Medical Immunology Immunobiology of HIV infection Jan 10, 2013 Medical Immunology Immunobiology of HIV infection Jan 10, 2013 Keith Fowke 539 BMSB

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 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

Total: 34.0 million [31.6 million – 35.2 million] Western & Central Europe [ – ] Middle East & North Africa [ – ] 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 [ – ] North America 1.3 million [1.0 million – 1.9 million] Latin America 1.5 million [1.2 million – 1.7 million] East Asia [ – 1.1 million] Caribbean [ – ] Adults and children estimated to be living with HIV  2010 UNAIDS 2011

Estimated number of adults and children newly infected with HIV  2010 Western & Central Europe [ – ] Middle East & North Africa [ – ] Sub-Saharan Africa 1.9 million [1.7 million – 2.1 million] Eastern Europe & Central Asia [ – ] South & South-East Asia [ – ] Oceania3300 [2400 – 4200] North America [ – ] Latin America [ – ] East Asia [ – ] Caribbean [9400 – ] Total: 2.7 million [2.4 million – 2.9 million] UNAIDS 2011 ~7,400 people HIV infected daily ~300 infected during this talk

Estimated adult and child deaths from AIDS  2010 Western & Central Europe 9900 [8900 – ] Middle East & North Africa [ – ] Sub-Saharan Africa 1.2 million [1.1 million – 1.4 million] Eastern Europe & Central Asia [ – ] South & South-East Asia [ – ] Oceania 1600 [1200 – 2000] North America [ – ] Latin America [ – ] East Asia [ – ] Caribbean 9000 [6900 – ] Total: 1.8 million [1.6 million – 1.9 million] UNAIDS 2011 ~4,900 people die daily ~200 die during this talk

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 in Manitoba Manitoba Health & Healthy Living Statistical Update on HIV/AIDS January 1985 –December 2007 ( In 2011 there are more than 1100 people in HIV Care in Manitoba

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

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

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; (2003)

HIV Structure exhiv.chat.ru

HIV Life Cycle Peterlin et al Nature Reviews Immunol 3; (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

Treating HIV Infection Peterlin et al Nature Reviews Immunol 3; (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.

Role of DCs in HIV Infection Nature Reviews Immunology 2; (2002)

Lymphatic System

The Kinetics of HIV Disease Progression 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)

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

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 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

The Kinetics of HIV Disease Progression 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)

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

CD4 APC Time 0 hrs Time 6 hrs CD8CD CD8 CD4 Detection of Apoptosis

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

HIV nef effects on CD4 and MHC I Peterlin et al Nature Reviews Immunol 3; (2003)

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

Kenya Nairobi Kisumu HIV prevalence 14% in in % in % in 2011 in adults Source UNAIDS Nairobi

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

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  -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

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

Immune Environment of Resistants is Different than HIV+ p  p=NS p  p=NS p=0.002 p=NS Resistant

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

Qualitatively Distinct Responses in RES Better Proliferative Responses Alimonti et al JID, resposneglo cpm (X10 3 ) ESNHIV + Nlow p24 peptides p=0.002 n = resposneglo cpm (X10 3 ) ESNHIV + Nlow p24 peptides p=0.002 n = resposneglo cpm (X10 3 ) RESHIV + Nlow p24 peptides p=0.002 n =

T CM higher in Resistants S Koesters

Two-phase model of HIV-resistance Mucosal

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

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

Whole Blood Res Neg T cell receptor signaling pathway:

Reproduced with permission T cell receptor signaling CD4+ whole blood

Zap70 Proteasome Stau1 Kif22 PP1 NF-κB CypA HIV Replication

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

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; (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, 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

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 C. Card Cervical LavageCervical Biopsy 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 T CM No exposure during activation phase 2. Maintain a quiescent phenotype at mucosa Stimulate mucosal Tregs Microbicides with anti-inflammatory activity

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

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 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

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