Novel rtM204 Mutations in HBV Polymerase Confer Reduced - - PowerPoint PPT Presentation

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Novel rtM204 Mutations in HBV Polymerase Confer Reduced - - PowerPoint PPT Presentation

Jul 21, 2023 17 likes •281 views

Novel rtM204 Mutations in HBV Polymerase Confer Reduced Susceptibility to Adefovir and Tenofovir Ke Zhang, Christian Bach, Yuchen Xia, Oliver Schildgen, Ulrike Protzer Institute of Virology Technische Universitt Mnchen / Helmholtz Zentrum

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SLIDE 1

Ke Zhang, Christian Bach, Yuchen Xia, Oliver Schildgen, Ulrike Protzer Institute of Virology

Technische Universität München / Helmholtz Zentrum München

Novel rtM204 Mutations in HBV Polymerase Confer Reduced Susceptibility to Adefovir and Tenofovir

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SLIDE 2

HBV antiviral drug resistance

1 9 9 1 Interferon alfa-2b 1 9 9 8 Lamivudine (LAM) 2 0 0 2 Adefovir dipivoxil (ADV) 2 0 0 5 Entecavir (ETV) peginterferon alfa-2a 2 0 0 6 Telbivudine 2 0 0 8 Tenofovir (TDF)

  • 5 nucleos(t)ide analogues are currently approved for chronic hepatitis B
  • They inhibit viral replication very efficiently, but do not eliminate HBV
  • Long term treatment may lead to selection of resistance mutations,

which largely limits the clinical efficacy of the treatment

→ Resistance testing is important to improve treatment monitoring and

  • ptimize antiviral therapy
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SLIDE 3

Establishment of a standardized phenotypic assay

Patient sample HBV clone sequence analysis and selection PCR cloning and direct sequencing Huh7 cell transfection + drug treatment (3 days) (LAM, ADV, ETV, TDF; 5 concentrations each) Automated HBV DNA extraction Selective qPCR and I C50 determination Day 1 Day 3 Day 4 -8 Day 8 Day 8

Shorter time frame, higher throughput; less labor intensive and more sensitive

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SLIDE 4

rtM204 mutations in vitro and in vivo

Fold resistance (FR): LAM ADV M204V: < 50 6.1 L180M+M204V: >1000 1.3

  • Mutations in YMDD motif at position rt204 (M204V/I) are well-known for

conferring resistance to LAM and ETV

  • M204V is selected together with compensatory mutation L180M in vivo

I n vitro fitness

W T M2 0 4 V L1 8 0 M + M2 0 4 V

YMDD

reverse transcriptase

  • Phenotypic assay:

M204V – but not L180M+M204V – shows reduced susceptibility to ADV and TDF in vitro

  • why not selected in nature? fitness
  • r bias?
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SLIDE 5

Further identified rtM2 0 4 mutations

  • M204S & L180M was first isolated from a LAM resistant patient

Bozdayi et al.; Journal of Viral Hepatitis 2003

  • M204A alone was observed in ADV resistant patients

Bartholomeusz et al., HEP DART 2005

  • M204Q was isolated together with rtA181T from a ADV resistant patient

von Boemmel et al.; ASSLD 2007

  • M204T was isolated with or without rtI169T from a ADV resistant patient

Wang et al.; ASSLD 2010

  • M204L was isolated from a TDF partial responder

Berg et al.; Gastroenterology 2010

  • M204K was isolated from a LAM/ADV cross-resistant patient

Schildgen et al.; Virology Journal 2010 BUT: no systematic phenotypic data available; the clinical relevance remains unknown YMDD

reverse transcriptase

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SLIDE 6

Phenotypic results of rtM204 variants

LAM IC50 FR ETV IC50 FR ADV IC50 FR TDF IC50 FR W T

0.1 1 0.003 1 1.3 1 0.6 1

M2 0 4 V

4.5 45 0.01 3 8.0 6.1 6.0 10

M2 0 4 I

> 100 > 1000 0.1 30 2.5 1.9 1.1 1.8

M2 0 4 S

1.0 10 0.003 1 2.6 2 2.4 4.0

M2 0 4 L

1.0 10 0.002 0.5 5.2 4 4.5 7.5

M2 0 4 A

1.0 10 0.003 1 1.6 1.2 0.8 1.3

M2 0 4 Q

8.0 80 0.003 1 2.4 1.8 2.5 4.0

M2 0 4 T

2.0 20 0.003 1 2.0 1.5 1.8 3.0

M2 0 4 K

50 500 0.005 1.8 > 128 > 1 0 0 > 64 > 1 0 0

N2 3 6 T

1.5 15 0.003 1 5.5 4.2 3.0 5.0

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SLIDE 7

rtM204K confers adefovir and tenofovir resistance

25 50 75 100 125 0.01 0.1 1 10 100 % viral replication Lamivudine [µM]

Lamivudine

WT M204K 25 50 75 100 125 1 2 4 8 16 % viral replication Adefovir [µM]

Adefovir

WT M204K 25 50 75 100 125 0.0005 0.005 0.05 0.5 5 % viral replication Entecavir [µM]

Entecavir

WT M204K 25 50 75 100 125 0.5 1 2 4 8 % viral replication Tenofovir [µM]

Tenofovir

WT M204K

FR: 5 0 0 FR: > 1 0 0 FR: 1 .8 FR: > 1 0 0

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SLIDE 8

rtM204Q isolated from an ADV resistant patient

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SLIDE 9

HBV replication capacity of rtM204 variants

M204K im pairs the viral replication capacity, while M204L and M204Q may increase it

W T M 2 4 V M 2 4 I M 2 4 S M 2 4 L M 2 4 A M 2 4 Q M 2 4 T M 2 4 K 50 100 150

Relative replication [%]

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SLIDE 10

Compensatory mutations for rtM204K?

Neither L180M, L80I, nor A181T, could restore viral fitness of rtM204K

WT M204K M204K + L180M M204K + L80I M204K + A181T 5 10 80 100 120

Relative replication [%]

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SLIDE 11

WT 5’- GTT ATA TGG ATG ATG -3’ V I W M M M204V GTT ATG TGG ATG ATG V M W M M M204I GTT ATA TTG ATG ATG V I L M M M204S GTT ATA GTG ATG ATG V I V M M M204L GTT ATC TGG ATG ATG V I W M M M204A GTT ATG CGG ATG ATG V M R M M M204Q GTT ATC AGG ATG ATG V I R M M M204T GTT ATA CGG ATG ATG V I R M M M204K GTT ATA AGG ATG ATG V I R M M

Term inal protein Spacer RT RNaseH Envelope PreS1 Pre S2 S

rtM204 sI195 / sW196

Hydrophobic Nonpolar Hydrophilic Positive charged

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SLIDE 12

HBsAg secretion of rtM204 variants

sW196R (rtM204A/ K/ Q/ T) confers a secretion defect

HBsAg secretion

WT M204V M204I M204S M204L M204A M204Q M204T M204K 20 40 60 80 100 120

HBsAg Secretion [%]

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SLIDE 13

sW196R/ rtM204Q blocks virion formation

sW196R produces only naked capsid particles

HuH-7 cells Transfection Ultrafilteration Concentration Density Gradient Fraction collection DNA purification rcDNA qPCR Centrification

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SLIDE 14

sW196R has a dominant negative secretion defect

WT (P-) M204K M204K (LMS-) M204K + WT (P-) M204K (LMS-) + WT (P-) 5 10 15

HBsAg (S/CO)

rtM204K, resulting in sW196R inhibits HBV virion secretion

WT (P-) M204K M204K (LMS-) M204K + WT (P-) M204K (LMS-) + WT (P-) 2.0× 104 4.0× 104 6.0× 104

HBV rcDNA

HBsAg secretion HBV virion secretion

P-: premature stop codon in polymerase gene LMS-: premature stop codon in L/ M/ S gene

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SLIDE 15

Infectivity of rtM204 variants on HepaRG cells

HuH-7 cells Transfection Heparin column purification and ultrafiltration Standardized virus input (MOI100) HepaRG cells Intracelluar/ extracellular DNA extraction Supernatant collection cccDNA rcDNA HBeAg Day 10 post-infection I nfection

HBsAg mutations are major determinant of infectivity

WT M204V M204I M204S M204L M204Q

50 100 150 200

cccDNA HBeAg Intracellular DNA Extracellular DNA

Relative to WT [%]

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SLIDE 16

Summary

  • Extensive in vitro analysis revealed novel rtM204 variants

conferring reduced susceptibility to adefovir and tenofovir

  • High resistance mutations: rtM204K
  • cross-resistant to lamivudine, adefovir and tenofovir
  • but at a high cost of viral fitness
  • high genetic barrier for selection of compensatory mutations
  • Clinical significance remains to be investigated
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SLIDE 17

Acknowledgement

HOPE consortium groups:

Cologne

  • Dr. Rolf Kaiser

PD Dr. Oliver Schildgen Leipzig

  • Dr. Florian van Bömmel
  • Prof. Dr. Thomas Berg

Giessen PD Dr. Dieter Glebe Saarbrücken

  • Dr. Sebastian Beggel
  • Prof. Dr. Thomas Lengauer
  • Prof. Ulrike Protzer
  • Dr. Christian Bach
  • Dr. Julie Lucifora

MSc Yuchen Xia Andrea Weicht Romina Bester Theresa Asen Kerstin Ackermann

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SLIDE 18

Thank you for your attention!

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SLIDE 19

Selective qPCR distingushing rcDNA from input plasmid

Principle Efficiency  Selective qPCR reduces plasmid detection by > 4 logs

5’ 5’

HBV rcDNA

Primer B Primer A

pCH-9 / 3 0 9 1

primer B primer A

1,00E+ 00 1,00E+ 01 1,00E+ 02 1,00E+ 03 1,00E+ 04 1,00E+ 05 1,00E+ 06 1,00E+ 07 1,00E+ 08 detected plasm id copy num ber input plasm id copy num ber normal qPCR selective qPCR

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SLIDE 20

Comparison between M204V and L180M+M204V

25 50 75 100 125 150 1 2 4 8 16 % viral replication Adefovir [µM]

Plasmid M204V treated with Adefovir

WT HBV HBV virus 25 50 75 100 125 0.5 1 2 4 8 % viral replication Tenofovir [µM]

Plasmid M204V treated with Tenofovir

WT HBV HBV virus 25 50 75 100 125 1 2 4 8 16 % viral replication Adefovir [µM]

Plasmid L180M+M204V treated with Adefovir

WT HBV HBV virus 25 50 75 100 125 0.5 1 2 4 8 % viral replication Tenofovir [µM]

L180M+M204V treated with Tenofovir

WT HBV HBV virus

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SLIDE 21

Comparable result from publication

  • Hepatology. 1998 Dec; 28(6): 1669-73.

Wild-type M204I M204V L180M L180M/ M204V

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SLIDE 22

Term inal protein Spacer Pol/ RT RNaseH A B C E D YMDD I ( G) I I ( F) GVGLSPFLLA

rtM2 0 4

2002 nt 2040 nt

YMDD 5’- C TGT TTG GCT TTC AGT TAT ATG GAT GAT GTG GTA TTG GG -3’ Y M D D YVDD C TGT TTG GCT TTC AGT TAT GTG GAT GAT GTG GTA TTG GG Y V D D YIDD GT TTG GCT TTC AGT TAT ATT GAT GAT GTG GTA TTG GG Y I D D YSDD GT TTG GCT TTC AGT TAT AGT GAT GAT GTG GTA TTG GG Y S D D YLDD C TGT TTG GCT TTC AGT TAT CTG GAT GAT GTG GTA TTG GG Y L D D YKDD GT TTG GCT TTC AGT TAT AAG GAT GAT GTG GTA TTG GG Y K D D YADD GT TTG GCT TTC AGT TAT GCG GAT GAT GTG GTA TTG GG Y A D D YTDD GT TTG GCT TTC AGT TAT ACG GAT GAT GTG GTA TTG GG Y T D D YQDD C TGT TTG GCT TTC AGT TAT CAG GAT GAT GTG GTA TTG GG Y Q D D

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SLIDE 23

Property of mutated rtM204 residues

Hydrophobic Nonpolar Hydrophilic Polar Positive charged

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A case report

M204K: newly isolated from a patient cross-resistant to Lamivudine & Adefovir Schildgen et al., Virology Journal 2010, 7: 167

ADV LAM

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SLIDE 25

M204L

25 50 75 100 125 0.01 0.1 1 10 100 % viral replication Lamivudine [µM]

M204L treated with Lamivudine

WT HBV HBV virus 25 50 75 100 125 1 2 4 8 16 % viral replication Adefovir [µM]

M204L treated with Adefovir

WT HBV HBV virus 25 50 75 100 125 0.0005 0.005 0.05 0.5 5 % viral replication Entecavir [µM]

M204L treated with Entecavir

WT HBV HBV virus 25 50 75 100 125 150 0.5 1 2 4 8 % viral replication Tenofovir [µM]

M204L treated with Tenofovir

WT HBV HBV virus

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SLIDE 26

M204A

25 50 75 100 125 1 2 4 8 16 % viral replication Adefovir [µM]

M204A treated with Adefovir

WT HBV HBV virus 25 50 75 100 125 0.5 1 2 4 8 % viral replication Tenofovir [µM]

M204A treated with Tenofovir

WT HBV HBV virus 25 50 75 100 125 0.01 0.1 1 10 100 % viral replication Lamivudine [µM]

M204A treated with Lamivudine

WT HBV HBV virus 25 50 75 100 125 0.0005 0.005 0.05 0.5 5 % viral replication Entecavir [µM]

M204A treated with Entecavir

WT HBV HBV virus