Phenotypic HCV-NS3-PI-Resistance Test Arevir Meeting, 11./12.04.2013 - PowerPoint PPT Presentation

Phenotypic HCV-NS3-PI-Resistance Test Arevir Meeting, 11./12.04.2013 Daniel Rupp, MD

Phenotypic HCV-NS3-PI-Resistance Assay Goal: Establishing a phenotypic NS3-PI resistance assay to characterize and monitor mutation development in patient derived samples Principle: Creating replicon-libraries containing patient-derived NS3-protease sequences with subsequent drug titration and IC50/IC90 calculation Arevir Meeting, Köln 11./12.04.2013

Characteristics of subgenomic HCV-Replicons bicistronic RNA-construct expression of Luciferase and HCV- Replication Kinetics non-structural-proteins (replicase) 1000 100 RLU (fold 4h) Luciferase activity reflects replication 10 Con1-ET Con1-deltaNS3Prot 1 0,1 0,01 0 24 48 72 Timepoint after Epo RNA Luciferase Arevir Meeting, Köln 11./12.04.2013

Characteristics of subgenomic HCV-Replicons bicistronic RNA-construct expression of Luciferase and HCV- Replication Kinetics non-structural-proteins (replicase) 1000 100 RLU (fold 4h) Luciferase activity reflects replication 10 Con1-ET Con1-deltaNS3Prot 1 0,1 0,01 Modify Replicons by introducing 0 24 48 72 Timepoint after Epo unique restriction sites for patients‘ sequence shuttling RNA Luciferase Arevir Meeting, Köln 11./12.04.2013

Phenotypic NS3-PI-Resistance Assay for gt1b-patients Introducing unique restriction sites enables to shuttle patient derived NS3-protease sequences ClaI AscI goal: create library that reflects the quasispecies-pool circulating in patients Replication Fitness Con1-ET-1xAsc ClaI AscI 120 100 % of Con1-ET 80 60 40 20 0 Con1-ET Con1-ET-1xAsc 100,00 43,08 Con1-ET: 4000x above background Con1-ET-1xAsc: 2200x above background Arevir Meeting, Köln 11./12.04.2013

Phenotypic NS3-PI-Resistance Assay for gt1b-patients ClaI AscI ClaI AscI ClaI AscI Replication Fitness - Con1-ET-1xAsc- Replication Fitness Con1-ET-1xAsc Mutants 120 120 100 100 % of Con1-ET-1xAsc % of Con1-ET 80 80 60 60 40 40 20 20 0 0 Con1-ET Con1-ET-1xAsc Con1-ET-1xAsc V36A T54A 100,00 43,08 100,00 37,83 54,92 Introducing the unique restriction-sites ClaI and AscI and known resistance mutations (V36A, T54A) only slightly reduce the replication fitness of Con1-ET-1xAsc Arevir Meeting, Köln 11./12.04.2013

Phenotypic NS3-PI-Resistance Assay for gt1b-patients RNA Luciferase 4h 0h 72h + DRUG Arevir Meeting, Köln 11./12.04.2013

Phenotypic NS3-PI-Resistance Assay for gt1b-patients RNA Luciferase 4h 0h 72h + DRUG Validation Resistence Mutants - Boceprevir Validation Resistence Mutants - Telaprevir 140 140 % compared to untreated % compared to untreated 120 120 100 100 Con1-ET-1xAsc 80 80 Con1-ET-1xAsc V36A V36A 60 60 T54A T54A 40 40 20 20 0 0 0,01 0,1 1 10 0,01 0,1 1 10 Concentration (µM) Concentration (µM) Telaprevir Boceprevir IC50 (nM) IC90 (nM) IC50 (nM) IC90 (nM) Con1-ET-1xAsc 178 1229 141 748 shift in IC50! V36A 1824 9220 383 489 T54A 860 2862 748 2609 Arevir Meeting, Köln 11./12.04.2013

Phenotypic NS3-PI-Resistance Assay for gt1b-patients Introducing NS3-protease sequences from treatment naive patients (1, 2; sera provided by Sandra Ciesek) can affect replication fitness… Replication fitness 120 100 % Con1-ET-1xAsc 80 60 40 20 0 Con1-ET- 1-1 1-2 2-1 2-2 1xAsc 100,00 46,85 33,93 75,48 105,28 Arevir Meeting, Köln 11./12.04.2013

Phenotypic NS3-PI-Resistance Assay for gt1b-patients Introducing NS3-protease sequences from treatment naive patients (1, 2; sera provided by Sandra Ciesek) can affect replication fitness… … but does not cause an shift in IC50/IC90. Replication fitness Telaprevir Boceprevir % Con1-ET-1xAsc 120 IC50 (nM) IC90 (nM) IC50 (nM) IC90 (nM) 100 80 Con1-ET-1xAsc 323 411 257 457 60 1-1 281 476 290 507 40 1-2 253 532 238 504 20 0 2-1 278 399 216 386 Con1-ET- 1-1 1-2 2-1 2-2 1xAsc 100,00 46,85 33,93 75,48 105,28 2-2 200 396 187 372 Boceprevir Titration Telaprevir Titration 160 140 140 120 120 % of untreated % of untreated 100 Con1-ET-1xAsc Con1-ET-1xAsc 100 gt1b 1-1 gt1b 1-1 80 80 gt1b 1-2 gt1b 1-2 60 gt1b 2-1 gt1b 2-1 60 gt1b 2-2 gt1b 2-2 40 40 20 20 0 0 0,01 0,1 1 10 0,01 0,1 1 10 Concentration (µM) Concentration (µM) Arevir Meeting, Köln 11./12.04.2013

Phenotypic NS3-PI-Resistance Assay for gt1a-patients Modifying the genotype 1a-Replicon H77 by introducing restriction sites for NS3- protease shuttling leads to a complete loss of replication competence . Solution: introduce the gt1a-protease into the gt1b-Replicon backbone to create a hybrid-construct. ClaI AscI gt1a gt1b Arevir Meeting, Köln 11./12.04.2013

Phenotypic NS3-PI-Resistance Assay for gt1a-patients Modifying the genotype 1a-Replicon H77 by introducing restriction sites for NS3- protease shuttling leads to a complete loss of replication competence . Solution: introduce the gt1a-protease into the gt1b-Replicon backbone to create a hybrid-construct. ClaI AscI gt1a gt1b Replication Fitness gt1b/1a Hybrid Vector 140 120 % of Con1-ET-1xAsc 100 80 60 40 20 0 Con1-ET-1xAsc Con1/H77 #4 #5 Con1-ET-1xAsc: 5000x above background 100,00 7,44 16,29 16,50 Con1/H77: 650x above background Arevir Meeting, Köln 11./12.04.2013

Phenotypic NS3-PI-Resistance Assay for gt1a-patients Modifying the genotype 1a-Replicon H77 by introducing restriction sites for NS3- protease shuttling leads to a complete loss of replication competence. Solution: introduce the gt1a-protease into the gt1b-Replicon backbone to create a hybrid-construct. ClaI AscI gt1a gt1b Replication Fitness gt1b/1a Hybrid Vector 140 120 % of Con1-ET-1xAsc 100 80 60 40 20 0 Con1-ET-1xAsc Con1/H77 #4 #5 100,00 7,44 16,29 16,50 Arevir Meeting, Köln 11./12.04.2013

Phenotypic NS3-PI-Resistance Assay Patient samples from Cologne (provided by Anna Marie Sikorski, Saleta Sierra-Aragón) gt1b Titration - Telaprevir Titration - Boceprevir 140 180 160 120 140 100 % untreated 120 Con1-ET-1xAsc % untreated 80 100 Con1-ET-1xAsc #26432 80 60 #26432 #25724 60 40 #25724 40 20 20 0 0 0,001 0,01 0,1 1 10 0,001 0,01 0,1 1 10 Concentration (µM) Concentration (µM) Telaprevir Boceprevir IC50 (nM) IC90 (nM) IC50 (nM) IC90 (nM) Con1-ET-1xAsc 298 703 174 794 #26432 275 6467 332 2490 #25724 317 1354 69 66131 Arevir Meeting, Köln 11./12.04.2013

Phenotypic NS3-PI-Resistance Assay Patient samples from Cologne (provided by Anna Marie Sikorski, Saleta Sierra-Aragón) gt1a Titration - Telaprevir Titration - Boceprevir 180 160 160 140 140 120 120 % untreated 100 % untreated 100 Hybrid Hybrid #10304 80 #10304 80 #3524 #3524 #10172 #10172 #10306 60 60 #10306 #9719 #9719 40 40 20 20 0 0 0,001 0,01 0,1 1 10 0,001 0,01 0,1 1 10 Concentration (µM) Concentration (µM) Telaprevir Boceprevir IC50 (nM) IC90 (nM) IC50 (nM) IC90 (nM) Hybrid 49 756 74 297 #10304 491 4621 2182 20863 #10306 70 695 78 271 #3524 109 2102 156 976 #10172 922 3202 524 1808 #9719 1792 17258 420 1629 Arevir Meeting, Köln 11./12.04.2013

Summary and Outlook drug titration works for gt1b and gt1a/b-Hybrid replicons resistance mutations in paternal replicons cause a shift in IC50/IC90 values sequentially taken samples show no difference in titration curves samples of clinically resistant patients show a shift in titration curves Arevir Meeting, Köln 11./12.04.2013

Summary drug titration works for gt1b and gt1a/b-Hybrid replicons resistance mutations in paternal replicons cause a shift in IC50/IC90 values sequentially taken samples show no difference in titration curves samples of clinically resistant patients show a shift in titration curves Tool for phenotypic monitoring of patients under NS3-Protease-Inhibitor treatment Arevir Meeting, Köln 11./12.04.2013

Acknowledgements Ralf Bartenschlager Ulrike Protzer Rolf Kaiser Thomas von Hahn Saleta Sierra-Aragón Sandra Ciesek Anna Marie Sikorski Arevir Meeting, Köln 11./12.04.2013