Pr Vincent Calvez. Faculté de Médecine Sorbonne Université UMR Sorbonne Université Inserm 1136 Service de Virologie Hôpital Pitié-Salpêtrière

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2 Pr Vincent Calvez Faculté de Médecine Sorbonne Université UMR Sorbonne Université Inserm 1136 Service de Virologie Hôpital Pitié-Salpêtrière

3 Pretreatment HIV Drug Resistance in the START Study Using Next Generation Sequencing Between , START enrolled 4,684 ART-naïve individuals in 35 countries Baseline NGS data at study entry was available for 3,365 participants; median age was 36 years, 23% female, median CD4 count 642 cells/μl, median HIV RNA 19,800 copies/ml, and median time since diagnosis was 0.9 years. 41.8% of specimens were collected between and 58.2% between HIV subtype was determined for 3076 samples, with 61% subtype B and 39% non-b. John D. Baxter et al, CROI 2018

4 Pretreatment HIV Drug Resistance in the START Study Using Next Generation Sequencing John D. Baxter et al, CROI 2018

5 Prevalence and Clinical Impact of Minority Resistant Variants to Integrase Inhibitors Poster 545 T Nguyen 1, DB Fofana 2, MP Lê 3, C Charpentier 4, G Peytavin 3, M Wirden 1, S Lambert-Niclot 2, N Desire 1, M Grude 5, L Morand-Joubert 2, P Flandre 5, C Katlama 6, D Descamps 4, V Calvez 1, E Todesco 1, AG Marcelin 1 1 Sorbonne Université, INSERM, Institut Pierre Louis d Epidémiologie et de Santé Publique (iplesp), AP-HP, Hôpital Pitié-Salpêtrière, Laboratoire de virologie, F Paris, France; 2 Sorbonne Université, INSERM, Institut Pierre Louis d Epidémiologie et de Santé Publique (iplesp), AP-HP, Hôpital Saint Antoine, Laboratoire de virologie, F Paris, France; 3 IAME, UMR 1137, INSERM, Université Paris Diderot, Sorbonne Paris Cité, AP-HP, Département de Pharmaco-Toxicologie, Hôpital Bichat-Claude Bernard, Paris, France; 4 IAME, UMR 1137, INSERM, Université Paris Diderot, Sorbonne Paris Cité, AP-HP, Laboratoire de Virologie, Hôpital Bichat-Claude Bernard, Paris, France ; 5 Sorbonne Université, INSERM, Institut Pierre Louis d Epidémiologie et de Santé Publique (iplesp), F Paris, France; 6 Sorbonne Université, INSERM, Institut Pierre Louis d Epidémiologie et de Santé Publique (iplesp), AP-HP, Hôpital Pitié-Salpêtrière, Service de maladies infectieuses, F Paris, France. TRANSPARENCY DECLARATIONS: None to declare

6 Impact of MiRVs? 3) MiRVs at baseline in patients with VF (n=34) and in those with VS (n=31) No difference in prevalence of baseline MiRVs in patients with VF (14.7%) and VS (12.9%) 34 patients with VF: 17 carried MaRVs at failure but not previously found at baseline. MiRVs at baseline in VF patients: not emerged at failure. Patients Treatment at Last viral load at Viral load MiRVs at MaRVs at INSTI C trough at failure failure baseline (cp/ml) at failure (cp/ml) baseline failure (ng/ml)-interpretation VF ETR/RAL BID S147G (1.8%) N155H (100%) 91-Effective VS EVG/c/FTC/TDF Q148H (13.1%) - - No emergence of MiRVs and no impact of baseline MiRVs on INSTI response

7 Integrase Inhibitor Resistance Selections Initiated with Drug Resistant HIV-1 K Andreatta et al CROI 2018 # 546

8 Integrase Inhibitor Resistance Selections Initiated with Drug Resistant HIV-1 K Andreatta et al CROI 2018 # 546

9 E157Q INTEGRASE POLYMORPHISM: IN VITRO PHENOTYPIC IMPACT AND IN VIVO VIROLOGIC OUTCOME Among >8500 IN sequences we showed an overall prevalence of E157Q polymorphism of 2.7% with heterogeneity of prevalence between the subtypes The prevalence of the E157Q polymorphism was more than three times higher in CRF02_AG than in B subtype sequences (5.6% versus 1.7%, p <0.0001) Based on the present data and on previous in vitro findings, in case of E157Q polymorphism: the most recommended INI might be dolutegravir in such patients elvitegravir should not be considered due to potential low-level resistance, especially in the context of CRF02_AG recombinant raltegravir seems adequate, since IC50 is not increased, but with the caveat that selection of E157Q has been described at VF in two case reports Charpentier et al, CROI 2018 # 547

10 Characterization of the Dolutegravir Monotherapy- Acquired S230R Resistance Mutation Hanh T Pham et al, CROI 2018 # 548

11 Baseline M184V Not Associated With Increased Probability of Virologic Failure in Virologically Suppressed Italian Patients Switching to Lamivudine-Based Dual Therapy Aim of the study was to compare virological efficacy of bpi or INI +3TC in patients with and without a history of M184V detection. Retrospective observational study performed in the ARCA database 436 Pts with HIV-RNA 50 cps/ml switching to DT (3TC+ PI/r or INI) and with at least one previous genotype were selected Primary endpoint: Time to virological failure in M184V+ and M184V Virological failure (VF): 2 VL >50 cp/ml or single value 200 cp/ml Gagliardi R, Ciccullo A, Borghetti A, et al. Impact of previous M184V on virologic outcome of switch to 3TC-based dual therapies. CROI 2018; Boston. Abstract 498.

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15 Conclusions In this retrospective analysis of virologically suppressed patients drawn from the Italian ARCA database, switching to lamivudine (3TC)-based dual therapy did not significantly increase time to virologic failure in patients with vs without baseline M184V mutation Significant risk factors for virologic failure were higher peak HIV-1 RNA and hepatitis B surface antigen (HBsAg) positivity Time to viral blips significantly shorter and probability of viral blips significantly greater in patients with M184V Significant risk factors for viral blips were historical detection of M184V and hepatitis C virus (HCV) coinfection Investigators hypothesized that results may be due to decreased HIV fitness precluding rebound, M184V protecting against selection for resistance mutations to dolutegravir, and longer virologic suppression disproportionately reducing reservoir of replication-impaired HIV (eg, variants with M184V)

16 Determinants of HIV-1 reservoir size and long-term dynamics under suppressive ART Bachmann N, et al. CROI 2018; Boston. Abstract 69LB.

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21 Phylogenetic Analysis Identifies Clusters of HIV-Infected Persons With High Transmission Rates 1.1 million adults and adolescents 13 years of age or older living with HIV infection in the United States Approximately 4 transmission events occur per 100 person-years (PY) Identifying active transmission patterns a key to prevention Targeted testing can identify previously undiagnosed persons and link them with care PREP and other prevention modalities can be provided along with community level interventions Current study used data generated by routine genotypic resistance testing to identify growing molecular clusters in the United States, assess transmission rates, and identify characteristics of persons in these clusters France AM, Panneer N, Ocfemia MCB, et al. Rapidly growing HIV transmission clusters in the United States, CROI 2018; Boston. Abstract O40.

22 Study design United States National HIV Surveillance System (NHSS), primary monitor of HIV infection trends in the United States HIV nucleotide sequence data reported to NHSS Between , sequence data reported from 27 jurisdictions Data available from > 280,000 people living with HIV Investigators identified priority clusters All sequence pairs compared to identify clusters of closely related pairs (ie, short genetic distance between sequences) Genetic distance threshold: 0.5% Priority cluster: 5 patients with HIV diagnosed in previous year For each priority cluster and across all priority clusters, transmission rate was estimated for the time of detection Demographic, clinical, and transmission risk characteristics were compared for those in vs those not in priority clusters

23 Conclusions N = 51,750 persons with HIV diagnosed between January 2013 and December 2016 who also had nucleotide sequence data 60 priority clusters identified involving 20 states in all regions of the United States Cluster size range (as of December 2016): 5-42 persons HIV transmission rate for priority clusters: 44/100 PY (range in clusters: /100 PY) > 10 x average transmission rate for United States Transmission risk categories in priority clusters disproportionately young men who have sex with men (MSM), especially young Hispanic MSM

24 A new B/CRF02 circulating recombinant spreading quickly in Paris area, France At January 2018, 49 infected patients were identified as presenting the unusual recombinant pattern. This pattern does not match the previous identified recombinant forms mixing subtype B and CRF02_AG. needs to be confirmed with additional near-full genome sequences and deeper phylogenetic analysis. All the virus identified with this new recombinant form are included in a recent transmission cluster in the PR-RT (branch support value >95% and maximum genetic distance <3.4%) as well as in all the other tested genes (data not shown). The corresponding patients were diagnosed in 2013 (n=2), 2014 (1), 2015 (8), 2016 (22) and 2017 (15). The tmrca analysis estimated the emergence of this cluster in July Wirden, et al. CROI 2018; Boston. Abstract 958.

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26 A new CRF02_AG/B recombinant, proposed as the CRF94_B02, has been identified and spread quickly in Paris area particularly among a very targeted population. This work shows that combining phylogenetic and demographic analysis can be useful to detect a specific area and/or population and, then, to trigger effective preventive actions allowing to break new active transmission clusters.

27 Unintegrated viral DNA involved in Dolutegravir resistance? Mbio, 2017 I. Malet, F. Subra, C. Richetta, H. Leh, C. Charpentier, G. Collin, D. Descamps, E. Deprez, V. Calvez, AG. Marcelin, O. Delelis WT HIV-1 Dolutegravir 500 nm mutant virus Analysis of the mutant Laï virus No mutation was selected in the integrase gene Sequencing allowed to highlight the presence of mutations in the 3 PPT region included in the nef gene as shown below WT sequence C G G G G G G Malet, et al. CROI 2018; Boston. Abstract 544. Mutated sequence T G C A G T

28 PCR cycles Replication of the virus Total viral DNA Viral replication of the 3 -ppt mutant Total viral DNA / ug of cellular DNA Quantification of integrated DNA WT virus Difference in average of 3.7 cycles d2 d3 d4 d5 WT 2 WT 2 + DTG 31 3'-PPT 200 3'-PPT DTG d7 d8 d9 d10 d11 Days post-infection mutant virus No difference d12 d13 d2 WT 2 d3 d4 WT 2 + DTG d5 3'-PPT 200 3'-PPT DTG D116 with Alu without Alu d7 d8 d9 Days post-infection d10 23 D WT 20 d4 WT 10 d4 WT 20 d3 WT 2 d5 15 3'-PPT 100 d7 3'-PPT 100 d7 + DTG 3'-PPT 200 d7 3'-PPT 200 d8 + DTG 3'-PPT 200 d8 3'-PPT 100 d8 3'-PPT 100 d8 DTG

29 Viral replication of the 3 -ppt mutant Sensitivity of the integrated viral DNA quantification 1,E+08 1,E+07 1,E+06 1,E+05 1,E+04 1,E+03 1,E+02 1,E+01 1,E+00 Ct=6,7 Ct=5,3 Ct=3,1 Ct=2, A known amount of integrated viral DNA = WT DNA 1 (pur), 2 (1/10), 3 (1/100), 4 (1/1000) was diluted in a constant amount of DNA from cells infected with the 3 -PPT mutant = 3 -PPT DNA We observed that the Ct decreased with the dilution but remained significant until a dilution of 1/1000 Quantification of DNA circles 40% 35% 30% 25% 20% 15% 10% Percentage of 2-LTR circles 70% 60% 50% 40% 30% 20% Percentage of 1-LTR circles 5% 10% 0% WT WT+DTG 3'-PPT 3'-PPT + DTG D116N No accumulation of 2-LTR circles 0% WT WT+DTG 3'-PPT 3'-PPT + DTG D116N Strong accumulation of 1-LTR circles

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63 Immune-based strategies to eliminate HIV reservoirs PROTECT ART Activate TLR7 Agonists Latently Infected CD4 T cells Immune effector cells Eliminate Engage Immune System Effector IgG, Bispecific Antibodies, Therapeutic Vaccines, TLR agonists, Anti-PD-1 antibodies CD8 cells NK cells MΦs MΦs, macrophages; NK, natural killer; PD-1, programmed cell death protein 1; TLR, toll-like receptor. Goal Combinations

64 TLR7 agonists Vesatolimod GS-9620 EC 50 (nm) TLR7 Fold TLR7 TLR8 Selectivity MYD88 TLR7 Endosome Plasmacytoid dendritic cells B cells GS-9620 (clinical) GS-986 (tool) IRF7 NFkB Selective, orally bioavailable Clinical trials phase I - healthy individuals (completed) - HBV viremic/aviremic (completed) - HIV aviremics (in-progress) IRF7 IRF7 Antiviral Cytokines IFNa/b, ISGs Nucleus NFkB Acute Phase Cytokines IL-1, IL-1RA, IL-6, ITAC Stimulate CD4 T cells (Activate HIV) Stimulate CD8 T cells, NKs, APCs (Eliminate HIV)

65 TLR7 Agonists in SIV+ Rhesus Monkeys on ART 11 Rhesus monkeys (A*01, B*08, B*17 negative) SIVmac251 infection ART initiated day 65 post-infection (TFV, FTC, DTG s.c. q.d.) >13 months ART before TLR7 agonist dosing Stop ART Group 1 (n=2) Placebo (Vehicle) EOW x 10 3 months EOW x 9 1 mo Group 2 (n=3) GS-986 (0.1 mg/kg) EOW x 10 3 months EOW x 9 1 mo Group 3 (n=3) GS-9620 (0.05 mg/kg) EOW x 10 3 months EOW x 9 1 mo Group 4 (n=3) GS-9620 (0.15mg/kg) EOW x 10 8 months Whitney et al., Sci. Transl. Med. (in press)

66 TLR7 Agonists Induce Transient Plasma Viremia V1 V Plasma SIV (log 10 RNA copies/ml) Placebo Plasma SIV RNA copies/ml GS-986 (0.1 mg/kg) GS-9620 (0.05 mg/kg) GS-9620 (0.15 mg/kg) Vehicle V1 V2 Time after TLR7 agonist dose (Hours) 0 Vehicle Viral blips observed in all TLR7-agonist treated animals Whitney et al., Sci. Transl. Med. (in press)

67 Plasma SIV RNA (log 10 copies/ml) TLR7 agonist treatment can lead to ART-free viral remission in rhesus monkeys Placebo GS-986 (0.1 mg/kg) GS-9620 (0.05 mg/kg) GS-9620 (0.15 mg/kg) Days After ART Stop Whitney et al., Sci. Transl. Med. (in press)

68 Evidence of SIV cure in 2 rhesus monkeys following TLR7 agonist treatment during ART Log SIV RNA copies/ml 1) Viral remission No viral rebound for 2 years after stopping ART 2) CD8 T cell depletion No viral rebound after CD8 T-cell depletion 3) Adoptive Transfer Peripheral blood and lymph node immune cells (10 8 ) transferred from controllers to naive monkeys Monkey 1 2 Pre-TLR7 Post-TLR Day post Adoptive Transfer

69 HIV Effector IgG Protect ART Activate Eliminate Latently Infected CD4 T cells HIV Envelope FcgRIIIA Effector IgG FcgRIIA NK cells ADCC MΦs ADCP MΦs, macrophages NK, natural killer

70 RESEARCH 25 th Conference on Retroviruses and Opportunistic Infections (CROI) Boston, MA March 6, 2018

71 PGT121 + TLR7 Agonist in SHIV-infected ART-Suppressed Rhesus Monkeys 44 rhesus monkeys infected IR with SHIV-SF162P3 ART (TDF/FTC/DTG) initiated at week 1 (day 7) Prolonged ART suppression x 96 weeks SHIV Infection Stop cart ART discontinued at week 130 (16 weeks after last PGT121/TLR7)

72 L o g D N A C o p i e s / L N M C PGT121 + GS-9620 Reduces Viral DNA in Lymph Nodes (Week 120) P = P = NS P = NS S h a m T L R 7 A b A b + T L R 7

73 Log SHIV RNA Copies / ml SHIV Plasma RNA Following ART Discontinuation Sham TLR7 11/11 Rebound (100%) 10/11 Rebound (91%) PGT121 PGT121+TLR7 9/11 Rebound (82%) 6/11 Rebound (55%) Days Following ART Discontinuation

74 Adoptive Transfer of 30x10 6 LNMC+PBMC From Aviremic Monkeys (Day 140) into Naïve Recipients Log SHIV RNA Copies / ml No rebound (4 from combo / 1 from PGT121) Transient rebound (2 from combo group) Days Following Adoptive Transfer