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Genotypic and Phenotypic characterization of ARV resistance in clade C HIV-1

Genotypic and Phenotypic characterization of ARV resistance in clade C HIV-1. Lieven J. Stuyver, PhD Tibotec Virco Virology BVBA ARTA Satellite symposium The potential role of HIV Drug Resistance Testing in Sub-Saharan Africa Sunday July 19 th 2009 10:15 – 12:15 hrs Mini Room 3.

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Genotypic and Phenotypic characterization of ARV resistance in clade C HIV-1

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  1. Genotypic and Phenotypic characterization of ARV resistance in clade C HIV-1 Lieven J. Stuyver, PhD Tibotec Virco Virology BVBA ARTA Satellite symposium The potential role of HIV Drug Resistance Testing in Sub-Saharan Africa Sunday July 19th 2009 10:15 – 12:15 hrsMini Room 3

  2. Global prevalence of HIV-1 genetic forms, expressed as a % of the total number of isolates.

  3. Question 1: HIV-1 protease and RT sequences are interpreted using data driven models. One model is VircoTYPE. Do the driving data in VircoTYPE cover all clades?

  4. Algorithms for the interpretation of HIV-1 genotypic drug resistance information Vercauteren et al., 2006

  5. The Virco gp database which drives vT predictions includes 11.5% non B subtypes, with a distribution similar to LANL van Vlijmen H. et al., XVIII IHDRW 2009

  6. Answer 1: HIV-1 protease and RT sequences are interpreted using data driven models. One model is VircoTYPE. Do the driving data in VircoTYPE cover all clades? In VircoTYPE, the current model parameters are mainly based on HIV-1 clade B information, but > 6000 non-B sybtype sequences are included. Several of the subtype sepcific differences are included (RT 106M; RT K65R). No data for the other algorithms

  7. Question 2: As a consequence of the mainly clade B composition of the in vitro phenotypic testing database, biological cut-offs (BCOs; border between drug susceptibility and resistance) are derived from clade B information. Should BCOs different for clade C viruses?

  8. Definition of a BCO derived from “genotypically wild type” HIV-1 genotype samples Verlinden Y . et al., XIV IHDRW 2005

  9. Minor differences in FC distribution of wild type isolates from subtypes B and C Van Houtte M. et al., EHDRW

  10. Answer 2: As a consequence of the mainly clade B composition of the in vitro phenotypic testing database, biological cut-offs (BCOs; border between drug susceptibility and resistance) are derived from clade B information. Should BCOs different for clade C viruses? Some minor differences in the FC distribution of wild type isolates could be observed, but the general pattern showed no important differences between clade B and C

  11. Question 3: Clade-specific polymorphisms that are conferring reduced susceptibility to ART do exist (included in IAS mutation list and VircoTYPE). In search of other variants that may affect drug susceptibility: Are clade C “genotypically wild type viruses ” with fold-change values > BCO (FC>BCO) present in the in vitro phenotypic testing database?

  12. Answer 3: Clade-specific polymorphisms that are conferring reduced susceptibility to ART do exist (included in IAS mutation list). In search of other variants that may affect drug susceptibility: Are clade C “zero profiles” with fold-change values > BCO (FC>BCO) present in the in vitro phenotypic testing database? Yes, by definition, since - samples with a FC>BCO = 2.5% of the population, and, - %>BCO is generally similar for subtype B and C. Genotypic pattern analysis is pending, and requires separating assay noise from real signal

  13. Question 4: HIV-1 recombinant viruses are built in the HXB2 (clade B) protease and RT-gene deleted backbone. Testing clade C clinical isolates always results in interclade recombinants. Is there an influence of the clade B HXB2 backbone on the clade C protease and RT fold-changes? CDB023: Generation of HIV-1 subtype C recombinant viruses for phenotypic analysis of protease and reverse transcriptase inhibitor resistance profiles David Nauwelaers, Margriet Van Houtte, Bart Winters, Lieven Stuyver Virco BVBA, Generaal De Wittelaan L11 B4, B-2800 Mechelen

  14. HIV-1 clade C phenotyping process using pHIV-1-C- ΔPRT backbone • TAT (1) • REV (2) Δ • REV (1) pHIV-1-C- ΔPRT backbone • VIF • GAG • NEF • ENV • 5' LTR • POL • VPR • VPU • TAT (2) • 3' LTR • p1 • PRT amplicon • p7 • p6 • Protease • Reverse Transcriptase The designed subtype C backbone (pHIV-1-C- ΔPRT) aligned with other subtype C reference sequences (Los Alamos) • 1755bp • FW Infusio • (2120-2149) • RV Infusion • (3847-3875) Process: Day 1 Day 2 Day 3 Day 4 PRT subtype C amplicon generation Pick StBl2 E. coli colonies and grow in liquid LB/Amp overnight • Transformation in • StBl2 E. coli cells • infusion Full subtype C genome pGEM-HIV-1-C ∆gprt Linearized with EcoRV and BstEII MiniPrep (plasmid DNA preparation) Day 5 restriction digest to check inserts Nucleofection (Amaxa) In MT4-eGFP cells Harvest virus, Titration, AVE Full subtype C genome Day 22-29 Day 7-21 Day 6

  15. HIV-1 clade C phenotyping process using pHIV-1-C- ΔPRT backbone • TAT (1) • REV (2) Δ • REV (1) pHIV-1-C- ΔPRT backbone • VIF • GAG • NEF • ENV • 5' LTR • POL • VPR • VPU • TAT (2) • 3' LTR • p1 PRT amplicon • p7 • p6 • Protease • Reverse Transcriptase • 1755bp • FW Infusio • (2120-2149) • RV Infusion • (3847-3875) Process: Day 1 Day 2 Day 3 Day 4 PRT subtype C amplicon generation Pick StBl2 E. coli colonies and grow in liquid LB/Amp overnight • Transformation in • StBl2 E. coli cells • infusion Full subtype C genome pGEM-HIV-1-C ∆gprt Linearized with EcoRV and BstEII MiniPrep (plasmid DNA preparation) Day 5 restriction digest to check inserts Nucleofection (Amaxa) In MT4-eGFP cells Harvest virus, Titration, AVE Full subtype C genome Day 22-29 Day 7-21 Day 6

  16. Drug FC values generated in both clade B and C viral backbones (388 data points) showed a high correlation • included in analysis • NRTIs • Zidovudine • Lamivudine • Didanosine • Stavudine • Abacavir • Emtricitabine • Tenofovir DF • NNRTIs • Nevirapine • Efavirenz • Etravirine

  17. Answer 4: HIV-1 recombinant viruses are built in the HXB2 (clade B) protease and RT-gene deleted backbone. Testing clade C clinical isolates always results in interclade recombinants. Is there an influence of the clade B HXB2 backbone on the clade C protease and RT fold changes? Drug FC values generated in both clade B and C viral backbones showed a high correlation

  18. Conclusions Genotypic analysis: The current algorithms are mainly based on HIV-1 clade B information Clade-specific polymorphisms were previously recognized and included in IAS mutation lists and vircoTYPE Phenotypic analysis: BCOs: no important differences between clade B and C Clade B and C “genotypically wild types” with FC>BCO: Search for "polymorphisms” linked to reduced susceptibility is ongoing. Challenge to separate assay “noise” from “real” signal.. Drug FC values generated in both clade B and C viral backbones showed a high correlation, but more testing is ongoing (part of ARTA). Preliminary conclusion considering everything: genotypic interpretation tools designed from clade B are also reliable tools for clade C (e.g. vircoTYPE has >6000 isolates included, no data for other algorithms…).

  19. Acknowledgements

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