Antiretroviral Drug Resistance Anna Maria Geretti

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

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

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 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 4

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 Guanosine to adenosine

Dominant quasispecies Escape from pressure Preserved fitness Within the quasispecies, positive (Darwinian) selection implies that one or more members of the quasispecies are better suited to a given environment while negative selection eliminates unfit variants In the absence of drug pressure, resistant mutants tend to disappear from the dominant quasispecies as they are outgrown by the fitter wild-type virus. The greater the impact on fitness the more rapid the disappearance 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 Single mutant Double mutant Triple mutant Thus, resistant variants pre-exist the introduction of drug pressure. Once drug pressure is introduced, if replication continues the resistant mutant will acquire a selective advantage and gradually emerge as dominant Evolution subsequently occurs so that multiple mutations come to coexist on the same genome Resistance mutations occur in regions of the viral genome that play a key role in virus replication and infectivity. As a result they come at a cost of reduced viral fitness, which can be described as the virus ability to replicate and infect in a given environment. Over time however, the virus tends to acquire further changes that compensate for the reduced fitness and restore virus replication at least partially. Thus we can say that the natural evolution of HIV under drug pressure is towards increasing resistance and preserved fitness Increasing number of mutations Accumulation of mutations on the same viral genome Initially reduced viral fitness Compensatory changes restore fitness

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 We can summarise these observations as follows

PCR Viral gene (e.g., RT) HIV RNA Plasma Sequencing Mutations RT M184V Methionine  Valine @ codon 184 of RT ATG / AUG  GTG / GUG This slides illustrates how genotypic resistance test is performed in the laboratory

escalating drug concentrations PCR Viral gene (e.g., RT) HIV RNA Plasma Culture with escalating drug concentrations Fold-changes in IC50 relative to wild-type M184V = 100 FC for 3TC Defective laboratory HIV vector (e.g., RT_) Infectious HIV

Detection of resistant mutants 0.001 0.01 0.1 1 10 100 Detected by ultrasensitive methods Mutation Frequency Detected by routine methods Natural background With this standard resistance testing method we can detected the most prevalent mutants. In research settings we can use more sensitive techniques (e.g., real-time PCR) to detect mutants present at levels between 10-20% and ~0.1%. Clinically it is not considered useful to detect rarer mutants (<0.1%) as these will be part of the “natural background” to be expected in all patients regardless of drug pressure We have increasing evidence that the low-frequency mutants present at levels between 10-20% and 0.1% matter, but no way of detecting them in routine diagnostic settings. Thus, expert and integrated interpretation of treatment history and resistance results is paramount.

Low-frequency resistance in the FIRST study Mutations Resistance test P Standard UDS NNRTI 7% 15% <0.001 NRTI 6% 14% PI 2% 5% 0.03 Any 28% N=258 Background.Minor (i.e., <20% prevalence) drug‐resistant human immunodeficiency virus (HIV) variants may go undetected, yet be clinically important. Objectives.To compare the prevalence of drug‐resistant variants detected with standard and ultra‐deep sequencing (detection down to 1% prevalence) and to determine the impact of minor resistant variants on virologic failure (VF). Methods.The Flexible Initial Retrovirus Suppressive Therapies (FIRST) Study (N = 1397) compared 3 initial antiretroviral therapy (ART) strategies. A random subset (n = 491) had baseline testing for drug‐resistance mutations performed by use of standard sequencing methods. Ultra‐deep sequencing was performed on samples that had sufficient viral content (N= 264). Proportional hazards models were used to compare rates of VF for those who did and did not have mutations identified. Results.Mutations were detected by standard and ultra‐deep sequencing (in 14% and 28% of participants, respectively;  ). Among individuals who initiated treatment with an ART regimen that combined nucleoside and nonnucleoside reverse‐transcriptase inhibitors (hereafter, “NNRTI strategy”), all individuals who had an NNRTI‐resistance mutation identified by ultra‐deep sequencing experienced VF. When these individuals were compared with individuals who initiated treatment with the NNRTI strategy but who had no NNRTI‐resistance mutations, the risk of VF was higher for those who had an NNRTI‐resistance mutation detected by both methods (hazard ratio [HR], 12.40 [95% confidence interval {CI}, 3.41–45.10]) and those who had mutation(s) detected only with ultra‐deep sequencing (HR, 2.50 [95% CI, 1.17–5.36]). Conclusions.Ultra‐deep sequencing identified a significantly larger proportion of HIV‐infected, treatment‐naive persons as harboring drug‐resistant viral variants. Among participants who initiated treatment with the NNRTI strategy, the risk of VF was significantly greater for participants who had low‐ and high‐prevalence NNRTI‐resistant variants. 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 13

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

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 The clinical implications of these observations can be summarised as follows 15

VL Ms S., 35 yr Δ HIV+ Dec 1997 M184V = 3TC, FTC Y181C = NNRTIs Wild-type Resistant M184V = 3TC, FTC Y181C = NNRTIs d4T 3TC NVP d4T 3TC NVP VL <50 M184V Y181C Testing should be undertaken on samples taken while the patient is receiving therapy

Transmitted drug resistance Stable after transmission Gradual reversion over time, sometimes incomplete Persistence at low frequency in plasma Persistence in latently infected cells Drug pressure Transmission Finally, a few considerations about transmitted drug resistance. We have discussed the dynamics of resistant mutants during treatment and seen that mutants selected during therapy disappear rapidly once therapy is interrupted Transmitted resistant mutants however behave differently and can persist for long periods in the absence of drug pressure. This cartoon explain the proposed model. HIV transmission leads to an infection which is initially highly homogenous. If the virus transmitted is resistant the infection will become established with a homogenously resistant virus. There is no wild-type virus that can rapidly out compete the resistant mutant. For the wild-type to occur the resistant mutants is required to back mutate, a process that is rather slow due to the fact that the transmitted virus is genetically constrained Reversion does occur gradually over time, but the resistant mutants will persist as minority species and archived resistance, with the potential for a long-lasting impact

Genetic barrier Residual activity Hypersusceptibility Key concepts Genetic barrier Residual activity Hypersusceptibility

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 19

Genetic barrier – A simplified overview Class ARVs Genetic barrier NRTIs ZDV/3TC, d4T/3TC ++ ABC/3TC, TDF/3TC + TDF/FTC NNRTIs EFV, NVP, ETV, RPV ETV, (RPV) +/++ PIs Boosted +++/++++ Fusion inhibitors T20 CCR5 antagonists MVC ++ (for R5 virus) Integrase inhibitors RAL, ELV 20

Common NRTI resistance patterns NRTIs Mutations ZDV d4T ABC ddI TDF 3TC FTC ZDV 3TC d4T 3TC M184V TAMs + TAMs d4T 3TC TDF 3TC TDF FTC K65R ABC 3TC L74V Y115F TAMs = thymidine analogue mutations: M41L, D67N, K70R, L210W, T215Y/F, L219Q/E

Resistance with first-line HAART Study ART wk Tests K65R L74V M184V TAMs 3rd drug GS903 n=299 TDF 3TC EFV 48 29 24% 41% EFVR 55% GS934 n=244 TDF FTC EFV 96 14 14% EFVR 71% GS934 n=243 ZDV 3TC EFV 31% 3% EFVR 62% CNA30021 n=770 ABC 3TC EFV 38 21% 47% EFVR 58% ABT418 n=190 TDF FTC LPV/r 23 17% PIR 0 SOLO n=190 ABC 3TC FPV/r 32 12% ARTEMIS n=343 TDF FTC DRV/r 31 6% STARTMRK n=281 TDF FTC RAL 13 38% RALR 33% ITT analysis RAL arm: Integrase from 12/39 VFs = 4 RALR*; RT in 13/39 VFs = 5 FTCR EFV arm: RT from 9/45 VFs = 5 EFVR; 2 FTCR Margot, HIV Med 2006; Margot, JAIDS 2009; Moyle, JAIDS 2005; Molina, IAC 2004; Gathe, AIDS 2004; Mills, AIDS 2009; Lennox, Lamcet 2009.

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 NNRTI resistance mutations emerge rapidly in patients with detectable viraemia during NNRTI therapy Single NNRTI mutations can cause high-level NNRTI resistance and cross-resistance Resistance mutations continue to accumulate 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 Patients with VL <50 copies/ml at wk 48 (ITT-TLOVR) 100 90 80 70 60 50 40 30 20 10 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

Activity of ETV with a weak backbone Study TMC125-C227: 2 NRTIs + ETV or PI Viral rebound was most apparent in individuals who had more than one NRTI mutation at baseline. In contrast, the subset of patients with no NRTI mutations demonstrated a more durable viral load suppression of about 1.75 log10 copies/mL below baseline as of week 16. Those patients with four or more NRTI mutations at baseline showed virtually no response to etravirine. The number of NNRTI mutations also had an impact on etravirine efficacy, and there was transient suppression in some individuals who had just one NNRTI mutation at baseline. ART-experienced, PI-naive patients with documented NNRTI resistance Ruxrungtham, HIV Med 2008

The genetic barrier of PIs in vitro 450 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) 400 350 300 Increase in PI selection concentration 250 200 150 100 50 100 300 500 700 900 1100 Time (days) De Meyer, Antimicrob Agents Chemother 2005; De Meyer, IHDRW 2006

VFs with developing mutations (%) Emergence of PI mutation with DRV/r vs LPV/r TITAN study: OBR + DRV/r or LPV/r TITAN 96 week analysis p0.05* p0.05* DRV/r LPV/r 40 24/72 (33%) 19/72 (26%) 30 6/39 (15%) VFs with developing mutations (%) 20 3/39 (8%) 10 Primary PI mutations1 NRTI RAMs1 *Exact Chi-Squared Test; TITAN 96 week analysis 1Johnson et al. Top HIV Med 2007; ART-experienced, LPV- and DRV-naive patients De Meyer, HIV-9 2008 27

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

How to calculate the GSS Genotypic Susceptibility Score RT mutations: M41L T215Y K103N Protease mutations: None Regimen: TDF 3TC LPV/r Resistance: Intermediate Susceptible Susceptible Score: 0.5 1 1 GSS ?

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

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 Pyrophosphate can also be an acceptor Antagonised by M184V, K65R, L74V Gotte, J Virol 2000

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 The clinical implications of these observations can be summarised as follows

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 a GSS ≥2 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 The clinical implications of these observations can be summarised as follows 33

Advanced Virology Workshop Are you curious about what to do when a patient suddenly stops NNRTI-based ART? What “undetectable” viral load really means? How we can easily predict HIV-1 tropism? Advanced Virology Workshop Ongoing Thank you 34