Profili di resistenza e barriera genetica dei nuovi trattamenti antiretrovirali Carlo Federico Perno Catania 10 Novembre 2015

Evolution of ARV drugs: Moving towards better and safer options Rilpivirine TDF/FTC/RPV (2011)

INSTI class showed the shortest median time of achieving virological suppression At 6 month of treatment around 80% of patients were suppressed N=8351 Time to viral suppression according to regimen class Time to viral suppression according to baseline VL Time to viral suppression (month) VL < 100,000 VL ≥ 100,000 N at risk VL<100,000 VL ≥ 100,000 Percent survival Log rank test < INSTI NNRTI PI PI INSTI N at risk NNRTI Time to viral suppression (month) Percent survival Log rank test < Khatchatourian et al EACS 2015

Santoro et al., Antivir ther 2013

 990 (13.0%) patients experienced virologic rebound ( CV > 50 copies/ml X2) N=7592 Median [IQR] follow-up time from cART initiation : 41.2 [23.8,61.5] months VL < 10,000 10, ,000 N at risk , ,000 VL > 500, Time to viral rebound (month) 100,000 ≤ VL < 500,000 VL ≥ 500,000 VL < 10,000 10,000 ≤ VL < 100, Time to virologic rebound according to baseline viral load Log rank test < Khatchatourian et al EACS 2015

Patients having pre-HAART viremia >500K copies/mL showed the highest probability of virological rebound (VL>50 copies/mL) by 4 years from the achievement of virological suppression Time (years from first date of VL<50 cps/mL) No. at risk Rebound as the first of 2 consecutive HIV-RNA >50 copies/mL Rebound as the first of 2 consecutive HIV-RNA >200 copies/mL Time (years from first date of VL<50 cps/mL) No. at risk 0-100K K K >500K Probability to reach virological rebound P<0.001P=0.716 Viremia Ranks 29% 12% Armenia et al., V ICAR 2013

Risk Factor: MSM Clinical Case: ID Patient infected with HIV-1 B subtype Age: 35 Sex: M 1 st Seropositivity: September 2010 On September 2010 Genotypic Resistance Test On September 2010 Genotypic Resistance Test (Viremia : 64,698 cps/ml; CD4: 501 cells/μl) (Viremia : 64,698 cps/ml; CD4: 501 cells/μl) Resistance mutations PR: M36I L63E RT: NONE GP-120(V3): T22A E25D Tropism: dual/mixed R5 (FPR*: 52.1%) Other mutations PR: T12A K14R E35D R41K D60E Q61E I62V I64V RT: K43Q K173R D177E R211K V245M A272P V276I I293V P294PT E297A Q334E *Geno2Pheno algorithm

Risk Factor: MSM Clinical Case: ID Patient infected with HIV-1 B subtype Age: 35 Sex: M 1 st Seropositivity: September 2010 Jan-13 Mar-13 May-13 Aug-13 Mar-14 Nov-14 Jan Viremia (log copies/ml) CD4 cell count (cells/ul) Undetectability threshold > cps/ml Sept 10- Jan 13 HIV drug naive On January 2013 VL: 43,366 cps/ml CD4: 432 cells/µl Jan - Aug 13 TDF/FTC/EFV (Atripla) Sept 13 - Jan 15 TDF/FTC/RPV (Eviplera) in out Other PR-RT mutations PR: T12A K14R E35D R41K D60E Q61E I62V I64V RT: D123N I135T K173R D177E I202V Q207H R211K V245M D250E A272P I293V E297A L303MR I309IM GRT January 2015 Plasma VL: 94 cps/ml CD4: 944 cells/ul PR: M36V L63E V77I RT: V90I E138K M184I K219R GP-120(V3): T22A, G24del, E25D Tropism: R5 (FPR*: 38.0%) *Geno2Pheno algorithm

Cumulative Genotypic Resistance to NRTIs and NNRTIs is Associated with a Higher Risk to Loose Virological Suppression in HIV-Positive Patients Switching to Tenofovir/Emtricitabine/Rilpivirine Single-Tablet Regimen D. Di Carlo 1, D. Armenia 1, A. Calcagno 2, F. Forbici 3, A. Bertoli 1, G. Berno 3, G. D’Offizi 3, E. Nicastri 3, A. Ammassari 3, R. Libertone 3, M. Zaccarelli 3, V. Ghisetti 2, M. Andreoni 4, F. Ceccherini-Silberstein 1, S. Bonora 2, G. Di Perri 2, A. Antinori 3, CF. Perno 3, MM. Santoro 1. 1 University of Rome Tor Vergata, Rome, Italy; 2 University of Turin, Department of Infectious Diseases, Turin, Italy; 3 L Spallanzani Hospital, Rome, Italy; 4 University Hospital Tor Vergata, Rome, Italy. 13 TH EUROPEAN HIV & HEPATITIS MEETING 2015 PE9/6

%6% 5%5% p=0.001 Time (Months) No. at risk Probability of experiencing virological rebound Virological rebound: the first of 2 consecutive viremia values >50 copies/mL, after TDF/FTC/RPV starting % Cumulative resistance No RTI res a NRTI/NNRTINRTI+NNRTI Patients followed until the last viremia measurement available under TDF/FTC/RPV. Median (IQR) viral load at virological rebound: 2.5 ( ); 4.1 ( ); 3.1 ( ). a Patients without RTI resistance, regardless the presence of PI resistance. Patients with cumulative NRTI+NNRTI resistance had a higher probability of experiencing virological rebound compared to those harboring NRTI or NNRTI resistance and to those without RTI-resistance.

…the risk of virologic rebound was higher in patients that achieved suppression after 6 months of treatment  990 (13.0%) patients experienced virologic rebound ( CV > 50 copies/ml X2) N=7592 Median [IQR] follow-up time from cART initiation : 41.2 [23.8,61.5] months VL < 10,000 10, ,000 N at risk , ,000 VL > 500, VS < 3 months VS 3-6 months N at risk VS > 6 months Time to viral rebound (month) 100,000 ≤ VL < 500,000 VL ≥ 500,000 VL < 10,000 10,000 ≤ VL < 100, Time to viral rebound (month) VS 3-6 months VS > 6 months VS ≤ 3 months Time to virologic rebound according to baseline viral load Time to virologic rebound according to time to viral suppression Log rank test < Khatchatourian et al EACS 2015

Di Biagio et al., J Med Virol 2014 Independent predictors of virological success at first line regimen are a higher baseline viral load, a weighted genotypic susceptibility score (wGSS) <3, male sex and the type of initial third drug employed (integrase inhibitor vs. boosted protease inhibitors).

DHHS, April INSTIs drug class has strengthened combined ART (cART)

Resistance profile and genetic barrier: RALTEGRAVIR

Evaluation of virological response and resistance profile in patients starting a first line HAART containing raltegravir in clinical practice Armenia et al, 1 st HIV forum:Integrase inhibitors

, Probability of achieving HIV-RNA <50 copies/mL % 88% 94% P<0.001 Viremia ranges (copies/mL) Time (Months) No. at risk By 12 months of treatment, in 103 drug-naïve patients the median time of achieving virological success significantly increased by increasing baseline viremia. A slightly lower probability of achieving virological success was observed in drug-naïve patients with baseline viremia >500,000 copies/mL Median time (95% CI) of achieving VS (months) (1.1 – 2.6) (2.3 – 3.7) (3.2 – 11.2) Armenia et al, 1 st HIV forum:Integrase inhibitors

ID Virological outcome Treatment Baseline HIV-RNA (copies/ml) Subtype Transmitted RAMs Days under RAL at failure HIV-RNA at failure (copies/ml) RAMs at failure NNRTINRTIPIINSTI 4555 No suppression RAL; FTC, TDF; ATV/r25,060B No suppression RAL; FTC, TDF297,262B M184V- T97A, Y143R, G163K Tardive suppression RAL; DRV/r634,929CRF01_AE M41L Tardive suppression RAL; FTC, TDF; DRV/r 829,983B ReboundRAL; DRV/r50,034CRF06_cpx Tardive suppression RAL; DRV/r375,990BM41L T97A No suppression RAL; DRV/r1,421,036CA62V N155H No suppression RAL; DRV/r; ABC800,100CRF02_AGND N155H No suppression RAL; 3TC; LPV/r5,289FND158395T97A Boldface indicates RAL primary RAMs. Among 9 drug-naïve patients with an available GRT at RAL failure 5/9 patients showed integrase resistance mutations. 1/9 showed FTC resistance Armenia et al, 1 st HIV forum:Integrase inhibitors

SusceptiblePotential resistanceResistance All drug-naïve patients failing raltegravir showed viruses fully susceptible to dolutegravir Drug-Naive (N=9) Genotipic susceptibility for INSTI at RAL failure Armenia et al, 1 st HIV forum:Integrase inhibitors

Resistance profile and genetic barrier: ELVITEGRAVIR

All elvitegravir failing patients with primary genotypic resistance-associated mutations to INSTI showed also the M184I/V mutation. Antiviral Therapy 2014

Kyto et al EACS 2015

Impact of baseline NRTI resistance mutations on virological suppression of HIV- 1 infected patients treated with elvitegravir/cobicistat/emtricitabine/tenofovir (E/C/F/T) Hall N, Allavena C, André-Garnier E, Morineau-Le Houssine P, Pineau S, Bouchez S, Bernaud C, Vivrel F, Boutoille D, Rodallec A, Jovelin T and Raffi F

Virological failures under E/C/F/T treatment Hall et al EACS 2015

The presence of pre-existing NRTI-RAM seems to negatively impact efficacy of E/C/F/T The rate of VL≥50c/ml at M6 in cART-experienced patients was 24% if NRTI- RAM were pre-existent vs. 5% in absence of NRTI-RAM (p=0.051) In case of virologic failure, major INSTI-RAM were detected in 3/3 patients (100%) E/C/F/T should be used with caution in patients with history of NRTI-RAM on RNA or DNA resistance testing, even in case of virologic suppression and in patients with history of failure on a suboptimal regimen including thymidinic NRTIs. Hall et al EACS 2015

%6% 5%5% p=0.001 Time (Months) No. at risk Probability of experiencing virological rebound Virological rebound: the first of 2 consecutive viremia values >50 copies/mL, after TDF/FTC/RPV starting % Cumulative resistance No RTI res a NRTI/NNRTINRTI+NNRTI Patients followed until the last viremia measurement available under TDF/FTC/RPV. Median (IQR) viral load at virological rebound: 2.5 ( ); 4.1 ( ); 3.1 ( ). a Patients without RTI resistance, regardless the presence of PI resistance. Patients with cumulative NRTI+NNRTI resistance had a higher probability of experiencing virological rebound compared to those harboring NRTI or NNRTI resistance and to those without RTI-resistance.

Resistance profile and genetic barrier: Dolutegravir

PROTOCOL-DEFINED VIROLOGIC FAILURE: TREATMENT-EMERGENT RESISTANCE TO WEEK 96 n (%)DTG 50 mg QD (N=411) RAL 400 mg BID (N=411) To Week 48Weeks 48–96To Week 48Weeks 48–96 Subjects with PDVF 20 (5)2 (<1)28 (7)1 (<1) Integrase genotypic results at baseline and time of PDVF 8 (40)2 (100)18 (64)1 (100) INI-resistant mutations 001 (6)0 Protease/reverse transcriptase genotypic results at baseline and time of PDVF 12 (60)2 (100)19 (68)1 (100) NRTI-resistant mutations 004 (21)*0 Adapted from Raffi F, et al. Lancet 2013;381:735–43 PDVF, protocol defined virologic failure (confirmed HIV-1 RNA ≥50 c/mL on or after Week 24) *One participant had INI-resistance mutations T97T/A, E138E/D, V151V/I, and N155H, and NRTI-resistance mutations A62A/V, K65K/R, K70K/E, and M184V; one participant had NRTI-resistance mutation M184M/I; one participant had NRTI-resistance mutation A62A/V; and one participant had NRTI-resistance mutation M184M/V Amongst DTG-treated subjects, no INI- nor NRTI-resistant mutations were detected through Week 96

DTG 50 mg +ABC/3TC QD (N=414) EFV/TDF/FTC QD (N=419) Week 0–48 1 Week 0–96 2 Week 0–144 4 Week 0–48 1 Week 0–96 2 Week 0–144 4 Subjects with PDVF, n (%) 18 (4)25 (6)39 (9)17 (4)25 (6)33 (8) PDVF genotypic population1122–916– Reverse transcriptase genotypic results at BL and PDVF 917–912– NRTI TE major mutations000 1 (K65R) 1 NNRTI TE major mutations000 4 (K101E, K103N, G190A)* 6 (K101E, K103N, G190A) ‡ Integrase genotypic results at BL and PDVF713–710– Integrase-resistant TE major substitution 0†0† 0†0† 0000 PROTOCOL-DEFINED VIROLOGIC FAILURE AND TREATMENT-EMERGENT RESISTANCE AT WEEK 48 AND WEEK 96 Amongst subjects receiving DTG + ABC/3TC, no integrase nor NRTI mutations were detected through Week 96

In other words…… - No mutations conferring resistance to dolutegravir appear at virological failure in integrase inibitors-naïve patients - No mutations conferring resistance to NRTI appear at virological failure in patients treated with regimens containing dolutegravir as first integrase inhibitor

Undetectability threshold > cps/ml Feb-12 Apr-12 May-12 Jun-12 Jul-12 Sep-12 Dec-12 Mar-13 Jul-13 Nov-13 Mar CD4 cell count (cells/ul) Oct Viremia (log copies/ml) in out Clinical Case 1 - Patient infected with HIV-1 B subtype Age 37 Sex M Risk Factor MSM CDC A First Seropositivity: Feb-12 (acute infection) Patient NAIVE From May 2012 to October 2015: DTG TDF/FTC Mar-15 Oct-15 GRT Feb-2012 VL: 93,896 cps/ml CD4: 421 cells/ul PR: A71T RT: None IN: None GP-120(V3): T2I R18S T22A E25Q Tropism*: Prevalence of a R5 tropic virus (FPR*: 43.5%) * Geno2pheno algorithm Other PR-RT mutations PR: I13V N37D L63P V77I I93L RT: I135T A272AV T286A I293V E297A IN: A23V S24G K111R T112A T125A V165I

CLINICAL CASE 2 Age: 55 years old Sex: Male HIV diagnosis: October 1992 HIV-1 subtype: B Tropism: X4 The patient started therapy on June 1990 Drugs administered from October 1992 to July 2013 NRTIs: ABC, AZT, DDI, D4T, TDF, 3TC NNRTIs: ETR PIs: DRV/r, FPV/r, LPV/r, RTV, SQV/r INI: RAL FIs: None CCR5-EI: None He had never achieved virological suppression CF Perno, personal communication

August 2013: viremia: 576,176 copies/mL; CD4: 123 cells/  l Genotypic resistance test In August 2013 the patient started DTG, 3TC, DRV/r (600/100) CASE 2 CF Perno, personal communication Resistance mutationsOther mutations Protease L10V Q58E A71V L90M I93LT12P L19I E34D I62VI L63C RT D67G T69D K70R K101E Y181CF G190S T215F K219Q K20R V60I K66R A98S I135T D177E V179S Q207E R211K F214L H221YH L228R Integrase N155HK14R S24G L28I S39CS L101I S119R T124AT I135V V201IV K211R

Clinical case 2 Aug-13 Sep-13 Nov-13 Dec-13 Jan-14 Mar-14 Jul-14 Dec-14 Apr-15 Jul-15 Undetectability threshold > cps/ml Viremia (log copies/ml) CD4 cell count (cells/ul) in out From August 2013 to July 2015: DTG 3TC DRV/r (600/100) GRT Aug-2013 (from plasma) VL: 576,176 cps/ml CD4: 123 cells/ul PR: L10V Q58E A71V L90M I93L RT: D67G T69D K70R K101E Y181CF G190S T215F K219Q IN: N155H Other PR-RT mutations PR: T12P L19I E34D I62VI L63C RT: K20R V60I K66R A98S I135T D177E V179S Q207E R211K F214L H221YH L228R IN: K14R S24G L28I S39CS L101I S119R T124AT I135V V201IV K211R CF Perno, personal communication

WHY DOES DOLUTEGRAVIR PREVENT THE APPEARANCE OF MUTATIONS NOT ONLY IN THE INTEGRASE, BUT ALSO IN REVERSE TRANSCRIPTASE AGAINST NRTI?

If a single mutation is sufficient to confer high level of resistance Drug has LOW genetic barrier HIGH likelihood of clinically meaningful resistance Genetic barrier: The type and the number of mutations required by HIV to develop a fully resistant virus. CF Perno, personal communication If a substantial number of mutations is required to confer high level of resistance Drug has HIGH genetic barrier LOW likelihood of clinically meaningful resistance

What about dolutegravir genetic barrier in RAL-failing patients?

Dolutegravir monotherapy

Dolutegravir monotherapy in patients with suppressed HIV viremia. C.Katlama 1,2, C.Soulié 2,3, C.Blanc 1, A.Denis 1,F.Caby 1,2,L.Schneider 1,2, MA.Valantin 1,2,R.Tubiana 1,2,M.Kirstetter 1, E.Valdenassi 1,G.Peytavin 4,V.Calvez 2,3, AG.Marcelin 2,3 1 Department of infectious diseases, 2 UMR S 1136, INSERM et Sorbonne Universités, UPMC Paris 06 3 Department of Virology Hospital de la Pitié-Salpêtrière, 4 Pharmacology Department Bichat hospital, Paris.

Study Design MonoDTG HIV RNA <50 cp/ml on ART Switch to mono DTG 50 mg QD May 14 to Jan patients enrolled W12 n=28 W12 n=28 W24 n=27 W24 n=27 28 patients Age : 48 y ART duration :17 years [11-20] Viral suppression : 6.6 years [ ] CD4 T cells: 624 /mm 3 [ ] CD4 T cells nadir:215 /mm 3 [ ] DNA :195 cp /10 6 PBMC [94-641] Baseline ART Triple drug ART 10 (36%) Dual ART 9 (32%) Mono ART (DRV/r) 9 (32%) INI containing regimen 7 (25%) Katlama C et al. EACS 2015, Oral PS4/4 1 mono DTG stop

Virological failure 1 Pt 1 PH : Female 35y CD4 nadir (/mm 3 ): 262 ART duration: 7 years Viral suppression duration : 6.7 months Prior INI : ETV+ RAL 8 month stop for pregnancy No blip Baseline CD4 nadir (/mm 3 ): 525 ART TDF/FTC/DRV/r DNA (cp10 6 PBMC): 310 HIV CRF06_cpx recombinant Genotype DNA Day 0 RT : wild type Protease : 20I, 36I, 69K, 89M MonoDTG Resistance INI DNA No RNA No DNA No RNA E138K / G140A, Q148R R DTG EVG RAL Katlama C et al. EACS 2015, Oral PS4/4 mutations E138K, G140A, Q148R

Virological failure 2 Pt 2 BP: Male 56 y CD4 nadir (/mm 3 ): 60 ART duration: 18 years Viral suppression duration : 17 months Baseline CD4 nadir (/mm 3 ): 1108 ART : TDF/FTC/EVG no blip DNA (cp/10 6 PBMC): 1459 HIV B subtype Genotype DNA Day 0: RT wild type; Protease 69Q, 77I Genotype RNA week 13: RT wild type ; Protease 69Q, 77I L74* polymorphism found in 12% naive MonoDTG INI Resistance Katlama C et al. EACS 2015, Oral PS4/4 DNA L74I* mutations L74 I, E92Q RNA L74I, E92Q R EVG RAL

Virological Failure 3 Pt 3 KJ: Male 57y CD4 nadir (/mm 3 ): 233 ART duration: 6 years Viral suppression duration : 5.8 years Baseline CD4 /(mm 3 ): 940 ART TDF/FTC/RAL (Blip 37cp) DNA (cp/10 6 PBMC): 174 HIV subtype B Genotype RNA week 24: wild type RT; 15VProtease MonoDTG INI ResistanceDNA None --- Katlama C et al. EACS 2015, Oral PS4/4 Resistance 155H RNA 155H R EVG RAL

Dolutegravir Monotherapy in HIV-infected Patients with Sustained Viral Suppression: A 24-week Pilot Study 15 th EUROPEAN AIDS CONFERENCE Barcelona, 22 October 2015 Jhon Rojas 1, Jose L Blanco 1, Montserrat Lonca 1, Berta Torres 1, Marta Parera 1, Ana Gonzalez 1, Josep Mallolas 1, Amparo Tricas 1, Laura Moreno 1, Federico Garcia 2, Jose M Gatell 1, and Esteban Martinez 1. 1 Hospital Clínic, University of Barcelona, Barcelona, Spain; and 2 Hospital Universitario San Cecilio, Granada, Spain.

Baseline characteristics (N=37) Age 56 (50-62) Women 18 (55%) Years since HIV diagnosis 19 (17-23) AIDS-defining events 13 (39%) Years with VL below detection threshold 8 (4-13) CD4 cells (/mm 3 ) 596 ( ) CD8 cells (/mm 3 ) 990 ( ) CD4/CD8 ratio 0.64 ( ) Data are median (interquartile range) unless othrwise expressed Rojas et al EACS 2015

ART discontinued and replaced by DTG monotherapy PI/r-based 22 (67%) DRV/r (n=17) LPV/r (n=1) NNRTI-based9 (27%) NVP+TDF/FTC (n=3) NVP+ABC/3TC (n=1) RPV/TDF/FTC(n=1) INSTI-based2 (6%) RAL+NVP (n=1) EVG/cTDF/FTC (n=1) ATV/r+MVC (n=2) DRV/r+MVC (n=1) ABC/3TC+ATV (n=1) EFV/TDF/FTC (n=1), EFV+ABC/3TC (n=1) ETV+TDF/FTC (n=1) ETV+ABC/3TC (n=1) Rojas et al EACS 2015

Virological response At 24 weeks, the therapeutic efficacy of dolutegravir monotherapy was 97% (95% CI 83%-100%) (ITT non-completer=failure) One patient had low-level virological failure after 4 weeks (88 copies/mL, and confirmed as 155 copies/mL). –52 yrs man, prior multi-drug abuse, HIV-infected for 12 years, 10 ART regimens –Previous RT- and PR- GRTs had shown the following mutations: 62V,106I, 118I, 15V, and 63P. Prior VF to a RAL-containing regimen without INSTI-GRT by the time of this VF. –Abstinent of drug abuse for the last 2 years, he maintained viral suppression with DRV/r monotherapy. –He was also receiving psychotropic (palinperidone, trazodone, and lorazepam), antihypertensive (losartan), and lipid-lowering (pitavastatin) therapy. Rojas et al EACS 2015

Patient with virological failure (cont.) After detecting virological failure at week 4, this patients was recommended to increase dolutegravir dose to 50 mg BID, but he continued 50mg QD. Plasma HIV-1 RNA remained detectable at 24 weeks (79 copies/mL, and confirmed as 101 copies/mL). HIV RNA genotypic resistance tests at 4 and 24 weeks detected no integrase mutations. HIV DNA genotypic resistance tests detected no integrase mutations at 4 weeks, but 118R was detected in 7% of the integrated-DNA in PMBC at 24 weeks Rojas et al EACS 2015

CONCLUSIONS The integrase inhibitors represent the newest and most promising class in antiretroviral therapy The majority of patients can take advantage of therapy based on integrase inhibitors, provided that the patients are properly studied, and adequate history of previous treatments is considered before taking decisions. Resistance is still an issue in our patients, though in a mode different than in the past. Preserving future therapeutic options requires choosing the right regimen for each single patients, based upon his/her characteristics, and that of the virus that we are fighting