What if HIV Could not Develop Resistance to a New Integrase Inhibitor when that Compound Is Used in First- Line Therapy? Mark A Wainberg McGill University AIDS Centre McGill University AIDS Centre Jewish General Hospital

Disclosures I have received honoraria from AbbVie, Bristol Myers Squib, Gilead, Janssen,Merck, ViiV

Antiretroviral Drug Approval: AZT ddI ddC d4T 3TC SQV RTV IDV NVP NFV DLV EFV ABC APV LPV/r TDF ENF ATV FTC FPV TPV DRV ETR RAL MVC RPV EVG DTG

Antiretroviral Drugs: 2014 nucleoside/tide RTIs (NRTIs) zidovudine (ZDV, AZT)zidovudine (ZDV, AZT) didanosine (ddI)didanosine (ddI) stavudine (d4T)stavudine (d4T) lamivudine (3TC)lamivudine (3TC) abacavir (ABC)abacavir (ABC) emtricitabine (FTC)emtricitabine (FTC) tenofovir (TDF)tenofovir (TDF)NNRTIs nevirapine (NVP)nevirapine (NVP) delavirdine (DLV)delavirdine (DLV) efavirenz (EFV)efavirenz (EFV) etravirine (ETR)etravirine (ETR) rilpivirine (RPV)rilpivirine (RPV) entry inhibitors (EIs) enfuvirtide (T-20, fusion inhibitor)enfuvirtide (T-20, fusion inhibitor) maraviroc (MVC, CCR5 antagonist)maraviroc (MVC, CCR5 antagonist) integrase inhibitors (IIs) raltegravir (RAL)raltegravir (RAL) elvitegravir (EVG)elvitegravir (EVG) dolutegravir (DTG)dolutegravir (DTG) protease inhibitors (PIs) saquinavir (SQV)saquinavir (SQV) ritonavir (RTV)ritonavir (RTV) indinavir (IDV)indinavir (IDV) nelfinavir (NFV)nelfinavir (NFV) lopinavir/r (LPV/r)lopinavir/r (LPV/r) atazanavir (ATV)atazanavir (ATV) fosamprenavir (FPV)fosamprenavir (FPV) tipranavir (TPV)tipranavir (TPV) darunavir (DRV)darunavir (DRV)

Antiretroviral Activity: HIV RNA change (log 10 c/mL) 1994: 2-NRTI Therapy 1997: HAART including 2-NRTI + PI 1987: AZT Monotherapy 24-week responses Fischl, NEJM, 1987Eron, NEJM, 1995 Hammer, NEJM, 1996 Gulick, NEJM, 1997 Cameron, Lancet, 1998

3-Drug Combination ART: AM4PM12 MID fasting (1 hour before/2 hours after meals) 1 liter of hydration/day AZT + 3TC + IDV

Approved Single Tablet Regimens TDF/FTC/EFV (2006) TDF/FTC/RPV (2011) TDF/FTC/EVG/cobi (2012) 7 years: Cassetti HIV Clin Trials :164 Cassetti IAS 2008, #TUPE weeks: Cohen AIDS 2013;27: weeks: Wilkin AIDS Res Hum Retro 2012;28: weeks: Rockstroh JAIDS 2013;62:483 Zolopa JAIDS 2013;63: weeks: Wohl ICAAC 2013, Clumeck EACS 2013, Kulkarni CROI 2014

Recommended Regimens – ALL VL NNRTI-basedNNRTI-based tenofovir/emtricitabine/efavirenztenofovir/emtricitabine/efavirenz PI-basedPI-based tenofovir/emtricitabine + atazanavir/rtenofovir/emtricitabine + atazanavir/r tenofovir/emtricitabine + darunavir/rtenofovir/emtricitabine + darunavir/r INSTI-basedINSTI-based tenofovir/emtricitabine + raltegravirtenofovir/emtricitabine + raltegravir tenofovir/emtricitabine/elvitegravir/cobitenofovir/emtricitabine/elvitegravir/cobi tenofovir/emtricitabine + dolutegravirtenofovir/emtricitabine + dolutegravir abacavir/lamivudine + dolutegravirabacavir/lamivudine + dolutegravir U.S. DHHS Guidelines 5/1/14

ART Responses in British Columbia Gill CID 2010;50:98 Year N = 87% 65%

Palella F, et al. NEJM, Use of protease inhibitors Deaths Deaths per 100 Person-Years Therapy with a Protease Inhibitor (% of patient-days)

Patient Substitutions de codon associées à une pharmacorésistance Mutations - INTIMutations - INNTIMutations - IP 1 41L; 67N; 69N; 70R; 74V; 184V; 215F; 219Q 100I; 103N 10I; 36I; 54V; 63P; 71V; 73S; 82V; 90M 2 41L; 184V; 215Y103N, 179E 48V; 63P; 71V; 73S; 77I; 82A; 90M 3 Aucune103N 10I; 54V; 63P; 71V; 82T; 84V; 90M 4 184V; 215Y103N77I 5 184V108I20R, 77I 6 41L; 67N; 210W; 215YAucune63P; 71V; 73S; 90M 7 41L; 215Y101EAucune 8 41L; 215Y101EAucune Brenner, B. et coll., AIDS, 18(12), le 20 août 2004, p. 1653–1660. INTI : Inhibiteurs nucléosidiques de la transcriptase inverse INNTI : Inhibiteurs non nucléosidiques de la transcriptase inverse IP : Inhibiteurs de la protéase Exemples de transmission de virus multirésistants dans les cas d’une primo-infection par le VIH-1

Cohen C, et al. 6th IAS 2011; Abstract TULBPE032 ECHO and THRIVE double-blind study designs 48 weeks primary analysis 96 weeks final analysis N=690 patients RPV 25mg qd + TDF/FTC + EFV placebo qd (N=346) EFV 600mg qd + TDF/FTC + RPV placebo qd (N=344) ECHO (TMC278-C209) 1:1 *Investigator’s choice: TDF/FTC; AZT/3TC; ABC/3TC N=678 patients RPV 25mg qd + 2 N(t)RTIs* + EFV placebo qd (N=340) EFV 600mg qd + 2 N(t)RTIs* + RPV placebo qd (N=338) THRIVE (TMC278-C215) 1:1 RPV was non-inferior to EFV in confirmed response (viral load <50 copies/mL, ITT-TLOVR) at Week 48 (primary objective) 1 RPV had a more favourable safety profile but higher virologic failure rate than EFV 1 RPV is approved in the US as a single-agent tablet 2 and a qd fixed-dose, single-tablet regimen with TDF/FTC is under development 3 1 Cohen CJ, et al. XVIIIth IAC Abstract THLBB206 2 FDA label for EDURANT TM (rilpivirine) tablets Mathias A, et al. XVIIIth IAC Abstract LBPE17 ITT = intent-to-treat; TLOVR = time-to-loss of virologic response

Rilpivirine RPV vs EFV

Cohen C, et al. 6th IAS 2011; Abstract TULBPE032 ≤100K Patients (%) RPV Responders Discontinued due to other reasons † VF eff Discontinued due to AE/death EFV 4.0 (–1.7, 9.7) § 8480 RPV N’=368 EFV N’=329 >100K Patients (%) 75 RPV N’=318 EFV N’= (-12, 1.5) § 70 Non responders Pooled ECHO and THRIVE: ITT-TLOVR outcome at Week 96 by baseline VL Responses by baseline CD4 cell count were (≥200 cells/mm 3 ): RPV 82% vs EFV 79%, (≥50–<200 cells/mm 3 ): RPV 71% vs EFV 75% and (<50 cells/mm 3 ): RPV 56% vs EFV 69%

Cohen C, et al. 6th IAS 2011; Abstract TULBPE032 Pooled ECHO and THRIVE: summary of resistance findings 1 Tambuyzer L et al. Antivir Ther 2009;14:103–9 2 Johnson VA et al. Top HIV Med 2009;17:138–45 * One VF res occurred after Week 96 in RPV group (E138K, K219E, M184I); † At least one emergent NNRTI RAM (from the NNRTI RAM list) 1 or IAS-USA N(t)RTI 2 RAM RPV N=686 EFV N=682 Time of failure All* Up to Week 48 Week 48to 96* All Up to Week 48 Week 48 to 96 VF res with resistance data, n No emergent NNRTI 1 and N(t)RTI RAMs 2, n (%) 35 (41)24 (36)11 (61)19 (45)11 (39)8 (57) Any emergent NNRTI 1 or/and N(t)RTI RAMs 2, n (%) 51 (59)43 (64)7 (39)23 (55)17 (61)6 (43) Any emergent † NNRTI RAMs 2, n (%) 46 (53)39 (58)6 (33)20 (48)16 (57)4 (29) Most frequent NNRTI RAM, n (%) E138K 31 (36) E138K 27 (40) E138K 3 (17) K103N 14 (33) K103N 11 (39) K103N 3 (21) Any emergent † N(t)RTI RAMs 1, n (%) 48 (56)41 (61)6(33)11 (26)9 (32)2 (14) Most frequent N(t)RTI RAM, n (%) M184I 32 (37) M184I 27 (40) M184I 4 (22) M184V 6 (14) M184V 6 (21) M184I 2 (14)

Compensatory effect of E138K on the replication capacity (RC) of M184I/V. The RC of both E138K and M184I are each decreased by 3-fold compared to wild-type and decreased by 2-fold for M184V. There is no difference in RC of double mutants E138K/M184I or E138K/M184Vc compared to wild-type.

Transmitted NNRTI resistance rising in the post-HAART era

What is the proper definition of: Genetic barrier for resistance? a. Number of mutations needed for a minimal level of resistance b. Number of mutations needed for resistance > 10-fold c. Time to development of clinically relevant resistance d. Duration of exposure to drug in order for resistance to occur e. Number of viral replicative cycles required to overcome inhibition by a drug. f. All of the above

Number of mutations needed for a minimal level of resistance or resistance > 10-fold Class Drug Mutation Fold-change NNRTI Nevirapine Y181C >100 Efavirenz K103N >100 Efavirenz K103N >100 N(t)RTI 3TC M184V >100 Tenofovir K65R Tenofovir K65R ≈ 3-fold Abacavir L74V 3-5 fold PI Darunavir >10 mutations 5-20 fold Lopinavir >8 mutations 5-20 fold

Time to development of clinically relevant resistance 1. Single dose NVP. Weeks? 2. Triple therapy. Only in cases of adherence? 3. With PIs, resistance based on mutations in PR is rare, even in cases of viral load rebound. What about cleavage site mutations in gag? Monitoring of cleavage site mutations is uncommon. 4. Oftentimes, compensatory mutations serve to increase levels of resistance while simultaneously restoring viral fitness that may have been impaired by a primary mutation, e.g. (a) zidovudine, the various TAM pathways; (b) the 148/140 mutational pathway for raltegravir and elvitegravir.

5. Does the concept of genetic barrier apply to transmitted resistance? E.g. K103N, TAMs, M184V (-the latter is commonly deselected after transmission).

The new era of integrase inhibitors: potent and well- tolerated drugs

14th European AIDS Conference; October 16-19, 2013; Brussels, Belgium FLAMINGO (ING114915) Study Design Clotet et al. EACS 2013; Brussels, Belgium. Abstract LBPS4/6. Primary endpoint: proportion with HIV-1 RNA <50 c/mL at Week 48, FDA Snapshot analysis, -12% non-inferiority (NI) margin Secondary endpoints: antiviral activity, safety, tolerability, health outcomes and viral resistance HIV+ ART-naive VL ≥1,000 c/mL Stratified by screening plasma HIV-1 RNA (≤ vs >100,000 c/mL) and background dual NRTI (ABC/3TC or TDF/FTC*) Week 96 analysis Randomization Week 48 analysis DRV/r 800 mg/100 mg QD + 2 NRTIs DTG + ART Open-label randomized phase DTG 50 mg QD + 2 NRTIs DTG 50 mg QD + 2 NRTIs Extension phase *Investigator selected backbone of choice

14th European AIDS Conference; October 16-19, 2013; Brussels, Belgium Results were confirmed in per protocol analysis: 91% DTG versus 84% DRV/r, ∆ (CI): 7.4 ( ) Proportion (95% CI) of Individuals With HIV-1 RNA <50 c/mL Over Time – Snapshot Clotet et al. EACS 2013; Brussels, Belgium. Abstract LBPS4/6. DTG: 90% DRV/r: 83% Week BL Proportion (%) 95% CI for difference a Favours DRV/r Favours DTG -20%020% % Test for superiority: P=0.025

Resistance to INSTIs in clinical trials in treatment-naïve patients Treatment Major resistance mutations detected by genotyping in treatment-naïve patients failing therapy Minor resistance mutations Raltegravir Y143 N155H Q148 Multiple Elvitegravir T66I E92Q N155H Q148 Multiple DolutegravirNONE RALTEGRAVIR Cooper et al., NEJM, 2008 Sichtig et al, JAC, 2009 Canducci et al, AIDS, 2009 Hatano et al, JAIDS, 2010 ELVITEGRAVIR Sax et al, Lancet, 2012 DeJesus et al, Lancet, 2012 DOLUTEGRAVIR vanLunzen et al., Lancet Infect. Dis., 2012

Major resistance pathways against INSTIs (clinical and tissue culture data) Resistance pathways Fold resistance RALEVGDTG Y143 pathway Y143C<10<2 Y143R<50<2 T97A/Y143C>100<2 T97A/Y143R>100<2 L74M/T97A/Y143G<50ND<2 L74M/T97A/E138A/Y143C<20ND<2 N155 pathway N155H<50 <2 E92Q/N155H<100>100<10 L74M/N155H<50 <2 Q148 pathway Q148H<20<10<2 Q148K<100 <2 Q148R<50<100<2 E138K/Q148H<10<20<2 E138K/Q148K>100 <20 E138K/Q148R>100 <10 G140S/Q148H>100 <20 G140S/Q148K<10<100<2 G140S/Q148R>100 <10 E138A/G140S/Y143H/Q148H>100ND<50 Quashie et al., Curr. Opin. Infect. Diseases, in press

SAILING CROI A study in which Dolutegravir was shown to be superior to Raltegravir in treatment-experienced integrase inhibitor- naïve subjects. The R263K mutation was present in two individuals who either rebounded or did not achieve virologic suppression to <50 c/ml.

Disclaimer The following hypothesis has been formulated by our research group and is not necessarily subscribed to by either ViiV Healthcare, Inc. or GlaxoSmithKline, Inc.

Subtype-specific mutations selected in vitro with dolutegravir HIV-1 subtype Most common mutations selected with dolutegravir BR263K, H51Y CG118R, H51Y Quashie, Mesplède et al., Journal of Virology, 2012

The R263K mutation confers low-level resistance to dolutegravir in cell culture GenotypeIC 50 fold change* R263K2.5 to 6 *Methodological differences (EC 50 for wild-type ≈1-6nM) Quashie, Mesplède et al., Journal of Virology, 2012

CONFIDENTIAL – NOT FOR DISSEMINATION INI DISSOCIATION FROM WT IN-DNA COMPLEX AT 37°C Adapted from Hightower KE, et al. Antimicrob Agents Chemother 2011;5:4552–9 INIk off (s -1 )t 1/2 (h) DTG 2.7 x RAL 22 x EVG 71 x K off, dissociation rate; t 1/2h, half-life in hours DTG dissociated more slowly from a WT IN-DNA complex at 37°C compared with RAL and EVG DTG dissociation was eight times longer than RAL and 26 times slower than EVG DTG dissociated more slowly from a WT IN-DNA complex at 37°C compared with RAL and EVG DTG dissociation was eight times longer than RAL and 26 times slower than EVG DTG RAL EVG Relative binding Time (h)

The R263K mutation decreases integrase activity in cell-free assays Quashie, Mesplède et al., Journal of Virology, 2012

The addition of H51Y to R263K further decreases IN strand transfer activity A B

The addition of H51Y to R263K further decreases HIV-1 infectivity

Virus Weeks 8-15 WTM184V H51YM184I R263KM184I H51Y/R263KNone G118RNone H51Y/G118RNone

Effect of the H51Y and R263K mutations on integrase structure. Effect of residues at position 51 and 263 on local side-chain electrostatic interactions and side-chain mobility of: A. IN wt (turquoise backbone); B. IN H51Y (purple backbone); C. IN R263K (salmon backbone) and D. IN H51Y/R263K (Dark green backbone). Mesplède et al. Retrovirology 2013 R263K and H51Y/R263K affect integrase structure

No compensatory mutations in regard to DTG resistance and viral fitness have developed over more than four years in culture.

Selection of HIV-1 containing R263K with RAL and EVG Time (weeks) Initial MutationAcquired Mutations EVG30R263KM50I, T66I RAL30R263K --- RAL38 ---T97A/T, Y143H/Y, Q148K/Q RAL41 ---T97A, Y143R

Replication Capacity of HIV Containing Various Combinations of INSTI Resistance Mutations Mutation(s)% fitness E92Q≈ 75% Y143≈ 72% Q148≈ 75% N155≈ 75% R263K≈ 70% G140/Q148≈ 95% R263K/H51Y≈ 25% R263K/E138K≈ 25% R263K/Q148R<5% R263K/Y143C<5% R263K/E92Q<5%

Clinical resistance has occurred in regard to every HIV drug developed until now and therefore must continue to occur for all future medications as well.

Approaches Being Used to Purge HIV Reservoirs 1. Histone deacetylase (HDAC) inhibitors 2. Farnesyl transferase inhibitors 3. Antibody and immunological approaches 4. Others

Administration of vorinostat disrupts HIV-1 latency in patients on antiretroviral therapy NM Archin, AL Liberty, AD Kashuba, SK Choudhary, JD Kuruc, AM Crooks, DC Parker, EM Anderson, MF Kearney, MC Strain, DD Richman, MG Hudgens, RJ Bosch, JM Coffin, JJ Eron, DJ Hazuda, DM Margolis Nature 487, (26 July 2012) doi: /nature11286

Rapid seeding of the viral reservoir prior to SIV viraemia in rhesus monkeys JB Whitney, AL Hill, S Sanisetty, P Penaloza- MacMaster, J Liu, M Setty, L Parenteau, C Cabral, J Shields, S Blackmore, JY Smith, AL Brinkman, LE Peter, SI Mathew, KM Smith, EN Borducchi, DIS Rosenbloom, MG Lewis, J Hattersley, B Li, J Hesselgesser, R Geleziunas, ML Robb, JH Kim, NL Michael, DH Barouch Nature 512, (7 August 2014) doi: /nature13594

At the very least, a drug that does not easily select for drug resistance should be intolerant of ongoing HIV transmission so long as patients remain adherent to therapy.

Thank you to our funding agencies

Bluma Brenner Hongtao Xu Dimitri Coutsinos Jerry Zaharatos Maureen Oliveira Thibault Mesplède Peter Quashie Kaitlin Anstett Vincent Cutillas Said Hassounah Acknowledgements

Thank you for slides Roy Gulick Bluma Brenner Paul Volberding

Thank you Merci Gracias