HIV Drug Resistance Program NCI–Frederick Targeting ‘Residual HIV’ In Its Reservoirs: Where We Are And Where Do We Go? HIV Reservoirs Workshop Vienna, Austria July 17,2010 Frank Maldarelli, M.D., Ph.D.

Time (weeks) HIV-1 RNA HIV Response to Antiretroviral Therapy Detection limit ARV

Decay Kinetics of Viral Infected Cells HIV-1 Infected Cells Half life of infected cells (days) 1 Longer 14 Detection limit Activated Lymphocyte Longer lived cells Macrophage? R 0 ≥ 1

Identifying the source of HIV viremia during suppressive antiretroviral therapy is essential to eradication Stable reservoirs NEW STRATEGIES NEEDED HIV production from reservoirs is NOT blocked by ARV therapy IMPROVED ARV NEEDED Active replication cycles Infected cell Uninfected cell HIV production from active replication is blocked by ARV therapy X

Quantitative Measures For Clinical Studies of HIV Reservoirs HIV nucleic acid analysis HIV population genetics

Quantitative Measures For Clinical Studies of HIV Reservoirs HIV nucleic acid analysis HIV population genetics

Single Copy Quantitation of HIV-1 Viremia Real time PCR assay Linear quantitation copies HIV-1 RNA Limit of detection 0.2 copies /ml plasma Does NOT measure a biological activity Assay is NOT FDA approved

Percent maintaining virologic response nelfinavir lopinavir/ritonavir 21% Failure 44% Failure Week p<0.001, Cox proportional hazards model Superior Efficacy of Lopinavir/ritonavir over Nelfinavir Abbott Study Does a difference in antiviral potency impact viremia on therapy? Walmsley, S. N. Engl. J. Med., 2002 Selected 130 patients (67 NFV, 63 LPV/r) Remained <50 copies/ml following wk 24

Log 10 viral RNA (copies/ml) Distribution Rank (Percentile) nelfinavir NNRTI Viremia on Therapy is Independent of Regimen lopinavir/ritonavir Median Mean

Correlation Between Baseline and Persistent Viremia at Week 60 HIV-1 RNA copies/ml

Persistent Viremia in Patients on Suppressive ART: Longitudinal Analysis Abbott M Study Long term observational study lopinavir/r treated patients (N=40) D4T/3TC/ lopinavir/ritonavir therapy Long term evaluation ≥ 7 y

Late Stage HIV-1 RNA Decay Occurs in at Least Two Phases Week Plasma HIV-1 RNA (log 10 copies/mL) Longitudinal analysis reveals an additional third and fourth phase of viral decay T 1/2 = 63 Weeks T 1/2 = ∞ Mixed effects model

HIV-1 RNA (copies/ml) Time Probing the mechanism of chronic viremia using antiretroviral intensification NO Ongoing Replication Ongoing Replication 30 day Intensification Intensification Enrollment Suppressed in commercial assays>1 y SCA ≥ 1 copy/ml No prior ARV resistance

NNRTI or PI Intensification Does NOT Decrease Persistent Viremia Dinoso et al., 2009

Raltegravir Intensification Does NOT Decrease Persistent Viremia Raltegravir HIV-1 RNA (log 10 copies/ml plasma) Time (days) Pre- Intensification Post- Intensification McMahon, CID, 2010

Antiretroviral intensification DOES NOT reduce HIV-1 plasma viral RNA levels EFV ATV/r LPV/r RVR Selected patient population ARV Intensification Does NOT Decrease Persistent Viremia

But… Nature Med LTR Circles 13/45 RTG 0/24 Control

Detecting HIV Replication in Reservoirs Anatomic compartmentalization is NOT well understood CNS GALT GU Anatomic Reduced ARV Penetration = Ongoing Replication Charter Study Best et al., AIDS 2009 Wong, Brain 2006 Genetically Distinct Populations

Detecting HIV Replication in Reservoirs Anatomic compartmentalization is NOT well understood CNS GALT GU ACTG 5201 Open Label Pilot of Regimen Simplification Swindells JAMA 2006 Wilkin, J.Inf.Dis. 2009ENTRY N=36 N=36 Suppressed≥ 48 weeks on combination ARV Suppressed≥ 48 weeks on combination ARVINTERVENTION: REGIMEN SIMPLIFIED TO r/ATZ ALONE REGIMEN SIMPLIFIED TO r/ATZ ALONERESULTS: 31/34 suppressed at 24 weeks 31/34 suppressed at 24 weeks 97% of all time points <50 c/ml 97% of all time points <50 c/ml Resistance did not emerge in most with rebound Resistance did not emerge in most with rebound SCA Detected increased viremia in rebound SCA Detected increased viremia in rebound NOT in patients with continued suppression NOT in patients with continued suppression Similar clinical data in randomized studies of r/darunavir monotherapy vs combination ARV (MONET), and r/Kaletra monotherapy vs combination therapy

Characteristics of HIV During Suppressive Therapy Persistent Viremia Quantifyable in c. 80% of patients Relatively stable steady state Third phase decline (t 1/2 c.63 wk) and fourth phase (no decline) with prolonged therapy Level of viremia is NOT correlated with drug regimen ARV therapy is potent and suppresses HIV >10 4 -fold Level of viremia IS correlated with level of pretherapy viremia Dynamic changes in HIV replication are reflected in level of viremia and detectable using SCA

Quantitative Measures For Clinical Studies of HIV Reservoirs HIV nucleic acid analysis HIV population genetics

Genetic Analysis of HIV RNA To Detect Ongoing Replication Pretherapy During therapy Divergence NO Ongoing Replication NO genetic evidence of ongoing replication during ARV suppression Ongoing Replication Divergence

Genetic Analysis of HIV RNA To Detect Ongoing Replication NO genetic evidence of ongoing replication during ARV suppression Time (days)

Analysis of HIV Viremia After Prolonged Suppression Composition of the plasma virus during suppressive therapy Persaud JAMA 2000

HIV Drug Resistance Program NCI–Frederick 10 1 HIV-1 RNA (copies/ml) CD4 (cells/µl) HIV-1 Genetic Diversity During ARV Therapy D4T/3TC/EFV Time on Study (days) Similar Genetic Diversity and Population Structure Before and After Initiation of Antiretroviral Therapy NO genetic evidence of ongoing replication during ARV suppression

Analysis of HIV Viremia After Prolonged Suppression Bailey et al., 2006 Predominant Plasma Clone (PPC) HIV cellular DNA HIV in plasma HIV from resting CD4 Distribution of HIV diversity Repeated isolation Repeated isolation Identical sequence Identical sequence NOT present in resting NOT present in resting CD4 CD4 NOT major constituent of NOT major constituent of cellular DNA cellular DNA Loss of other shorter lived cells exposed rare PPC-producing cell(s)? Pool of cells undergoing expansion?

Characteristics of HIV During Suppressive Therapy HIV population genetics HIV populations are genetically diverse Do not undergo genetic bottleneck upon introduction of ARV Genetic variation is markedly restricted during suppressive therapy Suggest little or no active replication during therapy

Requirements Maintain suppression of active HIV-1 replication Continue ARV during eradication Dual approach Target cells with low level HIV-1 production Ensure activation of cells with “latent” HIV infection Permanent silencing for durable effect Eradication Strategies Critical Test of Eradication: Interrupt Antiretrovirals

Detecting HIV during suppressive therapy and eradication strategies Sensitive detection systems Single copy nucleic acid detection RNA DNA IUPM Genetic analyses Robust performance characteristics Poisson limitations Patient selection and characterization is essential Useful assays are essential to ensure patient safety Eradication Strategies

Acknowledgments NIAID/CCMD Clinic H. C. Lane H. Masur R. Davey M. Polis J. Kovacs J. Mican I. Sereti S. Migueles A. O’Shea C. Rehm R. Dewar S. Mitchell J. Metcalf Clinical Fellows HIV Drug Resistance Program S. Hughes J. Coffin M. Kearney A. Wiegand V. Boltz W. Shao J. Spindler H. Mens S. Yu N. Urban F. Cossarini C. Poethke Karoll Cortez University of Pittsburgh J. Mellors D. McMahon J. Jones Tufts University John Coffin Karolinska Institute S. Palmer S. Palmer Abbott Lab. M. King S. Brun D. Kempf G. Hanna Johns Hopkins University J. Dinoso S. Gange R. Silicano Patient Volunteers Patient Volunteers

Stimulate HIV expression from latently infected cells HDAC and other approaches to remodel chromatin Specific HIV induction Immune modulators Target infected cells with low level replication Inhibit cellular activation Direct cytotoxic therapy Gene therapy approaches Transplantation approaches Replacement of bone marrow with HIV resistant donor Heller et al., 2009 NOT widely applicable ARV discontinuation Clinical success will require surveillance Eradication Strategies

antigen stimulation Status of HIV Infected Cell During Therapy Constitutive HIV Replication Inducible HIV Replication U3 RU5 “LATENT” U3 RU5 Chromatin Remodeling Target HIV Directly Target HIV Indirectly Activate Chromatin Remodeling Nature of reservoir requires distinct approaches to eradication +1 HIV mRNA +1 HIV mRNA Transcription Factors Transcription Factors

HIV Drug Resistance Program NCI–Frederick 10 1 HIV-1 RNA (copies/ml) CD4 (cells/µl) HIV-1 Genetic Diversity During ARV Therapy D4T/3TC/EFV Time on Study (days) Similar Genetic Diversity and Population Structure Before and After Initiation of Antiretroviral Therapy NO genetic evidence of ongoing replication during ARV suppression

HIV Reservoirs: Distinct Subsets Diverse Activation Signalling Central Memory Transitional Memory

HIV Eradication Anti-Latency Strategies U5 R +1 AP-1 ATF/CREB AP-3 NFAT NRE AP-3 NFAT

HIV Eradication Anti-Latency Strategies R U5 AP-1 ATF/CREB AP-3 NFAT NRE AP-3 NFAT C/EBPNF-κB SP TATA SP U3 +1 TAR

HIV Eradication Anti-Latency Strategies R U5 AP-1 ATF/CREB AP-3 NFAT NRE AP-3 NFAT C/EBPNF-κB SP TATA SP U3 +1 TAR TBP associated factors

HIV Eradication Anti-Latency Strategies R U5 AP-1 ATF/CREB AP-3 NFAT NRE AP-3 NFAT C/EBPNF-κB SP TATA SP U3 +1 TAR SP/KLF Zn ++ Finger binding

HIV-1 Suppression by Transplant Hutter et al., NEJM, HIV RNA copies/ml Chemotherapy Conditioning/Transplant ARV Conditioning/Transplant Engraftment with ΔCCR5 No viremia off ART but leukemic failure Second transplant controlled leukemia Multiphase HIV decay to therapy Elimination of reservoir by replacement AND… Graft vs HIV infected cell effect? All latent infected cells undergo activation OR All infected cells are detectable by graft

HIV Eradication Strategies Neoplastic diseases therapy as paradigm Successful especially when tumor burden is substantial Relevance to low frequency targets like HIV infected cells depends on specificity

Status of HIV Infected Cell During Therapy Constitutive HIV Replication U3 RU5 Target HIV Directly Nature of reservoir requires distinct approaches to eradication +1 HIV mRNA Transcription Factors Targeting Low Level HIV Production Anti-CD45 Ro Zeta chain therapy Pseudomonas exotoxin targeting Env

antigen stimulation Status of HIV Infected Cell During Therapy Inducible HIV Replication “LATENT” U3 RU5 Chromatin Remodeling Target HIV Indirectly Activate Chromatin Remodeling Nature of reservoir requires distinct approaches to eradication +1 HIV mRNA Transcription Factors Excellent models in vitro Cell lines Lymphocytes ex vivo Numerous potential strategies Integration site selection Chromosome modeling Valproate Transcriptional approaches Post transcriptional approaches Active agents with potential Disrupt nucleic acid sites required for activation Disrupt nucleic acid- activator interactions Modulate activation and expression of activators

antigen stimulation Status of HIV Infected Cell During Therapy “LATENT” U3 RU5 Chromatin Remodeling Nature of reservoir requires distinct approaches to eradication, unless we just target everything +1 HIV mRNA Transcription Factors