Treatments to modulate HIV reservoirs:A virological and immunological cross-talkJean-Pierre Routy M.D.McGill UniversityMontrealProspects for Eradication:Determinants of Viral Reservoirs

HIV reservoirsViral definition:The persistence of transcriptionally silent but replication-competent HIV, in the presence of HAARTCellular definition:Latently-infected resting memory cells harboring an integrated form of the viral genome that lacks the ability to produce viral proteins, in the presence of HAARTReservoir of latently infected cells:Resting memory CD4+ T cells Monocyte/Macrophage lineageYet unidentified source: Brennan et al J virol 2009;June 2009Anatomic reservoir: CNSStability of such reservoir after years on HAART

Strategies are needed to decrease and ultimately eradicate latently infected cells

Viral model: Low levels of ongoing viral replication could lead to de novo infection of memory cellsContinuous replenishment of the reservoirProvirus has to become transcriptionally activeHAART not fully potent:Anatomic reservoir (CNS)?Cellular model:Infected monocytes/macrophagesIntrinsic stability of latently infected CD4 cells (survival)Central memory CD4 cells survive for yearsPro-survival pathways are triggered (transcriptional factor FOXO3a)van Grevenynghe et al. Nat Med. 2008; 14:266 Cellular proliferation depends on:Antigen-induced (TCR)Cytokine-induced (homeostatic)

Proposed mechanisms for HIV reservoir persistence

Virologic model in 2009 Recent data no longer support this model:Reservoir sizes are established before therapyAbsence of genetic evolution in viral reservoir Presence of a stable long-lived cellular reservoir Ultrasensitve PCR: 1 copy/mL. Palmer S et al. PNAS 2008;105:3879 Failure of treatment intensification:Dinoso JB et al. PNAS 2009;106:9403Absence of anti-integrase effect (Raltegravir) on reservoir:Sedaghat A et al. Antiviral therapy 2009; 14:263Ghandi et al. Raltegravir Intensification does not reduce low-level residual viremia. ACTG A5244

Cellular model in 2009Memory CD4 cells are phenotypically and functionally heterogeneous:Central Memory: Self renewal, persistenceCD45RA-, CCR7+, CD27+ Transitional Memory: CD45RA-, CCR7-, CD27+Effector Memory: Immediate effector function, short term survival:CD45 RA-, CCR7-, CD27-Late differentiated Memory: CD45 RA+, CCR7-, CD27-

CD4 cell depletion drives the reservoir sizeChaumont et al. Nat Med June 2009

CD4/CD8 ratio and duration of viremia drive the reservoir sizeChaumont et al. Nat Med June 2009

CD4 cells drive the size and the location of the HIV reservoirChaumont et al. Nat Med June 2009Central memoryTransitory and effector memory

CD4 proliferation and immune activation drive the size and location of the HIV reservoirKi67: ProliferationPD-1: T cell ActivationChaumont et al. Nat Med June 2009LowLow

Central and transitional memory CD4 cells are driven by different mechanismsReservoir of patients with moderate CD4 recovery (< 500) consists mainly of transitory memory CD4 cells: Immune activation and proliferationHomeostatic proliferation (IL-7)Reservoir of patients with adequate CD4 recovery ( > 500) consists mainly of central memory CD4 cells: Low-level antigen driven proliferation (TCR)Intrinsic T-cell survival (FOXO3a): Stem cell-like

TCM cells resemble chronic myeloid leukemia (CML) stem cells treated with Imatinib (Gleevec) a tyrosine kinase inhibitorKikuchi S et al. Cancer Science 2007; 98:1949 Hui et al. Mol. and Cell. Biology 2008; 28:5886

Persistence of reservoirsNot due to lack of HAART potencyDriven by:Immune activation and T cell proliferationT cell survivalTreatments to modulate HIV reservoirs should be based on:HIV latencyT cell dynamics

Why treat the HIV reservoir?Cons:CD4 memory cells are rare and do not contribute to pathogenesis HAART has improved and is less toxic Failure of initial attempt with HAART and IL-2 Risky intervention Priority for:Increasing HIV screeningEarly HAART initiation to decrease AIDS and non-AIDS events

Pros:Lifelong adherence to therapyUnknown effects of long-term treatmentStigmatization due to HIV will last for lifeIf we never try we will never succeed The Berlin patient

2 transplantations for AMLAtriplaHutter G et al NEJM 2009; 360: 692Monocyte and CNS reservoirs not targetedLong-term Control of HIV by CCR5 Delta32/Delta32 Stem Cell Transplantation

Reducing the HIV reservoir:Treatment objectivesA stepwise approach:Reducing the HIV reservoir by: Increasing CD4 recovery (central vs. effector CD4 memory)Reducing immune activation should contribute to reducing non-AIDS events (inflammation)ART-free remission: No evidence of disease or symptomsCure:No evidence of disease or symptomsEliminating every functional virionEliminating every infected cell

A risk/benefit approach is necessary to conduct clinical research on the reservoirAnimal model:Monkey model: Dinoso et al. J Virol July 2009Treatment during primary HIV infection:CCR5 inhibitor, anti-integrase, IL-7Immunoreconstitution strategy:Recovery of CD4 and CTL functionHIV-specific targeted therapy:Anti-HIV latency strategies with HAART alone or associated with:Immuno-reconstitution therapyTherapeutic vaccineNon-HIV specific therapy:Cytotoxic approach: lymphoid with or without myeloablation

IL-7: potential anti-latency and Immunoreconstitution therapyInterleukin-7:Inducing in vitro HIV expression without T cell activation: Archin et al Curr Opin HIV AIDS 2006;1:134Increasing nave and central memory T cells: Levy et al J Clin Invest 2009;119:997Enhancing ex vivo CTL function: Sereti et al. Blood 2009; 113: 6304Early immune gut effect in monkeys: Nascimbeni et al Blood 2009; 113: 6112

Alone or in combination with:Therapeutic vaccine: in mice with cancers. Pellegrini et al Nat Med 2009; 15: 528Monoclonal antibody with immunotoxin or intelligent radiotherapy

IL-7 therapy:Mobilization of HIV (bleep) and HIV-specific T cellsVLIL-7

Strategies to enhance CTL functionEnhancing CTL (CD8) functionDendritic cell therapy to increase antigen (HIV) presentation to CTLArgos Therapeutics, CTN # 239TRF6 and fatty acid metabolism modulator:Metformin and anti-cancer therapeutic vaccine in micePearce et al. Nature June 2009Enhancing CTL function by modulating exhaustion: CTLA 4: Kaufmann et al. J Immunol 2009;182: 5891IL-21 pathway: Frolick A et al. Science 2009; 324: 1576IL-10 pathway: Brokman MA et al. Blood 2009; 114:346Program death-1 (PD-1): Trautmann et al. Nat Med. 2006; 12:1198, Said et al. MOAA205, IAS 2009

Enhancing CTL function by modulation of PD-1 pathwayPD-1 pathway:Highly expressed on specific anti-HIV CD8 cellsEnhanced expression also on non-specific T cells PD-1high CD4 cells harbor larger HIV reservoir Anti-PD-1 antibody:Enhanced ex vivo CTL functionOngoing studies blocking PD-1 pathway:Cancer patients: Berger et al 2008; 14: 3044HCV-infected patients: Nakamoto PloS Pathog 2009; Fev 27Auto-immunity: Reynoso et al J Immunol 2009; 182: 2102

MicroRNAs and HIV latency

HIV-specific strategy:Shock and kill" Hypothesis proposed by D. H. Hamer Curr HIV Res 2004, 2:99-111Consist of 2 treatment phases:"shock" phase: Drugs which can reactivate HIV from latency in combination with ART to block viral spread, without increasing T cell activation "kill" phase (cell destruction):Natural means: immune response, viral cytopathogenicity Drugs which induce cell death: Monoclonal antibodies, immunotoxin, radio-labeled antibodiesShock" phase: 2 classes of drugsHistone deacetylase inhibitors (HDACIs)NFKB-independent activators

Shock and kill" strategies: Histone deacetylase inhibitorsTo induce HIV activation:Pilot studies with valproic acid:Yes and no: Lehrman and Margolis al. Lancet 2005; 366: 549No: Siliciano et al. JID 2007;195: 833 Randomized controlled study: CTN # 205Deacetylase inhibitor and Prostratin:Synergistic effect in vitro Reuse et al. Plos One 2009;4:6093Class/isoform-selective HDACIs: In vitroAssociated with glutathione-synthesis inhibitor (BSO) Savarini et al. Retroviral 2009; 6:52Followed by expected cell killing: CTL and or viral killing?

Shock and kill" strategies: NFKB independent HIV activatorHIV LTR integrates the function of a multitude of transcription factors NFKB-dependent: Cell activation and proliferationNFKB-independent: Transcriptor factor VII Ets-1: in vitroInduces transcription from the HIV TLR leading to HIV production without inducing T cell activation Yang et al. PNAS 2009;106: 6321

Non-HIV specific cell strategies for memory CD4 cellsCentral memory CD4 cells:Direct cell destruction:Transitory blockade of immune stem cell specific receptorsiRNA for transcription factors (FOXO3a)Transitory and effector memory CD4 cells:Immune activation modulators: Microbial translocation: Germ modifiers TLRs modifiers: Chloroquine, CTN # 246 IRF-7 modifiers Anti-homeostasis factors:Anti IL-7: Chomont et al. Nat Med July 2009

Patients with malignancy: Berlins patient-like therapy Objectives:Myelo and T cell ablation with cytotoxic agentsFollowed by repopulation of the immune system with HIV resistant cells Important limitations:CCR5 homozygosity: 3% of human populationStem cell registries not assessing CCR5 statusAllo stem cell transplant: Graft versus Host effect

New stem cell strategies:Resistance-conferring genes into stem cells before transplantation to induce CCR5 defective receptor Once-in-a-lifetime treatmentAutologous source; Zinc finger nucleases: Perez et al. Nat Biotechnol. 2008; 26:808Clinical stem cell therapy in AIDS-related lymphoma: Von Laer et al. J Gene Med 2006;8:658 Autologous stem cell transplantEx vivo infected cell purging:Phototherapy for activated T cellsAnti-CCR5 or other cell entry inhibitor

Direct in vivo gene transfer without prior cytoreductive therapy to achieve a sustained antiviral effectStrategies limited to patients with malignancy: Berlins patient-like therapy

ConclusionLikely no ongoing viral replication with HAARTStrategy could include mono or combination therapy with HAARTEarly HAART represents the easiest intervention to control reservoir size (CD4 nadir effect)Ethical considerations:Risk/benefit to participate in therapeutic trials a 4-step approach:Primary HIV infection Immunoreconstitution HIV-specific strategiesNon-HIV specific strategiesTissue sampling: Leukopheresis, lymph node and gut biopsiesNew commitment:Infrastructure for translational research International collaborationIAS initiative

AcknowledgementUniversit de Montral: Nicolas Chomont Mohamed El FarLydie TrautmannFrancesco ProcopioBader Yassine-DiabGenevieve BoucherPetronela AncutaCcile TremblayElias HaddadRafick SekalyUniversit McGill:Rachid BoulasselRichard LalondeMarina KleinNorbert GilmoreMark WainbergCTN:Joel SingerJacquie SasAbbott Canada:Nabil Ackad Argos therapeutics:Charles NicoletteCytheris:Michel MorreThrse Croughs

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