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Immunity not Luck:
Comprehensive studies of Mechanisms of
HIV Resistance in Highly Exposed
Uninfected Women
VIDI Symposium January 2009
Objective: To undertake an exhaustive analysis of immunologic and
genetic factors that mediate HIV-1 resistance with the goal of a more
complete understanding what constitutes protective immunity against HIV
infection.
Within a population there is
heterogeneity in susceptibility to
infection and disease due to infectious
agents
Beginning with Jenner, most
vaccines have been developed
through at least a basic
understanding of natural acquired
immunity to infection
Resistance to HIV-1 Infection
1 .0
Survival Probability
Highly-exposed
persistently
seronegative.
3 years follow, HIV-1
serology and PCR
negative, Active in
sex work.
10% of highly
exposed sex
workers resistant
to HIV-1.
.8
.6
.4
.2
0 .0
0
2
4
Decline
in HIV-1
Incidence
with1 8
6
8
10
12
14
16
follow up in the Pumwani Cohort.
Years of Followup
Adapted from Fowke et al Lancet 1996; 348: 1347–51
20
Altered susceptibility to HIV is not unique
London DCs
Milan IVDUs
Florence DCs
Seattle DCs
Montreal DCs
Montreal IVDUs
Toronto DCs
Beijing DCs
Barcelona DCs
Chandigarh DCs
New Jersey DCs
Baltimore DCs
Baltimore IVDUs
WIHS multi-centre high risk women
MACS multi-centre high risk men
Fajara CSW
Fajara DCs
Cambodia DCs
Addis Ababa CSW
Entebbe DCs
Abidjan CSW
Moshi Bar workers
Chiang Mai CSW
Nairobi CSW
Lusaka DCs
Bangui DCs
Lilongwe EUs
Durban EUs
Discordant couples
Highly exposed persistently seronegative
Other
Bangkok DCs
Ho Chi Minh City IVDUs
What do we know about Resistance?
(Bold are new findings/findings being expanded by GC studies)

Ten per cent of highly exposed sex workers are resistant to HIV-1. Resistance
is likely cross-reactive to multiple clades of HIV-1. (Lancet 1996, McKinnon JAIDS
2005, Land AIDS Res 2008)



Known genetic mechanisms of resistance (-CCR5 mutation) do not explain
resistance to infection. (Plummer Immunol Ltr 1999). PBMC from resistant women
are susceptible to infection in traditional infection assays. (Fowke AIDS Res 1998)
Resistance (and susceptibility) correlates with specific MHC Class I and Class II
HLA alleles. (MacDonald JID 2000, 2001, Luo-submitted). Several new protective
and susceptibility MHC class II alleles have been identified (LaCap AIDS
2008, Hardie AIDS 2008, Hardie AIDS 2008). Evidence that natural selection for
protective alleles and that MHC Class I heterozygotes have an
advantage is emerging. (Luo-submitted)
HIV resistance associates with polymorphisms in the
immunoregulatory protein IRF-1 and a reduced level of IRF-1 protein
expression. (Ball AIDS 2007). IRF-1 is a known transcriptional activator of HIV
replication and HIV replication is reduced in subjects with protective
genotypes (Ji – submitted)
What do we know about Resistance ? (II):

Resistance is associated with broadly cross reactive CTL to HIV-1.
These systemic CTL responses my recognise different epitopes. (RowlandJones JCI 1998, Fowke Immun Cell Biol 2000, Kaul Immunol Ltrs 2001, Rowland-Jones Immunol Ltrs
2001,)


Continued HIV-1 exposure through sex work appears to be necessary for
persistence of detectable levels of CTL and protection against infection (Kaul JCI
2001). Systemic HIV specific IFN- CTL responses alone do not correlate with
protection against infection in a prospective study (Kaul AIDS 2004).
CD4+ T cell responses in resistant women are qualitatively distinct
from those in HIV infected women and had a stronger proliferative
response, but weaker IFN- response than HIV infected patients (Trivedi
FASEB J 2001, Alimonte JID 2005).

Divergent central and effector CD8 T cell responses to HIV target
specific epitopes (McKinnon JI 2007, JAIDS 2008), These responses also differ in
HIV resistant women (Alimonte Immune Cell Bio 2006).
What do we know about Resistance ? (III):

HIV specific CTL responses were detected in the genital tract of HIV
resistant women. Genital tract CTL responses were more frequent and
of a greater magnitude than in the systemic compartment (Kaul JI 2000,
AIDS 2003).



Some resistant women have HIV-1 specific IgA mucosal antibody that mediate
HIV-1 neutralization and inhibit HIV-1 transcytosis. gp120 specific monoclonal
antibodies have cloned from these women (Kaul AIDS 1999, Berry Hyb Hybrid 2003).
However, HIV specific IgA does not correlate with resistance in the
majority of resistant women (Horton –accepted).
HIV resistant women have elevated T cell recruitment to the genital
tract, and elevated RANTES levels in their genital mucosa, neither of
which was reflected in the systemic compartment (Iqbal JID 2005).
Resistance is associated altered innate cytokine responsiveness to TLR
stimulation, and reduced TLR expression, especially at the FGT. (Lester
AIDS 2008, Lester–in prep)
Nature of HIV Resistance
Immunology
Innate
Adaptive
Humoral
Genetics
Host
Cellular
TLRs
Mucosal Circulating
Innate antiviral
factors
Resistance/Susceptibility
Markers
Gene Expression
SNPs
B Cells
CD8
Receptor
Polymorphism
CD4
HLA
Mucosal
Immunoglobulins;
Tcm/Tem cellular responses;
HIV
Innate factors
Immune activation
Replication




Goal 1: To characterize correlates of protective immune responses
to HIV in the systemic and mucosal compartments of resistant and
susceptible women:
Goal 2: To identify genetic and innate factors associated with
resistance in resistant women and their families
Goal 3: To determine how genotypes/phenotypes identified above
will determine immune responsiveness to a model antigenic
challenge of a live, attenuated mucosal vaccinogen.
Goal 4: To determine if genotypes/phenotypes associated with
resistance, or with a favourable response to the model vaccinogen
protect against HIV infection in a prospective study of HIV
seroconversion in sexworker and non-sexworker cohorts.
Clustering of HIV-1 Resistance in Families
within the Pumwani CSW Cohort


A case control study of HIV-1 seroprevelence comparing
prostitutes related to a resistant woman to those related
to a susceptible woman.
CSW’s related to a resistant CSW were 10 fold less likely
to be HIV positive than those related to an HIV infected
CSW.
Index Seronegative Seropositive
Resistant Families
55
18
15
Susceptible Families 89
10
83
(OR-0.1, CI95% 0.07-0.26, p<0.00001)
A non-biased genome wide SNP screen to map
genes associated with resistance
(Ma Luo - PhD)

SW cohort: 127 resistant women; 600+ HIV Infected


640 kindred
The plan:



127 resistant women (sw)
400 positive (susceptible) women (sw)
80 relatives of each
TCAG: The Canadian centre for human genome and disease researchMicroarray Facility Toronto Hospital for Sick kids


50K Xba 240 GeneChip
250K Nsp GeneChip


Human mapping 100K (Hind 240 and Xba 240)
Human mapping 500K (Nsp and Sty)

168 samples on 50K Xba (74 HIV-S, 93 HIV-R)

Preliminary analysis C/G rs1552896 (p<1x10-9, p<9.5x10-5 corrected)

117 samples on 250K Nsp (61 HIV-S, 56 HIV-R)

Preliminary analysis C/G rs1889050 (p<1.7x10-8, p<1.4x10-3
corrected)
Association of FREM1 with Resistance to HIV-1
Infection in the Pumwani Sexworker Cohort
(James Sainsbury PhD)

2 SNP’s on independent platforms associate with resistance to
infection.

Both rs1552896 and rs1889050 occur in intronic regions of FREM1

Association confirmed in >1000 samples from the Pumwani sex
worker cohort (p<0.001).


Validated in low risk cohort (MCH), with protective genotypes
occurring at a higher frequency in HIV-1 negative individuals
(p<0.05).
Linkage between rs1552896 and rs1889050 in the HIV-1 resistant
group
FREM1


FRAS1 related extracellular matrix 1
Mouse homologue Frem1





Smyth et al., 2004
Mutations in Frem 1 associated with blebbed mouse phenotypes
blebs mice are a model of Human Fraser Syndrome
Fras1/Grip1/Frem1 pathway responsible for epidermal
adhesion during embryonic development (but not for
postnatal development)
Secreted (lacks a transmembrane domain) forming part
of the extracellular milieu

Ma Luo 2007



CSPG domain – interaction with collagen, Fras 1.
Calx-beta domain – calcium binding domain
regulates the activity of both the CSPG domains
and the C-type lectin domain.
C-type lectin domain – carbohydrate binding
domain
Ma Luo, 2007
FREM1 is relatively highly expressed in mucosal
tissues and especially the FGT
Semi-quantitative expression of FREM1
splice variants: HIV-R vs HIV+

Total RNA





Whole Blood
PBMCs
Standardized Total RNA in Reverse Transcription (1 µg)
PCR specific to each FREM1 splice variant A,B, C, D, E, F,
G.
(Semi) quantified via ethidium bromide stain and
agarose gel electrophoresis
Gene expression profiles of HIV Resistant
sex workers
E M Songok PhD


Goal 2: To identify genetic and innate factors associated with
resistance in resistant women and their families
2.3: Determine by gene expression analysis if there are genes
differentially expressed in resistant women and their families
Outcome



1474 genes: 819 down, 655 up (FC 1.3, p
=0.0001)
1214 (79%) could be annotated by their
Gene Ontology term (GO-TERM)
390 genes (25%) could be mapped to
known signalling pathways (KEGG)
Gene expression profiling in HIV Resistants
CD4 T cells
Res
Whole Blood
Negs
Res
Neg
Key genes impacted insulin/glycolysis pathway
suggest Type 2 diabetes





DPP4
IRS-1
G6PDH
GA3PDH
PGM-1
DPP4
EXPRESSION
p= 0.0000014
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
-1.5
-2.0
CONTR
RES
INSULIN RECEPTOR SUBSTRATE 1
EXPRESSION
P=0.0001
1.5
1.0
0.5
0.0
-0.5
-1.0
-1.5
-2.0
-2.5
-3.0
MCH
HIVR
But..preliminary Random Blood glucose levels…
Blood sugar levels
25
20
p=0.91
15
10
5
0
HIV NEG
HIV RES
n=55 each
HIV POS
Observations



A gene signature pattern on HIV
resistance exists
More than 50% of genes earlier identified
by various workers found in our screening
Evidence of association of HIV resistance
with genetic traits of Type 2 diabetes
T cell Function in HIV Resistance:
The Role of Immune Quiescence
Keith Fowke
Universities of Manitoba and Nairobi
Assessment of T cell Function

Gene expression analysis

Purified CD4+ T cells – Paul McLaren


Whole Blood – Martim Songok



9 Res, 9 High-risk negatives
23 Res, 19 Low-risk negatives
Used Affymetrix U133 Plus 2.0
T cell functional assays



Cytokine production
Cellular activation markers
Regulatory T cells
HIV-Dependent Host Factors in CD4 cells
Gene
Symbol
Fold
P Value
Description
PPP1CA
-2.19
0.00001
protein phosphatase 1
STAU
-1.67
0.00002
staufen, RNA binding protein
KIF22
-1.88
0.00005
kinesin family member 22
RELA
-1.40
0.0009
NFkB subunit (p65)
ITGA5
-1.79
0.004
integrin, alpha 5
CYPA
-1.43
0.009
peptidylprolyl isomerase A (cyclophilin A)
NFKB1
-1.71
0.01
NFkB subunit (p50)
Down Regulated Genes involved in HIV replication
•Brass et al (Science 2008) used siRNA functional analyses, 260 HIV-dependent
Host Factors
•In CD4 T cell array – 45 were shared, 42 lower expression in Res
•In Whole Blood array - 109 (42%) were present, most lower expression
•Cellular machinery does not favour HIV replication
T cell receptor signalling in Whole Blood
Neg
Res
Antigen Presentation Pathway in Whole Blood of HIV-RES
Res
HLA-A
Neg
HLA-C
HLA-B
CCR5
P = NS
p 0.04
p 0.01
p 0.01
0.3
2
-0.0
-0.1
-0.2
-0.3
-0.4
EXPRESSION
0.25
0.1
EXPRESSION
EXPRESSION
EXPRESSION
0.2
0.4
0.3
0.2
0.1
-0.0
-0.1
-0.2
-0.3
-0.4
-0.5
-0.6
0.00
-0.25
1
0
-1
-0.50
-0.5
-2
-0.75
CONTR
RES
CONTR
RES
CONTR
RES
CONTR
Similar finding for NK receptors (KIR), TLRs (7 and 8), IRFs (1 and 7)
RES
Assessment of T cell Function

Gene expression analysis

Purified CD4+ T cells


Whole Blood



9 Res, 9 High-risk negatives
23 Res, 19 Low-risk negatives
Used Affymetrix U133 Plus 2.0
T cell functional assays



Cytokine production
Cellular activation markers
Regulatory T cells
HIV Resistants have Normal Recall Responses
Activation (CD69) is lower in HIV resistant subjects
•HIV does not replicate efficiently in quiescent cells
•Fewer HIV target cells
Immune Quiescence in HIV Resistants

Lower
Lower
Lower
Lower

Normal Antigen recall function – not immune suppression

OVERALL immune cells seem to be resting or quiescent

We have termed this phenotype Immune Quiescence



overall gene expression, CD4+ T cells and whole blood
gene expression in HIV and T cell receptor pathways
resting PBMC cytokine production
level of cellular activation on T cells
Evidence of Immune Quiescence in other cohorts

Dutch cohort HIV-R Men (Koning et. al. J. Immunol. 2005)


Abijan cohort of HIV-R CSW


↓ IA (HLA-DR, CD38, CD70)
↓ CD69 following allo-stim
Central African Rep



(Jennes et. al. Clin Exp. Immunol. 2006)
(Begaud et. al. Retrovirology 2006)
low I.A. on CD4 (DR, CCR5)
Reduced HIV susceptibility in unstimulated PBMC
Difference lost when PHA stimulated cells
What is driving Immune Quiescence?



Regulatory T cells
T cells CD4+, CD25+, FoxP3+
Function to suppress activation of immune cells
Tregs are higher in PBMC of HIV resistant subjects
Two phase model of HIV resistance
IQ phase
CMI phase
HIV-specific CMI
Treg
Susceptible
Exposure
Quiescent cell
(low susceptibility)
Activated cell
(high susceptibility)
Treg
Infection
“Susceptible”
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, inhib NF-κB
Resistant
Exposure
No Infection
“Resistant”
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. Confirm pathway changes at protein level
4. Validate in other cohorts
Mucosal proteomics of HIV-1resistance
Overview

Goal of project:

Identify innate factors in cervicovaginal
mucosa differentially expressed in HIVresistant and HIV-susceptible women by
proteomic techniques
“Known” innate anti-HIV-1 factors
in mucosal fluid
α-defensins
Lysozyme
SLPI
Thrombospondin
RANTES Calprotectin
Lactoferrin
Histone H2A
Cystatins
Trappin-2?
Mucins
β-defensins
Histatins
Cathepsin B
agglutinins
Cervical mucosa protein profiling


“Unbiased” approaches to examine
differential protein expression in cervical
lavage fluid
Proteomic platforms being used:

Traditional gel-based technique


2D-DIGE
Mass spectrometry based techniques
SELDI-TOF
 2D LC FTICR-MS

2D-DIGE platform results
“Identification of differentially expressed
proteins in cervical mucosa of HIV-1-resistant
Kenyan sex workers”
A. Burgener, J. Boutilier, C. Wachihi, J.
Kimani, M. Carpenter, T. Ball, F. Plummer. J.
Proteome Research. August 2008.
Differentially expressed protein spots in HIVResistant CVL fluid Underexpressed
Overexpressed
A
B
A
B
Immune
response
13%
Ion transport
14%
Anti apoptosis
14%
Ion
homeostasis
14%
Cell proliferation
13%
Protease
inhibitor
74%
Cell
organization
29%
Immune
response
29%
Biological function of differentially expressed proteins in
cervical lavage of HIV-1-resistant women.
Overexpressed in HIV-1-resistant women
Swiss-Prot
accession
number
Q86VF3
Q86WO3
Q6ZW52
Protein
Serpin B3
Serpin B4
Alpha 2-macroglobulin like-1 protein
P10599
P01040
P31151
Q9UIV8
P30740
Protein MW (kDa)
44.6
45
Thioredoxin
Protein biological function
Cysteine protease inhibitor
Serine protease inhibitor
Elastase inhibitor, broad-spectrum endopeptidase
inhibitor
Cell proliferation
Cystatin A
S100A7 protein
Serpin B13
Serpin B1
Cysteine protease inhibitor
Innate immune response, epidermis development
Cysteine protease inhibitor
Elastase/Cathepsin G inhibitor
11
11
44
43
161
12
Underexpressed in HIV-1-resistant women
Swiss-Prot
accession
number
P52566
P00738
Q6GMX2
P60709
P02647
P01024
Q08188
O00299
Protein
Rho GDP dissociation inhibitor
Haptoglobin
IGHA1 protein
Actin beta
Apolipoprotein A1
Complement component 3
Transglutaminase 3
Chloride intracellular channel 1
Protein biological function
Anti-apoptosis, cell motility, cell adhesion
Ion homeostasis, defense response
Immune response
Cell organization
Lipid carrier
Immune response
Cell organization
Chloride transport, signal transduction
Protein MW (kDa)
23
45
51
42
31
187
77
27

Majority of upregulated proteins are
antiproteases

Cystatin A – cysteine protease inhibitor

Known Anti-HIV-1 factor:




Serpins B1, B4, B13

inhibitors of Cathepsin G, an inflammatory protease


Chemoattractant for monocytes/neutrophils, stimulates T-cells,
enhances HIV-1 infectivity, degrades RANTES
Serpin B1 and A2ML1



Blocks viral processing?
Blocks epithelial-lymphocyte interactions
Inhibition of Vif function? (essential for HIV-1 replication)
Inhibitors of human neutrophil elastase (HNE)
May antagonize HNE function and help maintain epithelial layer
Overall these could contribute to an anti-inflammatory
environment -> protective against HIV-1 infection?

Many downregulated proteins involved with
HIV-1

Complement component 3:


Rho dissociation inhibitor:


mediates HIV-1-infected cell migration though tight
junctions
Beta-actin:


chemoattractant involved in immune response
binds HIV-1 reverse transcriptase and may be
involved in HIV-1 secretion
Apolipoprotein A:

surprisingly is known to bind gp41 of HIV-1 and act
as a competitive inhibitor
Conclusions


Proteomics data on CVL generally
consistent with immune quiesence
hypothesis
Identification of possible microbicides
Ultimate Goal: Inform HIV Vaccine/Therapeutics

How will an understanding of HIV resistance
contribute to a new vaccine/treatment?




New epitopes or targets for vaccine (Th, CTL)
Microbicide (HIV inhibitory molecules at the FGT or
systemic)
Novel approaches to immunization – Treg vaccine?
Adjuvants/immunoregulation (alternate gene
expression?)
Collaborators






Blake Ball, Joshua Kimani, Ma Luo, (U Manitoba)
 Genomics, Proteomics, Clinical
Keith Fowke (U Manitoba)
 T cell Immunology
Walter Jaoko, Benson Estamble (U Nairobi)
 Clinical; Challenge experiment
Rupert Kaul (U Toronto)
 Mucosal Immunology
Ken Rosenthal (McMaster University)
 Innate Immunology
Rafick Sekaly (U Montreal)
 Tetramers, TCR-Vb, ‘omics’
Manitoban Project Staff