for your attention !