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HIV+ Long-Term Nonprogressors: Understanding Natural Immune Control of HIV

HIV+ Long-Term Nonprogressors: Understanding Natural Immune Control of HIV. Stephen A. Migueles, M.D. HIV-Specific Immunity Section, Laboratory of Immunoregulation, NIAID, NIH, Department of Health & Human Services. Overview. LTNP Host Genetic Factors: HLA Associations

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HIV+ Long-Term Nonprogressors: Understanding Natural Immune Control of HIV

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  1. HIV+ Long-Term Nonprogressors: Understanding Natural Immune Control of HIV Stephen A. Migueles, M.D. HIV-Specific Immunity Section, Laboratory of Immunoregulation, NIAID, NIH, Department of Health & Human Services

  2. Overview • LTNP • Host Genetic Factors: • HLA Associations • Host Immune Response Factors • HIV-Specific CD8+ T Cells

  3. Patient 1 • 46 year-old African American medical assistant from New Orleans diagnosed with HIV infection in 1989. • Her only risk factor was unprotected heterosexual intercourse. • Past medical history was significant for hypertension and depression. • At the time of her diagnosis, she was divorced and living with her three children, all HIV-negative and healthy. • She remained clinically well over time with stable, “low-normal” CD4 counts and “undetectable” viral loads despite no ART. • Her PCP referred her to NIH in 2004. • She was displaced by Hurricane Katrina and had a relapse of her depression. • Her CD4 counts have been stable and her HIV RNA levels have remained <50 copies/ml.

  4. Patient 2 • 44 year-old African American barber from Washington, D.C. diagnosed with HIV infection in 1986 and was lost to follow-up because “I wasn’t feeling bad.” • Risk factors were IDU and unprotected heterosexual intercourse. • He was incarcerated in 2004 and found to be infected with HCV 1a. • In 2006, his transaminases were 4x the upper limit of normal and his HCV load was 4.7 million IU/ml. • CD4 count was 1,314 cells/ml with HIV RNA levels <50 copies/ml. • Liver biopsy (6/07) revealed moderate portal inflammation, moderate piecemeal necrosis and focal mild peri-sinusoidal fibrosis. • SVR was achieved with 48 weeks of pegylated interferon--2b and ribavirin therapy. • He is antiretroviral naïve, yet has not experienced any opportunistic diseases. • He has maintained normal CD4 counts and his HIV RNA levels have been <50 copies/ml in 26 out of 27 determinations.

  5. Patient 3 • 73 year-old Caucasian divorced, retired businessman from Florida reported a rash and high fevers 2 weeks after after unprotected intercourse with a male acquaintance in 1987. • His sexual partner’s long-term companion had recently died from AIDS. • The sexual partner eventually died from fulminant AIDS-related Pneumocystis jiroveci pneumonia. • His past medical history was remarkable for ankylosing spondylitis, ulcerative colitis, type II DM and BPH. • A psychic friend told him that he would not die from HIV, had important “work” to do and should enroll in a study. • Since 1998, his course had remained stable with a CD4 nadir of 748 cells/ml and HIV RNA levels <50 copies/ml (31/32 measurements), except for one “blip” to 1,787 copies/ml in 1998. • He is antiretroviral naïve.

  6. Why Study HIV+ LTNP? • As the AIDS pandemic continues to expand, the need for an effective HIV vaccine has never been greater. • Durable control of HIV replication does occur in rare individuals (<0.5% of the infected population) called long-term nonprogressors (LTNP), HIV controllers, elite controllers or elite suppressors. • Understanding the mechanisms of control in LTNP is likely to provide critical information for development of effective vaccines and immune-based therapies.

  7. Protocol 02-I-0086: Leukapheresis Procedures to Study HIV-Specific Immunity Long-Term Nonprogressor (LTNP)

  8. Benefits of Research Participation for LTNP • Remuneration • $100 for 2-pass leukapheresis; $200 for 4-pass • Insight into their disease course • Contribute to an important cause • Paying tribute to lost loved ones • Beacons of hope for their children and future generations

  9. LTNP Case Definition • HIV+ by standard antibody tests • Healthy with stable clinical course • Negative history of opportunistic diseases • Stable, non-declining T cell counts • Set point HIV RNA levels <50 copies/ml • No antiretroviral or immunomodulatory treatment

  10. Characteristics of LTNP

  11. Mechanisms of Non-Progresive HIV Infection

  12. Belief Systems and Behaviors of LTNP • “I exercise religiously and eat well.” • “I smoke 2 packs a day, drink 2 liters of diet Coke a day and never exercise. I have no clue!” • “I was feeling depressed and drank rat poison when I was in my 20’s. I’m convinced that’s what made my immune system different.” • “I drink only the most expensive red wines from France!” • “It’s because of my family’s support and my positive frame of mind.” • “God’s given me a gift for whatever reason; it’s got nothing to do with what I do. I’m just blessed.”

  13. Proposed Mechanisms of Non-progressive HIV Infection • Virus Factors • Inactivating mutations in nef, tat, rev or vpr • CTL-induced escape mutations • Host Genetics • Heterozygosity for CCR5 32 deletion • Protective HLA class I alleles • Host Immune Response Factors • Adaptive immune system • Effective humoral responses • HIV-specific CD4+ T cell responses • HIV-specific CD8+ T cells responses

  14. Proposed Mechanisms of Non-progressive HIV Infection • Virus Factors • Inactivating mutations in nef, tat, rev or vpr • CTL-induced escape mutations • Host Genetics • Heterozygosity for CCR5 32 deletion • Protective HLA class I alleles • Host Immune Response Factors • Adaptive immune system • Effective humoral responses • HIV-specific CD4+ T cell responses • HIV-specific CD8+ T cells responses

  15. Protective Heterozygote advantage A*32 B*14 B*27 B*44 B*51 B*57 B*58 Cw*08 Homozygosity for Bw4 Bw4-80Ile alleles/KIR3DS1 B*57 supertype/KIR3DL1 Susceptible A*24 B*3 B*8 B*35-Px Cw*04 Cw*16 HLA and HIV Infection Buchbinder et al., 1992; Klein et al., 1994; Goulder et al., 1996; Kaslow et al., 1996; Keet et al., 1999; Carrington et al., 1999; Hendel et al., 1999; Migueles et al., 2000; Flores-Villanueva et al., 2001; Martin et al., 2002; Leslie et al., 2004; Martin et al., 2005

  16. HLA and Disease Association • B*27 • Ankylosing spondylitis • Reactive arthritis • Arthritis associated with psoriasis, Crohn disease and ulcerative colitis • B*57 • B*5701-Cw*0602 and psoriatic arthritis • B*5701 and Abacavir Hypersensitivity Syndrome (AHS) • Evidence for cellular immune basis, specifically CD8+ T cell responses (Chessman et al., 2008)

  17. Soluble Antiviral Factors (IFN-, TNF-, CAF) HIV T Cell Receptor HIV Peptide-HLA Class I CD8+ T Cell Cytotoxic Granules GrB Perforin Cytotoxicity Pathway (Perforin, Granzymes) The Immunologic Synapse HIV-Infected Target Cell

  18. Soluble Antiviral Factors (IFN-, TNF-, CAF) HIV T Cell Receptor HIV Peptide-HLA Class I CD8+ T Cell Cytotoxic Granules GrB Perforin Cytotoxicity Pathway (Perforin, Granzymes) The Immunologic Synapse HIV-Infected Target Cell

  19. 25 CD69+ IFN-+ CD8+ T cells (%) Tat Rev 20 Nef Env Pol 15 Gag 10 5 0 3 4 5 7 9 10 17 6 8 25 1 20 21 19 27 15 107 106 29 14 108 2 105 104 102 101 103 B A C D LTNP <50 SP 3,996-7,774 Progressors 2,486-249,966 Treated 1,008-7,873 Migueles et al., Proc Natl Acad Sci, 2000 Gea-Banacloche et al., J Immunol, 2000 Migueles and Connors, Imm Letters, 2001 High Frequencies of HIV-Specific CD8+ T Cells Persist in Patients with Poor Restriction of Virus Replication

  20. Qualitative Features of HIV-Specific CD8+ T Cells

  21. CFSE-Labeled Effectors Fluorescent Dye to Track CD8+ T Cell Proliferation Targets (CD4+ T Cells)

  22. 0.27 99.7 CD8+ T Cell Proliferation to Autologous HIV-Infected CD4+ T Cells is Greater in LTNP Uninfected CD4 Targets Infected CD4 Targets 4.67e-3 100 1.58 98.4 Progressor CD8 75.6 24.4 LTNP Migueles et al., Nature Immunol, 2002 CFSE

  23. Infected Uninfected Day 1 0 26.5 1.19e-3 27 Day 2 1.49e-3 5.94e-3 73 73.5 0.02 22.6 3.2e-3 22.6 Day 3 0.019 77.4 0.027 77.3 7.36e-3 17 2.93 21.7 Day 4 0.046 23.1 14.1 22.3 0.037 83 0.3 75.1 0.81 62.9 0.019 76.8 Day 5 43.5 17.7 0.052 18 0.023 81.9 1.04 37.8 Day 6 0.03 18.9 63.7 18.7 Perforin CFSE 0.044 81.1 0.32 17.2 CD8+ T Cell Proliferation Is Coupled with Perforin Expression LTNP Net Perforin Expression Perforin+ CD8+ T Cells (%) Day #

  24. 87.9 85.6 58.4 61.5 45.7 46.4 Day 6 HIV-Specific CD8+ T Cells of LTNP Exhibit Greater Up-Regulation of Cytotoxic Proteins than Cells of Progressors P<0.01 P=0.004 P>0.5 GrB Perforin CD107 LTNP Progressors

  25. Fluorogenic GrB Substrate Cytotoxic Granules “Day 6 or “Day 0” CD8+ T Cell GrB Perforin 6 hours 1 hour GrB Activity ICE Infection by p24 (E:T) IFN-(E:T) In Vitro Cytotoxicity Assay for Measuring Granzyme B Activity in Live Cells and Infected Cell Elimination (ICE) Effector Target HIV HIV-Infected CD4+ T Cell

  26. Quantitative and Qualitative Models of a Recall Response of HIV-Specific CD8+ T Cells Memory Cell Effector Cell Antigen Recognition Primarily Quantitative Expansion Cytolytic Antigen Recognition Quantitative and Qualitative Expansion Poorly Cytolytic

  27. Granzyme B Target Cell Activity Infected CD4 Elimination P<0.001 P<0.001 P<0.001 P<0.001 P<0.001 P=0.01 GrB Substrate+ Targets (%) Infected CD4 Elimination (%) 100 50 40 75 30 50 20 25 10 0 0 LTNP Prog Rx<50 LTNP Prog Rx<50 LTNP Prog Rx<50 LTNP Prog Rx<50 D#0 D#0 D#0 D#0 D#0 D#0 D#6 D#6 D#6 D#6 D#6 D#6 Perforin+ CD8+ T cells D#6 (%) 100 Infected CD4 Elimination D#6 (%) 70 60 75 50 40 50 30 R=0.79, P<0.001 R=0.92, P<0.001 20 25 10 0 0 0 10 20 30 40 50 0 10 20 30 40 50 60 70 GrB Substrate+ Targets D#6 (%) GrB Substrate+ Targets D#6 (%) Day 6 HIV-Specific CD8+ T Cells of LTNP Mediate Greater Cytotoxicity of HIV-Infected CD4+ T Cell Targets than Cells of Progressors Migueles et al., Immunity, 2008

  28. Two Models Demonstrating Similar or Disparate Per-Cell Cytotoxic Capacity of HIV-Infected CD8+ T Cells LTNP P>0.5 P<0.001 100 100 Progressors Infected CD4 Elimination D#6 (%) Infected CD4 Elimination D#6 (%) 80 80 60 60 True Effector:Target Ratio 40 40 20 20 0 0 0 5 10 0 5 10 15 20 15 20 25 25

  29. 100 80 60 40 20 0 0 5 10 15 20 25 Day 6 HIV-Specific CD8+ T Cells of LTNP Mediate Greater Cytotoxicity of HIV-Infected CD4+ T Cell Targets on a Per-Cell Basis than Cells of Progressors LTNP LTNP Progressors Rx<50 Infected CD4 Elimination D#6 (%) P<0.001 P<0.001 (IFN-+ CD8+ T Cells) Effector:Target (p24+) Ratio Migueles et al., Immunity, 2008 Migueles et al., J Virol, 2009

  30. Model of HIV-Specific CD8+ T Cell-Mediated Control of Virus Replication

  31. Summary/Conclusions • Immune-mediated control is associated with HLA B*57, but not with increased frequencies of HIV-specific CD8+ T cells. • HIV-specific CD8+ T cell proliferation in LTNP is significantly greater than the responses observed in progressors. • Expanded HIV-specific CD8+ T cells of LTNP have increased expression of the proteins contained within cytotoxic granules. • Following this lytic granule loading, HIV-specific CD8+ T cells of LTNP exhibit extraordinary cytotoxic capacity on a per-cell basis and eliminate HIV-infected CD4+ T cell targets by the efficient delivery of functional GrB. • These HIV-specific functions distinguish patients who maintain control of HIV and should be considered in the next generation of HIV vaccines.

  32. Acknowledgements CMRS/LIR Julia Rood Amy Berkley Daniel Mendoza Tiffany Guo Christine Osborne Alex Compton Rohan Joshi Kristin Weeks Prasanna Jagannathan Andy Patamawenu Alisha C. Laborico Claire W. Hallahan JoAnn Mican Richard T. Davey Mark Connors H. Clifford Lane Anthony S. Fauci SAIC/NCI/Frederick Hiromi Imamichi Frank Maldarelli Sarah Palmer Ann Weigand Clinics Nancy Cogliano-Shutta Margaret Lloyd Mary McLaughlin Richard Kwan Gregg Roby Julia A. Metcalf Sara Stallings Catherine Rehm NIH HLA Laboratory Sharon Adams SAIC/NCI/Frederick Beverly Z. Packard Akira Komoriya *The Patients

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