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. 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J Exp Med 198(12): 1909-1922. 84 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 92 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