Optimizing Phenotypic HBV Drug Resistance Testing for High - PowerPoint PPT Presentation

Optimizing Phenotypic HBV Drug Resistance Testing for High Throughput Ke Zhang, Rolf Kaiser, Ulrike Protzer Institute of Virology

Treatment Options for Chronic Hepatitis B Approved for HBV Unlabeled Investigational • Interferon alfa-2b Phase III Phase III • Lamivudine • Clevudine • Emtricitabine / • Adefovir Combination with • Entecavir Phase II Tenofovir* • Peginterferon alfa-2a • Pradefovir • Telbivudine • Valtorcitabine • Tenofovir DF • Amdoxovir • ANA 380 • Racivir *Approved by the FDA for treatment of HIV infection.

Long-Term Therapy May Lead to the Emergence of Resistant Viruses Cumulative Genotypic Resistance Rates

Antiviral Resistance Testing Clinical Resistance Clinical Resistance Genotypic Resistance Genotypic Resistance Testing Testing Phenotypic in- in -vitro vitro Phenotypic Resistance?? Resistance??

Antiviral Resistance Testing Phenotypic Assays � IC50: Concentration of drug that inhibits 50% of viral replication � Fold resistance: Ratio of the IC50s of the tested virus and the reference virus (wt) Fold Resistance = WT L180M+A181T Mutant IC 50 WT IC 50 Antiviral drug resistance in vitro can be described in terms of: -HIGH (>100 fold increase in IC50) -INTERMEDIATE (10-100 fold increase) -LOW-LEVEL (2-9 fold increase)

Strategy of HBV Genome Cloning for Phenotyping Modified from Günther et al. J Virol 1995 and Yang, H et al. Antiviral Res 2004

Strategy of Sub-genome Cloning for Phenotyping BspEI EcoRI SphI RsrII Terminal Spacer Pol/RT RNaseH protein 845 a.a. GVGLSPFLLA YMDD I(G) II(F) A B C D E LAM resistance rtL80V/I rtV173L rtM204V/I/S rtL180M rtN236T ADV resistance rtA181T/V rtl233V ? ETV resistance rtL180M rtM204V/I rtT184S/A/I/L rtS202G/C rtM250I/V LdT resistance rtL80V/I rtL180M rtM204I TDF resistance rtA194T rtM204V rtL180M Allen MI, et al. Hepatology. 1998;27:1670-1677. Qi X, et al. J Hepatol. 2004;40(suppl 1):20-21. Tenney D, et al. Antimicrob Agents Chemother. 2004;48:3498-3507. Tyzeka [package insert]. Lai CL, et al. Gastroenterology. 2005;129:528-536. Schildgen O, et al. N Engl J Med. 2006;354:1807- 1812. Locarnini S. 2006 IDRW. Abstract P2.

PCR Amplification of HBV Whole Genome, POL Gene and RT Region from Patient Serum Genome POL RT M S1 S2 S3 M S1 S2 S3 M S1 S2 S3 3kb 2.5kb 1kb M: DNA Ladder; S1: 10 8 copies/ml, Genotype D; S2: 10 6 copies/ml, Genotype A; S3: 10 4 copies/ml, Genotype D

Mutation Detection Direct Sequencing vs. PCR Cloning-Sequencing y n 80% 90% 95% x 20% 7-8 10-11 13-14 10% 15-16 21-22 28-29 5% 31-32 44-45 58-59 1 - (1-x) n ≥ y Requirement: x: Proportion of mutant Proportion of mutant virus ≥ 20% y: Possibility of being detected n: Minimal clones sequenced

Phenotypic Testing on Genotypic Resistance Mutation • PCR-Cloning of HBV full genome / pCH-9/200 (with clinical HBV insert) sub-genome into pCH-9 • Confirm correct clones by sequencing • Transfect into HuH7 cells, treat with 5 concentrations of LAM, ADF, ENT…. • Analyze HBV replication by Southern blotting or selective qPCR • Calculate replicative capacity, IC50 and fold resistance

Selective qPCR for Differentiating HBV Genomic rcDNA from Plasmids Principle Efficiency

Clinical Examples: rtL180M+T184S+M204V and rtL180M+A181V mutant HBV - Chronic hepatitis B, sequential treatment with LAM and ADF - Secondary treatment failure to LAM and to ADF Fold Increase >100: HIGH Fold Increase 10-15: INTERMEDIATE

Fold Increase of Phenotypic Resistance to Lamivudine In Vitro Supernatant Cell Lysate Literature* Wild -type 1 1 1 A181V 2 4 2-6 L180M+A181V 10 15 / L180M+T184S+M204V - >100 >1000** * Chin et al. Antimicrob Agents Chemother 2001; 45:2495-2501. * Delaney et al. Antivir Chem Chemother 2001; 12:1-35. ** Result from the mutation L180M+M204V

Summary � HBV resistance testing is of increasing importance � A phenotypic assay for large scale analysis in a standardized fashion is needed � Plasmid constructs and a selective real-time PCR based method for analysis were developed � First validation with clinical samples has started

Acknowledgments Institute of Virology, TUM / Helmholtz Zentrum München Prof. Dr. Volker Bruss Gregor Ebert Dr. Julie Lucifora Universität zu Köln Beate Schittl Christian Bach Dr. Roland Kaiser Maria Fraune Prof. Dr. Tobias Göser Thanks for your attention!