University of Ghana http://ugspace.ug.edu.gh QR185.8 C95 N16 bite C.l G375309 University of Ghana http://ugspace.ug.edu.gh CYTOKINE AND ANTIBODY RESPONSES TO M ALARIA VACCINE CANDIDATE ANTIGENS, PLASMODIUM FALCIPARUM GLUTAM ATE RICH PROTEIN (GLURP) AND M EROZOITE SURFACE PROTEIN (MSP3) IN GHANAIAN CHILDREN BY HELENA NARTEY THIS THESIS IS SUBM ITTED TO THE UNIVERSITY OF GHANA, LEGON, IN PARTIAL FULFILLM ENT FOR THE REQUIREM ENT FOR THE AW ARD OF A M ASTER OF PHILOSOPHY DEGREE IN ZOOLOGY. MAY 2005 University of Ghana http://ugspace.ug.edu.gh DECLARATION The work described in this thesis was performed by me at the Immunology Department o f Noguchi M emorial Institute for Medical Research (NMIMR), University o f Ghana, Legon, under the supervision o f Dr. Daniel Dodoo o f Immunology Department, NMIMR, and Prof. Dominic Edoh o f Zoology Department, University o f Ghana, Legon. References cited in this thesis have fully been acknowledged. Helena Nartey Signature Dr. Daniel Dodoo Signature ignature II University of Ghana http://ugspace.ug.edu.gh DEDICATION I dedicate this work to God Almighty, and to my dear parents M adam Margaret Otoo and Mr. Jacob Benjamin Nartey for their numerous sacrifices. Ill University of Ghana http://ugspace.ug.edu.gh ACKNOW LEDGEM ENT Sincere thanks to the Almighty God, for helping me complete this work. I acknowledge my supervisors, Dr. Daniel Dodoo o f Immunology Unit, NMIMR, and Professor Dominic Edoh o f the Zoology Department, University if Ghana, for their guidance and knowledgeable contributions made towards this work. I also thank Dr. Michael Theisen o f The State Serum Institute, Denmark for co-supervising my thesis and the indispensable contributions he made to help complete this work. I extend my appreciation to Professor Akanmori, Dr. Ben Gyan, and Dr. Mike Ofori o f Immunology Unit, NMIMR, for the various ways in which they contributed to this work. My sincere gratitude to all staff and colleagues o f Immunology Unit, NMIMR, especially Mrs. Anastasia Aikins, Mr. Asamoah Kusi, Mr. Gerald Laryea, Mr. Kakra Fredua-Agyeman and Mr. James Kretchy for the diverse ways they assisted me to complete this work. I also thank Dr. Langbong Bimi o f the Zoology Department, University t f Ghana, for his assistance. I further extend my gratitude to Mr. Nathaniel Lamptey for his support and encouragement. This study was funded by WHO/TDR Re-entry grant (A10830). IV University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS Declaration...................................................................................................................................... H Dedication....................................................................................................................................... HI Acknowledgement......................................................................................................................... IV Table o f Contents.............................................................................................................................V List o f Tables and Figures........................................................................................................... VII List o f A bbreviations......................................................................................................................IX A bstract............................................................................................................................................... X 1.0 INTRODUCTION.............................................................................................................3 2.0 LITERATURE REVIEW .............................................................................................11 2.1 Epidemiology o f m alaria.............................................................................................11 2.1.1 The burden o f m alaria................................................................................................. 11 2.1.2 M alaria distribution in Africa.....................................................................................12 2.2 L ifecyc le ........................................................................................................................ 13 2.3 Case definition o f clinical m alaria ............................................................................14 2.4 M alaria im m unity......................................................................................................... 15 2.4.1 Innate immune response..............................................................................................15 2.4.2 Acquired im m unity ......................................................................................................15 2.4.2.1 Humoral immune response........................................................................................ 15 2.4.2.2 Cell m ediated im m unity..............................................................................................18 2.5 M alarial antigens.......................................................................................................... 25 2.5.1 Pre-erythrocytic stage antigens................................................................................. 26 2.5.2 Asexual blood stage antigens.....................................................................................27 2.5.3 Immune responses against GLURP and M SP3......................................................29 3.0 M ATERIALS AND M E T H O D S............................................................................... 32 3.1 Study area and study population...............................................................................32 3.2 Study procedures.......................................................................................................... 3 4 3.2.1 A ntigens......................................................................................................................... 3 4 V University of Ghana http://ugspace.ug.edu.gh 3.2.1.1 Sequences o f an tigens..................................................................................................35 3.2.2 Continuous cultivation o f malaria parasites............................................................ 37 3.2.3 Preparation o f the crude malaria schizont antigen.................................................39 3.2.4 Antibody m easurem ents...............................................................................................40 3.2.5 Cellular assays................................................................................................................42 3.2.5.1 Lymphocyte cultivation and proliferation a ssay s ................................................ 42 3.2.5.2 Cytokine m easurem ents............................................................................................. 44 3.3 Statistical analysis.........................................................................................................45 4.0 R E SU L T S.......................................................................................................................... 46 4.1 The pattern o f malaria in the cohort..........................................................................46 4.2 Antibody responses to GLURP, MSP3 and GLURP-MSP3 hybrid antigens..48 4.2.1 IgG / IgM responses to GLURP, MSP3 and GLURP-MSP3 hybrid antigens.48 4.2.2 IgG subclass responses to GLURP, MSP3 and GLURP-MSP3 antigens........50 4.2.3 Antibody responses to GLURP and MSP3 in relation to a g e .............................52 4.2.4 Antibody responses to GLURP and MSP3, and protection from m alaria .......53 4.2.4.1 Age related exposure and protection from clinical m alaria ................................ 57 4.3 Antibody responses to GLURP-MSP3, GLURP and MSP3 an tigens..............58 4.4 Complementary antibody responses to GLURP and M S P 3 ...............................60 4.5 T-cell responses to GLURP peptides....................................................................... 61 4.5.1 Cytokine responses in Ghanaian children and adults............................................61 4.5.2 Cytokine responses in relation to a g e ...................................................................... 66 4.5.3 Cytokine responses in relation to protection from m alaria ................................. 6 6 4.5.4 Cytokine ratios in relation to malaria infection...................................................... 68 4.5.5 Relationship between humoral and cellular responses......................................... 70 5.0 D ISC U SSIO N ................................................................................................................... 71 5.1 CO N CLU SIO N .............................................................................................................81 6.0 REFER EN C ES.................................................................................................................82 VI University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Table 3.1: Amino acid sequences o f GLURP peptides and MSP3 antigen................35 Table 4.1: Association o f antibody responses with age o f Ghanaian children.......... 52 Table 4.2: IgG and IgM responses in Ghanaian children protected or susceptible to m alaria.................................................................................................................... 54 Table 4.3: IgG subclass responses to GLURP and MSP3 in Ghanaian children protected or susceptible to m alaria...................................................................55 Table 4.4: Cytophilic to non-cytophilic antibody ratios in children protected or susceptible to malaria from Dodow a............................................................... 56 Table 4.5: Cytokine levels in exposed and unexposed individuals..............................65 Table 4.6: Cytokine levels in Ghanaian children susceptible to, or protected from clinical malaria.....................................................................................................67 Table 4.7: Cytokine ratios in Ghanaian children susceptible to or protected from clinical malaria.....................................................................................................69 LIST OF FIGURES Fig 2.1: The life cycle o f the P. falciparum parasite...................................................25 Fig 2.2: GLURP IgG and ADCI mechanism in vitro................................................. 29 Fig 3.1 Amino acid sequences o f GLURP-MSP3 hybrid antigen......................... 36 Fig. 4.1: Prevalence o f P. falciparum parasitaemia in D odow a...............................47 Fig 4.2: IgG and IgM responses in the three antigens in Ghanaian children......... 49 Fig 4.3: IgG subclass responses to Glurp, MSP3 and Glurp-MSP3 antigens in Ghanaian children................................................................................................ 51 Fig. 4.4: Correlation o f GLURP-MSP3 hybrid to GLURP or MSP3 antigens.......59 Fig 4.5: Cytokine levels in Ghanaian children and Danish controls........................62 Fig 4.6: Cytokine levels in Ghanaian adults and Danish controls........................... 64 VII University of Ghana http://ugspace.ug.edu.gh LIST OF ABBREVIATIONS ADCI Antibody-dependent cell mediated inhibition AMA1 Apical M erozoite Antigen 1 CSP Circumsporozoite Surface Protein ELISA Enzyme-linked immunosorbent assay Fc Fragment o f crystallization FBS Foetal bovine serum GLURP Glutamate Rich Protein IFN-y interferon-y Ig Immunoglobulin IgG Immunoglobulin G IgM Immunoglobulin M IL Interleukin LSA Liver Stage Antigen MHC M ajor Histocompatibility Complex MOH M inistry o f Health MSP1 M erozoite Surface Protein 1 MSP3 M erozoite Surface Protein 3 NK cells Natural Killer Cells OD Optical Density OPD Ortho-phenylenediamine P. falciparum plasmodium falciparum PBMC Peripheral Blood Mononuclear Cells PBS Phosphate Buffered Saline PfEM Pl P. falciparum Erythrocyte Membrane Protein PHA Phytohaemaglutin PMA Phorbol 12-myristate 13-acetate PPD Purified protein derivative o f tuberculin RBC Red blood cell SCID Severe combined immunodeficiency Tc T-cytotoxic University of Ghana http://ugspace.ug.edu.gh TGF P Tumour growth factor P, Th cells T helper cells TNFa. Tumour necrosis factor WHO W orld Health Organization IX University of Ghana http://ugspace.ug.edu.gh ABSTRACT GLURP and MSP3 are targets for antibodies involved in antibody-dependent cellular inhibition that may lead to protective immunity against malaria. Comparative assessment o f IgG and subclass responses to GLURP and MSP3 in relation to immunity against malaria will provide relevant information that will be useful in future malaria vaccine development. Several studies have led to the recent recognition o f the importance o f T- cell in the generation o f long-term antibody responses in m alaria infection. Therefore, cellular responses to GLURP and MSP3 must be investigated to get a better understanding o f the roles the various cytokines play in conferring immunity against malaria. The antibody responses to GLURP and MSP3 were measured by Enzyme-linked immunosorbent assay (ELISA) in samples obtained from a cohort o f 300 children, 3-15 years o f age. The plasm a samples came from a previous longitudinal morbidity survey carried out over a period o f 18 months (1994-1995) covering two m alaria transmission seasons, in which children were classified as susceptible or resistant to malaria. In addition, peripheral blood mononuclear cells (PBM C’s) were stimulated in culture with GLURP peptides and cytokines were measured. The pattern o f IgG subclass responses to both antigens was similar, indicating higher prevalence for cytophilic antibodies than non-cytophilic antibodies. The association between antibody levels and protection was statistically significant for GLURP IgG (P=<0.001) and MSP3 IgG (P=<0.001), and for cytophilic IgG l and IgG3 X University of Ghana http://ugspace.ug.edu.gh (0.009 >p>0.001) for both GLURP and MSP3. However, when the effect o f age was adjusted for in a logistic regression model, GLURP IgG and IgG l responses were associated with protection in Ghanaian children. For cellular responses to GLURP peptides, higher cytokine responses were raised in antigen-stimulated cultures o f exposed individuals than in non-exposed individuals. The study found no significant association between cytokine responses in protected Ghanaian children and those susceptible to malaria (p>0.05). Furthermore, there was no correlation between antibody and cytokine responses in Ghanaian children. These results confirm the association between cytophilic antibodies against GLURP and MSP3 and protection from clinical malaria. The complementarity o f antibodies responses against both antigens supports their use as a hybrid in a future m alaria vaccine. The study showed cytokine responses against some GLURP peptides, suggesting that there are T- cell epitopes within the antigen. The knowledge o f the level o f cellular and antibody responses to GLURP and MSP3 o f P. falciparum, and possibly the combined effect of these two antigens will be very useful in future malaria vaccine development. XI University of Ghana http://ugspace.ug.edu.gh CHAPTER 1 1.0 INTRODUCTION M alaria is a protozoan disease, which is transmitted through the bite o f a female anopheles mosquito. There are four species o f the genus plasmodium that infect humans. These are Plasmodium falciparum , Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae. These plasmodia species cause four kinds o f malaria with different disease presentations. P. vivax causes the benign or tertian form o f malaria, responsible for about 43% o f cases worldwide. It is prevalent throughout the tropics and many temperate regions. P. malariae causes quartan malaria, which is responsible for about 7% o f malaria cases in the world. Likewise P. ovale causes tertian malaria, a rare and milder form o f infection, and confined to only tropical Africa and subtropics. P falciparum is the most common malaria parasite in Africa, responsible for about 50% o f all malarial cases worldwide, and causes the most lethal form o f malaria, especially in children, pregnant women and travelers from non-malaria endemic countries. Plasmodium falciparum infection is the major cause o f morbidity and mortality in malaria endemic regions (Osuntokun, 1983). The clinical symptoms o f human P. falciparum infections include chills, fever, headache, nausea, vomiting, and diarrhoea. Severe malaria complication manifests as cerebral malaria, severe anaemia, convulsions and respiratory distress, occurring mainly in children under 5 years o f age. In Africa, studies suggest an annual mortality o f 1.0-2.5 million, majority o f which are children below five years (Snow et al., 1999; WHO, 2002). However, there is an increasing trend in m alaria morbidity and mortality that can mainly be attributed to drug resistant parasite strains and insecticide resistant vectors, thus leading to an increase in 3 University of Ghana http://ugspace.ug.edu.gh the social and economic burden o f the disease. M alaria is said to be a disease o f the poor, as well as a cause o f poverty in Africa. The annual economic growth in malaria endemic countries has been shown to be lower than in countries without malaria (Roll back malaria, 2001). M alaria also has a direct impact on A frica’s human resources, due to mortality and loss o f productivity associated with the illness. In addition, it affects children’s education and social development as the sickness causes absenteeism from school. The greatest challenges facing malaria eradication in Africa is drug resistance, and the development o f insecticide resistance by the mosquito vector (Greenwood, 2002). With the advent o f drug resistant parasites and insecticide resistant mosquitoes, much emphasis has been placed on developing a malaria vaccine. Development o f a m alaria vaccine or other new immunotherapies may lead to a decrease in malaria morbidity and mortality especially in children. This can be possible by understanding the complex life cycle, and the interactions o f the parasite with the host’s immune system. The complete life cycle o f P. falciparum involves an invertebrate host, which is a female anopheles mosquito and an intermediate vertebrate human host. There are three stages o f the life cycle in the human host namely the pre-erythrocytic, erythrocytic and gametocytic stages. The parasite is mainly intracellular in all the stages with only brief periods o f extracellular existence prior to sporozoite invasion o f liver cells, or merozoite invasion o f erythrocytes. However, both antibody and T-Cell responses are elicited against all the 3 stages o f the parasite in the human host. During the pre-erythrocytic stage, antibodies are raised against the sporozoites that may prevent their entrance into the liver cells. Nevertheless, some sporozoites manage to enter into the liver cells, which leads to induction o f cytokine responses by the host immune 4 University of Ghana http://ugspace.ug.edu.gh system, thus inhibiting the development o f the parasites in the liver cells. Furthermore, antibodies raised against merozoites may prevent the invasion o f parasites into the erythrocytes, and lead to splenic clearance o f infected red blood cells. At the gametocytic stage, antibodies and cytokines are raised against the gametocytes and may thus prevent their development into sporozoites. The multiplication o f parasites o f the erythrocytic stage is responsible for the clinical symptoms o f malaria, and there is therefore a lot o f focus on this stage, with the view o f identifying antigens important for protective immune responses. Generally, the human immune system is divided into innate and acquired systems. The innate immunity is inherent and always present to protect the individual from foreign materials and pathogens. It consists o f body surfaces and internal organs such as the skin and mucous membrane, other components include interferons, phagocytic cells, macrophages and microglial cells o f the central nervous system. Cells o f the innate immune system detect the presence o f pathogens first and send signals to the adaptive immune system during infection. The acquired immune system on the other hand, consists o f antigen specific cells that supplements the protection provided by the innate immune system. They express cell surface molecules that are capable o f recognizing a wider range o f antigens. The two arms o f acquired immunity are humoral immune response, which is mediated by B cells, and cellular immune response, mediated by T- cells. The B cells produce soluble antibodies, which are heterogeneous mixture o f serum globulins (immunoglobulins) that circulates in the body, recognize and bind specific microbial antigens for destruction. The antigen-antibody reaction activates the complement system, which consists o f serum enzymes resulting in the lysis o f microbes 5 University of Ghana http://ugspace.ug.edu.gh (Benjamini et al., 2000). The cell-mediated immune response consists o f T lymphocytes, which bears antigen receptors called T cell receptors. T lymphocytes circulate to antigen directly to destroy it in the context o f MHC class I or II reactivity. W hen an antigen is presented by macrophages or other antigen presenting cells, it results in the activation and proliferation o f T helper cells (Th cells) cells as well as T-cytotoxic (Tc) cells. Th cells function by releasing cytokines, which activates signals for B cells leading to the production o f antibodies to eliminate extra cellular pathogens. Th cells can be divided into two functional subsets (Thi and Tjg), and these secrete different cytokines. These cytokines are soluble low molecular weight proteins produced by various cells o f the innate and acquired immune system. They are chemical messengers that convey information between cells, regulate the development, differentiation o f the effector cells and modulate immune responses. These cytokines include interferons, interleukins, tumour necrosis factor and other proteins. T cells also release cytokines that lead to delayed-type hypersensitivity reaction. The cytotoxicity mechanism o f cell-mediated immunity may be either through antigen specific T lymphocytes (CD 8 + cells), bearing T cell receptors or may involve nonspecific cells like Natural Killer cells (NK cells) and macrophages. These cytotoxic T cells play important role in directly recognizing and eliminating infected cells and antigens. TH-cell differentiation in the presence o f antigens leads to the synthesis and secretion o f a range o f cytokines like IL-2, which would eventually lead to pathogen destruction. Cytokines produced by activated Th cells can influence the activity o f B cells, NK cells, macrophages, granulocytes and the entire network o f cells in the immune system (Benjamini et al., 2000). 6 University of Ghana http://ugspace.ug.edu.gh M alaria infection results in both activation o f antibody and cellular immune responses from the host. These immune responses are regulated by the innate and adaptive immune systems (Perlmann et al., 2002). T-cells and cytokines are also known to be involved in the immune regulation and effector phases o f malaria immunity through T helper cells (Weidanz et al., 1988; Riley et al., 1988; Luty et al., 1999). Epidemiological survey performed in m alaria endemic communities has shown that gradual acquisition o f partial clinical immunity to malaria in individuals is due to repeated exposure to malaria parasites. Infants bom to immune mothers are protected from the disease in their early months as a result o f in utero acquisition o f maternal antibodies (Marsh, 1992). Antibodies have been shown to play a major role in immunity against the blood stages, the stage responsible for clinical malaria (Dodoo et al., 2000; Okech et al., 2004). Several studies have associated naturally acquired protection against m alaria in endemic populations, with immune responses to particular m alaria parasite antigens (Bouharoun- Tayoun et al., 1990; Egan et al., 1996; John et al., 2004; Okech et al., 2004; Oeuvray et al., 1994; Theisen et al., 1998 and 2001). It is also known that there is participation o f cell-mediated effector mechanisms in the establishment and maintenance o f protective immunity (Good et al., 1998). Cell mediated immune responses against malaria is thought to be protective against both pre-erythrocytic and erythrocytic stages o f the parasite (Troye-Blomberg et al., 1994). The T-helper cells, which secrete cytokines, are believed to be important for the outcome o f infection in human and animal models, either leading to pathology or protection (Omer et al., 2000; Shear et al., 1990; Stevenson et al., 1990). The balance 7 University of Ghana http://ugspace.ug.edu.gh between pro-inflammatory cytokines and anti-inflammatory cytokines may be important in m alaria infection and immunity (Troye-Blomberg et al., 2002). Cell-mediated immune responses are effected through cytokines. It is important to elucidate the role played by cytokines in protective immunity, and whether certain cytokines influence the production o f cytophilic antibodies. A number o f malaria antigens have been identified and characterized (Figure 2.1), and may be targets for protective immunity to malaria (Warrell, 1993). These antigens include, the M erozoite Surface Protein 1-19 (M S P I19), the M erozoite Surface Protein 3 (MSP3), the Circumsporozoite Surface Protein (CSP), P. falciparum Erythrocyte Membrane Protein (PfEM Pl), Apical M erozoite Antigen 1 (AMA1), Liver Stage Antigen 1 (LSA1) and the Glutamate Rich Protein (GLURP). This study focused on GLURP and MSP3, which have been identified as targets for antibodies involved in antibody-dependent cellular inhibition (ADCI). Several studies on antibody responses to GLURP and MSP3 have shown the importance o f GLURP and M SP3-speciflc antibodies, more especially, cytophilic antibodies in mediating immunity against malaria (Theisen et al., 1998; Dodoo et al., 2000; Oeuvray et al., 1994 and 2000; Theisen et al., 2001). Since GLURP and MSP3 are effective in ADCI, there is the need for baseline immunological data in malaria endemic regions that will provide rational for including both antigens in a future multivalent m alaria vaccine. Furthermore, to be an effective m alaria vaccine, it may be required that GLURP induces T-cell responses to regulate efficient and long lasting antibody responses under natural infection. However, little has been done with regards to cell-mediated immune responses to GLURP, in relation to the pattern o f cytokine responses (either pro-inflammatory or anti­ inflammatory). Since regulation o f antibody production by B-cells is T-cell mediated 8 University of Ghana http://ugspace.ug.edu.gh and involves cytokines, it is important to elucidate cytokine(s) that may influence production o f cytophilic antibody responses (IgGl and IgG3) that has been consistently found to be associated with protection from malaria (Dodoo et al., 2000; Oeuvray et al., 2000; Theisen et al., 2001). This study therefore aims to characterize T-cell cytokine responses to selected peptides o f GLURP, and to assess which specific cytokine profiles are associated with cytophilic antibodies in Ghanaians. This would give a better understanding o f the role o f cytokine responses to GLURP and immunity against malaria. In addition, this study also aims to compare antibody levels raised individually against GLURP or MSP3 and a hybrid GLURP-MSP3 antigen in a cohort o f Ghanaian children. These antigens are on phase I clinical trials, yet not much has been done to compare the two antigens in their level o f antibody response in m alaria infections. The knowledge o f the level o f antibody responses to GLURP and MSP3 o f Plasmodium falciparum , and possibly the combined effect o f these two antigens will be very useful in future m alaria vaccine development. Furthermore, characterizing cellular responses to these antigens will provide much needed information regarding which epitopes to include in the GLURP vaccine that will ensure long lasting and protective antibody production. The study therefore hypotheses that GLURP induces cellular responses and antibody responses that are protective, and specific cytokine(s) induced by components o f GLURP will enhance cytophilic antibody production. Objectives 9 University of Ghana http://ugspace.ug.edu.gh The objectives are 1. To measure and compare immunoglobulin G (IgG), and subclass (IgGl-IgG4) responses to GLURP, MSP3 and Hybrid (GLURP-MSP3) in Ghanaian children. 2. To assess and compare the association between individual antibody responses to GLURP, MSP3, and that o f the hybrid GLURP-MSP3. 3. To assess in vitro cellular responses to synthetic peptides o f GLURP (LR129, LR130, M R186, M R187), as measured by stimulation and production o f cytokines (interferon gamma, interleukin-2, interleukin-4 and interleukin-10) in Ghanaian children. 4. To compare cytokine and antibody responses to GLURP in relation to protection, and assess which cytokine(s) may be correlated with cytophilic antibodies measured in Ghanaian children. 10 University of Ghana http://ugspace.ug.edu.gh CHAPTER 2 2.0 LITERATURE REVIEW 2.1 Epidemiology of malaria M alaria mostly occurs in Africa, South East Asia, The Caribbean, Eastern Mediterranean, W estern Pacific, Latin America and parts o f Europe (WHO, 2002), and it presents major socio economic problems. The global prevalence o f malaria is 515 million episodes in 2 0 0 2 , resulting in 1 million deaths annually and most o f these are in children under five year old (Snow et al., 2005). About 90% o f m alaria mortality occurs in Sub-Saharan Africa, this is because the majority o f infections are caused by Plasmodium falciparum , the most dangerous o f the four human m alaria parasites. Moreover, Anopheles gambiae, which is the most effective vector is widespread in Africa and it is very difficult to control. Children and pregnant women living in malaria transmission areas are at the highest risk o f malaria mortality and morbidity. Previous and recent reviews have shown that malaria causes about 10 -2 0 % o f all deaths in children under five years o f age in Africa and kills a child every 30 seconds (Binka et al., 1994; Jaffar et al., 1997; Greenwood, 1999; WHO, 2002; Africa M alaria Report, 2003). 2.1.1 The burden o f malaria The burden o f m alaria on health in all malaria endemic countries in Africa is enormous. Up to 25-40% o f all outpatients clinic visits are due to malaria, and 20-50% o f all hospital admissions are as a result o f malaria infection (Africa M alaria Report, 2003). Malaria infection poses a big burden on the socioeconomic status o f people in endemic areas (Gallup and Sachs, 2001; Shephard et al., 1991). Child malaria mortality rates 11 University of Ghana http://ugspace.ug.edu.gh have been shown to be higher in poorer households, since poor people are at risk o f becoming infected frequently. This has been shown in a demographic surveillance carried out in Tanzania, in which under five years mortality due to malaria was found to be 39% higher in the poorest socioeconomic group (Mwageni et al., 2002). A survey done in Zambia also found a significantly higher prevalence o f malaria infection among the poor population (Zambia Roll Back Malaria Report, 2001) and similar observation was made in Northern part o f Ghana (Akazili, 2002). These could be due to settlement conditions o f poor families, un-affordability o f mosquito treated nets, and inability to pay for effective malaria treatment. 2.1.2 Malaria distribution in Africa There is a wide distribution o f malaria in Sub Saharan Africa, approximately 63% of people in Sub Saharan Africa live in malarious areas. M alaria is endemic throughout Zambia, an estimate o f 4.8 million (32%) cases o f malaria each year among a population o f about 10 million. 35.6% hospital admissions is due to m alaria and 14.8% mortality rate. Chloroquine resistance to malaria has been estimated between 20-50% in Zambia (RBM/WHO, 2000; Zambia Fact Sheet, 2001). 90% o f the Ugandan country is highly endemic, with an estimated 5.3 million cases o f malaria per year for a total population of 21 million. Outpatient m alaria cases are between 25-40%, and 20% hospital admissions and 9-14% death cases is attributable to malaria infection (RBM/W HO, 2000). In Rwanda 1.2 million cases o f malaria is reported every year. In Kenya, an estimated 8.2 million cases o f m alaria are reported annually for a total population o f 30 million (Kindermans, 2002), resulting in the death o f about 26000 children under five years everyday. In Tanzania, malaria is a leading cause o f mortality and morbidity with 16 12 University of Ghana http://ugspace.ug.edu.gh million cases every year. It is estimated that 93.7% o f Tanzanian’s 32.8 million people are at risk o f the disease. In Ghana, malaria is hyperendemic, P. falciparum accounts for 90% o f infection, P. malariae (9.9%) and P. ovale (0.1%). Clinical malaria results in 42­ 45% o f outpatient hospital visits and 22% o f under-5 year old mortality (Binka et al., 1994; RBM, 2005). The morbidity o f m alaria has increased yearly due to deteriorating health systems, insecticide and drug resistance, climatic changes and civil war. The greatest challenges facing m alaria eradication in Africa is drug and insecticide resistance, since malaria control in Africa is based mainly on chemotherapy (WHO, 1999; White, 1999; Trape, 2001; Greenwood, 2002). Resistance to chloroquine is now widespread in 80% o f the 92 countries where malaria continues to be a major killer, while resistance to other drugs o f choice for treatment is increasing (RBM/WHO, 2000). Development o f a malaria vaccine or other new immunotherapy’s may lead to a decrease in m alaria morbidity and mortality especially in children. This can be possible by understanding the complex life cycle, and the interactions o f the parasite with the immune system o f the host. 2.2 Life cycle The complete life cycle o f P falciparum involves both the mosquito and the human host (Figure 2.1). Briefly, malaria transmission begins with the bite o f an infected female anopheles mosquito, which injects sporozoites into a human host during feeding. These sporozoites enter the blood circulation, and invade the hepatocytes, where they undergo asexual stage o f reproduction for 9-16 days. The liver cells then rupture to release merozoites into the bloodstream. These merozoites invade the red blood cells, reside in the parasitophorous vacuole, undergo development to the early ring stage 13 University of Ghana http://ugspace.ug.edu.gh trophozoite, late trophozoite, and by mitotic division mature into the schizont stage that contains about 32 merozoites. The schizonts are released into the blood when the red blood cells rupture and invade new red blood cells for repetitive intra-erythrocytic cycle. After a number o f intra-erythrocytic cycles, the merozoites develop and, differentiate into infective female and male gametocytes, which are ingested by the mosquito during a blood meal. Sexual reproduction o f gametocytes takes place in the midgut o f the mosquito, resulting in the production o f zygote, which develops into oocysts through the process o f sporogony to release sporozoites. The sporozoites invade the salivary glands, and enter into man when the infected female mosquito feeds on man and the cycle continues. 2.3 Case definition o f clinical malaria Clinical disease caused by the parasite is due to asexual multiplication o f blood stage parasites, therefore a lot o f focus has been put on the erythrocytic stage with the view of identifying antigens targeted for protective immune responses. The clinical manifestation o f P. falciparum infection includes chills, fever, headache, nausea, vomiting, and diarrhoea. Major complications o f P. falciparum malaria in children are cerebral malaria, severe anaemia, convulsions and respiratory distress. The definition of clinical malaria is usually based on the microscopic detection o f malaria parasitemia, fever, and febrile temperature > 37.5 °C. It has been established that fever due to malaria is induced by the release o f parasite toxins when schizont infected red blood cells rupture to release merozoites (Kwiatkowski et al., 1989). Hence, malaria disease is said to be directly caused by parasite multiplication that leads to febrile temperatures. The use o f febrile temperature > 37.5 °C and parasitaemia at various levels results in highly sensitive and specific malaria case definition (Armstrong et al., 1994). It should however 14 University of Ghana http://ugspace.ug.edu.gh be noted that individuals in highly endemic areas may have parasites without fever or other symptoms associated with malaria. 2.4 Malaria immunity 2.4.1 Innate immune response The innate immune system mediates a nonspecific protection through monocytes, macrophages, dendritic cells, natural killer (NK cells) cells, eosinophils, neutrophils, mast cells, complement and acute phase proteins. It also includes physical barriers like epithelial layers, and anti microbial substances on these surfaces. In innate immunity, neutrophiles, mononuclear phagocytes and NK cells are known to play a role in malaria infections. NK cells have been shown to be involved in the lyses o f P. falciparum infected erythrocytes in vitro. They elicit the production o f cytokines such as interferon- y (IFN-y), which activates macrophages leading to phagocytosis o f invading foreign particles (Orago et al., 1991; Artavanis-Tsakonas; Riley, 2002). In addition, innate immune mechanisms by NK cells leads to the stimulation o f IFNy, which limits the initial phase o f parasite replication, this has also been demonstrated in studies done on murine m alaria (Doolan and Hoffmann 1999; Fell and Smith, 1998; Mohan et al., 1997). 2.4.2 Acquired immunity 2.4.2.1 Humoral immune response M alaria infection induces strong humoral immune responses through the production of high concentrations o f immunoglobulins (Ig), especially IgG and IgM as well as IgE. The extent o f protective immunity acquired against malaria infection in humans and mice has been shown to be associated with the level o f antibody against the asexual blood stage antigens (Piper et al., 1999; Hirunpetcharat et al., 1998; Astagneau et al., 15 University of Ghana http://ugspace.ug.edu.gh 1995). The importance o f antibody in malaria immunity is evident from the protection conferred to neonates and infants by malaria specific antibodies acquired by mothers (McGregor et al., 1963; Sabchareon, et al., 1991). Passive transfer o f monoclonal antibody against plasmodium parasite antigens conferred protection in naive mice (Spencer et al., 1998; Narum et al., 2000). Although various immunoglobulins may confer protection to individuals with malaria, IgG is the most important o f all. The use o f purified immunoglobulin from sera o f African adults in clinical trials to treat some sick children drastically reduced clinical symptoms and parasitaemia (Bouharoun- Tayoun et al., 1990). This has established that immunoglobulin G (IgG) is a main component o f protection against the asexual blood stage o f P. falciparum (Druilhe et al., 1994). Cytophilic antibodies have been shown to play critical role in anti-malaria immunity. They may act in collaboration with monocytes and macrophages by attaching to certain parasite antigens by their Fc receptors and lead to phagocytosis or release o f toxic factors that kill infected cells. Studies done on the role o f subclass responses in naturally acquired immunity are important. Increased levels o f cytophilic antibodies (IgGl and IgG3 subclasses) have been found in individuals protected from malaria (Bouharoun- Tayoun et al., 1992; Sarthou et al, 1997; Oeuvray et al., 2000). Further studies done by Aribot et al., 1996 revealed the association between high levels o f parasite specific IgG3 and malaria. In addition, higher levels o f IgG3 antibodies have been found in certain populations and its association with malaria infection has been reported (Aribot et al., 1996 and Rzepezyk et a l, 1997; Ndungu et al., 2002). It has also been revealed that 16 University of Ghana http://ugspace.ug.edu.gh repeated m alaria infections are associated with elevations in total IgE and its regulation in T-cell activities (Perlmann et al., 1994). Mechanism o f antibody response: Antibodies are known to mediate protection against malaria, through various mechanisms. Results from studies performed in vivo suggest that one mechanism may confer clinical immunity to m alaria by antibody interruption o f parasite multiplication (M cGregor et al., 1964; Sabchareon et al., 1991). Antibodies against blood-stage merozoite antigens may block parasite invasion o f erythrocytes or make them susceptible to phagocytosis, leading to reduction in parasitemia (Blackman et al., 1994; Holder et al., 1992). Other mechanisms are clearance o f infected erythrocytes from circulation by antibodies binding to their surface via Fc receptors and its elimination from the body (Udeinya, 1981; Bouharoun-Tayoun et a l, 1990 and 1995). Some o f the parasites elicit antibodies that form clumps or rosettes which the immune system recognize and clear them from circulation by opsonization or phagocytosis (Treutiger et al., 1992). Antibody dependent cell-mediated cytotoxicity o f parasites may be elicited through cytophilic antibodies, as well as parasite inhibition by effector cells like neutrophils and monocytes (Bouharoun-Tayoun et al., 1990; Groux and Gysin, 1990). Parasite agglutination and indirect effects like the antibody dependent cellular inhibition (ADCI) (Oeuvray et al., 1994) are other immune mechanisms that may protect against P. falciparum malaria. The ADCI associated killing o f parasites is mediated by cytophilic antibodies, which act in collaboration with monocytes to release soluble factors from macrophages and monocytes to destroy parasites (Bouharoun-Tayoun et al., 1995). The malarial parasites also mount up immune evasion mechanisms. These mechanisms involve antigenic variation, since so many different parasite antigens are 17 University of Ghana http://ugspace.ug.edu.gh presented to the immune system. Others are manipulation o f the host immune response that could contribute to pathological changes, polymorphism o f parasite protein and competition between protective and non-protective responses (Troye-Blomberg et al., 1999). 2.4.2.2 Cell mediated immunity Although antibody plays a major role in malaria immunity, T-cells and cytokines are also known to be involved in the immune regulation and effector phases o f anti malaria immunity through T helper cells (Weidanz et al., 1988). Cell mediated immune responses against m alaria is thought to be protective against both pre-erythrocytic and erythrocytic parasite stages (Troye-Blomberg and Perlmann, 1994). The importance o f cytokines in conferring protective immunity to malaria infection in animal models has been documented (Kobayash et al., 1996; Shear et al., 1990; Stevenson et al., 1990). Several studies have revealed cellular mechanisms such as lymphocyte proliferation, IFNy production, activation and killing o f parasites by macrophages when peripheral blood mononuclear cells (PBMC) from immune individuals were stimulated with malaria antigens in vitro (Troye-Blomberg et al., 1984 and 1985; Ballet et al., 1985; Brown et al., 1986; Ockenhouse et al., 1984). W hen similar studies were done on non- immune individuals infected with P. falciparum, it revealed a decreased cellular recognition o f plasmodial antigens (Ho et al., 1988; Theander et al., 1986). T-cells that regulate antibody production are also involved in both inflammation and its regulation via cytokine production. The differentiation o f TH-cells into their subsets ThI and Th2 may have important biological and immunological implications towards the susceptibility or resistance to particular diseases or infection (Troye-Blomberg and Perlmann, 1994). 18 University of Ghana http://ugspace.ug.edu.gh Thus different subsets o f Th cells play different roles in terms o f inflammation or anti inflammation in malaria infection. M echanisms o f cellular immune response: Studies have revealed that ThI and Th2 cells are responsible for the regulation o f antibody mediated immunity and cell-mediated immunity. This is evident from a study in which T-cells regulated antibody production via ThI and TH2 induced cytokine production (W eidanz et al., 1988). ThI cells secrete cytokines like interleukin-2 (IL-2), interferon (IFN) y, Tumour growth factor (TGF) (3, and tumour necrosis factor (TNF) a. These cytokines activates macrophages that helps in opsonization through IgG antibodies, promotes inflammatory response leading to tissue injury, and mediates delayed hypersensitivity reactions (Abbas et al., 1996). Th2 cells secrete cytokines like IL-4, IL-5, IL-6 , IL-10, and IL-13. These cytokines stimulates the proliferation o f mast cells and eosinophils, and provides help to B-cells during infection (Abbas et al., 1996). These Th cells cross-regulate the differentiation and activities o f each other through the cytokines that they produce. Studies conducted revealed that Th2 secreted cytokines regulate the functional activity, and production of ThI secreted cytokine in P. falciparum infection (Ho et al., 1995). The ThI secreted cytokines are known to down regulate the development o f Th2 cytokines, whereas the Th2 cells also secrete cytokines that down regulates ThI cell maturation (Troye- Blomberg and Perlmann, 1994). For instance, IFN y secreted by ThI cells inhibits the development and proliferation o f Th2 cells, as well as IL-4 and IL-10 secreted by Th2 cells inhibit the development o f ThI cells. It has been shown that cytokines like IL-1, IL-6 , IFN-y, and TN F-a may be protective by inducing parasite killing by macrophages 19 University of Ghana http://ugspace.ug.edu.gh and neutrophils (Kumaratilake and Ferrante, 1994; Taylor-Robinson et al., 1993; Troye- Blomberg et al., 1999). 2.4.2.2.1 Role o f cytokines in malaria immunity The role of ThI secreted cytokines: ThI cells are responsible for cell-mediated immunity, by activating macrophages and other effector cells to release inflammatory cytokines. A number o f cytokines play a significant role in the development o f acute or chronic inflammatory response to P. falciparum antigens. IFNy plays an important role in resistance to blood stage malaria infection through enhanced activities o f macrophages. W ork done by Luty et al., 1999, have shown an association between IFNy responses to the liver and blood-stage antigens, and resistance to m alaria re-infection. W hen macrophages were activated with IFNy, it resulted in the activation o f phagocytic cells and killing o f m alaria parasites (Ockenhouse et al., 1984). Studies done with human serum have shown a correlation between IFNy levels with resistance to P. falciparum m alaria (Deloron et al., 1991; Riley et al., 1988). In addition, it has been demonstrated that IFNy concentrations increased in plasma o f individuals with symptomatic malaria infection than in asymptomatic individuals (M shana et al., 1991). Furthermore, high levels o f IFNy production by antigen stimulated PBMC in vitro correlates with reduced risk o f clinical malaria in Ghanaian children (Dodoo et al., 2002). An association between IFNy production from peripheral blood mononuclear cells (PBM C’s) in response to soluble antigens o f P falciparum and increased risk o f malarial 20 University of Ghana http://ugspace.ug.edu.gh infection has been demonstrated (Riley et al., 1991). PBM C’s from children with mild Plasmodium falciparum infection produced high levels o f IFNy when stimulated in vitro with malaria antigen. These children indicated a lower risk o f re-infection when cleared from parasitaemia by drug treatment. Whereas children with severe malaria showed lower levels o f IFNy, when their PBM C’s were stimulated in vitro with malaria antigens, which makes them more susceptible to re-infection (Luty et al., 1999). In animal models, IFNy-deficient mice with P. c. chaubaudi infection resulted in increased malaria morbidity and mortality (Su and Stevenson, 2000). This indicates the important role that IFNy plays in protection against malaria infection. Other research in malaria have revealed an increased expression o f IFNy during parasite resolution, as well as decreased IL-2 production, suggesting a regulatory mechanism o f cytokines in malaria infection (W inkler et al., 1998). Although IFNy is known to be essential in the resolution o f primary infection, its over-production predisposes individuals to severe immunopathology (Waki et al., 1992). IL-2 cytokine, which is secreted by ThI cells, induces proliferation o f antigen-primed cells and enhances activity o f NK cells. A study done in which PBM C’s were stimulated with malaria-specific antigens during acute malaria infection, revealed no detectable levels o f IL-2 production. This suggests that there may be defective cell mediated immune response to these malaria specific antigens during acute P. falciparum infection, which might have resulted in immune unresponsiveness (Ho et al., 1988). Measurement o f plasma IL-2 levels in some West African children showed no significant association o f IL-2 response and malaria infection. Furthermore, studies conducted in a malaria- endemic community in West Africa revealed no association between plasma soluble IL- 21 University of Ghana http://ugspace.ug.edu.gh 2 receptor levels and in vitro proliferative responses o f peripheral blood mononuclear cells (PBM C’s) to some m alaria antigens. In contrast, a high soluble IL-2 receptor was detected in the plasma o f malaria infected individuals (Riley et al., 1993), a funding which did not support the idea o f immune suppression in malaria infection from previous studies (Ho et al., 1988). The role o f Th2 secreted cytokines: Th2 cells regulate humoral immunity by providing help to B cells in antibody production. Cytokines such as interleukin-4 (IL-4) and interleukin-10 (IL-10), which are Th2 secreted cytokines are known to elicit anti­ inflammatory responses to malarial antigens. IL-10 cytokine has been shown to regulate the functional activity and the production o f T hl cytokine (Ho et al., 1995 and 1998). It is also known to induce B-cell proliferation, immunoglobulin production, leading to the development and maturation o f anti-malaria antibodies (Moore et al., 1993). It has also been reported that higher IL-10 levels were found in children and adults with clinical malaria than healthy controls (Joao et al., 1997; Peyron et al., 1994; and Deloron et al., 1994). Other studies done to compare specific cytokine responses o f patients with or without malaria in Malawi revealed higher serum IL-10 levels in malaria patients than in other patients (Jason et al., 2001). Experiments done have also shown an increased IL- 10 as well as TN Fa levels during malaria infection (W enisch et al., 1995). Othoro et al., 1999 predicted that higher plasma IL-10 over TN Fa levels might provide protection against severe m alaria anemia by down regulating the immuno-pathologic effects o f the later. The same study revealed that children with mild malaria had significantly higher IL-10 to TN Fa ratios than children with severe malaria. Furthermore, TNFa to IL-10 ratios were found to be significantly lower in patients with severe malaria anaemia than 22 University of Ghana http://ugspace.ug.edu.gh in patients with uncomplicated malaria (Kurtzhals et al., 1998). This means that the lack or insufficient IL-10 response in comparison to TN Fa may predispose an individual to severe malaria. Studies done with animal models also suggest the significant role that IL-10 plays in down regulating severe malaria. Although IL-10 plays a role in humoral immunity, it has been shown to play other roles, for instance in an in-vitro model, IL-10 was found to decrease the production o f IL-6 and TN Fa and IFNy secretion and function (Moore et al., 1993; de W aal et al., 1991). These findings support the idea that IL-10 is a critical factor in down regulating the pathogenesis o f severe malaria. Interleukin-4 (IL-4), a Th2 secreted cytokine induces the proliferation and differentiation o f B cells. Studies have demonstrated that IL-4 production by CD4+ T cells is frequently associated with serum antibodies (Troye-Blomberg et al., 1990; Riley et al., 1991; Kulane et al., 1997), suggesting that IL-4 plays a role in certain anti- malarial antibody responses. The finding o f high serum IL-4 levels in parasitemic individuals in a holoendemic P. falciparum region (M shana et al., 1991) indicates the significant role that IL-4 plays in clearing parasites. However, IL-4 has been shown to facilitate parasite survival by suppressing anti-parasite activities by macrophages (Kumaratilake and Ferrente, 1992). Research conducted by W inkler et al., 1998 revealed no direct involvement o f IL-4 in the clearance o f parasitemia, since IL-4 was shown to suppress macrophage anti-P falciparum activity in vitro (Kumaratilake and Ferrente, 1992). The percentage o f lymphocytes making IL-4 cytokines in adults was found to be significantly lower in malaria patients than those without malaria infection in an endemic area (Jason et al, 2001). In addition, the lack o f IL-4 production in malaria patients found in a number o f studies suggests that IL-4 may not be a critical factor in 23 University of Ghana http://ugspace.ug.edu.gh the pathogenesis o f malaria (Othoro et al., 1999). There has been a limitation into studies in IL-4 responses in human infection because it has been difficult to detect IL-4 cytokine in supernatants (Kurtzhals et al., 1992). 24 University of Ghana http://ugspace.ug.edu.gh 2.5 M alarial antigens There have been attempts to identify and characterize antigens on the asexual stage o f the parasite (Figure 2.1), that may be o f importance in the development o f protective immunity to malaria (Warrell, 1993). Some o f the antigens found on P falciparum include, the merozoite surface protein 1-19 (MSP 119), the M erozoite surface protein 3 (MSP3), the Circumsporozoite Surface Protein (CSP), P. falciparum Erythrocyte Membrane Protein (PfEM Pl), Apical Merozoite Antigen 1 (AMA1), Liver stage antigen (LSA) and the Glutamate Rich Protein (GLURP). Good e t a l., 1988 (Modified) Fig 2.1 The life cycle of the P. falciparum parasite indicating stage specific antigens. 25 University of Ghana http://ugspace.ug.edu.gh In a number o f recent immuno-epidemiological studies, some antigens have been identified as possible targets o f protective antibody-immunity to malaria. These include GLURP, MSP1-19, AM A1, and MSP3, which are now currently in phase I clinical trials (Dodoo et al., 2000; Oeuvray et al., 2000; Theisen et al., 2001). Also, CSP (Hoffman et al., 1986), LSA and PfEM Pl (Bull et al., 1998) have been studied in association with protection. 2.5.1 Pre-erythrocytic stage antigens Circumsporozoite surface protein (CSP) antigen is found on the surface o f matured sporozoites. W hen host antibodies bind to CSP on sporozoites, the CSP-antibody complexes are shed through a process called the circumsporozoite protein reaction and may serve as defense against host immunity. Antibody directed against CSP can inhibit sporozoite invasion into liver cells (Hoffman et al., 1986). Liver Stage Antigen (LSA) Liver stage antigens are processed and presented by the liver cell. This leads to recognition by T-cytotoxic cells and killing o f the infected cell. It evokes the stimulation o f T cells to produce cytokines such as y-interferon and IL-10 that ultimately lead to the death o f the intracellular parasite. Thus a pre-erythrocytic stage vaccine will prevent the establishment o f parasites in the blood stage and will provide protection against clinical malaria. 26 University of Ghana http://ugspace.ug.edu.gh 2.5.2 Asexual blood stage antigens The M erozoite Surface Protein 1 found on the surface o f blood stage merozoites is a possible m alaria vaccine candidate. A conserved 19-kDa part o f the protein is attached to the merozoite during erythrocyte invasion and expressed by the parasite during the early ring stages. Antibodies raised against the conserved fragment may block merozoite invasion into erythrocytes and inhibit parasite multiplication (Holder et al., 1992). The Merozoite Surface Protein-3 (MSP3) is a 48KDa protein; antibodies raised against MSP3 antigen protect subjects clinically from m alaria through ADCI mechanism. Cytophilic antibodies raised against this antigen can clear parasites by opsonization or phagocytosis (Bouharoun-Tayoun et al., 1995). Plasmodium falciparum Erythrocyte Membrane Protein-1 (PfEMP-1) is a highly variant antigen o f approximately 300 kDa expressed on surface o f erythrocytes infected with late schizont stage o f the parasite. It is coded for by about 50 genes o f the ‘var’ multigene family (Smith et al., 1995). PfEMP-1 has been identified as the molecule responsible for intra-vascular sequestration o f P. falciparum infected blood cells. The role o f PfEMP-1 in cytoadherance o f infected erythrocyte antibodies to variants o f this protein have been reported to occur in people living on malaria endemic regions. (Baruch et al., 1997; Biggs et al., 1990). The Glutamate Rich Protein (GLURP) is a 200-kDa parasite protein found on the surface o f merozoites, located in the parasitophorous vacuole o f exo-erythrocytic, erythrocytic and mature schizont-infected erythrocytes (Borre et al., 1991). GLURP is synthesized during all stages o f the parasite in the host and is found on the surface o f 27 University of Ghana http://ugspace.ug.edu.gh newly released merozoites (Borre et al., 1991). GLURP has been characterized molecularly (Borre et al., 1991), and several studies done on antibody responses to GLURP indicate the importance o f anti-Glurp antibodies, more especially, cytophilic antibodies in mediating immunity against malaria (Dodoo et al., 2000; Oeuvray et al., 2000; Theisen et al., 1998 and 2001). 28 University of Ghana http://ugspace.ug.edu.gh 2.5.3 Immune responses against GLURP and MSP3 A large number o f parasite antigens play important role in conferring protection to malaria infection. GLURP and MSP3 have been identified as target antigens for antibodies involved in antibody dependent cellular inhibition mechanism (Figure 2.2) (Oeuvray et al., 1994; Theisen et al., 1998). Human monocytes can cooperate with antibodies to exert this ADCI mechanism in vitro (Bouharoun-Tayoun et al., 1990; Khusmith et al., 1983; Oeuvray et al., 1994; Theisen et al., 1995) and also, in humanized SCID mouse model (Badell et al., 2000). High levels o f GLURP and MSP3- specific antibodies were associated with acquired protective immunity to malaria (Bouharoun-Tayoun et al., 1995; Dodoo et al., 2000; Oeuvray et al., 1994). Furthermore, high affinity-purified human IgG antibodies to GLURP have been found to promote a strong monocyte-dependent inhibition o f P. falciparum in vitro (Theisen et al., 1998). G L U R P -s p e c if ic hum an IgG antibod ies inhibit parasite growth in v itro in collaboration with m o n o cytes malaria immune Ig G 1 D ru ilh e e t al. A n ti-G L U R P IgG R O -IgG > R 2 IgG T h e is e n , M . , et. a l. 1998. In fe ct. I m m u n . 6 6 :1 1 -1 7 Fig 2.2: GLURP IgG and ADCI mechanism in vitro 29 University of Ghana http://ugspace.ug.edu.gh In Ghana, GLURP IgG l and IgG3 responses were significantly associated with protection from P. falciparum malaria infection in children (Dodoo et al., 2000). These data on GLURP and MSP3 suggests that cytophilic antibodies against both antigens contributed to the development o f clinical immunity in W est African children (Oeuvray et al., 1994 and 2000). Previous studies have shown that purified human IgG antibodies against non-repeat epitopes o f GLURP inhibit parasite growth in vitro in a monocyte-dependent manner (Theisen et al., 1998 and 2001). Further studies conducted in Asia have shown an association between cytophilic antibodies against MSP3 and protection against clinical malaria (Soe et al., 2004). The levels o f human IgG antibodies found against GLURP peptides were significantly associated with the absence o f disease in Ghanaian children (Theisen et al., 2001). In addition, these peptides were demonstrated to be targets o f cytophilic antibody responses in individuals living in endemic areas. T-cell responses to these peptides also revealed that T-cell epitopes were detected in LR 67 and LR 6 8 peptides. This suggests that the non-repeat region o f GLURP may contain T-helper-cell epitopes that are recognized by individuals exposed to malaria (Theisen et al., 2001). There is evidence that production o f an anti P falciparum antibody in vitro requires T-cell-derived signals from cytokines (Garraud et al., 1999). It is still unknown which cytokines induce cytophilic antibody production in humans to enhance protective immunity against malaria. This present study aims to characterize cell-mediated and responses to selected GLURP peptides (LR129, LR130, MR186 and MR187) and antibody responses to the recombinant Non-repeat N-terminal antigen, R0. In addition, to identify cytokines 30 University of Ghana http://ugspace.ug.edu.gh associated with GLURP, and to assess which specific cytokine profiles are associated with cytophilic antibodies in a cohort o f Ghanaians. Most studies done on GLURP were on antibody responses, which do not provide much information on cytokine responses to synthetic peptides and recombinant antigens o f GLURP. M uch information is needed regarding which epitopes to include in the GLURP and MSP3 vaccine being developed, which will ensure long lasting and protective antibody production. 31 University of Ghana http://ugspace.ug.edu.gh CHAPTER 3 3.0 M ATERIALS AND METHODS 3.1 Study area and study population Plasma and cell samples obtained in a previous immuno-epidemiological project conducted in Dodowa were used for the study. Dodowa is a semi rural town approximately 50km from Accra, mainly a subsistence fanning community and has a population o f about 6,500. It has two rainy seasons; the major rainy reason is from May- August, and the minor one from October-November, followed by a dry season from December to April. Although malaria transmission is perennial, it is highest during or immediately after the rainy season (high-transmission season) and lowest during the dry season (low-transmission season). It has been estimated that individuals in Dodowa are exposed to about 20 infective bites per year, and 98% o f the infections are due to P. falciparum (Afari et al., 1995). Dodowa can thus be described as an area of hyperendemic and seasonal malaria transmission. The transmission is stable because it does not vary considerably from year to year. Plasma and peripheral blood mononuclear cells (PBM C’s) were obtained from a cohort o f 300 children, 3-15 years o f age prior to and at the end o f the malaria transmission season for immunological studies. A longitudinal morbidity survey was done over a period o f 18 months covering two malaria transmission seasons. The children were enrolled into the study after informed consent has been obtained from the parent of each child. Those included in the study were children who do not carry the sickle cell trait or genotype. Each child was visited weekly, during which physical examination was done, and the body temperature measured. In the case o f fever above 37.5°C or other 32 University of Ghana http://ugspace.ug.edu.gh symptoms o f malaria, a malaria blood slide was made and treatment offered if the slide was positive for malaria parasites. Children were said to have malaria if they had febrile temperature above 37.5°C and parasitemia > 5000|j.l in blood. In the case o f severe symptoms like convulsions, respiratory distress and severe anaemia, the child was referred to the hospital. Children with asymptomatic malaria i.e. parasitemia without clinical symptoms, were not treated. During the clinical and parasitological follow up, once a month, a blood smear was prepared by finger pricking from all children irrespective o f symptoms to identify asymptomatic malaria infections. The parents or guardian and children were encouraged not to start self-treatment or seek treatment elsewhere unless the field team was not available. They were also encouraged to inform the research team if they did otherwise. 33 University of Ghana http://ugspace.ug.edu.gh 3.2 Study procedures 3.2.1 Antigens The P falciparum GLURP recombinant protein RO and peptides from the N-terminal non-repetitive region o f GLURP, LR129, LR130, MR186 and M R187, and LR55 from MSP3 antigen were kindly supplied by Dr. Michael Theisen from the State serum Institute, Denmark. Amino acid sequences o f GLURP peptides, MSP3, and the GLURP- MSP3 hybrid are shown in Table 3.1 and Figure 3.1. Crude malaria schizont antigens were prepared and used in stimulation assay to assess responses to total blood stage soluble antigens. 34 University of Ghana http://ugspace.ug.edu.gh L_U LL l LUU U LU L<: KLLUU LX U LU O Q LLlU > Z OLU QZ Z —} L_UJ L C* U < Q _l LU 2i | O CO 2 CzO o g £1 “LU x | %*> <3 ... *LU 9> Z > w LU ZO s * -- ^ HI ^ X z LU C/3 < ^ C^D Q9- z > 20] Ca/3 0lu- CL1- /3 >Z 5 CO ^ n a^ co O — U CD * £ LU CLUO □ LU L > £ i s X w LU U Z□ < Z S> o* LU Q t o z 1 __j co _i — a£ Z 2 § aZ C* L i w Ci/3 ! O i lu p iZ. i 1 “LU z z ^—1 CL/3l Z< LLUU 1L-U XZ a lu v; i s * * hCD a clq_l: C*O W -1 v s i^ s i X LCUO 2O; Qz LU g LU o- O z Z £3 mO CJ lu< cl z* Oo CD LU d H X ^ 1) <2 > ouj Q£- 2< °u- oa a*: LaU 0__1 lu wu3 a ocQJr a Z Cz/3 1LU *c<3 1-/D L—UI —Zi oCO CO O CO > a*: Q>- a CO C\ TJ o CO CO 00a 5 _ :j a: a: imn 5 _j |tNo C£ e r> CO aQo _i c at l 2 2 a. w W cu *-1 Cd Q w Cu X CO Q w Q D-i Oj Cd Oi M W H C£1 Q H < w Q Eh U. 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Oi 1—3 Cu i—l w CO U t/5 w Q > a Cu 2 MCO CO Oi < > Hq CO Da CO W Q CO W cu H > w Pm ca C£1 Cd M 2 2CO Cu 2 OI CU CD r2j OS X X CO a w 2 2 Da 2 £3 Qa a OI a Q hi - > > cu < > w w w Da P O a 2 Cd ►-a C£l n a cu gCO w X 2 M CD 2 o i a s co 2 (X Z pc; 2 C J w H w cu H a H M CO Cu 2 co Z u M CU >- 2 > o u o M Cd Da Ehs Q CO Oi a Da q cu 2 CO Q Cd j w 22 co Cd Oi > X w3 CO H Cd M 2 Da CO O' Cu 2 2 2 H > >QJ 1— 1 Eh 2 EhC« CO Cd X Da 1— 1 Q O Ol X SG 2 X Cd CO < CO u CO 2 w CU OI J S3 Eh 2 O Q 5J> *-1 >H 2 H Dao Q Q X CO w a a M w > r,lcc CO X a < s E X M w OI DaW a i—i 2 Da » Oj CO Eh cu Q X H 'O CD Eh cu a »-a Da CU OJ O Cd Cu 2 CO > u M cu cu cu Q Da css cc w M H CO o^1 (-1 CO M Eh cs - a 23 w CU J 2 Da 111 aj c^ CO aCd 2 w Da !S2 X Cd is: C£l 3 Cd Eh 2 X cu Cu O CO O > .-1 Eh O Eh ID Q h-3 < Da HW Cd OJ CU X X fH P$ a a 5 w Q 2 Q W Q 2 O CO X tt a Cd Q Cl] > X CO J* Sa3. pl5A pAM(3l EEKEEENEKKKEQEKEQSNENNDQKKDM EAQNLISKNQNNNEKNVKEAAESIM KTLAGLIKGNNQIDSTLKDLVEELSKYF University of Ghana http://ugspace.ug.edu.gh 3.2.2 Continuous cultivation of malaria parasites In vitro cultivation o f P. falciparum was done using cryopreserved infected erythrocytes, by the continuous culture technique with slight modifications. Frozen laboratory strain parasites (3D7) were removed from liquid nitrogen, thawed quickly in a water bath pre­ set at 37°C. The mixture was centrifuged at 1500 rpm for 10 minutes and the supernatant discarded. Equal volume o f thawing mixture (3.5% sodium chloride in sterile distilled water) was added, thoroughly mixed, and the suspension spun again at 1500 rpm for 10 minutes. The supernatant was removed and 1ml o f complete parasite medium (consisting o f RPM I-1640, Albumax (Gibco BRL, USA), Gentamycin and L-glutamine at 10|ig/ml, (Sigma) were added to the parasite culture, and resuspended for washing. After washing and removal o f supernatants, the parasites were added to culture flasks containing 5ml o f complete parasite culturing medium and 200|il 0 + packed red blood cells to provide optimum conditions for growth. The culture medium was gassed with a special gas mixture (2% O2, 5.5% CO2 and 92.5% N 2) and incubated at 37 °C. The culturing medium was changed routinely to prevent changes in pH and accumulation of toxic substances that may inhibit parasite growth. This was done by gently removing the supernatant from the parasite culture and then mixed thoroughly. After thorough mixing o f the infected red blood cells in the culture flask, a thin slide preparation was made by placing a drop o f parasite culture on a slide and smeared to a thin smooth layer. The slide was then fixed in methanol for about 10 minutes and stained in 10% Giemsa buffer for 15 minutes, after which the Giemsa stain was washed o ff with water and the slide dried. The erythrocytes were counted under the microscope (Olympus BH2 Microscope) 37 University of Ghana http://ugspace.ug.edu.gh at 100X magnification. The parasitemia was estimated, and the various parasite stages identified and recorded. The rate o f parasite growth is estimated by: % parasitaemia = Number o f parasite infected RBC / Total number o f red blood cells x 100 When parasitemia level was about 2.5% with a high number o f the late trophozoites and schizonts, a sub-culture was made by transferring 1.5ml o f parasite culture into a bigger culture flask for optimal growth. Then 1ml o f packed RBC and 20ml o f complete parasite medium added and gassed. The cultures were maintained until the parasites matured to the schizont stage with parasitemia o f approximately 5% or higher for use in the preparation o f crude schizont antigen. 38 University of Ghana http://ugspace.ug.edu.gh 3.2.3 Preparation o f the crude malaria schizont antigen Crude malaria schizont antigen was prepared as soluble antigens o f P falciparum. Erythrocytes infected with P falciparum in the late trophozoite and mature schizont stages (5% parasitaemia or higher) were separated from uninfected and ring-stage infected erythrocytes by magnet-activated cell sorting (MACS; Miltenyi-BioTec, Bergisch Gladbach, Germany), (Miltenyi et al., 1993). The MACS column was flushed initially with 2% foetal bovine serum (FBS) in phosphate buffered saline (PBS). Infected erythrocytes from in vitro cultures were mixed and passed gradually through a size C MACS column mounted with a 0.9mm x 40mm needle (Becton, Dickinson, Fraga, Spain). The column was further washed with 2% foetal bovine serum (FBS) in PBS until no erythrocytes were seen in the eluate. The erythrocytes infected with parasites in the late developmental stages were retained specifically due to their high content o f paramagnetic haem ozoin (Paul et al., 1981). The column was removed from the magnetic field and the late stage infected erythrocytes eluted by washing the column with buffer (2.0% FCS in PBS). The eluate collected was centrifuged at 2000 rpm for 10 minutes and the supernatant discarded. The cells were resuspended in 3ml wash medium, and 50^1 o f cell suspension was then added to 950|il o f wash medium (20-fold dilution) and pipetted into the slide chamber (Neubauer improved 0.100mm, Germany) for counting under a microscope. The schizont enriched RBC preparation is estimated by: Number o f RBC/ml = Number o f cells counted X dilution factor X 104 Number o f fields After counting, the cell suspension was adjusted to a concentration o f 5.0 x 106 cells /ml by adding complete parasite medium. Parasite lysate antigen was prepared from the 39 University of Ghana http://ugspace.ug.edu.gh eluate enriched for the schizont stage by 3 cycles o f freezing and thawing. The parasite antigens from the lysed RBC released into solution was centrifuged at high speed o f 16000 rpm for 30 minutes to remove the cellular debris. The supernatant containing the parasite soluble antigens was removed and stored at -80 °C until required. The prepared crude antigen was used as controls in stimulation assays. 3.2.4 Antibody measurements Antibody levels in plasma against GLURP and MSP3 antigens were measured by an indirect ELISA method (Sousa et al., 1998). Ninety-six well microtitre plates (Maxisorp; Nunc, Denmark) were coated (0.1M NaHCo3 pH 9.6) overnight at 4°C with lOOjal/well o f goat anti-human F(ab)2 antibody (Sigma, Aldrich) in PBS and 0 .1M N aCl at 2 jig/ml in columns 1-2 o f each plate. Then 100)il/well o f 0.2(ig/ml recombinant GLURP-R0, and 1 jig/ml LR55 (State Serum Institute, Denmark) were coated in columns 3 to 12 o f each Elisa. The plates were washed four times in washing buffer containing 0.5M NaCl in Phosphate-buffered saline (PBS) and 0.1% Tween 20. This was done by dispensing washing buffer into all the wells and allowing it to stand for 1 minute before discarding and padding the plates to dry. The plates were blocked for 1 hour with 150(xl/well o f blocking buffer containing 3% milk powder in PBS-Tween 20. Two-fold serial dilutions o f the reference standard were prepared in dilution buffer (1% milk powder in PBS and 0.1% Tween-20) for each isotype and subclass at various starting concentrations; purified polyclonal IgG (BP055) at 50ng; purified human IgM kappa (BP090) at 2000ng; purified human IgG l kappa (BP078) at 500ng; purified human IgG2 kappa (BP080) at 50ng; purified human IgG3 kappa (BP082) at lOOOng; and purified human 40 University of Ghana http://ugspace.ug.edu.gh IgG4 kappa (BP084) lOOOng. All standard antibodies were from The Binding Site, Birmingham UK. The standards were added at 1 OOfxl/well in columns 1-2 to be captured by the anti-Fab. Afterwards, 10 0 |al/well o f diluted test plasm a samples 1/200 (IgG and IgM) and 1/50 (IgG subclasses) in serum dilution buffer (1% m ilk powder in PBS and 0.1 % Tween-20 and 0.02% Na azide) were added to antigen-coated portions o f the plate, and incubated for two hours. The secondary antibodies added were peroxidase- conjugated goat anti-human IgG (H I7007, Catlag, 1 mg/ml) and Peroxidase-conjugated rabbit anti-human IgM (H15007, Catlag, lm g/m l) at l|ig /m l, (1:3000 in dilution buffer at 100)j.l/well). For IgG subclasses, polyclonal sheep anti-human peroxidase conjugated subclass-specific antibodies (The Binding Site, Birmingham, UK) were added at 100nl/well, for IgG l (AP006, lm g/m l) at 1:1000, IgG2 (AP007, lm g/m l) at 1:1000, IgG3 (AP008, lm g/m l) at 1:2000 and IgG4 (AP009, lm g/m l) at 1:800 dilutions. After one hour o f incubation, the level o f bound secondary antibody in the test sample was quantified by colouring with 0.4 mg/ml o f o-phenylenediamine (OPD) substrate with Hydrogen peroxide (H2O2), (Sigma-Aldrich, St. Louis, USA) in citrate buffer (100|il/well) for 30 minutes in the dark. The reaction was stopped by adding 50|il o f 3M H2SO4. The plates were washed four times with PBS-Tween between each incubation step. The optical density (OD) at 492nm was determined by ELISA plate reader (Multiskan Ascent, Labsystems, Helsinki-Finland). 41 University of Ghana http://ugspace.ug.edu.gh 3.2.5 Cellular assays 3.2.5.1 Lymphocyte cultivation and proliferation assays Frozen Peripheral Blood Mononuclear Cells (PBMC) from 56 children, and 21 Ghanaian adults were thawed quickly at 37 °C, washed by adding 30 ml o f washing medium (RPMI 1640, 5% Fetal Bovine Serum (FBS), L-glutamine at a concentration o f 10ng/ml and streptomycin-penicillin (5ng/ml) and centrifuged at 1500rpm for 10 minutes. The supernatant was discarded and the cell pellets resuspended in washing medium and centrifuged at 1500rpm for 10 minutes. The washing and centrifugation steps were repeated and after discarding the supernatant, the cell pellets were then resuspended in 3 ml o f complete parasite medium (RPMI 1640 supplemented with 10% Fetal Bovine Serum (FBS), 4ml o f 10 p.g/ml L-glutamine and 5ml o f 5|ig/ml streptomycin-penicillin) and enumerated under the microscope (Olympus BH2 Microscope) at 40X magnification. The cell concentration was estimated as follows, Num ber o f cells/ml = Num ber o f cells counted X dilution factor X 104/ml Number o f fields After counting, the cell suspension was adjusted to 1.0 x 106 cells /ml by adding complete parasite medium. The 96-well round-bottom plates (Nunc, Denmark) were dosed initially with 20|il o f GLURP peptide antigens (LR129, LR130, MR186, and MR 187) for a final concentration o f 10jig/ml. For the positive control antigens, purified protein derivative o f tuberculin (PPD, Sigma), and phytohaemaglutin (PHA, Sigma), at a final concentration o f 5|j.g/ml and 2.5|ig/ml were used respectively. The crude schizont antigen was tested at a concentration o f 4.0 x 105 cells/well, 150,000 cells in a total 42 University of Ghana http://ugspace.ug.edu.gh volume o f 150(j.l o f complete culture medium were added to each antigen dosed well. Cultures were done in triplicates and the plates were incubated at 37°C in a humidified atmosphere containing 5% CO2. Parallel cultures were done for the various cytokines, and IL-2 was harvested on day 2. On day 5, some cultures were stimulated with Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, P8139) and Calcium Ionophore Hemicalcium (Sigma-Aldrich, C9275) mixture at 1 ng/ml and 15|ig/ml respectively to enhance the detection o f IL-4 in the culture supernatants. After 24 hours, supernatants from triplicate cultures were then pooled for IL-4 measurement. The other parallel cultures were harvested on day 5 for analysis o f in vitro measurement o f IL-10, IFNy by ELISA. The harvested supernatants were stored in -80°C until needed for cytokine measurements. 43 University of Ghana http://ugspace.ug.edu.gh 3.2.5.2 Cytokine measurements Measurement o f in vitro cytokine production was done by ELISA using culture supernatants o f antigen stimulated, and unstimulated cells. IFNy, IL-2, IL-4 and IL-10 were detected in culture supernatants by double sandwich ELISA according to manufacturer’s protocol. Briefly, microtitre plates were coated (1 x PBS and 0.001% phenol red) at 50fj.l/well o f anti-cytokine capture antibody at 4|ig/ml (IL-10, IL-4), and 2^g/ml for IL-2. IFNy (MABTACH AB) capture antibody was coated with 0.1M NaHCCh buffer, pH 8.2 at 2(ig/ml and incubated overnight at 4°C. Plates were washed 4 times in washing medium (PBS plus 0.05% Tween 20, pH 7.4), followed by blocking with 150|_il/well o f blocking medium (5% heat inactivated Foetal Bovine Serum (FBS) in PBS) for 1 hour at room temperature to block non-specific binding. After this step, the plates were washed 2 times. Cytokine standards (BD PharMingen, San Deigo) at starting concentrations between 2 0 0 0 pg/ml—4000 pg/ml were prepared in two-fold serial dilutions in standard diluent (RPMI 1640 plus 1% NHS) and 50 ^il/well added. Then, 50 ^I/well of unstimulated and antigen stimulated culture supernatants were added in duplicates and incubated for 2 hours at room temperature on a rocker. After washing four times, 50 (j,l/well o f biotinylated anti-human monoclonal cytokine antibodies were added at l|J.g/ml, but 6 |ig/ml for IFNy, and incubated for 45 minutes (room temperature). Plates were washed five times and avidin peroxidase (1:1000) was added at 50|il/well for 30 minutes at room temperature. For IFNy plates, 50|il/well o f streptavidin alkaline phosphase in 5% FCS in PBS was added at 2.5|xg/ml and incubated for 45 minutes 44 University of Ghana http://ugspace.ug.edu.gh on a rocker. Microtitre plates were washed eight times afterwards to remove excess unbound enzyme conjugates. The plates were developed with 0.4 mg/ml of o- phenylenediamine (OPD) substrate with Hydrogen peroxide (H2O2), (Sigma-Aldrich, St. Louis, USA) in citrate buffer for 30 minutes to obtain colour, the reaction was stopped with 2.5 M H2S04 and absorbance read at 492nm for IL-4, IL-2 and IL-10 ELISA’s. Whereas, for IFNy, 100|i 1/well of phosphate substrate (Blue Phos ™, Kirkegaard & Perry Laboratories, USA) (mixture of equal volumes of solutions A and B) were added and incubated for 20 minutes in the dark. The plates were stopped with stop solution (Blue Phos ™) and absorbance read at 630nm. 3.3 Statistical analysis All data were entered into micro-soft excel program, and the Sigma stat statistical software (Jandel Scientific, San Rafael, Calif.) used for data analysis. Spearman rank correlation was used to assess the strength of association between age of Ghanaian children and antibody responses to GLURP, MSP3 and the hybrid, and also the correlation between individual GLURP or MSP3 antibody responses and antibody responses to the GLURP-MSP3 hybrid. In addition, spearman rank correlation was used to estimate the correlation between antibody responses and cytokine levels. Mann Whitney’s test was used to compare antibody responses in children categorized as susceptible to or protected from clinical malaria. R-sept and STATA 8.0 program (Stata Corporation, Texas, USA) were used for Logistic regression analysis to correct for the confounding effect of age. Differences were considered statistically significant if the P value <0.05. 45 University of Ghana http://ugspace.ug.edu.gh CHAPTER 4 4.0 RESULTS 4.1 The pattern of malaria in the cohort Amongst the cohort of 300 children ages between 3 to 15 years, parasite infection seem to be stable, with about 50 percent of the children being asymptomatically infected at any particular time (Fig 4.1). However, the incidence o f clinical malaria was lowest prior to the rainy season (April), and was highest during the peak transmission season (July-September). Using the case definition of malaria as fever above 37.5 and parasiteamia above 5000 parasites/(il of blood, 63 children were categorized as having at least one episode of malaria (susceptible), and 202 children did not have any episode of malaria during the transmission season (protected). Ten children had fever above 37.5 and parasitaemia below the cut-off of 5000 and were categorized as indeterminate. The protected and susceptible groups o f children were used in subsequent statistical analysis. The median age of the susceptible children was significantly lower than that of the protected children (median age-susceptible = 5years, median age-protected = 8years, p < 0 . 0 0 1 ) 46 University of Ghana http://ugspace.ug.edu.gh P o in t p re va le n ce of P .fa lcip arum parasitaem ia a n d m alaria in c id e n ce rate d u rin g A p ril to N o v e m b e r 1994 Fig. 4.1: Prevalence o f P. falciparum parasitaemia in one transm ission season in Dodowa (Dodoo et al., 1999). 47 University of Ghana http://ugspace.ug.edu.gh 4.2 Antibody responses to GLURP, MSP3 and GLURP-MSP3 hybrid antigens 4.2.1 IgG / IgM responses to GLURP, MSP3 and GLURP-MSP3 hybrid antigens Isotype and IgG subclasses responses to recombinant GLURP, MSP3 and GLURP - MSP3 hybrid antigens were measured by ELISA in plasma samples of 275 children out of an original cohort o f 300 children residing in Dodowa. Fifty plasma samples obtained from healthy Danish adults who have never been exposed to malaria were also tested as controls. Prevalence of positive responses was defined as any response above the mean antibody level of the control plasmas plus two standard deviations (Mean+2SD). The prevalence of IgG and IgM responses to GLURP antigens were 48% and 58% respectively. The isotype responses to MSP3 were 38% for IgG and 20% for IgM. The prevalence o f IgG and IgM responses to the hybrid GLURP-MSP3 were 81% and 60% respectively (Fig 4.2). 48 University of Ghana http://ugspace.ug.edu.gh Prevalence of IgG and IgM Responses to GLURP, MSP3 and GLURP-MSP3 Hybrid 100 Antibodies Fig 4.2: IgG and IgM responses in all three antigens in Ghanaian children. 49 University of Ghana http://ugspace.ug.edu.gh 4.2.2 IgG subclass responses to GLURP, MSP3 and GLURP-MSP3 antigens The IgGl-IgG4 subclass responses raised against GLURP were 29%, 1%, 9% and 4% respectively, whiles subclass responses to MSP3 were 27%, 0%, 13%, and 4% for IgGl- 4, respectively. The prevalence of cytophilic antibody levels (IgGl and IgG3) were higher in all the three antigens than the non-cytophilic antibodies (IgG2 and IgG4), (Fig 4.3). Among the three antigens, GL-MSP3 Hybrid had significantly higher antibody responses to the IgG subclasses. 50 University of Ghana http://ugspace.ug.edu.gh P re v a le n c e o f Ig G s u b c la s s re s p o n s e s to G L U R P P r e v a le n c e o f Ig G s u b c la s s re s p o n s e s to M S P 3 Fig 4.3: Graph of IgG subclass responses to GLURP, M SP3 and G LURP-M SP3 antigens in G hanaian children. 51 University of Ghana http://ugspace.ug.edu.gh 4.2.3 Antibody responses to GLURP and MSP3 in relation to age Isotype and IgG subclass responses to both GLURP and MSP3 significantly correlated with age (0.54>r>0.31, p<0.0001) (Table 4.1). Table 4.1: Association of antibody responses with age of Ghanaian children. Antigen Correlation Coefficient (r) Significant Level GLURP IgG 0.414 <0.0001 IgM 0.486 <0.0001 IgGl 0.512 <0.0001 IgG2 0.473 <0.0001 IgG3 0.347 <0.0001 IgG4 0.470 <0.0001 MSP3 IgG 0.541 <0.0001 IgM 0.310 <0.0001 IgGl 0.393 <0.0001 IgG2 0.367 <0.0001 IgG3 0.520 <0.0001 IgG4 0.453 <0.0001 Table 4.1: done with spearman’s rank-order correlation. 52 University of Ghana http://ugspace.ug.edu.gh 4.2.4 Antibody responses to GLURP and MSP3, and protection from malaria IgG and IgM antibody levels of GLURP and MSP3 were compared between individuals categorized as susceptible or protected form malaria, and the data showed that antibody levels were significantly higher in protected individuals than those susceptible to malaria (p<0.05) (Table 4.2). IgG subclass responses to both GLURP and MSP3 in the protected individuals were significantly higher than in the susceptible individuals (0.03>p>0.001, Table 4.3). 53 University of Ghana http://ugspace.ug.edu.gh Table 4.2: IgG and IgM antibody responses in Ghanaian children protected or susceptible to malaria. Antigens Antibody levels (Median and Interquartile range) Protected (n=202) Susceptible (n=63) P-Valuea P-Valueb GLURP IgG 545.0(153.3-2224.4) 152.0 (51.3 -541 .0) <0.001 0.046 IgM 1422.8 (857.5-3450.2) 874.7 (541.3 - 1589.4) <0.001 0.089 MSP3 IgG 437.9 (229 .8 - 1385.8) 240.2 (97.7-521.9) <0.001 0.118 IgM 380.2 (233.3 -636 .3) 287.6(140.0-530.0) 0.021 0.258 P-value “ done with Mann-W hitney rank-sum test P-value bMultiple Logistic Regression Analysis (r-Sept); Data was transformed to normality using various equations. Data in ng/mL P>0.05 NOT Significant. 54 University of Ghana http://ugspace.ug.edu.gh Table 4.3: IgG subclass responses to GLURP and MSP3 in Ghanaian children protected or susceptible to malaria. Antigen Antibody levels (Median and Interauartile ranee") Protected (n=202) Susceptible (n=63) P-Value3 P-Value” GLURP IgGl 123 .9(30 .0- 1177.1) 30.0 (3 0 .0 - 133.3) <0.001 0.050 IgG2 1.8 (1 .0 -9 .9 ) 1.0 (1.0 - 2.3) <0.001 0.167 IgG3 16.0(10.0-47.4) 15.0(8 .0-20.6) 0.009 0.201 IgG4 14.5 (8 .0 -56 .4 ) 8 .0 (5 .0 -16 .3 ) <0.001 0.013* MSP3 IgGl 120.0(36.1 -808.6) 37 .2 (20 .0 - 167.0) <0.001 0.105 IgG2 1.79(1.15-2.90) 1 .47(1 .0-2 .43) 0.032 0.576 IgG3 14.5(10.0-165.3) 10.0(10.0-15.7) <0.001 0.126 IgG4 13.7(10.0-35.7) 10.0 (7 .1 -15 .7 ) 0.001 0.541 From Mann-Whitney rank-sum lest. P-value 4 Multiple Logistic Regression Analysis (r-Sept); Data was transformed to normality using various equations. Data in ng/ml. P>0.05 NOT Significant. ’ data had extremely low OD’s (92% o f OD’s below 0.2). Antibody ratios in relation to protection from clinical malaria: The cytophilic versus non-cytophilic antibody ratios (IgGl + IgG3) : ( IgG2 + IgG4) o f the two antigens were compared between protected and susceptible individuals in relation to protection. The data indicated significantly higher ratios in protected compared to susceptible children with regards to responses against MSP3 (P=0.006), but not for GLURP. However, no significant difference was found when the ratios were compared after correcting for age (Table 4.4). 55 University of Ghana http://ugspace.ug.edu.gh Table 4.4: Cytophilic to non-cytophilic antibody ratios in children protected or susceptible to malaria from Dodowa. Antibody Ratios (Median and interquartile ranee) Antigens Protected (n=202) Susceptible (n=63) P-Valuea P-Value6 GLURP 7.68 (4 .2 -21 .6 ) 6.3 (4 .9 -10 .0 ) 0.254 0.596 MSP3 7.85 (3 .62-46 .4) 4 .9(2 .78-12 .7 ) 0.006 0.075 P-value ° By Mann-Whitney rank-sum lest. P-value b Multiple Logistic Regression Analysis (r-Sept); Data was transformed to normality using various equations. Data in ng/ml P>0.05 NOT Significant 56 University of Ghana http://ugspace.ug.edu.gh 4.2.4.1 Age related exposure and protection from clinical malaria Anti-GLURP or anti-MSP3 antibody levels and age were significantly associated with protection, and therefore require correction to establish a true relationship between the specific antibody responses and immunity to malaria. The data was therefore re­ analyzed using a logistic regression model in which both age and antibody levels were included as explanatory variables for the outcome of infection, whether susceptible or protected. This analysis showed a significant association with protection for GLURP IgG (P=0.04) and IgGl and IgG4 subclasses (P=0.05; P=0.01, respectively) (Table 4.3). However, IgG4 responses though statistically significant, the OD’s were extremely low (92% below 0.2) comparable to the background levels. Although the majority of individuals with OD above 0.2 belong to the protected group. After correcting for the confounding effect of age-related exposure, isotype and IgG subclass responses to MSP3 were not significantly associated with protection (P>0.05, Table 4.2 and 4.3). 57 University of Ghana http://ugspace.ug.edu.gh 4.3 Antibody responses to GLURP-MSP3, GLURP and MSP3 antigens Antibody responses to either GLURP or MSP3 were compared to that of a recombinant hybrid antigen, GLURP-MSP3 in view of the fact that a future hybrid vaccine antigen is anticipated. The results showed a significant relationship between IgG antibody levels of GLURP-MSP3 versus GLURP and MSP3 antibody levels (r=0.756, 0.695: P= 0.0001) respectively. There was also a significant correlation between IgGl and IgG3 responses to the hybrid antigen and that of GLURP (r=0.696, P= 0.0001; r=0.363, p=0.0001, respectively) or to that of MSP3 (r= 0.713, p=0.0001; 0.676; P= 0.0001, respectively), (Fig 4.4). 58 University of Ghana http://ugspace.ug.edu.gh IgG A n tib o d y L ev els o f G L U R P -M S P 3 H y b r id v e rsu s G L U R P IgG A n tib o d y Levels o f H y b rid G L11RP-M SP3 v e rsu s M S P3 vs GL11RP-M SP3 H y b rid IgG AU (pg/m l) G L U R P -M S P 3 IgG A ll (pg /m l)GLM3G g l g - Plot 1 Regr Subclass A ntibody Levels of G LU RP-M SP3 versus G LU RP S u b c l a s s A n t i b o d y L e v e l s o f H y b r i d G L U R P - M S P 3 v e r s u s M S P 3 ■ GLM3G1 vs Q_G1 G L U R P -M SP J IgG1 AU (pg/ml) — PIO11 Ret* • GLM3G1 vs M3G1 G L U R P -M S P 3 Ig G 1 A U (p g /m I) ---- Plot 1 Rogf S i i J d a s s A ntib o d y L e v d s o f Q U R P -M S P 3 H ybrid v e r b is GLURP S u b c l a s s A n l i h o d y L e v e l s o f G L U R P - M S P 3 H y b r i d v e r s u s M S P 3 • GLM3G3 V. M3G3 G LU RP-M SP3 lgG 3 AU (pg/ml) • ojjDffl v» C3J33 GLURP-NOP31gG3AUlp^n^| ---PlQl 1 ReflT ---- PWlRey Fig. 4.4: Correlation o f G LURP-M SP3 hybrid and G LURP or M SP3 antigens. 59 University of Ghana http://ugspace.ug.edu.gh 4.4 Complementary antibody responses to GLURP and MSP3 The antibody responses to GLURP and MSP3 were compared to characterize the pattern o f responses in individual samples. The pattern o f responses showed that some individuals responded to GLURP only, others to MSP3 only and some responded to both antigens. The prevalence of IgG, IgGl and IgG3 responses to GLURP were 48%, 29% and 9%, respectively whiles that of MSP3 were 41%, 27% and 13%, respectively. When responder frequencies were analyzed for either GLURP or MSP3, a larger proportion of responders were indicated; 60% for IgG, 52% for IgGl and 20% for IgG3, thus indicating a level of complementarity in inducing antibody responses to GLURP and MSP3. 60 University of Ghana http://ugspace.ug.edu.gh 4.5 T-cell responses to GLURP peptides 4.5.1 Cytokine responses in Ghanaian children and adults Cryo-preserved peripheral blood mononuclear cells (PBMC) from a selected number of children were tested for cellular responses against GLURP specific peptides (LR129, LR130, MR186 and MR187). Control antigens included P. falciparum crude antigens, PHA and PPD. Fourteen PBMC’s obtained from healthy adults from Denmark who have never been exposed to malaria were used as control cells. IFN y, IL-10, IL-2 and IL-4 responses to GLURP peptide stimulated cultures of Ghanaian children were higher compared to the Danish controls (Fig 4.5). However, cultures of both Ghanaian and Danish volunteers stimulated with control antigen (PPD) produced elevated levels of cytokines (Fig 4.6). 61 University of Ghana http://ugspace.ug.edu.gh G ra p h o f I F N G a m a Levels in G h a n a ia n C h ild re n F ro m Dodow a G ra p h O F IL -1 0 Levels in Ghanaian Children I n ■ B „ ^ J f jr J t J ° < V 4 # S t i m u l a t i n g A n t i g e n s IFN y levels in children from Dodowa and Danish controls. S ti m ula t i ng Anti gens IL-10 levels in children from Dodowa and Danish controls G r a p h of IL-2 Levels in G h a n a ia n Children G r a p h o f I L - 4 L e v e l s i n G h a n a i a n C h i l d r e n S t i m u l a t i n g A n t i g e n s S t i m u l a t i n g A n t i g e n s IL-2 levels in children from Dodowa and Danish controls IL-4 levels in children from Dodowa and Danish control Fig 4.5: Cytokine levels in exposed (EXP) and non-exposed groups (CTRL). 62 University of Ghana http://ugspace.ug.edu.gh The cytokine responses in the exposed individuals and non-exposed individuals were, however, not statistically significant (P> 0.05) for IL-10, IL-4 and IL-2 in the stimulated cultures, except for IL-2 levels in PPD stimulated cultures (P= 0.002). Furthermore, the differences in IFNy levels to P. falciparum crude schizont antigen, PPD, LR129 and LR130 stimulated cultures in the Ghanaian children were statistically significant (P<0.050) than levels in Danish controls (Table 4.5). In addition to data from the Ghanaian children, PBMC’s of 21 healthy Ghanaian adults were stimulated with GLURP peptides and control antigens and cytokine levels were measured in culture supernatants. In the Ghanaian adults, cytokine levels in stimulated cultures were significantly higher (p>0.05) than levels in cultures from adult Danish controls (Fig 4.6). 63 University of Ghana http://ugspace.ug.edu.gh G r a p h o f IF N g a m a levels in G h a n a i a n a d u lt s G r a p h o f IL -1 0 levels in G h a n a i a n a d u l t s . f f m / / C £F - - P ,vv < ? '• j , v o v ^ ^ -*• v o ' ^ ^ 00.05 is not statistically significant. 65 University of Ghana http://ugspace.ug.edu.gh 4.5.2 Cytokine responses in relation to age PPD and PHA-induced IFNy and PPD-induced IL-2 levels significantly correlated with age of Ghanaian children (r= 0.372, P= 0.004 and r= 0.430, P=0.001, r= -0.279, P= 0.037, respectively). However, for the GLURP peptide and crude antigen stimulated cultures, there was no significant association with age for the other cytokines measured (r = -0.06 to -0.30, P >0.05). 4.5.3 Cytokine responses in relation to protection from malaria Of the 4 GLURP specific peptides tested (LR129, LR130, MR186, MR187), MR186- induced IFNy was significantly higher in the protected Ghanaian children compared to that in the susceptible children (p=0.003). None of the other cytokine levels including IL-10, IL-2 and IL-4 measured in the peptide and crude antigen stimulated cultures correlated with protection (p>0.05, Table 4.6). 66 University of Ghana http://ugspace.ug.edu.gh Table 4.6: Cytokine levels in Ghanaian children susceptible to, or protected from clinical malaria Antigens Cytokine levels (Median and interquartile range) SusceDtible (n=17) Protected (n=39) Sic. Level IFN GAMA CRUDE 60.0(55.0-- 100.0) 60 .0 (60 .0 - 100.0) 0.527 PPD 912.4 (334. 1-2913.3) 1343.8 (767.7-3211.7) 0.318 PHA 3472.7 (3143.6-4019.3) 4000.0(3000.0-5000.0) 0.470 LR129 60.0(55.0-- 100.0) 60.0(55.0 - 100) 0.755 LR130 60.0 (58.8-- 100.0) 60 .0 (60 .0 - 100.0) 0.708 MR186 60.0(55.0--63.1) 100.0(60.0- 154.7) 0.003 MR187 60.0(55.0-- 100.0) 60.0 (6 0 .0 - 100) 0.305 IL-10 CRUDE 30.0(25.0--71.5) 30.0 (25 .0-54.8) 0.979 PPD 57.3 (33.9--196.5) 73.1 (4 4 .2 - 120.8) 0.972 PHA 394.8 (297.2 - 608.7) 605.7 (336.3 -939 .9) 0.187 LR129 30.0 (25.0--42.5) 30.0 (26 .0-50 .0) 0.611 LR130 30.0(25.0--40.0) 30.0 (25 .0-50 .0) 0.327 MR186 30.0(25.0--40.0) 30.0(26 .0-50 .0) 0.438 MR187 40.0(28.8--42.5) 30.0 (26 .2-81 .9) 0.845 IL-4 CRUDE 243.0(102.2 - 620.0) 366.6 (46.5 - 808.40 0.397 PPD 551.5 (152. 7-1193.6) 752.5 (9 6 .2 - 1622.8) 0.539 PHA 136.6 (89.6 - 208.4) 182.6(114.0-334.5 0.134 LR129 187.5 (69.0 -479.9) 361.6 (85.8 -745.9) 0.289 LR130 227.9(104. 3 -468.0) 351.9(88.6-817.5) 0.226 MR186 191.4 (93.0 - 844.5) 420.4 (78.2-731.3) 0.630 MR187 219.1 (88.4 - 502.2) 325.4 (74.3 - 860.9) 0.363 IL-2 CRUDE 25.0(25.0- 30.0) 25.0(25 .0-30 .0) 0.986 PPD 30.0(25.0-- 49.4) 30.0 (25 .0-59.9) 1.00 PHA 894.9 (638.6 - 1527.3) 1049.8 (746.1 - 1765.4) 0.407 LR129 25.0 (25.0- 26.3) 25.0 (25 .0-30.0) 0.788 LR130 25.0(25.0--27.8) 25.0(25 .0-30 .0) 0.957 MR186 25.0(25.0- 26.3) 25.0(25 .0-27 .9) 0.789 MR 187 25.0 (25.0 - 26.3) 25.0(25 .0-25 .0) 1.00 Table 4.6: Median values (pg/ml) o f cytokine responses o f Ghanaian children susceptible or protected from malaria. P>0.05 is not statistically significant. University of Ghana http://ugspace.ug.edu.gh 4.5.4 Cytokine ratios in relation to malaria infection The capacity to regulate inflammation has been previously associated with protection from clinical malaria, however, ratios of pro-inflammatory cytokines (IFN y and IL-2) to anti-inflammatory cytokines (IL-10 and IL-4) assessed in this study were not significantly associated with protection from malaria (p>0.05, Table 4.7). 68 University of Ghana http://ugspace.ug.edu.gh Table 4.7: Cytokine ratios in Ghanaian children susceptible to or protected from clinical malaria Antigens Cytokine Levels (Median and interquartile ranee) Susceptible (n=17) Protected (n=39) Sis. Level IFNy/IL-10 CRUDE 2.0(1 .50 -2 .20 ) 2 .0 (1 .28-2 .65) 0.229 PPD 7.9 (3.18-33.63) 13.8 (5 .2-43 .20) 0.465 PHA 8.89 (3 .5 - 11.43) 5.54 (3 .20-8 .73) 0.176 LR129 2 .0 (1 .43-2 .25) 2 .0 (1 .0 -2 .72 ) 0.533 LR130 2 .2 (1 .89-2 .45) 2 .0 (1 .2 -2 .95 ) 0.796 MR186 2 .0 (1 .67-2 .20) 2 .2 (1 .74 -4 .0 ) 0.157 MR187 2 .0 (1 .50-2 .20) 2 .0 (1 .58-2 .40) 0.796 IL-2/IL-4 CRUDE 0.16(0 .06-0 .49) 0.07 (0.03 - 0.30) 0.173 PPD 0.10(0 .04-0 .19) 0.07 (0 .03-0 .60) 0.605 PHA 9.49 (5 .2 7 - 12.60) 6.32 (3 .56-9 .18) 0.181 LR129 0.16(0 .06-0 .38) 0.08 (0.03-0 .36) 0.363 LR130 0.11 (0.06-0 .23) 0.08 (0.03-0 .41) 0.293 MR186 0.11 (0.03-0 .23) 0.06 (0.03-0 .38) 0.782 MR187 0.11 (0.05 -0 .28) 0.08 (0.03 -0 .73 ) 0.527 IFNy/IL-4 CRUDE 0.39(0 .13-0 .67) 0.21 (0 .0 8 - 1.52) 0.340 PPD 1.16(0.62-4.49) 1.95 (0.86-9 .47) 0.593 PHA 28.73 (15.30-42.50) 22.96(10.80-27.86) 0.187 LR129 0.30(0 .17-0 .84) 0.23 (0 .0 9 - 1.38) 0.533 LR130 0.43 (0.13-0 .68) 0.28 (0 .0 9 - 1.39) 0.612 MR186 0.38 (0.08-0 .70) 0.40 (0 .0 9 - 1.50) 0.433 MR187 0.27 (0 .1 2 - 1.67) 0.24 (0 .0 8 - 1.67) 0.593 IL-2/IL-10 CRUDE 0.83 (0 .3 6 - 1.0) 0.83 (0 .5 4 - 1.0) 0.873 PPD 0.52 (0 .24-0 .83) 0.51 (0 .32-0 .73) 0.735 PHA 2.34(1 .17-4 .05) 1.52(1.07-2.68) 0.345 LR129 0.83 (0 .6 2 - 1.0) 0.83 (0 .58-0 .96) 0.702 LR130 0.83 (0.63 - 1.0) 0.83 (0 .5 2 - 1.0) 0.363 MR186 0.83 (0.63 - 1.0) 0.83 (0 .6 0 - 1.0) 0.412 MR187 0.63 (0 .62-0 .88) 0.83 (0 .31-1 .0 ) 0.776 Table 4.7: Cytokine ratios o f Ghanaian children susceptible to or protected from malaria. P>0.05 is not statistically significant. 69 University of Ghana http://ugspace.ug.edu.gh 4.5.5 Relationship between humoral and cellular responses It is known that cell mediated immune responses generally regulate antibody responses, thus GLURP induced cytokine responses in the selected Ghanaian children were related to antibody responses to GLURP recombinant antigens. The data however, could not demonstrate any relationship between cytokine and antibody responses to GLURP (-0.09 0.03, p>0.05) as a whole, or in relationship with immunity from malaria (0.01 < r>0.07, p>0.05). 70 University of Ghana http://ugspace.ug.edu.gh CHAPTER 5 5.0 DISCUSSION Several malaria immuno-epidemiological studies have shown that antibodies play important role in protective immunity against malaria (Aribot et al., 1996; Baruch et al., 1997; Bouharoun-Tayoun et al., 1990; Bull et al., 1998; Dodoo et al., 1999 and 2000; Hoffman et al., 1986; Holder et al., 1992; Oeuvray et al., 1994 and 2000; Okech et al., 2004; Smith et al., 1995; Soe et al., 2004; Theisen et al., 1998 and 2001). Although both cellular and antibody responses have been shown to be important for immunity to malaria, antibody seem to promote effector cell mechanism against the malaria parasite, and thus protection from disease (McGregor et al., 1963; Sabchareon, et al., 1991). Multiplication of blood stage parasites are responsible for clinical malaria, and of the several identified blood stage antigens, Plasmodium falciparum glutamate rich protein (GLURP) and merozoite surface protein (MSP3) have been unequivocally demonstrated to be important in immunity against malaria. They have been associated with acquired protective immunity to malaria in many field studies (Bouharoun-Tayoun et al., 1995; Dodoo et al., 2000; Oeuvray et al., 1994 and 2000; Soe et al., 2004; Theisen et al., 1998 and 2001). Affinity purified antibodies from both GLURP and MSP3 have been shown to inhibit parasite growth in vitro in the antibody dependent cellular inhibition (ADCI) assay, which involves monocytes and macrophages that release factors to indirectly kill parasites (Bouharoun-Tayoun et al., 1995; Oeuvray et al., 1994). Previous immuno-epidemiological study in Ghana was done with a subset of a larger cohort, and focused on only serological responses to GLURP. In the present study, the larger cohort was assessed for antibody responses to both GLURP and MSP3 and cell University of Ghana http://ugspace.ug.edu.gh mediated responses to overlapping peptides of GLURP. It is important to assess immune responses to both GLURP and MSP3, since both antigens are being considered in a future vaccine, either as single antigens or as a hybrid construct. The prevalence of Isotype and IgG subclass levels to GLURP and MSP3 antigens in plasma was significantly high in Ghanaian children. For both GLURP and MSP3 antigens, the prevalence of cytophilic antibody responses (IgGl and IgG3) were higher than non-cytophilic antibodies (IgG2 and IgG4). This agrees with several studies done to show the critical role cytophilic antibodies play in individuals protected from malaria (Aribot et al., 1996; Luty et al., 1994; Ndungu et al., 2002; Oeuvray et al., 1994 and 2000; Soe et al., 2004). In a previous study done in Ghana, the prevalence of GLURP (RO)-specific cytophilic antibodies (52%-89%) were higher than non-cytophilic antibodies (7%-51 %), (Dodoo et al., 2000). In another study conducted in Senegal, antibody responses to GLURP (R0) showed a similar trend in which cytophilic antibodies were higher in protected children than susceptible children (Oeuvray et al., 2000). The antibody prevalence against GLURP and MSP3 antigens were found to be significantly associated with age of Ghanaian children (P<0.0001, r= 0.54>r>0.31). This occurrence has been frequently reported of antibody responses to various P. falciparum blood stage antigens (al-Yaman et al., 1995; Aribot et al., 1996; Dodoo et al., 2000; Dziegiel et al., 1993; Oeuvray et al., 2000). This shows how partial protective immunity is acquired as individuals mature in age. Various studies conducted in endemic areas have shown an age-dependent increase in malaria immunity and a decrease in clinical 72 University of Ghana http://ugspace.ug.edu.gh malaria episodes (al-Yaman et al., 1995; Deloran et al., 1987; Marsh and Greenwood 1992). Several studies have confirmed the important role GLURP and MSP3-specific antibodies play in conferring protection against malaria. The children were therefore categorized into susceptible and protected groups to assess the role of antibodies in these groups. The data generated revealed a significant higher antibody levels in the protected children than susceptible children (P<0.05), indicating a significant association between the antibody levels and protection. A study done by Theisen et al., (2001), showed higher GLURP-specific antibodies in protected individuals in than susceptible individuals. In addition, a study conducted in Asia, also showed elevated levels of cytophilic antibodies against MSP3 antigen in protected individuals than in susceptible children (Soe et al., 2004). Furthermore, cytophilic to non-cytophilic antibody ratios have also been related to clinical malaria in immuno-epidemiological studies (Dodoo et al., 2000) with the view that the interactions between the types of antibodies may be more important than relating individual cytophilic antibodies to clinical outcome. From this study, when the cytophilic to non-cytophilic ratios were compared, the cytophilic antibody ratio was higher in protected group than in susceptible individuals. However, the difference was statistically significant between protected and susceptible groups for MSP3 (P=0.006), but not significant for GLURP-specific antibody responses (P>0.05), probably due to higher non-cytophilic antibody levels to GLURP. This was not in agreement with previous work done in which cytophilic to non-cytophilic antibody ratios against GLURP (R0) were significantly higher (p=0.002) in the protected group than in susceptible individuals (Dodoo et al., 2000). Oeuvray et al., (2000) have also 73 University of Ghana http://ugspace.ug.edu.gh demonstrated the association of high cytophilic antibody ratios and ADCI activity in vitro, indicating that incorporation of cytophilic antibodies and monocytes is important for parasite clearance. In this present study, however, after correcting for the effect of age in a logistic regression model, GLURP-specific IgG and IgGl were associated with protection (P<0.05). None of the MSP3 IgG and subclass levels were significantly associated with protection against malaria when the confounding effect of age was corrected (P>0.05). This is in contrast with work done in which levels of IgG and cytophilic antibody subclass responses against GLURP and MSP3 were associated with protection after the effect of age was adjusted (Aribot et al., 1996; Dodoo et al., 2000; Oeuvray et al., 2000). Interestingly, GLURP-specific IgG4 was found to be associated with protection in this study, this is not consistent with what has been documented by others in which non- cytophilic antibodies were not associated with protection (Aribot et al., 1996; Dodoo et al., 2000; Oeuvray et al., 2000; Soe et al., 2004). It may also be possible that the measured GLURP-specific IgG4 levels in this study associated with protection may not be of biological significance, since 92% of the measurements were comparable to the background levels. From this study, some individuals, who did not respond to GLURP, responded strongly to MSP3 antigens. This suggests that in each individual, antibodies could be raised against either GLURP or MSP3 or both antigens at the same time to enhance protection against malaria. This complementarity of responses to GLURP and MSP3, which could lead to more effective immunity against malaria, was in agreement with a study done in 74 University of Ghana http://ugspace.ug.edu.gh Asia in which there was complementary antibody responses to GLURP and MSP3 antigens (Soe et al., 2004). Hence, it would be important to consider combining both antigens in a future vaccine against malaria. Antibodies raised against GLURP and MSP3 have been shown to be protective, it is therefore important to look at their combined responses. Hence, epitopes of these two antigens were genetically coupled into a recombinant hybrid protein, GLURP-MSP3 in view of the fact that a future hybrid vaccine antigen is anticipated. Higher antibody levels were raised against GLURP-MSP3 hybrid in Ghanaian children compared to the individual GLURP and MSP3 antigens. Furthermore, there was a correlation between antibody levels of GLURP-MSP3 hybrid with MSP3 or GLURP antigens in Ghanaian children (P<0.05). This is consistent with a study done in which plasma of Liberian adults showed higher antibody levels to the hybrid antigen compared to individual GLURP and MSP3-specific antibody levels in vitro (Theisen et al., 2003). This suggests that the hybrid protein might have produced adequate presentation of the GLURP and MSP3 epitopes or antigenic determinants. Furthermore, in the same study, upon immunizing mice with GLURP, MSP3 and the hybrid antigens, antibody responses to hybrid protein exceeded that of individual GLURP or MSP3-specific antibody responses (Theisen et al., 2003). Hence these two antigens when combined into a hybrid vaccine, may elicit higher antibody responses, which could lead to protective immunity against malaria. 75 University of Ghana http://ugspace.ug.edu.gh Several studies have led to the recognition of the importance of T-cell in the generation of long-term antibody responses in malaria infection (Ballou et al., 1987; Good et al., 1988). Further studies have shown that cell-mediated immune responses play important role in malaria infection (Troye-Blomberg et al., 1994). T-cell responses to GLURP peptides also revealed that T-cell epitopes were detected in LR67 and LR68 peptides. This suggests that the non-repeat region of GLURP may contain T cell epitopes that are recognized by individuals exposed to malaria (Theisen et al., 2001). Previous study has been shown that production of an anti P. falciparum antibody in vitro requires T-cell- derived signals from cytokines (Garraud et al., 1999). This study further assessed the in vitro cellular responses in PBMC culture supernatant of children stimulated with some overlapping peptides of GLURP by the production of cytokines. The data showed lower cytokine responses to the antigen-stimulated cultures of the Danish controls who have never been exposed to malaria, compared to the antigen-stimulated cultures of exposed children. The cytokine responses observed in the Ghanaian children to the peptide antigens could be due to the role of memory T-cells acquired through repeated exposure to P. falciparum infection. Studies done by Theander et al., (1997), have shown similar results in which cytokine responses were higher in PBMC’s of exposed individuals than controls from non-endemic area after stimulating with P. falciparum synthetic MSP2 peptide. From this study however, although cytokine responses in exposed group were higher than in unexposed group, the difference between stimulated cultures of exposed and non-exposed groups for IL-10, IL-4 and IL-2 levels were not statistically significant 76 University of Ghana http://ugspace.ug.edu.gh (P>0.05). Only PPD induced IL-2 level (P=0.002), crude, PPD and LR129 induced-IFNy levels were significantly different in the two groups (P<0.05). The level of cytokines produced in the peptide-stimulated cultures of the exposed group could be due to exposure to P falciparum infection. Studies have shown that P. falciparum parasites are able to induce the production of several cytokines (Allan et al., 1995; Jakobsen et al., 1993). Surprisingly, from this study, a single Danish control sample produced IFNy in crude antigen stimulated cultures. This is in agreement with studies done in which unexposed individual responded positively to malarial antigens (Jacobsen et al., 1993; Rzepczyk et al., 1989). Previous study done by Gabrielsen et al., (1982) has demonstrated that responses to crude malaria antigens may be due to other mitogenic reactions. The cytokine responses to malarial antigens in unexposed individuals may be due to previous exposure to cross-reacting antigens from other infections (Hviid et al., 1992 and Jacobsen et al., 1993). Although these children are in a malaria endemic area and their cells were expected to have generated cytokine production after stimulating with malaria antigens, only a small proportion of individuals responded to all the peptide antigens. Generally, the low cytokine responses observed in Ghanaian children could be due to lack of T-cell epitopes and MHC restriction. High crude antigen-induced cytokine responses (IL-2, IL- 4, IL-10 and IFNy) were observed in most of the cultures of Ghanaian children. The reason could be that the crude antigen has a repertoire of other P. falciparum antigens, which elicit a wide range of cytokine responses. This is in agreement with studies conducted in which significant levels of IL-2 and IFNy were seen when stimulated with crude antigens compared with other purified malarial antigens or peptides (Butcher et 77 University of Ghana http://ugspace.ug.edu.gh al., 1990 and Riley et al., 1988). Cytokine levels between Ghanaian adults and children revealed a higher IFNy levels from antigen stimulated cultures of adults than in children. There were more positive responders to IL-10 induced cytokines in children than in adults; however the levels of IL-4 and IL-2 were not significantly different in adults and children. Studies conducted have shown that T-cell responses to malaria antigens were higher in children than in adults (Jakobsen et al., 1993), this was in agreement with what was observed in IL-10 induced cytokines produced. However, it contrasts with elevated IFNy levels found in adults than in children in this study. IL-10 induction may be higher in children as anti-inflammatory responses, whilst higher IFNy levels in adults may reflect immune status. There was no significant relationship between cytokine levels of peptide stimulated cultures and age of Ghanaian children (p>0.05). Previous studies conducted in Ghana, found no correlation with induced cytokine responses with age in children (Dodoo et al., 2002). A study conducted in Kenya found an association between IFNy responses to pre- erythrocytic antigens (LSA1 and MSP1) and age of children (Chelimo et al., 2003), however, IL-10 levels to LSA1 were not age dependent in another study (John et al., 2000). This suggests that some cytokine responses may be age-dependent and thus relate to age specific acquisition of immunity, whiles others may be related to the pathogenesis of malaria infection. Cytokine responses were compared between Ghanaian children categorized into susceptible or protected from malaria. Of the four peptides tested, MR186-induced IFNy showed a significant association with protection. This agrees with a previous study that 78 University of Ghana http://ugspace.ug.edu.gh associated high malaria specific IFNy levels with reduced risk of clinical malaria (Dodoo et al., 2002; Chelimo et al., 2003). Previous studies have demonstrated the important role IFNy plays in resistance to blood and liver- stage malaria infection and its association with protective immunity (Riley et a l, 1988; Luty et al., 1999). The levels of IL-4 response were higher in protective group than in susceptible children, however, the difference was not statistically significant. However, some previous studies have shown that the induction of IL-4 against malaria could lead to protective immunity against malaria (Smythe et al., 1988; Clark et al., 1989). In contrast, IL-4 has been shown in other studies to facilitate parasite survival by suppressing anti-parasite activities of macrophages (Kumaratilake and Ferrente, 1992). Various studies have shown significantly higher levels of IL-10 in children with malaria infection than healthy controls (Joao et al., 1997). Other studies done to compare specific cytokine responses of patients with or without malaria in Malawi revealed higher serum IL-10 levels in malaria patients (Jason et al., 2001). However, this study could not show any significant difference (P>0.05) in IL-10 levels between sick and protected individuals. IL-2 levels between the sick and protected children were not significantly different. This is in agreement with studies done involving IL-2, in which PBMC’s stimulated in response to malaria-specific antigens during acute malaria infection, revealed no detectable levels of IL-2 production. This suggests that there may be defective cell mediated immune response to these malaria specific antigens during P falciparum infection, which might have resulted in immune unresponsiveness (Ho et al., 1988). 79 University of Ghana http://ugspace.ug.edu.gh It has been shown that a balance between pro-inflammatory and anti-inflammatory cytokines plays a critical role in determining the outcome of clinical malaria (Troye- Blomberg and Perlmann, 1994). From this study, the ratio of pro-inflammatory cytokines (IFNy and IL-2) and anti-inflammatory cytokines (11-10 and IL-4) between sick and protected children showed no significant association with protection (P>0.05), which agrees with previous studies conducted by Dodoo et al., (2002), that failed to establish a significant association between cytokine ratios and clinical malaria. Furthermore, when the cellular and antibody responses to GLURP were compared, to assess which cytokine(s) correlate with cytophilic antibodies, the data did not show any significant association between cytokine levels or ratios and cytophilic antibodies (P>0.05). The low proportion of individuals responding to the GLURP peptide antigens by the production of cytokines could be due to MHC restriction (Theander et al., 1992). 80 University of Ghana http://ugspace.ug.edu.gh 5.1 CONCLUSION In conclusion, cytophilic antibodies raised against both GLURP and MSP3 were associated with protection in Ghanaian children. There was an association between antibodies and age; however it was not significant for all antigens when the effect of age was corrected in a logistic regression model. In addition, antibody responses to GLURP and MSP3 antigens were complementary, suggesting that individuals could raise antibodies against either GLURP or MSP3 or both antigens and thus afford protection against malaria for a larger population. This finding provides evidence for the inclusion of these two antigens in a future multivalent antigen development. IFNy responses to the GLURP peptide MR186 correlated with protection, and this suggests that there are T- cell epitopes in GLURP. The study did not however find an association between cytokine responses and antibody responses. The study has confirmed the importance of cytophilic antibody responses to GLURP and MSP3 in malaria immunity and that the combination of both antigens in a future vaccine is promising. GLURP specific T-cell cytokine responses found in the study further indicates its potential as a future malaria vaccine. 81 University of Ghana http://ugspace.ug.edu.gh 6.0 REFERENCES Abbas, A.K., Murphy, K.M. and Sher, A. (1996). Functional diversity of helper T lymphocytes. Nature, 383; 787-793. Afari, E.A., Appawu, M., Dunyo, S., Baffoe-Wilmot, A., Nkrumah, F.K. (1995). Malaria Infection, Morbidity and Transmission in two Ecological zones in Southern Ghana. Afri. J. Health Sci. 2:312-6. African Malaria Report 2003; WHO/UNICEF: WHO/CDS/MAL/2003.1093. Retrieved from www.unicef.org/publications/index 7936.html Akazili, J. (2002). Costs to households of seeking malaria care in the Kassena-Nankana District of Northern Ghana. In: Third MIM Pan-African Conference on Malaria, Arusha, Tanzania, 17-22 Bethesda, MD, Multilateral Initiative on Malaria: abstract 473. Allan, R.J., Beattie, P., Bate, C., Van Hensbroek, M.B., Morris-Jones, S., Greenwood, B.M., Kwiatkowski, D. (1995). Strain variation in tumor necrosis factor induction by parasites from children with acute falciparum malaria. Infect Immun.63 (4): 1173-5. Al-Yaman, F., Genton, B., Anders, R., Taraika, J., Ginny, M., Miller, S. (1995). Assessment of the role of the humoral response to plasmodium falciparum MSP2 compared to RESA and Spf66 in protecting Papua New Guinean children from clinical malaria. Parasite Immunol, 17:493-501. Amstrong, S. J.R.M., Smith, T., Alonso, P.L., Hayes, R.J. (1994). What is clinical malaria? Finding case definition for field research in highly endemic areas. Parasitol. Today, 10:439-342. Aribot, G., Rogier, C., Sarthou, J.L., Balde, A.T., Druilhe, P., Roussilhon, C. (1996). Pattern of immunoglobulin isotype response to plasmodium falciparum blood-stage 82 University of Ghana http://ugspace.ug.edu.gh antigens in individuals living in a holoendemic area of Senegal, (Dielmo, West Africa). Am J Trop M ed Hyg 54:449-457. Astagneau, P., Roberts, J. M., Steketee, R. W., Wirima, J. J., Lepers, J. P., Deloron, P. (1995). Antibodies to a Plasmodium falciparum blood-stage antigen as a tool for predicting the protection levels of two malaria-exposed populations. Am. J. Trop. Med. Hyg. 53:23. Artavanis-Tsakonas, K. and Riley, E.M. (2002). Innate immune response to malaria: rapid induction of IFN-gamma from human NK cells by live Plasmodium falciparum- infected erythrocytes. J. Immunol. 15;169(6):2956-63. Aucan, C., Traore, Y., Tall, F., Nacro, B., Traore-Leroux, T., Fumoux, F., and Rihet, P. (2000). High immunoglobulin G2 (IgG2) and low IgG4 levels are associated with human disease resistance to plasmodium falciparum malaria. Infect. Immun. 68:1252­ 1258. Badell, E., Oeuvray, C., Moreno, A., Soe, S., van Rooijen, N., Bouzidi, A., and Druilhe, P. (2000). Human malaria in immunocompromised mice; an in vivo model to study defense mechanisms against P. falciparum. J. Exp. Med. 192, 1653-1660. Ballet, J.J., Druilhe, P., Vasconcelos, I., Schmitt, C., Agrapart, M., and Frommer, D., (1985). Human Lymphocyte response to plasmodium falciparum merozoite Antigens. A functional assay o f protective immunity? Trans. R. Soc. Trop. Med. Hyg. 79,497. Ballou, W.R., Hoffman, S.L., Sherwood, J.A., Hollingdale, M.R., Neva, F.A., Hockmeyer, W.T., Gordon, D.M., Schneider, I., Wirtz, R.A., Young, J.F. (1987). Safety and efficacy of a recombinant DNA Plasmodium falciparum sporozoite vaccine. Lancet. 6; 1(8545): 1277-81. 83 University of Ghana http://ugspace.ug.edu.gh Baruch, D.I., Ma, X.C., Singh, H.B., Bi, X., Pasloske, B.L. and Howard, R.J. (1997). Identification of a region of PfEMP that mediates adherence of Plasmodium falciparum infected erythrocytes to CD36: Conserved function with variant sequence. Blood 90, 3766-3775. Biggs, B.A., Gooze, L., Wycherley, K., Wilkinson, D., Boyd, A.W., Forsyth, K.P., Edelman, L., Brown, G.V., Leech, J.H. (1990). Knob-independent cytoadherence of Plasmodium falciparum to the leukocyte differentiation antigen CD36. J Exp M ed1 11 (6): 1883-92. Binka, F. N., Morris, S. S., Ross, D. A., Arthur, P., Aryeetey, M, E. (1994). Patterns of malaria morbidity and mortality in children in northern Ghana. Trans R. Soc. Trop. Med. Hyg. 88: 381-385. Blackman, M. J., Scott-Finnigan, T. J., Shai, S., Holder, A. A. (1994). Antibodies inhibit the protease-mediated processing of a malaria merozoite surface protein. J. Exp. Med. 180:389 Benjamini, E., and Leskowitz, S. (2000). Immunology A short Course. Second Edition. Wiley-Liss Inc. New York, USA. Borre, M.B., Dziegiel, M., Hough, B. (1991). Primary structure and localization of a conserved immunogenic P. falciparum GLURP expressed both the pre-erythrocytic and erythrocytic stages of the vertebrate life cycle. Mol Biochem Parasitol: 49: 119-32. Bouharoun-Tayoun, H., Attanth, P., Sabchareon, A., Chongsuphajaisiddhi, T., Druilhe, P. (1990). Antibodies that protect humans against Plasmodium falciparum blood stages do not on their own inhibit parasite growth and invasion in vitro, but act in cooperation with monocytes. J. Exp Med. 172: 1633-1641. 84 University of Ghana http://ugspace.ug.edu.gh Bouharoun-Tayoun, H. and Druilhe, P. (1992). Plasmodium falciparum malaria: Evidence for an isotype imbalance, which may be responsible for delayed acquisition of protective immunity. Infect immunol. 60:1473-1481. Bouharoun-Tayoun, H., Oeuvray, C., Lunel, F., Druilhe, P. (1995). Mechanisms underlying the monocyte-mediated antibody-dependent killing of plasmodium falciparum asexual blood stages. J. Exp Med. 182: 409-418. Brown, J., Greenwood, B.M., and Terry, R.J., (1986). Cellular mechanism in recovery from acute malaria in Gambian children. Parasite Immunol. 8, 551. Bull, P. C., Lowe, B.S., Kortok, M., Molyneux, S., Newbold, C. I., Marsh, K. (1998). Parasite antigen on the infected red cell surface are targets for naturally acquired immunity to malaria. Nature'. 4: 358-360. Butcher, G.A. (1990). In vitro responses of human peripheral blood mononuclear cells to Plasmodium falciparum antigen. Int. J. Parasitol. 20 (2):211-6. Chelimo, K., Sumba, P.O., Kazura, J.W., Ofulla, V.A., Chandy, C.J. (2003). Interferon- gama responses to Plasmodium falciparum liver stage antigen-1 and Merozoite surface protein-1 increase with age in children in a malaria holoendemic area of Western Kenya. Malaria Journal. 2:37. Clark, J.T., Donachie, S., Anandi, R, Wilson, C.F., Heidrich H.G., Mcbride, J.S. (1989). 46-53 Kilodalton glycoprotein from the surface of Plasmodium falciparum merozoites Mol. Biochem. Parasitol. 32: 15-24. Deloron, P., Le Bras, J., and Coulaud, J. P. (1987). Antibodies to the P fl55 antigen of Plasmodium falciparum measurement by cell-ELISA and correlation with expected immune protection. Amer. J. Trop. Med. Hyg. 37; 22-26. 85 University of Ghana http://ugspace.ug.edu.gh Deloron, P., Chougnet, C., Lepers, J.P., Tallet, S., Coulange, P. (1991). Protective value of elevated levels of y interferon in serum against exoerytrocytic stages of P. falciparum. J.Clin. Microbiol. 29:1757-1760. Deloron, P., Dumont, N., Nyongabo, T., Aubry, P., Astagneau, P., Ndarugirire, F., Menetrier-Caux, C., Burdin, N., Brelivert, J. C. and Peyron, F. (1994). Immunologic and biochemical alterations in severe falciparum malaria: Relation to neurological symptoms and outcome. Clin. Infect. Dis., 19: 480-485. de Waal-malefyt, R., Abrams, J., Bennet, B., Figder, C.C., and de Vries J.E., (1991). Interleukin 10 inhibits cytokine synthesis by human monocytes, An auto regulatory role of IL-10 produced by monocytes. J. Exp. Med. 174,1209-1220. Dodoo, D., Theander, T. G., Kurtzhals, J.A.L. (1999). Levels of antibody to conserved parts o f Plasmodium falciparum merozoite surface protein 1 in Ghanaian children are not associated with clinical protection from malaria. Infect. Immun. 67:2131-7. Dodoo, D., Theisen, M., Kurtzhals, J.A., Akanmori, B. D., Koram, K. A., Jepsen, S., Nkrumah, F. K., Theander, T. G., and Hviid, L. (2000). Naturally acquired antibodies to the glutamate-rich protein are associated with protection against Plasmodium falciparum malaria. J. Infect. Dis. 181:1202-1205. Dodoo, D., Omer, F.M., Todd, J., Akanmori, B.D., Koram, K.A., Riley, E.M. (2002). Absolute levels and ratios of proinflammatory and anti-inflammatory cytokine production in vitro predict clinical immunity to Plasmodium falciparum malaria. J Infect Dis. 185 (7): 971-9. Doolan, D. L., and Hoffmann, S. L. (1999). IL-12 and NK cells are required for antigen- specific adaptive immunity against malaria initiated by CD8+ T cells in the Plasmodium falciparum yoelii model. J. Immunol. 163: 884. 86 University of Ghana http://ugspace.ug.edu.gh Druilhe, P., and Perignon, J.L. (1994). Mechanisms o f defense against P. falciparum asexual blood stages in humans. Immunol Lett. 41(2-3): 115-20. Dziegiel, M., Rowe, P., Bennett, S., Allen, S.J., Olerup, O., Gottschau, A., Borre, M., Riley, E.M. (1993). Immunoglobulin M and G antibody responses to Plasmodium falciparum glutamate-rich protein: correlation with clinical immunity in Gambian children. Infect Immun. 61 (l):103-8. Egan, A.F., Morris. J., and Bamish, G. (1996). Clinical immunity to plasm odium falciparum malaria is associated with serum antibodies to the 19kDa C-terminal fragment of the merozoite surface antigen, PfMSPl. J. Infect. Dis. 173: 765-769. Fell, A.H., and Smith, N.C. (1998). Immunity to asexual blood stages o f plasmodium: is resistance to acute malaria adaptive or innate? Parasitol Today, 14:364-369. Gallup, J. L., and Sachs, J.D. (2001). The economic burden o f malaria. Am J. Trop. M ed Hyg (Suppl 1): 84-96. Garraud, 0 ., Diouf, A. I., Holm, A., Perraut, and S. Longacre. (1999). Immune responses to P. falciparum -merozoite surface protein 1 (MSP1) antigen. II. Induction of parasite specific IgG in unsensitized human B-cells after in vitro T-cell priming with MSP 119. Immunology 97: 497-505. Grau, G., Taylor, T., Molyneux, M. E. (1989). Tumour necrosis factor and disease severity in children with falciparum malaria. N. Engl. J. Med. 320: 1586-1591. Greenwood B. (1999). Malaria mortality and morbidity in Africa. Bull World Health organ 77: 617-618. Greenwood, B. and Mutabingwa, T. (2002). Malaria in 2002. Nature. 415(6872):670- 672. 87 University of Ghana http://ugspace.ug.edu.gh Gabrielsen, A.A., Jensen, J.B. (1982). Mitogenic activity of extracts from continuous cultures of Plasmodium falciparum. Am. J. Trop. Med. Hyg. 31:441-448. Good, M.F., Quakyi, LA., Riley, E.M., Houghten, R.A., Menan, A., Ailing, D.W., Berzofsky, J.A., Miller, L.H. (1988). Human T-cell recognition of the circumsporosoite protein of Plasmodium falciparum; Immunodominant T-cell domains map to the polymorphic regions of the molecule. Proc. Natl. Acad. Sci. U.S.A. 85: 1199 -1203. Good, M.F., Kaslow, D.C., Miller, L.H. (1998). Pathways and strategies for developing a malaria blood-stage vaccine. A m u Rev Immunol, 16:57-87. Groux, H., and Gysin, J., (1990) Opsonization as an effector mechanism in human protection against asexual blood stages of plasmodium falciparum : Functional role of IgG subclasses. Res. Immunol., 141: 529-542. Hirunpetcharat, C., Stanisic, D., Lui, X.Q. (1998). Intranasal immunization with yeast- expressed 19 kD carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein 1 (yM SPli9) induces protective immunity to blood stage malaria infection in mice. Parasite immunol. 20: 413. Ho, F., Schollardt, T., Snape, S., Looareesuwan, S., Suntharasamai, P., and White, N.F. (1998). Endogeneous interleukin 10 modulates proinflammatory response in plasmodium falciparum malaria. J. Infect. Dis. 178: 520-525. Ho, M., Webster, H.K., Green, B., Looareesuwan, S., Kongchareon, S., White, N.J. (1988). Defective production of and response to IL-2 in acute human falciparum malaria. J. Immunol. 141(8):2755-9. Ho, M., Sexton, M. M., Tongtawe, P., Looareesuwan, S., Suntharasamai, P. and Webster, K. (1995). Interleukin-10 inhibits tumour necrosis factor production but not antigen-specific proliferation in acute P. falciparum malaria. J. Infect. Dis. 172: 838-44. 88 University of Ghana http://ugspace.ug.edu.gh Hoffman, S.L., Wister, R., Ballou, W.R., Hollingdale, M.R., Wirtz, R.A., Schneider, I., Marwoto, H.A., Hockmeyer, W.T. (1986). Immunity to malaria and naturally acquired antibodies to the circumsporozoite protein of Plasmodium falciparum. N.Eng. J. Med. 315 :601-606. Holder, A.A., Blackman, M.J., Burghaus, P.A., Chappel, J.A., Ling, I.T., McCallum- Deighton, N., Shai, S. (1992). A malaria merozoite surface protein (MSPl)-structure, processing and function. Mem Inst Oswaldo Cruz; 87 Suppl 3:37-42. Hviid. L.. Jakobsen, P.H., Abu-zeid, Y.A., Theander, T.G. (1992). T-cell responses in malaria. APMIS . 100 : 95-106. Hviid, L. (1998). Clinical disease, immunity and protection against Plasmodium falciparum malaria in populations living in endemic areas. Exp. Rev. Mol. Med. http://www-ermm.cbcu.cam.ac.uk/lhc/txt0011hc.htm. Jaffar, S., Leach, A., Greenwood, A. M., Jepson, A., Muller, O., Ota, M. O., Bojang, K., Obaro, S., and Greenwood, B. M. (1997). Changes in the pattern o f infant and childhood mortality in upper river division, The Gambia, from 1983-1993. Trop M ed Int Health 2: 28-37. Jakobsen, P.H., Hviid, L.,Theander, T.G., Afare, E.A., Ridley, R.G., Heegaard, P.M.H., Stuber, D., Dalsgaard, K, Nkrumah, F.K. (1993). Specific T-cell recognition of the merozoite proteins Rhoptry - Asscoiated Protein 1 and Erythrocyte Binding Antigen 1 of Plasmodium falciparum . Infect. Immun. 61:268-273. Jason, J., Lennox K.A., Okey, C.N., Bell, M., Ian, B., Joshua, L., Kazembe, P.N., Hamish, D., Bharat, P., Byrd, M. G., Eick, A., Han, A., and Jarvis, W.R. (2001). Cytokines and Malaria Parasitemia. Clinical Immunology. 100 (2), 208-218. 89 University of Ghana http://ugspace.ug.edu.gh Joao, L.B., Guido, V., Marc, W., and Van Marc, E. (1997). Cytokine levels during mild and cerebral falciparum malaria in children living in a mesoendemic area. Tropical Medicine and International health. 2: (7) 673-679. John, C.C.. Sumba, P.O., Ouma, J.H., Nahlen, B.L., King, C.L., Kazura, J.W. (2000) Cytokine responses to Plasmodium falciparum liver-stage antigen 1 vary in rainy and dry seasons in highland Kenya. Infect Immun. 68(9): 5198-204. John, C. C.. O ’Donnell, R.A., Sumba, P. O., Moormann, A. M., de Koning-Ward, T. F., King, L. C., Kazura, J. W., and Crabb, B. S. (2004). Evidence that Invasion-Inhibitory Antibodies Specific for the 19-kDa Fragment of Merozoite Surface Protein-1 (M SP-I19) Can Play a Protective Role against Blood-Stage Plasmodium falciparum Infection in Individuals in a Malaria Endemic Area of Africa. The Journal o f Immunology, 173: 666­ 672. Kindermans. Jean-Marie, (2002). Press Dossier; Changing national malaria treatment protocol in Africa. Nairobi, Kenya. Retrieved from www.accessmed.msf.org. Kobayash, F., Morii, T., Matsui, T., Fujino, T., Watanabe, Y., Weidanz, W., and Tsuji M., (1996). Production of IL-10 during malaria caused by lethal and nonlethal variants of Plasmodium yoelii. Parasitol. Res. 82: 385-391. Kulane, A., Siddique, A.B., Perlmann, H., Ahlborg, N., Roussilhon, C., Tall, A., Dieye, A., Perlmann, P., Troye-Blomberg, M. (1997). T and B-cell responses of malaria immune individuals to synthetic peptides corresponding to non-repeat sequences in the N-terminal region o f the Plasmodium falciparum antigen Pfl55/RESA. Acta Trop; 68(1): 37-51. Kumaratilake, L.M., Ferrante, A., Jaeger, T., and Rzepczyk, C.M., (1992). Effect of cytokines, complement and antibody on the neutrophil respiratory burst and phagocytic response to plasm odium falciparum merozoites. Infect. Immun. 60: 3731-3738. 90 University of Ghana http://ugspace.ug.edu.gh Kumaratilake, L. M. and Ferrante, A., (1994). T-cell cytokines in malaria: Their role in the regulation o f neutrophil and macrophage-mediated killing of Plasmodium falciparum asexual blood forms. Res. Immunol. 145: 423-436. Kurtzhals. J.A. L., Hansen, M.B., Hey, A. S., and Poulsen, L.K. (1992). Measurement of antigen-dependent interleukin-4 production by human peripheral blood mononuclear cells; introduction o f an amplification step using ionomycin and phorbol myristate acetate. J. Immunl. M ethods, 156: 239-245. Kurtzhals, J.A., Adabayeri, V., Goka, B.Q., Akanmori, B.D., Oliver-Commey, J.O., Nkrumah, F.K., Behr, C., Hviid, L. (1998). Low plasma concentrations of interleukin 10 in severe malarial anaemia compared with cerebral and uncomplicated malaria. Lancet 351: 1768-1772. Khusmith, S., and Druilhe, P. (1983). Antibody-dependent ingestion of P. falciparum merozoites by human blood monocytes. Parasite Immunol. 5(4):357-68. Kwiatkowski, D., Cannon, J.G., Manogue, K.R., Cerami, A., Dinarello, C.A., and Greenwood, B.M. (1989). Tumour necrosis factor production in falciparum malaria and its association with schizont rupture. Clin Exp Immunol. 77:361-6. Luty, A.J.F., Lell, B., Schmidt-Ott, Lehman, L.G., Luckner D., Greve, B., Matousek, P., Herbich, K., Schmid, D., Migot-Nabias, F., Deloron, P., Nussenzweig, R.S., and Kremsner, P.G. (1999). Interferon-7 responses are associated with resistance to reinfection with P. falciparum in young African children. J. Infect. Dis. 179; 980-988. Mahanty, S., Allan, S., and Louis, H.M., (2003). Progress in the development of recombinant and synthetic blood-stage malaria vaccines. J. Expt. Bio. 206: 3781-3788. 91 University of Ghana http://ugspace.ug.edu.gh Marsh, K., and Greenwood, B.M. (1992). Malaria-A neglected disease? Parasitology. 104: 553-569. Mshana, R.N., Boulandi, J., Mshana, N.M., Mayombo, J., and Mendome, G. (1991) Levels of IL-lp, IL-4, IL-6 , TNFa and IFNy in plasma o f healthy individuals and malaria patients in a holoendemic area. J.Clin. Lab. Immunol., 34:131-139. McGregor. I.A., Carrington, S.C., Cohen, S. (1963). Treatment of East African Plasmodium falciparum malaria with West African human gammaglobulin. Trans. R. Soc Trop M ed Hyg. 57:170-175. McGregor. I. A. (1964). The passive transfer of human malarial immunity. Am. J. Med. Hyg 13: 237-239. ^ Miltenyi, S., Muller, W., Weichel, W., Radbruch, A. (1993). High gradient magn&r\i 'X \ separation with MACS. Cytometry, 231:238-1990. Mohan, K., Moulin, P., and Stevenson, M. M. (1997). Natural killer cell cytokine production, not cytotoxicity, contributes to resistance against blood-stage Plasmodium chaubaudi AS infection. J. Immunol. 159: 4990. Moore, K.W., O ’Garra, A., de-Waal Malefyt R., Vierra, P., and Mosmann, T.R. (1993). Interleukin 10. Annu. Rev. Immunol. 11: 165-190. Mwageni, E. (2002). Household wealth ranking and risks of malaria mortality in rural Tanzania. In: Third M IM Pan-African Conference on Malaria, Arusha, Tanzania. Multilateral Initiative on Malaria: abstract 12. Narum, D.L., Ogun, S. A., Thomas, A.W., Holder, A.A. (2000). Immunization with parasite-derived apical membrane antigen 1 or passive immunization with a specific University of Ghana http://ugspace.ug.edu.gh monoclonal antibody protects BALB/c mice against lethal Plasmodium yoelii yoelii YM blood stage infection. Infect. Immun\ 68:2899. Ndungu, F. M.. Bull. P. C., Ross. A., Lowe, B. S., Kabiru, E., and Marsh, K. (2002). Naturally acquired immunoglobulin (Ig)G subclass antibodies to crude asexual Plasmodium falciparum lysates: Evidence for association with protection for IgGl and disease for IgG2. Parasite Immunol. 24:77-82. Ockenhouse. C.F., Schulman, S., and Shear, H.L., (1984). Induction of crisis forms in the human malaria parasite plasmodium falciparum by y-interferon-activated, monocyte- derived macrophages. J. Immunol. 133: 1601. Oeuvray. C.. Bouharoun-Tayroun, H., Grass-Masse, H., Bottius, E., Kaidoh, T., Aikawa, M.. Filgueira, M.C., Tartar, A., and Druilhe, P. (1994). Merozoite surface protein 3; a malaria protein inducing antibodies that promote plasmodium falciparum killing by corporation with blood monocytes. Blood. 84; 1594-1602. Oeuvray. C.. Theisen, M., Rogier, C., Trape, J.F., Jepsen, S. and Druilhe, P. (2000). Cytophilic immunoglobulin responses to Plasmodium falciparum glutamate-rich protein are correlated with protection against clinical malaria in Dielmo, Senegal. Infect. Immun. 68:2617-2620. Okech, B.A., Corran, P.H., Todd, J., Joynson-Hicks, A., Uthaipibull, C., Egwang, T.G., Holder, A.A., Riley, E.M. (2004). Fine specificity of serum antibodies to Plasmodium falciparum merozoite surface protein, PfMSP-l(19), predicts protection from malaria infection and high-density parasitemia. Infect Immun. 72(3): 1557-67. Omer, F. M., Kurtzhals, J.A., and Riley, E. M. (2000). Maintaining the immunological balance in parasitic infections: a role for TGF-beta? Parasitol. Today 16: 18-23. 93 University of Ghana http://ugspace.ug.edu.gh Orago, A.S.S., Facer, C.A. (1991). Cytotoxicity of human natural killer (NK) cell subsets for plasmodium falciparum erythrocytic schizonts: Stimulation by cytokines and inhibition by neomycin. Clin Exp Immunol', 86:22-29. Osuntokun, B. O. (1983). Malaria and the nervous system. Afr. J. M edSci. 12: 165-172. Othoro, C., Lai, A.A., Nahlen, B., Koech, D., Orago, A.S.S., and Udhajakumar, V., (1999). A low interleukin 10, tumor necrosis factor ratio is associated with malaria anemia in children residing in a holoendemic malaria region in western Kenya. J. Infec. Dis. 179: 279-282. Paul, F.. Roath, S., Melville, D., Warhurst, D.C., and Osisanya, J. O. (1981). Separation of malaria infected erythrocytes from whole blood; use of a selective high gradient magnetic separation technique. Lancet: 2: 70-71. Perlmann, H.M., Helmby, H., Hagstedt, M., Carlson, J., Larsson, P.H., Troye-Blomberg, M., Perlmann, P. (1994). IgE elevation and IgE anti-malarial antibodies in plasmodium falciparum malaria: Association of high IgE levels with cerebral malaria. Clin. Exp Immunol 97:284-292. Perlmann, P., Troye-Blomberg, M., (eds) (2002). Malaria Immunology. Chem Immunol. Basel, Karger. 80: 229-242. Peyron, F., Burdin, N., Ringwald, P., Vuillez, J.P., Rousset, F., Banchereau, J. (1994). High levels of circulating IL-10 in human malaria. Clin. Exp. Immunol. 95; 300-303. Piper, K.P., Hayward, R.E., Cox, M.J., Day, K.P. (1999). Malaria transmission and naturally acquired immunity to PfEMP-1. Infect. Immun.; 67:6369. Roll Back Malaria / World Health Organization Country Updates. (2000). WHO/CDS/RBM/2000.24. James J. Banda. 94 University of Ghana http://ugspace.ug.edu.gh www.http://rbm.who.int/cmc upload/0/000/015/163/countrvUpdates.pdf Roll Back Malaria Monitoring and Evaluation. (2005). Retrieved from: www.http://rbin.who.int/wmr2005/profiles/ghana.pdf Riley, E.M., Andersson, G., Otoo, L.N., Jepsen, S., Greenwood, B.M. (1988). Cellular immune responses to Plasmodium falciparum antigens in Gambian children during and after an acute attack of falciparum malaria. Clin Exp Immunol, 73 (1): 17-22. Riley. E.M., Jakobsen, P.H., Allen, S.J., (1991). Immune response to soluble exoantigens o f plasm odium falciparum may contribute to both pathogenesis and protection in clinical malaria: evidence from a longitudinal, prospective study of semi- immune African children. Eur. J. Immunol. 21; 1019-1025. Riley, E.M., Rowe, P., Allen, S.J., Greenwood, B.M. (1993). Soluble plasma IL-2 receptors and malaria Clin Exp Immunol. 91: 3 :495-9. Rzepczyk, C.M., Ramasomy, R., Match, D.A., Ho, P.C., Battistutta, D., Anderson, K.L. Parkinson, D., Doran, T.J., Honeyman, M. (1989). Analysis of human T-cell response to two Plasmodium falciparum merozoite surface antigens. Eur. J. Immunol. 19: 1797­ 1802. Rzepezyk, C.M., Hale, K., Woodroffe, N., Bobogare, A., Csurshes, P., Ishii, A., Ferrante A. (1997). Humoral immune responses of Solomon Islanders to the merozoite surface antigen 2 of plasm odium falciparum show pronounced skewing towards antibodies of immunoglobulin G3 subclass. Infect Immun. 65: 1098-1100. Sabchareon, A., T. Burnouf, D. Outtara, P. Attanath, H. Bouharoun-Tayoun, P. Chantavanich, C. Foucault, T. Chongsuphajaisiddhi, and P. Druilhe., (1991). Parasitologic and clinical human response to immunoglobulin administration in falciparum malaria. Am. J.Trop.Med.Hyg. 45:297-308. 95 University of Ghana http://ugspace.ug.edu.gh Sarthou, J.L., Angel, G., Aribot, G., Rogier, C., Dieye, A., Balde, A.T., Diatta, B., Seignot, P., Roussilhon, C. (1997). Prognostic value of anti-plasmodium falciparum specific immunoglobulin G3, cytokines and their soluble receptors in West Africa patients with severe malaria Infect. Immun. 65:3271-3276. Shear. H.. Ng, C., and Zhao, Y., (1990). Cytokine production in lethal and non-lethal murine malaria. Immnol. Lett. 25: 123-127. Shephard. D. S., Ettling, M.B., Brinkmann, V., and Saurbom, R. (1991). The economic cost of malaria in Africa. Trop M ed Parasitol 42: 199-203. Smith. J.D.. Chitnis, C.E., Craig, A.G. (1995). Swithches in expression of Plasmodium falciparum var genes correlate with changes in antigenic and cytoadherant phenotypes of infected erythrocytes. Cell; 82: 101. Symthe. J.A., Coppel, R.L., Brown, G.V, Ramasamy, R., Kemp, D.J., Andrews, R. F. (1988). Identification of two integral proteins of Plasmodium falciparum. Proc. Nate. Acad. Sci. 85: 5195-5199. Snow, R.W., Guerra, C.A., Noor, A.M., Myint, H.Y., and Hay, S.I. (2005). The global distribution of clinical episodes of Plasmodium falciparum malaria. Nature 434 (7030):214-217. Spencer, V.L.M., Ogun, S. A., Fleck, S.L. (1998). Passive immunization with antibodies against three distinct epitopes of Plasmodiun yoelii merozoite surface protein 1 supresses parasitemia. Infect. Immun,; 66: 3925. Stevenson, M., Tam, M., and Nowotarski, M. (1990). Role of interferon gamma and tumor necrosis factor in host resistance to plasmodium chabaudi. As. Immunol. Lett. 25: 115-121. 96 University of Ghana http://ugspace.ug.edu.gh Soe, S., Theisen, M., Roussilhon, C., Khin-Saw-Aye, and Druilhe, P. (2004). Association between protection against clinical malaria and antibodies to merozoite surface antigens in a hyper-endemic area of Myanmar: complementarity between responses to MSP3 and GLURP. Infect. Immun.72:247-252. Su, Z.. and Stevenson. M.M. (2000). Central role of endogenous gamma interferon in protective immunity against blood-stage Plasmodium chabaudi AS infection. Infect. Immun. 68: 4399. Taylor-Robinson, A.W., Philips, R.S., Seveme, A., Moncada, S., Liew, F.Y. (1993). The role of Thl and Th2 cells in rodent malaria infection. Science. 260: 1931-1934. Theander. T.G.. Bygbjerg, I.C., Andersen, B.J., Jepsen, S., Kharazmi, A., Odum, N. (1986). Suppression of parasite specific response in Plasmodium falciparum malaria. A longitudinal study of blood mononuclear cell proliferation and subset composition. Scand. J. Immunol. 24: 73-81. Theander, T.G. (1992). Defence mechanisms and immune evasion in the interplay between the human defence system and Plasmodium falciparum. Dan M ed Bull. 39:49 63. Theander, T.G., Hviid, L., Dodoo, D., Afari, E.A., Jensen, J.B., Rzepczyk, C.M. (1997). Human T-cell recognition of synthetic peptides representing conserved and variant sequences from the merozoite surface protein 2 of Plasmodium falciparum. Immunol Lett. 58(1): 1-8. Theisen, M., Dodoo, D., Aissatou Toure-Balde, Soe Soe, Giampietro Corradin, K. A. Koram, J.A. Kurtzhals, L. Hviid, T. G. Theander, B. D. Akanmori, Mohamedou Ndiaye, and P. Druilhe. (2001). Selection of Glutamate-Rich Protein Long Synthetic Peptides for 97 University of Ghana http://ugspace.ug.edu.gh Vaccine Development: Antigenicity and relationship with Clinical Protection and Immunogenicity. Infect. Immun. 69; 5223-5229. Theisen, M., Soe, S., Oeuvray, C., Thomas, A. W., Vuust, J. S. Danielsen, S. Jepsen, and P. Druilhe. (1998). The glutamate-rich protein (GLURP) of Plasmodium falciparum is a target for antibody-dependent monocyte-mediated inhibition of parasite growth in vitro. Infect. Immun. 66 : 11-17. Theisen. M., J. Vuust, A. Gottschau, S. Jepsen, and B. Hogh. (1995). Antigenicity and Immunogenicity o f recombinant GLURP of P. falciparum expressed in Escherichia coli. Clin. Diagn. Lab. Immunology 2:30-34. Trape Jean-Francios (2001). The public health impact of chloroquine resistance in Africa. Am. J. Trop. Med. Hyg. 64 (1, 2) S, 12-17. Treutiger. C.J., Hedlund, I., Helmby, H., Carlsson, J., Jepson, A., Twumasi, P. (1992). Rosette formation in plasmodium falciparum isolate and anti-rosette activity in sera from Gambians with cerebral or uncomplicated malaria. Am. J. Trop. Med. Hyg. 46: 503-510. Troye-Blomberg, M., Romero, P., Patarroyo, M.E., Bjorkman A., and Perlman P. 1984 Regulation of the immune response in Plasmodium falciparum malaria; Proliferative response to antigen in vitro and subset composition of T-cells from patients with acute infection or from immune donors. Clin. Exp. Immunol. 58: 380. Troye-Blomberg, M., Andersson, G., Stoczkowska, M., Shabo, R., Romero, P., Patarroyo, M.E., Wigzell, H., Perlmann, P. (1985). Production of IL-2 and IFN-gamma by T cells from malaria patients in response to Plasmodium falciparum or erythrocyte antigens in vitro. .1 Immunol. 135(5):3498-504. Troye-Blomberg, M., Riley, E.M., Labikin, L., Holmberg, M., Perlmann, H., Anderson, V., Heusser, C.H., Perlmann, A. (1990). Production by activated human T-cells of 98 University of Ghana http://ugspace.ug.edu.gh interleukin-4 but not interferon-gamma is associated with elevated levels of serum antibodies to activating malaria antigens. Proc. Natl. Acid. Sci. U.S.A. 87: 5484-5488. Troye-Blomberg. M. and Perlmann, P., 1994. Malaria immunity: an overview with emphasis on T-cell functions; In Molecular Immunological Considerations in Malaria Vaccine Development. Good, M. F. and Saul, A. J. Boca CRC Press, pp 1—46. Troye-Blomberg, M., Weidanz, W. P. and Van der Heyde, H. C. (1999). The role of T- cells in immunity to malaria and the pathogenesis of disease; In Wahlgren, M., Perlmann. P. (eds.): Malaria: Molecular and Clinical Aspects. Amsterdam, Harwood Academic. 403-438. Troye-Blomberg M. (2002). Genetic regulation of malaria infection in humans. Chem. Immunol. 80. 243-252. Udeinya, I.L., Schmidt, J.A., Aikawa, M., Miller, L.H., Green, I. (1981). Falciparum malaria-infected erythrocytes specifically bind to cultured human endothelial cells. Science; 213:555-557. Waki, S., Uehara, S., Kanbe, K., Onok, Suzuki, M., Nariuchi, H. (1992). The role of T- cells in pathogenesis and protective immunity to murine malaria. Immunol. 75:646-651. Warrell, D.A. (1993). Clinical features of malaria, In H.M. Gillis and D.A. Warrell (ed.), Bruce-Chwatt’s essential malariology. Edward Arnold, London, England, p. 35-45. Weidanz, W.P., Brake, D.A., Cavacini, L.A. and Long, C.A. (1988). The protective role ofT cells in immunity to malaria. Adv .Exp. Med. Biol. 239:99-111. Wenisch, C., Parschalk, B., Narzt, E., looareesuwan, S. and Graninger, W. (1995). Elevated serum levels o f IL-10 and IFN in patients with acute plasmodium malaria. Clin. Immunol. Immunopathol. 74: 115-7. 99 University of Ghana http://ugspace.ug.edu.gh White, N. (1999). Antimalarial drug resistance and mortality in falciparum malaria. Trop Med. Intern Health 7: 469-470. Winkler, S., Martin, W., Karin, B., Daniela, S., Alexander, A., Wolfgang, G., and Kremsner. P. (1998). Reciprocal regulation of Thl and Th2-cytokine producing T-cells during clearance o f parasitemia in plasmodium falciparum malaria. Infect. Immunol. 66 (12) 6040-6044. WHO, The World Health Organization Report, Geneva: (1999); 49. World Health Report (2002). Reducing risks, promoting healthy life. Geneva, World Health Organization. Zambia Roll Back Malaria Report baseline study undertaken in 10 sentinel districts, (July-August 2001). Zambia, RBM National Secretariat. 100