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

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

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

Characteristics of HIV-1 infection 1.High virus replication rate ( virus particles / day) 2.Rapid virus clearance (T ½ cells producing virus: <1 day; T ½ free virus in plasma: a few hrs) 3.Virus latency (1:10 6 resting CD4 T cells) 4.Chronic immune activation and inflammation 5.CD4 T-cell depletion ( cells lost daily) 6.Progressive immune deficient state 7.Continuous virus genetic evolution Wong 1997; Chun 1997; Siliciano 2003; Strain 2003; Han 2007

Mechanisms of HIV genetic evolution 1.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

Dominant quasispecies Escape from pressure Preserved fitness rapid turnover rapid adaptation

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

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 EvolutionEmergence Single mutant Double mutant Triple mutant

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 evolution of resistance and cross-resistance Resistant mutants often display reduced fitness but compensatory changes emerge over time that partially restore virus fitness

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

Plasma HIV RNA PCR Defective laboratory HIV vector (e.g., RT _ ) Infectious HIV Culture with escalating drug concentrations Fold-changes in IC 50 relative to wild-type M184V = 100 FC for 3TC Viral gene (e.g., RT)

Detection of resistant mutants Detected by ultrasensitive methods Mutation Frequency Detected by routine methods Natural background

Low-frequency resistance in the FIRST study MutationsResistance testP StandardUDS NNRTI 7%15%<0.001 NRTI 6%14%<0.001 PI 2% 5%0.03 Any 14%28%<0.001 N=258 Risk of failure of first-line NNRTI-based ART in patients with NNRTI resistance Bulk resistance: HR 12.4 [ ] UDS resistance: HR 2.5 [ ] Siemen, JID 2009 USD = Ultra deep sequencing

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

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 patients treatment history

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

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

Genetic barrier Key concepts

Genetic barrier to resistance 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 Resistant Wild-type

Genetic barrier – A simplified overview ClassARVsGenetic barrier NRTIs ZDV/3TC, d4T/3TC++ ABC/3TC, TDF/3TC+ TDF/FTC++ NNRTIs EFV, NVP, ETV, RPV+ ETV, RPV+/++ PIsBoosted+++/++++ Fusion inhibitorsT20+ CCR5 antagonistsMVC++ (for R5 virus) Integrase inhibitorsRAL, ELV+

Common NRTI resistance patterns NRTIsMutationsZDVd4TABCddITDF3TCFTC ZDV 3TC d4T 3TC M184V TAMs + TAMs d4T 3TC TDF 3TC TDF FTC M184V K65R ABC 3TC M184V L74V K65RY115F TAMs = thymidine analogue mutations: M41L, D67N, K70R, L210W, T215Y/F, L219Q/E

Resistance with first-line HAART StudyARTwkTestsK65RL74VM184VTAMs3 rd drug GS903 n=299TDF 3TC EFV482924%041%0EFV R 55% GS934 n=244TDF FTC EFV %0EFV R 71% GS934 n=243ZDV 3TC EFV %3%EFV R 62% CNA30021 n=770ABC 3TC EFV48383%21%47%0EFV R 58% ABT418 n=190TDF FTC LPV/r %0PI R 0 SOLO n=190ABC 3TC FPV/r %0PI R 0 ARTEMIS n=343TDF FTC DRV/r %0PI R 0 STARTMRK n=281TDF FTC RAL %0RAL R 33% Margot, HIV Med 2006; Margot, JAIDS 2009; Moyle, JAIDS 2005; Molina, IAC 2004; Gathe, AIDS 2004; Mills, AIDS 2009; Lennox, Lamcet 2009.

1 st and 2 nd generation NNRTIs NevirapineEfavirenzEtravirine 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

Activity of ETV with a strong backbone DUET studies: OBR (with DRV/r) + ETV or Placebo % p<0.0001* Responders (%) ± 95% CI Time (weeks) Patients with VL <50 copies/ml at wk 48 (ITT-TLOVR) ETV + OBR (n=599) Placebo + OBR (n=604) 61% Katlama, AIDS 2009 ART-experienced patients with documented NNRTI and PI resistance

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

The genetic barrier of PIs in vitro De Meyer, Antimicrob Agents Chemother 2005; De Meyer, IHDRW 2006 Time (days) Increase in PI selection concentration DRV (R41T, K70E) RTV (G16E, M46I, V82F, I84V) SQV (G48V, A71V, G73S, I84V, L90M) NFV (L10F, D30N, R41K, K45I, M46I, V77I, I84V, N88D) APV (L10F, V32I, L33F, M46I, I47V, I50V) LPV (L10F, L23I, M46I, I50V, I54V, L63P, V82A) TPV (L33V, M46L, V82T) ATV (L10F, V32I, M46I, I62V, A71V, I84V, N88S)

Emergence of PI mutation with DRV/r vs LPV/r TITAN study: OBR + DRV/r or LPV/r 1 Johnson et al. Top HIV Med 2007; Primary PI mutations 1 NRTI RAMs 1 VFs with developing mutations (%) 40 DRV/r LPV/r p 0.05* 6/39 (15%) 24/72 (33%) 3/39 (8%) 19/72 (26%) p 0.05* *Exact Chi-Squared Test; TITAN 96 week analysis De Meyer, HIV TITAN 96 week analysis ART-experienced, LPV- and DRV-naive patients

Hypersusceptibility Key concepts

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 P P P P P P P P P P P P P P P P Gotte, J Virol 2000

Residual activity Key concepts

Response Resistance 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 Resistance as a continuum

Partial treatment interruption in patients with resistance reveals residual activity Deeks, CROI 2005

Key principles of resistance Resistance moves along a continuum and 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

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

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