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

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 Universität München / Helmholtz Zentrum München

HBV antiviral drug resistance • 5 nucleos(t)ide analogues are currently approved for chronic hepatitis B • They inhibit viral replication very efficiently, but do not eliminate HBV 1 9 9 1 2 0 0 2 2 0 0 6 Interferon alfa-2b Adefovir Telbivudine dipivoxil (ADV) 1 9 9 8 2 0 0 5 2 0 0 8 Lamivudine Entecavir (ETV) Tenofovir (TDF) (LAM) peginterferon alfa-2a • 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 optimize antiviral therapy

Establishment of a standardized phenotypic assay Day 1 Day 3 PCR cloning HBV clone and direct sequence analysis Patient sequencing and selection sample Day 4 -8 Huh7 cell transfection + drug treatment ( 3 days ) Shorter time frame, higher throughput; (LAM, ADV, ETV, TDF; less labor intensive and more sensitive 5 concentrations each) Day 8 Day 8 Automated HBV Selective qPCR DNA extraction and I C 50 determination

rtM204 mutations in vitro and in vivo  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  Phenotypic assay : I n vitro fitness M204V – but not L180M+M204V – shows reduced susceptibility to ADV and TDF in vitro  why not selected in nature? fitness or bias? L1 8 0 M W T M2 0 4 V + M2 0 4 V Y M DD Fold resistance (FR): LAM ADV M204V: < 50 6.1 L180M+M204V: >1000 1.3 reverse transcriptase

Further identified rt M2 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 Y M DD BUT: no systematic phenotypic data available; the clinical relevance remains unknown reverse transcriptase

Phenotypic results of rtM204 variants LAM ETV ADV TDF IC 50 FR IC 50 FR IC 50 FR IC 50 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

rtM204K confers adefovir and tenofovir resistance Lamivudine Adefovir FR: 5 0 0 FR: > 1 0 0 125 125 % viral replication % viral replication 100 100 75 75 50 50 25 25 0 0 0 1 2 4 8 16 0 0.01 0.1 1 10 100 Lamivudine [µM] Adefovir [µM] WT M204K WT M204K Tenofovir Entecavir FR: 1 .8 FR: > 1 0 0 125 125 % viral replication % viral replication 100 100 75 75 50 50 25 25 0 0 0 0.5 1 2 4 8 0 0.0005 0.005 0.05 0.5 5 Entecavir [µM] Tenofovir [µM] WT M204K WT M204K

rtM204Q isolated from an ADV resistant patient

HBV replication capacity of rtM204 variants 150 Relative replication [%] 100 50 0 T V I S L A Q T K 4 W 4 4 4 4 4 4 4 0 0 0 0 0 0 0 0 2 2 2 2 2 2 2 2 M M M M M M M M M204K im pairs the viral replication capacity, while M204L and M204Q may increase it

Compensatory mutations for rtM204K? 120 Relative replication [%] 100 80 10 5 0 WT M204K M204K + L180M M204K + L80I M204K + A181T Neither L180M, L80I, nor A181T, could restore viral fitness of rtM204K

rtM204 Term inal Spacer RT RNaseH protein PreS1 Pre S Envelope S2 sI195 / sW196 WT 5’- GTT AT A TG G ATG ATG -3’ V I W M M M204V GTT AT G TG G ATG ATG V M W M M M204I GTT AT A TT G ATG ATG V I L M M M204S GTT AT A GT G ATG ATG V I V M M M204L GTT AT C TG G ATG ATG V I W M M Hydrophobic Nonpolar M204A GTT AT G CG G ATG ATG V M R M M M204Q GTT AT C AG G ATG ATG V I R M M M204T GTT AT A CG G ATG ATG V I R M M Hydrophilic M204K GTT AT A AG G ATG ATG Positive charged V I R M M

HBsAg secretion of rtM204 variants HBsAg secretion 120 HBsAg Secretion [%] 100 80 60 40 20 0 M204L WT M204V M204I M204S M204A M204Q M204T M204K sW196R (rtM204A/ K/ Q/ T) confers a secretion defect

sW196R/ rtM204Q blocks virion formation HuH-7 cells Density Gradient Ultrafilteration Fraction collection rcDNA qPCR Concentration DNA purification Centrification Transfection sW196R produces only naked capsid particles

sW196R has a dominant negative secretion defect HBsAg secretion HBV virion secretion 6.0 × 10 4 15 HBsAg (S/CO) HBV rcDNA 4.0 × 10 4 10 2.0 × 10 4 5 0 0 WT (P-) M204K M204K (LMS-) M204K + WT (P-) M204K (LMS-) + WT (P-) WT (P-) M204K M204K (LMS-) M204K + WT (P-) M204K (LMS-) + WT (P-) P-: premature stop codon in polymerase gene LMS-: premature stop codon in L/ M/ S gene rtM204K, resulting in sW196R inhibits HBV virion secretion

Infectivity of rtM204 variants on HepaRG cells HuH-7 cells HepaRG cells Heparin column purification Intracelluar/ extracellular cccDNA and ultrafiltration DNA extraction rcDNA Standardized virus input Supernatant collection HBeAg (MOI100) I nfection Day 10 Transfection post-infection 200 cccDNA Relative to WT [%] 150 HBeAg Intracellular DNA Extracellular DNA 100 50 0 WT M204V M204I M204S M204L M204Q HBsAg mutations are major determinant of infectivity

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

Acknowledgement Prof. Ulrike Protzer HOPE consortium groups: Cologne Dr. Christian Bach Dr. Rolf Kaiser Dr. Julie Lucifora PD Dr. Oliver Schildgen MSc Yuchen Xia Leipzig Dr. Florian van Bömmel Andrea Weicht Prof. Dr. Thomas Berg Romina Bester Theresa Asen Giessen Kerstin Ackermann PD Dr. Dieter Glebe Saarbrücken Dr. Sebastian Beggel Prof. Dr. Thomas Lengauer

Thank you for your attention!

Selective qPCR distingushing rcDNA from input plasmid Principle Efficiency 1,00E+ 08 detected plasm id copy num ber 1,00E+ 07 primer A 1,00E+ 06 1,00E+ 05 1,00E+ 04 HBV 1,00E+ 03 rcDNA pCH-9 / 3 0 9 1 1,00E+ 02 Primer B primer B 1,00E+ 01 5’ 1,00E+ 00 Primer A 5’ input plasm id copy num ber normal qPCR selective qPCR  Selective qPCR reduces plasmid detection by > 4 logs

Comparison between M204V and L180M+M204V Plasmid M204V treated with Adefovir Plasmid M204V treated with Tenofovir 125 150 % viral replication % viral replication 125 100 100 75 75 50 50 25 25 0 0 0 1 2 4 8 16 0 0.5 1 2 4 8 Tenofovir [µM] Adefovir [µM] WT HBV HBV virus WT HBV HBV virus Plasmid L180M+M204V treated with L180M+M204V treated with Tenofovir Adefovir 125 125 % viral replication % viral replication 100 100 75 75 50 50 25 25 0 0 0 1 2 4 8 16 0 0.5 1 2 4 8 Tenofovir [µM] Adefovir [µM] WT HBV HBV virus WT HBV HBV virus

Comparable result from publication Wild-type M204I M204V L180M L180M/ M204V Hepatology. 1998 Dec; 28(6): 1669-73.

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

Property of mutated rtM204 residues Hydrophobic Nonpolar Hydrophilic Polar Positive charged

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