Role of mutations outside the RT-domain of HBV polymerase on - - PowerPoint PPT Presentation

▶
role of mutations outside the rt domain of hbv polymerase
SMART_READER_LITE
LIVE PREVIEW

Role of mutations outside the RT-domain of HBV polymerase on - - PowerPoint PPT Presentation

Dec 07, 2022 271 likes •608 views

AREVIR-GenaFor-Meeting, Bonn, 05. May 2010 Role of mutations outside the RT-domain of HBV polymerase on antiviral resistance PD Dr. Dieter Glebe Institut fr Medizinische Virologie Justus-Liebig-Universitt Gieen HBV Polymerase

slide-1
SLIDE 1

“AREVIR-GenaFor-Meeting”, Bonn, 05. May 2010

PD Dr. Dieter Glebe

Institut für Medizinische Virologie Justus-Liebig-Universität Gießen

Role of mutations

  • utside the RT-domain of

HBV polymerase on antiviral resistance

slide-2
SLIDE 2

HBV Polymerase

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

  • analogues. Gastroenterology, 2009, modified

Primary Resistance- mutations Secondary- resistance mutations

slide-3
SLIDE 3

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

  • analogues. Gastroenterology, 2009; 137:1593-1608

Adapt treatment based on genotypic information

HBV genotypic assay

slide-4
SLIDE 4

g2p[hbv] – Input Page

Look at www.genafor.org

slide-5
SLIDE 5

g2p[hbv] – Result Page

Look at www.genafor.org

slide-6
SLIDE 6

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),
  • 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

slide-7
SLIDE 7

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

slide-8
SLIDE 8

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

Wildtype

pCH9-3091

Wildtype

pCH9-3091 pCH9-3091

Wildtype

Transfection of HuH7-cells

  • incubate over night
  • wash cells

pCH9-3091

Wildtype

pCH9-3091

Wildtype

pCH9-3091 pCH9-3091

Wildtype

pCH9-3091 pCH9-3091 pCH9-3091

  • incubation for 3 days
  • purify DNA from supernatant
  • quantify DNA with Real time PCR

HBV resistance: from genotype to phenotype

slide-9
SLIDE 9

Adefovir Entecavir Lamivudin Tenofovir

no drug

  • 72 datapoints for every clinical isolate

HBV resistance: from genotype to phenotype

slide-10
SLIDE 10

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

slide-11
SLIDE 11

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-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-Lamivudin

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

Lamivudin [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

slide-12
SLIDE 12

HBV-Polymerase rtM204I

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

  • analogues. Gastroenterology, 2009; 137:1593-1608

S: sensitive; R: resistance; I: intermediate 1,0 mg/d Telbivudin 245 mg/d Tenofovir disoproxil 0,5 mg/d Entecavir 10 mg/d Adefovir dipivoxil 100 mg/d Lamivudin Nucleotide analogs Nucleoside analogs

aus: Schaefer, Glebe, Gerlich. Hepatitis B Virus; in: Doerr & Gerlich, Medizinische Virologie

HBV resistance: from genotype to phenotype

slide-13
SLIDE 13

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

Lamivudin

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

Lamivudin [nM] relative replication

Entecavir

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

Entecavir [nM] relative replication

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

HBV-Polymerase rtM204I

1 24 3 >10000

HBV resistance: from genotype to phenotype

slide-14
SLIDE 14

Tenofovir

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

Tenofvir [nM] relative replication

Lamivudin

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

Lamivudin [nM] relative replication

Entecavir

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

Entecavir [nM] relative replication

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

Phenotypic in vitro resistance assay

HBV-Polymerase rtL180M,rtM204V

2,5 21,5 3,3 >10000

slide-15
SLIDE 15

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

  • analogues. Gastroenterology, 2009; 137:1593-1608

S: sensitive; R: resistance; I: intermediate 1,0 mg/d Telbivudin 245 mg/d Tenofovir disoproxil 0,5 mg/d Entecavir 10 mg/d Adefovir dipivoxil 100 mg/d Lamivudin Nukleotide analogs Nukleoside analogs

aus: Schaefer, Glebe, Gerlich. Hepatitis B Virus; in: Doerr & Gerlich, Medizinische Virologie

HBV-Polymerase rtL180M,rtT184A,rtM204V

HBV resistance: from genotype to phenotype

slide-16
SLIDE 16

Tenofovir

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

Tenofvir [nM] relative replication

Lamivudin

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

Lamivudin [nM] relative replication

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

Entecavir

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

Entecavir [nM] relative replication

HBV-Polymerase rtL180M,rtT184A,rtM204V

2,5 153 3,2 >10000

HBV resistance: from genotype to phenotype

slide-17
SLIDE 17

Lamivudin

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

Lamivudin [nM] relative replication

Entecavir

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

Entecavir [nM] relative replication

Tenofovir

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

Tenofovir [nM] relative replication

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

Phenotypic in vitro resistance assay

HBV-Polymerase rtN236T

4 1 6 2,3

slide-18
SLIDE 18

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

  • analogues. Gastroenterology, 2009; 137:1593-1608

S: sensitive; R: resistance; I: intermediate 1,0 mg/d Telbivudin 245 mg/d Tenofovir disoproxil 0,5 mg/d Entecavir 10 mg/d Adefovir dipivoxil 100 mg/d Lamivudin Nukleotide analogs Nukleoside analogs

aus: Schaefer, Glebe, Gerlich. Hepatitis B Virus; in: Doerr & Gerlich, Medizinische Virologie

HBV-Polymerase rtA181V

HBV resistance: from genotype to phenotype

slide-19
SLIDE 19

Entecavir

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

Entecavir [nM] relative replication

Lamivudin

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

Lamivudin [nM] relative replication

Adefovir

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

Adefovir [nM] relative replication

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-Polymerase rtA181V

4,5 1,5 4 18

HBV resistance: from genotype to phenotype

slide-20
SLIDE 20

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

  • analogues. Gastroenterology, 2009; 137:1593-1608

S: sensitive; R: resistance; I: intermediate 1,0 mg/d Telbivudin 245 mg/d Tenofovir disoproxil 0,5 mg/d Entecavir 10 mg/d Adefovir dipivoxil 100 mg/d Lamivudin Nukleotide analogs Nukleoside analogs

aus: Schaefer, Glebe, Gerlich. Hepatitis B Virus; in: Doerr & Gerlich, Medizinische Virologie

HBV-Polymerase rtA181V,rtN236T

HBV resistance: from genotype to phenotype

slide-21
SLIDE 21

Entecavir

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

Entecavir [nM] relative replication

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

Lamivudin

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

Lamivudin [nM] relative replication

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

HBV-Polymerase rtA181V,rtN236T

13,5 1 15 17,5

HBV resistance: from genotype to phenotype

slide-22
SLIDE 22

Phenotypic in vitro resistance assay

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

  • analogues. Gastroenterology, 2009; 137:1593-1608

S: sensitive; R: resistance; I: intermediate 1,0 mg/d Telbivudin 245 mg/d Tenofovir disoproxil 0,5 mg/d Entecavir 10 mg/d Adefovir dipivoxil 100 mg/d Lamivudin Nukleotide analogs Nukleoside analogs

aus: Schaefer, Glebe, Gerlich. Hepatitis B Virus; in: Doerr & Gerlich, Medizinische Virologie

HBV-Polymerase rtA181V,rtN236T…

slide-23
SLIDE 23

Tenofovir

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

Tenofovir [nM] relative replication

Lamivudin

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

Lamivudin [nM] relative replication

Entecavir

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

Entecavir [nM] relative replication

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

HBV-Polymerase mutations

~100 10 30 200

HBV resistance: from genotype to phenotype

slide-24
SLIDE 24

Resistance factor

LAM ENT ADF TDF

1 1 1 1

HBV polymerase ORF

Phenotypic resistance testing in in vit vitro

wt

rtA181V rtN236T

mut

slide-25
SLIDE 25

Resistance factor

LAM ENT ADF TDF

1 1 1 1 220 10 93 26

HBV polymerase ORF

rtA181V rtN236T

Phenotypic resistance testing in in vit vitro

wt mut

Very strong resistance against ADF, TDF

slide-26
SLIDE 26

Resistance factor

LAM ENT ADF TDF

1 1 1 1 220 10 93 26

HBV polymerase ORF

rtA181V rtN236T

Phenotypic resistance testing in in vit vitro

wt mut

slide-27
SLIDE 27

Resistance factor

LAM ENT ADF TDF

1 1 1 1 220 10 93 26 19 2 10 3

HBV polymerase ORF

rtA181V rtN236T

Phenotypic resistance testing in in vit vitro

wt mut

Reduced resistance against all antivirals Influence of external domains

slide-28
SLIDE 28

Resistance factor

LAM ENT ADF TDF

1 1 1 1 220 10 93 26 19 2 10 3 13 1 8 4

HBV polymerase ORF

rtA181V rtN236T

Phenotypic resistance testing in in vit vitro

wt mut

Similar reduced resistance against all antivirals No influence of additional mutations in RT domain

slide-29
SLIDE 29

Resistance factor

LAM ENT ADF TDF

1 1 1 1 220 10 93 26 19 2 10 3 13 1 8 4 66 2 15 12

HBV polymerase ORF

rtA181V rtN236T

Phenotypic resistance testing in in vit vitro

wt mut

Increased resistance against LAM, ADF, TDF Influence of external mutations on mutated RT domain

slide-30
SLIDE 30

Resistance factor

LAM ENT ADF TDF

1 1 1 1 220 10 93 26 19 2 10 3 13 1 8 4 66 2 15 12 2 1 1 1

HBV polymerase ORF

rtA181V rtN236T

Phenotypic resistance testing in in vit vitro

wt mut

No resistance at all No influence of external mutations on wt RT domain

Interacting mutations within 3D structure of HBV polymerase

slide-31
SLIDE 31

Institute of Medical Virology Justus-Liebig-University Giessen, Germany Andreas Geipel Pia Seiz 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

slide-32
SLIDE 32

“AREVIR-GenaFor-Meeting”, Bonn, 05. May 2010

Institut für Medizinische Virologie Justus-Liebig-Universität Gießen

Thank you for your attention !