Epitope Specificity and Polyfunctional CD4+ Responses in HIV-1 Highly Exposed
Seronegative Versus Infected Female Commercial Sex Workers
Marion Wangui Kiguoya
A thesis submitted in partial fulfillment for the Degree of Master of Science in
Immunology in the Jomo Kenyatta University of Agriculture and Technology
2010
DECLARATION
This thesis is my original work and has not been presented for a degree in any other
University.
Signature.......................................................................
Date:………………
Marion Wangui Kiguoya
This thesis has been submitted for examination with our approval as University
Supervisors
Signature ………………………………………………
Date:…………………
Dr. Rebecca Waihenya
JKUAT, Kenya.
Signature ………………………………………………
Date:…………………
Dr. Helen Lydia Kutima
JKUAT, Kenya.
Signature ………………………………………………
Date:…………………
Dr. Keith Fowke
University of Manitoba, Canada.
Signature ………………………………………………
Dr. James Mwanjewe
UON, Kenya
i
Date:…………………
DEDICATION
I dedicate this thesis to my dear parents John Karaka and Margaret Waringa who
have been instrumental in shaping my life and for all the support, sacrifice and
prayers to succeed and to enable me complete my studies. To my sisters, Silvia and
Diana, my brothers Edward and Eric. To my loving Grandfather Rufas Karaka
Manjeru for his prayers and for showing me the importance of education. Finally to
my daughter Isabelle Margaret Waringa and all my friends who have been patient
and understanding during the period of research.
ii
ACKNOWLEDGEMENTS
My gratitude goes to the Universities of Manitoba and Nairobi for sponsoring the
study through the Bill Gates Foundation grant and for giving me time and resources
to carry out this research.
My sincere Thanks to my institution supervisors; Drs. Keith Fowke and James
Mwanjewe for their patience, interest, indefatigable support, guidance and
encouragement throughout the study period. God bless you. You made a change in
my life.
To my mentors and University Supervisors; Drs. Rebecca Waihenya and Helen
Kutima for their patience, wisdom, professional guidance and encouragement
throughout.
To the department post graduate coordinators, Drs Shadrack Muya and Zipporah
Osiemo for their wisdom and admirable humility guidance and encouragement.
To the Project Managers; Drs. Larry Gelmon and Blake Ball, Laboratory Manager;
David Mburu and Clinical Director Joshua Kimani for the invaluable support and
guidance.
To the entire Kenya Aids Control Project staff for their support, the data Team,
Festus Muriuki, Tony Kariri , Ricky Kitsao, Richard Gichuhi and Kevin Kamau.
Clinical Team Dr. Makobu Kimani , Elizabeth Bwibo, Jane Kamene and Njoki
Mwangi. To my friends and colleagues in the Laboratory; Anne Maingi , Jemima
Nyakio, Jane Njeri, Erastus Irungu, Peter Muthoga , Billy Nyanga , Nancy Kayere,
iii
Cecilia Maitha and Wendy Adhiambo for the encouragement and moral support
during the entire study.
Lastly to the Majengo ladies for their dedicated and consistent participation in this
longitudinal cohort study. Above all, Thanks to God Almighty for good health,
wisdom and grace for making everything possible.
iv
TABLE OF CONTENTS
DECLARATION
i
DEDICATION
ii
ACKNOWLEDGEMENTS
iii
TABLE OF CONTENTS
v
LIST OF FIGURES………………………………………………………………..vii
LIST OF APPENDICES
ix
LIST OF ABBREVIATIONS
x
ABSTRACT
xi
v
CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW
1
1.1Background Information
1
1.2 HIV and AIDS Etiology
5
1.3 HIV Geographical Distribution and Epidemiology
9
1.5 HIV Pathogenesis
13
1.6 Treatment and Management of HIV/AIDS
15
1.7 Immune Responses to HIV-1 Infection
18
1.8 Cytokines of Interest
20
v
1.9 Genetic Resistance Mechanisms to HIV-1
23
1.10 Evidences of Other Immune Mechanisms in the Majengo Cohort
24
1.11 STATEMENT OF THE PROBLEM
25
1.12 JUSTIFICATION OF THE STUDY
26
1.13 HYPOTHESES
27
1.13NULL HYPOTHESES
27
1.13 General Objective-
28
1.13.1 Specific Objectives –
28
CHAPTER TWO: MATERIALS AND METHODS
29
2.1 Study site
29
2.2 Ethical consideration
30
2.3 Sampling.
30
2.4 Blood collection and separation
32
2.5 Synthetic Peptides and Antibodies
33
2.6 Stimulation and staining
35
2.7 Flow Cytometry
37
2.8 Data Analysis
38
CHAPTER THREE: RESULT…………………………………………….............38
3.1 Gating strategies
40
3.2 Epitope Specificity
46
vi
CHAPTER FOUR; DISCUSSION…………………………………………..53
4.1 Background information…………………………………………….…….53
4.2 Epitope specificity………………………………………………..………..56
4.3Polyfunctionality and Proliferation………………………………………..59
4.4 Conclusion…………………………………………………………...........62
4.5 Recommendations…………………………………………………………63
REFERENCES………………………………………………………………..65
APPENDICES…………………………………………………………………85
vii
LIST OF FIGURES
Figure 1.1
Kaplan Meier plot
3
Figure 1.2
HIV Life cycle
8
Figure1. 3
Global HIV distribution…………………………………………10
Figure 1.4
HIV structure
11
Figure 1.5
Graph of HIV progression
13
Figure 1.6
Immune responses to HIV infection
19
Figure 2.5a
Designing of the peptide pools
34
Figure 2.5b
Overlapping of peptides
35
Figure 2.6
Showing experiment layout
37
Figure 2.7
Emission spectra of the LSR
38
Figure 3.1
Scatter diagram for total lymphocytes
41
Figure 3.2
Scatter diagram for CD4+ gating.
41
Figure 3.3
Scatter diagram for negative IFNγ production gating
42
Figure 3.4
Scatter diagram for positive IFNγ production gating
42
Figure 3.5
Scatter diagram for negative IL-2 production gating
43
Figure 3.6
Scatter diagram for positive IL-2 production gating
43
Figure 3.7
Scatter diagram for negative TNF-α production gating
44
Figure 3.8
Scatter diagram for positive TNF-α production gating
viii
44
LIST OF APPENDICES
APPENDIX 1:
Ethics/consent form
85
APPENDIX II:
Ethical clearance
102
APPENDIX III:
Peptide pools
103
APPENDIX IV:
Bench set up for PBMC isolation
108
APPENDIX V:
Staining of the cells
108
APPENDIX VI:
Acquisition of data on the LSR
109
APPENDIX VII;
Analysis of data on the LSR
109
APPENDIX VIII:
Clients during a focus group meeting
110
APPENDIX IX:
Entire Project team
110
ix
LIST OF ABBREVIATIONS AND ACRONYMS
AIDS
Acquired immunodeficiency Syndrome
APC
Allophycocyanin
APC
Antigen Presenting Cells
ART
Antiretroviral Therapy
BSL3
Biosafety level 3 laboratory
CCR5
Cystein-Cystein Linked Chemokine receptor type 5(- chemokine
receptor)
CD
Cluster of Differenciation
CD4
Cluster of Differenciation Antigen number 4
CD4+
CD4 positive effector T lymphocytes.
CFSE
Carboxyflourescein diacetate succinimidyl ester
CMV
Cytomegallo Virus
CTL
Cytotoxic T Lymphocytes
DAART
Directly administered antiretroviral therapy
DC- SIGN
C type lectin receptor on macrophages and DC
DC
Dendritic Cells
DMSO
Dimethyl Sulfoxide
DNA
Deoxyribonucleic Acid
dsRNA
Double stranded RNA
EBV
Epstein Barr Virus
EI
Entry Inhibitor
x
ELISA
Enzyme Linked Immunosorbent Assay
env
Envelope
ESN
Exposed seronegative
FACS
Fluorescent Activated Cell Sorting
FITC
Flouresceine Isothio Cyanate
FSC
Forward Scatter
gag
Group Antigen Specific.
Gp 120
A sugar containing glycoprotein of approximately 120 daltons
Gp 160
A sugar containing glycoprotein of approximately 160daltons
Gp 41
A sugar containing glycoprotein of approximately 41 daltons
HAART
Highly active antiretroviral therapy
HEPS
Highly Exposed Persistently Seronegative
HIV-1
Human Immunodeficiency Virus type 1
HLA
Human Leukocyte Antigen
HR1
Gp41 dormain one
HR2
Gp41 dormain two
ICTV
Ithaca College Television
IFNγ
Interferon gamma
IL -2
Interleukin 2
LSR
Light amplification by stimulated emission of radiation
MARP
Most at risk population
xi
MHC
Major Histocompatibility Complex
NARTI
Nucleoside Analogue Reverse Transcriptase Inhibitor
nef
Negative Factor Gene
NKT
Natural Killer T cell
NNRTI
Non Nucleoside Reverse Transcriptase Inhibitor
P17
HIV-1 Protein 17
P24
HIV-1 Protein 24
P6
HIV-1 Protein 6
P7
HIV-1 protein 7
PBMCs
Peripheral Blood Mononuclear Cells
PBS
Phosphate Buffer Saline
PCR
Polymerase Chain Reaction
PE
Phycoerythrin
PerCP
Peridin chlorophyll protein
pol
HIV-1 polymerase gene
R5
CCR5 receptor using virus
Rev
Regulator of viron gene
RNA
Ribonucleic Acid
RT
Reverse Transcriptase
SSC
Side Scatter
STI
Sexually Transmitted infections
xii
Tat
Trans- Activator of Trancription
TCR
T Cell receptor
Th
T helpers
TLR
Toll-like receptors
TNF α
Tumor Necrosis Factor alpha
UNAIDS
United Nations Program on HIV /AIDS
V3 region
The third variable region on the gp 120 subunit
Vif
Viral infectivity factor
Vpr
Viral protein regulatory gene
Vpu
Viral protein unknown gene
Vpx
Viral protein x gene
WHO
World Health Organization
xiii
ABSTRACT
A long standing study of low social economic status commercial sex workers, in the
Majengo cohort of Nairobi has demonstrated variable susceptibility to HIV-1, where
some of the highly exposed subjects remain persistently seronegative, indicative of
resistance to HIV -1. There has been intense interest in understanding the mechanism
responsible for this phenomenon. Understanding the specific immune responses
conferring protection from infection in individuals exposed to HIV-1 is critical for
vaccine design. However, if both HIV -1 infected and resistant individuals have
HIV-1 specific T helper responses, what is unique about the later group that protects
them from infection? This study evaluated the protective immunity in light of
polyfunctional immune responses due to specific selective peptide recognition by in
vitro stimulation of the subjects. The peptide pools were designed from the entire
HIV-1 Clade -A genome giving 778 overlapping peptides grouped into 20 peptide
pools. Each pool had 40 peptides apart from pool 20 which had 18 peptides. The
immune responses were measured by a 4 laser 10 color LSR II flow Cytometer from
a total of 104 subjects. The Peripheral Blood Mononuclear Cells were obtained by
ficoll centrifugation method of cell separation. PBMCs were stimulated with peptide
pools of HIV -1 and the responses measured for pro-inflammatory cytokines
production at day three and for proliferation at day six of incubation at 37oC and 5%
CO2. The data was analyzed to ascertain polyfuctionality due to epitope specifity
done using the chi squared and Student t tests. The resistant group showed significant
levels of IFNγ, TNF –α, IL-2 production and proliferation, especially in response to
peptide pools 1, 12, 13 and 14 (Env, P24, P31 and P2P7P1, P6 P7, Protease and REV
peptide pools). The difference was significant at 95% CI with a p value of 0.0001.
xiv
There was no correlation between IFN-y production and proliferation using students
T test at a P value 0.0001. Pool numbers 8 (gag and nef) and 17(Rt and Tat) showed
polyfunctional immune responses while in the initial screening the two did not show
high IFN-γ production thus the need to repeat the polyfunctinality capturing the
entire genome to capture all epitopes that are specific. It is evident that there could be
selective preferential epitope recognition in the resistant group that could be
responsible for the unique polyfuctionality. Future directions include breaking down
the specific peptide pools with unique responses confirming polyfuctionality due to
epitope specificity. Polyfuctionality and epitope specificity correlates could be used
to determine efficacy of HIV-1 vaccine by mimicking the effective responses of
those who appear resistant.
xv
CHAPTER ONE:
1.0 INTRODUCTION AND LITERATURE REVIEW
1.1 Background Information
In all populations of infectious diseases biology, it is characteristic that there is
variability in susceptibility to infection and disease caused by microbial agents.
Among susceptible individuals exposed to an infection, not all become infected and
among the infected individuals, not all proceed to the same level of development of
the disease and some do not get infected at all. It is logical that such variability in
susceptibility to infection and disease would apply to infection and disease with HIV
-1. Longitudinal studies in women from the Majengo Sex worker cohort have
provided early data that there might be biologically mediated resistance to infection
(Fowke et al., 1996 and Rowland et al., 1998). Indirect evidence that not all HIV-1
exposed individuals become persistently infected come from studies demonstrating
HIV-1 specific cellular immune responses in high risk exposed seronegative
individuals (Traver et al., 1995; Paxton et al.,1996; Smith et al.,1997 and Beyrer et
al., 1999). Several studies have shown that Peripheral Blood Mononuclear cells
(PBMC’s) from presumably HIV-1 exposed but apparently uninfected individuals
(Yarchoan et al., 1989; Krasinski et al., 1990 and Clerici et al., 1992) secrete
cytokines and have unique proliferative capabilities (Clerici et al., 1992; Baellini et
al., 1995).
1
In a study of risk factors for HIV-1 infections the mean duration of prostitution in the
Majengo Sex worker cohort was inversely related to the risk of HIV-1 infection
(Simonsen et al., 1990). This is illogical as 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. These
women have intense exposure to HIV-1 through their occupation, and although
condom use is frequent (>80 % of sexual encounters) their risk of acquiring HIV-1
infection is enormous (Ngugi et al., 1988). Mathematical modeling by Kaplan Meir
(Plummer et al., 1999) shows that it is not by chance alone that some women remain
HIV-1 uninfected; statistically these women should be infected if all the women were
equally susceptible. (Figure 1.1)
In this plateau HIV incidence is not by a chance phenomenon and is not related to
sexual behaviors, altered susceptibility to factors increasing the risk of HIV- 1
infection of seronegative HIV-1 infection.
As shown in figure 1.1, despite the
intense exposure, the risk of HIV-1 seroconversion in the cohort gradually declines
and the longer a woman remains uninfected, the less likely she is to seroconvert to
HIV-1. The probability of remaining HIV-1 seronegative increases with increasing
duration of follow up (Plummer et al., 1999).
2
1.0
.8
.6
.4
.2
0.0
0
2
4
6
8
10
12
14
16
18
20
Years of Followup
Fig 1.1 Kaplan Meir plot of the probability of remaining HIV-1 seronegative
with follow up in the Majengo cohort (Plummer et al., 1998)
Several other groups of Highly Exposed Persistently Seronegative (HEPS)
individuals (discordant couples, gay discordant and commercial sex workers) who
appear to be protected against HIV-1 infection have now been described (Travers et
al., 1995; Paxton et al., 1996; Beyrer et al., 1997 and Smith et al., 1997). These
groups have reported HIV-1 specific CTL responses in individuals who are exposed
but not infected with HIV-1. MHC class I restricted CTL to HIV- 1 have been
reported in the uninfected children born of HIV-1 mothers (Cheynier et al., 1993 and
Rowland et al ., 1993) ,uninfected regular heterosexual partners of HIV-1 infected
individuals, (Langlade et al.,1994) health-care workers with occupational exposure
3
to HIV-1 (Pinto et al.,1995) and HIV-1 uninfected prostitutes in Gambia (Roland et
al.,1995). These findings have led to the suggestions that cellular rather than
systemic humoral immunity may be important in protection against HIV-1 infection.
Improved methods have continually been sought to analyze single cytokine
production and cell lines the techniques include ELISPOT, Hybridization,
Immunochemistry, Limiting dilution analysis and single cell PCR (Carter et al.,
1997). These techniques have significant drawbacks requiring either high technical
proficiency or tedious collection and analysis.
Flow Cytometry is a powerful analytical technique in which individuals cells are
simultaneously analyzed for several parameters, including size and granularity as
well expression of surface and intracellular markers defined by fluorescent (Parks et
al ., 1989; Jung et al ., 1993; Prussin et al., 1995 and Carter et al., 1997). Majority of
the instruments manufacturers employ an air cooled argon gas laser emitting a
monochromatic beam of light fixed at 488 nm at 15mW of power. As the particles or
cells flow in a single file past the intersection of the beam, light is scattered in
various directions. If there is a flourochrome labeled monoclonal antibody associated
with the cell, it becomes excited by the laser and a fluorescent emission results. The
resulting signals are processed to gather information about the relative size of the cell
(Forward scatter) and its shape or internal complexity (Side scatter) as well as
diversity of cellular structures and antigens (fluorescence).
4
The cytometer itself is set up and monitored routinely with a quality control program
utilizing a series of unlabelled and fluorescently labeled calibration particles as a
reference check on instrument (Rafael, 2001). LSR II flow cytometer tool is fitted
with 4 lasers capable of analyzing upto 18 different parameters measuring and
characterizing cells and cellular constituents as they travel in a stream. Flow
cytometer is the most flexible yet powerful bench top analyzer available from BD
sciences. Innovative technology in the BD LSR II optics and digital electronics has
created a more sensitive flow cytometer that yields more information from each
sample. It has the following distinctive features four Fixed-Aligned Lasers,
revolutionary new optics collects more light and High-Performance BD FACSDiva
Software.
1.2 HIV and AIDS Etiology
Human immunodeficiency virus (HIV) is a member of the genus lentivirus part of
the family Retroviridae (ICTV, 2006). HIV causes acquired immunodeficiency
syndrome (AIDS), a condition in humans in which the immune system begins to fail,
leading to life-threatening opportunistic infections. AIDS is the most severe
acceleration of infection with HIV. HIV is a primate retrovirus that primarily infects
vital cells of the human immune system particularly CD4+ T cells (a subset of T
cells), macrophages and dendritic cells. It directly and indirectly destroys CD4+ T
cells (Alimonti et al. 2003). Once HIV has destroyed CD4+ T cells to a level below
200 of these cells per microliter (µL) of blood (Feldman, 2005), cellular immunity is
5
lost allowing for opportunistic infections. CD4+ T cell depletions differ in the
different phases of disease progression (Hel et al., 2006).
Acute HIV infection progresses over time to clinical latent HIV infection and then to
early symptomatic HIV infection and later to AIDS, which is identified either on the
basis of the amount of CD4+ T cells remaining in the blood, and/or the presence of
opportunistic infections (Lipman et al., 2003). Lentiviruses are characteristically
responsible for long duration illnesses with long incubation periods (Levy et al
.,1993). Lentivirus is transmitted as single stranded, positive sense enveloped RNA
viruses. Upon entry to the target cell, the viral RNA genome is converted to double
stranded DNA by a virally encoded reverse transcriptase that is present in the virus
particle. The viral DNA is then integrated into cellular DNA by virally encoded
integrase; along with host cellular co factors (Smith et al., 2006), in order for the
genome to be transcribed. After the virus has infected the cell, two pathways are
possible; either the virus becomes latent and the infected cell continues to function or
the virus becomes active and replicates.
After HIV-1 enters the body, it then hijacks the host cell’s cellular machinery to
reproduce thousands of copies of its own. HIV enters the macrophages and CD4+ T
cells by adsorption of glycoprotein on its surface to receptors on the target cell
followed by fusion of the viral envelope with the cell membrane and the release of
the HIV- capsid into the cell (Chan and Kim, 1998; Wyatt and Sodroski, 1998). The
interactions of the trimetric envelope complex gp160 protein, involves CD4 and a
6
chemokine receptor either CCR5 or CXCR4 on the cell surface. The gp160 contains
binding dormains for both CD4 and chemokine receptors (Chan and Kim, 1998)
The first step in fusion involves the affinity attachment of the CD4 binding dormains
of gp120 to CD4. Once gp120 is bound to the CD4 protein, the envelope completely
undergoes a structural change, exposing the chemokine binding domains of gp120
and allowing them to interact with the target chemokine receptor. This allows for a
more stable two step attachment, which allows the N- terminal fusion peptide gp41
to penetrate the cell membrane. Repeat sequences in gp41, HR1 and HR2 then
interact, causing the collapse of the extra cellular portion of gp41 into a hairpin
(Chan and Kim, 1998)
This loop structure brings the virus and the cell membranes close together, allowing
fusion of the membranes and subsequent entry of the viral capsid. HIV can infect
dendritic cells by this CD4-CCR5 route, though another route using mannose –
specific- C- type lectin receptors such as DC-SIGN can also be used. Dendritic cells
(DC) are one of the first cells encountered by the virus during sexual transmission.
They are thought to play an important role by transmitting HIV to T cells once the
virus has been captured in the mucosa by DCs (Pope and Haase, 2003).
Figure 1.2 is a summary of the HIV replication cycle, the different enzymes involved
at different steps of the process.
7
Integrated RNA complex once
virus is internalized.
Immature HIV viruses
Fig 1.2: HIV replication cycle (Doc Kaisers, 2008)
8
1.3 HIV Geographical Distribution and Epidemiology
HIV has become one of the world’s most serious health and development challenges
since the first cases were reported in 1981. AIDS is now a pandemic (Kallings et al.,
2008). More than 25 million people have died of AIDS and over 33 million are
currently living with HIV /AIDS (UNAIDS/WHO, 2008). While cases have been
reported in all regions of the world, almost all those living with HIV (96%) reside in
low and middle income countries particularly Sub-Saharan Africa (UNAIDS, 2006).
HIV infection is becoming endemic in sub-Saharan Africa, which is home to just
over 10% of the world’s population but has more than 60% of all people living with
HIV. The adult (ages 15-49) HIV prevalence rate is 7.2% (range: 6.6 - 8.0%) with
between 20.9 million and 24.3 million people currently living with HIV. However,
the actual prevalence varies between regions (UNAIDS/WHO, 2008).
Figure 1.3 is a representative of the worldwide distribution of HIV-1 pandemic
where Sub-Saharan Africa and Asia are the most hit with high prevalence rates.
9
Fig 1.3: HIV Global distribution
(www.who.int/global_HIV_map_2007_fullscr.jpg)
In figure 1.4 the generalized HIV structure is represented, where the env which has
the gp120 and gp41, the enzyme reverse transcriptase are seen, they are involved in
the replication process.
10
Fig 1.4: HIV structure (Gelderblom;1997)
1.4 HIV Classes and Structure
HIV is different in structure from other retroviruses. It is roughly spherical
(McGovern et al., 2002) with a diameter of about 120 nm. It is composed of two
copies of positive single-stranded RNA that codes for the virus's three genes (gag,
pol, and env,) which contain information needed for structural proteins ( tat, rev, nef,
vif, vpr, vpu, and tev which is only present in HIV-1 isolates) code for proteins
controlling the ability of HIV to infect cells, produce new copies or cause disease. It
is enclosed by a conical capsid composed of 2,000 copies of the viral protein p24
(Chan et al., 1997). The single-stranded RNA is tightly bound to nucleocapsid
proteins, p7 and enzymes needed for the development of the virion such as reverse
transcriptase, proteases, ribonuclease and integrase. A matrix composed of the viral
protein p17 surrounds the capsid ensuring the integrity of the virion particle. This is,
in turn, surrounded by the viral envelope which is composed of two layers of fatty
11
molecules called phospholipids taken from the membrane of a human cell when a
newly formed virus particle buds from the cell. Embedded in the viral envelope are
proteins from the host cell and about 70 copies of a complex HIV protein that
protrudes through the surface of the virus particle.
The protein, Env, consists of a cap made of three molecules called glycoprotein (gp)
120, and a stem consisting of three gp41 molecules that anchor the structure into the
viral envelope (Chan et al., 1997). This glycoprotein complex enables the virus to
attach to and fuse with target cells to initiate the infectious cycle (Chan et al., 1997).
Both these surface proteins, especially gp120, have been considered as targets of
future treatments or vaccines against HIV (National Institute of Health, 1998).
There are two strains of HIV known to exist; HIV-1 and HIV-2. HIV -1 is the virus
that was initially discovered, it is more virulent and is relatively easily transmitted, it
is the cause of majority of HIV infections globally. HIV-2 is less transmittable or less
virulent and is largely confined to West Africa (Reeves et al., 2002). Three groups of
HIV have been identified on the basis of differences in env: M, N, and O (Thompson
et al., 2002).
Group M is the most prevalent and is subdivided into eight subtypes (or clades),
based on the whole genome, which are geographically distinct (Carr et al., 1998).
The most prevalent are subtypes B (found mainly in North America and Europe), A
and D (found mainly in Africa), and C (found mainly in Africa and Asia); these
subtypes form branches in the phylogenetic tree representing the lineage of the M
12
group of HIV-1. Infection with HIV-1 is associated with a progressive decrease of
the CD4+ T cell count and an increase in viral load.
1.5 HIV Pathogenesis
The stage of infection can be determined by measuring the patient's CD4+ T cell
count, and the level of HIV in the blood. HIV infection has basically four stages:
incubation period, acute infection, latency stage and AIDS. The initial incubation
period upon infection is asymptomatic and usually lasts between two and four weeks.
The second stage, acute infection, which lasts an average of 28 days and can include
symptoms such as fever, lymphadenopathy (swollen lymph nodes), pharyngitis (sore
throat), rash, myalgia (muscle pain), malaise, and mouth and esophageal sores. The
latency stage, which occurs third, shows few or no symptoms and can last from two
weeks to twenty years and beyond. AIDS, the fourth and final stage of HIV infection
shows as symptoms of various opportunistic infections (Holmes et al., 2003). The
various opportunistic infections manifest in the various systems of the body. The
manifestations include Pneumocystis jirovecii Pneumonia (PCP), Tuberculosis (TB),
Kaposi's sarcoma (KS), Cryptosporidiosis, Candida, Cytomegalovirus (CMV),
Isosporiasis, Toxoplasmosis, Cryptococcosis, Non-Hodgkin’s lymphoma, Varicella
Zoster and Herpes simplex.
Figure 1.5 shows a generalized graph of the relationship between HIV copies (viral
load) and CD4 counts over the average course of untreated HIV infection however
any particular individual's disease course may vary considerably.
13
Fig 1.5: The relationship between HIV copies and CD4 during untreated HIV
infection (Roitt et al., 2007)
Infection with HIV occurs by the transfer of blood, semen, vaginal fluid, preejaculate, or breast milk. Within these bodily fluids, HIV is present as both free virus
particles and virus within infected immune cells. The four major routes of
transmission are unsafe sex, contaminated needles, breast milk, and transmission
from an infected mother to her baby at birth (Vertical transmission). Screening of
blood products for HIV has largely eliminated transmission through blood
transfusions or infected blood products in the world. Acquired Immune Deficiency
syndrome or acquired immunodeficiency syndrome (AIDS) is a set of symptoms and
14
infections resulting from the damage to the human immune system caused by the
human immunodeficiency virus (HIV). This condition progressively reduces the
effectiveness of the immune system and leaves individuals susceptible to
opportunistic infections and tumors. This transmission can involve anal, vaginal or
oral sex, blood transfusion, contaminated hypodermic needles, exchange between
mother and baby during pregnancy .
Many factors affect the rate of progression. These include those that influence the
body's ability to defend against HIV such as the infected person's general immune
function (Clerici et al., 1996 and Morgan et al., 2002). Older people have weaker
immune systems, and therefore have a greater risk of rapid disease progression than
younger people. Poor access to health care and the existence of coexisting infections
such as tuberculosis also may predispose people to faster disease progression
(Gendelman et al., 1986; Bentwich et al., 1995 and Morgan et al., 2002)
1.6 Treatment and Management of HIV/AIDS
There is currently no vaccine or cure for HIV or AIDS (Robb et al., 2008). The only
known method of prevention is avoiding exposure to the virus. However, a course of
antiretroviral treatment administered immediately after exposure, referred to as postexposure prophylaxis, is believed to reduce the risk of infection if begun as quickly
as possible (Fan et al., 2005). ARV's slow down the reproduction rate of HIV. Once
the virus is reproducing at a slower rate, it is less able to harm the immune system.
ARVs have four main functions; ensuring maximum and lasting controls of the HIV
15
viral load ,restoring and protecting the immune function of the body by allowing
CD4 cells to replenish in number, reducing HIV related illness’ and deaths and
providing a long run to improve the quality of life for people who are infected. The
different type of ARV target different stages of HIV replication (Fan et al., 2005).
Nucleoside and Nucleotide reverse transcriptase inhibitors (NRTI) inhibit reverse
transcription by being incorporated into the newly synthesized DNA and preventing
its further elongation. Non –nucleoside reverse transcriptase inhibitors (NNRTI)
inhibit reverse transcriptase by binding to this enzyme thus interfering with its
functioning. Protease inhibitors (PIs) target viral assembly by inhibiting the activity
of protease that is vital in the final assembly of new virions (Fan et al., 2005).
Integrase inhibitors inhibit the integrase enzyme, which is responsible for integration
of viral DNA into the DNA of the infected cell (Robb et al., 2008) Entry inhibitors
(or fusion inhibitors) interfere with the binding, fusion and entry of HIV-1 to the
hosts’ cell by blocking one of several targets. Maturation inhibitors inhibit the last
step in gaga processing in which the viral capsid polyprotein is cleaved, hence
blocking the conversion of the polyprotein into mature capsid protein(Robb et al.,
2008).
In the absence of antiretroviral therapy, the median time of progression from HIV
infection to AIDS is nine to ten years, and the median survival time after developing
AIDS is only 9.2 months (Morgan et al., 2002). However, the rate of clinical disease
progression varies widely between individuals, from two weeks up to 20 years
16
(Morgan et al., 2002). HIV is genetically variable and exists as different strains,
which cause different rates of clinical disease progression (Quinones et al., 1998;
Kaleebu et al., 2002 and Campbell et al., 2004). There are current WHO ARV
guidelines according to the WHO staging (WHO, 2008).
Current treatment for HIV infection consists of highly active antiretroviral therapy
(HAART) (Department of Health and Human Services, 2005). This has been highly
beneficial to many HIV-infected individuals since its introduction in 1996 when the
protease inhibitor-based HAART initially became available (Palella et al., 1998).
Current HAART options are combinations (or "cocktails") consisting of at least three
drugs belonging to at least two types, or "classes," of antiretroviral agents. Typically,
these classes are two nucleoside analogue reverse transcriptase inhibitors (NARTIs
or NRTIs) plus either a protease inhibitor or a non-nucleoside reverse transcriptase
inhibitor (NNRTI). New classes of drugs such as Entry Inhibitors (EIs) provide
treatment options for patients who are infected with viruses already resistant to
common therapies, although they are not widely available and not typically
accessible in resource-limited settings. AIDS progression in children is more rapid
and less predictable than in adults, particularly in young infants, hence more
aggressive treatment is recommended for children than adults. In developed countries
where HAART is more readily available, doctors assess their patients thoroughly:
measuring the viral load, how fast CD4 declines, and patient readiness to start ARV
treatment. They then decide when to recommend starting treatment. Standard goals
of HAART include improvement in the patient’s quality of life, reduction in
17
complications, and reduction of HIV viremia below the limit of detection but it does
not cure HIV (Blankson et al., 2002).
1.7 Immune Responses to HIV-1 Infection
The immune system has two T- helper cell types; Th1 helps generate cytotoxic T cell
responses while Th2 helps to generate an antibody response. The balance in
contribution of these two paths to an immune response depends upon the particular
infectious organism and the particular background of the infected individual. Once
HIV has entered the body, the immune system initiates anti HIV antibody and
cytotoxic T cell production. The immune response is weakened with time as the
memory T cells (CD4 + CCR5+) are destroyed. Figure 1.7 shows a representation of
both immune systems kicking off: With the recognition of the type 1/type 2
dichotomy in the T helper cell responses to infection (Mosmann et al., 1986 ;
Cherwinski et al., 1987) the concept that cellular effector mechanism may mediate
protective immunity to HIV-1, in the absence of systemic humoral immunity, has
attracted intense interest in the recent past (Bretscher et al., 1992; Clerici et al., 1994
;Yang et al, 1996 ;).
The immune response to any infectious microorganisms adopts either humoral
(antibody mediated) against extracellular pathogens or cell mediated immune
response to intracellular pathogens as summarized in figure 1.7. There is interplay of
the two arms of the immune system whereby Th cells at some point could either
18
stimulate B cells giving rise to memory cells and plasma cells or stimulate CTL
giving rise active cytotoxic cells and memory cells.
Fig 1.6: Immune responses to HIV infection (Wesley Longman., 1999)
19
1.8 Cytokines Of Interest in This Study
Cytokines are small proteins, peptides or glycoproteins which are secreted by certain
cells of the immune system which carry signals locally between cells and thus have
an effect on other cells. They are a category of signaling molecules that are used
extensively in cellular communications. They stimulate immune cell proliferation
and differentiation and are often secreted by immune cells that have encountered a
pathogen, thereby activating and recruiting further immune cells to increase the
system's response to the pathogen (Cannon, 2000). Cytokines are produced by many
different cells causing the target cell to produce a certain reaction depending on the
cell and the cytokine. Often the target cell produces other cytokines in response to
the initial cytokine; this complicated relationship is called the cytokine network. This
network is one of the most important ways that the immune system communicates
and orchestrates appropriate responses to microbial infections. Most cytokines are
produced by T –lymphocytes cells and a lesser degree by monocytes and
macrophages. (Raffi, 1995). In this research looked at IFN- γ, IL-2 and TNF-α.
There is growing evidence that initial HIV infection disrupts the normal balance of
cytokines by causing levels of certain cytokines to rise. Cytokine imbalances may
also help HIV to target CD4+ cells and lymph nodes, leading to the progressive
immunosupression and the opportunistic infections that follow (Raffi, 1995). The
shift in cytokine balance is called Th-1/Th-2 theory. Th-1 cytokine produced in
20
response to the initial HIV infection include IFN- γ, IL-2 and IL-12. During the
asymptomatic period of HIV infection the levels of these Th-1 cytokines remain
high. (Cannon, 2000). Continuous HIV replication results in a state of generalized
immune activation persisting throughout the chronic phase of HIV infection (Appay
et al., 2008). Immune activation, which is reflected by the increased activation of
immune cells and release of proinflammatory cytokines, results from the activity of
several HIV gene products and the immune response to ongoing HIV replication.
The cytokines of interest include Interleukin-2, (IL-2), Interferon gamma, (IFN-γ)
and Tumor Necrosis Factor alpha(TNF-α) which are proinflammatory cytokines that
promote systemic inflammation (Appay et al ., 2008).
Interleukin-2 (IL-2) is a type of cytokine immune system signaling molecule, which
is a leukocytotrophic hormone that is instrumental in the body's natural response to
microbial infection and in discriminating between foreign (non-self) and self. IL-2
mediates its effects by binding to IL-2 receptors, which are expressed by
lymphocytes, the cells that are responsible for immunity. IL-2 is normally produced
by the body during an immune response (Cantrell et al., 1984; Smith., 1988).
Antigen binding to the T cell receptor (TCR) stimulates the secretion of IL-2, and the
expression of IL-2 receptors IL-2R. The IL-2/IL-2R interaction then stimulates the
growth, differentiation and survival of antigen-selected cytotoxic T cells via the
activation of the expression of specific genes (Beadling et al., 1993 and Beadling et
al ., 2002).
21
Interferon-gamma (IFN-γ) is a dimerized soluble cytokine that is the only member
of the type II class of interferons. The importance of IFN-γ in the immune system
stems in part from its ability to inhibit viral replication directly but most importantly
derives from its immunostimulatory and immunomodulatory effects. IFN-γ is
produced predominantly by natural killer (NK) and natural killer T (NKT) cells as
part of the innate immune response, and by CD4 and CD8 CTL effector T cells once
antigen-specific immunity develops (Schoenberg and Wilson, 2007). IFN-γ can be
expressed by all cells and is also known as immune interferon. It is serologically
distinct from Type I interferon in that it is acid-labile, while the type I variants are
acid-stable. IFN-γ has antiviral, immunoregulatory, and anti-tumor properties
(Schroeder et al., 2004). It alters transcription in up to 30 genes producing a variety
of physiological and cellular responses. Amongst the effects are : increase antigen
presentation of macrophages, activate and increase lysosome activity in
macrophages, suppress Th2 cell activity, cause normal cells to increase expression of
class I MHC molecules, promote adhesion and binding required for leukocyte
migration, promote NK cell activity and activating antigen presenting cells.
Tumor necrosis factor-alpha (TNF-α) is a cytokine involved in systemic
inflammation and is a member of a group of cytokines that stimulate the acute phase
reaction. The primary role of TNF is in the regulation of immune cells. TNF is also
able to induce apoptotic cell death, to induce inflammation, and to inhibit tumor
genesis and viral replication (Locksley et al., 2001). TNF is produced mainly by
macrophages, but they are produced also by a broad variety of other cell types
22
including lymphoid cells, mast cells, endothelial cells, cardiac myocytes, adipose
tissue, fibroblasts, and neuronal tissue
1.9 Genetic Resistance Mechanisms to HIV-1
There has been intense interest in the recent past in understanding mechanisms
responsible for resistance to HIV-1 infection. The infected person's genetic
inheritance plays an important role to resistance and susceptibility. In humans, the
CCR5 gene location on the short (p) arm at position 21 on chromosome 3 is a
chemokine co-receptor important in HIV attachment to the host CD4 protein.
Individuals with the homozygous CCR5-Δ32 variation are resistant to infection with
certain strains of HIV (Tang et al., 2003). CCR5-Δ32 is a deletion mutation of gene
that has a specific impact on the function T cells (Galvani et al., 2003).
Several types of genetically mediated resistance to HIV-1 infection and disease are
now known. Cellular resistance to HIV-1 infection has been shown to be related to
homozygosity for a 32 base pair deletion mutation in the HIV co receptor molecule
CCR5 (-32-CCR5) (Lui et al.,1996 and Goth et al., 1998). This molecule acts as a
co -receptor for macrophage tropic isolates of HIV-1 (Paxton et al., 1996). However
the -32-CCR5 has not been detected in African populations and is not found in
HIV-1 resistant women of the Majengo sex worker cohort (Fowke et al.,1998).
Neither polymorphisms in the promoter region of CCR5 (Yang et al., 2003) CCR2
641, SDF-1 (Anzala et al.,1998), overproduction of chemokines or altered CCR5 or
CXCR4 expression levels (Fowke et al.,1998) account for this HIV-1 resistance in
the Majengo sex workers cohort. It is unlikely that other HIV-1 receptors or co23
receptor mutations are responsible for this resistance in the Majengo cohort as their
PBMCs have been shown to be readily infected with multiple HIV-1 strains as well
as primary HIV-1 isolates in-vitro (Smith et al., 1997).
Studies of the role of HLA alleles in controlling HIV-1 progression also suggest an
important role for the immune system in controlling infection. Combinations of
Major Histocompactibility Complex (MHC) alleles, for example HLA –B13(Winkler
et al., 1998) and HLA-A2 (Macdonald et al.,2008) and Tap gene variants associate
with resistance to infection and a reduced risk of HIV-1 disease progression (Kaslow
et al.,1996 and Lui et al.,1996). MHC class 1 heterozygosity and rarity provides
advantage in survival with HIV -1 infection, suggesting that diversity of CTL
responses may protect against HIV-1 related disease indicating the importance of the
immune response in controlling HIV-1 infection (Carrington et al., 1999 and
Trachtenberg et al., 2003).
1.10 Evidence of Other Immune Mechanisms in the Majengo Cohort
Rosenberg et al., (1997) have shown evidence in the ability of cellular immune
response to control virus levels among long term non progressors, HIV-1 specific T
helper responses which were inversely correlated with viral load. The detection of
HIV-1 specific cellular responses in the resistant commercial sex workers suggests
that these women have had exposures to HIV-1 sufficient to induce cellular immune
response but insufficient to induce antibody.. However the two studies do not show
whether these responses protect on subsequent exposure, rather they just suggest that
24
some individuals are able to clear an HIV infection (Fowke et al., 2000). There is
need to investigate populations where there are high levels of exposure to HIV- 1
providing sufficient opportunity for HIV- 1 infection yet there are evidences that
these individuals are not infected and the fact that there are evidences of CTL
responses which may contribute to this protection.
There is evidence for protection against HIV-1 infection (Fowke et al., 1996) from
observational epidemiological studies done from this cohort, which could be
independent of exposure (Fowke et al., 1996). In the Majengo cohort the mean
duration of prostitution is considerably shorter in HIV-1 infected women (Simonsen
et al., 1990). In the sexual partners of HIV-1 infected hemophiliacs and the sexual
partners of persons infected through blood transfusions, the risk of HIV-1 infection
is, to some degree independent of sexual exposure (Peterman et al., 1988). It has also
been shown that HIV-2 infected prostitutes are partially protected against HIV-1
infection (Travers et al., 1995).
1.11 STATEMENT OF THE PROBLEM
HIV /AIDS has reached pandemic proportions and although the available treatment
slows down the course of the disease by reducing both mortality and morbidity of the
infection; there is currently no vaccine or cure. HAART neither cures the patient nor
does it uniformly remove all the symptoms. Research to improve current treatments
include decreasing side effects of current drugs , further simplifying drug regimens to
improve adherence and determining the best sequence of regimens to manage drug
25
resistance. Only a vaccine however would be able to halt the pandemic as it would
cost less in prevention strategies and would not require a daily treatment. The failure
of vaccine candidates to protect against HIV infection and progression to AIDS has
led to a renewed focus on the biological mechanisms responsible for HIV resistance
in most at risk populations (MARP) (Travers et al., 1985; Paxton et al., 1986; Smith
et al., 1987 and Beyrer et al., 1999). Studies in women from the Majengo Sex
Worker cohort have provided early data that there might be biologically mediated
resistance to HIV infection, where the first evidence of protection against HIV-1
infection originated (Fowke et al ., 1996). Mathematical Models have shown that this
resistance is not by chance alone and in actual statistical sense most of the women in
the cohort should be infected through their intense exposure from their occupation.
If both HIV infected and HIV resistant women in this extensively studied Sex worker
cohort have specific T helper responses, what is unique about the later group that
protects them from infection? How can CTL protect against infection in one instance
and not in another? While it seems likely that the systemic and mucosal responses
detected in the resistant women may be part of mediating this protection, then why
do some individuals develop immune responses that provide protection, while others
in the same cohort become infected with HIV?
1.12 JUSTIFICATION OF THE STUDY
HIV/AIDS menace has reached pandemic proportions there is a dire need need of a
therapeutic vaccine. Efforts to develop HIV-1 vaccines are hindered by the absence
26
of models of naturally occurring protective immunity to HIV-1 including lack of
vaccines to induce broad neutralizing antibodies and rapid rate of mutations in HIV
virus. There is an intense interest in understanding the mechanism responsible for the
resistance to HIV-1 by some of the women in the Majengo cohort. Characterization
of immune responses to HIV-1 that protect against infection would help provide a
focus for HIV-1 vaccine research and could speed the development of effective
vaccines. If CTL responses are in part responsible for the protection, it is then
important to examine and understand the role of MHC alleles. Alternatively,
protective alleles may have peptide presentation properties from the entire or
conserved region of HIV -1 genome. Enhanced susceptible alleles may indicate that
these alleles present immunodorminant, non protective peptide epitopes. This study
is set to evaluate the protective immunity in light of polyfuctionality and
proliferation due to specific HIV-1 Epitope recognition from the entire HIV -1
genome. Where a preventive or curative therapeutic model vaccine can be cloned in
light of polyfuctionality due to epitope specificity. Elucidations of the protective
mechanisms and factors mediating their development may be important in studying
the protective immunity against HIV-1 and ultimately in the development of HIV-1
vaccine.
1.13 HYPOTHESES
1.13 NULL HYPOTHESES
a) There is no preferential Epitope recognition to HIV -1 peptides unique in
the resistant as compared to the infected commercial sex workers.
27
b) There are no polyfunctional responses of specific peptides unique in the
resistant group that may be responsible for the resistance.
1.13 General Objective
To determine the epitope specificity and polyfunctional CD4+ responses in HIV-1
exposed seronegative versus infected female commercial sex workers.
1.13.1 Specific Objectives –
1. To determine the specific peptides showing high immune responses in terms
of cytokine production in the ESN,Positives and Newly negative.
2. To compare polyfuctionality in cytokine production in the ESN,Positives and
Newly negative groups.
3. To compare proliferation in relation to polyfuctionality in all the
ESN,Positives and Newly negative. groups.
28
CHAPTER TWO:
2.0 MATERIALS AND METHODS
2.1 Study site
The study was carried out at Majengo sex Worker cohort Clinic and University of
Nairobi Institute of Tropical Infections and Diseases Flow Cytometry laboratory.
Study Population
The population involved female sex workers in the cohort which was established in
1985 comprising over 3000 women enrolled with above 700 women who are
currently active in the cohort. All female commercial sex workers in the area were
eligible for participation in the study. HIV-1 resistant women have been defined as
having been followed up in the Majengo cohort for three or more years, continuing
sex work and persistently HIV-1 seronegative and negative by HIV-1 PCR.
Currently there are slightly over 100 such individuals. HIV-1 negative non –resistant
sex workers (newly negative ) are HIV -1 seronegative prostitutes that are newly
enrolled in the cohort who do not meet the definition of resistance since they have
not been followed up for more than three years. A portion of them may ultimately
become resistant Control populations were the HIV-1 seronegative newly negative
sex workers for comparison. The control group was to determine the factors
surrounding the immunologic correlates of HIV-1 resistance.
The definition of resistant women was any woman who was confirmed by
performing HIV-1 Negative by Vironistika and Detect ELISA assays before
commencing the stimulation assays. Current reproductive health data including
29
pregnancy, contraceptive methods, date of last menses, douching practices and other
genital hygiene practices were captured through an interview (Appendix 1)
Inclusion criteria
Every commercial sex worker enrolled in the cohort was eligible to enter the research
as long as they gave an informed consent.
Exclusion Criteria
Any woman who was not enrolled in the cohort could not participate in the cohort.
2.2 Ethical consideration
Ethical clearance from the KNH Ethical Review committee was obtained before the
study commenced where the study subjects gave an informed consent before the
study commenced in accordance with the Declaration of Helsinki on human subjects’
research. (See attached on the appendix I).
2.3 Sampling.
A
Randomized Design was used to develop a sequence sampling plan; this was
done using a mathematical model on the population dispersion, determining of
economic threshold of the resistant and positive population. The following formula
by Fleiss, (1981) was used to get the sample size.
30
104
Fleiss JL Statistical Methods for Rates and Proportions (2nd edition). Wiley: New
York, 1981.
Where;
P1 is probability of being HIV positive
P2 is probability of being HIV negative (resistant)
P-bar is p corrected
q is 1-p
r is the ratio of positive to negative
31
n’1 is the number of subject required for positive
n’2 is the number of subject required for negative
n1 is the number of subject required for positive after continuity correction
n2 is the number of subject required for negative after continuity correction
α is the level of precision.
Z is the standard normal.
After implementing the formulae the sample size was 104
A total of 33 ESN, 30 newly negative, and 41 positives were randomly
choosen to participate in the cohort.
2.4 Blood collection and separation
Approximately 20 ml of peripheral blood was obtained in Heparin containing tubes
by sterile vein puncture from the subjects for functional immunological assay. Blood
was processed on the same day within three hours of collection to provide lymphoid
cells for cellular immunobiology assays. Peripheral Blood Mononuclear Cells
(PBMCs) were isolated by Ficoll Hypaque (Pfizer, New York, NY) density gradient
centrifugation Method according to Imami et al., (2002). The cells were harvested in
RPMI 1640 medium supplemented with 10 % fetal calf serum, Penicillin,
Streptomycin, L- Glutamine (final concentrations IU/ml, 100µg/ml and 2mM
respectively). The cells were then counted to ensure a concentration of 1 X 106 cells
per milliliter (ml). Stimulation of the cells followed immediately after isolation to
ensure viability of the cells.
32
2.5 Synthetic Peptides and Antibodies
Flourochrome – labeled antibodies: PE-Cy5 anti-human CD3, PerCp anti-human
CD8, PECy7 conjugated anti-human CD62L (L selectin), APC anti-human CD4 and
CD45RA- ECD (BD Biosciences ) were used for surface staining, while PE antihuman IFN Gamma, Pacific Blue anti-human TNF- α and FITC Rat anti-human IL 2 (BD Biosciences) were used for Intracellular staining . All antibodies were bound
to flourochrome dyes that enabled them to be detected by the LSR II flow cytometer.
The volumes added were ascertained by titration assays. The antibody panels used
analyzed the production of the three cytokines (IFN-γ, IL-2 and TNF-α) and was
used as a measure of polyfuctionality. HIV-1 Clade A peptide pools containing
between 11 and 16 amino acids (average15-mer) peptides spanning the entire
proteome; were synthesized via F-moc chemistry (Sigma -Genosys). The peptide
pools were designed from the entire HIV-1 Clade A proteome giving 778
overlapping peptides grouped into 20 pools (see appendix III). Each pool had 40
peptides (except pool 20 which had 18 peptides) that were resuspended in DMSO at
a concentration of 2 mg/ml .The peptides were then diluted in RPMI 1640 making a
working stock of 0.2 µg/µl. A final concentration of the peptides stimulation was 2
ng/ul.
A total of 20 peptides were designed from the 778 peptides of the entire HIV-1
genome as color coded in figure 2.5.a each pool had 40 peptides except pool 20
which had 18 peptides.
33
1
2
3
4
5
6
7
8
9
10
NEF M
GAG
ENV
ENV
ENV
ENV
ENV
GAG
NEF
GAG
P15
NEF
NEF M
GAG
P17
11
12
13
14
15
16
17
18
19
20
VPR
P17
P24
P6
P2P7P1
P7
REV
P31
TAT
RT
VIF
VPU
RT
Protease
P31
RT
P24
VIF
P6
VPR
REV
TAT
Fig 2.5.a The design of the 20 peptide pools designed from 778 peptides
The pools from the entire 778 peptides of HIV-1 genome were designed in an
overlapping manner as shown in figure 2.5.b. Pool 1 comprised peptides from Env
184-1 to Env 184, while pool 2 comprised of Env 184-41 to Env184-80 consequently
pool 3 comprised of Env184-81 to Env184-120. This then followed up to the 20th
pool which comprised of Vpr 20-19 to Vpr17 - (see appendix III).
34
Pool 1
Pool 2
Pool 3
Fig 2.5.b Overlapping of the 20 peptides designed from the HIV-1 genome.
The peptides were reconstituted in DMSO to be used at a final concentration of
2µg/mL in the entire experiment. Dimethyl sulfoxide is an organ sulfur compound
important in preservation. Surface staining and intracellular staining was performed
using previously published protocols with minor modifications after titration (Imami
et al., 2002). All staining assays were done on freshly isolated PBMCs.
2.6 Stimulation and staining of the PBMCs
The isolated PBMCs were aliquoted at this juncture and half were stained with CFSE
dye (a flourescein derivative that is cell permiant and non fluorescent) to monitor
proliferation of the cells at day 6, while the other half was used to monitor cytokine
production at day 3. Stimulation was done with 1 X 106 cells per tube 2 µg/l of the
designed peptide pools from the entire HIV -1 Genome. They were then co-cultured
with CD28 and CD49 co-stimulants at concentrations of 10µl/ml in culture medium
to enhance the immune responses. Staphylococcus enterotoxin B super antigen was
35
used as a positive control incubated at 370 C at 5% CO2 incubator for the respective
days. Tubes containing media alone were used as negative controls.
The cells were then harvested at the appropriate time points (Day 3 and day 6) by
resuspending the cells in Phosphate Buffer Solution (PBS) containing 2% fetal calf
serum (FACS wash) solution after which the cells were spun at 1700 X g for ten
minutes. The supernatant was then poured off and the cells transferred from the falcon
tubes to a 96 well V shaped microtitre plate for staining. The cells were then washed in
10% perm wash (BD BIOSCIENCES). The cells were consequently fixed using 75 µl
cytofix stabilizing fixative (BD BIOSCIENCES) and then incubated in the dark at 4
0
C for 30 minutes. After permeabilization the cells were washed twice using FACS
wash and then spun at 1700 X G for ten minutes. The cells were stained for
intracellular cytokines; IFN-γ, IL-2, TNFα .The cells were transferred from the
microtitre plate to unsterile falcon tubes for analysis. Epitope specificity of the CD4+
cells refers to the activated threshold immune response to a defined concentration of a
peptide pool.
Figure 2.6 briefly shows the steps employed in the experiment layout. Whole blood
was processed to isolate PBMCs which were aliquoted into half for use at Day 3 and
6 for where stimulation with the designed peptides. On the respective days the cells
were harvested and stained on day 3 for intracellular cytokine staining and day 6 for
CFSE staining for proliferation. The staining and harvesting of cells was conducted
according to standard procedures (Imami et al., 2002).
36
BLOOD
PBMCs
DAY 3
DAY 6
HARVEST / STAIN
FLOW CYTOMETRY
Fig 2.6: The experiment layout of the sample processing
2.7 Flow Cytometry
The data was acquired using BD LSR II flow cytometer and BD FACSdiva software
V5.0.1. Over 30,000 events as lymphocytes were acquired per sample analysis
(Imami et al .,2002). Forward scatter area versus Side scatter height parameters were
used to exclude cell debris and only lymphocytes were cells gated. CD3+ cells and
CD4+ cells were gated against the lymphocytes. Functional analysis was achieved by
putting gates against each cytokine expressed on the CD4+ T cells.
Figure 2.7 shows the emission maximas of the different flourochromes used in the
intracellular cytokine staining processes.
37
R-PE
ECD
578nM 613nM
APC
660nM
PE-Cy7
760nM
Emission
FITC
525nM
500nM
600nM
700nM
800nM
FITC-Flouresceine Isothio Cyanate
PE-Phycoerythrin
ECD-Texas red dye
APC- Allophycocyanin
Fig 2.7: An Emission spectra extract showing commonly used dyes
2.8 Data Analysis
Data was analyzed using the FACs Diva Software V5.0.1 (BD Biosciences, San Jose
USA). The data was then transferred to excel and graph pad programs where Chi
squared test and students T test were used for comparison of sample proportions and
means, to evaluate polyfuctionality due to specific epitopes in the three groups at a
confidence intervals of 95%.
38
CHAPTER THREE:
3.0 RESULTS
3.1 Preamble
The frequency of HIV-1 specific CD4+ T cell epitope specificity and
polyfuctionality in the commercial sex workers was evaluated on the basis of their
ability to secrete IFN-γ, IL-2, and TNF-α after stimulation with the 20 peptide pools.
Proliferation was measured using CFSE dye to compare quantity of cell division.
This dye was used to show the proliferation of the CD4+ cells and the loss of
intensity of CFSE in the subsequent progenies at day 6 (incubation was at 370 C at
5% CO2 ). Staphylococcus Enterotoxin B (SEB) is super antigen that was used as a
positive control. It is in a class of antigen which causes non specific activation of T
cell polyclonal activation and massive cytokine release. Co- stimulants CD28 and
CD49 are highly potent that were used in the entire experiment and were able to
stimulate T lymphocyte population expressing T cells antigen receptors (TCR)
belonging to particular Vβ families. The use of the super antigen and the co
stimulants gave a remarkable result which was demonstarted by Nagla et al., (1997)
who showed MHC class II dependent peptide antigen versus super antigen
presentation to T cells. Golgi plug was used just before day three harvest of cells to
enable detection of intracellular cytokines. All functions were measured
simultaneously by multi color parameter LSR II equipment.
39
Ideally it was expected that the positive women were to secrete more cytokines in
terms of polyfuctionality as they are living with the virus stimulating with the HIV-1
specific peptide and hence would be expected to give high secondary immune
responses. Interestingly the resistant women gave significantly higher polyfunctional
responses and proliferation which was not seen in the newly negative women. This
was indicative of certain unique correlate of immune protection in the resistant
group.
3. 2 Gating strategies
The flow cytometer LSR II is a multi parameter tool that permits the detailed
analysis of markers of cellular differentiation; it is used in developmental systems
and in assessments of vaccine driven immune responses as it permits the
simultaneous evaluation of cell phenotype and function. Cells were double stained
for surface staining and intracellular staining to assess the relative proportions and
the phenotypes of the subpopulations within the PBMCs. Auto fluorescent controls
and appropriate color compensations were also included for each flourchrome. A
minimum of 30,000 events were recorded.
In order to get the CD4+ cells producing the cytokines ,polyfuctionality
and
proliferation, it was necessary to obtain pure populations of CD4+ cells as the parent
lineage cells, from which IFN-γ, IL-2 and TNF-α producing CD4+ cells were gated
against. This was achieved by gating these specific populations in the fluorescent
scatter diagrams of interest as illustrated below.
40
Figure 3.1 showed the gating strategy used to select the lymphocytes (showed in
green) from all the events acquired in every tube. A total of 30,000 events against the
gated lymphocytes were acquired at a single acquisition.
Fig 3.1 Total lymphocytes acquired in a media alone (control) tube in one of the
patients
Figure 3.2 showed CD3+CD4+ cells that were gated against total lymphocytes cells
acquired shown in green. The pool of cells shown in green are CD3+CD4- cells
while the pool of cells shown in red are CD4-CD3- cells. This was a representative
of the gating scheme employed for identification of HIV-1 Clade A specific CD4+ T
cells response for patient.
Fig 3.2: Scatter diagram for CD4+ cells gated against total lymphocytes in a
patient
41
Figure 3.3 shows a negative control (media alone) scatter diagram of CD4+ cells
producing INF-γ, where the number of cells on the right stained CD4+IFN- γ+ are
fewer than those produced in Figure 3.4 which shows more cells on the right
stained CD4+IFN- γ+ representing a positive control (SEB) response to a patient
CD4+ cells producing INF-γ.
Fig 3.3 Scatter diagram for negative control (media alone tube) for IFNγ
production gating in a patient
Fig 3.4: Scatter diagram for positive control (SEB tube) for INF-γ production
gating in a patient
Figure 3.5 shows a negative control (media alone) scatter diagram of CD4+ cells
producing IL-2 , where the number of cells on the right stained CD4+IL-2 + are
fewer than those produced in Figure 3.6 which shows more cells on the right
42
stained CD4+IL-2 + representing a positive control (SEB) response to a patient
CD4+ cells producing IL-2.
Fig 3.5: Scatter diagram for a negative control (media alone tube) IL -2
production gating in a patient
Fig 3.6: Scatter diagram for a positive control IL -2(SEB tube) production
gating in a patient
Figure 3.7 shows a negative control (media alone) scatter diagram of CD4+ cells
producing TNF-α , where the number of cells on the right stained CD4+ TNF-α + are
fewer than those produced in figure 3.8 which shows more cells on the right
stained CD4+ TNF-α + representing a positive control (SEB) response to a patient
CD4+ cells producing TNF-α.
43
Fig 3.7: Scatter diagram for a negative control (media alone tube) TNF-α
production gating in a patient
Fig 3.8: Scatter diagram for a positive control TNF-α (SEB tube) production
gating in a patient
Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) was used as a marker of
proliferation; it is fluorescein derivative which is cell permeate and non fluorescent,
it binds to free amines resulting in long live fluorescent adducts. CFSE is partitioned
equally among daughter cells with each division this allows differential staining of
cells. The proliferative index is the sum of the cells in all generations divided by the
calculated number of original parent cells; it is very useful for comparing quantity of
cell division (proliferation). The PBMCs loaded with CFSE as per manufacturer‘s
protocols (Molecular probes).
44
Figure 3.9 shows a negative control (media alone) scatter diagram of CD4+ cells
loaded with CFSE dye, while Figure 3.10 shows a positive control (SEB) response to
a patient CD4+ cells loaded with CFSE dye. The proliferation of the cells was seen
on the left where figure 3.1.10 shows more cells showing the quantity of cell division
due to the dye than in the media alone as shown in figure 3.1.9.
3.9:Scatter diagram for negative control (media alone tube) for CFSE Loaded
cells gating in a patient
Fig 3.10: Scatter diagram for positive control (SEB) CFSE Loaded cells gating
in patient.
45
3.3 Epitope Specificity
The initially screening for epitope specificity was done using IFN-γ with the positive,
resistant and newly negative women. Figure 3.21 shows IFN-γ production responses
with the 20 peptides in the resistant group, where pools 1, 12, 13 and 14 coincided
with ENV, P24, P31, P2P7P1, P6P7 and Protease. These pools are highlighted with
the white dotted columns.
Resistant group
Resistant
Response % Control
2200.00
1200.00
1769
499
1780
1851
1952
1943
751
1601
1938
1764
1362
1394
1942
19
1860
18
1356
17
1817
16
12
11
10
1940
15
1808
1552
14
1622
887
13
1668
1573
Neg
1635
9
8
7
6
5
4
3
2
1
ati v
e
200.00
889
1923
Fig 3.11: Scatter plot showing IFN-γ responses in the resistant group
Figure 3.12 shows production of IFN-γ in the HIV-1 positive women, where the
responses did not concentrate around any pool.
46
Positive
group
Positive
Response % Control
1000.00
500.00
2028
2773
2185
2446
2558
2801
2614
2561
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
Nega tiv
e
0.00
2604
FIG 3.12: IFN-γ responses in the positive group
The same observation was made in the newly negative women where IFN-γ
production was not concentrated around any pool.
Figure 3.13 shows production of IFN-γ in the HIV-1 Newly negative women, where
the responses did not concentrate around any pool either.
47
Newly negatives group
FIG 3.13: IFN-γ responses in the newly negative group
3.4 Correlation between IFN- γ production and proliferation
The resistant group shows more IFN-γ production and proliferation, especially in
response to peptide pools 1, 12, 13 and 14. Students T test was used to test the
correlation between the IFN-γ production and proliferation unique in the resistant
group as shown in figure 3.14
48
IFN-у Production
Response % Control
10000
1000
Res vs Neg
0.0158
P<0.0001
Res vs Pos
0.0485
P<0.0001
Neg vs Pos
0.1383
0.0075
100
10
Res
Neg
Pos
CSFSE Proliferation
Prolif: Pool 13
Response % Control
10000
1000
Res vs Neg
0.6030
P<0.0001
Res vs Pos
0.4347
0.0004
Neg vs Pos
0.0576
0.2766
100
10
Res
Neg
Pos
Fig 3.14: Resistant group clearly showing HIV-1 specific IFN-y production as
well as HIV-1 specific proliferation.
There was a presumption was that there is a positive correlation between Interferon
gamma production and proliferation. However in Figure 3.15 it was seen that there
was a negative correlation between IFN-γ production and proliferation where the
PBMCs were stimulated with pool 1. In the other pools there was basically no
49
correlation between the cytokine production and proliferation of the cells. Students T
test was used to test the correlation between the IFN-γ production and proliferation
unique in the Resistant group. The difference was significant at a confidence interval
of 95% using student T test p value 0. 0001.
180
160
IFNg Production
140
120
100
80
60
40
20
0
0
100
CFSE
200
300
400
500
600
Proliferation
proliferation
PPPpPPROLIFERATIO
N
Fig: 3.15 The correlation between IFN-y production and proliferation the
resistant group
3.5 Polyfuctionality Results
Consequently the peptides showing high IFNγ production immune responses in the
resistant group; that is peptides 1, 12, 13 and 14 were analyzed for polyfuctionality
of IFNγ, IL-2, TNF-α . Pools 8 and 17 were added randomly in the panel to make a
total of 24 pools from a total of 240 peptides. In figure 3.16 the pie charts show the
polyfuctionality representation between the resistant versus the newly negative group
50
where the resistant group showed more polyfuctionality (ability to co express more
than one cytokine) than the negative group. Where the yellow represent co
expression of all three cytokines, red represents expression of any two cytokine while
the blue show the production of a single cytokine.
Fig 3.16: Comparison of polyfunctional responses between the resistant women
and Newly Negatives women
51
In figure 3.17 the pie charts show the difference between the resistant and the
positive groups, where the resistant group shows more polyfuctionality (ability to co
express more than one cytokine) in most of the pools. Where the yellow represent co
expression of all three cytokines, red represents expression of any two cytokine while
the blue show the production of a single cytokine.
Fig: 3.17: Compares Polyfuctionality responses between Resistant and the
positive
Pools 8 (Gag and Nef) and 17 (Rt and Tat) showed more of three cytokine
production in the resistant group than in the negative or the positive groups, however
the initial screening did not show high IFN-γ production.
52
CHAPTER FOUR:
4.0 DISCUSSION
The continued global spread of HIV-1, despite the intervention programs of
comprehensive care to the infected populations, and with HIV having reached
pandemic proportions, makes the development of HIV-1 vaccines an urgent priority.
Efforts to develop the vaccine are hampered by the absence of models of naturally
occurring protective immunity to HIV-1 and difficulty to develop broad spectrum
neutralizing antibodies against HIV-1. Characterization of immune responses to
HIV-1 that protect against infection would help to provide a focus for HIV-1 vaccine
research and could speed the development of effective vaccines.
Efforts to an effective HIV vaccine have been hampered by the extreme genetic
diversity and evolutionary capabilities of HIV (Goulder et al., 2004). In order to
develop effective HIV-1 vaccines and curative treatment strategies; it has become
clear that understating resistance mechanisms will be essential. With the recognition
of the type 1/type 2 dichotomy in the T helper responses to infection (Mosmann et
al., 1996 and Cherwinski et al., 1997), the concept that cellular effector mechanisms
may mediate protective immunity to HIV-1, in the absence of humoral immunity, has
gained confidence over the recent past (Fowke et al., 2002).
The study was set to evaluate the protective immunity in light of polyfuctionality and
proliferation due to specific epitopes from HIV -1 genome. To address the above
flow cytometry was employed. The cytokines used targeted Th-1 (IFN-γ and IL-2)
53
which are associated with a cellular immune responses, which are produced in
response to intracellular pathogens and Th-2 which are associated with a humoral
immune response, which are produced in response to extracellular pathogens that
intrude outside body cells and body fluids (Raffi, 1995).
There is a strong evidence that cell mediated immunity is crucial in the control of
established chronic virus infections, such as Cytomegallo Virus (Doherty et al.,
2001) and Epstein Barr Virus infection (Callan et al., 2003). Vaccine induced cellmediated immune responses have also been shown to control chronic disease in the
Simian Immunodeficiency virus (SIV) model of AIDS (Barouch et al., 2000, Amara
et al., 2001, Belyakov et al., 2001,Rose et al., 2001). Cellular immunity to HIV-1
infection plays an important role in the control of infection (McMichael et al., 2001)
4.2 Epitope Specificity
For the initial screening experiment the whole HIV-1 clade A was used since it
posses multiple epitopes and the Majengo cohort and thus population posses
typically Clade A, the cohort has highly HIV exposure with up to 500 unprotected
sexual exposures to HIV-1 infected men in over 13 years of follow up (Fowke et al.,
1996).
Recently there have been great advances in the ability to direct and characterize
antigen –specific T cells. Small overlapping peptides that span an entire protein of
interest are being used to determine the number of antigen –reactive T cells within
clinical samples by intracellular flow cytometry (Kern et al., 1998, 1999, 2002; Addo
54
et al., 2001; Altfeld et al., 2001 and Betts et al.,2001).In this study where a total of
778 overlapping peptides were employed .
The production of IFN-γ is routinely used as a marker of for antigen specific T cell
activity ,its secretion constitutes a first line of antiviral defence as T-cells release
IFN-γ immediately after stimulation with antigen (Kaech et al.,2002) .Results of
IFN-γ production and proliferation in the initial screening experiment revealed that
there is preferential epitope specifity unique in the resistant group to pools 1, 12, 13
and 14 coinciding with pools from Env, P24,P31 and P2P7P1, P6 P7, Protease and
REV peptide pools . This was not seen in the positive and Newly Negative group as
shown in figures already presented in chapter three.
In a study to determine the polyfunctional CD4+ T cells against HIV-1 Gag it was
seen that the large majority of gag-specific CD4 T lymphocytes cannot be identified
by the sole expression of interleukin-2 and or interferon-gamma, which is early
impaired (Nemes et al., 2010). This then means there is a need to evaluate the
various other HIV-1 epitopes which accord protective immunity as seen in the
resistant women.
Current research has shown that MHC alleles play a significant role in the possible
mechanisms of resistance. T cell responses to HIV-1 are known to be critical in
control of HIV -1 infection and the loss of these responses correlates with increased
viremia and disease progression (Anzala et al.,1998). Combinations of MHC alleles
for example HLA –B13 and HLA-A2 and Tap gene variants are associated with
55
resistance to infection and a reduced risk of HIV-1 Disease progression (Kaslow et
al.,1996 ; Lui et al.,1996); (Winkler et al., 1998); (Macdonald et al.,2008). Several
host genetic factors have been associated with viral control and protection against
disease progression. Some report have shown that association of HLA-DRB1*13)
and or DQB1*06 with the Long term non progressors(LTNP ) ( Keet et
al.,1999,Malhotra et al.,2001 and Vyakarnam et al.,2004) Most frequently cited is
the enrichment of certain class 1 Human leucocyte Antigen (HLA) alleles in
particular
HLA-B27
and
B57
among
HIV
controllers
(Miguele
et
al.,2000,Carrington et al.,2003)and Den et al.,2004). MHC alleles recognizing
specific epitopes in the resistant group could be having alleles which recognize
specific HIV-1 epitopes responsible for eliciting polyfunctional immune responses.
This research demonstrates there was no correlation in IFNγ production to
proliferation in the resistant group (students T test with p value of 0.001) suggesting
polyfuctionality could be important. Similar results have been reported by Lyle et al.,
2007 who found several proliferative and IFN-γ responses to ENV epitopes and
reported no correlation between the magnitude of responses between cytokine
production and proliferative assays. The findings of this study support that IFN-γ was
appropriate for screening for epitope specificity but a comprehensive functional
characterization should follow as HIV- 1 vaccine candidate requires thorough
immunological evaluation to qualify polyfuctionality as a correlate of immune
protection.
56
CD4+ T cells play a key role in the generation of immunity by providing appropriate
aid for the development of humoral and cell mediated immune responses. A number
of studies support the association between HIV- specific CD4+ T cells responses,
such as proliferative capacity and IL-2 production, and the control of viral replication
and prevention of HIV- associated disease (Lyasere et al., 2003; Younes et al.,2003;
Pantealeo et al.,2004; Pantealeo et al.,2006 ; Harari et al.,2004).
4.3 Polyfuctionality and Proliferation
Several studies have demonstrated that production of IFN-γ alone is insufficient to
define a protective immune response (Boaz et al., 2002, Betts et al., and Makedonas
et al., 2006). IFN-γ signaling increases the sensitivity of virally infected cells to
apoptotic mechanisms, by promoting the expression of the TNF-α receptor on the
cell surface (Tsujimoto et al.,1986). The CD4+ T helper lymphocytes are critical in
the maintance of effective immunity against several infections. Virus –specific CD4+
T- cells proliferate and secrete cytokines for instance Il-2, that promotes antiviral
function from other arms of the immune system, especially CTL (Matloubian et al.,
1994). IL-2 is a protein growth factor that is secreted by T – cells to promote the
proliferation and differentiations of antigen specific T cells (Lyasere et al., 2003 and
Younes et al., 2003).
Antigen specific Il-2 production has been demonstrated in several experimental
systems to be a more accurate predictor of the protective capacity of responder T cell
population than IFN-γ secretion alone (Cousesens et al.,1995). The capacity to
57
produce IL-2 may reflect a stage of superior functional differentiation than the
secretion of IFN-γ. TNF –α has been shown, in combination with IFN-γ, to clear
HBV from hepatocytes and LCMV from acutely infected mice. CD4+ Lymphocytes
appear to be important correlate of immunity against HIV infection (Mestan et al.,
1986, Guidotti et al., 1999a and Guidotti et al., 1999b). In a study by Sandberg et
al.,(2001) stimulating PBMCs from normal human donors with CMV pp65 revealed
IFN-γ+TNF-α+ cells were the most prevalent in the responding CD8 + T –cell
population.
The term functionality describes the ability of cells to produce multiple cytokines
(Betts et al., 2006; Makedonas et al., 2006 ; Seder et al., 2008). Polyfunctional T
cells are generated after exposure to Influenza virus, Epstein –Barr virus, Varicella
zoster virus and Cytomegalovirus (Seder et al., 2008). In the mouse model of
Leishmania major, mice were administered several vaccine formulations and
subsequently challenged; mice that demonstrated the greatest protection were those
that produced the highest frequencies polyfunctional cells (IFN-γ+ TNF-α+ and IL2+) (Darrah et al., 2007)
Polyfunctional T- cells have been documented in HIV and Hepatitis vaccine
recipients (De rosa et al., 2004) as well as individuals vaccinated with vaccinia virus
(Precopio et al., 2007). In this study it has been shown that a predominance of
polyfunctional CD4+ T cells specific for the resistant more than the positives and
newly negative group. Virus specific CD4+ cells which produce multiple effector
functions simultaneously, such as IL-2 and IFNγ, have been shown to be a hallmark
58
of protective immunity in controlled viral infections, such as CMV and EBV.
Notably, in other studies the HIV- specific CD4+ T cells in subjects with non
progressive diseases were polyfunctional in EBV and CMV specific CD4+ T cells
responses (Harari et al., 2003). EBV- and CMV- specific T cell responses are
characterized by the presence of three functionally distinct populations; cells that
secrete IL-2 but not IFN-γ, cells that secrete both IL-2 and IFN-γ and cells that
secrete IFN-γ but not IL-2. The patterns of HIV-specific CD4+ T cells responses
during primary infection are characteristic of an effector response (Harari and Betts,
2008), that is IFNγ, TNFα secretion with limited IL-2 secretion or proliferation
capacity (Pantaleo and Harari, 2006; Harari et al., 2002).
Technological advancements in flow cytometry now permit the staining and
detection of up to 18 different markers on human T cells. Staining panels and
procedures have been developed to permit the examination of five T cell functions
simultaneously. In this study, the measurement of several functions concurrently
enabled the discernment of a fundamental difference between HIV-1 specific CD8+
T cell responses in the resistant women compared to the positive (HIV-1infected)
women.
When the experiment was redone including IL-2 and TNF- α in addition to IFN-γ
for intracellular staining at day Three. Figures 3.16 and 3.17 clearly showed a
significant difference in polyfuctionality between the resistant group and the
negatives and positive respectively with P values less than 0.001 using the chi
squared test. Hence it would be imperative to then break down the individual pools to
59
assess if any specific peptides elicit the polyfunctional cytokine production seen
uniquely in the resistant group.
Detection of HIV-1 specific IL-2, IFN-γ, and TNF- α secretion in the resistant group
indicates that the immune system must have encountered HIV-1 virus. MHC I
presentation is required for the development of CTL – detection of HIV- 1 specific
CTL in the resistant also indicates that these women had the replicating virus but
probably they are able to control it before it manifests to make the immune system is
able to produce antibodies against the virus. Having determined increased
functionality in the resistant group it is paramount to specifically ascertain that these
antigen specific cells are responsible in eliciting the polyfuctionality in responses to
peptide stimulation.
Having determined increased functionality in the resistant group it is paramount to
specifically ascertain that these antigen specific cells are responsible in eliciting the
polyfuctionality in responses to peptide stimulation.
The resistant group showed significant proliferative responses with the CFSE based
proliferation assay than the positive and negative groups. CD4+ T cell proliferation
in the positive and negative groups were not due to a general defect, because
stimulation with superantigen ( Staphylococcus Enterotoxin B ) yielded comparable
CD4+ cell proliferative responses in the two groups.
60
4.5 Conclusion
The identification of immune of immune correlates of protection is commonly
considered as the backbone of HIV vaccine research. Understanding the specific
immune responses conferring protection from infection in individuals in exposed to
HIV-1 is critical for Vaccine design. It is evident that there is selective preferential
epitope recognition unique in the resistant group that could be responsible for the
unique polyfunctional immune responses. This is an important correlate of immune
protection against HIV-1 that should be considered in the modeling of a HIV-1
vaccine.
It is likely that not every vaccine that stimulates HIV-1 specific T -cell immunity will
prove equally efficacious or even have the same correlate of protection. Although
evaluating polyfuctionality and proliferation due to selective preferential epitope
specificity to HIV-1 genome unique in the resistant people may dissect a protective
correlate of immune protection to HIV-1 in future trial of T – cell vaccines
stimulating vaccine. This will in turn help determine the breath of the vaccine
induced CD4+ T -cell response and the best combination of vaccines be cloned in the
prime boost immunization strategies which would stimulate an immune response
similar to that thought to confer protection from infection and disease progression in
the resistant (persistently exposed seronegative high risk persons). If a strong
correlate of protection to HIV-1 is found, then future trials of vaccines can use all a
cocktail of all the known correlates of protection against HIV-1 to streamline the
testing process of an ideal curative or preventive vaccine.
61
The surface antigens play an important role in the cell signaling leading to cell
proliferation and differentiation monitoring of surface expression markers may be
very useful. Results from the recent Thai trial have further emphasized the need to
identify new immune correlates. This will require continuous efforts in the field, new
studies and development of new methods to asses’ aspects of T- cell immune
responses. It is becoming clear that polyfunctional analysis would critical to the
accurate assessment of vaccine –induced T cell responses.
i.
The results revealed that there is preferential epitope specificity unique in the
resistant groups to pools 1, 12, 13, and 14 coinciding with pools from
Env,P24, P31 and P2P7P1, P6P7, Protease and Rev peptide pools. This was
not seen in the positive and newly negative group.This leads to rejecting the
null hypothesis that there is no preferential epitope recognition to the infected
commercial sex workers . Hence the alternative hypothesis is accepted that
there is preferential epitope recognition to HIV-1 peptides unique in the
resistant as compared to the infected commercial sex workers was accepted.
ii.
The
resistant
group
showed
an
increased
cytokine
production
(polyfunctional) in most of the HIV-1 peptide pools. This was not seen in the
positive and the newly negative group .This leads to rejecting the null
hypothesis that there are no polyfunctional responses of specific peptides
unique in the resistant group that may be responsible for the resistance. Hence
the alternative hypothesis is accepted that there are polyfunctional responses
62
of specific peptides unique in the resistant group that may be responsible for
the resistance.
iii.
There was no correlation in cytokine production to proliferation of the cells
though most of the proliferation was seen in the resistant group.
iv.
This lead to the conclusion that there could be another correlate of immune
protection in addition to polyfuctionality in the resistant group.
4.6 Recommendations
i.
Specific peptide pools should be broken down using the deconvulating
matrices confirming the epitope specificity.
ii.
The cytokine panel should be added to capture memory markers
cytokines/chemokines such as Rantes, MIP-1, IL-2 and others to confirm the
polyfuctionality.
iii.
Run peptide binding assays and stability Kinetics to confirm the binding of
the peptides to the MHC alleles.
iv.
Additional studies will be required to address issues such as role of innate
immunity in HIV control(Toll like receptors),the level of ongoing viral
replication and the replication competence and probably the impact of host
genetics on T cell infectectability and viral kinetics
v.
The entire experiment should be repeated using the entire HIV-1 clade A
genome to confirm polyfuctionality.
63
4.7 Limitations/challenge
The major limitation of this study was the inability to repeat the experiment with the
entire 20 pools from the HIV-1 Clade A genome when we added the cytokine panel
as this had antibody cost implications.
64
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APPENDICES
APPENDIX 1: Ethics /consent document
Comprehensive Studies of Mechanisms of HIV Resistance in Nairobi, Kenya
Majengo, SWOP, Korogocho and MCH Pumwani Research Clinics
Patient Information and Consent Form
This information will be communicated orally in English, Swahili or other Kenyan
dialect of potential participant’s preference.
Investigators:
Dr. Charles Wachihi, University of Nairobi, tel. 714851, PO Box 19676, Nairobi,
Kenya
Dr. Joshua Kimani,
University of Nairobi, tel. 714851, PO Box 19676, Nairobi,
Kenya
Dr. Jessie Kwatampora University of Nairobi, tel. 714851, PO Box 19676, Nairobi,
Kenya
Dr. Walter Jaoko,
University of Nairobi, tel. 714851, PO Box 19676, Nairobi,
Kenya
Dr. T. Blake Ball
University of Manitoba, 730 William Ave. Winnipeg, MB,
Canada 1-(204) 789-3202
Dr. Francis Plummer University of Manitoba, 730 William Ave. Winnipeg, MB,
Canada 1-(204) 789-2000
Ethics document cont’
85
Dr. Joanne Embree,
University of Manitoba, 730 William Ave. Winnipeg, MB,
Canada 1-(204) 789-3630
Dr. Keith R. Fowke, University of Manitoba, 730 William Ave. Winnipeg, MB,
Canada 1-(204) 789-3818
Dr. Rupert Kaul,
University of Toronto, 1 King's College Toronto, ON, Canada
1-(204) 416-978-860
BACKGROUND INFORMATION
The University of Nairobi and its collaborators from Canada have been working for
many years to fight the epidemics of AIDS and other sexually transmitted infections
that we are facing in Kenya. This basic science research program is conducting
studies to determine the relationship between immunity and susceptibility to sexually
transmitted infections (STI) with the goal of developing vaccines or treatments for
STIs. You are being asked to participate in this study because you are:
a) at a very high risk of acquiring an STI or are already infected with an STI
or
b) at a low risk of acquiring an STI; or
c) the relative of a person in group a) or b).
Ethics document continued’
86
The purpose of this research program is to determine if there are factors that could
protect individuals from acquiring sexually transmitted infections (STI) especially
HIV.
It is important to keep free of other sexually transmitted diseases, as the presence of
these infections may increase your risk of becoming infected with HIV. If you have
an STI, you should seek treatment for it as quickly as possible. However, sometimes
you may have an STD and not know it, because you may not have any symptoms and
thus advised to visit the clinic monthly for free check ups.
Why Is This Study Being Done?
This study is being done to find out why some people are more or less likely to get
the Human Immunodeficiency Virus (HIV), the virus that causes AIDS. There is
more and more evidence that the immune system in some people is able to protect
them against infection with HIV.
Since most people get HIV through sexual
exposure to an HIV infected partner, the first contact with the virus occur in the
genital tract, the vagina and cervix in women. We know from some of our previous
work that some women, who seem to be protected against HIV, have a special type
of immune response that it not present in women who get HIV. The purpose of this
study is to try to find out the targets of this immune response in the vagina, uterus
and cervix and to try to find out what is special about the immune system of these
Ethics document continued’
87
few individuals. This work may be helpful in eventually making a vaccine for HIV.
To help you understand what is involved in the study a drawing of the vagina, cervix
and uterus of the female genital tract is shown below.
Fallopian Tube
Uterus (womb)
Ovary
Cervix
Vagina
How Many People Will Take Part in the Study
About 3000 participants mainly women will take part in this study.
What Is Involved in the Study?
88
Ethics document continued’
You have been invited to voluntarily participate in this study because all are at risk of
becoming infected with STDs and HIV. Some sexual behavior especially among sex
workers or those who use sex as an income generating activity exposes those
involved or their partners to a higher risk of contracting HIV. If you now agree to
participate in the study, you will first be given additional counseling and information
on HIV risk reduction. Those who are sex workers will be given additional
counseling, advised on appropriate STIs prevention strategies and requested to
consider leaving prostitution. If a sex worker and you choose not to leave prostitution
at this time you will be asked to return to the clinic every month for free check ups.
Again, the results of these tests will be ready after one week or less, and you will be
informed of the results and given the correct treatment if you have an infection. You
will also be encouraged to come to the clinic for examination and treatment at any
other time that you feel ill. If you forget to return to the clinic for one of your
scheduled visits, a clinic staff member will contact you by phone, SMS or send one
of your friends to remind you of the missed appointment. All study participants will
also be encouraged to either retest for HIV or recheck their CD4/CD8 profiles
depending on HIV infection status every three months. In addition, we will store
specimens from your blood for future studies of the genes involved in resistance and
susceptibility to HIV and other infections.
Ethics document cont’
89
Clinic visits
First visit and semi-annual or Resurvey visits (All study participants)
1. We will ask you general questions about your life, about problems you are
having, and about your sexual history.
2. The doctor will examine your body, including your female parts.
3. Swab and washing from your vagina to look for germs and to collect samples for
studying your immune response.
4. Swab from your cervix to look from germs and to collect samples for studying
your immune response.
5. A thin plastic tube will be placed in your cervix (opening to your womb) to get
some of the mucous your cervix makes.
6. Urine to look for germs.
7. Three tablespoons of blood will be taken for testing syphilis and HIV and for
studying your immune response. We will inform you of your results at your one
month visit. We also will test your spouse for the HIV virus free of charge if
he/she wishes.
Monthly visits (Those in sex work or engaged in high risk sexual behaviors)
1. Questions will be asked about you, and what problems you are having.
90
Ethics document continued’
2. If you have any complaints the doctor will examine your body, including your
female parts.
3. Every third month all study participants will be encouraged to either retest for
HIV or recheck their CD4/CD8 profiles depending on individuals HIV infection
status.
Follow-up visits (All study participants)
1. You will be asked to return 3 to 7 days after every visit to be given you
laboratory results.
2. You will be treated for new infections, free of charge.
How Long Will I Be in the Study?
The study will last 5 years. Although we would appreciate if you stayed in the study
for the entire period you may choose to leave the study at any time without any
penalty to you.
What Are the Risks of the Study?
Risk of blood and cervical collection
This study requires the use of your blood. In order to get the blood we will need to
insert a needle into a vein in your arm so that the blood can be removed. There will
be some pain associated with the needle stick but this will be only for a short period
of time.
91
Ethics document continued’
There may be some bruising around the needle site and, although we will sterilize the
site to minimize infection, there is a very minimal risk of infection at the site. There
is also some discomfort associated with taking specimens from your cervix.
HIV test
Non-physical risks:
1. If you are HIV positive, learning so may cause you to become depressed. We
will counsel you about your HIV test results if you are negative or positive.
If you are HIV positive, we will refer you to another clinic for care and
treatment. We will also test your husband or boyfriend for the HIV virus if
he wants.
Risks of taking antibiotics / Antiretroviral
If we find that you have an STD or AIDS we will provide you with the appropriate
treatment. With any drug there is some potential for side effects. For the antibiotics/
antiretroviral you might receive, the following side effects are possible.
Very likely:
1. Sick to your stomach
2. Headache
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Ethics document continued’
3. Metallic taste in mouth
4. Diarrhea
5. If a woman - infection of your vagina by yeast (a white discharge with
itching). If this happens, we will give you medicine to put inside your
vagina to treat the yeast infection.
Less likely but serious:
1. Less than 1 person in 100 will have a severe allergic reaction to one of the
antibiotics/ antiretroviral.
Are There Benefits to Taking Part in the Study?
The benefits that you will get from this study are that you will be examined
regularly, and if you are found to have an STD or AIDS, you will receive appropriate
and effective medication. Medical care will also be provided for other illnesses that
you might have. You will also be informed about what you are suffering from, and
you will be informed about the future implications of these STDs and of HIV.
What about Confidentiality?
93
Ethics document continued’
Efforts will be made to keep your personal information confidential. We will record
your information only by a special number assigned to you. The number will only be
known to the clinic staff and yourself.
Organizations that may inspect and/or copy your research records for quality
assurance and data analysis include groups such as: the researchers, members of the
local and international ethics teams and the National Institutes of Health in the
United States of America. The research results will be published, but your identity
will remain secret.
What Are My Rights as a Participant?
Taking part in this study is voluntary. You may choose not to take part or may leave
the study at any time. Leaving the study will not result in any penalty or loss of
benefits to which you are entitled. If the participation in the study results in you
becoming ill, the study team will provide you with medical care for the problem for
free.
Although you will not be paid to participate in the study, you will be offered a small
payment of four hundred shillings (KSh 400) during the resurvey visits only to
compensate you for your transportation to the clinic and any other expenses you
might incur.
94
Ethics document continued’
We will also provide you with any new information and findings from the study that
may affect your health, welfare, or willingness to stay in this study.
All information that is obtained will be kept strictly confidential, and your identity
will not be known, except to those providing your medical care.
At the end of every year, we will be holding baraza’s at the different clinics to give
progress reports and share any new findings from the study with all members of the
different clinics.
WHOM DO I CALL IF I HAVE QUESTIONS OR PROBLEMS?
For questions about the study or a research-related injury, call or contact Drs.
Wachihi, Kwatampora, Barasa or any one of the researchers named above at the
Medical Microbiology Annex at the University of Nairobi
For questions about your rights as a research participant, contact Professor Bhatt,
who is the chairperson of the Ethical Review Committee at the University of Nairobi,
by calling 725452, or make an appointment to see her in the Department of
Medicine, at the University of Nairobi.
95
Ethics document continued’
Statement of Consent:
If you agree to participate in the study, please sign below.
I, ____________________, have read or have had read to me, the consent form for
the above study and have discussed the study with ____________________.
I understand that the following (check the box only if you fully understand and agree
with each statement):

the goals of this research program are to study resistance and susceptibility to
sexually transmitted infections

enrolment is completely voluntary and I can withdraw from the study at any
time

blood, cervical and vaginal specimens will be required for this study and may
be used for genetic studies
Ethics document continued’
96

any blood specimens previously collected may be used for this study
a portion of my blood, cervical and vaginal specimens will be stored for future
studies of the genes involved in resistance and susceptibility to HIV and other
infections.
I am willing to participate in the study.
Name of Study Participant______________________________________________
Signature/Thumb
print:
__________________________________
Date:
______________
For clinic staff:
I, _________________________________, have explained the nature and purpose
of
the
above
study
to
_____________________________________________________
Name
of
Clinic
___________________________________________________
Signature: __________________________________ Date: ______________
Assigned Study Number / Clinic Number ____ ____ ____
97
Staff:
Ethics document continued’
NB: All study participants will be issued with a copy of this information and consent
forms Standards of Medical Care for Participants in the Research Clinics
This document outlines the standard of medical care for all participants in the
Majengo, MCH Pumwani, Kindred, Kibera and Korogocho cohorts, regardless of
HIV-1 serostatus. It should be emphasized that any member of the said cohorts may
freely decline to take part in any cohort substudy, and that this decision will in no
way affect their access to this standard of care. All care outlined is provided free of
charge, thereby significantly improving health care access and outcomes for all
members of the cohorts.The nature of the medical care will vary depending on HIV-1
serostatus of the participants, as outlined below.
1. General medical care for all participants, regardless of HIV-1 status.

HIV and STD prevention services: provision of the male condom,
and peer-based and clinic-based counseling regarding safer sexual
practices.

Family planning services as directed in the Kenyan National
Family Planning Guidelines

98
Ethics document continued’

Rapid and effective treatment of sexually transmitted diseases in
accordance with the Kenya National Guidelines for the Syndromic
Management of Sexually Transmitted Diseases

Medical care for acute and chronic illnesses, both infectious and
non-infectious

Access to diagnostic testing in haematology, biochemistry,
infectious diseases, immunology, radiology

Prompt referral for specialist consultation and hospitalization
when indicated
2. Management of Opportunistic Infections in HIV-1 Infected Participants.

Primary Prophylaxis: Trimethoprim-Sulphamethoxazole (Septrin):
all participants with a CD4+ T cell count <200/mm3, for
prevention of PCP, toxoplasmosis and bacterial infections
(bacterial pneumonia, bacteremias, some bacterial diarrhoea),
according to National AIDS/STD Control Program (NASCOP)
Guidelines

Secondary Prophylaxis: Septrin: offered to all participants
regardless of CD4+ T cell count after an episode of PCP,
toxoplasmosis, or severe
99
Ethics document continued’
bacterial infection. Fluconazole: provided for secondary prevention of Cryptococcus
Treatment

Herpes. simplex/Herpes zoster infection: acyclovir

Candidiasis (oral, esophageal, vaginal): nystatin, clotrimazole,
Fluconazole

Tuberculosis (pulmonary or extra pulmonary): referral to
National TB Programme

Toxoplasmosis: referral for inpatient therapy

Cryptococcus: referral for inpatient therapy

PCP: Septrin (with prednisolone, if severe)

Kaposi’s Sarcoma: ARV and referral to Clinical Oncologist
3. Antiretroviral therapy.
Antiretroviral therapy rollout in Kenya is supported and directed by NASCOP
and The Ministry of Health. Kenya is a recipient of ARVs and infrastructure
support through the Presidents Emergency Plan for AIDS Relief (PEPFAR) a US
government international development initiative.
100
Ethics document continued’

ARV drugs and infrastructure support has been secured by the University
of Manitoba from NASCOP/PEPFAR and CDC PEPFAR to provide HIV
basic and ARV care for all cohorts members who are eligible as per the
“Guidelines to Antiretroviral Drug Therapy in Kenya” (NASCOP-2002).
Such medical treatment and its requisite follow-up, integrated with the
above standard of care, will also be provided at no cost.
101
APPENDIX II ; Ethical Clearance
102
APPENDIX III: Table 1 showing the 778 peptide pools from HIV-1 clade A
genome. (Sigma Genosys Company)
Pool 1
Pool 2
Pool 3
Pool 4
1
ENV 184-1
41
ENV 184-41
81
ENV 184-81
121
ENV 184-121
2
ENV 184-2
42
ENV 184-42
82
ENV 184-82
122
ENV 184-122
3
ENV 184-3
43
ENV 184-43
83
ENV 184-83
123
ENV 184-123
4
ENV 184-4
44
ENV 184-44
84
ENV 184-84
124
ENV 184-124
5
ENV 184-5
45
ENV 184-45
85
ENV 184-85
125
ENV 184-125
6
ENV 184-6
46
ENV 184-46
86
ENV 184-86
126
ENV 184-126
7
ENV 184-7
47
ENV 184-47
87
ENV 184-87
127
ENV 184-127
8
ENV 184-8
48
ENV 184-48
88
ENV 184-88
128
ENV 184-128
9
ENV 184-9
49
ENV 184-49
89
ENV 184-89
129
ENV 184-129
10
ENV 184-10
50
ENV 184-50
90
ENV 184-90
130
ENV 184-130
11
ENV 184-11
51
ENV 184-51
91
ENV 184-91
131
ENV 184-131
12
ENV 184-12
52
ENV 184-52
92
ENV 184-92
132
ENV 184-132
13
ENV 184-13
53
ENV 184-53
93
ENV 184-93
133
ENV 184-133
14
ENV 184-14
54
ENV 184-54
94
ENV 184-94
134
ENV 184-134
15
ENV 184-15
55
ENV 184-55
95
ENV 184-95
135
ENV 184-135
16
ENV 184-16
56
ENV 184-56
96
ENV 184-96
136
ENV 184-136
17
ENV 184-17
57
ENV 184-57
97
ENV 184-97
137
ENV 184-137
18
ENV 184-18
58
ENV 184-58
98
ENV 184-98
138
ENV 184-138
19
ENV 184-19
59
ENV 184-59
99
ENV 184-99
139
ENV 184-139
20
ENV 184-20
60
ENV 184-60
100
ENV 184-100
140
ENV 184-140
21
ENV 184-21
61
ENV 184-61
101
ENV 184-101
141
ENV 184-141
22
ENV 184-22
62
ENV 184-62
102
ENV 184-102
142
ENV 184-142
23
ENV 184-23
63
ENV 184-63
103
ENV 184-103
143
ENV 184-143
24
ENV 184-24
64
ENV 184-64
104
ENV 184-104
144
ENV 184-144
25
ENV 184-25
65
ENV 184-65
105
ENV 184-105
145
ENV 184-145
26
ENV 184-26
66
ENV 184-66
106
ENV 184-106
146
ENV 184-146
27
ENV 184-27
67
ENV 184-67
107
ENV 184-107
147
ENV 184-147
28
ENV 184-28
68
ENV 184-68
108
ENV 184-108
148
ENV 184-148
29
ENV 184-29
69
ENV 18469
109
ENV 184-109
149
ENV 184-149
30
ENV 184-30
70
ENV 184-70
110
ENV 184-110
150
ENV 184-150
31
ENV 184-31
71
ENV 184-71
111
ENV 184-111
151
ENV 184-151
32
ENV 184-32
72
ENV 184-72
112
ENV 184-112
152
ENV 184-152
33
ENV 184-33
73
ENV 184-73
113
ENV 184-113
153
ENV 184-153
34
ENV 184-34
74
ENV 184-74
114
ENV 184-114
154
ENV 184-154
35
ENV 184-35
75
ENV 184-75
115
ENV 184-115
155
ENV 184-155
36
ENV 184-36
76
ENV 184-76
116
ENV 184-116
156
ENV 184-156
37
ENV 184-37
77
ENV 184-77
117
ENV 184-117
157
ENV 184-157
38
ENV 184-38
78
ENV 184-78
118
ENV 184-118
158
ENV 184-158
39
ENV 184-39
79
ENV 184-79
119
ENV 184-119
159
ENV 184-159
40
ENV 184-40
80
ENV 184-80
120
ENV 184-120
160
ENV 184-160
103
Pool 5
Pool 6
Pool 7
Pool 8
161
ENV 184-161
201
GAG 113-17
241
GAG 113-57
281
GAG 113-98
162
ENV 184-162
202
GAG 113-18
242
GAG 113-58
282
GAG 113-99
163
ENV 184-163
203
GAG 113-19
243
GAG 113-59
283
GAG 113-100
164
ENV 184-164
204
GAG 113-20
244
GAG 113-60
284
GAG 113-101
165
ENV 184-165
205
GAG 113-21
245
GAG 113-61
285
GAG 113-102
166
ENV 184-166
206
GAG 113-22
246
GAG 113-62
286
GAG 113-103
167
ENV 184-167
207
GAG 113-23
247
GAG 113-63
287
GAG 113-104
168
ENV 184-168
208
GAG 113-24
248
GAG 113-64
288
GAG 113-105
169
ENV 184-169
209
GAG 113-25
249
GAG 113-65
289
GAG 113-106
170
ENV 184-170
210
GAG 113-26
250
GAG 113-66
290
GAG 113-107
171
ENV 184-171
211
GAG 113-27
251
GAG 113-67
291
GAG 113-108
172
ENV 184-172
212
GAG 113-28
252
GAG 113-68
292
GAG 113-109
173
ENV 184-173
213
GAG 113-29
253
GAG 113-69
293
GAG 113-110
174
ENV 184-174
214
GAG 113-30
254
GAG 113-70
294
GAG 113-111
175
ENV 184-175
215
GAG 113-31
255
GAG 113-71
295
GAG 113-112
176
ENV 184-176
216
GAG 113-32
256
GAG 113-72
296
GAG 113-113
177
ENV 184-177
217
GAG 113-33
257
GAG 113-73
297
NEF 44-1
178
ENV 184-178
218
GAG 113-34
258
GAG 113-74
298
NEF 44-2
179
ENV 184-179
219
GAG 113-35
259
GAG 113-75
299
NEF 44-3
180
ENV 184-180
220
GAG 113-36
260
GAG 113-76
300
NEF 44-4
181
ENV 184-181
221
GAG 113-37
261
GAG 113-77
301
NEF 44-5
182
ENV 184-182
222
GAG 113-38
262
GAG 113-78
302
NEF 44-6
183
ENV 184-183
223
GAG 113-39
263
GAG 113-79
303
NEF 44-7
184
ENV 184-184
224
GAG 113-40
264
GAG 113-81
304
NEF 44-8
185
GAG 113-1
225
GAG 113-41
265
GAG 113-82
305
NEF 44-9
186
GAG 113-2
226
GAG 113-42
266
GAG 113-83
306
NEF 44-10
187
GAG 113-3
227
GAG 113-43
267
GAG 113-84
307
NEF 44-11
188
GAG 113-4
228
GAG 113-44
268
GAG 113-85
308
NEF 44-12
189
GAG 113-5
229
GAG 113-45
269
GAG 113-86
309
NEF 44-13
190
GAG 113-6
230
GAG 113-46
270
GAG 113-87
310
NEF 44-14
191
GAG 113-7
231
GAG 113-47
271
GAG 113-88
311
NEF 44-15
192
GAG 113-8
232
GAG 113-48
272
GAG 113-89
312
NEF 44-16
193
GAG 113-9
233
GAG 113-49
273
GAG 113-90
313
NEF 44-17
194
GAG 113-10
234
GAG 113-50
274
GAG 113-91
314
NEF 44-18
195
GAG 113-11
235
GAG 113-51
275
GAG 113-92
315
NEF 44-19
196
GAG 113-12
236
GAG 113-52
276
GAG 113-93
316
NEF 44-20
197
GAG 113-13
237
GAG 113-53
277
GAG 113-94
317
NEF 44-21
198
GAG 113-14
238
GAG 113-54
278
GAG 113-95
318
NEF 44-22
199
GAG 113-15
239
GAG 113-55
279
GAG 113-96
319
NEF 44-23
200
GAG 113-16
240
GAG 113-56
280
GAG 113-97
320
NEF 44-24
104
Pool 9
Pool 10
Pool 11
Pool 12
321
NEF 44-26
361
NEF M-33
401
P17 28-10
441
P24 49-43
322
NEF 44-27
362
NEF M-34
402
P17 28-11
442
P24 49-44
323
NEF 44-28
363
NEF M-35
403
P17 28-12
443
P24 49-45
324
NEF 44-29
364
NEF M-36
404
P17 28-13
444
P24 49-48
325
NEF 44-30
365
NEF M-37
405
P17 28-14
445
P24 49-49
326
NEF 44-31
366
NEF M-38
406
P17 28-15
446
P2P7P1 17-1
327
NEF 44-32
367
NEF M-40
407
P17 28-16
447
P2P7P1 17-2
328
NEF 44-33
368
NEF M-42
408
P17 28-17
448
P2P7P1 17-3
329
NEF 44-34
369
NEF M-43
409
P17 28-18
449
P2P7P1 17-4
330
NEF 44-35
370
NEF M-44
410
P17 28-19
450
P2P7P1 17-5
331
NEF 44-36
371
P15 25-1
411
P17 28-20
451
P2P7P1 17-6
332
NEF 44-37
372
P15 25-2
412
P17 28-21
452
P2P7P1 17-15
333
NEF 44-38
373
P15 25-3
413
P17 28-22
453
P2P7P1 17-16
334
NEF 44-39
374
P15 25-4
414
P17 28-23
454
P2P7P1 17-17
335
NEF 44-40
375
P15 25-5
415
P17 28-24
455
P31 63-1
336
NEF 44-41
376
P15 25-6
416
P17 28-25
456
P31 63-2
337
NEF 44-42
377
P15 25-7
417
P17 28-26
457
P31 63-3
338
NEF 44-43
378
P15 25-8
418
P17 28-27
458
P31 63-4
339
NEF 44-44
379
P15 25-9
419
P17 28-28
459
P31 63-5
340
NEF M-1
380
P15 25-10
420
P24 49-1
460
P31 63-6
341
NEF M-2
381
P15 25-11
421
P24 49-2
461
P31 63-7
342
NEF M-3
382
P15 25-12
422
P24 49-3
462
P31 63-8
343
NEF M-4
383
P15 25-13
423
P24 49-10
463
P31 63-9
344
NEF M-5
384
P15 25-14
424
P24 49-11
464
P31 63-10
345
NEF M-6
385
P15 25-15
425
P24 49-12
465
P31 63-11
346
NEF M-7
386
P15 25-16
426
P24 49-13
466
P31 63-12
347
NEF M-8
387
P15 25-17
427
P24 49-14
467
P31 63-13
348
NEF M-9
388
P15 25-18
428
P24 49-22
468
P31 63-14
349
NEF M-10
389
P15 25-19
429
P24 49-23
469
P31 63-15
350
NEF M-11
390
P15 25-20
430
P24 49-24
470
P31 63-16
351
NEF M-12
391
P15 25-21
431
P24 49-25
471
P31 63-17
352
NEF M-21
392
P15 25-22
432
P24 49-26
472
P31 63-18
353
NEF M-22
393
P15 25-23
433
P24 49-27
473
P31 63-19
354
NEF M-23
394
P15 25-24
434
P24 49-28
474
P31 63-20
355
NEF M-24
395
P15 25-25
435
P24 49-29
475
P31 63-21
356
NEF M-25
396
P17 28-2
436
P24 49-30
476
P31 63-22
357
NEF M-26
397
P17 28-5
437
P24 49-31
477
P31 63-23
358
NEF M-28
398
P17 28-6
438
P24 49-33
478
P31 63-24
359
NEF M-29
399
P17 28-7
439
P24 49-34
479
P31 63-25
360
NEF M-30
400
P17 28-9
440
P24 49-35
480
P31 63-26
105
Pool 13
Pool 14
Pool 15
Pool 16
481
P31 63-27
521
P6 10-4
561
REV 26-8
601
RT 100-22
482
P31 63-28
522
P6 10-5
562
REV 26-9
602
RT 100-23
483
P31 63-29
523
P6 10-6
563
REV 26-10
603
RT 100-24
484
P31 63-30
524
P6 10-7
564
REV 26-11
604
RT 100-25
485
P31 63-31
525
P6 10-8
565
REV 26-12
605
RT 100-26
486
P31 63-32
526
P6 10-9
566
REV 26-13
606
RT 100-27
487
P31 63-33
527
P7 10-1
567
REV 26-14
607
RT 100-28
488
P31 63-34
528
P7 10-2
568
REV 26-15
608
RT 100-29
489
P31 63-35
529
P7 10-3
569
REV 26-16
609
RT 100-30
490
P31 63-36
530
P7 10-4
570
REV 26-17
610
RT 100-31
491
P31 63-37
531
P7 10-5
571
REV 26-18
611
RT 100-32
492
P31 63-38
532
P7 10-10
572
REV 26-19
612
RT 100-33
493
P31 63-39
533
PROTEASE 21-1
573
REV 26-20
613
RT 100-34
494
P31 63-40
534
PROTEASE 21-2
574
REV 26-21
614
RT 100-35
495
P31 63-41
535
PROTEASE 21-3
575
REV 26-22
615
RT 100-36
496
P31 63-42
536
PROTEASE 21-4
576
REV 26-23
616
RT 100-37
497
P31 63-43
537
PROTEASE 21-5
577
REV 26-24
617
RT 100-38
498
P31 63-44
538
PROTEASE 21-6
578
REV 26-25
618
RT 100-39
499
P31 63-45
539
PROTEASE 21-7
579
REV 26-26
619
RT 100-40
500
P31 63-46
540
PROTEASE 21-8
580
RT 100-1
620
RT 100-41
501
P31 63-47
541
PROTEASE 21-9
581
RT 100-2
621
RT 100-42
502
P31 63-48
542
PROTEASE 21-10
582
RT 100-3
622
RT 100-43
503
P31 63-49
543
PROTEASE 21-11
583
RT 100-4
623
RT 100-44
504
P31 63-50
544
PROTEASE 21-12
584
RT 100-5
624
RT 100-45
505
P31 63-51
545
PROTEASE 21-13
585
RT 100-6
625
RT 100-46
506
P31 63-52
546
PROTEASE 21-14
586
RT 100-7
626
RT 100-47
507
P31 63-53
547
PROTEASE 21-15
587
RT 100-8
627
RT 100-48
508
P31 63-54
548
PROTEASE 21-16
588
RT 100-9
628
RT 100-49
509
P31 63-55
549
PROTEASE 21-17
589
RT 100-10
629
RT 100-50
510
P31 63-56
550
PROTEASE 21-18
590
RT 100-11
630
RT 100-51
511
P31 63-57
551
PROTEASE 21-19
591
RT 100-12
631
RT 100-52
512
P31 63-58
552
PROTEASE 21-20
592
RT 100-13
632
RT 100-53
513
P31 63-59
553
PROTEASE 21-21
593
RT 100-14
633
RT 100-54
514
P31 63-60
554
REV 26-1
594
RT 100-15
634
RT 100-55
515
P31 63-61
555
REV 26-2
595
RT 100-16
635
RT 100-56
516
P31 63-62
556
REV 26-3
596
RT 100-17
636
RT 100-57
517
P31 63-63
557
REV 26-4
597
RT 100-18
637
RT 100-58
518
P6 10-1
558
REV 26-5
598
RT 100-19
638
RT 100-59
519
P6 10-2
559
REV 26-6
599
RT 100-20
639
RT 100-60
520
P6 10-3
560
REV 26-7
600
RT 100-21
640
RT 100-61
106
Pool 17
Pool 18
Pool 19
Pool 20
641
RT 100-62
681
TAT 21-2
721
VIF 42-21
761
VPR 20-19
642
RT 100-63
682
TAT 21-3
722
VIF 42-22
762
VPR 20-20
643
RT 100-64
683
TAT 21-4
723
VIF 42-23
763
VPU 17-1
644
RT 100-65
684
TAT 21-5
724
VIF 42-24
764
VPU 17-2
645
RT 100-66
685
TAT 21-6
725
VIF 42-25
765
VPU 17-3
646
RT 100-67
686
TAT 21-7
726
VIF 42-26
766
VPU 17-4
647
RT 100-68
687
TAT 21-8
727
VIF 42-27
767
VPU 17-5
648
RT 100-69
688
TAT 21-9
728
VIF 42-28
768
VPU 17-6
649
RT 100-70
689
TAT 21-10
729
VIF 42-29
769
VPU 17-7
650
RT 100-71
690
TAT 21-11
730
VIF 42-30
770
VPU 17-8
651
RT 100-72
691
TAT 21-12
731
VIF 42-31
771
VPU 17-9
652
RT 100-73
692
TAT 21-13
732
VIF 42-32
772
VPU 17-10
653
RT 100-74
693
TAT 21-14
733
VIF 42-33
773
VPU 17-11
654
RT 100-75
694
TAT 21-15
734
VIF 42-34
774
VPU 17-12
655
RT 100-76
695
TAT 21-16
735
VIF 42-35
775
VPU 17-13
656
RT 100-77
696
TAT 21-17
736
VIF 42-36
776
VPU 17-14
657
RT 100-78
697
TAT 21-18
737
VIF 42-37
777
VPU 17-15
658
RT 100-79
698
TAT 21-19
738
VIF 42-38
778
VPU 17-16
659
RT 100-80
699
TAT 21-20
739
VIF 42-39
660
RT 100-81
700
TAT 21-21
740
VIF 42-40
661
RT 100-82
701
VIF 42-1
741
VIF 42-41
662
RT 100-83
702
VIF 42-2
742
VIF 42-42
663
RT 100-84
703
VIF 42-3
743
VPR 20-1
664
RT 100-85
704
VIF 42-4
744
VPR 20-2
665
RT 100-86
705
VIF 42-5
745
VPR 20-3
666
RT 100-87
706
VIF 42-6
746
VPR 20-4
667
RT 100-88
707
VIF 42-7
747
VPR 20-5
668
RT 100-89
708
VIF 42-8
748
VPR 20-6
669
RT 100-90
709
VIF 42-9
749
VPR 20-7
670
RT 100-91
710
VIF 42-10
750
VPR 20-8
671
RT 100-92
711
VIF 42-11
751
VPR 20-9
672
RT 100-93
712
VIF 42-12
752
VPR 20-10
673
RT 100-94
713
VIF 42-13
753
VPR 20-11
674
RT 100-95
714
VIF 42-14
754
VPR 20-12
675
RT 100-96
715
VIF 42-15
755
VPR 20-13
676
RT 100-97
716
VIF 42-16
756
VPR 20-14
677
RT 100-98
717
VIF 42-17
757
VPR 20-15
678
RT 100-99
718
VIF 42-18
758
VPR 20-16
679
680
RT 100-100
TAT 21-1
719
720
VIF 42-19
VIF 42-20
759
760
VPR 20-17
VPR 20-18
107
APPENDIX IV: BENCH SET UP PBMC ISOLATION
APPENDIX V: STAINING OF THE CELLS
108
APPENDIX VI: ACQUIRING DATA ON THE LSR
APPENDIX VII: ANALYZING DATA ON THE LSR
109
APPENDIX VIII: CLIENTS DURING A FOCUS GROUP MEETING
APPENDIX IX: THE ENTIRE PROJECT
110
112