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Attachment B - Research Plan
TABLE OF CONTENTS
I.
Detailed Technical Plan
1
A.
B.
C.
D.
E.
F.
1
4
9
20
20
20
Background
Preliminary Studies
Methods and Experimental Design
Project Team
Management Plan
Product and Project Maturation Plans
References
21
II.
Organization Capacity
25
III.
Key Considerations
26
A.
B.
C.
D.
E.
F.
26
26
26
26
27
27
Plan for Managing Intellectual Property
Data Sharing Plan
Discussion of Ethical, Social or Cultural Implications of Research
Research on Human Subjects
Animal Research
Other Sensitive Research
IV.
Facilities & Resources Statement
27
V.
Budget Justification
28
I. DETAILED TECHNICAL PLAN
A. Background: It is a basic tenet of infectious disease biology that variability in susceptibility
to infection and disease caused by microbial agents is a characteristic of all populations. Among
susceptible individuals exposed to an infection, not all become infected and among infected
individuals, not all develop disease. It seems logical that such variability in susceptibility to
infection and disease would apply to infection and disease with Human Immunodeficiency Virus
type 1 (HIV-1) and considerable evidence has emerged that this is the case. Most vaccines against
infectious diseases were developed out of knowledge of the basis for protective adaptive immunity.
Smallpox vaccination grew out the knowledge that infection with cowpox protected milkmaids
against smallpox. Vaccines for polio and the childhood exanthems were developed out of an
understanding that natural infection confers highly protective immunity against repeat infection. If
we can understand what constitutes and results in protective immunity to HIV-1, we can likely
replicate it through vaccines. Studies aligned along these hypotheses conducted by our group
have already significantly informed HIV vaccine research, and further study of the unique
cohorts we follow will continue to do so.
Studies in women from the Pumwani Sex Worker Cohort provided early data that there might be
biologically mediated resistance to infection. In a study of risk factors for HIV-1 1infection, the
mean duration of prostitution was inversely related to the risk of HIV-1 infection . This is of
course illogical; the duration of exposure should be directly related to the risk of infection, unless
other factors are operative. One such factor would be population heterogeneity in susceptibility to
HIV-1 infection. In a highly exposed population, over time susceptible women would be infected
with HIV-1 and, through death or illness, be removed from the population. The remaining
population would be enriched for women less susceptible or relatively resistant to infection. Thus,
we hypothesized that there was biologically mediated resistance to HIV-1 infection among the
women in the Pumwani cohort. The first
evidence of protection against HIV-1
infection in
humans comes from this study2, which began in
1985 and continues through the present. Women
in the cohort have intense exposure to HIV-1
through their occupation and, although condom
use is frequent, their risk of acquiring HIV-1
infection is enormous. They currently have more
than 60 unprotected sexual exposures to HIV-1
infected sexual partners annually and many
hundreds over the time of follow up in the cohort.
As shown in Figure 1, despite this intense
exposure, the risk of HIV-1 seroconversion in the
cohort gradually declines and the longer a woman Figure 1. Decline in HIV-1 Incidence with
remains uninfected, the less likely she is to follow up in the Pumwani Cohort.
seroconvert to HIV-1. A small number (25% of
initially HIV-1 seronegative women and 10% of
all women enrolling in the Pumwani cohort) remain HIV-1 uninfected for prolonged periods (up to
17 years). Mathematical modelling shows that this is not related to chance alone and that in a
statistical sense, these women should be HIV-1 infected. Other studies show that this phenomenon
is not related to seronegative HIV-1 infection, to differences in sexual behaviour or the incidence
of other sexually transmitted diseases. From this, we concluded that a fraction of women in the
cohort are protected against HIV-1 through an unknown biologic mechanism.
1.0
Survival Probability
.8
.6
.4
.2
0.0
0
2
4
6
8
10
12
14
16
18
20
Years of Followup
Several other groups of highly exposed persistently seronegative (HEPS)
individuals who appear
to be protected against HIV-1 infection have now been described3,4,5,6, but this cohort is one of
the largest and most comprehensively studied. Studies of mechanisms of immune protection to
HIV infection in other HEPS cohorts present the problem of demonstrating that exposed
individuals are indeed protected against infection. From the beginning of the HIV-1 epidemic, it
was obvious that not all HIV-1 exposed individuals become infected. It is only through long-term
epidemiologic follow-up in HIV endemic areas that it is possible to predict sufficient exposure to
result in infection. Although other HEPS groups such as discordant couples or sex worker
cohorts have been described, few have been as extensively characterised as the Pumwani cohort.
There has been intense interest in understanding mechanisms responsible for resistance to HIV-1
1
infection. Several types of genetically mediated resistance to HIV-1 infection and disease are now
known. Cellular resistance to infection was reported by Paxton et al5 and shown to be related to
homozygosity
for a 32 base pair deletion mutation in the HIV co-receptor molecule CCR5 (-32CCR5)7,8. The -32-CCR5 has not been detected
in African populations and is not found in HIV-1
9
resistant
women
from
the
Pumwani
cohort
.
Neither
polymorphisms in the promoter region of
CCR510, CCR2 964I11, SDF-111, overproduction of chemokines or altered CCR5 or CXCR4
expression levels account for HIV resistance. It is unlikely that other HIV-1 receptor or coreceptor mutations are responsible for HIV-1 resistance in the Pumwani cohort as their PBMC
have been
shown to be readily infected with multiple HIV-1 strains, as well as primary HIV-1
isolates5. Thus known mechanisms of resistance do not account for HIV-1 resistance in the
Pumwani cohort.
While receptor mediated resistance to HIV-1 infection is important, there is interest in immune
mechanisms that may mediate protection against infection. T cell responses to HIV-1 are known
to be critical in control of HIV-112infection and the loss of these responses correlates with increased
viremia and disease progression . Studies of the role of HLA alleles in HIV-1 disease progression
also suggest an important role for the immune system in controlling infection. Combinations of
major histocompatibility complex (MHC) alleles, for example, HLA-B1813 and HLA-A214 and
TAP gene 15,
variants associate with resistance to infection and a reduced risk of HIV-1 disease
progression 16. MHC Class I heterozygosity and rarity provides advantage in survival with HIV1 infection, suggesting that diversity of CTL responses may protect against HIV-1 related disease
17,18
and indicating the importance of the immune response in controlling HIV-1 infection
.
Functional studies of22groups of HEPS and other exposed groups 23such as discordant couples19,20,21,
exposed sex workers
, accidentally exposed health care workers and uninfected children of HIV
positive mothers24,25, show that high proportions have evidence of T-cell responses to HIV-1.
Levy has 2reported CD8 mediated suppression of HIV-1 replication in exposed uninfected
individuals . As a result of these studies, it has been hypothesized that cellular immunity
to HIV1, rather than systemic humoral immunity is important in protection against infection26.
To examine the HIV-1 specific T helper cell responses among HIV-1 resistant women, we
compared cytokine 27
responses to HIV-1 env T helper epitopes to HIV-1 unexposed and HIV-1
infected individuals . HIV-1 resistant women produced IL-2 in response to env peptides and
gp120 greater than that produced by unexposed controls or HIV-1 infected individuals, indicating
that resistant women have adaptive T cell memory
to HIV-1. In further studies, we examined CTL
to HIV-1 antigens among resistant women29. Fifteen of 22 resistant women showed specific lysis
of the env vaccinia infected target compared to none of 12 HIV-1 unexposed controls (p<0.0003).
Since HIV-1 clades A and D are most prevalent in Nairobi, these 28
studies indicate that resistant
women likely have CTL that recognize relatively conserved epitopes . If CTL are responsible for
protection, these data would indicate that immune responses that cross protect against
heterologous viral challenge, the ultimate goal of HIV-1 vaccine research, are achievable. To
understand the specificity of the CTL response to HIV-1 among resistant women, we initiated
collaboration with Drs. Sarah Rowland-Jones and Andrew McMichael of Oxford University. In
these studies, we examined 22 resistant women for CTL directed at conserved HIV-1 peptide
epitopes restricted by their HLA alleles. Half of the women are positive in these assays 29.
Studies have also shown that these women have CTL to multiple epitopes and cross protective
immune responses to HIV-1.
If both HIV infected and HIV resistant individuals have HIV-specific T helper responses, what is
unique about the later that protects them from infection? The difference may well be immune
memory. Memory cells are long-lived, can expand at low levels in the absence of antigen
and are
key to providing secondary immune responses that vaccines require. Lanzavecchia 30 identified
two classes of memory cells based on their expression of CCR7. Effector memory (TEM) cells do
not express CCR7, therefore, do not reside in the lymph node but travel to non-lymphoid tissues.
In general, these cells rapidly produce effector molecules, such as IFN-γ or perforin/granzyme,
but lack the ability to proliferate. Central memory (TCM) cells express CCR7 and reside in
lymphatic tissue where they can be stimulated by APC. Functionally T CM do not have good
effector qualities but are able to proliferate extremely well. The importance of immune memory
was recently demonstrated in a study of occasional HIV-1 seroconversions in women who had
been defined as resistant. Combined epidemiologic and immunologic studies demonstrated that
the best correlate
of seroconversion in previously HIV-1 resistant women was a decrease in
sexual activity31, suggesting that constant boosting of immune memory may be necessary to
2
maintain protective immune responses. During HIV infection memory CD4 T cells are the
major latent reservoir 35for HIV32,33,34 and in acute HIV infection a significant impairment of T
helper function results . It is likely that HIV-specific T helper cells in resistant women may be
quantitatively and qualitatively different than those in infected persons. Memory
CD4 and CD8
T-cells, but not naïve cells, have a decreased half-life in HIV infection36 likely due to their
increased 37,38,39
susceptibility to apoptosis due to increased activation and expression of
CD95/Fas
. It is well know that HIV infection increases the amount of immune activation
and in this environment memory cells are susceptible to apoptotic cell death40. Thus, constant
immune activation by an active HIV-1 infection leads to a skewing of the memory phenotype.
The presence of CD4 T-cells that have proliferative potential in HIV resistant women leads us to
speculate that resistant women have more HIV-specific TCM than infected individuals, and that it
is the presence of specific memory populations that may, in part be mediating protective immune
responses to HIV-1.
A major gap in our understanding on immune responses in HIV-1 exposed uninfected
individuals has been the role of effector mechanisms in the genital tract. As initial contact
between HIV and the immune system occurs at this site, mucosal immune responses would be
critical in mediating protection against infection and if so, would be important in the
development of HIV-1 vaccines. This thesis is supported by animal models, in which HIV-1
specific CD8 genital lymphocyte
responses were shown to confer long-lasting protection against
mucosal HIV-1 challenge41. There have been relatively few studies of the mucosal immune
response to HIV-1 in humans due to difficulties in obtaining sufficient cervical mononuclear
cells (CMC’s) to conduct immune assays. Studies on genital tract immune responses in other
HEPS groups replicate what is already known about systemic responses to
HIV in our group and
others. There is evidence of an 43adaptive cellular response to HIV-142, mucosal CTL49 and
mucosal HIV suppressive factors . Some
have suggested that these CD8 mediated responses
derive from the systemic compartment89, although this is controversial due to the lack of
consistent cell-surface markers for mucosally targeted cells.
An important study by Mazzoli et al44 reported a high frequency of HIV-1 specific IgA in a
HEPS cohort in Italy. Prompted by these findings, we examined resistant women from our
cohort, HIV-1 infected
sex workers and lower risk controls for evidence of HIV-1 specific
mucosal antibody45. Virus specific IgA was present in the genital tract of 76% HIV-1 resistant
sex workers, 26% HIV-1 infected women, and 11% lower risk women (p <0.0001). No virus
specific genital tract IgG was detected in resistant women, while all infected women and 1 of 28
lower risk controls had detectable genital tract IgG 46(p<0.0001). Kristina Broliden’s group has
shown that these HIV specific IgA are neutralizing and can 47
neutralize multiple HIV strains including primary isolates as well 48as neutralizing across clades . This is very exciting data and
together with a report from Italy , provide some of the first evidence that cervical antibody
correlates with protection against HIV in humans. However 49
this data remains controversial as it
has been corroborated in some groups of exposed-individuals but this has not in others50.
While it seems likely that the systemic cellular and mucosal responses detected in resistant sex
workers are in part mediating protection, this leads to further questions. Why do some individuals
develop immune responses that provide protection, while others become infected with HIV-1? If
CTL responses are in part responsible for protection, it is logical to examine the role of MHC
alleles. Indeed we have found important correlations between resistance and susceptibility to HIV1 infection in our cohort and MHC Class I and Class II alleles. The HLA-A2-6802 supertype and
HLA-DR01 correlate with a 2-4 fold protection against infection, while HLA-A23 strongly
correlated with increased susceptibility to infection. The protective Class I association might occur
if the allele presents
peptide epitopes from highly conserved regions of HIV-1 and there is some
evidence for this32. Alternatively, protective alleles may have peptide presentation properties that
are more tolerant of mutation. Enhanced susceptibility alleles may indicate that these alleles
present immunodominant, non-protective peptide epitopes. The protection provided by HLADR01 could result if it results in specific T helper cell responses and the induction of CTL. For
instance the affinity of Class II binding helps to determine which type of cellular51,52,53
immune
responses develop to antigen and this phenomenon has been linked to human disease
. In
addition to these individual associations, we have recently been able to show a heterozygote
advantage in protection against HIV-1 infection, arguing for the importance of the cellular immune
responses in protection against HIV-1 infection.
3
The factors involved in the development of T helper cell responses are becoming clearer. The
major determinant of the Th
1-Th2 polarisation of a naïve T-cell is the cytokine environment in
which it encounters antigen54. However, the role of dendritic cells (DC) and the innate
immune
system in the generation of T helper cell responses has recently been elucidated 55. Innate
immunity is particularly important for defense of mucosal surfaces where the vast majority of
pathogens initiate infection. Vertebrates make use of an evolutionarily conserved pattern
recognition system of host defense to provide almost immediate protection against microbial
infection while adaptive immunity is being mounted. A recently described class of pattern
recognition receptors the Toll-like receptors (TLRs), has been shown to activate innate host
defenses, including the release of chemokines and cytokines, anti-viral factors, recruitment of
neutrophils, and activation of macrophages and DCs. Furthermore, it is now clear that TLR
signaling is critical for guiding the subsequent Th1-Th2 polarisation of T cells not only through
DC-T cell interaction, but through the cytokine
response elicited by TLR ligands such as LPS,
peptidoglycan, CpG motiff’s, and dsRNA56. To date, eleven TLRs have been identified in
humans. The most recent, TLR11 is primarily
expressed in the urogenital system and senses
bacterial infection in the kidney and bladder 57. It was also recently demonstrated that58the
natural
ligand for TLR7 in mice and TLR8 in humans is single-stranded RNA from viruses ,59 Indeed,
Heil et al81. showed that a GU-rich sequence from HIV-1 RNA boosted production of interferon from DCs. Perhaps DC in individuals who exhibit resistance to HIV-1 and the associated
immune responses are genetically or environmentally programmed to respond in fashion more
likely to drive Th1 type responses.
B. Preliminary Studies: In the almost twenty years since this study began, we have made
considerable progress in understanding the phenomenon of resistance and the role of immune
responses in mediating it. Based out of the University of Nairobi, our group has been working in
the Pumwani area since 1985 and has an enormous amount of data on sex workers from this area.
This open cohort has enrolled over 2200 women and women continue to enroll at the rate of
100/year. Of the entire cohort, 127 women have met the epidemiologic criteria for HIV
resistance and had immunologic assays performed. The follow up on this group is well over 900
person years and the follow up since first immunologic assays were performed is now mean 4.5
+ 2.4, total 451 person years.
B.1 Systemic Adaptive Immune Responses
B.1.1 Differential epitope recognition in HIV resistant vs. infected women. While CTL clearly
are correlated with HIV-1 resistance, a persistent puzzle has been that HIV-1 infected individuals
have extremely high levels of CTL to HIV-1. How can CTL protect against infection in one
instance and not in another? Recent results point to a potential answer. In characterizing CTL
responses to HIV-1 in resistant sex workers, we found that HIV-1
infected and resistant women
with the same HLA types recognize different peptide epitopes60. Furthermore, if resistant women
become HIV-1 infected, the immunodominant response switches. This finding is fascinating and
important for HIV-1 vaccine development. There may be important functional differences in CTL
elicited by different epitopes. These questions will be addressed in this project to comprehensively
determine specificity and functionality of CD8 responses in resistant women, and to determine if
similar responses can be induced in resistant women by a model vaccine challenge.
Survival Probality
1 .0
.9
.8
.7
0
2
4
6
8
10
Years of Follow up
Figure 2. Kaplan Meier Plot of Time to HIV seroconversion in
women with CTL to vaccinia-env (solid line) and women
negative for vaccinia-env CTL (dotted line) (p<0.05 one tail).
B.1.2 Prospective Correlates of HIV-1
Seroconversion. As well as assaying for
CTL using peptides in Elispots, we have
continued to use vaccinia-env constructs 61
as
CTL targets in a modified Elispot assay .
In examining correlates of protection in
resistant women the best correlate identified
is CTL to vaccinia-env. As shown in Figure
2, no seroconversions have occurred to date
in women with vaccinia-env CTL. Further
expansion of these studies will be required
to confirm this. Interestingly, there is no
similar correlation with CTL against gag,
pol and nef. This suggests that CTL to some
targets may be more important than others
4
and is one of the reasons that we will focus on defining gp120 epitopes by CTL from HIV
resistant women in continuation of these studies. Further evidence that all CTL may not be equal
comes from our study of formerly HIV-1 resistant women who become HIV-1 infected.
Immunodominant peptide epitopes recognized by HIV-1 resistant women differ from those
recognized by HIV-1 infected subjects. Moreover, after seroconversion immunodominant
epitopes switch to those most commonly recognized by HIV-1 infected individuals34. This may
reflect different functionality of CTL that recognize different epitopes.
ai
ve
N
D
4
C
C
D
4
Te
m
Te
m
D
4
C
C
D
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ai
ve
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D
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C
Te
m
D
4
C
Te
m
0
D
4
10
0
C
20
10
D
4
30
20
C
40
30
/td
50
40
Tc
m
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50
/td
60
Tc
m
B.1.3 Mapping of CTL env Epitopes Recognized by HIV-1 Resistant Women. As noted
above the best CTL correlate of protection in HIV resistant women is CTL to vaccine-env
constructs. Mapping the fine specificity of these responses may be important for understanding
the phenomenon of resistance and could significantly inform HIV vaccine development.
Research from our group and others suggest that fine-mapping of CTL epitopes in individuals
with low CTL precursor frequency such as vaccinees and HEPS groups is best conducted using
overlapping 9-mer libraries (R. Kaul submitted). In a recently completed pilot study of systemic
env CTL responses, we confirmed these findings, and identified two clade A env-peptide pools
that appear to be recognized more commonly in negative sex workers than positive sex workers.
Pool 9-nm is immunodominant in 3 of 5 HIV resistant responders, while immunodominant in 0
of 13 HIV+ responders (P=0.012) and sub-dominant in 5 HIV+ women. Pool 16-m is
recognized by 3 of 3 responding HIV resistant women, while recognized by only 2 of 14 HIV+
responders (P=0.015). Expansion of these findings and determination of individual epitopes
being recognized will provide further evidence of whether differential epitope recognition is
occurring and identify potential targets for vaccine development.
B.1.4 Immune Memory and Resistance.
Resistants
New ML negs
In terms of their role in HIV infection,
numerous studies have implicated T-helper
responses in being critical in containing
HIV 62,63
infection during the asymptomatic
phase
. T-helper responses have been
shown 64to65 correlate with anti-HIV
CTL
activity , and lower viral loads66. Our
recent studies have focused on T helper
responses in HIV resistant individuals and
controls and resulted in two significant
Figure 3. CD4 T cell memory phenotypes are altered in resistant women.
findings. The first is that the breadth of the
CCR7 and CD45RA expression was analysed in PBMC from 8 resistant
p24 epitopes to which the HIV resistant
women and 12 seronegative women. Resistant women had significantly
(p=0.036) more central memory CD4 T cells.
individuals responded was similar to HIV
infected controls. That is, in epitope
mapping experiments conducted on a Kenyan clade A p24 gene we found that there were no
over-represented or unique epitopes in the HIV resistant subjects, it is interesting that CD4
epitopes and CD8 epitopes appear to differ inthis manner. The second finding was that we were
able to determine that HIV resistant individuals had both HIV-specific short-term IFN-γ and
long-term proliferative responses. The proliferative capacity of the resistant women was 7.5 fold
greater than the infected women, suggesting that CD4 T cell memory in HIV resistant women is
different from infected subjects and that qualitative rather than quantitative difference in these
responses may be important. We have expanded these studies to begin examining the role of
CD4 memory subsets in HIV resistance. Figure 3 demonstrates that using multiparametric flow
cytometry to measure CD4 TCM and TEM subsets revealed that resistant women had elevated TCM
levels compared to HIV uninfected controls, suggesting that differences in T cell phenotype and
functionality are important in HIV resistance and may be representative of what a protective
immune response to HIV-1 is. We will expand these studies and determine if they are a
representative phenotype of resistant women, and to determine in our vaccine challenge whether
similar types of responses can be generated by resistant women and their kin.
B.2 Mucosal Adaptive Immune Responses
B.2.1 Mucosal CTL Responses To HIV-1. In spite of the obvious importance, the role of
mucosal cellular immune responses in resistance to HIV has only begun to be studied. We
recently reported on the frequency of CD8 IFN- responses to HIV-1 peptide epitopes in
resistant sex workers and controls. Matched CMC and PBMC assays were conducted. We found
HIV-1 specific cellular responses were present in the cervix of 4 of 7 seropositive, 11 of 18 HIV5
1 resistant sex workers and 0 of 6 low risk control women. A systemic response to the same
peptide epitope was found in 3 of 4 HIV-1 infected and 7 of 10 resistant sex workers67. In half
of the women with positive cervical responses, the IFN- secreting cell frequency in the cervix
was equivalent or higher than the frequency in simultaneously obtained PBMC suggesting
mucosal CTL responses are more vigorous and prevalent than systemic responses. Expansion
and further characterization of mucosal CTL responses is a major focus of the studies proposed.
B2.2 Genital Tract CD4 Cells
An impediment to research on
cellular immune responses in the
genital is the limited cell
numbers available to work with.
We have spent considerable
effort optimizing techniques for
cell recovery from endocervical
and vaginal sites resulting in
better recovery of endocervical
cells. In addition we began
describing characteristics of T
cells in the genital tract of HIV
Figure 4. Cervical T cells are elevated in HIV resistant sex workers compared
resistant women, comparing
to uninfected sex workers. Bars indicate median cell numbers observed in
them to HIV infected and low
CMC’s by cytometric analysis.
HIV exposure risk controls
Results of T cell phenotyping and chemokine analyses are quite dramatic. Overall HIV resistant
women show higher numbers of CD8 and CD4 T cells than HIV uninfected sex workers, and
low risk controls (Figure 4). We have also examined expression of cell surface markers CXCR4
and CCR5 on the surface of endocervial T cells from HIV resistant women and controls. Both
the percentage and proportion of CD4 cells expressing these markers are higher in HIV resistant
women compared to both HIV infected and low risk control subjects (p<.022 for both). These
preliminary findings suggest that CD4 T cells are highly important in protection against HIV . It
also indicates that there are ample HIV-1 susceptible targets in the genital tract of resistant
women HIV. Analysis of cytokines and chemokines in vaginal secretions from resistant women
show that these women demonstrate elevated RANTES levels compared to uninfected women
(p<0.005) potentially inhibiting HIV replication, or recruiting Type-1 CD4 lymphocytes, thus
explaining the elevated T cell numbers observed.
B.3
Innate Immune responses and HIV resistance
It is now clear that TLRs and innate immune activation are critical for guiding the nature and
subsequent Th1-Th2 polarisation of T cells. We compared production of cytokines important in
driving Th1 responses (IL-12, IFN-, IFN-) and immunoregulatory cytokines such as IL-10 in
response to TLR-2, 4 or 7 ligands in PBMC from resistant and susceptible women. These groups
exhibited markedly different cytokine responses to the TLR ligands tested, which was both
cytokine and TLR ligand dependent. HIV-resistant women had elevated IL-10 responses to TLR2 and 7 stimuli (p=0.001 and 0.021), elevated IL-12p40 to TLR-4 ligand, (p=0.009) and
depressed IFN- responses to TLR-4 and 7 stimuli (p=0.021 and 0.013). This is among the first
data to demonstrate that altered innate cytokine responses to TLR stimulation occurs in
immunologically distinct groups of humans. Due to the critical role of TLR signals in instructing
the adaptive response, these findings have
important implications in understanding
differential susceptibility to infectious diseases
such as HIV. Further data supporting these
points comes from studies carried out by our
collaborators; Ahkar et al. showing that local
activation of TLRs in the vaginal mucosa of
female mice can prevent genital infection
with
herpes simplex virus type 2 (HSV-2)68. Their
results showed that local, but not systemic,
delivery of CpG oligodeoxynucleotides (ODN),
the ligand for TLR9, to the vaginal mucosa
caused rapid proliferation and thickening of the
Figure 5. Kaplan-Meier plot of time to HIV-1 seroconversion and
6
number of identified resistant alleles identified in a given individual.
genital epithelium and activation of an innate antiviral state that protected against intravaginal
HSV-2 infection. They went on to show that HSV-2 entered CpG-treated genital mucosal
epithelial cells but did not replicate. Similarly, CpG ODN treatment of macrophage cell lines
expressing TLR9 in vitro significantly protected against HSV-2 infection. More recently, they
demonstrated that local delivery of dsRNA, the ligand for TLR3, to the vaginal mucosa protected
against genital HSV-2 infection (in press). Together these findings demonstrate a potent ability
of the innate immune system to prevent infection with a sexually-transmissible virus, and along
with our data of altered TLR responses in resistant women suggest potential mechanisms by
which HIV resistant women would be more likely to develop protective immune responses to
HIV-1. We will expand these experiments in the studies proposed here, and determine if innate
responses are predictive of better immune responses to our model vaccine.
B.4
Immunogenetics of HIV-1 Resistance
Earlier studies showing the correlation of individual MHC alleles with HIV-1 resistance clearly
indicate at least some genetic basis for the phenomenon. However, the MHC associations do not
explain the phenomenon entirely. One of our clinicians made the observation that there was
clustering of the resistance phenotype in sex worker families69 prompting us to establish an
epidemiologic study to determine if genetic factors in addition to MHC determine whether HIV-1
exposed individuals develop protective responses and escape persistent infection. Among sex
worker relatives of HIV-1 resistant index women from our cohort there was a high degree of intrafamily correlation of HIV-1 infection status, independent of HLA alleles (intraclass correlation
coefficient 0.31 and p values <0.001). Furthermore in non-sex worker relatives being related to an
HIV-1 resistant sex worker reduced the risk of HIV-1 by three fold (OR=0.32, CI95% 0.15- 0.7,
p<0.006), indicating a genetic component to resistance outside of HLA associations. To test the
hypothesis that HIV-1 resistance involves genes responsible for generation of type 1 and type 2
immune responses, we conducted a microsattelite analysis of the IL-4 gene cluster on archived
DNA from initially HIV-1 negative women from our sex worker cohort. One polymorphism in
the IRF-1 gene (termed IRF-1 179) was associated with a 2-fold reduction in the risk of incident
HIV-1 infection (Incidence rate ration 1.9, CI95% 1.4-2.6, p<0.0001). This association is
independent of HLA associations. This IRF-1 allele also appears to slow progression to AIDS
and lengthen survival in HIV-1 infected women enrolled in our studies. We have some
preliminary data from functional studies that this IRF-1 allele is associated with enhanced IFN-
-4 responses to HIV-1 in the T helper cell studies. Further work to fully
characterize the functional meaning of this IRF-1 allele and its relationship with IgA, T helper
cell responses, CTL and other immune responses in systemically and in the genital tract will be
undertaken in the studies proposed in this application.
In order to more completely understand the role of HLA and resistance we initiated highresolution sequence-based typing of class I and class II DRB genes on more than 800 women
including 120 resistant women. We confirmed previously reported associations and identified a
number of novel associations of class I and class II alleles with resistance and susceptibility to
HIV-1 infection. We were able to demonstrate the additive benefits of having multiple copies of
HLA types associated with resistance.
Genes upregulated in HIV resistant individuals
Women who had any single resistant alleles
N=5
Genbank ID
Common Name
Fold
p(2-tailed)
Description
were more than two fold protected against
J03143
IFNGR1
6.94
>0.0001 Interferon gamma receptor 1
HIV-1 infection (p=0.00001, Odds ratio:
NM_030751
TCF8
5.60
>0.0001 Transcription factor 8
2.45, CI95% 1.64-3.65) than women who
NM_004920
AATK
5.00
>0.0001 Apoptosis-associated tyrosine kinase have none of the identified resistant alleles.
NM_001926
DEFA6
4.65
>0.0001 Defensin, alpha 6
Furthermore, women who have two or more
AA282023
STAT5B
3.92
>0.0001 Signal transducer/activator of transcription
identified resistant alleles have additional
NM_004504
HRB
3.11
0.002 HIV-1 Rev binding protein
protection (Figure 5). Two thirds of HIV-1
NM_000572
IL10
2.69
0.007 Interleukin 10
resistant women in Pumwani cohort have at
least one of the identified resistant alleles
N=5
Genes downregulated in HIV resistant individuals
and possibly more resistant alleles will be
Genbank ID
Common Name
Fold
p(2-tailed)
Description
NM_001781
CD69
-3.90
>0.0001 CD69 antigen (T-cell activation antigen)identified once we complete high resolution
AA486367
STAT1
-3.65
0.0003 Signal transducer /activator of transcription
typing of other class II genes. These findings
NM_002198
IRF1
-2.76
0.005 Interferon regulatory factor 1
indicate a number of potential genetic
NM_000586
IL2
-2.72
0.006 Interleukin 2
mechanisms for resistance. We will be
NM_004513
IL16
-2.38
0.02 Interleukin 16
expand these studies in a number of
NM_002416
MIG
-2.33
0.02 Monokine induced by gamma interferon
comprehensive genetic analyses in this
NM_000576
IL1B
-2.16
0.03 Interleukin 1, beta
Table 1: Genes alternately regulated in HIV resistant Individuals revealed by microarray
analysis
7
proposal, as well as directly test if non-HLA genetic mechanisms associate with better
immunologic responses to a model vaccine.
B.5
Microarray expression analysis of CD4 lymphocytes from HIV resistant and
susceptible individuals
Microarray technology provides a powerful means by which to study the expression of thousands
of genes simultaneously. The Fowke lab has been conducting experiments utilizing gene array
technology for the past two years. To determine if there is a gene expression profile that could
be used as a correlate of the HIV resistant phenotype, they performed pilot studies on gene
expression of CD4 cells from 5 HIV resistant and 5 susceptible HIV negative women. Immune
microarray analysis of RNA from PBMC stimulated with media or a panel of antigens on a 4600
immune array revealed by cluster analysis that HIV resistant women have a similar pattern of
gene expression following antigenic stimulation with a common recall antigen, Candida
albicans. Even when stimulated with media alone, there were a number of genes that were
significantly differentially regulated in the HIV resistant women (Table 1). Although the sample
size was small, in this pilot study immunologically relevant genes such as α-definsin 6 and
interferon gamma receptor 1 are shown to be differentially regulated in HIV resistant women
(p<0.0001). Taken together these results indicate a genetic commonality between resistant
women, and suggest that a “resistant genotype” can be determined by genetic analysis.
Confirmation and expansion of these results will be a major focus of studies in both resistant
women and controls, but also in the Kindred cohort, which is biased in selection of individuals
most likely to have inherited traits associated with resistance (Goal 2.2). The hypothesis that a
resistance genotype correlates with a good outcome to mucosal viral challenge will be tested in
Goal 3.
B.6
Defining the Immunologic Microenvironment of the Genital Tract Using a
Proteomics Approach
A key goal of our work has been to understand how
the immunologic milieu (the totality of
immunogenetics, and cytokine and chemokine
influences on immune effectors) is involved in the
phenomenon of HIV-1 resistance. However, studies
of mucosal immunity in the human genital tract are
challenging because of limitations on amounts of
material available. However a proteomics approach
offers a solution to some of these problems. Surface
enhanced laser desorption ionization time of flight
(SELDI TOF) mass spectrometry70has been a powerful
for identifying unknown proteins .
HIV Negative
2500
3000
3500
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4500
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20
ML2001 - HIV Negativ e
0
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ML2012 - HIV Negativ e
0
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4500
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ML1997 - HIV Negativ e
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ML1997
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50
ML1989 - HIV Negativ e
ML1989
25
0
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3500
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ML1905 - HIV Negativ e
10
ML1905
0
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3918 daltons
HIV Resistant
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4500
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3500
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ML1376 - HIV Resistant
20
ML1376
10
0
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ML767 - HIV Resistant
ML767
10
0
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ML1275 - HIV Resistant
10
ML1275
0
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ML1622 - HIV Resistant
20
ML1622
0
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ML466 - HIV Resistant
10
ML466
0
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3500
-defensins
Expanding this work is a major focus of the work
proposed in this submission. This approach will not
only allow us to identify novel proteins that may be
ML2012
10
A-defensins
Protein chips can be used to determine protein
composition of all proteins in a biologic sample and
was used to recently characterize
the HIV inhibitory
properties of -defensins71. Preliminary data on
SELDI TOF use in characterizing genital tract
secretions of HIV resistant sex workers has yielded
some exciting results; proteins with the molecular
weight corresponding to the -defensins were found
to be present at higher levels in cervical secretions
from HIV resistant women compared to uninfected
controls and HIV infected sex workers respectively.
More interestingly, an unknown protein of mass 3918
Da was present at high levels in all HIV resistant
women. Several other protein peaks appear to differ
between the groups as well.
ML2001
10
4000
4000
4500
4500
3918 daltons
Figure 6. SELDI TOF analysis of endocervical secretions
8
associated with, and presumably playing a role in mediating resistance to infection by HIV-1, but
will also allow us to determine more precisely the presence of many of the previously described
anti-HIV factors such as as -defensins and β- chemokines and determinetheir relationship to
mucosal viral challenge
C. Methods and Experimental Design: From this work to date, we conclude that there is
resistance to HIV-1 among prostitutes who are continuously exposed to HIV-1. Resistance
correlates with MHC Class I and Class II alleles, with HIV-1 specific cellular systemic and
mucosal immune responses and HIV-1 specific mucosal IgA. While cellular and mucosal
immune responses may be important resistance mechanisms, the very important question of why
some individuals develop these responses, while others become infected, remains. The clustering
of HIV-1 resistance in families, and associations with polymorphisms in immunoregulatory genes,
all independent of MHC associations, suggests some other genetic factor is responsible, perhaps
working in concert with the protective MHC alleles. HIV-1 resistant women have some features of
a polarized type-1 systemic response to HIV-1. We hypothesize that the ability of resistant sex
workers to preferentially mount a type-1 immune response may develop from altered innate
responsiveness to TLR stimulation or an immunogenetic bias such that type-1 responses
preferentially develop.
In the research proposed here, we hypothesize that systemic as well as genital tract antibody and
cellular immune responses mediate protection against HIV-1 infection in resistant sex workers.
Further we hypothesize that these responses develop as a result genetic or acquired
immunoregulatory responses to HIV antigens. In the studies proposed in this submission we will
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 and test these associations in a vaccine model of mucosal immune
challenge.
Goal 1: To characterize correlates of protective immune responses to HIV in the systemic
and mucosal compartments of resistant and susceptible women by:
1.1
Determine the functional phenotype/specificity of HIV specific CD4 and CD8 T cells,
1.2
Characterise the frequency, specificity, neutralisation capacity and ability to inhibit
transcytosis of HIV specific IgA and IgG,
1.3
Determine if resistant women exhibit differential responsiveness to innate stimulation, or
altered innate receptor expression such that are more likely to develop protective immune
responses to HIV,
Goal 2: To identify genetic and innate factors associated with resistance in resistant women
and their families by:
2.1
Conducting a non-biased genome wide SNP screen to map genes associated with
resistance in both the SW and Kindred cohorts,
2.2
Determine if associations exist between resistance and polymorphisms in previously
identified genes important in susceptibility to HIV-1,
2.3
Determine by gene expression analysis if there are genes differentially expressed in
resistant women and their families,
2.4
By a mass spectrometry approach identify known and unknown innate factors in serum or
mucosa differentially expressed in resistant and susceptible women
Goal 3: To determine how genotypes/phenotypes identified above will determine immune
responsiveness to a model antigenic challenge of a live, attenuated mucosal vaccinogen.
3.1
The nature and specificity of immune responsiveness to the challenge vaccinogen will be
determined by a combination of assays utilised above (gene expression analysis,
proteomics, and T cell analysis of function and specificity),
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 serocoversion in sexworker and non-sexworker cohorts.
4.1
The presence of innate, genetic, or the development of HIV specific cellular immune
responses correlate with a reduced likelihood of HIV seroconversion in initially HIV
uninfected individuals at significant risk for HIV infection.
9
Experimental Plan: To address these goals, we will conduct nested cross sectional and cohort
studies among sex worker populations and their relatives in Nairobi. Studies proposed here will
be conducted on women enrolled in the Pumwani Sex Worker and Kindred Cohorts, in the
context of our University of Manitoba/University of Nairobi collaborative group. Study subjects
will be drawn from the following well-characterized populations.
The Pumwani Sex Worker Cohort: This cohort was established in 1985 to study the
epidemiology, biology and immunobiology of HIV-1 and STDs. The cohort currently comprises
over 2200 women, with new enrolment continuing at the rate of approximately 100 women per
year. These women have an extremely high risk of HIV-1 infection, with approximately 60%
of
women being HIV-1 seropositive at enrolment. Despite effective intervention programmes 72, the
annual incidence of HIV-1 infection among initially seronegative women is currently about 10%.
HIV-1 Resistant Women: We have defined resistance to HIV-1 infection as follow up in the
Pumwani cohort for three or more years, continuing sex work, and persistently HIV-1
seronegative and negative by HIV-1 PCR. Currently, we have identified 127 such individuals,
HIV-1 Negative Non-resistant Sex Workers: HIV-1 negative non-resistant prostitutes are those
women newly enrolled in the cohort who do not meet our definition of resistance – that is they
have not been followed for three years. A portion of them will ultimately become resistant. This
is a key group for determining the factors surrounding the immunologic correlates of HIV-1
resistance. There are 195 such women enrolled in the cohort and in follow up.
Kindred Cohort: This cohort was established to investigate genetic correlates of protection. We
initiated a case control family study of HIV-1 susceptibility phenotype among the prostitute and
non-prostitute relatives of resistant women. To date, 166 family groups with over 652 members
have been identified, 58 families with 241 members in which the index is a resistant sex worker
and 108 families with 411 members in which the index is an HIV-1 infected sex worker.
Control Populations: Several control populations are available to us for comparison with HIV-1
resistant women. These include several hundred HIV-1 infected sex workers from the Pumwani
cohort and several hundred HIV-1 uninfected low risk women enrolled in our HIV-1 Mother to
Child Transmission study.
Enrolment, Follow-up and Sampling of Subjects: Generally immune studies will be conducted
on all resistant members of the Pumwani sex worker cohort making this the largest examination
of immune responses to HIV-1 in a HEPS cohort to date. For genetic and proteomic studies we
will study women from the Pumwani cohort as well as members of the Kindred cohort. Over the
5 years of this proposed project, we anticipate recruitment of more than 100 newly enrolled
seronegative women of whom about 25 will ultimately meet our definition of resistance. All
women from the appropriate study groups will already be enrolled in the Pumwani cohort. They
will be individually approached about participating in the current study and the study
requirements will be explained to them. Women giving informed consent will be interviewed
regarding demographic and reproductive health variables. In addition to a general medical
history (including previous vaccinations and symptoms of allergy/atopy) a history of sexual
practices, such as condom usage, duration of prostitution and occurrence of previous sexually
transmitted diseases will be obtained. Current reproductive health data including pregnancy,
contraceptive methods, date of last menses, douching practices and other genital hygiene
practises will also be obtained.
Examination and STI Diagnosis: A general clinical and detailed gynaecologic exam will be
performed and specimens obtained for the diagnosis of sexually transmitted and genital tract
infections. Vaginitis and cervicitis will be determined syndromically by an experienced clinician
and confirmed via laboratory testing of vaginal discharge, plasma, and pap smears.
Endocervicitus and ulceration due to additional bacterial and viral STI’s such as Chlamydia, N.
gonorrhoea, T. palladum, H ducreii and HSV will be determined by a combination of diagnostic
PCR, serology and bacteriology.
Sampling for Immunologic, Genetic and Mass Spectrometry Analysis: Thirty ml of peripheral
blood will be obtained for serology for HIV-1, CD4 and CD8 counts and immunologic assays.
Genital tract specimens will be collected using a combination of lavage, cervical cytobrushing
10
and ectocervical sampling with a plastic spatula, a protocol that has been optimised for
maximum cellular recovery. Lavage samples will be obtained by washing the cervix with 2 ml
of sterile PBS and recovered from the posterior fornex region and placed on ice for transport to
the laboratory. Following lavage, CMC’s will be collected from the cervix by cytobrush as well0
as an ectocervical scraping. In the laboratory, lavage samples will be frozen immediately
at –70
for later assays. CMC’s will be essentially collected as previously described73. The specimens
will be collected at enrolment and every six months over the period of follow up.
Follow Up: After enrolment, women will be asked to return for follow up every month for a
review of interim history and examination for STI. Every six months complete sampling and reevaluation will be performed as previously described. Women enrolled in the cohort will be
provided with free clinical services and will be involved in condom promotion and STI
prevention programs based in the clinics. Based upon over 18 years of experience with working
in this cohort we expect approximately 10% loss to follow-up overall, and as this predominantly
occurs in new enrolee’s into the cohort we expect little loss to follow-up of resistant women.
1.1
Determine the functional phenotype/specificity of HIV specific CD4 and CD8 T cells
Rationale: The signal features of the immune response to HIV-1 in resistant individuals are
systemic and mucosal CTL and HIV-1 specific IgA in plasma and genital tract secretions. To
date we have only studied a minority of resistant women and many questions remain. How long
do CTL persist, what is their specificity, and what is their functional phenotype? CTL may in
part account for protection but we have limited understanding of why they develop. Studies of T
helper cell responses suggest that altered cytokine production, proliferative capacity and memory
phenotype may be involved in this genesis of these responses in HIV-1 resistant women. We
will expand our studies of resistant and susceptible women to perform a comprehensive analysis
of CD4 and CD8 specificity and function by epitope mapping CD4 and CD8 T cell responses by
Elispot and CFSE proliferation assays, determining their functional phenotype by multicolour
flow cytometry, and by determining diversity and breadth of responses by analysis of their TCR
repertoire. These experiments will more precisely characterize the true breadth and magnitude of
HIV specific reactivity in resistant women.
Assays for mapping CD4 and CD8 responses to HIV-1: Specimens for epitope mapping studies
will be collected from all HIV-1 resistant sex workers in our cohort (127 currently), 30 nonresistant HIV-1 seronegative sex workers, 30 HIV-1 infected sex workers, and 20 low risk HIV-1
uninfected women. For CD4 studies PBMC’s will be stimulated with peptide matrices composed
of pools of HIV-1 clade A peptides corresponding to HIV-1 proteins. These peptide pools will be
used to screen for HIV specific responses by Elispot and CFSE proliferation. We already have a
library representing gp120 and p24 that has been utilised in previous work. A 15-mer
library for
the entire HIV-1 genome overlapping by 11 will utilize approximately 1.2 x 107 cells. For CD8
studies, we have shown that 9-mer libraries overlapping by 1 are optimal for detecting weaker
CD8 responses such as those observed in vaccinees and HEPS individuals. Based upon
preliminary studies we will focus on gp120 to start, and expand to other HIV proteins as
appropriate. We already have a partial 9-mer library available for Clade A gp120. The number
of PBMC needed to perform these analyses will be similar to those used above. If enough cells
are not available we will stagger these studies to maximize efficiency. Reactive peptides will be
retested individually to verify stimulatory capacity. CD4 or CD8 T cell depletion will be
performed to confirm which T cell subpopulation is mediating responses.
Production of tetrameric class I and class II complexed to HIV epitopes: Identification and
characterization of antigen-specific T lymphocytes during the course of an immune response is
tedious and indirect. It has been always difficult to follow the fate of a T cell clone in vivo due to
the lack to an efficient tool. Our collaborators have developed MHC class-I and class-II
tetramers to identify CD4 and CD8 HIV specific T-cells. The use of tetramers combined with
CFSE or the phenotyping panels described below should permit a more accurate qualitative and
quantitative analysis of the anti-viral cellular immune response. These issues can best be
addressed using class I and II tetramer technology established in the Sékaly laboratory.
Determine the TCR repertoire diversity of HIV specific CD4 and CD8 HIV-specific cells. In
order to determine the diversity and the breadth of responses generated in resistant women two
approaches can be used to establish the parallel between HIV-specific responses: The first
11
approach will consist of identifying specific TCR-Vβs and HIV specific responses. We will use
anti-TCR antibodies to determine whether the detected HIV specific responses are oligoclonal or
polyclonal as detected by Elispot, CFSE and tetramer binding) and CD4 or CD8 amplified TCR
V s. The second will be to determine by heteroduplex tracking assays (HTA) and TCRβV
specific primers to determine
TCRβV phenotype and complexity. Such an experiment does not
require more than 105 cells. With the knowledge that the HTA assays can pick up a single clone
in about 10-50,000 cells we should have no problems in identifying dominant clonotypes form
such small number of cells. Moreover we will have significantly contracted the repertoire and
enriched for dominant clonotypes by sorting for tetramer positive cells.
Flow Cytometric Determination of Function/Phenotype of HIV specific CD4/8 cells: For
epitope stimulated responses it will be possible to confirm results generated in the Elispot or
CFSE assay with multi-parametric FACS analysis to measure cytokine secretion, function and
phenotype. For epitopes restricted by MHC alleles for which tetramers are available we will
determine the number of functional antigen specific cells by using HIV peptide tetramers to
detect antigen specific cells and their functional phenotype using intracellular cytokine staining
and CFSE proliferation. For these experiments, tetramer positive cells will be stained using the
panels described below. Flow cytometric analysis will be performed on an LSR flow cytometric
analyzer, which allows us to perform 10 colors staining. Functional phenotyping of HIV specific
cells will be conducted with memory,
Fl
Fluorochome
Memory
Function
Apoptosis
Activation
Phenotyping
function, apoptosis, activation and
Channel
/ Dyes
Panel
Panel
phenotyping panels as described in
1
AMCA 350
CD27
CFSE
CD45 RA
CD95
CD14
Table 2. By focusing our analysis on
2
Marina Blue
CCR7
CCR7
CCR7
the different functions of T cells,
3
FITC/ 7AAD
CD45RA
CD45RA
Annexin V
CD45RA
CD16
namely proliferation, IFN-γ and IL-2
4
PE
CD62L
IFN-γ
CCR7
CD38
CD19
production, we should be able to
5
PE-Cy5
CD28
IFNγ
7-AAD
CTLA-4
CD33
generate a more accurate picture of the
6
PerCP-Cy5.5
CD4
CD4
CD45RA
CD4
CD4
breadth of the HIV CD8 and CD4
responses after vaccination.
7
PE-Cy7
CD8
CD8
CD4
CD8
CD8
8
APC
CTLA-4
IL-2
CD8
HLA-DR
CD11c
9
APC-Cy7
CD3
CD3
CD95
CD3
CD3
Genital mucosal T cells: Cells isolated
from peripheral blood and from genital
10
Hoechst
Hoechst
Hoechst
tract samples will be used in Elispot
33342
33342
33342
assays, CFSE staining and Flow
Table 2: Immunophenotyping Panels for 10 Colour Flow
cytometry experiments similar to those
described above. Cervical response to
CTL and CD4 epitopes identified in PBMC will tested as described. Due to the paucity of
CMC’s available, screening of responses will be conducted on PBMC’s first, before testing
CMC responsiveness. The latter will likely be carried out at a subsequent visit due to the
difficulty in maintaining cervical cells while awaiting PBMC testing.
Data Analysis: CTL and CD4 responses will be compared across study groups in two ways. The
frequency of positive responses to particular epitopes will be compared across groups using
Odds ratios with 95% confidence intervals and exact p values. As these data are a measure of
CTL precursors it will also be possible to compare results across groups using nonparametric
statistical tests. The epitope specificity of the responses in PBMC and genital tract secretion will
be also be compared between resistant and HIV-1 infected women using Odd ratios with 95%
confidence intervals and exact p values. Multivariate analysis and logistic regression will also be
conducted to control for physiological factors that may affect these results, such as stage of
menstrual cycle, hormonal contraceptive use, or douching practices.
Potential Problems and Alternative Approaches: These studies are an extension of work we and
our collaborators are already doing, and no new techniques need to be developed. The major
challenges will be keeping study subjects in regular follow up and coping with the volume of
work. We have long experience with conducting these types of studies in Nairobi and an
excellent relationship with the cohort and surrounding community.
1.2
Characterise the frequency, specificity, neutralisation capacity and ability to inhibit
transcytosis of HIV specific IgA and IgG,
12
Rationale: Our initial data show that a high proportion of HIV-1 resistant women have HIV-1
specific IgA in their cervical secretions, saliva and plasma directed at a recombinant envelope
antigen. Further studies show that IgA antibody neutralizes both syncytium inducing and nonsyncytium inducing HIV-1 isolates, neutralizes across clades and inhibits transcytosis. Thus
further characterization of HIV specific neutralizing antibodies is of obvious relevance to
vaccine development. The main purpose of the IgA studies proposed here is to understand why
these responses develop in some individuals by correlating the presence of IgA antibody to other
immunologic responses and immunogenetic factors. To determine how these responses change
over time and whether they correlate with protection against HIV-1 infection, we will sample all
women with in the study subgroup at six-month intervals over the five-year study period. This
should permit the correlation of IgA antibody to HIV-1 among HIV-1 negative women with
concurrent infection with other STIs, immunogenetic factors and T helper cell responses, as well
as with protection against infection. The neutralizing activity of IgA antibody detected
is of
course of interest, as would the ability of these antibodies to inhibit transcytosis of HIV-174.
Isolation and quantitation of IgA and IgG from Plasma and Genital Tract Specimens: Specimens
for cervical antibody studies will be collected from all HIV-1 resistant sex workers in our cohort
(127 currently), non-resistant HIV-1 seronegative sex workers (195 currently), HIV-1 infected
sex workers (30 selected women) and low risk seronegative HIV-1 uninfected women (20
women attending family planning clinics in Nairobi). For IgA assays, IgG antibody will be
removed using an IgM anti-IgG antibody as previously described to prevent masking of IgA
responses. Cervical and systemic antibody levels against various HIV proteins will be
quantitated using a new bead array assay developed in the Fowke lab. In this assay various HIV
recombinant proteins are linked to beads with different levels of fluorescent dyes, exposed to
serum or lavage samples and developed with fluorchrome-linked antibodies against IgG or IgA.
Determination of HIV-1 Specific IgA, neutralization capacity, and ability to inhibit transcytosis:
Levels of HIV-1 specific
IgG and IgA will be measured in genital tract samples using methods
previously described45 by immunoblot, and by colorimetric bead analysis. HIV-1 specific IgA
and IgG will be quantitated as will the proportion of HIV-specific
dimeric IgA. Immunoblots
will be performed as previously described by our laboratory75 except that specific reactivity to
HIV-1 protein bands will be quantified by densitometric analysis. Neutralization profiles of76IgA
and IgG obtained from resistant and susceptible women will be determined as described in
collaboration with Dr. Ken Rosenthal. Neutralization titers will be compared among the various
study groups. For transcytosis assays primary endometrial and uterine cell cultures and the
human HEC-1 endometrial cell line will be seeded in polycarbonate membrane transwells and
cultured for until formation of tight junctions and a well-differentiated monolayer is achieved
and confirmed by electrical resistance across the membrane. Indicator cells will be placed in the
basolateral compartment of the transwells. The indicator cell line will be either Jurkat or MT-2
(X4 Virus), or U38 cells for R5 virus. For the assay itself, cultures will be treated with
monoclonal IgG or IgA antibodies or medium as a control for 24 hours,5 followed by addition of
cell-free HIV-1 (50-200 ng p24/well) or cell-associated HIV (2.5 x 10 cells/well) to the apical
chamber of transwells. Following 3 hours of co-incubation of epithelial cells (EC), the virus
inoculum will be removed by extensive washing of the ECs. Supernatants will be collected from
the indicator cells in the basolateral compartment at various times post-infection and p24 ELISA
will be done to determine the virus quantities. In addition to unlabelled HIV, we will be utilizing
HIV-1-GFP visualized under a confocal microscope.
Data Analysis: Plasma and genital tract antibody levels, Neutralisation capacity and transcytosis
capacity will be across study groups in two ways. Overall antibody concentrations and optical
densities will be compared between groups using nonparametric statistical tests (Mann-Whitney
U). The frequency of positive tests will be compared between groups using Odds ratios with
95% confidence intervals and exact p values.
Potential Problems and Alternative Approaches: These studies are straightforward and the
methodology has been worked out. As the majority of women in the cohort practice post-coital
douching (>95%), any potential contaminating effects of IgA in their partner’s semen can likely
be ruled out. The major problems will be in ensuring follow up of women.
1.3
Determine if resistant women exhibit differential responsiveness to innate
stimulation, or altered innate receptor expression such that are more likely to
13
develop protective immune responses to HIV,
Rationale: Although much of the focus of immune-mediated resistance of women in the
Pumwani cohort has focused on local adaptive immune responses, including HIV-specific
antibodies and T cell responses, it will be important to investigate the role of innate immunity,
particularly in the genital mucosa in these women. We have shown that HIV resistant women
exhibit differential responsiveness of cytokines important in the development of type-1 adaptive
responses (IL-10, IL-12, IFN-, IFN-) to a number of TLR ligands, including TLR-7,
previously shown to respond to HIV-1 RNA sequences. This suggests that they have altered
innate responses to HIV-1 and indicate a mechanism by which protective immunity to HIV-1
may develop.
Determine the responsiveness of PBMC, CMC and DC to TLR stimulation: We will determine
cytokine expression levels following stimulation by panel of TLR ligands in PBMC, CMC’s and
isolated DC from all HIV-1 resistant sex workers in our cohort (127 currently), non-resistant
HIV-1 seronegative sex workers (80 selected women), HIV-1 infected sex workers (30 selected
women), low risk seronegative HIV-1 uninfected women (30) and kindred of resistant women
(80). In addition, expression of TLRs will be assessed using real-time PCR on RNA isolated
from PBMC and mononuclear cells isolated from the genital mucosa following collection using
cervical cytobrush. Cells isolated from peripheral blood, genital tract and tissue samples will be
used in short-term ex-vivo culture to determine cytokine responsiveness to a panel of innate TLR
stimuli. Briefly, PBMC and CMC will be isolated and stimulated with peptidoglycan-TLR-2,
Poly I:C-TLR-3, LPS-TLR-4, flagellin-TLR-5, GU-rich ssRNA-TLR-7/8, CpG ODN-TLR9.
Cell culture supernatants will be collected and assayed for IL-6, 10, 12, IFN-, , TNF-, by
ultrasensitive ELISA’s. RNA will be collected from the remaining cells and tested for TLR
expression as described below.
Expression of Toll-like receptors in female genital tract: Collaborators in Dr. Rosenthal’s
laboratory have developed semi-quantitative RT-PCR for the whole panel of TLRs in both mice
and humans. They have began to examine the profile of mRNA expression of all 11 human
TLRs in primary cell cultures of human endometrial and cervical EC monolayers. They will use
these techniques to measure TLR expression in PBMC, CMC’s, DC and EC obtained from HIV
resistant and susceptible women. In order to correlate expression of TLRs expressed on primary
epithelial cultures with in vivo expression, they will utilize a combination of Laser Capture
Microscopy (LCM) with Real Time RT-PCR. Laser Capture Microscopy (LCM) has been
successfully used to isolate vaginal epithelium from female mouse genital tract tissue and more
recently from human genital tissue.
Data Analysis: Individual cytokine levels and cellular and genital tract TLR expression levels
will be compared across study groups in two ways. Comparisons will be made between study
groups by analyzing means, medians and proportions by t-tests, nonparametric testing and
controlling for potential confounding variables by logistic regression. TLR expression and
responsiveness will also be correlated with other IgA and CTL responses, protein profiles of
genital tract secretions, genetic factors and epidemiologic and behavioral data using parametric
and nonparametric statistical tests and correlated with HIV-1 infection risk in women who
seroconvert to HIV-1 other course of this study. We will control for potential confounding
physiologic variables such as stage of menstrual cycle, hormonal contraceptive use or douching
practices by multiple and or logistic regression as appropriate to the data.
2.1
Conducting a non-biased genome wide SNP screen to map genes associated with
resistance in both the SW and Kindred cohorts,
Rationale: The completion of the human genome project has allowed for detailed maps to be
created that span the entire genome. Single nucleotide polymorphisms, spaced evenly throughout
the genome, can be used to map loci associated with HIV resistance. Our research group has
data that suggests that there is a genetic component to resistance that is not completely explained
by HLA type, or by IRF-1 geneotype. These data suggest that there is a genetic component to
resistance. In this specific objective we will undertake a non-biased total genome SNP analysis
to discover genes linked to resistance to HIV.
14
Experimental Design: We have already collected genomic DNA on 640 Kindreds and 900 sex
workers and established B cell lines on 200 Kindreds and 400 sex workers. DNA will be
isolated from HIV-1 resistant sex workers (127 currently), HIV susceptible (infected-80)
women), HIV uninfected first degree relatives of both resistant (80) and susceptible (80) women
for single nucleotide polymorphism analysis. SNP frequency in these populations will be
measured using the Affymetrix GeneChip© mapping assay which simultaneously types a single
sample for greater than 10,000 SNPs spanning the entire human genome. DNA from all samples
will be restriction enzyme degraded, ligated into a generic amplification adapter, PCR amplified,
end-labeled and hybridized to the Affymetrix 10k GeneChip© prior to scanning and analysis.
Scanning and analysis of GeneChip© arrays will be carried out through the DNA core facility at
the National Microbiology Laboratories (NML) at Health Canada. Genomic areas containing
SNPs found to be highly associated with resistance to infection will be further analyzed either by
DNA sequencing of candidate genes within that area or further SNP typing using custom SNP
chips (Affymetrix) designed using public databases.
Data Analysis: Analysis will consist of determining SNPs found to be over or under-represented
in the HIV resistant population. After scanning Affymetrix GeneChip DNA analysis software
(GDAS) is used to make genotyping calls. Individuals can then be scored as having 0, 1, or 2
copies of a given SNP. Data can be easily exported from the affymetrix software to third party
programs for comparative analysis. This allows for comparison of both individual SNPs as well
as haplotype analysis. The NML DNA core facility has experttise in this form of analysis.
Further analysis of genomic areas of interest may be carried out in the same manner if focused
SNP chips are used or by sequence analysis.
Potential Problems and Alternative Approaches: GeneChip © technology has been widely used
and Affymetrix has been a reliable source for gene expression and genetyping technology for
over 10 years. As the GeneChip © technology only requires minimal sample material (250 ng of
genomic DNA) it is unlikely we will encounter any setbacks from a technical perspective.
2.2
Determine if associations exist between resistance and polymorphisms in previously
identified genes important in susceptibility to HIV-1,
Rationale: The clustering of HIV-1 resistance is independent of MHC alleles, but it is important to
understand how other potential genetic influences are affected by MHC susceptibility and
resistance alleles in the sex worker cohort as well as the Kindred cohort. We will thus complete
molecular MHC Class I and Class II genotyping on individuals enrolled in the family study. As
noted HIV-1 resistance in the sex worker cohort is associated with a particular allele of the IRF-1
gene. We have not yet examined this polymorphism in the families of resistant sex workers. In
addition to examining these genes we will continue to examine potential candidate genes as they
arise, either ourselves or through collaboration. If initial studies show a relationship between
other polymorphisms and resistance we would proceed to genotype the subjects in the family
study. In addition it is almost certain that additional genes involved in susceptibility to HIV-1
infection and disease will be identified by other groups and we will examine their association
with HIV-1 resistance in both the sex worker cohort and the kindred cohort as they arise.
Experimental Design: We will determine the HLA type and IRF-1 genotypes of all new
enrollee’s into the sex-worker cohort and those who have not been previously typed (approx 100
new enrollee’s). We will also type all members of the Kindred cohort related to a resistant
women (241) or an HIV susceptible women (411). DNA for both microsatellite analysis and
molecular HLA typing will be extracted from either buffy coats, PBMC, or from B-LCL. Using
primers and annealing temperatures specific for the specific microsatellites, PCR reactions will
be carried out in the presence of fluorescently labeled primer and resolved on an ABI 3100
genetic analyzer. The inheritance of specific microsatellite alleles specific to the resistant
families could indicate potential candidate genes responsible for the resistance phenotype.
Molecular HLA Class I and Class II typing will be performed using the sequence based approach
developed in our Group.
Sample Size and Data Analysis: The microsatellite analysis and MHC genotyping will be
performed on all subjects in the family study. The alleles of interest will be correlated with HIV-1
infection status, the presence of atopy and asthma and immunologic phenotype (as described
below). Standard epidemiologic and statistical techniques will be used to correlate HIV-1
15
phenotype and microsattelite polymorphisms on chromosome 5 and MHC alleles. The issue of
multiple comparisons, which is unavoidable in this type of study, arises here. The problem is
mitigated somewhat by the fact that we have an a priori hypothesis for the potential
polymorphisms and Class I and Class II alleles. For associations with no a priori hypothesis, we
will not correct for multiple comparisons but will report only associations with highly significant p
values (ie. p<0.005).
Limitations and Alternative Approaches: These studies present no technical challenge as our
laboratory is already proficient in the basic techniques and adequate resources for automated DNA
sequencing are available. The approach we have chosen seems to be the most efficient one based
on our findings to date.
2.3
Determine by gene expression analysis if there are genes differentially expressed in
resistant women and their families,
Rationale: Why are some women resistant to HIV infection and have been so for up to 15 years
while other become infected soon after initiating risky behavior? Our research group has data
that suggests that there is a genetic component to resistance that is not completely explained by
HLA type, or by IRF-1 geneotype. We have found that sex worker relatives of HIV resistant
woman were less likely to be HIV infected than those related to HIV infected woman. This
association extended beyond those involved in sex work and non-sexworker relatives of HIV
resistant women were half as likely to be HIV infected as the non-sexworker relatives of infected
women. These data suggest that there is a genetic component to resistance. In this specific
objective we will determine if the HIV resistant women have differential gene expression
profiles from CD4, CD8, and cells of the monocyte/macrophage lineage from PBMC as well an
unbiased analysis of whole blood and CMC’s.
Experimental Design: We will determine gene expression analysis of isolated cell populations
as well as whole blood DNA and CMC samples from all HIV-1 resistant sex workers in our
cohort (127 currently), non-resistant HIV-1 seronegative sex workers (80 selected women), HIV
uninfected first degree relatives of both resistant (80) and susceptible (80) women. We will
specifically isolate the specific cells from PBMC using a magnetic bead/FACS sorting
procedure. Whole blood samples and CMC samples will be obtained directly ex-vivo to provide
an unselected analysis platform. Once the cells are isolated RNA will be extracted mRNA will be
linearly amplified using the Ambion Message Amp aRNA kit. Linear amplification of mRNA
has been shown to increase the yield by over 40 fold and has not only been validated but shown
to improve sensitivity. cDNA from subjects and control standard RNA will be synthesized and
reciprocally labelled with fluorescent dyes (Cy3 and Cy5) and co-hybridized onto the human
19,000 gene array obtained from CANVAC and the Ontario Cancer Institute. Array
quantification will be done using Array Pro software and normalization and analysis will be
performed using Gene Traffic and Gene Spring software. The Fowke lab has experience with
both of these software packages.
Data Analysis: Analysis will consist of developing a “genes of interest” list and comparing the
expression within groups between each cell type examined and will include immunologically
relevant genes, such as those used to define memory, those involved in lymphocyte trafficking,
cytokines and their receptors, signal transduction molecules, factors that regulate signal
transduction, TLR’s, and transcriptional regulators. In addition to this hypothesis driven analysis
of the gene specific lists, we will also perform “data mining” which is a blinded look for genes
that are differentially regulated between the comparison groups. Any alterations in gene
expression levels suggested by array will be confirmed by RT-PCR. For the individuals for
whom we are performing the genomics study we will also obtain enough PBMC to perform the
detailed phenotyping and functional studies previously outlined. In this manner we can increase
the power of the study by correlating changes in gene expression data with functional parameters
such as memory phenotype, immune activation and antigen-specific responses.
Potential Problems and Alternative Approaches: The obvious potential problem is that we will
not be able to identify any differences in gene expression between resistant women and controls.
We believe that this is highly unlikely and as has been described, our pilot study based upon few
individuals has already identified a number of genes differentially expressed in resistant women
(Table 1).
16
2.4
By a mass spectrometry approach identify known and unknown innate factors in
serum or mucosa differentially expressed in resistant and susceptible women
Rationale: During sexual exposure to HIV-1 the immune system’s initial encounter with the
virus occurs in the complex environment of the genital tract. In resistant women we hypothesize
that the genital tract microenvironment has a bias that permits the development of protective HIV
specific responses and subsequent protection. Studying this environment in humans is
challenging because of the small amount of secretions, low numbers of cells, low concentrations
of cytokines and chemokines, the difficulty of working with important cell types and bacterial
contamination of the material. A proteomics approach using SELDI TOF allows us to overcome
many of these difficulties. Thus a more complete picture of the microenvironment than can be
obtained by measuring chemokines, cytokines or other proteins individually is produced. Since
the procedures require small amounts of material and are quite rapid once standardized they lend
themselves to repeated assays on individuals over time. An important advantage is that unknown
or unsuspected factors can also be examined using this approach. This approach has already been
used for the identification of alpha-defensins as anti-HIV factors and is being applied to the
identification of78biomarkers useful for diagnosing tissue rejection77, as well as determining stage
of breast cancer .
Experimental Procedures: We will collect cervical and serum samples for proteomic studies
from all HIV-1 resistant sex workers in our cohort (127 currently), non-resistant HIV-1
seronegative sex workers (195 currently), HIV-1 infected sex workers (30 selected women) and
low risk seronegative HIV-1 uninfected women (30). As well we will examine the proteomic
content of serum in 80 members of the Kindred cohort related to a resistant woman and 80
members related to a susceptible woman. Using Cytometric Bead Array analysis (CBA) we will
also determine systemic and vaginal cytokine and chemokine levels. Each proteomic sample
will be fractionated into 4 fractions in duplicate, based upon pH. Five microliters of each
fractionated sample will be spotted in duplicate onto three different protein chips with different
binding characteristics. Samples will be incubated for 10 minutes before being washed twice
with water before one microliter of matrix is added. The chips will then analysed using SELDITOF mass spectrometry on a ProteinChip Reader (Model PBS II), This protocol will give 12
different protein mass spectra of cervical mucosal secretions for each individual .
Data Analysis: The mass spectra obtained will be analyzed using ProteinChip Software v. 3.0
and compared between resistant and control women, and the kindred cohort, using Biomarker
Patterns™ Software system (Ciphergen). This software is designed to readily differentiate
between hundreds of biomarkers (peptide mass spectra peaks) simultaneously. Peak patterns
will be compared between groups on each of the 12 spectra obtained per individual.
Differentially expressed peptide peaks will be identified based upon their mass, and/or isolated
for peptide sequencing by tandem mass spectrometry. Peptide sequences will then be screened
through the appropriate human and other protein databases for identification and source. Novel
sequences will be further characterized through tandem mass spectrometry sequencing and
comparisons to human expressed sequence tag (EST) databases. More traditional assays such as
ELISA or Western blots will be used to supplement this approach where necessary.
Potential Problems and Alternative Approaches: The obvious potential problem is that we will
not be able to identify any differences between secretions obtained from resistant women and
controls. We believe that this is highly unlikely and as has been described, our pilot study based
upon few individuals has already identified at least 5-6 peaks that are present in resistant women,
but not controls.
3.1
The nature and specificity of immune responsiveness to the challenge vaccinogen
will be determined by a combination of assays utilised above (gene expression
analysis, proteomics, and T cell analysis of function and specificity),
Rationale: In order to determine unequivocally the correlates of protection from HIV infection,
an HIV challenge would be necessary. As this is obviously not possible, we will use an in vivo
viral challenge system to model in HIV resistant women and controls how the adaptive immune
systems responds to a mucosal infectious insult, and what role genetics, or innate responses play
in the development of these responses. In HIV resistant commercial sex workers, exposure to
17
HIV is almost exclusively through the vaginal mucosa. Therefore, to mimic as practically as
possible this situation, we will use a live attenuated influenza vaccine model delivered through
the anterior nares to generate a mucosal challenge. As HIV resistance clusters in families this
suggests a genetic component. To determine if there is a genetic predisposition that contributes
to resistance, HIV resistant women and their relatives will be characterized by gene array
analysis and functional immune assays following a controlled antigenic challenge. The
comparison group will be HIV susceptible women and their kindred.
The vaccine
challene: The FDA has approved the use of a cold adapted live-attenuated (CAIVT, FluMist® MedImmune Vaccines, Inc. Gaithersburg, MD) intranasal influenza vaccine for use
in healthy individuals (age 5 to 49). The master influenza virus was attenuated through
successive passages at lower temperatures. The A/Ann Arbor/6/60 virus, an influenza type A
H2N2 virus, and the B/Ann Arbour/1/66 virus, a type B virus, have been cold adapted and
through multiple mutations in the polymerase genes the attenuation has been shown to be stable.
Using these viruses as backbones, genetic reassortants have been constructed to introduce new
hemagglutinin (HA) and neuraminidase
(NA) genes into the attenuated virus and have been
shown to be genetically stable79. The current vaccine is composed of A/New Caledonia/20/99
(H1N1), A/Panama/2007/99 (H3N2) and B/Hong Kong/330/2001, however, because the exact
make-up changes for year to year we will use what ever strains are current at the time of80the
study (year 3). Clinical trials have shown the vaccine to be both immunogenic and effective 81.
The benefits of this vaccine for this study is it is FDA approved; live attenuated; delivered
mucosally; highly immunogenic; only requires one dose; assays are well developed to monitor
immune response; and provides real-life health benefit to those in the study.
Protocol: This study will evaluate the functional, proteomic and genomic immunological
response of 60 HIV resistant sex workers; 60 uninfected controls from the sex worker cohort, as
well as two first degree sex-matched relations of each resistant and susceptible woman (120 for
resistants and susceptibles) to maximize likelihood of genetic correlations. The sample size of
the newly enrolled group is sufficiently large to control for those that later go on to become HIV
resistant. The protocol will consist of vaccination at baseline with the live attenuated influenza
vaccine. The vaccine will be appropriate for the circulating strains that year and delivered by
aspirating 0.25ml of culture into each nostril. The follow-up visits will be at one week and one
month post vaccination based on our previous experience with influenza vaccination as a model
of immune activation82. At baseline and the two follow-up visits; 30 mls blood, a nasal swab,
saliva and cervical lavage secretions will be obtained for assessment of immune responses.
Standard samples and examinations will be given.
Measuring Adaptive Immune Responses: Using vaccine-matched live influenza viruses (to
measure CD8 responses) and recombinant baculovirus produced HA antigens (to measure CD4
responses) obtained from Dr. Li, adaptive immune responses to the vaccine challenge will be
measured using previously described protocols, except Flu specific antigens will be based upon
the vaccine strain in use. The immunophenotyping panels described will be used to confirm the
phenotype, memory status and activation profile of the responding cell. This will allow us to
correlate HIV resistant phenotypes or genotypes with strength, specificity and phenotype of T
cell responses to the vaccine challenge. We have extensive analysis in measuring T cell
responses to HIV-1 and experience
measuring influenza-specific cellular immune responses
following vaccine challenges83. Antibody responses will be determined by hemeagglutination
inhibition (HAI) assays and strain specific antibody titrations will be performed with vaccine
matched viral isolates. Pre-existing antibody titres will be determined at baseline and a 4 fold
increase in titres will indicate a positive response post vaccination. HAI titrations will be
determined in plasma samples by
Dr. Yan Li who directs is the Canadian national reference
laboratory for influenza studies84,85, 86,87,88. We will also measure nasal, salivary and vaginal
levels of influenza-specific IgG and IgA using our multiplex bead array assay with recombinant
HA antigens produced in Dr. Li’s lab. Again this approach will allow us to correlate HIV
resistant phenotypes or genotypes with strength and specificity of site specific humoral
responses.
Correlates of Innate and Genetic Responses: We will correlate altered innate cytokine responses
to TLR stimuli, or altered TLR expression with immunologic outcomes to the model vaccine.
We would hypothesize that individuals with an altered innate response associated with resistance
would be predictive of better immunologic responses to the model vaccine challenge. In a similar
18
manner we will correlate genetic factors associated with resistance such as the IRF-1 179
genotype or those identified by SNP analysis to responses to the model vaccine challenge.
Proteomic responses: Mucosal and plasma levels of cytokines and proteomic biomarkers
identified in Goals 2.4 will be determined by a combination of CBA’s and mass spectrometry in
both plasma and mucosal samples. This will determine a) if these biomarkers are elevated in
resistant women, suggesting a factor likely mediating immune responsiveness to the vaccine
challenge, or b) these markers are associated with a better immunologic outcome to the model
vaccine challenge suggesting an innate factor mediating immunity to a mucosal viral pathogen.
Genomics: As described above immune expression assays will be performed using a 19K gene
glass array. Gene expression analyses will be performed on whole blood immediately preserved
ex vivo and on influenza-stimulated T cells. PBMC will be isolated and stimulated for 24 hours
with media alone or with the vaccine-matched influenza viruses. Following stimulation CD4 and
CD8 T cell and monocyte populations will be separated as described. Microarray experiments
will be performed and analyzed as described above. This will determine a) if genes alternatively
expressed in resistant women to HIV are also alternatively expressed in response to the model
vaccine, suggesting mechanisms mediating immune responsiveness to the vaccine challenge, or
b) these markers are associated with a better immunologic outcome to the model vaccine
challenge suggesting genetic factor(s) mediating immunity to a mucosal viral pathogen.
4.1
The presence of innate, genetic, or the development of HIV specific cellular immune
responses correlate with a reduced likelihood of HIV seroconversion in initially HIV
uninfected individuals at significant risk for HIV infection.
Rationale: We have little information about what is required for the development of HIV
specific immune responses, how long they persist or what factors may contribute to their waxing
or waning. These kinds of data may be important in understanding how to design HIV-1
vaccines or in refining HIV-1 vaccine immunization strategies. Longitudinal study of women
newly enrolling in the Puwmani will allow observation of the development of CTL, T helper cell
responses and HIV-1 specific IgA responses in a proportion of women and how these correlate
with genetic factors, epidemiological, reproductive and behavioral variables. We will also, in a
few individuals, be able to correlate HIV-1 specific immune responses with incident HIV-1.
Unfortunately even with very effective prevention programs, HIV-1 incidence remains at about
10% a year in newly enrolling HIV-1 negative sex workers. There are also occasional infections
in HIV-1 resistant women. We estimate that over the five-years of follow up 50 new HIV-1
(65% prevalence of HIV-1 infection at entry, 100 enrollments per year, a 10% annual incidence
of HIV- 1 seroconversion and a 10% loss to follow up of HIV-1 uninfected women annually)
infections will occur. This will permit us to examine the events surrounding infections acquired
in the face of HIV-1 specific immune responses. There will also be the opportunity to correlate
genital tract and responses with protection against infection prospectively.
Experimental Plan: We will follow the cohort of HIV-1 resistant and non-resistant HIV-1
uninfected sex workers at monthly intervals as described. Specimens for IgA determination, and
proteomics assays will be obtained at baseline and at six-month intervals.
Data Analysis: Results of assays will be correlated with infections with other sexually
transmitted and urinary tract infections, the results of other immunologic assays, epidemiologic
data and reproductive health data and history. Comparisons between groups will be as described
above. We will collect data on a wide variety of potential confounding factors that have been
shown to effect immunity at the female genital tract such as the use of hormonal contraception,
concurrent STI, stage of menstrual cycle, time, frequency, and type of douching, pregnancy
status, and cervicitis and vaginitis. All of which have been shown to effect specific facets of
genital tract immune responses to HIV-1. This will allow at least cross sectional analysis of their
effects on HIV-1 specific immune responses.
Potential Problems and Alternative Approaches: This element of the proposed research presents
no technical challenges. The major problem is ensuring continued follow up and sampling of the
Pumwani cohort but as explained above we have experience on our side.
19
D. Project Team
Dr. Frank Plummer will responsible for overall scientific direction and progress of this proposal.
He will be responsible for study design, direction and data analysis.
Dr. Keith Fowke will oversee the CD4 and CD8 studies described. He will be responsible for
conducting the immunophenotyping and functional cellular assays described.
Dr. Ken Rosenthal will oversee the innate studies, and assist on the IgA transcytosis and
neutralisation assays.
Dr. Rafick Sekaly will be responsible for TCR analysis of CD4 and CD8 T cells and will assist
in Tetramer based analysis of CD4 and 8 Cell function.
Dr. Rupert Kaul will oversee the CD8 epitope mapping studies and provide assistance in
measuring immune responses at the genital tract.
Dr. Yan Li will be responsible for conducting the Flu specific antibody studies, as well as
provide vaccine-strain specific reagents for the immune assays. He will provide scientific
direction for the influenza vaccine challenge.
Dr. Joshua Kimani will be responsible for direction of the Pumwani sex-worker and Kindred
cohorts. He will be responsible for direction of clinical staff.
Dr. Walter Jaoko will oversee Kenyan laboratory based staff, be involved in study design and
implementation and provide contact with the Kenyan AIDS Vaccine Initiative and
International AIDS Vaccine Initiative
Dr. Ben Estamble, as director of the University of Nairobi Institute for Tropical and Infectious
Disease, will provide direction and oversight of all Kenyan based studies, be responsible for
negotiation with University of Nairobi officials, and the Kenyan government. Together with
Dr. Jaoko he will responsible for identifying future Kenyan collaborators and trainees.
Dr. Blake Ball will be responsible for the day-to-day operation and maintenance of the Grand
Challenge. He will assist in innate and antibody studies, and be responsible for proteomic
analyses.
Dr. Ma Luo will be responsible for conducting genetic studies, including high-throughput HLA
and IRF-1 typing, as well as overseeing SNP and other genetic analyses.
The described team members all have a history of working together in a collaborative group.
Dr’s Ball, Luo and Kimani are already part of Dr. Plummer’s research team. Dr’s Kaul and
Fowke have worked with the research group for 16 and 10 years, respectively, and are PI’s
within the Resistance and Susceptibility to Infections Research Group led by Dr. Plummer and
are actively involved in research in the Nairobi cohorts. Dr. Li is a colleague of Dr. Plummer at
Health Canada’s National Microbiology laboratories. Dr’s Rosenthal and Sekaly have
collaborated with Dr. Plummer’s group for many years, both independently, or as members of
the Canadian Network for Vaccines and Therapeutics (CANVAC). Dr’s Jaoko and Estamble are
long-time collaborators with Dr Plummer during his tenure at the University of Nairobi, and are
outstanding supporters of the University of Manitoba/University of Nairobi collaborative group,
E. Management Plan: This group has already been collaborating for a great many years, we
will continue to do so, and in the context of the Grand Challenge we will implement more
vigorous mechanisms to insure communication is maintained. Management and coordination
will be centred at the University of Manitoba. Meetings specific to Grand Challenge issues will
be scheduled on a regular basis. Monthly conference calls are already in place between
University of Manitoba and University of Nairobi. These will continue. We will schedule bimonthly conference calls between all researches to coordinate studies and experimental
procedures. Decision making will lie with Dr. Plummer, in consultation with those investigators
with experience most relevant to the decision in question. Resources will be allocated according
to need, and according to the detailed experimental plans provided. Focus will be maintained by
insuring lines of communication remain open and through scientific interaction.
F. Product and Project Maturation Plans: We do not anticipate any products, potential
inventions, or intellectual property to be developed to completion through the 5 year portion of
this grant. This project has the potential for commercially important findings, such as novel antiHIV factors, identification of vaccine targets, or identification of vaccine adjuvants.
Memorandums of Understanding between the University of Manitoba and the University of
Nairobi cover these issues and are reviewed on a regular basis. Additionally, the University of
Manitoba has an Industrial Liason Office skilled in management of potential IP issues, as well as
long-term product development.
20
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Commun Dis Rep 25:177.
ORGANIZATIONAL CAPACITY
In 1980, the University of Manitoba and the University of Nairobi embarked on a small
collaborative research program on chancroid and Haemophilus ducreyi. Over time, this
collaboration has been built into a major international initiative for the study of infectious
diseases. Centred on the initial University of Manitoba-University of Nairobi collaboration, this
initiative now includes scientists from nine institutions (five Canadian institutions including the
University of Manitoba, Health Canada’s National Microbiology Laboratory in Winnipeg, the
University of Toronto, the University of British Columbia, the Université de Montreal and its
partners in the CANVAC Network of Centres of Excellence (NCE) and Université Laval). In
addition to the University of Nairobi, the non-Canadian institutions include the University of
Oxford, UK, the University of Washington, U.S.A., and the University of Ghent, Belgium. This
collaboration is regarded internationally as the model for collaborative international research88.
The collaboration’s research interests are extremely broad, particularly in research on HIV and
sexually transmitted infections. This highly successful international collaboration was gradually
formed by building research capacities and programs on areas of strength and expansion into
areas of comparative advantage.
This collaboration has been enormously productive. Over the past 20 years, over 700 original
articles and hundreds of reviews, monographs and book chapters have resulted from this
collaboration. Among these are some of the most influential publications in the field of sexually
transmitted infection research. As evidence, the average Institute for Scientific Information (ISI)
citation count of the collaboration’s publications is 13.6, which compares favourably with an
average of 8.5 for all papers in the Science Citation Index. HIV/AIDS-related publications of the
Manitoba-Nairobi axis of the collaboration have been particularly highly cited. In a citation
analysis by ISI in December 2001, the collaboration’s AIDS papers were cited over 5000 times
with the average citation rate of the collaboration’s AIDS publications being 42.0 compared to
an expected journal average of 21.0. This is an outstanding record given that Science regarded
an average88 citation rate per paper of greater than 11 as being a “heavy hitter” as an AIDS
researcher . The average citation rate for the Group’s AIDS publications would top the Science
1996 list. More importantly, not only have the publications been highly cited, they have led to
several changes in global and national health policy, as well as changes in research direction for
HIV-1 epidemiological, immunological and vaccine-related research. As further evidence of the
impact of the collaboration, several members of the collaboration
- Kenyan and Canadian - were
profiled in a special issue of Science on AIDS in Africa88.
The collaboration’s research has been transformative, consistently producing results that
challenge conventions and shifts paradigms. These paradigm shifts include: the recognition of
the importance and mechanisms of heterosexual transmission of HIV; the importance of
commercial sex in HIV transmission dynamics and in HIV prevention; the role of male
circumcision in protecting against HIV infection; the significance of post-natal mother to child
HIV transmission through breastfeeding; and finally, the recognition that there is biologic
resistance to HIV infection, probably mediated immunologically. This body of work has had
much to do with shaping the current understanding of the global HIV pandemic. The importance
of the research contribution has been recognized nationally and internationally by many awards
and prizes to various members, including the Order of Canada, a Tier I Canada Research Chair,
CIHR (and MRC) scholarship, scientist and senior scientist awards, the American Venereal
Disease Association (achievement and career achievement awards), the I.S. Ravdin Award from
25
the American College of Surgeons, election to prestigious bodies such as the Royal Society of
Canada and the American Society for Clinical Investigation.
At the same time, these research findings have been translated by the collaborative group into
highly effective interventions, which now prevent many thousands of HIV infections in Africa,
India, Thailand and Cambodia. Simultaneously, the collaborative project has developed an
impressive cadre of young Kenyan, European, American and Canadian scientists.
III
Key Considerations:
A. Plan for Managing Intellectual Property Current Memorandums of Understanding (MOU)
between the University of Manitoba and Nairobi describe how IP issues will be handles between
the two institutions. In the event of a patentable product it is our intention to build upon this
MOU to recognise both the Kenyan collaborators who help conduct these studies, but also to
recognise the disadvantaged individuals who are the subjects of these studies. Our group has
considerable experience in working with these populations in the developing world in
development, intervention, program design. We would draw on that expertise to insure all are
equitably compensated in the event of patentable findings. We anticipate no likely issues with
obtaining materials, or expertise due to IP issues.
B. Data Sharing Plan: We plan on publishing these finding in scientific publications, and
sharing our results with others. This will include the deposition of sequence, biologic, or
immunologic data in the appropriate databases and repositories.
C. Discussion of Ethical, Social or Cultural Implications of Research: The concerns of
working with at risk and stigmatised populations are discussed below.
D. Research on Human Subjects
All of the women who will participate in this study are female sex workers living and working in
Nairobi, Kenya. All women will be of African ethnicity, citizens of Kenya, Tanzania and
Uganda. All of the study population will be derived from our continuing studies of STIs
epidemiology and immunobiology in the Puwmani Sex Worker Cohort. This is a long-standing
program involving 2200 women. The mean age of women enrolled in the study is predicted to
be approximately 30 and range from 18-50. HIV-1 and other STD are among the main health
problems that these women face. Thirty of the women involved in the study will be HIV-1
infected. Specific antiretroviral therapy is available in Kenya to those who can afford the cost.
Unfortunately, the women involved in this study will almost certainly be unable to afford such
costs. Our group is working with international donors and the pharmaceutical industry to
develop treatment options for the cohort. Despite the interventions described below,
approximately 10% of women will become HIV-1 infected annually.
Recruitment criteria for this study population will be:
1. Age greater than 18 years.
2. Willing to participate in frequent follow-up with blood letting, pelvic exams and testing
for HIV-1 infection.
3. Uterus and cervix are present.
4. In general good health, with CD4 counts over 400/mm3.
The research material collected from the study participants in the course of the study includes
records of a demographic, social and medical nature, blood samples and genital tract specimens
in the form of swabs, cervical aspirates and cervical cytobrushings. We will in addition establish
B-cell lines and a DNA bank consisting of specimens from all subjects. Most of the samples
collected are consistent with those collected during standard medical care of STD related
conditions. Samples that would not ordinarily be collected include genital lavages and cervical
cytobrushings. Additional specimens and additional visits solely for the purposes of the study
will be required in some instances.
All the study participants from the Puwmani cohort are enrolled in other studies. Subjects
enrolled in the present study will be nested within this cohort. To recruit participants, at the time
26
of clinic visits for routine care or in the course of other studies, potential participants in this study
will be asked to participate in this study. A study nurse, specially trained in counseling will do
this in a private interview/counseling room in the clinic. Prospective participants will be
informed about the purposes of the study and the nature of follow up and specimens collected
will be detailed. Informed written consent will be obtained. Individuals choosing not to enroll in
these studies will continue to be followed in the cohort and will have access to all services
provided by the program, including free care of STD and HIV-1 related problems, counseling for
HIV-1 and AIDS and supplies of condoms.
The most important risks involved in this project are those related to being identified as a
prostitute and being tested for HIV-1 infection, which are stigmatization, victimization and
psychological stress. This will not be unique to the women in these studies in that all members
of the Pumwani cohort experience these same risks. Data collection and storage are managed in
such a way as to minimize the risk of inappropriate disclosure of HIV-1 infection status of
participants. All data collected on study subjects in both cohorts will be recorded solely by a
subject number, known only to the clinic staff and the concerned individual. The identification
of study participants as being involved in sex work is more difficult to deal with. It is widely
known in the surrounding community that the clinic is solely for sex workers and thus in
attending the clinic all women de facto “declare” that they are involved in sex work. Since the
clinic is based in the community where the women reside and the community already most
probably knows their occupation, this may do little to increase the stigmatization associated with
sex work or further victimize the participants. Despite these potential concerns, there are
substantial benefits to participation in these studies. These include free access to a standard of
care for STD and HIV-1 related problems that is otherwise unavailable to these women, access to
condoms, access to HIV-1 counseling and community support programs and participation in
STD and HIV-1 intervention programs.
The main physical risk is the physical discomfort is associated with frequent bloodletting and
frequent pelvic examinations. These also bring significant benefits to the study participants in
that early detection and treatment of gonococcal and Chlamydial infections and other STD will
reduce the risk of complications, probably reduce susceptibility to HIV-1 and may reduce the
rate at which HIV-1 infected women develop immune deficiency.
The Puwmani study cohort has been approved by the National AIDS Committee, the National
Ethical and Scientific Review Committee of Kenyatta National Hospital and the University of
Manitoba Use Of Human Subjects In Research review committee. The protocol for additional
studies in women from the Pumwani cohort has been approved for both institutional review
boards for review.
E. Animal Research
None
F. Other Sensitive Research
None
IV
FACILITIES & RESOURCES STATEMENT
Laboratory: The U. Manitoba (UM) has been collaborating with the U. Nairobi (UN) for 22
yrs. Three diagnostic laboratories at these 2 institutions are available to this project. Lab staff
include 8 technologists & 7 support staff. Additional research staff will be hired for this project.
A strength of this proposal is that the majority of the functional immunology studies will be
performed on fresh specimens on site in Nairobi. We have laboratories at the University of
Nairobi and have performed studies there for the past 20 years.
Equipment currently available to
this project includes incubators, refrigerators, -20 & -700C freezers, centrifuges, biosafety
cabinets, spectrophotometers & liquid nitrogen tanks. The labs are experienced in the culture of
N. gonorrhoeae & C. trachomatis, nucleic acid detection of Chlamydia, HIV-1 antibody testing
27
& separation of PBMCs. A FACSCAN is available for flow cytometry. HLA typing using
serologic techniques has been performed for the past 9 yrs. Recently, HLA class I genotyping
facilities were added to the lab in Nairobi and high resolution HLA class I and class II
genotyping capabilities were added to the lab in Manitoba.
Continuing our tradition of technology transfer and capacity building at the University of
Nairobi, the Canadian Foundation for Innovation has recently approved a $3.7 million dollar
(CDN) to expand laboratory facilities at our University of Nairobi site. The expanded facilities
should be operational in early 2005. This will be a state-of-the-art facility that expands our
capacity, allowing for more sophisticated analyses on fresh specimens. The project includes a
BSL 3 lab with a level 4 glove box for level 3 and 4 emerging pathogen analysis and processing,
a non-aerosol level 3 lab for HIV culture work, an immunology lab for cellular immune assays, a
biorepository for storage and shipping of specimens, a genomics lab with an ABI 3100
sequencer, a flow cytometer lab with a 3 laser analysis flow cytometer (BD LSR II), bacteriology
lab, serology lab, and IT areas such as computer lab and bioinformatics suite. We have already
established mirrored servers in Nairobi and Winnipeg which allows for efficient and
synchronized data sharing.
The lab space available to this project on an unlimited basis at the University Manitoba includes
the Plummer and Fowke labs which are equipped for study of HIV and the human immune
response, including a Beckman Coulter Epics Elite 3 laser high speed flow cytometer in the BSL
3 lab, a BD FACScaliber flow cytometer, a confocal image facility, a genetic analysis facility
equipped ABI 310 and 3100 automated sequencers and real-time PCR capability as well as a
viral immunology core facility with a Packard cell harvester, a Packard Topcount 96 well
scintillation and luminescence detector, a Molecular Devices Spectramax UV
spectrophotometer/ELISA reader, an MVE 1520 HE 33,000 sample liquid nitrogen storage
facility, an ELISpot reader, and a Nikon inverted fluorescent/phase contrast microscope with
digital camera. We have obtained access to a Ciphergen ProteinChip® Biomarker System
through the University of Manitoba Proteomics group and through collaborations with Health
Canada (Dr. Plummer is currently Scientific Director General of the National Microbiology
Laboratories, Health Canada). Through co-applicants Drs. Sekaly and Rosenthal the equipment
necessary for studies on T cell receptor and innate immunity are possible.
Clinical:
The field studies for this project will be conducted at one of our Nairobi clinics.
The Majengo STD treatment and research clinic was established and is operated by the
University of Nairobi and University of Manitoba for research on the epidemiology & control of
STD and HIV-1 among women working as sex workers in the Pumwani area. The clinic consists
of a waiting area, an interview area, 2 examining rooms, a counselling room, a drug dispensary
and a wash-up room, and is staffed by 1 physician, 2 nurses, and 2 support staff. The clinic
routinely sees 25-35 women/day. The clinic is exclusively for the Pumwani STD/HIV-1 project
and 30% of the clinic resources are available for this project.
Computer: All data collection and storage will be automated using a laptop microcomputer
system. Data is entered on a daily basis using scanning equipment. In addition, the University of
Nairobi’s Department of Medical Microbiology has a sophisticated multiuser microcomputer
network, several microcomputers and appropriate expertise in data management and analysis. A
web-based database is currently being constructed to facilitate the real-time sharing of data
between Nairobi and Winnipeg, as well as being accessible to collaborators around the world.
Dr. Nagelkerke will supplement that expertise during periodic visits to Nairobi.
Office: Office facilities available to this project include adequate office space, secretarial
services, telephone and facsimile services contained in an annex to the University of Nairobi’s
Department of Medical Microbiology. These facilities are dedicated to STD/HIV-1 research.
V
BUDGET JUSTIFICATION
MAJOR ACTIVITY #1
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PERSONNEL
T. Blake Ball. Dr. Ball is an outstanding immunogeneticist recently recruited to be the HIV
Laboratory Manager for Dr. Frank Plummer. Dr. Ball will be responsible for the day-today supervision of technical personnel and graduate students employed for this project.
Dr. Ball will also be directly responsible for proteomic and innate assays.
John Rutherford. John Rutherford is a technologist will long experience with antibody
purification and cellular immunology. He will be responsible for purifying IgA from
genital tract secretions and plasma and performing IgG and IgA HIV-1 enzyme
immunoassays on study subjects. He will also be responsible for performing T helper
cell assays and two color ELISPOT assays.
Sue Ramdahin. Ms. Ramdahin is a technologist skilled in cellular immunology. She will be
responsible for performing flow cytometry studies. This will include cellular separation,
staining and proliferation assays.
Richard Lester. Dr. Lester is an Infectious Diseases fellow who will be based in Nairobi
effective January 1, 2005. He will be responsible for performing innate immune assays
on PBMC and genital tract cells, and the day-to-day supervision of Kenyan laboratory
personnel. He also participates in T helper cell and dendritic cell experiments.
Shehzad Iqbal. Mr. Iqbal is a PhD Candidate in the Department of Medical Microbiology,
University of Manitoba. He will be conducting flow cytometric analysis of CD8
specificity and function.
James Onyango. Mr. Onyango is a Kenya technologist who has long experience in flow
cytometry and cellular immunologic assays. He will be responsible for processing
genital tract and plasma specimens, separation of cell populations, storage of specimens,
shipping of specimens, flow cytometry on study subjects and assisting Ms. Ramdahin and
Dr. Kaul with T helper and CTL assays.
TBA – Junior Kenyan technician. This individual will be responsible for handling the cellular
material and performing the immunologic assays.
TBA – U Montreal Personnel. These individuals will be responsible for immune phenotyping
of CD4 and CD8 T cells, conducting TCR analysis, and tetramer based functional assays.
TBA – McMaster Personnel. These individuals will be responsible for innate studies, TLR
expression studies, and conducting transcytosis and neutralization assays.
FRINGE BENEFITS
The University of Manitoba provides benefits (@ 16% of base salary) and pay levy (@ 2.25% of
base salary) to full-time unionized employees. Students and trainees (ie., undergraduate and
graduate students, postdoctoral fellows, Infectious Diseases fellows, etc.) are not entitled to these
benefits. It should also be noted that this is not applicable for Kenyan personnel.
TRAVEL
Nine international return airfares (Canada-Nairobi) are requested for the collaborating
researchers from the three major centres (Manitoba, Montreal and McMaster) to conduct site
visits and the long-term field studies described in this proposal.
EQUIPMENT
The University of Montreal has requested a laptop computer and software for data analysis
during years 1-3 under Major Activity #1.
SUPPLIES – MEDICAL AND LABORATORY
Epitope mapping and functional phenotyping of CD4/CD8 T cells will require $30,000/year in
antibodies, cell cultures and peptide synthesis. We include $20,000/year for flow cytometry
services in Montreal and Winnipeg. Flow cytometry services are available to us at no cost due to
our CFI funded infrastructure award.
Humoral immune response assays will require.
$15,000/year in ELISAs, Western blots, antibodies, and antigens and plasticware. Innate
immune response assays will require $25,000/year in culture media, antigens, and plasticware.
TLR expression analysis will require $25,000/year in nucleotides, Taq polymerase, reagents and
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consumables. The costs stated above are based on the projected testing of 250 individuals per
annum for the proposed studies
CONTRACTED SERVICES
As discussed above, flow cytometry services in Winnipeg and Montreal are requested.
MAJOR ACTIVITY #2
Dr. Ma Luo will be involved with a 100% effort in this project. She is a PhD molecular
biologist. She will be performing the molecular class II genotyping and characterization
of the IRF-1 allele patterns among all study participants. She will oversee the proposed
SNP studies and coordinate transport of genetic specimens from Nairobi to Canada and
ensure their appropriate storage prior to processing.
TBA – Postdoctoral Fellow. This individual will be responsible for the gene expression
analysis, experiments and overseeing the two PhD candidate students who will be also
assigned to this project.
TBA – PhD Candidates. These individuals will be involved in the gene expression analysis
studies proposed, and/or the planned proteomic studies.
TBA – Kenyan Technicians. Two individuals will be involved in sample collection, processing
and shipment of proteomic and genetic material. Eventually, they will be trained to
perform these techniques in Nairobi.
TRAVEL
Four international return airfares (Winnipeg-Nairobi) are requested for the U Manitoba personnel
to conduct site visits and field studies described in this proposal.
EQUIPMENT
The proposed studies are very data intensive and require considerable analyses. We request
additional computer equipment anticipated to meet the needs of these proposed studies. We
include software and site licensing fees to account for the use of propreitory genetic analysis
software.
SUPPLIES – MEDICAL AND LABORATORY
Based upon our projected analysis of 200 individuals/year, funding is requested for SNP analysis
($125,000), for HLA sequencing and gene typing ($30,000), and for gene expression ($90,000)
and proteomic ($100,000) analyses. The majority of costs incurred for this project are based on
proprietary technology from Ciphergen® Biosystems and Affymetrix®. As such, these arrays
are exclusively developed by and sold through these companies only. We supplement this
technology with expertise from the University of Manitoba Proteomics Group.
CONTRACTED SERVICES
We request funding to contract out the design and development of focused SNP arrays likely to
be necessary based on the initial Affymetrix® screening. It is our experience that suitable
collaborators or contractors can be obtained.
MAJOR ACTIVITY #3
Dr. Charles Otieno is a Kenyan physician who has worked with the Pumwani Sex Worker
Cohort for the past 5 years. He will be responsible for patient enrollment and sample
collection from study subjects involved in the vaccine challenge project.
TBA – Kenyan Research Nurses. Three research nurses will be hired and 100% involved in
this project working in collaboration with the clinic physician to coordinate the collection
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of specimens, data entry and follow-up of patients enrolled in the vaccine challenge
project.
TBA – Kenyan Technicians. Two individuals will be involved in sample collection, processing
and shipment of proteomic and genetic material for the vaccine challenge project.
Eventually, they will be trained to perform these techniques in Nairobi.
TBA – Postdoctoral Fellow. This individual will be responsible for the immunophenotyping
experiments and overseeing the PhD candidate and Kenyan technicians who will be also
assigned to the vaccine challenge project.
TBA – PhD Candidates. These individuals will be involved in the gene expression analysis and
proteomic studies proposed for the vaccine challenge project.
TRAVEL
Two international return airfares (Winnipeg-Nairobi) are requested for the U Manitoba personnel
to conduct site visits and field studies described in this proposal. One international return airfare
from Nairobi-Winnipeg is also requested for the Kenyan physician to coordinate the study with
U Manitoba senior personnel.
SUPPLIES – MEDICAL AND LABORATORY
This supply justification is similar to that described in Major Activity #1, except studies will be
conducted on the smaller vaccine challenge study.
MAJOR ACTIVITY #4
Joshua Kimani. Dr. Kimani is a physician epidemioligist and the University of Manitoba field
director in Nairobi. He has worked with the Pumwani Sex Worker Cohort for the past 10
years and is expert in establishing and maintaining cohort studies. He will be responsible
for establishing follow up procedures for the study subjects involved in this project,
overseeing collection of clinical and epidemiologic data and day to day supervision of
data management.
TBA – Kenyan Technicians. Two individuals will be involved in sample collection, processing
and shipment of immune, proteomic and genetic material for the prospective study.
Eventually, they will be trained to perform these techniques in Nairobi.
TRAVEL
One international return airfare from Nairobi-Winnipeg is requested for the Kenyan physician to
coordinate the study with U Manitoba senior personnel.
EQUIPMENT
The proposed studies are data intensive and require considerable analyses. We request additional
computer equipment anticipated to track specimen shipments and coordinate sample and data
collection.
SUPPLIES – MEDICAL AND LABORATORY
This supply justification is similar to that described in Major Activity #1 and #2, except studies
will be conducted on the smaller prospective study.
MAJOR ACTIVITY #5
Administrative Assistance. A 0.5 FTE U Manitoba administrator and two 1.0 FTE Kenyan
administrators are requested to assistant with this project.
Kenyan Driver. A driver has been requested to transport the Kenyan personnel to/from the
study clinic sites as well as to perform contact tracing of patients to ensure long-term
follow-up.
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TRAVEL
Per the GCGHSB guidelines, travel support has been requested of the PI and 3 study
collaborators to attend meetings with the Scientific Board.
EQUIPMENT
We request computer equipment to assist with the administration of these projects.
OTHER SUPPLIES
We have requested funding for communication costs, publication costs, as well as vehicle fuel,
maintenance and insurance costs in project vehicles to be used in Nairobi. As well, funding has
been requested to support the shipment of samples between Nairobi, Kenya and Winnipeg,
Canada via air express courier and on dry ice. The University of Manitoba has many years
experience in shipping samples and supplies between the two sites and thus no delays or loss of
goods is anticipated.
CONTRACTED SERVICES
Funding has been requested for service maintenance contracts for an ABI 3100 genetic analyzer
and an LSR-II flow cytometer which are currently available to us through our CFI infrastructure
award.
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