medical immunology immunobiology of hiv infection jan 10, 2013 medical immunology immunobiology of...

Medical ImmunologyImmunobiology of HIV infection

Jan 10, 2013

Medical ImmunologyImmunobiology of HIV infection

Jan 10, 2013

Keith Fowke539 BMSB789-3818

fowkekr@cc.umanitoba.ca

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

Total: 34.0 million [31.6 million – 35.2 million]

Western & Central Europe

840 000[770 000 – 930 000]

Middle East & North Africa470 000

[350 000 – 570 000]

Sub-Saharan Africa22.9 million

[21.6 million – 24.1 million]

Eastern Europe & Central Asia1.5 million

South & South-East Asia4.0 million

Oceania54 000

[48 000 – 62 000]

North America1.3 million

Latin America1.5 million

East Asia790 000

[580 000 – 1.1 million]Caribbean200 000

[170 000 – 220 000]

Adults and children estimated to be living with HIV 2010

UNAIDS 2011

Estimated number of adults and childrennewly infected with HIV 2010

30 00030 000[22 000 – 39 000]

Middle East & North Africa59 00059 000

[40 000 – 73 000]

Sub-Saharan Africa1.9 million1.9 million

Eastern Europe & Central Asia

160 000 160 000 [110 000 – 200 000]

South & South-East Asia270 000270 000

[230 000 – 340 000]

Oceania33003300

[2400 – 4200]

North America58 000

[24 000 – 130 000]

Latin America100 000100 000

[73 000 – 140 000]

East Asia88 00088 000

[48 000 – 160 000]

Caribbean12 000

[9400 – 17 000]

UNAIDS 2011

~7,400 people HIV infected daily

~300 infected during this talk

Estimated adult and child deaths from AIDS 2010

9900[8900 – 11 000]

Middle East & North Africa35 000

[25 000 – 42 000]

Sub-Saharan Africa1.2 million

Eastern Europe & Central Asia

90 000 [74 000 – 110 000]

South & South-East Asia250 000

[210 000 – 280 000]

Oceania1600

[1200 – 2000]

North America20 000

[16 000 – 27 000]

Latin America67 000

[45 000 – 92 000]

East Asia56 000

[40 000 – 76 000]Caribbean

9000[6900 – 12 000]

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 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)

200020012002200320042005200620072008

Annual Number of Individuals Testing HIV+ in Manitoba

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

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

Fusion of HIV using CD4 and chemokine receptor

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

Treating HIV Infection

Three main sites for HIV drugsA. Reverse transcriptaseB. HIV proteaseC. HIV entryD. Integration

Main classes of HIV drugs

1. Nucleoside analogues (zidovudine) - A2. Non-nucleoside (nevaripine) - A3. Protease Inhibitors (indinavir) - B4. Chemokine Receptor Antagonists (maraviroc) – C5. Fusion Inhibitors – (enfuvirtide) - C6. Integrase Inhibitors - (elvitegravir) - D

Role of DCs in HIV Infection

Nature Reviews Immunology 2; 957-965 (2002)

Lymphatic System

The Kinetics of HIV Disease Progression

0 1 3 6 12 24 36 48 60 72 84 96 108 120 132 1440

CD4+ T cells

HIV CTL

Neut Ab

HIV viral load

Time Post Infection (Months)

Acute Phase

AsymptomaticPhase

Alimonti, Ball & Fowke, J GenVirol (in press)

Outline

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-2IL-12 IL-4

IL-4 IFN-

Dominant Cellular Immunity

Dominant Humoral Immunity

IFN- IL-4IL-5

IL-13IL-4, IL-10

Naive CD4+ Tcell

Activated CD4+ Tcell

Th2Th1

Suppression

The Kinetics of HIV Disease Progression

0 1 3 6 12 24 36 48 60 72 84 96 108 120 132 1440

CD4+ T cells

HIV CTL

Neut Ab

HIV viral load

Time Post Infection (Months)

Acute Phase

AsymptomaticPhase

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

CD8 CD4

25.8 0

8.212.3

49.528.3

CD8 CD4

Detection of Apoptosis

Relationship Between Apoptosis and CD4 or Virus Levels

0 100 200 300 400 500 600

HIV Titre (KEq/ml)

100% Apoptotic Nuclei

Spontaneous Mitogen

0 200 400 600 800 1000 1200 1400

CD4 Counts

% Apoptotic Nuclei

Spontaneous Mitogen

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

=sgp120

=p56lck

=P-p56lck

HIV nef effects on CD4 and MHC I

Outline

Nairobi

Kisumu

•HIV prevalence•14% in 1997•6.7 in 2003•8.5% in 2007•6.2% 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+

p0.001

p0.012

p=0.002

Resistant

HIV-specific CD4+ T cells in HESN

Fowke et al Immunology and Cell Biology, 2000

Qualitatively Distinct Responses in RESBetter Proliferative Responses

Alimonti et al JID, 2005

res pos neglo

ESN HIV+ HIVNlow

p24 peptides

p=0.002

n = 6 12 1

res pos neglo

ESN HIV+ HIVNlow

p24 peptides

p=0.002

n = 6 12 1

res pos neglo

RES HIV + HIV Nlow

p24 peptides

p=0.002

n = 6 12 1

TCM higher in Resistants

Resistants – CD4+ T cell Subset Distribution

CD4 Naï

CD4 Tcm

CD4 Tem

60 p<0.001 p=0.001

p<0.001

CD4 Naï

CD4 Tcm

CD4 Tem

CD4 Naï

CD4 Tcm

CD4 Tem

60 p<0.001 p=0.001

p<0.001

Resistants- CD8+ T cell Subset Distribution

CD8 Nai

CD8 Tcm

CD8 Tem

ells p<0.001

p=0.007

p<0.001

CD8 Nai

CD8 Tcm

CD8 Tem

CD8 Nai

CD8 Tcm

CD8 Tem

ells p<0.001

p=0.007

p<0.001

Group Differences in CD4+ Tcm

60 p = 0.031p = 0.036

p = 0.049p = 0.038

CD4 Naï

CD4 Tcm

CD4 Tem

60 p<0.001 p=0.001

p<0.001

CD4 Naï

CD4 Tcm

CD4 Tem

CD4 Naï

CD4 Tcm

CD4 Tem

60 p<0.001 p=0.001

p<0.001

CD8 Nai

CD8 Tcm

CD8 Tem

ells p<0.001

p=0.007

p<0.001

CD8 Nai

CD8 Tcm

CD8 Tem

CD8 Nai

CD8 Tcm

CD8 Tem

ells p<0.001

p=0.007

p<0.001

60 p = 0.031p = 0.036

p = 0.049p = 0.038

CD4 Naï

CD4 Tcm

CD4 Tem

60 p<0.001 p=0.001

p<0.001

CD4 Naï

CD4 Tcm

CD4 Tem

CD4 Naï

CD4 Tcm

CD4 Tem

60 p<0.001 p=0.001

p<0.001

CD8 Nai

CD8 Tcm

CD8 Tem

ells p<0.001

p=0.007

p<0.001

CD8 Nai

CD8 Tcm

CD8 Tem

CD8 Nai

CD8 Tcm

CD8 Tem

ells p<0.001

p=0.007

p<0.001

60 p = 0.031p = 0.036

p = 0.049p = 0.038

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 cellsCD4 T cells Whole BloodWhole BloodGene expression profiling in HIV ResistantsGene expression profiling in HIV Resistants

ResNegResNeg

McLaren et al JID 2010

Whole Blood

ResNegResNeg T cell receptor signaling pathway:

Reproduced with permission www.biorag.org

T cell receptor signaling

whole blood

Proteasome

Kif22PP1NF-κB

http://www.tibotec.com

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

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

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 Lavage Cervical 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

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 The 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

UNIVERSITY OF NAIROBI

Ongoing studies

1. Evaluation of Immune Quiescence at the genital mucosaa) Activation phenotypeb) 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 The 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

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

medical immunology immunobiology of hiv infection jan 10, 2013 medical immunology immunobiology of...

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