Time (weeks)

1. 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.

2. 105 104 103 102 Detection limit 101 16 8 0 4 HIV Response to Antiretroviral Therapy ARV HIV-1 RNA Time (weeks)

3. 108 107 106 105 104 Decay Kinetics of Viral Infected Cells Activated Lymphocyte HIV-1 Infected Cells Longer lived cells Macrophage? Detection limit R0 ≥ 1 1 Longer 14 Half life of infected cells (days)

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

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

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

7. Real time PCR assay Linear quantitation 1 - 106 copies HIV-1 RNA Limit of detection 0.2 copies /ml plasma Does NOT measure a biological activity Assay is NOT FDA approved Single Copy Quantitation of HIV-1 Viremia

8. Superior Efficacy of Lopinavir/ritonavir over Nelfinavir Abbott 98-863 Study Does a difference in antiviral potency impact viremia on therapy? Selected 130 patients (67 NFV, 63 LPV/r) Remained <50 copies/ml following wk 24 lopinavir/ritonavir 21% Failure 44% Failure nelfinavir Percent maintaining virologic response p<0.001, Cox proportional hazards model Week Walmsley, S. N. Engl. J. Med., 2002

9. Viremia on Therapy is Independent of Regimen 100 80 60 Distribution Rank (Percentile) lopinavir/ritonavir 0.53 0.51 40 Median Mean 20 nelfinavir 0.48 0.43 NNRTI 0.35 0.19 0 -0.5 0 0.5 1 1.5 2 2.5 Log10 viral RNA (copies/ml)

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

11. Persistent Viremia in Patients on Suppressive ART: Longitudinal Analysis • Abbott M97-720 Study • Long term observational study lopinavir/r treated patients (N=40) • D4T/3TC/ lopinavir/ritonavir therapy • Long term evaluation ≥ 7 y

12. Longitudinal analysis reveals an additional third and fourth phase of viral decay Late Stage HIV-1 RNADecay Occurs in at Least Two Phases T1/2 = 63 Weeks ∞ T1/2 = Mixed effects model 2.0 1.5 copies/mL) 1.0 10 0.5 Plasma HIV-1 RNA (log 0.0 -0.5 -1.0 0 60 120 180 240 300 360 Week

13. Probing the mechanism of chronic viremia using antiretroviral intensification Intensification 102 HIV-1 RNA (copies/ml) 101 NO Ongoing Replication 30 day 100 Ongoing Replication Enrollment • Suppressed in commercial assays>1 y • SCA ≥ 1 copy/ml • No prior ARV resistance Time

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

15. Raltegravir Intensification Does NOT Decrease Persistent Viremia Post-Intensification Pre-Intensification Raltegravir 0.92 0.73 HIV-1 RNA (log10 copies/ml plasma) Time (days) McMahon, CID, 2010

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

17. But… 2 LTR Circles 13/45 RTG 0/24 Control Nature Med 2010

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

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

20. Characteristics of HIV During Suppressive Therapy • Persistent Viremia • Quantifyable in c. 80% of patients • Relatively stable steady state • Third phase decline (t1/2 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 >104-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

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

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

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

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

25. HIV-1 Genetic Diversity During ARV Therapy Similar Genetic Diversity and Population Structure Before and After Initiation of Antiretroviral Therapy NO genetic evidence of ongoing replication during ARV suppression 1019.510 1030.723 1019.522 145242-12 1019.516 1019.57 1206.97 1115.89 1115.81 1115.810 1206.95 145241-9 145242-3 145241-10 145241-14 1115.816 1115.83 145241-13 1206.914 1115.820 1206.98 145242-1 1115.814 1030.71 0 50 100 150 200 250 300 1019.517 1019.52 Time on Study (days) 1030.716 0.001 substitutions/site D4T/3TC/EFV 107 1000 900 106 800 105 700 600 104 CD4 (cells/µl) HIV-1 RNA (copies/ml) 500 103 400 300 102 200 101 100 1 0

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

27. 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

28. Eradication Strategies • 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 Critical Test of Eradication: Interrupt Antiretrovirals

29. Eradication Strategies • 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

30. 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 Acknowledgments Patient Volunteers • Tufts University • John Coffin • Karolinska • Institute • S. Palmer • Abbott Lab. • M. King • S. Brun • D. Kempf • G. Hanna • Johns Hopkins • University • J. Dinoso • S. Gange • R. Silicano

31. Eradication Strategies • 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

32. Status of HIV Infected Cell During Therapy Chromatin Remodeling Transcription Factors Transcription Factors Nature of reservoir requires distinct approaches to eradication “LATENT” +1 +1 HIV mRNA HIV mRNA U3 R U5 antigen stimulation U3 R U5 Constitutive HIV Replication Inducible HIV Replication Target HIV Indirectly Activate Chromatin Remodeling Target HIV Directly

33. HIV-1 Genetic Diversity During ARV Therapy Similar Genetic Diversity and Population Structure Before and After Initiation of Antiretroviral Therapy NO genetic evidence of ongoing replication during ARV suppression 1019.510 1030.723 1019.522 145242-12 1019.516 1019.57 1206.97 1115.89 1115.81 1115.810 1206.95 145241-9 145242-3 145241-10 145241-14 1115.816 1115.83 145241-13 1206.914 1115.820 1206.98 145242-1 1115.814 1030.71 0 50 100 150 200 250 300 1019.517 1019.52 Time on Study (days) 1030.716 0.001 substitutions/site D4T/3TC/EFV 107 1000 900 106 800 105 700 600 104 CD4 (cells/µl) HIV-1 RNA (copies/ml) 500 103 400 300 102 200 101 100 1 0

34. HIV Reservoirs:Distinct Subsets Diverse Activation Signalling • Central Memory • Transitional Memory

35. HIV EradicationAnti-Latency Strategies +1 R AP-1 ATF/CREB AP-3 NFAT AP-3 NFAT NRE U5

36. HIV EradicationAnti-Latency Strategies TAR +1 TATA AP-3 NFAT AP-1 ATF/CREB AP-3 NFAT SP C/EBP NF-κB SP NRE U3 R U5

37. HIV EradicationAnti-Latency Strategies TAR +1 TATA AP-3 NFAT AP-1 ATF/CREB AP-3 NFAT SP C/EBP NF-κB SP NRE U3 R U5 TBP associated factors

38. HIV EradicationAnti-Latency Strategies TAR +1 TATA AP-3 NFAT AP-1 ATF/CREB AP-3 NFAT SP C/EBP NF-κB SP NRE U3 R U5 Zn++ Finger binding SP/KLF

39. HIV-1 Suppression by Transplant Hutter et al., NEJM, 2009 106 ARV ARV HIV RNA copies/ml 104 102 +548 -4 -206 +108 +332 Chemotherapy Conditioning/Transplant Conditioning/Transplant Multiphase HIV decay to therapy Engraftment with ΔCCR5 No viremia off ART but leukemic failure Second transplant controlled leukemia 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

40. 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

41. Status of HIV Infected Cell During Therapy Transcription Factors Nature of reservoir requires distinct approaches to eradication +1 HIV mRNA U3 R U5 Constitutive HIV Replication • Targeting Low Level • HIV Production • Anti-CD45 Ro • Zeta chain therapy • Pseudomonas exotoxin targeting Env Target HIV Directly

42. Status of HIV Infected Cell During Therapy Chromatin Remodeling Transcription Factors Nature of reservoir requires distinct approaches to eradication “LATENT” +1 HIV mRNA U3 R U5 antigen stimulation • 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 Inducible HIV Replication Target HIV Indirectly Activate Chromatin Remodeling

43. Status of HIV Infected Cell During Therapy Chromatin Remodeling Transcription Factors Nature of reservoir requires distinct approaches to eradication, unless we just target everything “LATENT” +1 HIV mRNA U3 R U5 antigen stimulation