1. Medical Immunology Immunobiology of HIV infection Jan 10, 2013 Medical Immunology Immunobiology of HIV infection Jan 10, 2013 Keith Fowke 539 BMSB 789-3818 fowkekr@cc.umanitoba.ca

2. Medical Immunology IMed 7190 Topic: HIV resistance Lecturer: Keith Fowke Objectives: –To discuss why HIV induces immune suppression –To discuss why some individuals are resistant to infection Expectations: –To list two main hypotheses why HIV infection leads to AIDS –To discuss the immunological and non-immunological methods of resistance to HIV infection

3. Outline Epidemiology of the disease HIV DiseaseHIV replication Why does HIV cause immunodeficiency? What does the CD4+ T cell do? Three types of T-helper cell. How does HIV decrease CD4+ levels? Apoptosis in HIV infectionHIV Resistance Mechanisms of Resistance

4. Total: 34.0 million [31.6 million – 35.2 million] Western & Central Europe 840 000 [770 000 – 930 000] Middle East & North Africa 470 000 [350 000 – 570 000] Sub-Saharan Africa 22.9 million [21.6 million – 24.1 million] Eastern Europe & Central Asia 1.5 million [1.3 million – 1.7 million] South & South-East Asia 4.0 million [3.6 million – 4.5 million] Oceania 54 000 [48 000 – 62 000] North America 1.3 million [1.0 million – 1.9 million] Latin America 1.5 million [1.2 million – 1.7 million] East Asia 790 000 [580 000 – 1.1 million] Caribbean 200 000 [170 000 – 220 000] Adults and children estimated to be living with HIV  2010 UNAIDS 2011

5. Estimated number of adults and children newly infected with HIV  2010 Western & Central Europe 30 000 [22 000 – 39 000] Middle East & North Africa 59 000 [40 000 – 73 000] Sub-Saharan Africa 1.9 million [1.7 million – 2.1 million] Eastern Europe & Central Asia 160 000 [110 000 – 200 000] South & South-East Asia 270 000 [230 000 – 340 000] Oceania3300 [2400 – 4200] North America 58 000 [24 000 – 130 000] Latin America 100 000 [73 000 – 140 000] East Asia 88 000 [48 000 – 160 000] Caribbean 12 000 [9400 – 17 000] Total: 2.7 million [2.4 million – 2.9 million] UNAIDS 2011 ~7,400 people HIV infected daily ~300 infected during this talk

6. Estimated adult and child deaths from AIDS  2010 Western & Central Europe 9900 [8900 – 11 000] Middle East & North Africa 35 000 [25 000 – 42 000] Sub-Saharan Africa 1.2 million [1.1 million – 1.4 million] Eastern Europe & Central Asia 90 000 [74 000 – 110 000] South & South-East Asia 250 000 [210 000 – 280 000] Oceania 1600 [1200 – 2000] North America 20 000 [16 000 – 27 000] Latin America 67 000 [45 000 – 92 000] East Asia 56 000 [40 000 – 76 000] Caribbean 9000 [6900 – 12 000] Total: 1.8 million [1.6 million – 1.9 million] UNAIDS 2011 ~4,900 people die daily ~200 die during this talk

7. Life Expectancy and HIV

8. 2008: 65,000 people living with HIV in Canada PHAC: Estimates of HIV Prevalence and Incidence in Canada, 2008

9. Annual Number of Individuals Testing HIV Antibody Positive 1985-2008 in Manitoba Manitoba Health & Healthy Living Statistical Update on HIV/AIDS January 1985 –December 2007 (http://www.gov.mb.ca/health/publichealth/cdc/surveillance/dec2007.pdf) In 2011 there are more than 1100 people in HIV Care in Manitoba

10. HIV in Manitoba 95 New Cases in 2011 Source: Manitoba HIV Program 2012 Report

11. Outline Epidemiology of the disease HIV DiseaseHIV replication Why does HIV cause immunodeficiency? What does the CD4+ T cell do? Three types of T-helper cell. How does HIV decrease CD4+ levels? Apoptosis in HIV infectionHIV Resistance Mechanisms of Resistance

12. A diagnosis of AIDS is made whenever a person is HIV positive and: he or she has a CD4+ cell count <200 cells/µL, or his or her CD4+ cells account for <14% of all lymphocytes, or that person has been diagnosed with one or more of the AIDS-defining illnesses listed below. AIDS-defining illnesses: Candidiasis of bronchi, trachea, or lungs Candidiasis, esophageal Cervical cancer, invasive* Coccidioidomycosis, disseminated Cryptococcosis, extrapulmonary Cryptosporidiosis, chronic intestinal (>1-month duration) Cytomegalovirus disease (other than liver, spleen, or lymph nodes) Cytomegalovirus retinitis (with loss of vision) Encephalopathy, HIV related# (see Dementia) Herpes simplex: chronic ulcer(s) (>1-month duration) or bronchitis, pneumonitis, or esophagitis Histoplasmosis, disseminated Isosporiasis, chronic intestinal (>1-month duration) Kaposi sarcoma Lymphoma, Burkitt Lymphoma, immunoblastic Lymphoma, primary, of brain (primary central nervous system lymphoma) Mycobacterium avium complex or disease caused by M kansasii, disseminated Disease caused by Mycobacterium tuberculosis, any site (pulmonary*or extrapulmonary#) Disease caused by Mycobacterium, other species, or unidentified species, disseminated Pneumocystis jiroveci (formerly carinii) pneumonia Pneumonia, recurrent* Progressive multifocal leukoencephalopathy Salmonella septicemia, recurrent Toxoplasmosis of brain (encephalitis) Wasting syndrome caused by HIV infection# Additional illnesses that are AIDS defining in children, but not adults Multiple, recurrent bacterial infections# Lymphoid interstitial pneumonia/pulmonary lymphoid hyperplasia

13. HIV Genes and Proteins Peterlin et al Nature Reviews Immunol 3; 97-107 (2003)

14. HIV Structure exhiv.chat.ru

16. HIV Life Cycle Peterlin et al Nature Reviews Immunol 3; 97-107 (2003) 1.HIV enters via CD4 2.RNA reverse transcribed into DNA 3.DNA integrates into host genome 4.Latency? 5.Replication produces proteins 6.Proteins assemble into new viruses

17. Treating HIV Infection Peterlin et al Nature Reviews Immunol 3; 97-107 (2003) Three main sites for HIV drugs A.Reverse transcriptase B.HIV protease C.HIV entry D.Integration A. C. B. Main classes of HIV drugs 1.Nucleoside analogues (zidovudine) - A 2.Non-nucleoside (nevaripine) - A 3.Protease Inhibitors (indinavir) - B 4.Chemokine Receptor Antagonists (maraviroc) – C 5.Fusion Inhibitors – (enfuvirtide) - C 6.Integrase Inhibitors - (elvitegravir) - D D.

18. Role of DCs in HIV Infection Nature Reviews Immunology 2; 957-965 (2002)

19. Lymphatic System

20. The Kinetics of HIV Disease Progression 01361224364860728496108120132144 0 2 4 6 8 10 12 CD4 + T cells HIV CTL Neut Ab HIV viral load Death Time Post Infection (Months) Relative Values Acute Phase Asymptomatic Phase AIDS Alimonti, Ball & Fowke, J GenVirol (in press)

21. Outline Epidemiology of the disease HIV DiseaseHIV replication Why does HIV cause immunodeficiency? What does the CD4+ T cell do? Three types of T-helper cell. How does HIV decrease CD4+ levels? Apoptosis in HIV infectionHIV Resistance Mechanisms of Resistance

22. Hallmark of HIV disease Loss of CD4+ T cells from peripheral blood What is the role of CD4+ T cells in the immune response?

23. CD4+ T helper cells: Conductors of the Immune System

24. Subsets of CD4+ T helper cells APC+Ag IL-2 IL-12 IL-4 IFN-  Dominant Cellular Immunity Dominant Humoral Immunity IFN-  IL-4 IL-5 IL-13 IL-4, IL-10 IFN-  Naive CD4+ Tcell Activated CD4+ Tcell Th2Th1 T reg Suppression Th17

25. The Kinetics of HIV Disease Progression 01361224364860728496108120132144 0 2 4 6 8 10 12 CD4 + T cells HIV CTL Neut Ab HIV viral load Death Time Post Infection (Months) Relative Values Acute Phase Asymptomatic Phase AIDS Alimonti, Ball & Fowke, J GenVirol (in press)

26. Mechanisms for CD4+ cell decline Direct Synctia formation (cell-cell fusion) Direct viral cytopathic effect Indirect Apoptosis/PCD Activation Induced Cell Death Autoimmune mechanisms Homology of viral proteins to self antigens Superantigen-mediated deletion Viral proteins acting as superantigens Type 1/Type 2 cytokine dysregulation

27. CD4 APC Time 0 hrs Time 6 hrs CD8CD4 9.20 090.8 25.80 074.2 8.212.3 079.5 0 49.528.3 22.2 CD8 CD4 Detection of Apoptosis

28. Fowke et al AIDS 11:1016, 1997

29. Apoptosis in HIV infection Mechanisms: gp120/41 - CD4 crosslinking, ↓ BCL-2, ↑CD95(Fas)/CD95L(FasL) gp120 induction of syncytia HIV protease activates caspase 8 and ↓ BCL-2 Tat – ↑ Caspase 8, Fas, FasL and ↓BCL-2 Vpr – membrane disruption of mitochondrion Nef - myristylated N-terminus interacts with TCR and leads to upregulation of Fas/L Fas/FasL – altered in T cells and monocytes due to nef AICD – increased Fas/FasL

30. CD4’s Role in Signal transduction T-Cell Activation

31. gp120-induced CD4-crosslinking Uninfected CD4 T cell CD4 cross-linking activates lck ↑ CD95(Fas) ↓ BCL-2 apoptosis = CD4 =sgp120 =p56 lck =P-p56lck

32. HIV nef effects on CD4 and MHC I Peterlin et al Nature Reviews Immunol 3; 97-107 (2003)

33. Outline Epidemiology of the disease HIV DiseaseHIV replication Why does HIV cause immunodeficiency? What does the CD4+ T cell do? Three types of T-helper cell. How does HIV decrease CD4+ levels? Apoptosis in HIV infectionHIV Resistance Mechanisms of Resistance

34. Kenya Nairobi Kisumu HIV prevalence 14% in 1997 6.7 in 2003 8.5% in 2007 6.2% in 2011 in adults Source UNAIDS Nairobi

35. UM’s contribution to HIV/AIDS 1.Heterosexual transmission of HIV 2.Mother to child transmission – incl breast milk 3.STI’s as significant risk factors 4.Directed interventions prevent new infections 5.Male circumcision clinical trial showed protection 6.HIV resistance

36. Majengo Clinic

37. Focus The health of commercial sex workers Provides Primary health care Trained physicians, nurses, pharmacist STI treatment HIV prevention education Condoms (male and female) HIV counseling HIV treatment Research

38. Majengo Clinic Staff Photos by Rich Lester & Keith Fowke

39. Majengo Clinic Clients

40. Majengo Clinic Baraza 2009 Photos by Rich Lester

41. Nairobi Sex Worker Study Pumwani cohort Est. in 1985, open cohort > 4000 women enrolled Average 4 clients/day most are HIV+ at entry, those not seroconvert within 2 yrs ~110 uninfected despite up to 500 unprotected exposures Exposure or co-factor determinants not different HIV resistance defined as: 1.No evidence of HIV infection 2.Still active in sex work 3.Followed in cohort for >7 years

42. HIV Resistance – Data Summary Resistance is not: ▫ Absolute ▫ Differing sexual practices ▫ Seronegative infection ▫ Decreased susceptibility to other infections ▫ Coreceptor polymorphisms ▫ enhanced  -chemokine production Resistance associates with: ▫ HIV-specific cellular immunity  CTL, CD4+ T cell responses in PBMC (Fowke et al.)  Mucosal CTL responses (Kaul et al.)  Qualitatively distinct responses  strong proliferation, weak IFN  (Alimonti et al.) ▫ Genetic basis for resistance  Familial association (Kimani)  Kindred of HIV-R more likely to remain HIV-negative (Kimani, Ball)  Polymorphisms associated with resistance, e.g. IRF-1 (Ji, Ball) Few data linking immune and genetic associations

43. HIV Resistance – Data Summary Resistance is not: ▫ Differing sexual practices ▫ Seronegative infection ▫ Decreased susceptibility to other infections ▫ Coreceptor polymorphisms ▫ enhanced  -chemokine production Resistance is: ▫ HIV-specific cellular immunity  CTL, CD4+ T cell responses in PBMC (Fowke et al.)  Mucosal CTL responses (Kaul et al.)  Qualitatively distinct responses  strong proliferation, weak IFN  (Alimonti et al.) ▫ Genetic basis for resistance  Familial association (Kimani)  Kindered of HIV-R more likely to remain HIV-negative (Kimani, Ball)  Polymorphisms associated with resistance, e.g. IRF-1 (Ji, Ball) Few data linking immune and genetic associations Hypotheses: Resistance is mediated by immune and genetic components HIV-R women will have HIV-specific T cell responses HIV-R women will have high levels of immune activation to fight infection

44. Immune Environment of Resistants is Different than HIV+ p  0.001 p=NS p  0.012 p=NS p=0.002 p=NS Resistant

45. HIV-specific CD4+ T cells in HESN Fowke et al Immunology and Cell Biology, 2000

46. Qualitatively Distinct Responses in RES Better Proliferative Responses Alimonti et al JID, 2005 0 2 4 6 8 10 resposneglo cpm (X10 3 ) ESNHIV + Nlow p24 peptides p=0.002 n = 6 12 1 0 2 4 6 8 10 resposneglo cpm (X10 3 ) ESNHIV + Nlow p24 peptides p=0.002 n = 6 12 1 0 2 4 6 8 10 resposneglo cpm (X10 3 ) RESHIV + Nlow p24 peptides p=0.002 n = 6 12 1

47. T CM higher in Resistants S Koesters

48. Two-phase model of HIV-resistance Mucosal

49. Assessment of T cell Function T cell functional assays ▫ Cytokine production ▫ Cellular activation markers Gene expression analysis ▫ Purified CD4+ T cells  9 Res, 9 High-risk negatives ▫ Whole Blood  23 Res, 19 Low-risk negatives ▫ Used Affymetrix U133 Plus 2.0

50. Res Negs CD4 T cellsWhole Blood Gene expression profiling in HIV Resistants Res Neg McLaren et al JID 2010

51. Whole Blood Res Neg T cell receptor signaling pathway:

52. Reproduced with permission www.biorag.org T cell receptor signaling CD4+ whole blood

53. Zap70 Proteasome Stau1 Kif22 PP1 NF-κB CypA http://www.tibotec.com HIV Replication

54. Assessment of T cell Function T cell functional assays ▫ Cytokine production ▫ Cellular activation markers Gene expression analysis ▫ Purified CD4+ T cells  9 Res, 9 High-risk negatives ▫ Whole Blood  23 Res, 19 Low-risk negatives ▫ Used Affymetrix U133 Plus 2.0

55. Baseline Cytokine production Resistant Negative

56. Resistants have normal recall responses Differences between HIV-R and HIV-N not observed after stimulation HIV-R women have normal recall responses but show lower baseline immune activation

57. HIV Replicates Better in an Activated Cell Peterlin et al Nature Reviews Immunol 3; 97-107 (2003)

58. Baseline T cell activation HIV replicates better in activated T cells HIV-R have fewer activated (CD69+) CD4+ and CD8+ T cells Card et al JID 2009

59. Immune Quiescence in HIV resistance Lower overall gene expression, CD4+ T cells and whole blood Lower gene expression in HIV and T cell receptor pathways Lower resting PBMC cytokine production Lower level of cellular activation on T cell Normal Antigen recall function – not immune suppression OVERALL immune cells seem to be resting or quiescent Termed this phenotype Immune Quiescence

60. Evidence of IQ in other cohorts Amsterdam cohort of HIV-R MSM (Koning et. al. J Immunol. 2005) –↓ frequencies of activated (HLA DR, CD38, CD70) CD4+ T cells and proliferating (Ki67) CD4+ and CD8+ T cells Abijan cohort of HIV-R CSW (Jennes et. al. Clin Exp. Immunol. 2006) –↓ CD69, IFN , MIP-1  and RANTES following allo-stimulation Hemophiliacs, highly exposed (Salkowitz et al Clin Imm 2001) –Low immune activation in exposed uninfecteds Discordant couples in Central African Republic (Begaud et. al. Retrovirology 2006) –↓ frequencies of activated (HLA DR, CCR5) CD4+ T cells –Reduced HIV susceptibility in unstimulated PBMC –Differences not observed when PHA stimulated cells were infected However, Clerici shows increased TLR activity associated with protection

61. T regs as IQ mediators HIV-R have elevated frequencies of regulatory T cells

62. Two phase model of HIV resistance

63. What is driving Immune Quiescence? Systemic Tregs correlate ? Transcriptional Factors ? Mucosal Factors

64. Two-phase model of HIV-resistance Mucosal

65. HIV replication in quiescent CD4+ Tcells?

66. HIV replication in quiescent cells Card et al Plos One 2012

67. HIV replication in quiescent cells Individuals vary in their ability to support HIV replication Ex vivo levels of T cell activation correlate with a ability to support HIV replication In the infected cultures, infected cells are more highly activated Among the infected cells, T regs are enriched

68. Evidence for IQ at mucosal surface?

69. Cervical Lavage Chemokine Levels Julie Lajoie, et al Mucosal Immunology 2012

70. Fewer Target cells at Mucosa C. Card Cervical LavageCervical Biopsy K. Broliden

71. Mucosal Immune Quiescence HESN have fewer CD4+CCR5+ T cells HESN have lower levels of the inflammatory cytokine IL-1a HESN have lower levels of the T cell migratory factors MIG and IP-10.

72. Mucosal IQ model

73. Role of Immune Quiescence in HESN Evidence of HIV-specific CD4+ and CD8+ T cell responses Lower levels of T cell activation Normal ability to respond to antigen Quiescent cells do not support HIV replication as well IQ phenotype extends to genital mucosa Fewer target cells – lower susceptibility to HIV

74. Two-phase model of HIV-resistance Mucosal 1. Vaccinate against HIV try to drive T CM No exposure during activation phase 2. Maintain a quiescent phenotype at mucosa Stimulate mucosal Tregs Microbicides with anti-inflammatory activity

75. Thanks CollaboratorsThe Funders Frank Plummer Blake Ball Ma Luo Joshua Kimani Walter Jaoko Ruey Su Aida Sivro Elijah Songok Paul McLaren Catherine Card Charles Wachihi Majengo Clinic staff MCH Clinic staff MHRC CIHR BM Gates Foundation U NIVERSITY OF N AIROBI

76. Ongoing studies 1.Evaluation of Immune Quiescence at the genital mucosa a)Activation phenotype b)Gene expression analysis 2.In vitro HIV infections of unstimulated PBMC 3.Use drugs to induce IQ in FGT 4.Validate in other cohorts

77. Thanks CollaboratorsThe Funders Frank Plummer Keith Fowke Blake Ball Ma Luo Joshua Kimani Walter Jaoko Ruey Su Aida Sivro Elijah Songok Paul McLaren Catherine Card Charles Wachihi Majengo Clinic staff MCH Clinic staff MHRC CIHR BM Gates Foundation U NIVERSITY OF N AIROBI

78. Laboratory of Viral Immunology

79. Winnipeg and Nairobi Research Teams

80. Majengo Clinic Staff and Clients Photo used with permission

81. The Hope for an HIV Vaccine

82. Take home message 1.HIV infects and kills the central cell of the immune system 2.HIV proteins can either promote or block apoptosis 3.HIV resistance is multi-factoral 4.Genetic, cell-mediated and immune quiescence mechanisms involved in resistance