Download Laboratory of Viral Immunology

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
p0.012
Resistant
p=NS
p=NS
p0.001
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