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

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

Jan 22, 2023 133 likes •297 views

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

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SLIDE 1

Optimizing Phenotypic HBV Drug Resistance Testing for High Throughput

Ke Zhang, Rolf Kaiser, Ulrike Protzer Institute of Virology

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SLIDE 2

Treatment Options for Chronic Hepatitis B

Approved for HBV Unlabeled Investigational

  • Interferon alfa-2b
  • Lamivudine
  • Adefovir
  • Entecavir
  • Peginterferon alfa-2a
  • Telbivudine
  • Tenofovir DF

Phase III

  • Emtricitabine /

Combination with Tenofovir* Phase III

  • Clevudine

Phase II

  • Pradefovir
  • Valtorcitabine
  • Amdoxovir
  • ANA 380
  • Racivir

*Approved by the FDA for treatment of HIV infection.

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SLIDE 3

Long-Term Therapy May Lead to the Emergence of Resistant Viruses

Cumulative Genotypic Resistance Rates

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Antiviral Resistance Testing

in in-

  • vitro

vitro Phenotypic Phenotypic Resistance?? Resistance?? Genotypic Resistance Genotypic Resistance Testing Testing Clinical Resistance Clinical Resistance

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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)

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)

WT L180M+A181T

Fold Resistance = Mutant IC50 WT IC50

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SLIDE 6

Modified from Günther et al. J Virol 1995 and Yang, H et al. Antiviral Res 2004

Strategy of HBV Genome Cloning for Phenotyping

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SLIDE 7

Strategy of Sub-genome Cloning for Phenotyping

845 a.a. Terminal protein Spacer Pol/RT RNaseH A B C E D YMDD GVGLSPFLLA I(G) II(F)

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.

rtL80V/I rtM204V/I/S LAM resistance rtV173L rtL180M rtA181T/V rtN236T rtl233V ? ADV resistance rtM204V/I rtS202G/C rtM250I/V rtT184S/A/I/L ETV resistance rtL180M rtM204I LdT resistance rtL80V/I rtL180M TDF resistance rtA194T rtM204V rtL180M

EcoRI SphI RsrII BspEI

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PCR Amplification of HBV Whole Genome, POL Gene and RT Region from Patient Serum

3kb

M S1 S2 S3

M: DNA Ladder; S1: 108 copies/ml, Genotype D; S2: 106 copies/ml, Genotype A; S3: 104 copies/ml, Genotype D

2.5kb 1kb

M S1 S2 S3 M S1 S2 S3

Genome POL RT

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Mutation Detection Direct Sequencing vs. PCR Cloning-Sequencing

Requirement: Proportion of mutant virus ≥20% n 80% 90% 95% 20% 7-8 10-11 13-14 10% 15-16 21-22 28-29 5% 31-32 44-45 58-59 x y

1 - (1-x)n ≥ y

x: Proportion of mutant y: Possibility of being detected n: Minimal clones sequenced

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Phenotypic Testing on Genotypic Resistance Mutation

  • PCR-Cloning of HBV full genome /

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

pCH-9/200 (with clinical HBV insert)

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Selective qPCR for Differentiating HBV Genomic rcDNA from Plasmids

Principle Efficiency

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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

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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

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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

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Acknowledgments

Institute of Virology, TUM / Helmholtz Zentrum München

  • Prof. Dr. Volker Bruss

Gregor Ebert

  • Dr. Julie Lucifora

Beate Schittl Christian Bach

Thanks for your attention!

Universität zu Köln

  • Dr. Roland Kaiser

Maria Fraune

  • Prof. Dr. Tobias Göser