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Medical Immunology Immunobiology of HIV infection Jan 10, 2013 Keith Fowke 539 BMSB 789-3818 [email protected] 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 Outline Epidemiology of the disease HIV Disease HIV 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 infection HIV Resistance Mechanisms of Resistance Adults and children estimated to be living with HIV 2010 Eastern Europe Western & Central Europe & Central Asia 840 000 1.5 million [770 000 – 930 000][1.3 million – 1.7 million] North America 1.3 million East Asia [1.0 million – 1.9 million] 790 000 Middle East & North Africa Caribbean 200 000 [170 000 – 220 000] Latin America 1.5 million [1.2 million – 1.7 million] [580 000 – 1.1 million] 470 000 [350 000 – 570 000] South & South-East Asia 4.0 million Sub-Saharan Africa [3.6 million – 4.5 million] [21.6 million – 24.1 million] Oceania 22.9 million 54 000 [48 000 – 62 000] Total: 34.0 million [31.6 million – 35.2 million] UNAIDS 2011 Estimated number of adults and children newly infected with HIV 2010 Eastern Europe Western & Central Europe & Central Asia 30 000 [22 000 – 39 000] North America 58 000 160 000 [110 000 – 200 000] [24 000 – 130 000] East Asia 88 000 [48 000 – 160 000] ~7,400 people HIV 59 000 infected daily Middle East & North Africa Caribbean 12 000 [9400 – 17 000] [40 000 – 73 000] South & South-East Asia 270 000 Sub-Saharan Africa [230 000 – 340 000] 1.9 million ~300 100 infected during this talk 000 3300 Latin America [73 000 – 140 000] [1.7 million – 2.1 million] Oceania [2400 – 4200] Total: 2.7 million [2.4 million – 2.9 million] UNAIDS 2011 Estimated adult and child deaths from AIDS 2010 Eastern Europe Western & Central Europe & Central Asia 9900 [8900 – 11 000] North America 20 000 90 000 [74 000 – 110 000] East Asia [16 000 – 27 000] 56 000 ~4,900 people die daily 35 000 9000 250 000 ~200 die during this talk Middle East & North Africa Caribbean [6900 – 12 000] Latin America 67 000 [45 000 – 92 000] [25 000 – 42 000] Sub-Saharan Africa 1.2 million [1.1 million – 1.4 million] [40 000 – 76 000] South & South-East Asia [210 000 – 280 000] Oceania 1600 [1200 – 2000] Total: 1.8 million [1.6 million – 1.9 million] UNAIDS 2011 Life Expectancy and HIV 2008: 65,000 people living with HIV in Canada PHAC: Estimates of HIV Prevalence and Incidence in Canada, 2008 Annual Number of Individuals Testing HIV Antibody Positive 1985-2008 in Manitoba In 2011 there are more than 1100 people in HIV Care 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) HIV in Manitoba 95 New Cases in 2011 Source: Manitoba HIV Program 2012 Report Outline Epidemiology of the disease HIV Disease HIV 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 infection HIV Resistance Mechanisms of Resistance 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 HIV Genes and Proteins Peterlin et al Nature Reviews Immunol 3; 97-107 (2003) HIV Structure exhiv.chat.ru HIV Life Cycle 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 Peterlin et al Nature Reviews Immunol 3; 97-107 (2003) Treating HIV Infection Three main sites for HIV drugs A. Reverse transcriptase B. HIV protease C. HIV entry D. Integration Main classes of HIV drugs 1. 2. 3. 4. 5. 6. C. B. A. Nucleoside analogues (zidovudine) - A Non-nucleoside (nevaripine) - A Protease Inhibitors (indinavir) - B Chemokine Receptor Antagonists (maraviroc) – C Fusion Inhibitors – (enfuvirtide) - C Integrase Inhibitors - (elvitegravir) - D D. Peterlin et al Nature Reviews Immunol 3; 97-107 (2003) Role of DCs in HIV Infection Nature Reviews Immunology 2; 957-965 (2002) Lymphatic System The Kinetics of HIV Disease Progression Acute Phase Asymptomatic Phase AIDS 12 10 CD4+ T cells 8 HIV CTL 6 Death Neut Ab 4 HIV viral load 2 0 0 1 3 6 12 24 36 48 60 72 84 96 108 120 132 144 Time Post Infection (Months) Alimonti, Ball & Fowke, J GenVirol (in press) Outline Epidemiology of the disease HIV Disease HIV 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 infection HIV Resistance Mechanisms of Resistance Hallmark of HIV disease • Loss of CD4+ T cells from peripheral blood What is the role of CD4+ T cells in the immune response? CD4+ T helper cells: Conductors of the Immune System Subsets of CD4+ T helper cells Th17 Naive CD4+ Tcell APC+Ag IL-4 IL-12 IL-2 IFN-g IL-4 IFN-g Th1 T reg Activated CD4+ Tcell Suppression Th2 IL-4, IL-10 IFN- g Dominant Cellular Immunity IL-4 IL-5 IL-13 Dominant Humoral Immunity The Kinetics of HIV Disease Progression Acute Phase Asymptomatic Phase AIDS 12 10 CD4+ T cells 8 HIV CTL 6 Death Neut Ab 4 HIV viral load 2 0 0 1 3 6 12 24 36 48 60 72 84 96 108 120 132 144 Time Post Infection (Months) Alimonti, Ball & Fowke, J GenVirol (in press) 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 Detection of Apoptosis CD4 APC CD8 9.2 CD4 12.3 0 8.2 Time 0 hrs 90.8 79.5 0 CD4 CD8 25.8 0 0 28.3 49.5 Time 6 hrs 74.2 0 22.2 0 Relationship Between Apoptosis and CD4 or Virus Levels 100 % Apoptotic Nuclei S pontaneous 100 % Apoptotic Nuclei S pontaneous Mitogen 80 Mitogen 80 60 60 40 40 20 20 0 0 -20 0 200 400 600 800 100012001400 CD4 Counts 0 100 200 300 400 500 600 HIV Titre (KEq/ml) Fowke et al AIDS 11:1016, 1997 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 CD4’s Role in Signal transduction T-Cell Activation gp120-induced CD4-crosslinking = CD4 =sgp120 =p56lck =P-p56lck Uninfected CD4 T cell CD4 cross-linking activates lck ↑ CD95(Fas) ↓ BCL-2 apoptosis HIV nef effects on CD4 and MHC I Peterlin et al Nature Reviews Immunol 3; 97-107 (2003) Outline Epidemiology of the disease HIV Disease HIV 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 infection HIV Resistance Mechanisms of Resistance Kenya Kisumu Nairobi Nairobi •HIV prevalence •14% in 1997 •6.7 in 2003 •8.5% in 2007 •6.2% in 2011 in adults Source UNAIDS 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 Majengo Clinic Majengo Clinic •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 Majengo Clinic Staff Photos by Rich Lester & Keith Fowke Majengo Clinic Clients Majengo Clinic Baraza 2009 Photos by Rich Lester 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 • HIV Resistance – Data Summary • Resistance is not: ▫ ▫ ▫ ▫ ▫ ▫ Absolute Differing sexual practices Seronegative infection Decreased susceptibility to other infections Coreceptor polymorphisms enhanced b-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 IFNg (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 HIV Resistance – Data Summary • Resistance is not: Hypotheses: ▫ ▫ ▫ ▫ ▫ Differing sexual practices Seronegative infection Resistance is mediated by immune Decreased susceptibility to other infections components Coreceptor polymorphisms enhanced b-chemokine production and genetic HIV-R women will have HIV-specific T cell responses • Resistance is: ▫ HIV-specific cellular immunity HIV-R women will have high levels of immune CTL, CD4+ T cell responses in PBMC (Fowke et al.) activation to fight infection Mucosal CTL responses (Kaul et al.) Qualitatively distinct responses strong proliferation, weak IFNg (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 Immune Environment of Resistants is Different than HIV+ p=NS p0.012 Resistant p=NS p=NS p0.001 p=0.002 HIV-specific CD4+ T cells in HESN Fowke et al Immunology and Cell Biology, 2000 Qualitatively Distinct Responses in RES Better Proliferative Responses p24 peptides 10 p=0.002 cpm (X103) cpm 8 6 4 2 0 res ESN RES n= 6 pos HIV++ 12 Nlow neglo HIVNlow 1 Alimonti et al JID, 2005 Resistants – CD4+ T cell Subset Distribution B Resistants- CD8+ T cell Subset Distribution p<0.001 C D 8 Group Differences in CD8+ Tcm p = 0.031 20 p = 0.036 Tcm as % of Total CD8+ T Cells 50 40 30 20 10 p = 0.049 p = 0.038 10 ne w M L ne g ne n= g + 25 M C H M C n= H n 13 eg ne w M L M C n= H n ne 11 eg n= g + 29 M C H ne g 0 0 R es is n= tan 8 ts M L n n= ew 12 ne g Tcm as % of Total CD4+ T Cells 60 D m /td Na i ve m /td C D 4 C D 4 Te Te m Tc C D 4 C D 4 Group Differences in CD4+ Tcm Te 0 C D 8 0 Te 10 m 10 m 20 C D 8 20 p=0.007 30 m 30 40 Tc 40 p<0.001 50 C D 8 50 C p<0.001 60 p=0.001 % of CD8+ T Cells p<0.001 Na ïv e % of CD4+ T Cells 60 R es i n= sta 11 nt s M L ne n= w 18 ne g A TCM higher in Resistants S Koesters Two-phase model of HIV-resistance Mucosal Assessment of T cell Function • 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 • T cell functional assays ▫ Cytokine production ▫ Cellular activation markers Gene expression profiling in HIV Resistants CD4 T cells Res Whole Blood Negs Res Neg McLaren et al JID 2010 Whole Blood Res Neg T cell receptor signaling pathway: T cell receptor signaling CD4+ whole blood Reproduced with permission www.biorag.org HIV Replication CypA Proteasome Zap70 Stau1 PP1 NF-κB Kif22 http://www.tibotec.com Assessment of T cell Function • 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 • T cell functional assays ▫ Cytokine production ▫ Cellular activation markers Baseline Cytokine production Resistant Negative 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 HIV Replicates Better in an Activated Cell Peterlin et al Nature Reviews Immunol 3; 97-107 (2003) 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 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 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, IFNg, MIP-1b 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 T regs as IQ mediators • HIV-R have elevated frequencies of regulatory T cells Two phase model of HIV resistance What is driving Immune Quiescence? Systemic Tregs correlate ? Transcriptional Factors ? Mucosal Factors Two-phase model of HIV-resistance Mucosal HIV replication in quiescent CD4+ Tcells? HIV replication in quiescent cells • Card et al Plos One 2012 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 Evidence for IQ at mucosal surface? Cervical Lavage Chemokine Levels • Julie Lajoie, et al Mucosal Immunology 2012 Fewer Target cells at Mucosa Cervical Lavage Cervical Biopsy C. Card K. Broliden 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. Mucosal IQ model 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 Two-phase model of HIV-resistance Mucosal 1. Vaccinate against HIV •try to drive TCM •No exposure during activation phase 2. Maintain a quiescent phenotype at mucosa •Stimulate mucosal Tregs •Microbicides with anti-inflammatory activity Thanks Collaborators • • • • • • • • • • • • • 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 The Funders • MHRC • CIHR • BM Gates Foundation UNIVERSITY OF NAIROBI 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 Thanks Collaborators • • • • • • • • • • • • • • 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 The Funders • MHRC • CIHR • BM Gates Foundation UNIVERSITY OF NAIROBI Laboratory of Viral Immunology Winnipeg and Nairobi Research Teams Majengo Clinic Staff and Clients Photo used with permission The Hope for an HIV Vaccine 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