UNIVERSITY OF GHANA EFFECT OF PARASITE DIVERSITY ON THE LEVELS AND QUALITY OF ANTIBODY RESPONSES TO PLASMODIUM FALCIPARUM IN AN AREA OF SEASONAL MALARIA TRANSMISSION BY ERIC KYEI-BAAFOUR (10168503) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON, IN PARTIAL FULLFILMENT OF THE REQUIREMENT FOR THE AWARD OF MASTER OF PHILOSOPHY (MPHIL) DEGREE IN ZOOLOGY JULY, 2015 University of Ghana http://ugspace.ug.edu.gh i DECLARATION I Kyei-Baafour Eric, hereby declare that except for references to other people’s work, which have duly been acknowledged, this thesis is the result of my own research conducted at the Immunology Department, Noguchi Memorial Institute for Medical Research, and at the Department of Animal Biology and Conservation Science, supervised by Prof. Ben A Gyan, and Dr. Kwadwo Asamoah Kusi both of Immunology Department, Noguchi Memorial Institute for Medical Research, and Dr. Langbong Bimi of the Department of Animal Biology and Conservation Science, University of Ghana. Neither all nor parts of this thesis have been presented for another degree elsewhere. Kyei-Baafour Eric _________________ _______________ (Candidate) SIGNATURE DATE Dr. Kwadwo Asamoah Kusi __________________ _______________ (Supervisor) SIGNATURE DATE Dr. Langbong Bimi _________________ _______________ (Supervisor) SIGNATURE DATE Prof. Ben Adu Gyan _________________ _______________ (Supervisor) SIGNATURE DATE University of Ghana http://ugspace.ug.edu.gh ii DEDICATION I dedicated this work to The Lord God Almighty. I also dedicate it to the three wonderful women in my life, my wife Sandra, my mother Alberta, and my daughter Elizabeth. University of Ghana http://ugspace.ug.edu.gh iii ACKNOWLEDGEMENTS I am highly indebted to God for how far he has brought me. I am indeed grateful for His mercies and favour. I am forever indebted to Prof. Ben Adu Gyan who used part of his project grant to pay my school fees. I am also grateful to my supervisors Dr. Kwadwo Asamoah Kusi, Dr. Bimi and Prof. Ben A. Gyan for their advice, constructive criticisms, and invaluable suggestions which propelled this work to its successful completion. Special thanks go to Dr. Kusi for having a big heart to contain all the troubles he had to go through with me in the course of this work. I thank him for his mentorship and guidance. I would like to thank Prof. Daniel Dodoo, the then head of Immunology Department, NMIMR, who encouraged me to apply for the M.Phil program. My special thanks also goes to Dr. Michael Ofori, Dr. Bright Adu, and Dr. Linda Amoah, all of the Immunology Department, NMIMR for their words of encouragement, and great suggestions. I would not have been able to complete this work without the invaluable contribution of Ms. Quratul-Ain Issahaque and Mr. Lukeman Osei Kwasi, national service personnel with the Immunology Department. May the Lord reward each of you greatly for all the sacrifices you made towards the completion of this work. I also express my highest appreciation to all my colleagues and staff of the Immunology Department-NMIMR, John, Helena, Alex, Owusu, and especially Kakra for the many ways they contributed to this work. I am deeply grateful to you all. Special thanks also go to Mr. Bernard Tonyigah, a fellow M.Phil student for the assistance he offered in performing PCR analysis. I would also like to thank my course mates for their support during the course work. University of Ghana http://ugspace.ug.edu.gh iv I also thank the Office of Research Support (ORS)-NMIMR, for providing financial assistance, through the post-graduate research scheme under the NMIMR Postdoctoral Programme, to purchase some of the reagents for this work. Finally, I am grateful to my wife Sandra, my mother Madam Alberta Afia Adu, and all my family members who in one way or the other helped me to complete my studies. University of Ghana http://ugspace.ug.edu.gh v Table of Contents DECLARATION .............................................................................................................. i DEDICATION ................................................................................................................. ii ACKNOWLEDGEMENTS ........................................................................................... iii Table of Contents ............................................................................................................. v List of Tables ................................................................................................................... ix List of Figures .................................................................................................................. x List of Abbreviations ....................................................................................................... xi ABSTRACT .................................................................................................................. xiv 1 CHAPTER ONE ....................................................................................................... 1 1.0 INTRODUCTION ............................................................................................. 1 1.1 Background ........................................................................................................ 1 1.2 Problem Statement ............................................................................................. 4 1.3 Justification: ....................................................................................................... 5 1.4 Main Objective: ................................................................................................. 6 1.4.1 Specific Objectives: .................................................................................... 6 CHAPTER TWO .............................................................................................................. 7 2.0 LITERATURE REVIEW .................................................................................. 7 2.1 Malaria: The disease .......................................................................................... 7 2.1.1 Brief History ............................................................................................... 7 2.2 The Parasite ....................................................................................................... 7 2.3 Life Cycle .......................................................................................................... 8 2.4 Malaria Situation ............................................................................................. 12 2.4.1 Global Malaria Situation .......................................................................... 12 University of Ghana http://ugspace.ug.edu.gh vi 2.4.2 Malaria in Ghana ...................................................................................... 12 2.4.3 Clinical manifestations of Malaria ........................................................... 14 2.4.4 Malaria Diagnosis, Prevention, and Treatment ........................................ 15 2.5 The Immune System ........................................................................................ 17 2.5.1 Immunity to Malaria ................................................................................. 17 2.5.2 Innate immunity ....................................................................................... 18 2.5.3 Acquired Immunity .................................................................................. 20 2.5.3.1 Cell-mediated immunity ................................................................... 20 2.5.3.2 Antibody-mediated immunity ........................................................... 22 2.6 Antigenic targets .............................................................................................. 25 2.6.1 Apical Membrane Antigen-1 (AMA1) ..................................................... 25 2.6.2 Merozoite Surface Protein 1-19 (MSP119) ................................................ 27 2.6.3 Cell-Traversal Protein for Ookinetes and Sporozoites (CelTOS) ............ 27 2.6.4 Circumsporozoite Protein (CSP) .............................................................. 28 2.7 Immune Evasion Mechanisms ......................................................................... 29 2.7.1 Antigenic Variation .................................................................................. 29 2.7.2 Allelic Polymorphism .............................................................................. 30 2.7.3 Intracellular Parasitism ............................................................................. 31 2.8 Strain Specific and Cross Reactive Immunity ................................................. 31 2.9 Multiplicity of Infection and Antibody Responses .......................................... 33 CHAPTER THREE ........................................................................................................ 35 3.0 MATERIALS AND METHODS .................................................................... 35 3.1 Ethics Statement .............................................................................................. 35 3.2 Study Area ....................................................................................................... 35 University of Ghana http://ugspace.ug.edu.gh vii 3.3 Study Design and Study Population ................................................................ 37 3.4 Sample Collection and Processing .................................................................. 37 3.5 Laboratory Measurements ............................................................................... 38 3.5.1 Parasitological Examination ..................................................................... 38 3.5.2 Malaria Antigens ...................................................................................... 38 3.5.3 Enzyme-linked Immunosorbent Assay (ELISA) ..................................... 41 3.5.3.1 Optimization and Standardization of ELISA .................................... 41 3.5.3.2 Determination of Total IgG levels .................................................... 41 3.5.4 Competition ELISA .................................................................................. 43 3.5.4.1 Determination of dilutions for Competition assay ............................ 43 3.5.4.2 Competition assay ............................................................................. 44 3.5.5 DNA Extraction ........................................................................................ 45 3.5.6 PCR Amplification ................................................................................... 46 3.5.7 Data Analysis ........................................................................................... 47 CHAPTER FOUR .......................................................................................................... 48 4.0 RESULTS ........................................................................................................ 48 4.1 Parasite Density and Proportions at the Sites .................................................. 49 4.2 Multiplicity of Infection and IgG Levels ......................................................... 50 4.3 Total IgG Levels in Plasma Samples between Sites ........................................ 52 4.4 Parasite Carriage and IgG Responses .............................................................. 54 4.5 Cross-reactive and Strain-specific Antibodies ................................................ 56 4.6 Cross Reactive and Strain Specific Responses at the Sites ............................. 60 CHAPTER FIVE ............................................................................................................ 63 5.0 DISCUSSION .................................................................................................. 63 University of Ghana http://ugspace.ug.edu.gh viii CHAPTER SIX .............................................................................................................. 67 6.0 Conclusions and Recommendations ................................................................ 67 6.1 Conclusions ..................................................................................................... 67 6.2 Recommendations ........................................................................................... 68 REFERENCE ................................................................................................................. 69 Appendixes ................................................................................................................... 100 7.1 Preparation of standard solutions and buffers. .............................................. 100 7.2 PCR Materials ................................................................................................ 102 University of Ghana http://ugspace.ug.edu.gh ix List of Tables Table 4. 1 Characteristics of study subjects for the Wet and Dry Seasons .................... 48 Table 4. 2 Proportion of individuals carrying parasites by microscopy ......................... 49 Table 4. 3 Proportion of individuals carrying parasites by PCR .................................... 49 Table 4. 4 Multiplicity of Plasmodium falciparum infection as assessed with MSP-2 marker ............................................................................................................................. 50 Table 4. 5 Mean multiplicity of Plasmodium falciparum infection between sites ........ 50 Table 4. 6 Residual anti-AMA1 IgG binding (minimum values) estimates for Adult and child plasma samples. ..................................................................................................... 59 University of Ghana http://ugspace.ug.edu.gh x List of Figures Figure 2. 1 Life cycle of Plasmodium species ............................................................... 11 Figure 3. 1 Map of Bongo District ................................................................................. 36 Figure 3. 2 Amino acid sequence alignment for the four AMA1 and three DiCo antigens ........................................................................................................................... 40 Figure 4. 1 Agarose gel photograph with a 100bp molecular weight DNA marker indicating multiple infection .......................................................................................... 51 Figure 4. 2 Comparison of anti-malarial IgG responses between the two sites. ............ 53 Figure 4. 3 Total IgG levels and parasitaemia between sites in the two seasons ........... 55 Figure 4. 4 Competition ELISA with plasma from a child and an adult ........................ 58 Figure 4. 5 Residual binding estimate for all competing antigens on 3D7 coated plates. ........................................................................................................................................ 61 Figure 4. 6 Residual binding estimate for all competing antigens on FVO coated plates. ........................................................................................................................................ 62 University of Ghana http://ugspace.ug.edu.gh xi List of Abbreviations ADCI Antibody Dependent Cellular Inhibition AMA1 Apical Membrane antigen-1 APC Antigen Presenting Cells AU Antibody units BC Before Christ CD4 Cluster of Differentiation 4 CD8 Cluster of Differentiation 8 CelTOS Cell Traversal Protein for Ookinetes and Sporozoites CSA Chondroitin Sulphate A CSP Circumsporozoite Protein DDT Dichlorodiphenyltrichloroethane DiCo Diversity Covering DNA Deoxyribonucleic Acid dNTP deoxynucleotide triphosphate EBA Erythrocyte Binding Antigen EGF Epidermal Growth Factor G-6PD Glucose-6-Phosphate Dehydrogenase GDP Gross Domestic Product GHS Ghana Health Service GMEP Global Malaria Eradication Program GPI Glycosylphosphatidyl inositols HbS Haemoglobin S HBs Hepatitis B surface antigen HLA Human Leukocyte Antigen IFN-γ Interferon gamma Ig Immunoglobulin IgA Immunoglobulin A IgD Immunoglobulin D IgE Immunoglobulin E University of Ghana http://ugspace.ug.edu.gh xii IgG Immunoglobulin G IgM Immunoglobulin M IL-1 Interleukin 1 IPTp Intermittent Preventive Therapy for pregnancy IRB Institutional Review Board iRBC infected Red Blood Cell IRS Indoor Residual Spraying kDa kilo Dalton LLIN Long Lasting Insecticidal Nets LSA Liver Stage Antigen mAB monoclonal antibodies MDA Mass Drug Administration MHC Major Histocompatibility complex MOI Multiplicity of Infection MSP Merozoite Surface Protein NHRC Navorongo Health Research Center NK cells Natural Killer cells nPCR Nested Polymerase Chain Reaction OD Optical Density PAM Pregnancy Associated Malaria PBMCs Peripheral Blood Mononuclear Cells PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction PfEMP1 Plasmodium falciparum erythrocyte membrane protein 1 RBC Red Blood Cell RBM Roll Back Malaria RDT Rapid Diagnostic Test RIFINs Repetitive Interspersed Family Protein STC Scientific and Technical Committee STEVOR Subtelomic Variable Open Reading frame University of Ghana http://ugspace.ug.edu.gh xiii SURFIN Surface-associated interspersed protein Tc cells T-cytotoxic cells TGF-β Transforming Growth Factor beta Th cells T-helper cells TNF-α Tumor Necrosis Factor Tregs T-regulatory cells UV Ultra violet VSA Variant Surface Antigen VSA Variant Surface Antigen WBC White Blood Cells WHO World Health Organization University of Ghana http://ugspace.ug.edu.gh xiv ABSTRACT The important role of antibody-mediated mechanisms in protection from clinical malaria has been demonstrated by passive transfer experiments but the targets of protective immunity are not clearly defined. A number of antigens are however in various stages of testing as possible vaccine candidates. Polymorphism in these antigens, which has been reported to be an immune evasion mechanism, has hampered the development of these antigens as vaccines since antibody responses against one allelic form of an antigen have been shown to be less effective against parasites that express a different allele of the same antigen. In animal studies, immunization with a mixture of allelic antigens induced cross-reactive antibodies that had greater and broader in vitro inhibition capacity compared to antibodies induced against the respective single antigens. This study therefore sought to determine the effect of parasite diversity on the levels and quality of antibody responses to P. falciparum in individuals living in an area of seasonal malaria transmission. Indirect ELISA was used to determine total IgG responses to AMA1-3D7, AMA1-FVO, MSP119, CSP, and CelTOS in stored plasma samples taken at two sites, one close to a dam and the other at least 20km away from the dam during the wet and dry season. Competition ELISA was used to determine the relative proportions of cross-reactive and stain-specific anti-AMA1 antibodies. Malaria parasites were detected in participant samples by both microscopy and molecular methods. The study found greater proportion of parasitaemic individuals at the dam site compared to those away from the dam during the dry season (p=0.0061), while proportions were similar in the rainy season. Generally, there were more multiple University of Ghana http://ugspace.ug.edu.gh xv infections per individual, described as the multiplicity of infection (MOI) in the wet season (60% of participants) compared to the dry season (40.3%, p=0.001). A similar trend was observed when MOI was compared between seasons for the non-dam site (p=0.001), but MOI was similar between the wet and dry seasons at the dam site Antibody levels to sporozoites antigens (CSP and CelTOS) were higher at the dam site compared to the non-dam site, irrespective of the season. No differences between sites were however observed for the blood stage antigens (AMA1 and MSP119). Antibody specificities to multiple AMA1 alleles were observed at sites with MOI greater than 1 and specificity to only the 3D7 allele was observed at sites with single infections. This data generally shows high levels of clinical immunity that is observed in high transmission areas may be associated more with infection by multiple parasite strains (hence a wider breadth of antibody responses) rather than high parasite burden. Consequently low levels of clinical immunity in low transmission areas may be the result of infection with one or a few parasite strains that may induce responses that is not as broad as is seen in high transmission areas. University of Ghana http://ugspace.ug.edu.gh 1 1 CHAPTER ONE 1.0 INTRODUCTION 1.1 Background Malaria is an infectious disease caused by a unicellular Haemosporozoan parasite of the genus Plasmodium, of which four species, namely P. vivax, P. malariae, P. ovale and P. falciparum cause disease in humans. Plasmodium knowlesi and P. cynomolgi which are known simian malaria parasites have recently been shown to also cause life- threatening human malaria (Cox-Singh et al., 2008, Luchavez et al., 2008, Ta et al., 2014). These parasites are transmitted to humans through the infective bite of a female Anopheles mosquito, which serves as the vector. The most severe forms of the disease such as cerebral malaria and severe malarial anaemia are however mainly associated with P. falciparum infection (Suh et al., 2004). High-risk groups include individuals with less developed immune systems, children under 5 years, and un-exposed travellers to malaria-endemic regions, pregnant women, especially during their first and second pregnancies, and those in endemic areas but are rarely exposed to infection(Miller et al., 1994). Infections with Plasmodium falciparum can cause a wide spectrum of illness ranging from apparently symptomless infections to severe forms and ultimately death. Some form of non-sterile clinical immunity is developed but only after repeated exposure to the parasite (Bull et al., 2002). About half of the world’s population is at risk of infection; with almost 198 million clinical cases, and about 584,000 deaths recorded worldwide in 2013 with P. falciparum infection accounting for about 91% of cases in sub-Saharan Africa (World Health Organization., 2014). Malaria is also a major cause of illness and death, University of Ghana http://ugspace.ug.edu.gh 2 particularly among children under 5 years of age, and primigravid pregnant women in Ghana accounting for about 34% of all outpatient illnesses, in 2010 (Ghana Health Service., 2010), with P. falciparum being the most prevalent parasite in Ghana. (Asante et al., 2011b, Ahmed, 1989). Current strategies to control malaria involve preventive measures that target the vector by the use of insecticides to spray breeding grounds, insecticide treated bed nets, and chemotherapeutic approaches where drugs are used to target the parasites. However the spread of drug-resistant parasite strains and insecticide-resistant mosquitoes have greatly hampered control efforts (Yadouleton et al., 2010, Marfurt et al., 2010), and a recent report linking climate change to the increased incidence of malaria in areas previously known to be malaria-free, such as higher altitudes, has also compounded the problem(Siraj et al., 2014). Vaccines are the most appropriate and cost effective means of controlling and eventually eliminating malaria as this has been used to effectively control or eliminate other diseases such as polio (Good, 2001), but this has not been successful due to a host of factors including: complex host-parasite interactions, polymorphism and antigenic variation, HLA restrictions, and the lack of suitable and potent adjuvants that would induce high-titre antibody responses(Good, 2001). In disease-endemic areas, immunity to malaria develops in a slow manner, with children under 5 years being very susceptible to clinical disease while adults beyond age 15 become semi-immune to malaria following repeated infection with the parasite. Immunity to malaria is mediated by components of both the innate and adaptive immune responses. The adaptive immune response is mediated by both cellular and University of Ghana http://ugspace.ug.edu.gh 3 antibody-mediated mechanisms and the crucial role of antibodies was demonstrated in passive transfer experiments that involved treating acutely sick individuals with immunoglobulins that had been purified from semi-immune adults (Cohen et al., 1961, McGregor, 1963, Sabchareon et al., 1991). Antibodies perform such roles as inhibition of red blood cell (RBC) invasion by merozoites, blocking cytoadherence of infected RBCs (iRBCs) to vascular endothelial cells, and enhancing phagocytic activity of monocytes and macrophages (Brown et al., 1982, Bouharoun-Tayoun et al., 1990, Wipasa et al., 2002a, Beeson et al., 2008). These antibodies are induced in response to parasite antigens, a number of which have been identified and are thought to be important targets of immunity. Some of these antigens include: Circumsporozoite surface protein (CSP), the Liver stage antigens (LSA), the Apical membrane antigen-1 (AMA1), the Merozoite surface proteins; MSP1, MSP2, MSP3, MSP4, MSP8, Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), Ring-infected erythrocyte surface antigen (RESA), Variant surface antigen (VSA), Erythrocyte binding antigen (EBA), and Plasmodium sp gametocyte surface antigens such as Pfg27, Pfs16, Pfs25 and Pfs230. An understanding of the mechanisms of acquisition of antibodies to these varied parasite antigens is essential for the development of interventions such as vaccines. While some field studies have found associations between antibodies to single antigens, or combinations of these antigens, with protection from clinical malaria (Riley et al., 1994, Dodoo et al., 1999, Cavanagh et al., 2004, Polley et al., 2006, Dodoo et al., 2008), other studies have failed to show these associations (Conway et al., 2000, Perraut et al., 2005). Thus the precise antigenic targets of protective immunity to malaria have not been properly defined and these conflicting findings though may University of Ghana http://ugspace.ug.edu.gh 4 reflect differences in design of these studies may also be attributable in part to allelic polymorphisms(Mackintosh et al., 2004). Antibody responses induced against one allelic form of a particular antigen have been shown to be less effective at limiting the multiplication of parasites that express a different allele of the same antigen (Supargiyono et al., 2013, Perraut et al., 2000). Allelic polymorphism is believed to be an immune evasion mechanism employed by parasites (Hisaeda et al., 2005, Ferreira and Hartl, 2007). Developing a broad based vaccine using the antigenic targets must take into consideration the strains circulating in a particular geographic area. Studies have shown individuals having high allele- specific responses, are mostly under 10 years of age (Cortes et al., 2005b). Another study showed greater antibody cross- reactivity in adults, reflecting a higher level of protective immunity in adults (Terheggen et al., 2014). Thus studying the induction of natural immune responses to these polymorphic antigens in individuals with varying levels of exposure to different parasite strains is necessary for shedding light on the acquisition of naturally acquired immunity to malaria. 1.2 Problem Statement Individuals in malaria endemic areas have partial immunity to malaria and this immunity is believed to develop rapidly in areas of high transmission but slow in low areas of transmission. Individuals living in low transmission areas are therefore more prone to clinical malaria attacks. Protection from blood stage infection is believed to be mediated mainly by anti-merozoite antibody responses and the functional quality of these responses is believed to be linked with polymorphism in the antigen targets and the frequency of exposure to parasites. It is generally believed that individuals University of Ghana http://ugspace.ug.edu.gh 5 accumulate a repertoire of malaria-specific antibodies with age and repeated exposure to different parasite strains over time. It is, however, unclear whether the antibody component of partial clinical immunity in high transmission areas is merely due to high parasite burden or is also dependent on the acquisition of broad antibody immunity to different strains of the parasite. 1.3 Justification: Allelic polymorphism represents a unique barrier to the development of broad acting subunit malaria vaccine since a potentially effective vaccine would have to induce broad acting immune responses that will be effective against the diversity of parasite strains that are known to circulate within any malaria endemic area. To be able to achieve this, a better understanding of the acquisition of broad clinical immunity especially in areas of high malaria transmission will be required Currently, obtaining a potent vaccine has been elusive and several factors have been adduced. One important factor is naturally occurring antigenic/allelic polymorphism which has been reported to be an immune evasion mechanism by the parasite (Ferreira and Hartl, 2007). Associating naturally acquired IgG responses to malaria specific- antigens and their alleles with protection from clinical malaria is a very crucial step towards vaccine production. Thus assessing antibody responses to these polymorphic antigens either individually or in combinations in relation to control of malaria infection and parasite multiplication is highly justified. Also, investigating the influence of parasite diversity on the levels of responses in populations with varying transmission intensity.is highly appropriate for vaccine formulators to determine the best strategies for formulating vaccines that can induce broad protective responses. University of Ghana http://ugspace.ug.edu.gh 6 1.4 Main Objective: The aim of this study was to investigate the effect of parasite diversity and disease transmission setting on the levels and quality of antibody responses to P. falciparum in individuals living in an area of seasonal malaria transmission. 1.4.1 Specific Objectives: i. To determine and compare specific IgG responses to four malaria antigens (CelTOS, AMA1, CSP and MSP119) in plasma samples collected in an area of seasonal malaria transmission. ii. To determine the association between anti-malarial antibody levels and corresponding blood parasitaemia. iii. To assess the effect of parasite diversity on the induction of cross-reactive and strain-specific anti-AMA1 antibodies at different transmission periods. University of Ghana http://ugspace.ug.edu.gh 7 CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 Malaria: The disease 2.1.1 Brief History The word malaria originated from the Italian word ‘mal’aria meaning ‘bad air’ because of its initial association with the smell from swamps. The disease has been known to humans for over 4000 years with the ancient Chinese describing some of the symptoms around 2700 BC in their writings. The Greek physician Hippocrates also noted most of the symptoms. Malaria devastated and caused decline in populations in many regions of the old world, until the medicinal Peruvian barks were used to treat fevers. The parasites that cause the disease were first discovered by Charles Alphonse Louis Laveran in 1880 (Cox, 2010, Cowman and Duraisingh, 2001). 2.2 The Parasite Malaria is a life threatening disease caused by a protozoan parasite of the genus Plasmodium. It is transmitted through the bite of the female Anopheles mosquito and characterized by fever with symptoms such as chills, headaches, malaise, vomiting, fatigue, and joint pains. These symptoms can result in many clinical manifestations ranging from asymptomatic infection through mild disease to severe complications such as cerebral malaria, and severe anaemia. Four Plasmodium species were originally known to cause malaria in humans. However it has recently emerged that two known simian parasites also cause the disease in humans, making a total of six Plasmodium species to cause human infections. These are Plasmodium falciparum, ovale, vivax, malariae, and the two simian parasites Plasmodium knowlesi and cynomolgi. (Ta et al., 2014, Cox-Singh et al., 2008, Luchavez et al., 2008). University of Ghana http://ugspace.ug.edu.gh 8 The most severe forms of the disease such as cerebral malaria and severe malarial anaemia are mainly caused by P. falciparum infection. However, the importance of the other species should not be underestimated, particularly P. vivax, which is the most widespread of the species in the world. Infection with P. vivax and P. ovale result in less severe symptoms. Relapses, however, can occur for up to three years, and chronic disease weakens the patient. The relapses occur due to the fact that the dormant liver stages (hypnozoites) of these parasites may reactivate. Plasmodium malariae is also widely distributed, but it is not as common as vivax malaria. Aside causing typical malaria symptoms during infection, P. malariae can also persist in the blood for very long periods, possibly decades, without ever producing symptoms. A person with asymptomatic P. malariae, however, can infect others, either through blood transfusion or mosquito bites. Two or more species of Plasmodium can infect a single individual at the same time (World Health Organization., 2010, Miller et al., 1994). 2.3 Life Cycle Plasmodium has a complex life cycle that involves interaction between the parasite, host, and vector. This cycle is made up of the sexual stage, which takes place in the mosquito, and the asexual stage, which occurs initially in the mosquito and continues in humans. Wipasa (Wipasa et al., 2002a), divided the stages in the human host into three categories namely: the pre-erythrocytic, erythrocytic and the gametocytic stages. The cycle in humans is initiated with the inoculation of sporozoites through the bite of the female Anopheles mosquito during feeding. Sporozoites migrate through the blood stream for some minutes before ending in the liver where invasion of liver cells (hepatocytes) is accomplished. Whilst in the liver they undergo asexual multiplication into schizonts. University of Ghana http://ugspace.ug.edu.gh 9 The erythrocytic stage begins with the bursting of schizonts out of hepatocytes as merozoites, to invade circulating erythrocytes or RBCs. The merozoites then develop into trophozoites within a vacuole formed by the internal membrane of the host red cell. The trophozoite feeds on haemoglobin by ingesting small amounts of red cell cytoplasm, which leads to formation of malaria pigment (haemozoin) as polymerized by-product of hemoglobin breakdown. RBC invasion begins by merozoites attaching themselves to specific receptors on the RBC membrane resulting in invagination of the membrane and causing the merozoites to move into the erythrocyte. While in the erythrocyte, the parasite resides in the parasitophorous vacuole and undergoes further development from the trophozoite stage to the schizont stage after many mitotic divisions. Each schizont releases up to 32 merozoites into the blood stream when the erythrocyte ruptures. Merozoites go through this intra-erythrocytic cycle by reinvading more erythrocytes when they escape the action of the immune system. This stage of the intra-erythrocytic cycle consists of asexual division, in which merozoites develop through ring, trophozoite and schizont stages, and gametogenesis where a small proportion of parasites differentiate into male and female gametocytes. Gametocytes can be taken up by the mosquito during a blood meal and differentiate into male and female gametes in the mid-gut of the mosquito. Then there is exflagellation forming zygote which further transforms into motile forms known as ookinetes. The ookinetes penetrate the epithelium of the gut and transform into oocysts in the epithelial lining. The oocysts contain large numbers of sporozoites and at maturity the sporozoites burst out and migrate to the salivary glands of the vector and can be transmitted to humans when the mosquito takes another blood meal. There are University of Ghana http://ugspace.ug.edu.gh 10 antigens produced by the parasite at every stage of the life cycle which have been identified as potential vaccine candidates. Though much progress has been made in the recent past to develop vaccines based on these identified candidates, for example the RTS’S vaccine, technical and immune- related difficulties have hindered vaccine development. Some of these difficulties include the cost of producing such vaccines, the complexity of the host parasite relationship, polymorphism and antigenic variation, and Human Leukocyte Antigen (HLA) restrictions where certain groups of individuals do not respond to some of these vaccine candidate proteins. Also the lack of suitable and potent adjuvants that would induce high-titre antibody responses is a major factor (Good, 2001). University of Ghana http://ugspace.ug.edu.gh 11 Figure 2. 1 Life cycle of Plasmodium species Source: (Winzeler, 2008) University of Ghana http://ugspace.ug.edu.gh 12 2.4 Malaria Situation 2.4.1 Global Malaria Situation Great efforts have been made over the last 60 years to combat the effect of malaria on human lives. According to a report published by the WHO (1956) malaria was controlled in the 1950s using the most potent tools available then: chloroquine, and dichlorodiphenyltrichloroethane (DDT) with considerable success. By the late 1970s malaria burden had considerably reduced (Wernsdorfer and Kouznetsov, 1980). However vector and parasite resistance to DDT and chloroquine, respectively, and non- commitment to fight the disease by countries in malarious areas led to a resurgence of the disease until the Global malaria control strategy ministerial conference was held in 1992 (Trape et al., 1998). Over the past decade, estimated malaria death rates have fallen by 42% in all age groups and 48% in children under 5 years of age (World Health Organization., 2011). In spite of these amazing achievements, about half of the world’s population is still at risk of the disease with about 207 million cases, a little over 660000 malaria attributable deaths recorded in 2013. Sub-Saharan Africa still bears considerable burden of the disease with about 80% of the clinical cases and 90% deaths occurring in the region. Most malaria deaths (about 70%) occur in children under 5 years of age and Plasmodium falciparum accounts for over 80% of malaria cases worldwide (World Health Organization., 2014). 2.4.2 Malaria in Ghana Malaria is a major cause of morbidity and mortality in Ghana, particularly among children under 5 years of age, and primigravid pregnant women, and the poor. In a study conducted in 2003 on the economic burden of malaria in Ghana, the disease was University of Ghana http://ugspace.ug.edu.gh 13 not only a health problem but a developmental as well as an economic problem. The financial hardships of the disease on households and the economy is enormous and the impact of the disease on real GDP growth is negative and decreases by -0.41% for every increase in malaria morbidity rate (Ankomah Asante and Asenso-Okyere, 2003). In 2010, malaria accounted for about 34% of all outpatient illnesses and about 37% of all hospital admissions. Malaria attributable deaths increased from 1.22% in 2009 to 1.44% in 2010, representing 19% of all deaths that were recorded. Infection rates in children are very high and peaks at about 80% in those between the ages of 5-9 years. Among pregnant women in 2006, as high as 13.7% of all admissions were as a result of malaria whilst 9% of them died from the disease (Ghana Health Service., 2010). Malaria is endemic throughout Ghana and varies with season being higher in the wet or rainy season where breeding of mosquitoes is favoured and lower in the dry season where breeding sites are few(Oduro et al., 2007, Owusu-Agyei et al., 2009). Ahmed (Ahmed, 1989) reported P. falciparum as the predominant cause of malaria in Ghana, accounting for over 90% of clinical cases. Another study in the Kassena Nankana District of northern Ghana showed that the disease accounted for about 41% of hospital deaths with parasitaemia around 71% in the high transmission season and 54.3% at the end of the low transmission seasons (Koram et al., 2000). Asante (Asante et al., 2011b) also reported 98.1% of parasites circulating in the middle forest belt of Ghana are P. falciparum. In a study conducted in the southern coastal savannah region, P. falciparum prevalence among pregnant women was 19.7% compared to the middle forest belt which had a prevalence of 35.1% (Ofori et al., 2009, Glover-Amengor et al., 2005). University of Ghana http://ugspace.ug.edu.gh 14 A study by Appawu and his group showed that Anopheles gambiae s.1 and Anopheles funestus constituted 94.3% of all the vectors sampled in the Kassena Nankana District in northern Ghana. They also reported the biting rates of the vectors to be about 36.7% bites per man per night in the irrigated areas and 5.2% in the non-irrigated lowland (Appawu et al., 2004). 2.4.3 Clinical manifestations of Malaria The clinical outcome of malaria depends on a host of factors including the parasite, host responses, geographic and social factors (Miller et al., 2002). Clinical manifestations of malaria cover a wide spectrum from when one is asymptomatic through mild disease to severe disease and eventually death, especially in young children, if untreated early and properly. An asymptomatic carrier could have the parasite in the blood for a long time without showing any symptom of the disease. Malaria infection can be categorized into two main groups based on the extent of disease severity. These are mild or uncomplicated malaria and severe malaria. Severe malaria includes cerebral malaria and severe malaria anaemia. Other forms of severe malaria include pulmonary edema, impaired consciousness, prostration, seizures, jaundice, haemoglobinuria; respiratory distress, (White, 2003). Uncomplicated malaria on the other hand present as mild non-specific febrile illness which can be resolved easily if appropriately treated. Some of the common symptoms of uncomplicated malaria are: common cold, chills, dizziness, abdominal discomfort, nausea, vomiting, headaches, cough, general body pains and mild diarrhea which can easily be treated. The other signs which may not be exclusive to malaria include: hepatosplenomegaly (enlargement of liver and spleen), pallor, orthostatic hypotension, tachycardia, jaundice (Murphy and Oldfield, 1996). However in severe malaria cases University of Ghana http://ugspace.ug.edu.gh 15 other symptoms are manifested such as: shock, acute renal failure, hypoglycaemia, metabolic acidosis, severe anaemia, multiple convulsions leading to coma. Still births, low birth weight, premature delivery, and miscarriages could also occur in pregnancy associated malaria (PAM) (Wipasa et al., 2002a). Disease symptoms are as a result of the exponential growth in the number of parasites in erythrocytes and the synchronous bursting of large numbers of these RBCs. These events release waste products of parasite metabolism and other pyrogens, and these are believed to trigger undesirable immune responses (Clark and Schofield, 2000, Good and Doolan, 1999). 2.4.4 Malaria Diagnosis, Prevention, and Treatment The ‘gold standard’ for diagnosing malaria is the use of light microscopy to observe Giemsa stained thick and thin blood films on a slide (Moody et al., 2000). Thick smear is used in identification of Plasmodium parasite whiles the thin smear is used for speciation. Though light microscopy is the ‘gold standard and recommended by WHO, accuracy depends on the quality of smear, and the competency level of the laboratory personnel conducting the test. Other diagnostic methods include the use of the rapid diagnostic test (RDT), florescent microscopy where the nuclei of the parasites are stained with fluorescent dye, and polymerase chain reaction (PCR) assay which is the most sensitive of the methods but expensive (Hanscheid and Grobusch, 2002). The discovery of the mode of transmission of malaria by Roland Ross and Batista et al led to various intervention programs to control and eliminate the disease. Most of these programs were aimed at vector elimination. Large scale vector control and mass drug administration programs were used in controlling the disease because of its success in controlling the disease during the construction of the Panama Canal (Najera et al., University of Ghana http://ugspace.ug.edu.gh 16 2011). The lunch of the Global Malaria Eradication Program (GMEP) in the mid-1950s succeeded in eliminating or reducing the burden of malaria in Europe, the Caribbean, North America, South Central America and some parts of Asia, but little success was achieved in Sub-Saharan Africa. The program was however discontinued due to a myriad of problems (Carter and Mendis, 2002, Lopez et al., 2006). The Roll Back Malaria program RBM launched in 1998 with the Global Malaria Action plan was to reduce malaria deaths to near zero, reduce global malaria cases by 75% and eliminate malaria in ‘new’ countries by the year 2015. Two main components of the control programme were: Vector control and chemotherapy. Vector control programs include the use of Long Lasting Insecticidal Nets (LLINs) and Indoor Residual Spraying (IRS). These two methods are the core of the vector control programs (World Health Organization., 2014). The second component of the control program is the use of potent drugs to reduce parasite numbers in the blood. The introduction of the intermittent preventive therapy for pregnant women (IPTp), for example, has helped to significantly reduced neonatal deaths resulting from malaria in pregnancy by about 61.3% (Menendez et al., 2010). Malaria treatment has faced some challenges such as resistance to the available antimalarial drugs. The major causes of resistance are the long term and inconsistent use of antimalarial drugs. WHO now recommends the use of Artemisinin-based combination drugs as the first line drugs to treat P. falciparum. However chloroquine is recommended to be used in areas where it is still effective to treat P. vivax. (World Health Organization., 2014) University of Ghana http://ugspace.ug.edu.gh 17 2.5 The Immune System The immune system comprises a network of specialised cells and organs that work together to protect the body from attack by ‘foreign’ substances including disease causing microbes such as viruses, bacteria, fungi, parasites, and other environmental substances. This complex system is highly organized and can recognise and destroy millions of microbes. The immune system is classified into innate and acquired systems. The innate system is made up of all those elements with which one is born including the skin barrier, mucous membranes, phagocytic cells like the neutrophils and macrophages. Other components of the innate immune system are NK cells and inflammatory responses. The body’s chemical environment such as pH and secreted fatty acids also forms part of the innate immune system. Acquired immunity is specific and can further be classified into humoral and cell-mediated system. The humoral system defends against infections in most body fluids whiles the cell-mediated arm uses cytotoxic lymphocytes to fight and kill infected cells. There is also the complement system which has components of both the innate and acquired immune systems and has three different pathways of activation to help fight infections. These are the classical, alternative, and lectin pathways. Both vertebrates and invertebrates depend on the innate immune system whilst only vertebrates use the adaptive system to fight against infections. (Coico and Sunshine, 2009). 2.5.1 Immunity to Malaria Immunity to malaria is the body’s ability to resist the disease. Studies have found that adults living in highly endemic areas have partial immunity to malaria though some may carry parasites in their blood (Druilhe and Khusmith, 1987). While some studies University of Ghana http://ugspace.ug.edu.gh 18 have found this immunity to be transferred to infants from their mothers from birth to about six months (Brabin et al., 1990, Klein Klouwenberg et al., 2005, Larru et al., 2009), others found no such association (Riley et al., 2000). Individuals living in malaria endemic areas over a period of time acquire immunity to malaria due to their frequent and repeated exposure to the parasites (Snow et al., 1997, Gupta et al., 1999, Marsh and Kinyanjui, 2006). Children living in malaria-endemic areas attain partial immunity against severe malaria as they grow, although they still suffer from uncomplicated malaria. Immunity to clinical disease in adults is believed to be partial and short-lived, and maintenance of this immune status requires the continuous presence of very low levels of parasites (Wipasa et al., 2002a, Doolan et al., 2009), a phenomenon known as premunition. Though mechanisms underlying protective immunity to parasite infection and clinical malaria are not fully understood in humans, studies in animal models and humans points to the involvement of both cellular and antibody mediated mechanisms(Good and Doolan, 1999, Doolan et al., 2009). In effect both innate and acquired immune mechanisms are mediators of host responses to infection with malaria (Bouharoun- Tayoun et al., 1995). 2.5.2 Innate immunity Innate immunity to malaria is the inherent or natural resistance to the disease. This form of immunity is the first line of defense against any infection and can be found in most vertebrates. Studies have found innate responses to malaria are very essential in limiting initial parasite replication in mice while in humans parasite growth can be modulated early in primary infections (Fell and Smith, 1998, Molineaux et al., 2002). University of Ghana http://ugspace.ug.edu.gh 19 Innate immunity can be achieved either by genetic, or cell-mediated immunologic mechanisms. Numerous genetic factors play major roles in innate resistance to malaria and this mostly affect the erythrocytes which are critical for parasite development. Some of these genetic factors include Sickle Cell trait (HbS) (Allison, 1954, Friedman, 1978, Aidoo et al., 2002) , Glucose-6-Phosphate Dehydrogenase (G-6PD) deficiency (Smith et al., 2002, Roth et al., 1983), and thalassaemias (Allen et al., 1997, Smith et al., 2002). These genetic factors confer some level of protection against malaria though they are usually seen as inherited genetic disorders of the hemoglobin generally referred to as haemoglobinopathies. These disorders, mostly found in individuals living in malaria endemic regions are usually associated with the development of erythrocytes, thus affecting parasite growth (Smith et al., 2002). Generally, erythrocytes of genetically protected individuals (individuals with sickle cell, alpha- and beta- thalassemia and G6PD deficiency) are vulnerable to repeated oxidation due to the release of H2O2 into the erythrocyte by P. falciparum which leads to an irreversible interaction between the oxidized haemoglobin and the red cell membrane. It is suggested that the irreversible oxidation could trigger mechanisms that reduce invasion of RBCs by the falciparum parasite, impair parasite survival and development within the cell, and accelerate infected erythrocyte clearance by phagocytosis (Destro Bisol, 1999). The cell-mediated immunologic mechanisms include phagocytosis of parasitized RBCs and merozoites by macrophages and neutrophils. Some of these immune cells release cytotoxic molecules such as cytokines, chemokines, and anti-parasite molecules such as nitric oxide that kill parasites, and others that help to opsonise parasitized cells. Some parasites are also cleared from the blood stream by the innate immune system (Smith et University of Ghana http://ugspace.ug.edu.gh 20 al., 2002, McGilvray et al., 2000). It has been demonstrated in vitro that components of blood-stage parasites, which include parasite-derived glycosilphosphatidylinositols (GPI), induce macrophages to produce IL-1, IL-6, and TNF-α (Scragg et al., 1999, Tachado et al., 1997). In addition some macrophages have also been shown to have the ability to present malaria antigens to T cells (Quin et al., 2001). Other innate mechanisms are the activation of the complement system through an alternative or the mannose-binding protein pathway and the cytotoxic activity of natural killer (NK) cells on host infected target cells (Fearon and Locksley, 1996). 2.5.3 Acquired Immunity 2.5.3.1 Cell-mediated immunity Though it has been establish that both humoral and cellular mechanisms contribute to the control of blood stage parasitaemia, the acquisition and maintenance of protective immunity depends largely on T-cell responses (Troye-Blomberg, 1994). There are two subpopulations of T-cells: T-helper (TH), and T-cytotoxic (TC). T helper and T cytotoxic cells can be separated from one another by the presence of either CD4+ or CD8+ glycoprotein receptors on their surfaces. T cells displaying the CD4 receptor generally function as helper T cells, whereas those displaying the CD8 receptor are cytotoxic T cells. Responses of both CD4+ and CD8+ T-cells are elicited on encountering parasite antigens in the precise major histocompatibility complex (MHC) context, by antigen presenting cells (APCs) such as dendritic cells, and macrophages. Such responses are very vital in the killing of parasites, and or inhibition of their growth (Chemtai et al., 1984, Good and Doolan, 1999, Frevert and Nardin, 2008). University of Ghana http://ugspace.ug.edu.gh 21 There are also different groups of CD4+ T-cells: T-helper 1 (Th1), and T-helper 2 (Th2) and this is based on the type of regulatory functions and the type of cytokines they produce. The Th1 cells activate cells such as macrophages to release inflammatory cytokines like TNF-α, IFN-γ and interleukin 2 (IL2), and are thus responsible for cell- mediated immunity. Th2 cells on the other hand produce IL10, IL4, IL5, IL6, and IL3 which aid the development of humoral immunity by activating B-cells to differentiate, proliferate and produce antibodies (Mosmann and Coffman, 1989, Abbas et al., 1996). In early acute infection with P. chabaudi, the prime cytokine produced is IFN-γ, a Th1 cytokine, and this has been shown to decline with decreasing parasitaemia and then replaced with Th2 cytokines such as IL10 and IL 4 in the later stages (Stevenson and Tam, 1993). This suggests that Th1 cells producing IFN-γ and IL-2 are important for controlling infection in its early phases, while Th2 cells, producing IL-4 and IL-10, together with antibodies, are essential for parasite clearance in later phases of infection (Troye-Blomberg et al., 1994). Thus both Th1 and Th2 T-cells play important roles in immunity to malaria at different stages of the disease and the balance between these two subsets is critical for the outcome of an infection (Wipasa et al., 2002b). Also, cytokines such as TNF-α, and IFN-γ may be produced on antigen stimulation, and may have anti-parasitic activity (Playfair and Taverne, 1987, Mordmuller et al., 1997). CD4+ T-cells play more important role than CD8+ though both carry αβ T cell receptors thus making role of CD8+ T cells in blood stage infections limited since antigen presentation cannot be done as the red blood cells carrying the parasites do not express MHC (Perlmann et al., 1998). Aside the crucial role played by CD4+ T-cells to protect the host by controlling parasitaemia, they can also be detrimental to the host (Amante and Good, 1997, Hermsen et al., 1998). University of Ghana http://ugspace.ug.edu.gh 22 Another important group of immune effector cells are the regulatory T cells (Tregs). These cells have been found to regulate cell mediated immunity in rodent malaria (Vigario et al., 2007, Amante and Good, 1997). High numbers of Tregs have been observed in the skin in the study state suggesting that Plasmodium specific Tregs are induced during skin stage infection (Guilbride et al., 2010). Inducible T regulatory cells are produced in the periphery and mainly induce the production of IL-10, IL-35, and TGF-β (Collison et al., 2007, Sakaguchi et al., 2009). Gamma delta T-cells (γδ) are T cells that have been demonstrated to inhibit parasite growth as their numbers increase in the first few days during malaria infection (Farouk et al., 2004), and that mice lacking γδ cells have been found to develop chronic parasitaemia following P. c. chabaudi infection (Seixas and Langhorne, 1999). Gamma delta (γδ) T-cells isolated from PBMCs from non-immune individuals inhibit parasite growth in vitro, and the number of γδ T-cells in the culture correlates with their inhibitory activities (Troye-Blomberg et al., 1999). 2.5.3.2 Antibody-mediated immunity Antibodies, the effectors of humoral immunity, are soluble proteins that are secreted by plasma cells that differentiate when B-cells are stimulated. Antibodies belong to a family of proteins called immunoglobulins (Igs) which have two distinct types of polypeptide chains; light and heavy chains, linked by disulphide bonds. There are five different types of immunoglobulins based on the type of heavy chain they possess, namely IgG, IgA, IgD, IgM, and IgE. IgG is further divided into subclasses: IgG1, IgG 2, IgG3, IgG4. Studies have shown that antibodies induced by malaria infections are mostly IgM, IgG and IgA (Targett, 1970). IgM is first produced during the primary phase of the immune University of Ghana http://ugspace.ug.edu.gh 23 response and is usually short-lived. In secondary responses, class-switching events lead to the production of high affinity IgG, though other antigens such as IgA and IgE are produced in the later stages of the disease (Collins et al., 1971). The main humoral mediators of protection from malaria are IgG. Anti-malaria antibodies, especially of the IgG isotype, have been shown to be very critical in controlling blood stage infections and symptoms of malaria. These protective antibodies are thought to target mostly merozoite surface antigens, erythrocyte invasion ligands and variant surface antigens expressed by P. falciparum infected erythrocytes (Bull et al., 1998, Good and Doolan, 1999). This has been demonstrated by the passive transfer of antibodies from adults who are semi-immune to malaria to treat clinically ill children (Cohen et al., 1961). In another study, pooled IgG from individuals living in various endemic areas in Africa was able to reduce parasitaemia and fever in patients with drug-resistance malaria (Sabchareon et al., 1991). Antibodies to merozoite antigens are believe to act by inhibition of merozoite invasion into new erythrocytes (Dutta et al., 2003a), blocking cytoadherence of infected RBCs (iRBCs) to endothelial cells (Singh et al., 1988, Taylor et al., 1992), complement- mediated opsonisation of infected erythrocytes (Groux and Gysin, 1990), enhancing phagocytic activity of monocytes and macrophages (Bouharoun-Tayoun et al., 1990), and complement blocking of erythrocyte invasion and erythrocyte stage replication of parasite (Boyle et al., 2015). Antibodies have also been found to neutralize parasite toxins (Schofield et al., 2002). It has also been shown that for individuals living in malaria endemic areas, naturally acquired immunity is largely dependent on the acquisition of a repertoire of specific antibodies directed against P. falciparum erythrocyte membrane protein-1(PfEMP-1) that are expressed on the infected RBC University of Ghana http://ugspace.ug.edu.gh 24 surface (Bull et al., 1998). The magnitude of protective immunity found in humans (Chizzolini et al., 1988, Piper et al., 1999), and monkeys (Egan et al., 2000) have been shown to correlate with the level of antibodies against asexual blood stage antigens, and are antibody isotype dependent. The IgG1 and IgG3 subclasses of IgG are cytophilic antibodies that are important in protective immunity against malaria (Bouharoun-Tayoun and Druilhe, 1992). It has also been reported that these cytophilic antibodies, which are the main effectors of Antibody Dependent Cellular Inhibition (ADCI), activates monocytes to secret soluble factors such as nitric oxide and cytokines such as TNF-α to inhibit parasite growth (Bouharoun-Tayoun et al., 1995). While some studies have found IgG2 to be protective (Aucan et al., 2000), others have found IgG2 and IgG4 to inhibit the opsonizing activity of IgG1 and IgG3 (Groux and Gysin, 1990). It has been reported that high levels of anti-malarial antibodies against pre-erythrocytic antigens are associated with reduced risk of developing malaria in endemic areas (John et al., 2005). Also several studies have associated high levels of anti-malarial antibodies to blood stage antigens and reduced risk of infection (Riley et al., 1992, Theisen et al., 1998, Dodoo et al., 2008, Dodoo et al., 2000). It has also been reported that antibodies whether acquired naturally, or through vaccination, are short lived (Maple et al., 2000). Immunity to malaria is parasite and strain specific, and clonal antigenic variation is common in P. falciparum (Hommel et al., 1983). The mechanism behind this antigenic variation is still not clear, but one very likely hypothesis is that there is a frequent on-going switching of variant surface antigens (VSA) in the parasite population, and an outgrowth of subpopulations with newer VSAs would occur when elicited antibodies are mainly against the other VSAs previously presented. The University of Ghana http://ugspace.ug.edu.gh 25 importance of VSAs in immunity can be shown by correlating the range of different anti-VSA antibodies to protection (Hviid, 2005). Pregnancy associated malaria (PAM) is caused by accumulation of iRBCs in the placenta. These parasites express a specific VSA that binds to chondroitin sulphate A (CSA), and the immune responses that are induced are parity dependent (Salanti et al., 2004). 2.6 Antigenic targets Antibodies have been shown to be crucial in acquired protective immunity to malaria. During the developmental stages of P. falciparum the parasites express certain proteins or antigens on their surfaces. Antibodies to these antigens confer protection through a variety of mechanisms (Groux and Gysin, 1990, Dutta et al., 2003a, Taylor et al., 1992, Singh et al., 1988, Theisen et al., 1998, Schofield et al., 2002). A number of these target antigens have been identified including: 2.6.1 Apical Membrane Antigen-1 (AMA1) AMA1 is one of the leading erythrocytic stage malaria vaccine candidates. It plays a crucial role in the invasion of erythrocytes and has also been found in the sporozoite and liver stages of the parasite (Silvie et al., 2004). AMA1 is a merozoite protein located in the micronemes of apicomplexans parasites (Remarque et al., 2008a). It is an integral membrane protein with a large N-terminal cysteine-rich ectodomain, followed by a single transmembrane domain and a short C-terminal cytoplasmic tail. Plasmodium falciparum AMA-1 is made of about 622 amino acids and is divided into three domains characterized by eight intra-domain disulphide bonds (Hodder et al 1996). With molecular weight of about 83 kDa, AMA1 is cleaved at the N-terminus yielding a mature protein of approximately 66 KDa (Narum and Thomas, 1994, Howell et al., University of Ghana http://ugspace.ug.edu.gh 26 2001). This 66 kDa AMA1 translocates to the merozoite surface and is cleaved proteolytically at one of two alternative sites resulting in two soluble fragments of 44 kDa and 48 kDa. The importance of the proteolytic processing is unknown, but it is suggested that it may be required for AMA1 to move to the parasite surface or to play its mediatory role in invasion or both (Howell et al., 2001). AMA1 has been shown to be essential for the survival of Plasmodium species within their host (Remarque et al., 2008a). AMA1 appears to play a vital role in erythrocyte invasion by participating in the re-orientation and attachment of the merozoite to the host erythrocyte cell surface (Triglia et al., 2000, Mitchell et al., 2004). Antibodies against AMA1 have been shown to inhibit merozoite invasion of erythrocytes in both in vitro and in vivo studies and also confer protection against parasite strains expressing the vaccine AMA1 allele in active immunization studies (Mitchell et al., 2004, Dutta et al., 2003b). The inhibitory effects are essentially species-specific with a large component of protection being strain-specific (Crewther et al., 1996). But to induce parasite-inhibitory activity AMA1 has to be folded correctly (Salvatore et al., 2002). Naturally acquired antibodies to AMA1 have been found to be associated with protection from clinical malaria in a population in coastal Kenya (Osier et al., 2008). AMA1 is highly polymorphic (Thomas et al., 1990, Remarque et al., 2008a) and this polymorphism is not due to repetitive sequence but is mainly as a result of single amino acid substitutions (Chesne-Seck et al., 2005). Animal studies have shown that antibodies to AMA1 obtained from one strain of Plasmodium inhibits the growth of a homologous strain effectively but inhibits other strains to various lesser degrees (Kennedy et al., 2002, Kocken et al., 2002, Kusi et al., 2009, Healer et al., 2004). University of Ghana http://ugspace.ug.edu.gh 27 2.6.2 Merozoite Surface Protein 1-19 (MSP119) MSP119 is located on the surface of the blood-stage merozoites and is first synthesized as a 200 kDa protein (MSP1) during blood stage schizogony. The various molecular weights that it is processed into include the C-terminal 42 kDa fragment. Majority of the fragments are chopped off before they invade erythrocytes (Holder et al., 1987). A 33kDa protein is processed from the C-terminal 42 kDa, which is further shed into a relatively conserved 19 kDa protein, known as the MSP119 (Holder et al., 1992). MSP119 is anchored to the merozoite membrane by glycosylphosphatidylinositol (GPI) (Blackman et al., 1996). There are 2 epidermal Growth Factor (EGF)-like domains, which may play critical role in merozoite invasion of erythrocytes (Holder et al., 1992). MSP119 has been shown to be an important malaria vaccine candidate and invasion of erythrocytes by parasites have shown to be blocked by this fragment and also inhibit parasite multiplication inside the erythrocyte (Hogh et al., 1995, Holder et al., 1992, O'Donnell et al., 2001). Although MSP1 has been found to be protective, others have found no association of MSP1 with protection from clinical malaria and also control parasite replication (Shi et al., 2007, Dodoo et al., 1999) 2.6.3 Cell-Traversal Protein for Ookinetes and Sporozoites (CelTOS) CelTOS is a 25kDa protein that is present in both the mosquito and human stages of the parasite and is localized to the micronemes of the sporozoite (Kariu et al., 2006). The protein is required by the parasite for motility and invasion of hepatocytes. CelTOS is a highly conserved protein and anti-PfCelTOS immune response were found to be protective against challenge from P. berghei (Bergmann-Leitner et al., 2010). Antibodies to CelTOS in mice have been shown to inhibit sporozoite motility and invasion of hepatocytes in vitro, and were also found to induce sterile protection in test animals (Bergmann-Leitner et al., 2011). CelTOS has also been found to induce cell- University of Ghana http://ugspace.ug.edu.gh 28 mediated immune responses where CelTOS-specific peptides stimulated high IFN-γ responses in PBMCs (Doolan et al., 2003). 2.6.4 Circumsporozoite Protein (CSP) CSP is the most abundant protein found on the surface of Plasmodium sporozoites and is essential to the survival of Plasmodium parasites as it is required for the development of infectious sporozoites in mosquitoes (Menard et al., 1997). The CSP of the 3D7 P. falciparum strain has 397 amino acids and is the most advanced and well documented of all the pre-erythrocytic vaccine candidates (Girard et al., 2007). CSP contains two major B cell epitopes consisting of tandem repeats. The most clinically advanced malaria vaccine, RTS, S, is a subunit vaccine consisting of the central repeat and C terminal regions of P. falciparum CSP fused to the hepatitis B surface antigen and co- expressed with free hepatitis B surface antigen to form a hepatitis B surface (HBs) Ag- like particle (Gordon et al., 1995). The results from phase III testing of the RTS, S vaccine has reported between 30-55% efficacy (Abdulla et al., 2013, Agnandji et al., 2011, Asante et al., 2011a). Although antibodies have been thought to be very important in protection from malaria, there is evidence to suggest that not all antibodies are protective. It has been reported that monoclonal antibodies (mAb) against MSP1 19 which inhibit RBC invasion by merozoites and prevent MSP-1 secondary processing, can be blocked by other mAb to the same antigen (Patino et al., 1997). Also polyclonal antibodies specific to MSP2, but not mAb specific to the same antigen, enhanced invasion of multiple merozoites into RBC (Ramasamy et al., 1999). These findings underscore the importance of finding epitopes that induce protective antibodies when designing a vaccine against malaria. University of Ghana http://ugspace.ug.edu.gh 29 2.7 Immune Evasion Mechanisms 2.7.1 Antigenic Variation Antigenic variation is the change in antigenic phenotype by regulated expression of different genes of a clonal population of parasites over the natural course of an infection (Doolan et al., 2009). Antigenic differences between parasite populations was first detected when mice harbouring chronic infections with P. berghei were found to be more susceptible to challenge with relapse parasites than to use the original parasite population (Cox, 1959). Another study with P. knowlesi showed a succession of antigenic variants with chronic infection of the erythrocytes (Brown and Brown, 1965). Plasmodium parasites evade the host immune system by varying the antigenic characters of the infected erythrocytes (Doolan et al., 2009). This evasion is achieved by having a large family of Plasmodium falciparum genes called var genes which encode a parasite derived protein called Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP-1) on the surface of infected erythrocytes. PfEMP-1 is a variant protein of approximately 300 kDa, expressed on the surface of RBC infected with late stage parasites and is coded for by approximately 50 genes of the var multigene family (Su et al., 1995, Smith et al., 1995). Antigenic variation of PfEMP1, through switching of the expression of different var genes, facilitates evasion of host immune responses, and specific variants of PfEMP1 have been found to mediate adhesion to chondroitin sulphate A (CSA) in pregnant women and to intercellular adhesion molecule 1 (ICAM1) in brain microvasculature (Gamain et al., 2004). PfEMP-1 has been implicated as a key target of naturally acquired immunity to malaria. University of Ghana http://ugspace.ug.edu.gh 30 Several studies have reported association between increased episodes of clinical malaria and a transient increase in the level of specific antibodies to variant surface antigens (VSAs) (Marsh and Howard, 1986, Giha et al., 1999). Studies have also found an association between the acquisition of antibodies to VSAs, which is age-dependent, and a decline in parasite density (Piper et al., 1999). In Ghana anti-VSA IgG levels have been correlated with protection from clinical malaria (Dodoo et al., 2001, Ofori et al., 2002). Reports have also been made from other epidemiological settings of correlation between anti-VSA antibody levels and protection from clinical malaria (Kinyanjui et al., 2004, Yone et al., 2005). As a result, clinical immunity probably develops once an individual acquires antibodies against multiple PfEMP1 variants in a natural setting, which may partly explain why natural immunity takes several years to develop (Wipasa et al., 2002a). VSAs have thus been proposed for malaria vaccine development (Chen, 2007). Other important antigenically variable antigens: are repetitive interspersed family proteins (RIFIN) (Kyes et al., 2001), Subtelomeric variable open reading frame proteins (STEVOR) (Cheng et al., 1998, Kaviratne et al., 2002), and surface-associated interspersed gene family proteins (SURFIN) (Winter et al., 2005). 2.7.2 Allelic Polymorphism Another immune evasion mechanism employed by Plasmodium parasites is allelic polymorphism. Polymorphisms are caused by variations in the sequence of the short tandem repeats, which is a characteristic feature of many malaria antigens and frequently constitute immunodominant regions (Bolad and Berzins, 2000). Studies have discovered that polymorphisms also result from point mutations in several antigens (Bull et al., 1999). University of Ghana http://ugspace.ug.edu.gh 31 Polymorphism has resulted in a situation where antibody responses induced against one allelic form of a particular antigen have been shown to be less effective at limiting the multiplication of parasites that express a different allele of the same antigen (Supargiyono et al., 2013, Perraut et al., 2000). For example MSP-1 is a leading malaria vaccine candidate antigen and is highly polymorphic (Qari et al., 1998), thus some antibodies to one MSP-1 allele may not fully recognize the other MSP1 alleles (Burns et al., 1989). Also, polymorphism severely affects T-cell recognition (Plebanski et al., 1999). T-cell recognition primarily depends on the amino acid sequence rather than protein conformation, which is often recognized by antibodies (Plebanski et al., 1999). The high genetic diversity in the Plasmodium parasite, mostly with the expression of variant surface antigens poses a considerable challenge to vaccine formulators. Most of the P. falciparum antigens currently under consideration for vaccine development have exhibited extensive polymorphisms in field isolates (Escalante et al., 1998, Barry et al., 2009). 2.7.3 Intracellular Parasitism Plasmodium parasites have evolved in deploying intracellular parasitism as a mechanism to escape host immune reaction. Anti-malarial antibodies that bind to free sporozoites or merozoites may not be able to access them once the parasites enter the host cells. Moreover, RBCs do not express MHC molecules on their surfaces thus merozoites escape recognition by CD8+ T cells (Hisaeda et al., 2005) 2.8 Strain Specific and Cross Reactive Immunity Strains results from allelic polymorphisms within a single parasite specie giving rise to different genotypes (McKenzie et al., 2008). Allelic polymorphisms in certain protein University of Ghana http://ugspace.ug.edu.gh 32 loci which produce antigenically different forms of the protein in different parasite strains underlie the concept of strain-specific immunity (Day and Marsh, 1991). Studies with animal models indicated that immunity to malaria is parasite strain- specific (Hommel et al., 1983, Jones et al., 2001). It has been shown in humans that a primary infection by one parasite strain elicited an immune response which was capable of protecting against that strain but not against infection by a different strain (Jeffery, 1966). In Aotus monkeys, repeated infections induced increasingly more rapid sterile immunity to homologous challenge (Jones et al., 2000). According to (Doolan et al., 2009), when immunity is induced experimentally using a homologous strain, it is very rapid but in areas of malaria endemicity, immunity is slow due to the challenge by different or heterologous strains. However in natural populations where malaria is endemic, significant strain-specific and cross-reactive inhibition of parasite multiplication was observed when homologous and heterologous sera from children were assessed for any inhibitory effects on parasite growth (Wilson and Phillips, 1976). In animal model experiments, it was established that immune sera from Aotus monkeys contained antibodies that blocked or reversed cytoadherence in vitro and were isolate-specific (Udeinya et al., 1983). Also, chronic infections of RBCs with P. knowlesi were shown to consist of a succession of antigenically distinct strains (Brown and Brown, 1965), and sera from rhesus macaque monkeys immune to one P. knowlesi strain did not react with cells infected with another strain (Brown et al 1968). Newbold (Newbold et al., 1992) showed using field isolates and laboratory clones that the predominant agglutinating antibody response in humans was variant-specific and that cross-reactive antibodies to different serotypes University of Ghana http://ugspace.ug.edu.gh 33 were rare. It was however demonstrated in a hyperendemic area in India that cross- reactive antibodies against VSA can be found in adults (Chattopadhyay et al., 2003). Also sera from children living in malaria endemic areas of different geographic regions did not agglutinate cells infected with different strains in agglutination assays. Sera from immune adults contained antibodies that cross-reacted by agglutination majority or all of the different isolates and also reacted with infected cells from most children (Marsh et al., 1986, Forsyth et al., 1989). Also Cortes (Cortes et al., 2005a), found significant allele-specific antibodies in children than in adults while (Terheggen et al., 2014) showed greater antibody cross- reactivity in adults, reflecting a higher level of protective immunity in adults. The passive administration of IgG from immune adult West Africans to children resident in East Africa, or Thailand, demonstrated the importance of cross-reactive antibodies in reducing parasitaemia (Cohen et al., 1961, McGregor, 1963, Sabchareon et al., 1991). 2.9 Multiplicity of Infection and Antibody Responses Malaria parasites have been found to be highly diverse genetically and that an individual may carry different parasite clones at a given time (Babiker et al., 1991, Branch et al., 2001). When multiple parasite clones simultaneously infect an individual, it is known as multiplicity of infection. To assess multiplicity of infection, polymorphic genes are genotyped. The merozoite surface proteins 1 and 2 are mostly used for the genotyping. The proteins are encoded by the single-copy genes msp-1 and msp-2 which are polymorphic and a parasite isolate with more than one MSP allele is thus considered multiple infections University of Ghana http://ugspace.ug.edu.gh 34 (Viriyakosol et al., 1995). The block two region of MSP1 and the block 3 region of MSP2 are the targeted regions. The block 2 region in msp-1 is in the family of three alleles: K1, Mad20 and RO33. The K1 variants and that of Mad20 have been found to differ in tripeptide or hexapeptide repeats, on the other hand the RO33 family does not contain repeats, but exhibits considerable heterogeneity (Miller et al., 1993). For the MSP2 block 3 region, the alleles are grouped into two families, the IC3D7 and FC27 (Smythe et al., 1990). The region 2 of Glutamate rich protein (GLURP) is also used as a molecular marker to detect parasite (Borre et al., 1991). Multiplicity of infection has been found to be influenced by age and season in central Ghana (Agyeman-Budu et al., 2013). In that MOI decreases with increasing age (Ntoumi et al., 1995). Increased transmission has generally been found to correlate with progressive increase in the average number of malaria parasite clones (Babiker et al., 1997). Multiplicity of infection also varies with malaria transmission season being higher in high transmission season and low in low transmission season. In areas where malaria is endemic, the average number of malaria parasite strains per person correlates with level of transmission (Arnot, 1998, Babiker et al., 1999). Also high parasite density have been correlated with MOI after malaria transmission (Vafa et al., 2008), and could be an indicator of immune status/premunition (Smith et al., 1999, Arnot, 1998), transmission indicator (Babiker et al., 1999), and an indicator for evaluating control interventions (Mayengue et al., 2009). High parasite diversity have been reported in high transmission season in some cases (Kiwanuka, 2009). University of Ghana http://ugspace.ug.edu.gh 35 CHAPTER THREE 3.0 MATERIALS AND METHODS 3.1 Ethics Statement Ethical and scientific approvals for the study were granted by the Institutional Review Board (IRB) and the Scientific and Technical Committee (STC) of Noguchi Memorial Institute for Medical Research (NMIMR) and the Navrongo Health Research Centre (NHRC) respectively. The study participants and/or their parents/legal guardians gave written informed consent before enrolment into the study. 3.2 Study Area Stored plasma and filter paper samples from a previous study conducted in Bongo in the Bongo District of the Upper East Region of Ghana were used in this study. The Bongo District is located in Northern Ghana and shares boundaries with Burkina Faso in the north, Kassena-Nankana District in the west, Bolga Municipal and Talensi- Nabdam to the south, and Bawku West District in the east. Bongo has a size of about 459 sq km. The land is flat with few hills in the south and east. The District has a tropical climate with savanna vegetation and has a rainy season from May to November, with maximum rainfall recorded in August and September, and dry season from December to April the following year. There is one large irrigation dam, the Vea irrigation dam, and other smaller dams scattered across the District (www.geradsn.org) The total population of the district is 84,545. (Ghana Statistical Service., 2010) The study area has marked seasonal malaria transmission that overlaps with rainfall and University of Ghana http://ugspace.ug.edu.gh 36 vector distribution patterns and malaria transmission is therefore high from June through November and comparatively lower between January and April. Figure 3. 1 Map of Bongo District Source: (http://recas-ghana.com/campus.html) University of Ghana http://ugspace.ug.edu.gh 37 3.3 Study Design and Study Population The original larger study was a cross-sectional community-based survey which was conducted close to the end of the rainy season in November/December 2012 and a second similar survey conducted at the end of the dry season in April 2013. Participants for the study were selected from two communities in the Bongo district; the Gowrie/Vea community and the Soe community. Gowrie and Vea are located around the Vea irrigation project with a large dam, and the Soe community is located about 20 km away from the irrigation project site. The Bongo district is one of the districts in northern Ghana that is mapped by the demographic surveillance system of the Navrongo Health Research Centre and has a regularly updated database. Approximately 200 volunteers were recruited from each of the two communities; thus 400 samples were collected per season and about 800 samples in total for both surveys. This current study was curved out of the larger study described, and about 300 stored plasma samples and corresponding filter paper samples per season were used. 3.4 Sample Collection and Processing About 3ml of venous blood was drawn from each participant into collection tubes that had EDTA as anti-coagulant. Filter paper blood blot, thick and thin film blood slides were obtained from all participants for each survey. Basic clinical and demographic data of participants were also captured by questionnaire. The venous blood was centrifuged using the Harrier 18/80 refrigerated centrifuge (MSE UK Ltd, UK) at 2000rpm for 10 minutes and the plasma taken and stored at -20oC. Thick and thin blood slides were stained with 10% Giemsa for Plasmodium parasite examination. Plasma samples were later transported to the Immunology Department, NMIMR in a box containing solid carbon dioxide (dry ice) for immunological analysis. University of Ghana http://ugspace.ug.edu.gh 38 3.5 Laboratory Measurements 3.5.1 Parasitological Examination Light microscopy was used to examine and quantify malaria parasites against 500 WBCs. The parasite density was then calculated using the formula: Parasite density = (Count x 8000)/500. Where ‘count’ is number of asexual blood stage parasites counted and 8000 and 500 are constants representing number of RBCs and WBCs respectively. 3.5.2 Malaria Antigens Four P. falciparum antigens, CSP and CelTOS from the sporozoite stage and the typical blood stage antigens AMA1 and MSP119 were used in this study. For AMA1, four natural alleles (from the 3D7, FVO, CAMP and HB3 parasite strains), and a mixture of three synthetically designed diversity covering (or DiCo) AMA1 antigens were used in competition assays (Kusi et al., 2010, Remarque et al., 2008b). The three DiCo antigens were used as a single equimolar mixture termed DiCo mix. All antigens were expressed and purified under GMP conditions. The CSP protein of the 3D7 strain and containing 19 of the 38 NANP repeats was expressed in an Escherichia coli system (Porter et al., 2013). The 3D7 strain CelTOS protein is comprised of 174 amino acids including an N-terminal six-histidine tag within a 16-amino acid linker which was also expressed in E.coli (Bergmann-Leitner et al., 2010). The 3D7 strain AMA1 protein is comprised of amino acids 83 to 531 of the AMA1 ectodomain and was also expressed in E. coli (Dutta et al., 2002). The full AMA1 ectodomain protein from the FVO strain of P. falciparum which is about 75kDa containing 622 amino acid residues was expressed in P. pastoris (Kocken et al., 2002). The natural allelic forms from the P. falciparum strains HB3, 3D7 and CAMP were University of Ghana http://ugspace.ug.edu.gh 39 expressed in Pichia pastoris by a similar methodology described by (Faber et al., 2008). The differences in the amino acid sequence of the various AMA1 antigens (Dico1, Dico 2, Dico 3 and AMA1 from the FVO 3D7 HB3 and CAMP parasite strains) are presented in Figure 3.2 University of Ghana http://ugspace.ug.edu.gh 40 Figure 3. 2 Amino acid sequence alignment for the four AMA1 and three DiCo antigens Source (Kusi et al., 2010) University of Ghana http://ugspace.ug.edu.gh 41 3.5.3 Enzyme-linked Immunosorbent Assay (ELISA) 3.5.3.1 Optimization and Standardization of ELISA Antigen coating concentration, plasma dilution, and enzyme-conjugate dilution were optimized for the four recombinant antigens, CelTOS, AMA1, CSP and MSP1(19) by ELISA checker-board titrations. In addition, optimization was done to determine the starting dilution of standard pool required for standard curves that convert sample ODs on test plates to concentration in arbitrary units (AU). All antigens tested were optimized and shown to be stable for at least two weeks, when antigen-coated plates were refrigerated (2 to 8°C). Plasma dilutions were also optimized and shown to be stable for 6 weeks when diluted in 1% milk/PBS with 0.02% sodium azide as preservative. 3.5.3.2 Determination of Total IgG levels Total IgG levels to the malarial recombinant antigens CSP, celTOS, MSP119, AMA1- FVO, and AMA1-3D7 were measured by indirect ELISA. Briefly, 100μl/well of a 1.0μg/ml antigen solution in coating buffer (plain PBS, pH 7.4) was pipetted into wells of a 96-well microtitre ELISA plate (Maxisorp Nunc, Denmark). Coated plates were kept in a refrigerator at 4°C overnight. Plates were then washed four times in washing buffer (PBS with 0.1% Tween-20) with 30 seconds incubation between each wash using the Biotek ELx 405 automated ELISA plate washer (Biotek Instruments, Winooski, VT; USA). The washed plates were padded dry on a tissue paper and blocked with 200μl blocking buffer (PBS with 5% milk powder, 0.1% Tween-20) and incubated at room temperature for 1 hour. Plates were then washed four times in washing buffer and plasma samples diluted at 1:1000 in sample dilution buffer (PBS with 1% milk powder, 0.1% Tween-20 and University of Ghana http://ugspace.ug.edu.gh 42 0.02% Na-azide) was added at 100μl/well in duplicates. To control for inter-assay and day-to-day variations in the standardized ELISA procedure, each assay (ELISA plate) included a calibration curve obtained by a 3-fold serial dilution of pooled hyper immune plasma (Standard pool) known to be positive for total IgG to the specific malaria antigens tested (CSP, CelTOS, MSP119, AMA1-FVO, AMA1-3D7). For CSP and CelTOS, the pool was diluted at 1:50, 1:100 for MSP119, and 1:10000 for AMA1- FVO and AMA1-3D7. A buffer blank (plasma dilution buffer) served as control for detection of background responses. The plates with the samples and standard pool plasma were then incubated at room temperature for 1 hour in a humidified chamber after which they were washed four times in washing buffer. Secondary antibody, goat anti-human IgG (H+L), HRPO conjugated (Invitrogen Corporation, Camarillo, CA; USA), diluted at 1:10,000 in conjugate dilution buffer (PBS with 1% milk powder, 0.1% Tween-20), was added at 100μl/well. The plates with the conjugates were incubated for 1 hour at room temperature after which they were washed four times in wash buffer and padded dry. Bound secondary antibody was quantified by incubation with ready to use TMB (3, 3’, 5, 5’-Tetramethylbenzidine) substrate (Kem-En-Tec Diagnosis A/S, Taastrup, Denmark) for 5 minutes in the dark. The enzymatic reaction was stopped with 100μl/well of 0.2 M H2SO4. Optical density (OD) was read at 450 nm with a reference wavelength of 630 nm using a Biotek EL 808 ELISA plate reader (Biotek Instruments, Winooski, VT; USA). Optical density (OD) values for the test samples were converted into antibody units (AU) with the standard reference curves generated for each ELISA plate using a Microsoft Excel-based four parameter logistic curve-fitting application (ADAMSEL b040, Ed Remarque© 2009). University of Ghana http://ugspace.ug.edu.gh 43 Samples were re-tested if the coefficient of variation between duplicate optical densities were higher than 30% and plates were also re-tested if the R-square value of the standard curve was less than 95%. 3.5.4 Competition ELISA 3.5.4.1 Determination of dilutions for Competition assay Competition ELISA was done to determine relative specificities of antibodies against 2 AMA1 antigens (AMA1-FVO, AMA1-3D7) using a modified version of the protocol described by (Kusi et al., 2009) Competition assay was performed first by estimating dilution that yields approximately 2AU based on the ELISA procedure described above and these dilutions were used for the competition assay. The concentration of antigens to use in the competition assay was determined by serially diluting the competing antigens and it was realised that at 30μg/ml almost all antigens are depleted after competing with same antigen as the coating antigen. The assay was also used to determine the concentration at which much of the other competing antigens would be depleted by using two plasma samples, one from a 6 year old child, and the other from a 45 year old adult. AMA1-3D7 and AMA1-FVO were used as coating antigens. The competition antigens were serially diluted three (3) fold from a high concentration of 30μg/ml to 0.04μg/ml; the last well had no competing antigen. Competing antigens were added first followed by the diluted samples at 50μl/well. Plates were incubated for 1 hour at room temperature in a humidified chamber and developed as describe above. ODs for the wells with competing antigens were expressed as a percentage of OD values from wells without competing antigens as percentage antibody depletion by the competing antigens. University of Ghana http://ugspace.ug.edu.gh 44 The percentage depletion was then plotted with a predicted percentage curve with a least square approximation using a four-parameter logistic function: Y = (100-Y min) + Y min 1 + e(Xmid-X)sc Results were reported as percentage depletion/Residual (minimum) binding value. 3.5.4.2 Competition assay Microtitre plates (96 well) were coated with each antigen (AMA1-FVO and AMA1- 3D7) at 1μg/ml with 100μl/well coating buffer, incubated overnight and blocked as described above. 50μl/well of competing (alleles) antigens (AMA1-3D7, AMA1-FVO, AMA1-CAMP, AMA1-HB3, and AMA1 –DICO MIX) were diluted at 30μg/ml and first added to the wells in duplicates. All five competing antigens were added on one plate with a standard plasma pool titrated 3 fold, with a highest dilution of 1:10,000. Appropriately diluted plasma sample each was added at 50ul/well to the competitor antigens to co-incubate. For any competing antigen added per sample, another 50μl/well of the same sample was also added in duplicate wells without competing antigens to be used to calculate percentage inhibition by the competing antigens. Plates were incubated at room temperature for 1 hour and developed as described above. OD values were converted to arbitrary units and expressed as a percentage of AU values from wells without soluble antigens using the formula below: Percentage antibody depletion = Average AU with competing antigen X 100% Average AU without competing antigen University of Ghana http://ugspace.ug.edu.gh 45 3.5.5 DNA Extraction DNA was extracted from stored filter paper (Watman 3MM, Watman Int. Ltd, England) using a modified version of the Chelex extraction protocol described by Wooden (Wooden et al., 1993). A 3 mm square piece of a blood blot on filter paper was cut out with a sterile punch into labeled 1.5ml microfuge tubes. About 1ml of autoclaved 1X PBS was added followed by 50μl of 10% saponin solution. The tube was vortexed (Stuart Scientific, UK) gently, and incubated at 4oC overnight. The tubes were then centrifuged at 14000 rpm for 30 seconds and the supernatant discarded. One milliliter (1ml) 1X PBS without saponin was added to the tubes and inverted several times and incubated at 4oC for 30 minutes. The tubes were again centrifuged at 14000 rpm for 30 seconds and the supernatant discarded leaving pellets. The pellets were re-suspended in 100μl of sterile distilled water and centrifuged at 14000rpm for 30 seconds and the supernatants discarded. The pellets were again re- suspended in 100μl of sterile distilled water followed 50μl of 20 % w/v Chelex-100 resin suspension in deionized water. The tubes were vortexed and incubated at 95°C using a Techne heating block, (Bibby Scientific Ltd, UK) for 10minutes with vortexing at 2minutes intervals. After incubation, the tubes were centrifuged at 14,000 rpm for 5mins and then the supernatant (DNA) was transferred into pre-labeled 0.5 ml microfuge tubes for use as template in PCR applications. The concentration of the genomic DNA was determined using Nanodrop 2000C spectrophotometer (Labteck International, UK) and stored at -20°C until ready for use. University of Ghana http://ugspace.ug.edu.gh 46 3.5.6 PCR Amplification The oligonucleotide primers used in this study were picked from already published sequences by (Smythe et al., 1990), to amplify the polymorphic block 3 region of the MSP-2 gene. Polymerase Chain Reaction (PCR) method was adapted from (Snounou and Singh, 2002, Snounou, 2002) with slight modifications. Block 3 region of the MSP2 gene was amplified using the published sequence specific primers by (Smythe et al., 1990): forward primer, (5'- ATGAAGGTAATTAAAACATTGTCTATTATA-3'), and the reverse primer (5'- CTTTGTTACCATCGGTACATTCTT-3'). Template DNA was amplified using nested PCR, with primers specific to the IC3D7 allelic family: forward- (5'- GCT TAT AAT ATG AGT ATA AGG AGA A -3’), reverse (5' - CTG AAG AGG TAC TGG TAG A- 3’). Separate reactions were performed for each pair of primary and nested primers. For the primary reaction, the following cycling conditions were used: denaturation at 94°C for 2 minutes preceded 30 amplification cycles: denaturation for 30 seconds at 94°C, annealing for 1 min 30 seconds at 55°C, and extension for 1 min at 72°C. The last extension was carried out for 5 minutes. The family specific (IC3D7 family) nested reaction was performed with the following conditions: denaturation at 94°C for 2 minutes preceded 30 amplification cycles: denaturation for 30 seconds at 94°C, annealing for 1 min 30 seconds at 58°C, and extension for 1 min at 72°C. The last extension was also carried out for 5 minutes. Both primary and nested reactions were done in a 25ul final volume, containing 2.5ul 10X reaction buffer, 2uM MgCl2, a 200uM concentration of a mixture of the four dNTPs, a 300nM concentration of each of the two appropriate primers, and 1U of Taq DNA University of Ghana http://ugspace.ug.edu.gh 47 polymerase (Sigma-Aldrich, St. Louis, MO, USA). In the first reaction, 5ul of Chelex- extracted DNA was added as a template, and 1ul of the first PCR product was added in the second reaction. All reactions had a genomic DNA from 3D7 laboratory strain which was used as positive control, and molecular grade water was used as negative control. The PCR products were analysed by electrophoresis on 2% agarose with ethidium bromide as the stain with a standard 100pb ladder. The DNA (bands) was visualized using UV trans-illumination, and fragments obtained were compared by size with a ladder loaded onto the gel. Positive samples were recorded with their band size and the number of bands per sample which refers to the clones in a sample. 3.5.7 Data Analysis Optical density OD values were converted to antibody units using a four parameter logistic fit, ADAMSEL (developed by Remarque). Mann-Whitney (Non parametric) test was used to determine differences in antibody levels between the sites and seasons. Percentage antibody depletion/residual binding (or minimum) values in competition assay, and their matching confidence intervals were generated by a 4-parameter logistic fit with least squares approximation using the R statistical package (R Development Core Team, 2008). Multiplicity of infection was calculated by adding all the clones per individual and finding their mean in each cohort. Chi-square test was used to compare multiplicity of infection between the two sites and across seasons. University of Ghana http://ugspace.ug.edu.gh 48 CHAPTER FOUR 4.0 RESULTS A total of 560 stored plasma samples from volunteers with ages ranging from 1-70 years were used in this study with 257 being males, and 303 females. During the rainy/wet season, 232 samples were obtained and 328 samples from the dry season. Forty-five percent of the samples obtained for the wet season were from males and 46% of dry season samples were from males. Mean age between the wet (17.1±0.9 years) and dry (16.8±0.7 years) seasons was not significantly different (p = 0.937, Mann-Whitney test). Stored filter paper samples were also used to determine parasitaemia by polymerase chain reaction (PCR). In the wet season 154 filter paper samples were used and in the dry season 134 filter paper samples were used. The overall median parasite density by microscopy among the subjects between the two seasons was also not significantly different (p=0.971, Mann-Whitney test). At enrolment during the rainy season, 79 subjects (34.1%) were carrying malaria parasites by microscopy, and 51 (15.5%) had parasites during the dry season (Table 4.1). Table 4. 1 Characteristics of study subjects for the Wet and Dry Seasons Season P value Characteristics Wet Season Dry Season No. of Subjects 232 328 Sex Males (%) 105 (45) 152 (46) Females (%) 127 (55) 176 (54) Age (SE) 17.1±0.9 16.8±0.7 0.937 Parasite Density (SE) 2703.2±1001.8 1872.1±474.6 0.529 Parasite Carriage (%) 79 (34.1) 51 (15.5) University of Ghana http://ugspace.ug.edu.gh 49 4.1 Parasite Density and Proportions at the Sites Parasite detection was done by microscopy (459 samples) and PCR in a subset of participants (290) whose filter paper samples were available. Samples were grouped into two based on proximity to the dam site and proportion of parasites compared between the sites. For both microscopy and PCR there was no difference in the proportions that were parasite positive at the two sites during the wet season. However the proportion that was parasite positive at the dam site was significantly greater than that at the non-dam site in the dry season (Tables 4.2 and 4.3). For samples that were parasite positive there was no significant difference in median parasite density between the dam sites and non-dam sites at any of the two seasons (p>0.05 in both cases). Table 4. 2 Proportion of individuals carrying parasites by microscopy Season Dam Total No. of Subjects No. parasitaemic No. Non- parasitaemic P value* Yes 92 27 65 0.21 Wet No 139 52 87 Yes 155 26 129 0.0061 Dry No 73 24 49 * P value obtained after Chi-square test for differences in proportion Table 4. 3 Proportion of individuals carrying parasites by PCR Dam Total No. of Subjects No. parasitaemic No. Non- parasitaemic P value Season Yes 67 40 27 0.4520 Wet No 89 48 41 Yes 69 66 3 <0.0001 Dry No 65 28 37 * P-value obtained after Chi-square test for differences in proportion. Filter paper blood samples were not available for all participants, hence differences in the number of subjects tested by microscopy and PCR. University of Ghana http://ugspace.ug.edu.gh 50 4.2 Multiplicity of Infection and IgG Levels Multiplicity of infection (MOI) is the concurrent infection an individual has at any time point. The overall mean MOI of the genotypes per infection between the wet and dry seasons were 1.76 and 1.46 (p=0.001) respectively (Table 4.4). When samples were categorized based on proximity to the dam, there was no difference in the MOI between the wet season and the dry season in individuals close to the dam and those who live far away. However significant differences were observed in the MOI values in the dry season between the sites, and also the dry season had lower MOI compared to the wet season. Also 60.2% of samples in the wet season had multiple infections compared to 40.3% in the dry season. Figure 4.1 shows a gel photograph with DNA bands that indicate whether a sample has single or multiple parasite clones. Table 4. 4 Multiplicity of Plasmodium falciparum infection as assessed with MSP-2 marker Number of PCR Fragments Wet Season (%) Dry Season (%) p value 1 43 (39.8) 70 (58.8) 2 49 (45.4) 43 36.1) 3 15 (13.9) 5(4.2) 4 1 (0.9) 1 (0.8) Multiplicity of infection 1.76 1.46 0.001* Table 4. 5 Mean multiplicity of Plasmodium falciparum infection between sites Wet Season Dry Season *P value Dam site 1.8 1.6 0.242 Non-Dam Sites 1.7 1.0 <0.001 #P value 0.599 <0.001 P values obtained following comparison between season (*) or between sites (#) by the student t test University of Ghana http://ugspace.ug.edu.gh 51 Figure 4. 1 Agarose gel photograph with a 100bp molecular weight DNA marker indicating multiple infection L 100bp molecular weight marker NC Negative control S1, S2 &S3 Samples with single infections PC Positive control (single infection) University of Ghana http://ugspace.ug.edu.gh 52 4.3 Total IgG Levels in Plasma Samples between Sites Levels of anti-malarial antibodies were compared between the two study sites for each season. The levels of antibodies to the two blood stage antigens, AMA1 and, MSP119, were not significantly different between the sites for both the wet and dry seasons. However the levels of antibodies to the two sporozoite antigens, CSP and CelTOS, were significantly different between the two sites for both seasons (Figure 4.2). University of Ghana http://ugspace.ug.edu.gh 53 A M A 1 - 3 D 7 A M A 1 - F V O 1 9 M S P 1 C S P C e l T O S 0 1000 2000 3000 Wet Season p<0.001 p<0.001 NON-DAM DAM Parasite antigens A n t i b o d y U n i t s A M A 1 - 3 D 7 A M A 1 - F V O 1 9 M S P 1 C S P C e l T O S 0 2000 4000 6000 8000 Dry Season p=0.024 p=0.004 Parasite antigens A n t i b o d y U n i t s Figure 4. 2 Comparison of anti-malarial IgG responses between the two sites. Error bars are standard error of the mean (SEM). University of Ghana http://ugspace.ug.edu.gh 54 4.4 Parasite Carriage and IgG Responses Total IgG responses were categorized according to season, proximity to the dam, and parasitaemia (Figure 4.3). The wet season the data showed no significant differences in the IgG levels to all the parasite antigens in samples with and without parasites across dam sites. A similar result was obtained during the dry season where differences were not found between the parasitaemic and non-parasitaemic groups. However the parasitaemic group was much younger (mean age = 12.9 ± 1.3 years) than those without parasites (19.2 ± 1.2 years, p=0.0011). University of Ghana http://ugspace.ug.edu.gh 55 . Wet Season Dam site A M A 1 - 3 D 7 A M A 1 - F V O 1 9 M S P 1 C S P C E L T O S 0 1000 2000 3000 4000 Parasite antigens A n t i b o d y U n i t s Wet Season Non-dam Site A M A 1 - 3 D 7 A M A 1 - F V O 1 9 M S P 1 C S P C E L T O S 0 1000 2000 3000 4000 non-parasititaemic parasitaemic Parasite antigens A n t i b o d y U n i t s Dry Season Dam Site A M A 1 - 3 D 7 A M A 1 - F V O 1 9 M S P 1 C S P C E L T O S 0 2000 4000 6000 8000 Parasite antigen A n t i b o d y U n i t s Dry Season Non-dam sites A M A 1 - 3 D 7 A M A 1 - F V O 1 9 M S P 1 C S P C E L T O S 0 2000 4000 6000 8000 Parasite antigen A n t i b o d y U n i t s Figure 4. 3 Total IgG levels and parasitaemia between sites in the two seasons University of Ghana http://ugspace.ug.edu.gh 56 4.5 Cross-reactive and Strain-specific Antibodies Competition ELISA was used to determine whether naturally acquired anti-AMA1 antibodies in the different age groups were more strain-specific or cross-reactive. Four natural alleles of AMA1 (3D7, FVO, HB3, and CAMP), expressed as recombinant antigens, and three in-silico designed AMA1 proteins known as Diversity-Covering (or DiCo) proteins, were used in the assay. An equimolar mixture of the three DiCo proteins, referred to as DiCo-mix, was used as a positive control in all assays. Initially, cross-reactive and strain-specific antibodies in the plasma of two study participants (a 6 year old child and a 45 year old adult) against the AMA1-3D7 and AMA1-FVO allelic antigens were determined. This was to prove the concept that antibody responses in children are most likely to be skewed towards strain specificity since they would have had limited exposure to diverse parasite populations. Adult antibody responses, in contrast, may be more focused on the common antigenic components of the numerous parasite variants they are likely to have been exposed to, and would hence be more cross-reactive in nature. Cross reactive antibodies are identified in competition ELISA on the basis of their depletion from an allelic antigen-coated plate by a soluble competitor allele that is co- incubated with the diluted plasma sample. Strain-specific antibodies (against the coated allele) cannot be depleted by the competitor allele in a similar system and therefore bind to the coated allele. Thus if the same allelic antigen is used for plate coating and as a competitor antigen, it is expected that there will be near complete depletion of antibodies. If two different allelic forms of the antigen are used for coating and competition respectively, only the fraction of antibodies that is cross reactive between these allelic forms will be depleted by the competitor antigen. Antibody depletion by University of Ghana http://ugspace.ug.edu.gh 57 competitor antigens was expressed as residual binding (minimal) value. Thus for example a 20 residual binding value means 20% antibodies are bound to the coated antigens and are therefore specific to the coating antigen relative to the competitor antigen while the rest 80% in solution are cross reactive and share common epitopes. On the basis of this principle, assays were performed with either AMA1-3D7 or AMA1-FVO as the coating allele and for each coated plate; AMA1-3D7, AMA1-FVO, AMA1-HB3, AMA1-CAMP and DiCo mix were used as competitor antigens. The depletion patterns show that the child plasma sample does have anti-AMA1 antibodies that may be 3D7 strain-specific, especially relative to the FVO, HB3 and CAMP AMA1 alleles (Figure 4.4 A & C, Table 4.6) whilst the adult plasma sample has a higher proportion of cross-reactive antibodies as all AMA1 alleles depleted statistically similar proportions of anti-AMA1 antibodies (Figure 4.4 B & D, Table 4.6). The DiCo mix control in all cases depleted similar proportions of antibodies as the homologous 3D7 antigen (over 95% since the highest residual binding observed was 4.4% for the adult sample assayed on the 3D7 AMA1-coated plate (Figure 4.4, Table 4.6). Based on the above interpretation, 60 samples with high responses to both AMA1-3D7 and AMA1- FVO antigens were selected from each season and the assay was run with only the highest competing antigen concentration of 30µg/ml. The data was categorised into single and multiple clones and the mean residual (minimal) binding values plotted University of Ghana http://ugspace.ug.edu.gh 58 Figure 4. 4 Competition ELISA with plasma from a child and an adult Assay on AMA1-3D7 coated plate. The experimentally determined data (points) were fitted to a 4- parameter logistic (lines). Plot A is the curve for the plasma from a child and plot B is the curve for the plasma for an adult. For the AMA1-FVO coated plates, plot C is for a child and plot D for adult. University of Ghana http://ugspace.ug.edu.gh 59 Table 4. 6 Residual anti-AMA1 IgG binding (minimum values) estimates for Adult and child plasma samples. Coating Antigen Plasma Sample Source Competitor Antigens 3D7^ FVO CAMP HB3 DICO MIX 3D7 Child Adult -39.0# (-81.0-2.9) 10.8* (6.4-15.1) 18.3* (7.7-28.9) -182.7# (-1686.8-1.3) 3.6 (1.5-5.7) 3.0 (-0.5-6.5) 1.9 (1.9-9.7) 4.3 (-2.9-11.5) -3.1# (-27.0-20.8) 4.4 (3.0-5.8) FVO Child Adult 3D7 -91.5# (-403.9-221.0) FVO^ -4.1* (-12.8-4.5) CAMP -61.1# (-278.2-156.1) HB3 -187.3# (-1769.1-1.4) DICO MIX -7.6# (-24.3-9.1) -3.4# (-15.1-8.3) 3.5 (-11.6-4.5) -2.1# (-10.7-6.5) -6.5# (-22.6-9.7 -2.5# (-8.6-3.6) Residual binding (minimum) value with 95% confidence intervals Residual binding values were generated with a four parameter logistic fit with least square approximation. These values are predicted minimum values based on measured values for competitor antigens. ^homologous competitor antigen, ie the same competitor antigen was used for coating the ELISA plate for that assay. * Antibody depletion levels for these competitor antigens are significantly different from that of the homologous competitor antigen for the same plasma sample. #Negative values indicates that the curves (minimum values) have not yet reached a plateau, but the confidence interval includes zero and hence the minimum values are not significantly different from zero (Fig 9 C). University of Ghana http://ugspace.ug.edu.gh 60 4.6 Cross Reactive and Strain Specific Responses at the Sites On the basis of the explanation of the preliminary competition ELISA data above, the complete data for each season was compared between the two sites. For both the wet and dry seasons, assays on the 3D7-coated plate showed the presence of 3D7-specific since the other 3 competing antigens (FVO, CAMP and HB3) depleted significantly less of the antibodies recognized by the 3D7 antigen at both sites (Figure 4.5). For assays done on FVO coated plates, the dry season data for the dam site only showed anti-FVO specificity with respect to the 3D7 and CAMP alleles (Figure 4.6 A) while the wet season data demonstrated the presence of FVO-specific antibodies with respect to the 3D7, CAMP and HB3 competing antigens (Figure 4.6 C). However, the non-dam site data showed that the anti-FVO antibodies were also recognized by all other AMA1 alleles in the dry season (Figure 4.6 B), suggesting that anti-FVO AMA1 antibodies at that site were mostly cross-reactive. Wet season data for the non-dam site however shows anti-AMA1 FVO specificity with respect to the 3D7 and CAMP alleles (Figure 4.6 D). These collectively suggest that there is a generally a high proportion of 3D7- specific antibodies compared to FVO-Specific antibodies, irrespective of the study site and transmission season. University of Ghana http://ugspace.ug.edu.gh 61 Dry Season Dam Site 3D7 FVO CAMP HB3 DICOMIX 0 50 100 150 * * * Competing AMA1 antigens % R e s i d u a l b i n d i n g Dry Season Non-dam sites 3D7 FVO CAMP HB3 DICOMIX 0 20 40 60 80 100 ** ** ** Competing AMA1 antigens % R e s i d u a l b i n d i n g Wet Season Non-dam Site 3D7 FVO CAMP HB3 DICOMIX 0 20 40 60 80 100 * * * Competing AMA1 antigens % R e s i d u a l b i n d i n g Wet Season Dam site 3D7 FVO CAMP HB3 DICOMIX 0 20 40 60 80 100 * * * Competing AMA1 antigens % R e s i d u a l b i n d i n g A B C D Figure 4. 5 Residual binding estimate for all competing antigens on 3D7 coated plates. The 3D7 competing antigen is expected to have the least residual binding since it will deplete the most antibodies from the coated plate. University of Ghana http://ugspace.ug.edu.gh 62 Dry Season Dam Site 3D7 FVO CAMP HB3 DICOMIX 0 50 100 * * Competing AMA1 antigens % R e s i d u a l b i n d i n g Dry Season Non-dam sites 3D7 FVO CAMP HB3 DICOMIX 0 20 40 60 80 100 Competing AMA1 antigens % R e s i d u a l b i n d i n g Wet Season Dam site 3D7 FVO CAMP HB3 DICOMIX 0 20 40 60 80 100 ** ** ** Competing AMA1 antigens % R e s i d u a l b i n d i n g Wet Season Non-dam Site 3D7 FVO CAMP HB3 DICOMIX 0 20 40 60 80 100 * * Competing AMA1 antigens % R e s i d u a l b i n d i n g A B C D Figure 4. 6 Residual binding estimate for all competing antigens on FVO coated plates. The FVO competing antigen is expected to have the least residual binding since it will deplete the most antibodies from the coated plate. University of Ghana http://ugspace.ug.edu.gh 63 CHAPTER FIVE 5.0 DISCUSSION Anti-malarial antibodies are an important component of immune response to P falciparum infection in humans and the induction of the right quality of antibodies is important for protection against infection. In malaria endemic areas, adults beyond a certain age are partially protected from clinical disease and this protection is mediated in part by antibodies following repeated infection. These antibodies in adults have been proposed to be an accumulation of different strain specificities following infection with diverse parasites or are more cross-reactive in nature as the immune response is focused on epitopes that are common to the diverse infecting parasites (Doolan et al., 2009). Irrespective of which of these proposed mechanisms is true the quality of antibodies induced within a given population would depend on the transmission dynamics and the parasite diversity within that population. This study was aimed at investigating the effect of parasite diversity and disease transmission pattern on the levels of antibody responses to P. falciparum in individuals living in an area of seasonal malaria transmission. Two blood stage antigens (MSP119 and seven variants of AMA1) and two liver stage antigens (CSP and CelTOS), were used for antibody analysis in this study since they are amongst the leading vaccine candidate antigens currently in pre-clinical or clinical testing. These antigens were tested against stored plasma samples from individuals living in two sites; one in close proximity to an irrigation dam and the other at least 20 kilometres away from the dam. Parasites were detected by microscopy and PCR and for both methods, the proportions of study participants who were parasite-positive were significantly greater at the dam site compared with those at the site away from the dam (non-dam site) during the dry University of Ghana http://ugspace.ug.edu.gh 64 but not the wet season (Tables 4.2 and 4.3). This may be a direct consequence of the presence of the dam, which serves as possible breeding grounds for disease vectors and results in higher levels of disease transmission at this site, relative to the non-dam site. Thus although the area has been generally described as one with seasonal transmission (Appawu et al., 2004) malaria transmission in communities around the dam may not be as seasonal. Diversity of infecting parasites was investigated by molecular typing of the block 3 region of the MSP 2 gene and the results showed the number of infections per person ranged from 0 to 4 in both seasons and the mean multiplicity of infection (MOI) was generally higher in the wet season compared to the dry season (1.76, and 1.46, p=0.001, Table 4.4). Comparing MOI between study sites there was a statistically significant difference between sites during the dry but not the wet season. (Table 4.5). It has been shown that in malaria endemic regions parasite diversity in high malaria transmission areas are high and that individuals could carry multiple genotypes (clones) but the opposite pertains in low endemic areas with most infections being monoclonal (Peyerl-Hoffmann et al., 2001, Babiker et al., 1997, Haddad et al., 1999). This therefore supports the conclusion that transmission at the dam site may be higher than at the non-dam sites during the dry season. Comparison of antibody levels between study sites showed that there were statistically significant difference in anti-sporozoite (CSP and CelTOS) antibody levels between the two sites for both transmission seasons whilst no such differences were observed for the two blood stage antigens (AMA1 and MSP119) (Figure 4.2). This observation may be explained by the fact that antibodies to AMA1 and MSP1, due to the cyclic nature of the blood stage infection, may persist for much longer periods while antibodies to the University of Ghana http://ugspace.ug.edu.gh 65 sporozoite antigens are short lived and are boosted only when there are new infectious bites (Campo et al., 2011, Torres et al., 2008, Kusi et al., 2014). This finding therefore supports the use of sporozoites antigens for monitoring malaria transmission Though differences in the proportions of infected individuals between the two study sites were observed, the median parasite densities for infected individuals were not significantly different by study site or season. Comparison of antigen specific antibody levels between parasitaemic and non- parasitaemic individuals within the different study sites did not show any significant differences (Figure 4.3). Thus antibody levels were not dependent on the presence of parasites at the sampling time point. It is possible that individuals who were non- parasitaemic may have recently recovered from an infection Apical membrane antigen, AMA1 has been found to be a promising blood stage vaccine candidate antigen because of the critical role it plays in erythrocyte invasion (Remarque et al., 2008a). But this potential has been dampen due to extensive polymorphism (Marshall et al., 1996). In a study by (Kusi et al., 2009), rabbits immunized with alleles of AMA1: 3D7, FVO HB3, and a mixture of these three antigens elicited high levels of cross-reactive antibodies to the antigen mixture compared the single allele immunization and concluded that cross-reactive antibodies were to epitopes that are shared by the three antigens. Thus the competition ELISA assay could be used to distinguish between responses to a single antigen and responses to simultaneous multiple antigens. Application of this to human infection is however limited by the fact that the infecting parasite strain history may not be known. Nevertheless this concept was tested in two naturally exposed humans and the data showed the possibility of detecting strain University of Ghana http://ugspace.ug.edu.gh 66 specific antibodies in the child and a more cross-reactive antibodies in the adult (Figure 4.4) While this data is based on just two samples two main conclusions can be drawn; i) that adults with numerous exposures to parasites may have a more cross-reactive repertoire of antibodies compared to children who have had only a few parasite exposures, corroborating previous findings (Cortes et al., 2005a, Kusi et al., 2012), and ii) the competition as described for the rabbit data can be applied to human samples for the detection of strain specific and cross-reactive antibodies. Applying the assay to samples from the two sites using 3D7 coated plates the 3D7 competing antigen always depleted significantly more antibodies compared to the FVO, CAMP and HB3 alleles irrespective of the study site or season (Figure 4.5), suggesting that there were 3D7 specific antibodies (relative to the other competing antigens) at both study sites. Assays done on the FVO coated plates however showed high proportions of cross-reactive antibodies in the dry season at the non-dam sites since the FVO competing antigen depleted similar quantities of antibodies as the other competing antigens (4.5 B). This therefore suggest a relationship between antibody specificity and MOI; antibody specificities to the both 3D7 and FVO AMA1 alleles were observed where the MOI was greater than 1 but specificity to the only the 3D7 allele was observed where the MOI was 1. This observation can be explained by the fact that individuals with multiple infections may be better protected because of the production of cross-reactive responses whiles individuals with single infection are not because of the production of antibodies specific to only the single clone hence introduction of a new clone could easily cause disease. This could explain why in high transmission settings some individuals are better protected compared to low transmission areas (Rogier and Trape, 1993, Doolan et al., 2009). University of Ghana http://ugspace.ug.edu.gh 67 CHAPTER SIX 6.0 Conclusions and Recommendations 6.1 Conclusions Data from the current study shows the multiplicity of infection was greater than one (MOI > 1) at the dam site irrespective of the season, and at the non-dam site only during the wet season. This suggests that MOI is high in areas of moderate to high transmission and similar findings have been described in other areas. This observation is possibly linked with the availability of water, most likely as possible breeding sites for the mosquito vectors. Thus despite the label of the larger study area being one with seasonal malaria transmission, disease transmission in communities around the dam, on the basis of the MOI estimated in this study may have transmission levels as high as occurs during the wet season. Communities around such large water bodies could therefore experience year round malaria transmission with high diversity of parasites. If these findings are confirmed, it will provide a basis for targeted control of malaria. It was also observed that when transmission intensity was relatively higher (as determined by the estimated MOI > 1), antibodies that were specific to either the 3D7 or FVO allele of AMA1 were elicited, while mostly 3D7 AMA1-specific antibodies were elicited (MOI = 1) under periods of low transmission. If this observation is confirmed for other polymorphic antigens, it could partly explain the difference in acquired malaria immunity between moderate to high transmission areas and areas with very low transmission. In addition, levels of antibodies to sporozoite antigens, but not those to blood stage antigens, were different between the wet and dry seasons, reflecting possible exposure to a greater number of infectious bites during the wet University of Ghana http://ugspace.ug.edu.gh 68 season. This finding supports the use of anti-sporozoite antibodies as markers for monitoring malaria transmission in endemic areas. 6.2 Recommendations 1. Future studies should include an entomological component to directly assess the vector population and possible differences in inoculation rates between communities around such large water bodies and those that are a good distance from it. 2. Genotyping was limited to the use of a single genetic marker (3D7 strain-specific primers) and that future studies must include primers that can be used to pick other strains of the parasite in order to determine the actual circulating parasite clones and how they influence antibody responses. 3. The use of other polymorphic antigens to assess antibody responses is important for confirmation for conclusions drawn on the possible effect of AMA1 diversity on immune responses 4. In vitro functional studies such as growth inhibition assays involving purified IgG must be conducted in future to determine the functional quality of antibodies and the relative contributions of strain-specific and cross-reactive antibodies to parasite invasion of red cells. University of Ghana http://ugspace.ug.edu.gh 69 REFERENCE Abbas, A. K., Murphy, K. M. & Sher, A. 1996. Functional diversity of helper T lymphocytes. Nature, 383, 787-793. 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A., Bockarie, M., Reeder, J. C., Cowman, A. F. & Crabb, B. S. 2001. Antibodies against merozoite surface protein (MSP)-1(19) are a major component of the invasion-inhibitory response in individuals immune to malaria. J Exp Med, 193, 1403-1412. Oduro, A. R., Koram, K. A., Rogers, W., Atuguba, F., Ansah, P., Anyorigiya, T., Ansah, A., Anto, F., Mensah, N., Hodgson, A. & Nkrumah, F. 2007. Severe falciparum malaria in young children of the Kassena-Nankana district of northern Ghana. Malar J, 6, 96. Ofori, M., Ansah, E., Agyepong, I., Ofori-Adjei, D., Hviid, L. & Akanmori, B. 2009. Pregnancy-associated malaria in a rural community of ghana. Ghana Med J, 43, 13-8. Ofori, M. F., Dodoo, D., Staalsoe, T., Kurtzhals, J. A., Koram, K., Theander, T. G., Akanmori, B. D. & Hviid, L. 2002. Malaria-induced acquisition of antibodies to Plasmodium falciparum variant surface antigens. Infect Immun, 70, 2982-8. Osier, F. H., Fegan, G., Polley, S. D., Murungi, L., Verra, F., Tetteh, K. 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G. & Luty, A. J. 2005. Immunoglobulin G isotype responses to erythrocyte surface-expressed variant antigens of Plasmodium falciparum predict protection from malaria in African children. Infect Immun, 73, 2281-7. University of Ghana http://ugspace.ug.edu.gh 100 Appendixes 7.1 Preparation of standard solutions and buffers. Unless otherwise stated, all standard solutions for ELISAs were prepared with double distilled water (dd H2O), while water in PCRs solutions were obtained commercially. Blocking Buffer (PBS with 5 % milk powder, 0.1% Tween-20) To prepare 500ml, 1 tablet of PBS was added to a beaker containing 500ml deionised water and mixed using a magnetic stirrer. 0.5ml of tween-20 was added and then 25.0g of skimmed milk was then added and stirred until all were in solution. Plasma Dilution Buffer (PBS with 1 % milk powder, 0.1% Tween-20 and 0.02% Na- azide) To prepare 500.0ml of the plasma dilution buffer, 1 PBS tablet was added to a beaker containing 500.0ml deionised water and mixed on a magnetic stirrer. After the tablet was dissolved, 5.0 g of skimmed milk, 0.5 ml of Tween-20 and 1.0 ml of 10 % Na- azide solution were added and the solution stirred until a homogeneous mixture was obtained. The 10 % Na-azide solution was prepared by adding 40.0ml of deionised water to 4.0g of Na-azide. University of Ghana http://ugspace.ug.edu.gh 101 Conjugate Dilution Buffer (PBS with 1% milk powder and 0.1% Tween-20) 500.0ml of conjugate dilution buffer was prepared by adding 1 tablet of PBS a beaker containing 500.0ml deionised water on a magnetic stirrer to mix. 5.0 g of skimmed milk and 0.5ml of Tween-20 was added. Washing Buffer (PBS with 0.1% Tween-20) 5L washing buffer was prepared by adding 10 tablets of PBS to a flask containing 5L deionised water and stirred until all is in solution. 5.0ml of Tween-20 was then added while still stirring. Colour Solution [TMB (3, 3’, 5, 5’-Tetramethylbenzidine)] Ready to use TMB plus2 (3, 3’, 5, 5’-Tetramethylbenzidine) solution was obtained from commercially from the manufacturer (Kem-En-Tec Diagnosis A/S, Taastrup, Denmark). Stop Solution (0.2M H2SO4) 500.0ml of stop solution, 10.0ml of 10.0M H2SO4 was added to 490.0ml of deionised water and the solution shaken to mix. It was then cooled to room temperature and kept in the hood until required University of Ghana http://ugspace.ug.edu.gh 102 7.2 PCR Materials Dry Primers used in PCR reaction were reconstituted with commercially acquired nuclease free waster (DEPC Treated Water, Invitrogen, CA, USA) as instructed by the manufacturer (Eurofins MWG Operon). The reconstituted primers were diluted to a working concentration of 10μm using commercially obtained water. Gel loading buffer Commercially obtained gel loading buffer (GelPilot DNA loading dye 5X, Qiagen,) was obtained and stored at 4oC until required. DNA Ladder DNA molecular weight size markers Direct Load 100bp DNA molecular weight marker was obtained commercially and stored at -20oC until required. Agarose gel To prepare a 2%w/v agarose gel, 4.0g of agarose powder (SeaKem®GTG® Agarose, Lonza, Rockland, ME, USA) was put into flask and X1 TAE was added to make a volume of 200ml. It was heated in microwave oven for 4 minutes to dissolve. 3ul of Ethidium Bromide (AppliChem, Damstadt, Germany) was added. The gel was then cast to set in a chamber with comb to make the wells. University of Ghana http://ugspace.ug.edu.gh