Antiretroviral Drug Resistance

1. Antiretroviral Drug Resistance Anna Maria Geretti UCL Medical School & Royal Free Hampstead Medical School, London

2. Host-related factors • Adherence • Tolerability • Immunity • Genetics • Drug-related factors • Potency • PK properties • Genetic barrier • Virus-related factors • Viral load • Drug susceptibility • Fitness Persistent virus replication during HAART Drug pressure Emergence and evolution of drug resistance

3. Attachment Fusion Release of RNA Assembly Reverse transcription Integration Transcription Maturation and budding

4. Characteristics of HIV-1 infection High virus replication rate (109-1010 virus particles / day) Rapid virus clearance (T½ cells producing virus: <1 day; T½ free virus in plasma: a few hrs) Virus latency (1:106 resting CD4 T cells) Chronic immune activation and inflammation CD4 T-cell depletion (108-109 cells lost daily) Progressive immune deficient state Continuous virus genetic evolution Wong 1997; Chun 1997; Siliciano 2003; Strain 2003; Han 2007

5. Mechanisms of HIV genetic evolution • RT-driven mutagenesis • Rate: ~1 wrong incorporation per genome round • All possible point mutations generated daily 2. APOBEC-driven mutagenesis • Deamination of cytosine residues in nascent DNA • GA hypermutation 3. Recombination • Rate: 7-30 per genome round • Hybrid virus progeny produced from different strains

6. Dominant quasispecies Escape from pressure Preserved fitness • rapid turnover • rapid adaptation

7. Consequences of HIV genetic variability • At the population level • Continuous emergence of new variants • At the patient level • Escape from immune pressure • Escape from drug pressure • Increased fitness and pathogenicity • Challenge for diagnostic and monitoring assays

8. Emergence Evolution Single mutant Double mutant Triple mutant Emergence and evolution of resistance • Increasing number of mutations • Accumulation of mutations on the same viral genome • Initially reduced viral fitness • Compensatory changes restore fitness

9. Key principles of resistance • Drug-resistant mutants are selected (not created) by drug pressure if virological suppression is incomplete • Ongoing virus replication under drug pressure leads to the evolutionof resistance and cross-resistance • Resistant mutants often display reduced fitness but compensatory changes emerge over time that partially restore virus fitness

10. PCR Viral gene (e.g., RT) HIV RNA Plasma Mutations RT M184V Methionine  Valine @ codon 184 of RT ATG / AUG  GTG / GUG Sequencing

11. 100 Detected by routine methods 10 Detected by ultrasensitive methods 1 Mutation Frequency 0.1 0.01 Natural background 0.001 Detection of resistant mutants

12. Low-frequency resistance in the FIRST study N=258 • Risk of failure of first-line NNRTI-based ART in patients with NNRTI resistance • Bulk resistance: HR 12.4 [3.4-45.1] • UDS resistance: HR 2.5 [1.2-5.4] USD = Ultra deep sequencing Siemen, JID 2009

13. Resistant Wild-type Drug pressure Limit of detection 20-30%

14. Key principles of resistance • Once drug pressure is removed, resistant mutants are outgrown by fitter wild-type virus and become undetectable by routine tests • Resistance test results obtained after therapy is discontinued are not reliable • Resistant mutants persist at low frequency in plasma and are “archived” in latently infected cells • Resistance is long-lasting • Resistance test results must be interpreted in the context of the patient’s treatment history

15. Ms S., 35 yr Δ HIV+ Dec 1997 Resistant Wild-type d4T 3TC NVP d4T 3TC NVP VL M184V M184V Y181C Y181C <50 M184V = 3TC, FTC Y181C = NNRTIs

16. Transmission Drug pressure Transmitted drug resistance Stable after transmission Gradual reversion over time, sometimes incomplete Persistence at low frequency in plasma Persistence in latently infected cells

17. Key concepts Genetic barrier

18. Genetic barrier to resistance Resistant Wild-type Defined by: Number of mutations required to compromise activity Impact of each mutation on drug susceptibility Interactions between mutations Fitness cost of resistance Drug concentration

19. Genetic barrier – A simplified overview

20. Common NRTI resistance patterns TAMs = thymidine analogue mutations: M41L, D67N, K70R, L210W, T215Y/F, L219Q/E

21. Resistance with first-line HAART Margot, HIV Med 2006; Margot, JAIDS 2009; Moyle, JAIDS 2005; Molina, IAC 2004; Gathe, AIDS 2004; Mills, AIDS 2009; Lennox, Lamcet 2009.

22. 1st and 2nd generation NNRTIs Nevirapine Efavirenz Etravirine Major resistance mutations L100I, K101E/P K103N/S V106A/M E138K, V179F Y181C/I/V, Y188L/H/C, G190A/S/E F227C, M230L, K238T Rilpivirine

23. Patients with VL <50 copies/ml at wk 48 (ITT-TLOVR) Activity of ETV with a strong backbone DUET studies: OBR (with DRV/r) + ETV or Placebo 100 90 80 70 60 50 40 30 20 10 0 ETV + OBR (n=599) Placebo + OBR (n=604) 61% Responders (%) ± 95% CI 40% p<0.0001* 0 2 4 8 12 16 20 24 32 40 48 Time (weeks) ART-experienced patients with documented NNRTI and PI resistance Katlama, AIDS 2009

24. Activity of ETV with a weak backboneStudy TMC125-C227: 2 NRTIs + ETV or PI ART-experienced, PI-naive patients with documented NNRTI resistance Ruxrungtham, HIV Med 2008

25. DRV (R41T, K70E) TPV (L33V, M46L, V82T) ATV (L10F, V32I, M46I, I62V, A71V, I84V, N88S) LPV (L10F, L23I, M46I, I50V, I54V, L63P, V82A) APV (L10F, V32I, L33F, M46I, I47V, I50V) NFV (L10F, D30N, R41K, K45I, M46I, V77I, I84V, N88D) SQV (G48V, A71V, G73S, I84V, L90M) RTV (G16E, M46I, V82F, I84V) The genetic barrier of PIs in vitro 450 400 350 300 Increase in PI selection concentration 250 200 150 100 50 0 0 100 300 500 700 900 1100 Time (days) De Meyer, Antimicrob Agents Chemother 2005; De Meyer, IHDRW 2006

26. Key concepts Hypersusceptibility

27. P P P P P P P P P P P P P P P P Mechanisms of NRTI resistance: Primer unblocking • T215Y-mediated resistance • Hydrolytic removal of the chain-terminating NRTI enables DNA synthesis to resume • The pyrophosphate donor in most cells is ATP • M184V antagonizes the process • 3TC partially restores susceptibility to ZDV,d4T and TDF in the presence of TAMs • 3TC antagonizes the emergence of TAMs Gotte, J Virol 2000

28. Key concepts Residual activity

29. Resistance as a continuum Lower cut-off = Level of resistance beyond which response begins to fall off Upper cut-off = Level of resistance beyond which clinical response is lost Zone of intermediate response Response Resistance

30. Partial treatment interruption in patients with resistance reveals residual activity Week 2 change in VL 1.0 Change in plasma viral load 0.5 0.0 –0.5 NRTI PI NNRTI T20 Discontinued treatment class Deeks, CROI 2005

31. Key principles of resistance • Resistance moves along a continuumand increasing numbers of mutations lead to progressive loss of responses • Residual activity is possible despite the presence of extensive resistance(best evidence for the NRTIs) • Resistance carries a fitness cost that reduces viral replication • Antagonistic effects between mutations can have beneficial effects

32. Clinical implications for patients with treatment failure • The likelihood of drug-resistance depends upon the drug, the regimen and the level of adherence • When selecting a new regimen, aim for at least 2 fully active drugs • Avoid functional monotherapy with drugs that have a low genetic barrier • If options are limited, exploit residual activity and hypersusceptibility effects – continue the NRTIs rather than stopping therapy

33. Thank you