University of Ghana http://ugspace.ug.edu.gh BOQ!LNLMS£̂ QKJ.55.-.5. m m k . . . . liiS&sJfiaft A ffE S s m ta University of Ghana http://ugspace.ug.edu.gh STUDIES ON CELLULAR IMMUNE RESPONSES OF PERIPHERAL BLOOD MONONUCLEAR CELLS FROM INDIVIDUALS IN AN ENDEMIC RURAL COMMUNITY TO A SYNTHETIC PLASMODIUM FALCIPARUM MEROZOITE ANTIGEN A Thesis Presented to the Board of Graduate Studies of The University of Ghana Legon By Daniel Dodoo In Partial Fulfillment of the Requirements for the Degree of Master of Philosophy From Department of Animal Science Faculty of Agriculture University of Ghana Legon February, 1994 University of Ghana http://ugspace.ug.edu.gh DECLARATION I certify that this work has not been submitted for a degree at any other University, and I further declare that the work embodied in it is my own. ..... DANIEL DODOO (Student) PROF. R.K.G. ASSOKU (Supervisor) / ' . T.G. THEANDER (Co-Supervisor) PROF. R.K.G. ASSOKU Dean of Graduate Studies and Head of Animal Science Dept. University of Ghana Legon. i University of Ghana http://ugspace.ug.edu.gh DEDICATION This thesis is dedicated to the glory of God, and to my dear wife Madeleine for her encouragement and sacrifice. University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENTS I wish to express my sincere thanks to all individuals and institutions which helped me in diverse ways to complete this work. First, I thank my supervisors, Prof. R.K.G. Assoku, Dean of Graduate Studies and Head of the Animal Science Department, University of Ghana, and Prof. T.G. Theander, Head of the Malaria Research Group, at the Centre for Medical Parasitology (C.M.P.), Denmark, whose rich experiences and deep knowledge on the subject helped immensely in the successful execution of the work. Dr. E.A Afari, head of the Epidemiology Unit, Noguchi Memorial Institute for Medical Research, (N.M.I.M.R.), is duly acknowledged for his help in organizing recruitment of individuals and sample collection at the study area. Also, I am very grateful to Dr. Jorgen Kurtzhals, Visiting Scientist from C.M.P., for his immense help, guidance, and criticisms during the preparation of this manuscript. I would also like to express my sincere thanks to Dr. Lars Hviid, Senior Research Fellow, C.M.P., Denmark for guidance and help during the conduction of the experiments, and Dr. K.M Bosompem, Research Fellow, Parasitology unit, N.M.I.M.R, Legon for helping with important suggestions on the final preparation and submission of the manuscript. The present work was carried out at the Noguchi Memorial Institute for Medical Research, (N.M.I.M.R.), Legon, of whose Director (Prof. F. Nkrumah) I am most grateful, and the Centre for Medical Parasitology (C.M.P.), University of Copenhagen, Denmark. I am thankful to DANIDA for the financial support for this work. iii University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS PAGE NO. TITLE PAGE ........................................................................................................ i DECLARATION ................................................................................................ i DEDICATION........................................................................................................ ii ACKNOWLEDGEMENTS .................................................................................. iii LIST OF FIG U RES............................................................................................. vi LIST OF TABLES ............................................................................................. vii LIST OF APPENDICES..................................................................................... xi SUMMARY ........................................................................................................ xii CHAPTER 1........................................................................................................... 1 1.1 Introduction..................................................................................... 2 1.2 Objectives of the Study ................................................................. 9 CHAPTER 2 ........................................................................................................... 11 2.1 Classification and identification of Plasmodium sp e c ie s ........... 12 2.2 Life cycle of the malaria parasite ................................................ 12 2.3 Human m a la ria ............................................................................... 14 2.4 Genetic resistance to m alaria ........................................................ 16 2.5 Development of protective immunity to malaria ....................... 17 2.6 Anti-disease Immunity.................................................................... 17 2.7 Anti-parasite Immunity ................................................................. 18 2.8 Parasite evasion of the immune system ........................................ 19 2.9 T-cell activation and the major histocompatibility complex . . . 20 2.10 Regulatory T-cell subsets ........................................................... 21 2.11 The relative roles of B- and T-cells in m alaria.......................... 21 2.12 T-cell responses to malaria an tigens.......................................... 22 2.12.1 Responses to pre-erythrocytic Plasmodium s ta g e s ......................................................................... 23 2.12.2 Responses to erythrocytic Plasmodium stages . . . . 24 2.13 The Impact of Malaria Infection on the Immune System . . . . 25 2.14 In vitro analysis of cellular immune responses in malaria . . . 29 2.15 Asexual malaria vaccine candidate antigens ............................ 31 2.15.1 Merozoite Surface Antigen 1 ..................................... 32 2.15.2 Merozoite Surface Antigen 2 ..................................... 3 3 2.16 Subunit malaria vaccines and MHC restriction......................... 34 2.17 Previous and ongoing vaccine t r i a l s .......................................... 3 5 University of Ghana http://ugspace.ug.edu.gh CHAPTER 3........................................................................................................... 37 3.1 Study a r e a ........................................................................................ 38 3.2 Target population............................................................................ 38 3.3 Blood sampling, isolation and storage of peripheral blood mononuclear c e lls .................................................................... 38 3.4 Microscopy ..................................................................................... 39 3.5 R eagents.......................................................................................... 40 3.5.1 MSA-2 p e p tid e s ........................................................... 40 3.5.2 Control an tigens........................................................... 40 3.5.3 Cell activators .............................................................. 41 3.6 Proliferation assay and generation of IFN- 7 containing supernatants ............................................................................ 43 3.7 Generation of interleukin-4 containing supernatants ................. 44 3.8 Determination of the effects of freezing and storage method on functional characteristics of PBMC ............................... 44 3.9 Preparation of reagents for cytokine E L IS A ............................... 45 3.9.1 Preparation of Protein A sepharose co lum n.............. 45 3.9.2 Purification of rabbit IgG ........................................... 45 3.9.3 Biotinylation of polyclonal rabbit anti-IFN- 7 or IL-4 IgG antibody ................................................... 46 3.10 Cytokine ELISA ............................................................................ 47 3.10.1 P rocedu re .................................................................... 47 3.11 Statistics ....................................................................................... 49 CHAPTER 4........................................................................................................... 50 4.1 Prevalence of malaria parasites among the subjects.................... 51 4.2 Lymphoproliferative responses...................................................... 51 4.3 Cytokine production....................................................................... 59 4.3.1 Pattern of IFN- 7 and IL-4 production....................... 62 4.4 Overall response of PBMC to any of the MSA-2 peptides . . . 62 4.5 Determination of the effects of freezing and storage method on proliferation and IFN- 7 a s sa y s ........................................ 62 CHAPTER 5........................................................................................................... 69 5.1 Discussion....................................................................................... 70 CHAPTER 6 ........................................................................................................... 77 6.1 Conclusion....................................................................................... 78 6.2 Recommendations ......................................................................... 78 REFERENCES..................................................................................................... 80 v University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES 1 Proliferative response to a peptide from the merozoite surface antigen (MSA-2) termed (GI) by peripheral blood mononuclear cells (PBMC) from Ghanaian adults, Ghanaian children and Danish adu lts .......................................................................................... 53 2 Proliferative response to a peptide from the merozoite surface antigen (MSA-2) termed (G2C) by peripheral blood mononuclear cells (PBMC) from Ghanaian adults, Ghanaian children and Danish adu lts................................................... 54 3 Proliferative response to a peptide from the merozoite surface antigen (MSA-2) termed (G4) by peripheral blood mononuclear cells (PBMC) from Ghanaian adults, Ghanaian children and Danish ad u lts .................................................................... 55 4 Proliferative response to positive control antigen purified protein derivative (PPD) by peripheral blood mononuclear cells (PBMC) from Ghanaian adults, Ghanaian children and Danish a d u lts .................................................................................. 56 5 Proliferative response to positive control antigen tetanus toxoid (TT) by peripheral blood mononuclear cells (PBMC) from Ghanaian adults, Ghanaian children and Danish adults..................... 57 6 Interferon gamma (IFN-7 ) production induced by MSA-2 peptides GI, G2C and G4 in PBMC from Ghanaian adults and Danish a d u lts .................................................................................. 60 7 Interleukin 4 (IL-4) production induced by MSA-2 peptides GI, G2C, and G4 in PBMC from Ghanaian adults and Danish adults ..................................................................................................... 61 8 IL-4 versus IFN- 7 production induced by MSA-2 peptides GI, G2C and G4 in PBMC from Ghanaian adults .................................. 64 9 Comparison of proliferative response induced by tetanus toxoid (TT) in frozen and fresh PBMC ............................................. 65 10 Comparison of interferon gamma (IFN-7 ) production induced by tetanus toxoid (TT) in frozen and fresh PBMC ............................... 6 6 University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES 1 Schematic representation of merozoite surface antigen (MSA-2) derived peptides used in the study......................................... 42 2 Prevalence of P. falciparum among Ghanaian adults and children..................................................................................................... 52 3a Frequencies of responses to merozoite surface antigen (MSA-2) derived peptides, purified protein derivative (PPD), and tetanus toxoid (TT) ....................................................................... 58 3b Statistical analysis of the frequencies of responses to merozoite surface antigen (MSA-2) derived peptides, purified protein derivative (PPD), and tetanus toxoid (TT)........................ 58 4 Interferon gamma (IFN-7 ) production in purified protein derivative (PPD) and tetanus (TT) stimulated peripheral blood mononuclear cells (PBMC) isolated from Ghanaian and Danish donors................................................................................... 67 5 Interleukin 4 (IL-4) release from purified protein derivative (PPD) and tetanus (TT) stimulated peripheral blood mononuclear cells (PBMC) isolated from Ghanaian and Danish donors .............................................................. 67 6 Frequencies of responses to merozoite surface antigen (MSA-2) in at least one of assays of T-cell responses used in the study ................................................................................................ 6 8 vii University of Ghana http://ugspace.ug.edu.gh ABBREVIATIONS AMA Apical membrane antigen APC Antigen presenting cell CPM - Counts per minute CSP Circumsporozoite protein CTL Cytotoxic lymphocytes DMSO Dimethylsulphoxide DTH Delayed type hypersensitivity EBA Erythrocyte binding antigen ELISA Enzyme-linked immunosorbent assay FCS Fetal calf serum GLURP Glutamine rich protein gm Gramme(s) HLA Human leucocyte antigen IFN- 7 Interferon gamma Ig Immunoglobulin IL- Interleukin- IU International units k Kilo kDa Kilodalton 1 Litre(s) LPS Lipopolysaccharide MHC Major Histocompatibility Complex min Minutes University of Ghana http://ugspace.ug.edu.gh ml Millitre(s) mm millimetre(s) MRC Medical Research Council MSA - Merozoite surface antigen Hg Microgram H1 Microlitre(s) MW Molecular weight NHS Normal human serum NIH National Institute of Health PBMC Peripheral blood mononuclear cells PBS Phosphate buffered saline pH Negative logarithm base 10 of hydrogen ion concentration PI Production index PMA Phorbol myristate acetate PPD Purified protein derivative of Tuberculin RAP-1 Rhoptry associated protein 1 RESA Ring infected erythrocyte surface antigen RPMI (1640) Rosewell Parke Memorial Institute Medium 1640 SERP Serine rich protein SI Significant index sIL-2R Soluble interleukin 2 receptor Spag Soluble purified P. falciparum antigen SPOD Peroxidase labelled streptavidin ix University of Ghana http://ugspace.ug.edu.gh TDR Tropical Disease Research Thl T helper 1 Th2 T helper 2 TNF-a Tumour necrosis factor alpha TT Tetanus toxoid WHO World Health Organization WRAIR Walter Reed Army Institute of Research X University of Ghana http://ugspace.ug.edu.gh LIST OF APPENDICES 1 The life cycle of human malaria parasites...........................................120 2 Schematic representation of the development of clinical and anti-parasite immunity in hyperendemic areas. The three curves represent the incidence of deaths from malaria, clinical attacks of malaria, and malaria parasitaemia, respectively................................................ 1 2 1 xi University of Ghana http://ugspace.ug.edu.gh SUMMARY Several Plasmodium spp. antigens have been found to be associated with protection against malaria and a few have been tested in the field in human volunteers. However, these efforts have achieved limited success, and as a result the search for additional more potent malaria vaccine components continues. The work reported in this thesis was executed with the objective of determining the suitability of three peptides obtained from the merozoite surface antigen, MSA-2, as putative sub-unit malaria vaccine components. Previous studies have suggested that a malaria vaccine capable of inducing long-lasting protective immune responses would most likely require incorporation of antigens expressing both B-and T-cell epitopes. In this respect the MSA-2 molecule which expresses both B- and T-cell epitopes has been shown to induce immune responses that eliminates mortality due to P. chabaudi malaria and significantly reduces parasitaemia in mice. It is, however, necessary to determine the protective ability of MSA-2 in man, since immunity induced in the animal model is not necessarily identical to the situation in humans. Investigations of this nature in man requires the use of peripheral blood mononuclear cells (PBMC) for in vitro assays. Clinical examination of the subjects included in this study, namely Ghanaian children and Ghanaian adults from a malaria endemic area, and unexposed control Danish adults, revealed no signs of clinical malaria at the time of blood sampling. However, P. falciparum parasites were found in blood samples of some of the Ghanaians with a significantly higher prevalence in the children (49%) than in adults (12%). xii University of Ghana http://ugspace.ug.edu.gh Initial experiments with fresh and cryo-preserved PBMC showed no significant differences (P< 0.005) in the ability to proliferate and to produce interferon gama (IFN-7 ) upon stimulation with tetanus toxoid (TT) as antigen. Cryo-preserved PBMC were therefore used in the experiments. In vitro lymphoproliferative assays using three MSA-2 peptides, namely GI, G2C and G4 which contain known T-cell epitopes showed significantly higher responses (P<0.03) in the exposed Ghanaian adults compared with the children and Danish adults. Thus, approximately 30% of cultures from Ghanaian adults responded to each of the peptides, and one each of the cultures from the Ghanaian children (5%) and Danish adults (6 %) responded to a single peptide. However, similar experiments using the control antigens, purified protein derivative of Tuberculin (PPD) and TT revealed no significant differences in lymphoproliferative responses between the three groups (P>0.05). This implies that the observed differences in the response to the MSA-2 peptides may be due to differences in immunological memory related to the level of prior exposure to malaria. Interferon-gamma (IFN-7 ) was detected in 32%, 36% and 29% of PBMC cultures from the Ghanaian adults following stimulation with the GI, G2C and G4 peptides, respectively. In all, 61% of these cultures produced IFN - 7 in response to at least one of the peptides. None of the cultures from Danish adults produced IFN-7 . The difference was statistically significant (P< 0.001), even though using the control antigens, PPD and TT, PBMC cultures from both groups produced similar amounts of IFN-7 . Interleukin-4 production was also detected in response to each of the peptides in PMBC cultures of only the Ghanaian adults. However, the number of University of Ghana http://ugspace.ug.edu.gh responding cultures was lower, and there was no statistical difference between the two groups. Similar to the results obtained for IFN-y production, there was no statistical difference between the Ghanaian and Danish adults with respect to IL-4 production in responses to control antigens. With one exception, PMBC cultures from Ghanaian adults produced only IFN-y or IL-4 in response to the MSA-2 peptides. In all, 82% of PMBC cultures from the exposed Ghanaian adults responded to MSA-2 peptides by proliferation or cytokine production. In contrast, only 14% of PMBC cultures from the unexposed Danish adults responded. The ability of the MSA-2 peptides to selectively stimulate PBMC of exposed Ghanaian adults seems to support the immunogenicity of the T-cell epitopes within the MSA-2 peptides in some individuals following natural exposure to malaria parasites. Furthermore, the association of these responses with the expected immunity of Ghanaian adults may suggest that MSA-2 T-cell epitopes could play a role in protective immunity against malaria. xiv University of Ghana http://ugspace.ug.edu.gh CHAPTER 1. INTRODUCTION 1 University of Ghana http://ugspace.ug.edu.gh 1.1 Introduction Malaria is arguably the most important tropical parasitic disease (TDR, 1985). It is a significant cause of abortion, still birth, child mortality, and death in pregnant women. It also causes impaired growth in children and loss of productive activity in adults (TDR, 1987). Extrapolations from epidemiological studies conducted in Africa indicate that the disease is responsible for not less than 1 . 0 to 2 . 8 million deaths annually, mainly among children below the age of five years (WHO, 1993). The enormous number of lives and labour lost together with the cost of treatment of patients, exerts a negative impact on development and thereby make malaria a major social and economic burden (TDR, 1993). Malaria is caused by protozoan parasites of the genus Plasmodium of which four species, P. falciparum, P. vivax, P. malariae and P ovale are responsible for the disease in man. These parasites are transmitted by Anopheles mosquitoes which act as vectors of the disease. Vivax malaria covers the widest geographic area, including temperate, tropical and subtropical zones. However it does not occur in large areas of tropical Africa. On the other hand ovale malaria is found mainly in tropical Africa, and malariae malaria is widely distributed, but it is not as common as vivax malaria (TDR, 1987). Falciparum malaria is the most lethal and frequently occurring form of the disease throughout the tropics and subtropics. In all about 300 million people are believed to be infected worldwide with malaria parasites, with 90% of them living in tropical Africa. Of these, about 120 million develop clinical disease which is mostly caused by P. falciparum. This parasite is also responsible for over one third of the infections in the rest of the 2 University of Ghana http://ugspace.ug.edu.gh world. According to TDR (1993), nearly 40% of the world’s population, over two billion people, are exposed to the risk of malaria infection. Human malarial infection begins with the bite of an infected female Anopheles mosquito. Injected sporozoites migrate through the bloodstream to the liver where they invade hepatocytes, and undergo a phase of maturation and asexual reproduction. Numerous asexual progeny, known as merozoites leave ruptured liver cells, enter the bloodstream and invade circulating erythrocytes. This invasion initiates the erythrocytic phase of the life cycle of the parasite. Parasites in the red cells multiply in species-characteristic fashion and break out of the host cells synchronously. Successive broods of merozoites appear at 48-hour intervals with P vivax, P. ovale, and P falciparum or every 72 hours with P malariae to initiate a new intra-erythrocytic cycle. During the erythrocytic cycles, some parasites differentiate into male or female gametocytes. This initiates the sexual cycle which can only continue with the sporogonic phase in the vector (Bruce-Chwatt, 1985). During a typical malaria infection, the various stages of the parasite that occur in the mammalian host present a wide variety of antigens to the host’s immune system. However, only a small proportion of the antigens are believed to be capable of stimulating protective immune responses. Some of the other antigens are either irrelevant to protection or may even induce undesirable host responses (TDR, 1985). Furthermore, stage-specific as well as strain-specific differences in the immunity to Plasmodium infections have been reported (Howard, 1987; Riley e ta l., 1993). 3 University of Ghana http://ugspace.ug.edu.gh Both antibody-dependent and antibody-independent immune effector mechanisms appear to be involved in naturally acquired protective immunity to malaria. As early as the 1950’s, Colbourne (1955) reported that this immunity appears to depend upon maintenance of immunological memory in the presence of low level parasitaemia, inhibiting new infections or maintaining the infections at a low level without clinical symptoms (Bruce-Chwatt, 1963). The ability of antibodies to give protection against Plasmodium is evident from the protection conferred to neonates and infants by maternally derived antibodies and from clinical treatment trials with immune serum or purified immunoglobulins (Cohen et al., 1961; McGregor et al., 1963). The total level of Plasmodium specific antibodies in individuals residing in areas of high malaria endemicity has been found to be age dependent. It increases during childhood and reaches maximum levels in early adult life (McGregor et al., 1970). It has been observed by several workers that the antibodies produced are directed against all the stages of the parasite that occur in the mammalian host, namely the sporozoites, blood stages and gametocytes (Nardin et al., 1979; Cohen and Butcher, 1971; Mendis et al., 1987). However, no significant correlation has been found between total anti- plasmodium antibody levels and malaria infection or clinical disease in individuals studied over long time periods. Thus, in general antibody levels have been found to be more indicative of previous infection than of functional acquired immunity (Voller, 1971; McGregor, 1986; Marsh et al. , 1989). Consequently, Weidanzand Long (1988) hypothesized that the development of protective immunity depends on the acquisition of a critical number of T-cells specific for malaria antigens which subsequently control the immune response through regulation of macrophages, B- 4 University of Ghana http://ugspace.ug.edu.gh cells, production of substances toxic to the infecting parasites and direct cytotoxicity (Ockenhouse and Shear, 1983, 1984; Kabilan et al., 1987; Sinigaglia et al., 1987; Hoffman et al., 1989a; Schofield et al., 1987a,b). Two subsets of helper T-cells (CD4 positive T-cells), Thl and Th2 have been characterized based on the pattern of their cytokine production (Mossman et al., 1986). Thl cells produce IFN-7 , IL-2, and lymphotoxin among other soluble mediators, whereas Th2 cells secrete IL-4, IL-5, IL-6 , and IL-10. Functionally, Thl cells have mainly been associated with cellular immune responses such as delayed type hypersensitivity (DTH) reactions. On the other hand, Th2 cells have been related to B-cell help leading to antibody production (Mossman and Coffman, 1989). Recently both Thl and Th2 cells have been found to play a protective role during P. chabaudi chabaudi infection in mice (Taylor-Robinson et al., 1993). Epidemiological studies in areas with stable endemic P. falciparum malaria have shown that parasitaemia is most common in young children and that the incidence of parasitaemia declines with age. Christophers (1924) suggested that this trend provides evidence of gradual acquisition of specific immune responses which inhibit parasite growth and eliminate blood stage parasites. However, McGregor (1960) and Gilles (1961), reported that individuals living in malaria endemic areas may pass through 5 stages before immunity is acquired. According to them, stage 1 involves 0 2 month old infants. These children who are generally protected against malaria by maternal antibodies, have low incidence of parasitaemia which is normally below 10%. Stage 2 involves 2 6 months old infants. The first 5 University of Ghana http://ugspace.ug.edu.gh clinical attacks of malaria which are normally mild are encountered in this group which have a high incidence of parasitaemia (Gilles, 1961). After this age stage 3 is reached during which repeated malaria attacks associated with severe clinical illness disrupt the normal pattern of growth. Profound anaemia and high mortality rates have also been reported among members of this group (McGregor et al., 1956). Stage 4 includes older children among whom there is a rapid decline in severity and frequency of clinical episodes, despite the persistence of relatively high parasitaemia (Ziemann, 1924). This stage represents the first phase of clinical immunity (or antitoxic immunity). The fifth and final stage comprises adolescents and adults who experience fewer and milder clinical attacks. The immunity in this group is not sterile as adults living in holoendemic areas will often harbour low parasite levels even though they exhibit no clinical symptoms (Bruce-Chwatt, 1963). The age at which an individual passes through each stage is dependent upon the endemicity of malaria in the area. Based on these epidemiological findings, individuals living in areas of high malaria endemicity may be categorized into, "non-immune" young children, "semi-immune" older children arid "immune" adults. The rapid emergence of parasite strains which are resistant to several anti- malarial drugs and failure of most malaria control programs utilizing the present repertoire of available tools has prompted the call for a vaccine against malaria (TDR, 1993). There has been considerable progress in defining malaria antigens, a few of which seem to play a role in protective immunity. Furthermore, genes that control the expression of some of the identified protective antigens have been characterized (TDR, 1993). However, in the attempt to develop vaccines against 6 University of Ghana http://ugspace.ug.edu.gh malaria, researchers have concentrated on aspects of humoral immunity, thereby neglecting cell-mediated immunity. With this observation in mind, the British Medical Council and the Malaria Immunology Scientific Working Group of WHO- TDR have emphasized the importance of investigating the role of T-cells in development and acquisition of immunity (MRC, 1989; TDR, 1985). In addition, several workers including Troye-Blomberg and Perlmann (1988) and Weidanz and Long (1988) argued that although B-cells are the source of antibody producing plasma cells, B-cell differentiation and antibody production is regulated by T-cells. Hence, to induce efficient and long-lasting antibody responses which can be boosted by natural infections, the parasite antigens used in vaccines must contain epitopes recognized by regulatory T-cells as well as B-cells (TDR, 1987). Subsequently, the importance of cell mediated immunity in protection against malaria has been established by studies with rodents in which T-cell mediated immunity to merozoites was shown to play an important role in protection (Jayawardena et al., 1982). T-cell responses to P falciparum merozoite surface antigens in mice and rhesus monkeys have been shown to be under genetic control, that is MHC- restriction (TDR, 1987). Studies in man, although not conclusive, also appear to suggest MHC-restriction of T-cell responses to P. falciparum antigens (Quakyi et al., 1989). These findings may suggest that future malaria vaccines should contain several T-cell epitopes, if the majority of vaccinated subjects are to respond favorably (TDR, 1987). This would mean that there is the need for thorough understanding of the nature and function of responses induced by malaria specific immunodominant T-cell epitopes. Furthermore, the rational development of malaria 7 University of Ghana http://ugspace.ug.edu.gh vaccines requires an understanding of the mechanisms underlying both protective immunity and the immunopathological complications associated with the disease (TDR, 1985). The present study, therefore, aimed at investigating in vitro T-cell responses to one of the putative malaria vaccine components, the merozoite surface antigen 2 (MSA-2) in Ghanaians exposed to the risk of malaria. Responses to three peptides from different regions of the molecule all containing aminoacid sequences (epitopes) which can stimulate T cells from immune individuals (Rzepczyk et al. , 1989) were used for this purpose. 8 University of Ghana http://ugspace.ug.edu.gh 1.2 Objectives of the Study 1) To study in vitro lymphoproliferative T-cell responses to MSA-2 peptides GI, G2C and G4, in peripheral blood mononuclear cells (PBMC) of children and adults from a malaria endemic Ghanaian community. 2) To compare in-vitro lymphoproliferative T-cell responses to MSA-2 in PBMC of subjects from a malaria endemic area of Ghana with those of unexposed Danish controls. 3) To examine the nature of the T-cell response to the MSA-2 peptides in Ghanaian adults with respect to IFN-y and IL-4 production. 9 University of Ghana http://ugspace.ug.edu.gh Justification The study of cellular immune responses to the MSA-2 antigen in Ghanaian children who are highly susceptible to malaria and Ghanaian adults who are less susceptible to the disease, would provide useful information needed to determine the suitability of MSA-2 as a vaccine candidate antigen. 10 University of Ghana http://ugspace.ug.edu.gh CHAPTER 2. LITERATURE REVIEW 11 University of Ghana http://ugspace.ug.edu.gh 2.1 Classification and identification of Plasmodium species Based on morphological and biological criteria malaria parasites are classified under the phylum Protozoa, the subphylum Sporozoa, the class Telosporea, the subclass Coccidia, the order Eucoccidia, suborder Haemosporina, family Plasmodiidae, and genus Plasmodium (Honigeberg et al., 1964; Levine, 1973). More than 100 Plasmodium species are known to infect a wide range of hosts including reptiles, birds, rodents and primates, with each parasite species exhibiting a narrow host specificity. Four species, namely, P. falciparum, P.vivax, P. ovale and P. malariae, are responsible for the disease in man. However, malaria caused by P. falciparum is the most lethal, resulting with the highest morbidity and mortality. These human infective Plasmodia are easily distinguished by their morphological appearances using ordinary light microscopy of Giemsa or Field stained blood films. 2.2 Life cycle of the malaria parasite Plasmodium spp. undergo complex life cycles, alternating between a vertebrate host and an arthropod vector. Malaria parasites are transmitted to humans through the bite of infective female mosquitoes belonging to the genus Anopheles. In this process, infective sporozoites are introduced into the skin of a susceptible host through the saliva of the vector. In the host, the injected sporozoites migrate through the bloodstream to the liver where they invade liver cells within half an hour after transmission to begin the exoerythrocytic hepatic stage of the life cycle. In the liver cells the 12 University of Ghana http://ugspace.ug.edu.gh parasites undergo asexual multiplication by which they develop through different stages that culminates in the formation of thousands of merozoites. With falciparum malaria, about 30,000 merozoites may be released from an infected liver cell 7-12 days after invasion. The released merozoites in turn invade erythrocytes to initiate the asexual erythrocytic stage of the life cycle. In the erythrocyte the parasite develops through ring, trophozoite and schizont stages with species distinguishing characteristics (that can be used in differential diagnoses). The intra-erythrocytic parasite is surrounded by a parasitophorous vacuole membrane. A great deal of parasite protein synthesis takes place inside infected erythrocytes especially during the trophozoite stage of development. During this development, the parasite modifies host cells in several ways so as to enhance its own survival. In the red cell, asexual division within the schizont stage results with about 8-24 daughter merozoites which are finally released following red cell rupture to initiate subsequent intra-erythrocytic cycles. Generally, the intra-erythrocytic cycle tends to become synchronous after a few cycles. In the case of P. falciparum, P.vivax and P ovale, the generation time has been determined to be 48 hours whilst that of P. malariae is 72 hours. The release of parasites into the blood stream is normally accompanied by fever which therefore becomes periodic because of the synchronous nature of merozoite release. In a susceptible host this asexual erythrocytic cycle may be repeated many times, during which a few invading parasites differentiate into sexual forms, (gametocytes) which alone can infect the insect vector. Plasmodium species infect the mosquito following ingestion of infective gametocytes in the blood of an infected vertebrate. In the gut of the mosquito 13 University of Ghana http://ugspace.ug.edu.gh gametocytes are released from infected erythrocytes to begin the sexual stage of the life cycle. First, the gametocytes differentiate into male and female gametes and the male microgamete fertilizes the female macrogamate resulting in the formation of a zygote. Shortly after fertilization, meiosis takes place leading to genetic recombination, thus creating new haploid parasite variants. Indeed, apart from the zygote all the parasite stages that occur in the vector and the vertebrate hosts are haploid. The zygote develops into a mobile ookinete which penetrates the peritrophic membrane surrounding the bloodmeal and the gut endothelium, to which it attaches. The ookinete then transforms into an oocyst which attaches to the outside of the mosquito gut wall suspended in the haemocoele. Inside the oocyst a massive multiplication of parasites takes place resulting in the formation of hundreds of thousands sporozoites. The sporozoites invade the mosquito salivary glands and mature into infective forms which alone can initiate infections in the vertebrate host. It takes about 10-11 days at 25°C for gametocytes to develop into infective sporozoites (reviwed by Theander, 1992; Riley et al., 1993). 2.3 Human malaria The clinical manifestations of human P. falciparum infections include chills, fever, headache, nausea, vomiting and diarrhoea. A prominent feature of the febrile response is its tendency of periodicity. In areas with stable malaria transmission clinical symptoms of malaria are normally confined to children and pregnant women (Theander, 1992). Greenwood (1987; 1991) observed that the incidence of clinical malaria in rural areas of the Gambia is in the range of 1-5 annual attacks per child, with each clinical attack 14 University of Ghana http://ugspace.ug.edu.gh having a mortality rate of about 0.5% (Greenwood, 1990). Nevertheless, cerebral malaria and severe anaemia with mortality rates of 15-30% are responsible for most deaths resulting from malaria in tropical Africa. Severe anaemia is especially prevalent in the younger children whereas cerebral malaria is more frequent in slightly older children (Greenwood, 1991, Brewster, 1990). It has been proposed that severe malaria may partly be due to exposure to more virulent parasite strains with additional properties including the expression of variant antigens that are new to the hosts immune system (Clark, 1989). However, the immune response of the host may also contribute to the pathogenesis of severe malaria, since the symptoms including, fever, nausea, vomiting, rigor, headache and thrombocytopenia, are similar to the physiological effects of certain cytokines including TNF-a (Clark, 1991). Immigrants and tourists as well as children and adults living in areas of unstable malaria transmission are all susceptible to clinical attacks. In such immunologically naive individuals malaria parasitism is nearly always associated with disease (Marsh, 1992). In non-endemic areas, the diagnosis of clinical malaria may thus be made on the basis of fever and a positive blood film. In malaria endemic areas, a case definition is extremely difficult even though clinical malaria is often defined as fever with temperatures greater than 37.5°C associated with more than 2500 parasites per ^1 blood. The definition has limitations since there appears to be no strict correlation between parasite burden and clinical symptoms. Thus children with very low parasitaemia sometimes have symptoms while other children with much higher parasitaemia have none (Marsh, 15 University of Ghana http://ugspace.ug.edu.gh 1992). To assume that a child needs to present with both fever and reasonably high parasitaemia to be declared ill from malaria may therefore exclude some true cases (Schellenberg et al., 1994). Nevertheless, in the presence of asymptomatic P. falciparum infections, fever could be due to other disease conditions like pneumonia and influenza. Hence one needs to apply strict exclusion criteria to rule out other disease conditions presenting with fever when diagnosing clinical malaria in endemic areas. 2.4 Genetic resistance to malaria Some red cell abnormalities including the sickle cell trait, alpha thalassemia, beta thalassemia, ovalocytosis, andglucose-6 -phosphate-dehydrogenase deficiency have been associated with various degrees of resistance to malaria (Weatherall, 1987). This is partly because high prevalence rates of these abnormalities are found in areas where malaria is endemic (Allison, 1954; ; Flint et al., 1986). It is so far established that the sickle cell trait confers protection against clinical malaria and especially against severe malaria but not against infection as such (Hill, 1991). HLA antigens have also been linked with protection against severe clinical malaria in Gambian children. In this respect, the class I antigen, HLA-Bw53 was reported to be associated with protection against both cerebral malaria and severe anaemia while the class II antigen HLA DRwl3 was associated with protection against severe anaemia (Hill, 1991). 16 University of Ghana http://ugspace.ug.edu.gh 2.5 Development of protective immunity to malaria Studies in malaria endemic populations on the acquisition of immunity to malaria have shown that the decline in the levels of parasitaemia is usually preceded by a decline in morbidity and mortality from malaria. In a study conducted in the Gambia where P. falciparum transmission was seasonal but relatively stable from year to year, malaria parasite rates were found to decline only after the age of 10 to 12 years (Riley et al., 1990a). In the same study clinical disease (fever associated with parasitaemia) was found to peak at 6 years of age. However peak mortality was found to occur in children aged 4 years (Greenwood et al., 1987). Hence the incidence of severe disease and death from malaria declines rapidly in young children at a time when they still harbour considerable parasite loads. In agreement, Playfair et al. (1990) and indeed Sinton (1939) proposed that clinical or anti-disease immunity may be distinct from anti-parasite immunity. It is likely that different protective mechanisms are involved in these two separate machanisms of protective immunity and different antigens may be involved in their induction (Playfair et al., 1990). 2.6 Anti-disease Immunity Some of the soluble exoantigens released into circulation during rupture of schizont infected erythrocytes have been shown to share chemical and biological characteristics with bacterial endotoxins such as lipopolysaccharide (LPS) (Jakobsen et el., 1987). These exoantigens directly trigger monocytes and macrophages to release endogenous pyrogens like interleukin-1 (IL-1) and tumour necrosis factor (TNF) (Bate et al., 1989; Taverne et al., 1990; Jakobsen et a l., 1991), which have 17 University of Ghana http://ugspace.ug.edu.gh been implicated in the pathogenesis of malaria (Clark and Cowden, 1991). It has been shown in mice that antibodies against these exoantigens can block the induction of pyrogenic cytokines and reduce severity of clinical symptoms (Clark and Cowden, 1991). Playfair et al. (1990) proposed that a similar situation may exist in humans. Thus, anti-disease immunity is believed to exist in humans with relatively high parasitaemia without any clinical disease. 2.7 Anti-parasite Immunity Immunity against malaria also depends on the ability to control parasite multiplication. This has been shown to involve both antibody dependent and antibody independent effector mechanisms (Troye-Blomberg and Perlmann, 1988; Weidanz and Long, 1988). For example, antibody against the circumsporozoite protein (CSP) can inhibit the entry of sporozoites into liver cells (Hollingdale, 1984), and the prevalence of such antibodies has been shown to increase with age in endemic populations (Hoffman et al., 1986). Also, sporozoite and liver stage antigen specific cytotoxic T-lymphocytes (CTL) have been demonstrated in vitro in mice (Kumar et al., 1988; Hoffman et al., 1989b). However, the importance of cytotoxic responses against infected hepatocytes in the development of immunity in humans have not been established. Also Interferon gamma (IFN-7 ) released by immune T-cells, have been shown to mediate killing of intrahepatic parasites in vivo (Ferreira et al., 1986). It is also known that antibody against merozoites can block their invasion into erythrocytes in vitro (Wahlin et al., 1984) and cytophilic antibodies can opsonize parasites for phagocytosis (Lunel and Druilhe, 1989). Furthermore, 18 University of Ghana http://ugspace.ug.edu.gh agglutinating antibodies can immobilize free merozoites (Green et al., 1981). Antibodies can also block the adherence of mature parasitized erythrocytes to capillary endothelium and thereby facilitate their clearance from circulation and reduce the risk of cerebral malaria (David et a l., 1983). In these processes, T-cells are believed to provide help for antibody production through release of cytokines such as IL-4, IL-5 and IFN- 7 (Weidanz and Long, 1988). Cytokines such as IFN-7 , activate macrophages to phagocytose infected erythrocytes and thereby kill malaria parasites (Brown and Kreier, 1986). A different aspect of anti-parasite immunity is the finding that antibodies to gamete surface antigens (Carter et al., 1985) and gamete-specific T-cells can block transmission of malaria parasites to mosquitoes (Harte et a l., 1985; Naotunne e ta l , 1991). 2.8 Parasite evasion of the immune system Malaria parasites are intracellular during most of their life cycle. As a result, they are exposed to the extra cellular environment for only brief periods of time. This situation helps them to evade much of the host’s defence mechanisms. Also, antigenic variation involving antigens which may be crucial to parasite survival, and inhibition of immune responses as well as variation of parasite proteins on the surface of infected cells such as erythrocytes (Hommel, 1983), are important evasive strategies employed by the parasite to slow down the development of immunity against malaria. Roberts (1992) observed that parasite antigens expressed on the surface membranes of infected erythrocytes may be linked with cytoadherence properties. Also, Marsh and Howard (1986) argued that 19 University of Ghana http://ugspace.ug.edu.gh the expression of parasite antigens on red cell membrane may slow down development of immunity against different strains of the malaria parasite. 2.9 T-cell activation and the major histocompatibility complex Antigen specific T-lymphocytes recognize degraded antigen on antigen presenting cells (APC), in the form of complexes with polymorphic cell surface proteins encoded by the major histocompatibility complex (MHC) (Male et al., 1991). In humans, the MHC is referred to as Human Leukocyte Antigens (HLA) of which two major types are involved in antigen presentation. These are the Class I and Class II antigens. The HLA antigens involved in antigen presentation determines the type of T-cells that recognize the antigen and become activated. This is because antigens presented in association with MHC Class I molecules are recognized by CD8 + T-cells, whilst antigens presented in association with MHC Class II molecules are recognized by CD4+ T-cells. The expression of CD4 and CD8 on mature T-cells is mutually exclusive. It has been determined that the CD4+ T-cell subset roughly corresponds to the functionally defined helper/inducer subset whereas CD8 + T-cells are mainly cytotoxic (Imboden and Weiss, 1988; Haas et al., 1990). The parts of a peptide recognized by T-cells are reffered to as T-cell epitopes. T-cell epitopes normally contain a linear row of 9-15 amino acids (Roit, 1991). Following activation some T-cells eventually differentiate into memory T- cells with a characteristic pattern of cell-surface molecules (or markers) (Sanders et al., 1988). On subsequent encounter with antigen, memory T-cells proliferate, releasing different cytokines which effect various biological functions (Sanders, et 20 University of Ghana http://ugspace.ug.edu.gh al., 1988). These include help to B-cells for antibody production and stimulation of natural killer cells and monocytes (Sinigaglia et al., 1987; Ockenhouse and Shear, 1983). 2.10 Regulatory T-cell subsets Analysis of murine T-cell clones has revealed that CD4+ cells can be divided into two subpopulations, based on their repertoire of cytokine production (Mossman et al., 1986). Upon activation, CD4+ cells of the T-helper 1 subset (Thl) produce IL-2 and IFN- 7 among other cytokines, while cells of the T-helper 2 subset (Th2) produce IL-4 and IL-5 (Mossman and Coffman, 1989). Although the relationship between these cell types is not clear, it appears that Thl cells mediate certain antibody independent responses and Th2 cells provide help for specific antibody production (Taylor-Robinson et al., 1993). In the murine malaria model using P. chabaudi, Thl cells have been shown to be important in the early phase of infection, whereas the final clearance of the parasite load, coincides with the appearance of malaria-antigen specific antibody mediated effector mechanisms (Langhorne et al., 1990). 2.11 The relative roles of B- and T-cells in malaria Experiments with laboratory rodents have contributed greatly to our understanding of the relative importance of the protective roles of T- and B-cells (Jayawardena, 1981, Weidanz and Long, 1988; Del Giudice et al., 1988). These experiments have shown that there is a large variation in T- and B-cell requirements, depending on the strain and species of the infecting Plasmodium 21 University of Ghana http://ugspace.ug.edu.gh parasites and the genetic constitution of the mice (Jayawardena, 1981; Long, 1988). Correlation between total anti-malaria antibodies and protection is poor, indicating that many of the formed antibodies do not have any protective value. However, passive transfer of protection with IgG from immune sera has been demonstrated in both humans (Cohen et al., 1961; McGregor et al., 1963), and in different animal models (Weidanz and Long, 1988). In addition, several parasite specific monoclonal antibodies have been shown to either protect or block transmission of rodent malaria (Hollingdale et al., 1984; Miller, et al., 1984). However, even in those experimental models in which the protective importance of antibodies is well established, it is agreed that maintenance of immunity is under the control of T-cells (Weidanz and Long, 1988). In general, anti-malaria antibodies are of T-dependent isotypes (Weidanz and Long, 1988; Troye-Blomberg and Perlmann, 1988), and T-cells also control the antibody independent effector systems involved in parasite clearance such as help for generation of antigen specific cytolytic cells (Hoffman et al. , 1989a), production of cytokines (Schofield et al., 1987a,b; Sinigaglia et. al, 1987), and activation of non-lymphoid cells leading to intracellular killing (Ockenhouse and Shear, 1983, 1984). 2.12 T-cell responses to malaria antigens There is good evidence from experimental animal models that cellular immune responses are involved in protective immunity to malaria. In some models, thymectomized animals failed to become immune whilst intact animals developed a long lasting immunity (Brown et al., 1968; Weinbaum et al., 1976). In the case of P. yoelii infections in mice, maximum protection depended on the cooperation 22 University of Ghana http://ugspace.ug.edu.gh between T and B lymphocytes (Mogil, 1987), whereas immunity to P. chabaudi adami appears to be independent of antibody (Grun and Weidanz, 1981). Although the extent to which animal models parallel the human response to malaria infection is not clear, accumulating evidence indicate that T-cells play an important role in the human response (Troye-Blomberg and Perlmann, 1988; Weidanz and Long, 1988). Thus, for example, stage specific malaria antigens have been shown to be recognized in vitro by T cells of individuals from malaria endemic regions (Hviid e ta l., 1992). 2.12 . 1 Responses to nre-ervthrocvtic Plasmodium stages Donors from malaria endemic areas often possess sporozoite antigen- reactive T-cells, directed against the circumsporozoite (CSP) antigen, a major surface protein of sporozoites (Dame et al., 1984; Good et al., 1988). In mice control of sporozoite infection is partly effected by CD8 + CSP-specific CTL response against infected hepatocytes (Schofield et al., 1987b; Hoffman et al., 1989b). However, the importance of cytotoxic responses against infected hepatocytes in the development of immunity in man is not known. As reported by Schofield (1987a), and Mellouk (1987), the best established human response against infected liver cell involves the cytokine IFN-7 . 23 University of Ghana http://ugspace.ug.edu.gh 2.12.2 Responses to ervthrocvtic Plasmodium stages Most malaria parasite antigens exposed to the immune system are from asexual blood stages. These include the ring infected erythrocyte surface antigen, Pfl55/RESA, which is a polypeptide originating from merozoites (Perlmann et al. , 1984; Coppel et al., 1984). This antigen is deposited into the erythrocyte surface membrane during parasite invasion. Pf 155/RESA has been shown to induce specific T-cell response in malaria exposed individuals. This response includes proliferation, cytokine production and T-cell dependent antibody production (Kabilan et al., 1988). Different human T-cell response types to Pfl55/RESA have been characterized (Kabilan et al., 1988), suggesting the existence of functionally distinct human T-cell subsets similar to Thl and Th2 type T-cell described in mice (Troye-Blomberg et al., 1990). Several T-cell epitopes have been identified in the P. falciparum major merozoite surface protein (MSA-1), using synthetic or recombinant peptides and T-cells from people of malaria endemic areas (Hviid et al., 1992). Responses to variable regions of MSA-1 by T-cells from Gambian donors were found to increase with age, whereas responses to conserved regions decreased (Hviid et al., 1992). Moreover both proliferation and IFN- 7 production in response to the C-terminal part of the molecule appeared to be associated with resistance to clinical disease and high parasitaemia. Another P falciparum merozoite surface antigen (MSA-2), has been shown to contain T-cell epitopes both in the constant and variant regions of the antigen (Rzepczyk et al., 1989, 1990), and to induce lymphoproliferation in malaria exposed donors in an age dependent way by both proliferation and IFN- 7 24 University of Ghana http://ugspace.ug.edu.gh production. Other asexual blood stage antigens which contain T-cell epitopes that induce specific proliferation and cytokine production from T-cells of malaria exposed donors include soluble purified P. falciparum antigen (Spag), purified from culture supernatant (Jepsen and Andersen, 1981; Jakobsen eta l., 1990, 1991; Hviid et al., 1990); glutamine rich protein (GLURP) and serine rich protein (SERP), which are soluble proteins stored in the parasitophorous vacuole and released during schizont rupture (Borre et al., 1991; Roussilhon, et al., 1990). No evidence of T-cell effector mechanism against parasitized erythrocytes has been found, which is not surprising considering the scarcity of MHC class I antigens on erythrocytes. Cytokines such as IFN- 7 do not have direct inhibitory effect on blood stage P falciparum parasites (Hviid et al., 1988; Ferreira et al., 1986), but might be involved in the immune responses through activation of monocytes (Ockenhouse and Shear, 1984). T-cells from malaria exposed individuals from the Gambia have been found to respond to crude gametocyte antigen, whilst unexposed individuals responded weakly (Riley et al., 1990). T-cells from 40% of the same exposed Gambian donors responded to affinity purified gametocyte antigen Pfs 48/45, whilst non-exposed donors were completely unresponsive (Riley et al. 1990) 2.13 The Impact of Malaria Infection on the Immune System There is much evidence to suggest that acute malaria leads to a temporary state of reduced immunocompetence (Houba, 1988; Goodnewardene et al., 1990; Hviid et al., 1992; Riley et al., 1993). Acute malaria is associated with increased 25 University of Ghana http://ugspace.ug.edu.gh susceptibility to salmonellosis (Bennett and Hook, 1959; Mabey et al., 1987), and other bacterial diseases (Greenwood, 1974), as well as reactivation of chronic or latent viral infections such as those caused by Herpes zoster (Cook, 1985), H. simplex (Scott, 1944), and Epstein-Barr virus (Whittle et a l., 1984, 1990). Children with malaria parasites also tend to respond less well to some vaccines, such as those against Clostridium tetani, Salmonella typhi, and group C meningococci, compared to uninfected children (Greenwood et a l , 1972; Williamson and Greenwood, 1978). However, Smedman et al. (1986) found the responses to live attenuated measles vaccine to be higher in malaria infected children. This observation was suggested to be due to prolonged survival of the virus within immunocompromised Plasmodium infected children (Smedman, et al., 1986). Also, Greenwood et al. (1988) found that children living in highly endemic areas who were protected from infection by chemoprophylaxis were less susceptible to other infectious diseases than unprotected children. Investigations with such protected children have revealed superior cellular proliferative and IFN-y responses to malaria antigens (Otoo et al., 1989). Nevertheless, the reduction in overall childhood mortality in children protected by chemoprophylaxis was found to be considerably greater than the expectation from prevention of deaths due to malaria alone (Greenwood et al., 1988). This may suggest that malaria infection predisposes to death from other diseases (Greenwood et al., 1988). Moreover, children protected by chemoprophylaxis against malaria responded better than unprotected children to routine childhood vaccinations (McGregor and Barr, 1962). It is, therefore, generally accepted that malaria patients are more susceptible to other infectious diseases because of reduced ability to mount effective immune 26 University of Ghana http://ugspace.ug.edu.gh responses. Interestingly, however, P. falciparum infection has been found to result in low or absent in vitro lymphoproliferative responses in some apparently immune individuals (Troye-Blomberg et a l., 1983a, 1984, Bygbjerg et al., 1986; Ho et al., 1986, Webster et al., 1988, Hviid et al., 1991). This lack of responsiveness appears to be associated with the mere presence of parasites in the blood, since some individuals with asymptomatic parasitaemia have also been found to be unresponsive (Theander et al., 1986b; Hviid et a l., 1990). This kind of observation led Riley et al. (1988) to conclude that infection does not affect only proliferative responses but also lymphokine (IL-2 and IFN-y) secretion. Nevertheless, some malaria-specific T-cells are present in the peripheral circulation during acute disease and can be activated. This may explain the success of Sinigaglia et al. (1985) and Pink et al. (1987) in isolating T-cell clones specific for defined P. falciparum antigens from the blood of malaria patients. The lack of in vitro response of PBMC to malaria antigens on the other hand contrasts with the high levels of soluble IL-2 receptor (sIL-2R) and soluble CD8 antigen found in sera of malaria patients (Riley et al., 1993; Hviid et al., 1991a; Hviid et al., 1991b; Josimovic-Alasevic et al., 1988; Kremsner et al., 1989; Nguyen-Dinh and Greenberg, 1988; Deloron et al., 1989). These markers suggest that cellular activation does take place and hence the reduced responses in PBMC may be due to reallocation of specific T-cells to other sites. Evidence from studies in mice show that activated T-cells and antigen presenting cells (APC) migrated to the spleen and liver during acute malaria (Dockrell et al., 1980; Playfair and De Sousa, 1982; Kumararatne et a l , 1987). More recently, Langhorne and Simon-Haarhus (1991), clearly demonstrated the presence of 27 University of Ghana http://ugspace.ug.edu.gh malaria specific T-cells in the spleen but not in the peripheral blood of mice during acute infection with P. chabaudi. However, responding cells could be found in both spleen and peripheral blood after treatment. It is likely that a similar phenomenon may occur in P. falciparum infected humans. This is likely to be true since lack of T-cell responsiveness is most pronounced in patients with enlarged spleens (Wyler, 1976; Greenwood et al., 1977). Furthermore, it has been reported that T-cells with high expression of the surface antigen LFA-1, which is known to be involved in cellular adhesion, are transiently lost from circulation in such patients (Hviid et al., 1991b) although the molecule is upregulated on P. falciparum stimulated T-cells in vitro (Hviid et al., 1993b). However, the extent of decrease in circulating T-lymphocytes during acute malaria appears to suggest that the loss cannot be attributed solely to depletion of malaria-specific T-cells. Some of the re-allocated cells may recognize epitopes that cross-react with malaria antigens or may be activated in a nonspecific fashion by T-cell derived cytokines. Mueller et al. (1989) have argued that cells activated in such a nonspecific manner may be induced to express activation markers and to sequester in the spleen. Loss of PBMC responses in malaria patients may also be partly due to activation of "suppressor" CD8 + cells. Activation of suppressor T-cells has been reported in mice infected with P. berghei (Lelchuk et al., 1981), P vinckei (Chilbert et al., 1981), and P. chabaudi (Russo and Weidanz, 1988). The ratio of CD8 + to CD4+ T-cells in the peripheral blood of patients with acute P. falciparum malaria is higher than normal (Theander et al., 1986; Troye-Blomberg et al., 1983), and high levels of soluble CD8 antigen are found in the sera of malaria patients (Kremsner and Bienzle, 1989; Elhassan et al., 1994). CD8 + T-cells 28 University of Ghana http://ugspace.ug.edu.gh obtained from immune individuals during the malaria transmission season has been shown to suppress proliferative responses to Plasmodium antigens in vitro, further supporting the role of CD8 + suppressor cells (Theander et al., 1993). A low molecular mass glycoprotein isolated from P. berghei infected erythrocytes can suppress primary antibody responses to T-dependent but not T-independent antigens in vivo (Khansari et al., 1981; Srour et al., 1988), and P. falciparum schizont extracts can suppress in vitro lymphoproliferative responses to purified malaria antigens and other soluble antigens (Riley et al., 1989). The precise parasite components that induce these suppressive effects, and the manner in which the suppression is mediated have not as yet been adequately characterized. 2.14 In vitro analysis of cellular immune responses in malaria T-cell responses to Plasmodium antigens in humans have been demonstrated in vitro by lymphocyte proliferation as well as by induction of cytokines such as IFN-y and IL-4 in peripheral blood mononuclear cells (PBMC) of adults from malaria endemic areas and in individuals recovering from clinical malaria (Ho et al., 1990; Riley et al., 1988; Troye-Blomberg et al., 1990). These responses have been shown to be long lasting, being detectable years after initial exposure (Bygbjerg et al., 1985; Ho and Webster, 1989). Hoffman et al. (1989a) found a positive correlation between in vitro proliferative T-cell responses to a specific epitope and resistance to P. falciparum malaria. It has been reported by several authors that in vitro exposure of T-cells from P. falciparum primed donors to crude or defined parasite antigens result in 29 University of Ghana http://ugspace.ug.edu.gh the release of IFN-y mainly by CD4+ cells (Chizzolini et al., 1990; Troye- Blomberg et al., 1990; Chougnet et al., 1990; Mshana et al., 1990). IFN-y is an important T-cell derived regulatory lymphokine known to increase the expression of MHC class II antigens and to activate macrophages. It is one of the factors believed to be important for the induction of cell mediated immunity to the parasite (Shear et al., 1989; Stevenson et al., 1990). IFN-y has been demonstrated in sera of patients with recent acute malaria (Rhodes-Feuillette et al., 1985). Recently, serum IFN-y was shown to be higher in non-parasitaemic donors than in donors with parasitaemia, suggesting a protective effect of that cytokine in vivo (Deloron et al., 1991). In vitro studies have shown that the target for IFN-y mediated inhibition appears to be the infected hepatocyte (Ferreira et a l., 1986, Maheshwari et al., 1986). In addition, IFN-y may also act through activation of macrophages and other effector cells (Meis and Verhave, 1988). Another cytokine produced during Plasmodium infections is IL-4, which has been reported to be important in T-cell help for antibody production (Howard et al., 1983). However, Liew et al., (1991) reported that IL-4 and IL-10 may inhibit the effect of IFN-y on macrophage activation. The proliferative response to malaria antigens in vitro is usually limited to the CD4+ T-cell subset (Mshana et al., 1991) although proliferating CD8 + T- cell clones recognizing malaria antigens have been obtained (Sinigaglia et al., 1987). Recent evidence suggest that different T-cell subsets may respond to malaria antigen stimulation by either proliferation, lymphokine production or both (Troye- Blomberg et al., 1990). This has been shown by the lack of association between these parameters (Troye-Blomberg et a l., 1985, 1990; Kabilan et al., 1988, 1990). 30 University of Ghana http://ugspace.ug.edu.gh It is important, therefore, not to measure only proliferation or lymphokine production in the effort to determine antigen specific T-cell responses, but to measure both and also determine the functional characteristics of the activated cells in relation to the type of cytokine released. This is of particular importance in selection of vaccine immunogens. 2.15 Asexual malaria vaccine candidate antigens Since the clinical manifestations of malaria are caused mainly by the asexual erythrocytic stages several authors have proposed the use of antigens from these stages in the development of a sub-unit vaccine against malaria (Howard and Pasloske, 1993). Such vaccines could induce a response against the extracellular parasite stages exposed to the host immune defence (Bruce-Chwatt, 1985). Many asexual malaria antigens are polymorphic with multiple alternative antigenic forms, often characterized by sequences of tandem amino acid repeats (Howard and Pasloske, 1993; Roberts et al., 1993). A range of antigenically conserved portions of the peptides will probably have to be included in a vaccine, in view of the capacity of malaria parasites for antigenic variation and the potential problem of MHC restriction (Howard and Pasloske, 1993). This will help minimize parasite avoidance of vaccine elicited immune responses and hopefully ensure protective responses in all individuals despite HLA differences. To date several proteins expressed by merozoites, including merozoite protein 1 (MSA-1), merozoite protein 2 (MSA-2), apical membrane antigen (AMA), and the erythrocyte binding antigen (EBA), have been identified as putative malaria vaccine components (reviewed by Romero, 1992). Of these, the merozoite surface antigens 31 University of Ghana http://ugspace.ug.edu.gh 1 and 2 are considered to be among the best vaccine candidate antigens for human trials (Howard and Pasloske, 1993). 2.15.1 Merozoite Surface Antipen 1 The MSA-1 is a high molecular weight protein that is synthesized during schizogony (Holder, 1988). It is a proteolytic fragment with an approximate molecular weight of 200 kDa, that is processed into fragments of 83, 42, 38, 28-30 and 19 kDa in vivo. The fragments can be demonstrated on the surface of mature merozoites (Holder, 1988; McBride and Heidrich, 1987). With the exception of the 19 kDa C-terminal fragment which is carried through into newly invaded erythrocytes, most of the other fragments are shed before or during red cell invasion (Blackman et al., 1990). The amino acid sequence of MSA-1 varies between isolates and can be divided into three regions, namely a variable region, an isolate specific region and a highly conserved dimorphic region (Tanabe et a l., 1987; Peterson et al., 1988). Riley and colleagues (1992) found that antibodies to the dimorphic regions of MSA 1 are prevalent in the sera of individuals exposed to P. falciparum infection. Also, using synthetic peptides and T-cells from donors of malaria endemic areas or MSA-1 specific T-cell clones, several workers including Crisanti et al. (1990) identified T-cell epitopes in the dimorphic regions of the molecule. T-cell proliferative responses and antibodies to the conserved regions of MSA-1 have been shown to correlate with clinical immunity (Riley et al., 1992). The possibility of using MSA-1 as vaccine antigen is supported also by the finding that a conserved 42 kDa C terminal fragment induced antibodies that inhibited growth of P falciparum in vitro (Chang et al., 1992) 32 University of Ghana http://ugspace.ug.edu.gh 2.15.2 Merozoite Surface Antigen 2 Another merozoite surface antigen is MSA-2 which is a glycosylated and myristilated protein (Ramasamy, 1987). It has a strain dependent molecular weight ranging from 35 to 56 kDa (Smythe et al., 1988; Clark et al., 1989; Ramasamy, 1987; Fenton et al., 1989). MSA-2 has been shown to be dimorphic (Fenton et al., 1991). Sequence analysis shows the C- and N- terminal regions to be highly conserved, whereas a large central region is variable (Thomas et al., 1990; Marshall et al., 1992). Anders et al. (1993) reported the presence of both repetitive and non repetitive sequences in the variable regions of this molecule, and antibodies to both repetitive and nonrepetitive regions of MSA-2 were prevalent in sera of people living in malaria endemic areas. MSA-2 was one of the antigens identified in immune complexes formed at the surface of merozoites when antibodies in immune serum were used to inhibit merozoite dispersal (Lyon et al., 1986). A monoclonal antibody against an epitope within a repeat region of MSA-2 was found to block red cell invasion by merozoites in vitro (Saul et al., 1989). MSA-2 has been shown to contain T-cell epitopes which are clustered in the variant parts of the antigen. Subsequently, proliferation of T-cells from individuals of malaria endemic regions have been found to be induced by MSA-2 repetitive and non-repetitive sequences (Rzepczyk et al., 1990, 1992). However, it is not known whether naturally acquired immune responses to repetitive and non­ repetitive regions of MSA-2 could protect against infection in man (Anders et a l., 1993). Saul et al. (1992) demonstrated in mice that peptides from the conserved regions of MSA-2 can protect against P. chabaudi infections. 33 University of Ghana http://ugspace.ug.edu.gh 2.16 Subunit malaria vaccines and MHC restriction Immunity to P. falciparum malaria is complex, and the mechanisms of protective immunity are not fully understood (Hviid et al., 1990). It is, however, believed that a suitable vaccine against malaria would most certainly have to be a subunit vaccine, with components expressing both T-cell epitopes and B-cell epitopes essential for eliciting long lasting immune responses against natural infections (Riley et al., 1991). Moreover, the immune response should preferable occur in all individuals and be directed against epitopes that do not vary between different parasite strains (Riley et al., 1991). However, several studies in mice (Del Giudice et al., 1986; Good et al., 1986, 1988; Lew et al., 1989) and in human populations (Good et al., 1988b, Chizzolini et al., 1988; Carter et al., 1989; Quakyi et al., 1989; Zevering et al., 1990) revealed a wide spread non­ responsiveness to sporozoite, merozoite and gamete vaccine candidate antigens. These differences in immune responses in a population may be due to MHC class II restriction (Good et al., 1988a). It could thus be assumed that only a limited number of MHC haplotypes would be able to present a particular epitope to the T- cells. This would seriously compromise the effectiveness of subunit malaria vaccines. MHC Class II molecules are encoded in the human leukocyte antigen D (HLA D) region and include at least three families of gene products; HLA-DR, DQ and -DP. These are polymorphic proteins capable of interacting with only discrete number of peptides (Babbitt et al., 1986, Buus et al., 1987). MHC genes exhibit an unusually high degree of allelic variation and unlike laboratory mice, human populations are outbred and frequently heterozygous at each genetic locus. 34 University of Ghana http://ugspace.ug.edu.gh On the other hand, it is argued that due to the complex polymorphism of the human HLA complex and the finding of "promiscuous" peptides able to bind a wide range of class II molecules (Sinigaglia et al., 1988, 1990; Ho et al., 1990), unresponsiveness in humans caused by MHC restriction might not be a major constraint for subunit vaccine development. Recent reports also suggest that peptides binding to a particular class II molecule share a common structure (Sette, 1989; O’Sullivan et al., 1991) and it might therefore be possible to select multi­ determinant peptides with the capacity to bind many MHC types. 2.17 Previous and ongoing vaccine trials The first clinical trials for the safety and immunogenicity of anti-P falciparum sporozoite vaccines were undertaken in human volunteers in the United States by the Walter Reed Army Institute of Research (WRAIR) and the National Institute of Health (NIH), Bethesda. Results of the WRAIR/NIH trial indicated that the first prototype vaccine was well tolerated but less immunogenic in humans than in mice and rabbits (TDR, 1987), and it failed to demonstrate vaccine efficacies greater than 20-30% (TDR, 1993). The failure of this vaccine was probably due to the inclusion of only B-cell epitopes for antibody production (Ballou et al., 1987). Since then the importance of T-cell epitopes has been recognized for B-cell help and memory (Good et al., 1988c). One synthetic candidate vaccine, the SPf6 6 against P falciparum malaria, was developed by the scientific group led by Dr. M.E. Patarroyo in Bogota, Colombia. The SPf6 6 has been field tested in more than 20,000 people in Latin America (Sempertegui et al., 1994). The vaccine appeared to be safe and could 35 University of Ghana http://ugspace.ug.edu.gh stimulate antibody responses in a majority of individuals immunized. Reports from one randomized, placebo-controlled double-blind trial carried out in Colombia suggest that the vaccine had an efficacy of about 40-60% in certain selected age groups (Patarroyo et al., 1992; TDR, 1993). Recently additional trials of this vaccine have been carried out in Tanzania (Alonso et al., 1994a; TDR, 1993). The results of this trial showed the efficacy of the vaccine to be about 31% (Alonso et al., 1994b). Other field trials of "SPf6 6 " are in progress in the Gambia, Thailand and Colombia (TDR, 1994). In addition to SPf6 6 , at least five parasite antigens with promising vaccine potential are being prepared for use in human trials over the next few years. These promising antigens are the merozoite surface antigen 1 (MSA-1), the apical membrane antigen (AMA-1), the serine rich antigen (SERA), the erythrocyte binding antigen (EBA), and Pfs 25, a molecule that could induce immune response capable of blocking infection in mosquitoes and thus put a break on malaria transmission (TDR, 1994). 36 University of Ghana http://ugspace.ug.edu.gh CHAPTER 3. MATERIALS AND METHODS 37 University of Ghana http://ugspace.ug.edu.gh 3.1 Study area The study was conducted with human subjects from Gomoa Onyadze, a village in the coastal region of Ghana, about 80km west of Accra, Ghana. This coastal area has a stable all year round malaria transmission. In a previous study, Afari et al. (1993) estimated malaria parasite prevalence rates as high as 40-60% in children and considerably lower in adolescents and adults. 3.2 Target population Blood samples were collected from 57 healthy adults, aged 16 years and above, and 21 children between the ages of 5 and 16 years from the study area. Control blood samples were obtained from 18 healthy Danish adults who had never lived in any malaria endemic area. Blood sampling was done after informed consent had been obtained. 3.3 Blood sampling, isolation and storage of peripheral blood mononuclear cells Twenty milliliters of venous blood from each donor was drawn aseptically into heparinized vacutainer tubes (Becton-Dickinson Ltd, Rutherford, NJ, USA) containing 400IU of heparin, and transported immediately to the laboratory. Blood processing was also done under sterile conditions. Sixteen milliliters of Ficoll- Paque (Lymphoprep, Nyegaard, Oslo, Norway), a density centrifugation medium, was transferred into 50ml Leucosep tubes (Greiner and Sohne, Germany), and centrifuged briefly to allow the Lymphoprep to go through its separating disc. The heparinized blood was then poured into the Leucosep tube and centrifuged at 38 University of Ghana http://ugspace.ug.edu.gh 800 x g for 15 min, resulting in separation of a top plasma layer; a second layer of peripheral blood mononuclear cells (PBMC); a third layer of Lymphoprep above and below the separating disc; and a bottom layer of red blood cells and granulocytes. The plasma was removed and stored at -20°C, whilst the mononuclear cells were transferred into a 50ml centrifuge tube and washed twice in washing medium consisting of RPMI 1640 supplemented with 5 % heat inactivated foetal calf serum (FCS), (Gibco, Grand Island, N.Y, USA) by centrifugation at 250xg for 10 min. The supernatant was discarded and the cell pellet resuspended in 1.5ml of enriched washing medium containing 10% FCS. The number of mononuclear cells was determined by counting in an improved Neubauer Counting Chamber using methyl violet stain, which is used in assesing nulear morhology when counting fresh cells (Hviid et al. , 1993a). The cell density was adjusted to between 8 and 12 million cells per ml with enriched washing medium. Seven hundred and fifty microliter of the cell suspension was added to cryo-preservation tubes and an equal volume of freezing medium (composed of 55 % RPMI 1640, 25 % FCS and 20% dimethyl sulfoxide (DMSO)), added to each tube just before freezing with a computer controlled cryo-freezing device (Hviid et al., 1993a). It took 60 min for the samples to cool to -140°C, after which they were transferred directly into liquid nitrogen at -196°C until use. 3.4 Microscopy Thick and thin blood films were prepared on glass slides from the blood samples collected. These were stained with Giemsa stain and screened for malaria 39 University of Ghana http://ugspace.ug.edu.gh parasites, using an Olympus microscope under oil immersion at xlOOO magnification. The different Plasmodium species were identified by their morphological characteristics as observed under the microscope. 3.5 Reagents 3.5.1 MSA-2 peptides Peptides of the P falciparum merozoite surface antigen (MSA-2) were supplied by Dr. C. Rzepczyk of the Queensland Institute of Medical Research, Brisbane, Australia. The peptides were synthesized by the simultaneous multiple peptide synthesis technique, using derivatized amino acids (Omni Biochemicals, National City, CA, USA) on benzhydrylamine resin (Multiple Peptide Systems, Solanos Beach, U.S.A) as described by Houghten (1985). Synthesized peptides were purified by reverse phase high performance liquid chromatography, using acetonitrile gradient with 0.1% trifluoroacetic acid as counter-ion (Jones, 1991). The MSA-2 peptides used in the study were the GI, G2C, and G4 as shown in Table la. Each peptide was used for stimulation of T-cell proliferation at final concentrations of 0.3/xg/ml, 3/xg/ml, 6 /xg/ml and 12/tg/ml. 3.5.2 Control antigens Purified protein derivative of tuberculin (PPD) and tetanus toxoid (TT) were used in this study as positive control recall antigens. These antigens were obtained from the Statens Serum Institute, Copenhagen, Denmark and used at final concentrations of 12/xg/ml purified PPD and 4/xg/ml purified TT, respectively. 40 University of Ghana http://ugspace.ug.edu.gh 3.5 .3 Cell activators Antigen specific production of IL-4 was measured by a method employing an amplification step using phorbol 12-myristate 13-acetate (PMA) (Sigma Chemical Corporation, MO, USA) and Ca-ionophore (ionomycin, Calbiochem, CA, USA) at final concentrations of 50ng/ml and ImM, respectively (Kurtzhals et al., 1992). 41 University of Ghana http://ugspace.ug.edu.gh TABLE la THE MSA2 PROTEIN OBTAINED FROM FC27 STRAIN OF PLASMODIUM FALCIPARUM GI REPEAT REPEAT G2 G3 G4 Conserved Variant region Conserved N-terminus C-terminus TABLE lb THE MSA-2 PEPTIDES USED FOR THE STUDY Peptide Sequence Region in MSA2 GI NESKYSNTFINNAYNMSIR Conserved N-terminus G2C TAADTPATESISPSPPC" Variant region G4 RNNHPQNTSDSQKECTDGNK Conserved C-terminus ’This cystein is not part of the MSA2 sequence 42 University of Ghana http://ugspace.ug.edu.gh 3.6 Proliferation assay and generation of IFN-y containing supernatants Frozen PBMC were retrieved from liquid nitrogen and quickly thawed in a waterbath at 37°C. The cells were immediately washed twice by centrifugation (250.xg for 10 min) in a washing medium made of RPMI 1640 supplemented with 5 % heat inactivated pooled normal human serum (NHS) from non-immune donors, 58.4/ng/ml of L-glutamine, 20IU/ml of penicillin and 20/*g/ml of streptomycin. Cell viability after storage was found to be greater than 90% as determined by trypan blue exclusion. The PBMC were cultured in the presence of MSA-2 peptides using culture medium consisting of RPMI 1640 supplemented with 15% NHS, 58.4/xg/ml L-glutamine, 20IU/ml penicillin and 20/zg/ml streptomycin. The cultures were initiated with 100,000 PBMC in 150/xl of culture medium in each well of 96-well roundbottomed microtiter plates (Nunc, Roskilde, Denmark). Twenty microliter of each antigen diluted in culture medium was added to triplicate wells for each PBMC sample to give the appropriate concentrations. Control unstimulated cultures received 20/ttl of culture medium without antigen. The cultures were incubated at 37°C in a humidified atmosphere containing 5 % C 0 2 for 7 days. Twenty- four hours prior to termination of cultures, the cells were pulsed with 185 mBq/ml of 3H- thymidine (New England Nuclear, Boston, MA, USA), added in 20/ul of culture medium per well. Culture supernatants from the triplicate wells were pooled and stored at -20°C for later determination of IFN-y. The cells were harvested onto glassfibre filters and the incorporation of 3H-thymidine into DNA determined by liquid scintillation spectrometry as measured in counts per minute (CPM). For each concentration of the peptides the median CPM was calculated. The CPM and cytokine production recorded were from the triplicates which gave the highest proliferative response. Control unstimulated cells were set up in four replicates consisting of triplicate wells. The highest background incorporation of 3H- 43 University of Ghana http://ugspace.ug.edu.gh thymidine and the highest IFN-y concentration produced by a triplicate set of unstimulated wells were recorded and used in estimation of stimulation index. In order to define a positive proliferative response to an antigen, two equations involving a stimulation index (SI) and an increment value (5), were used: CPM, _ . „ . , g m i ' = _________ (antiGenstim.) 5 “ CPM(anti?enstim, j ~CPM(un_gclm j A proliferative response was considered positive when SI was greater than 2 and 5 was greater than 1000CPM. 3.7 Generation of interleukin-4 containing supernatants To measure IL-4 production in antigen-stimulated cultures, an experimental set similar to the one described for IFN-y production was used, except that the cells in each well were pulsed with 20/tl mixture of 10^M ionomycin and 500ng/ml PMA in the last 24 hours before culture supernatants were harvested (Kurtzhals et al., 1992). 3.8 Determination of the effects of freezing and storage method on functional characteristics of PBMC In order to correctly identify the effect of the MSA-2 peptides on the PBMC of Ghanaian and Danish donors, experiments were first conducted to determine the effect of freezing on immunological functional characteristics of PBMC. In these experiments, PBMC obtained from healthy Danes were each divided into 44 University of Ghana http://ugspace.ug.edu.gh two, and one half frozen and stored for at least 2 hours using the procedure described earlier (see, section 3.3). The frozen cells were retrieved and together with the unfrozen batch prepared for proliferative and cytokine analysis. The stimulating antigen used was TT and the cytokine measured was IFN-y. T-cell proliferation and IFN-y production of the previously frozen and unfrozen PBMC were then compared. 3.9 Preparation of reagents for cytokine ELISA 3.9.1 Preparation of Protein A sepharose column Polyclonal rabbit IgG antibodies against human IFN-y and IL-4 were purified by Protein A affinity chromatography. Protein A sepharose gel (Pharmacia, Uppsala, Sweden) was treated as recommended. Briefly, the gel was added to PBS, pH 7.4, mixed and allowed to settle. Half of the supernatant was decanted away and the gel resuspended before adding it carefully to a column (PD-10; bed volume:9.1 ml, bed height:5 cm) (Pharmacia, Uppsala, Sweden). The packed gel was then rinsed with PBS after which 0 .1M glycine, pH 2.4 was added. The column was then again rinsed with PBS before application of 2 % (w/v) bovine serum albumin in PBS. The gel was kept soaked in this solution while the column was left to stabilize for an hour at room temperature. 3.9.2 Purification of rabbit IgG Polyclonal anti-cytokine antibodies had been induced in rabbits by immunization with either recombinant IFN-y or IL-4. The anti-cytokine containing rabbit sera were a generous gift from Dr. K. Bendtzen, University of Copenhagen. After washing of the columns sera were added and recirculated for 2 hours in PBS to allow binding of IgG to the protein A. The gel was then washed carefully with PBS until all the colour of the rabbit 45 University of Ghana http://ugspace.ug.edu.gh serum had completely disappeared, indicating that unbound protein had been removed. Five hundred microliter of 0 .1M glycine, pH 2.4 was then added to the column and collected in a fraction tube containing TRIS buffer. Similarly, eleven other fractions were collected, each with 500/xl of glycine. The protein concentrations in the fractions were measured using a Biorad protein detection reagent (Biorad, Munich, Germany) to identify fractions containing the eluted IgG. The protein containing fractions were pooled and divided into two equal portions. One half was dialyzed against PBS, pH 7.4 and the other against 0.1M NaHC03, pH,8 .4 at 4°C overnight. The protein fraction dialyzed against PBS, was preserved in 0.1% (w/v) sodium azide and stored at 4°C and used for coating of micro ELISA plates. 3.9.3 Biotinvlation of polyclonal rabbit anti-IFN-y or IL-4 IgG antibody Rabbit anti-IFN-y or anti-IL-4 IgG dialyzed against NaHC03 were conjugated to biotin using a stock biotin solution consisting of 50mg/ml of hydroxysuccinimidobiotin in DMSO. The concentration (C) of biotin required for conjugating the antibody was determined from the equation: Cbiotin = Ca„«^y x 1.821 and the appropriate concentrations were obtained by dilution of the stock solution with 0.01M PBS, pH 7.2. The volume (V) of this biotin solution required to conjugate the antibody was determined from the equation: VbiotiI1 =(Vantibody/20). This volume of biotin solution was added slowly in drops to the antibody solution whilst shaking, after which the mixture was incubated at room temperature for 4 hours in the dark, with continuous shaking. The biotinylated antibody was then dialyzed against excess PBS for 5 days with daily changes of the buffer. The prepared conjugate was added with an equal volume of 46 University of Ghana http://ugspace.ug.edu.gh glycerol and stored at -20°C. 3.10 Cytokine ELISA IFN-y and IL-4 were measured in the generated culture supernatants by double sandwich-ELISA using rabbit anti-human IFN-y or IL-4 described by Kemp et al. (1992) with biotin-avidin amplification. Due to shortage of reagents, the cytokine assays were carried out on supernatants generated from MSA-2 stimulated PBMC of 28 of the Ghanaian adult donors and 7 Danish adults. Supernatants generated from PBMC stimulated with control peptides (PPD and TT) of 18 adult Danes were analyzed for the two cytokines. 3.10.1 Procedure Maxisorb micro ELISA plates (Nunc, Rockilde, Denmark) were coated with anti-cytokine antibody. The antibodies were diluted in PBS, pH 7.4 to give 3.7 /ig/ml of anti-IFN-y or 4.0 ixglm\ anti-IL-4, and lOOjiil volumes dispensed into each well. To coat, the plates were sealed and incubated at 4°C for 3 days, after which they were washed 3 times with PBS-tween (washing buffer) and stored for at least two weeks at 4°C. The concentrations of coating and biotinylated antibodies used in the ELISA were arrived at by checker board titration of coating and biotinylated antibodies against standard IFN-y and IL-4. Selected concentrations were those that gave the best distinction between low cytokine concentrations and blank wells, and which required the lowest amount of antibody. When ready for analysis, plates were washed thrice with washing buffer and then incubated with 150/d/well blocking buffer consisting of 2% (w/v) human serum albumin in PBS, pH 7.4 at 37°C for 2 hours. Again, the plates were washed 3 times with washing buffer. Samples were diluted 1:1 with incubation buffer (consisting of 650/*! of rabbit serum, 0.2mM 47 University of Ghana http://ugspace.ug.edu.gh polyethylene glycol, 6 .8 mM NaCl, 1 drop of Tween 20 in 15.5 ml PBS; pH 7.4) and incubated lOO^l/well in duplicates at 4 °Covernight. For estimation of cytokine concentrations in the samples, serial dilutions of known concentrations of purified standardized native cytokines generated from cultures of human PBMC were made in incubation buffer, diluted 1 :1 in culture medium and incubated as described for the test samples (thus ensuring equivalent amounts of culture medium and buffer in each well). The plates were then washed 4 times to remove excess unbound protein and incubated with 100/zl/well of biotinylated anti-cytokine antibody diluted 1:1000 for IFN-y or 1:1400 for IL-4 in incubation buffer. The plates were once again washed 4 times with washing buffer to remove excess unbound antibody conjugate and further incubated with 1 0 0 ^ 1/well of peroxidase labelled streptavidin (SPOD) diluted 1:2000 with incubation buffer at room temperature on a shaker for 30 mins. This was followed by another 4 times wash after which substrate solution consisting of 66.7% 1,2-phenylenediamine dihydrochloride, 0.1M citric acid-phosphate buffer, pH 5.0 and 0.0125% H20 2, was added 100/xl/well. The plates were incubated in the dark for 10-15 min. during which the reaction (colour development) was monitored and stopped with lOOptl/well of 2.5M H2S0 4 when there was visible difference between the most diluted standard and blank wells. The optical densities were recorded at 492nm using an automated ELISA plate reader. Standard curves of absorbance against standard cytokine concentrations on a semi-log graph were used to estimate the concentration of cytokines in test samples. In order to define a positive cytokine response a cytokine production index (PI) was calculated as: P ' Im - & t o k i n e lmtlBanatia^ - C y t o k i n e {ua_atlaA C y t o k i n e {unratjam) 48 University of Ghana http://ugspace.ug.edu.gh Cytokine production was considered positive when the production index was greater than 2 . The assays were calibrated to detect IL-4 within the range of 63-10,OOOpg/ml, and IFN- 7 within the range of 1-64 IU/ml (specific activity of the reference protein being 2xl0 8IU/mg). The ELISA did not cross-react with IL-1, IL-2, TNF, GM-CSF, or IL-6 . The IFN - 7 ELISA did not cross-react with IL-4, and the IL-4 ELISA did not cross react with IFN - 7 (Kurtzhals et al., 1992). 3.11 Statistics The Chi-square test for un-paired data was used for comparison of proportions. Ratios between proportions were approximated by odds ratios due to the cross-sectional study design. Binomial confidence intervals were calculated when applicable. Paired data were compared using rank correlation analysis. Calculations were done using Sigmastat 1.02 (Jandel Scientific Corporation, 1986-1992) and Epilnfo 5.1 (U.S.D, Inc. Stone Mountain, G.A., U.S.A.) computer programmes. Two-tailed P-values <0.05 were considered significant. 49 University of Ghana http://ugspace.ug.edu.gh CHAPTER 4. RESULTS 50 University of Ghana http://ugspace.ug.edu.gh 4.1 Prevalence of malaria parasites among the subjects None of the individuals examined had any signs of clinical malaria at the time of blood sampling. Nevertheless, as shown in Table 2, P. falciparum was frequently found in the blood samples of the Ghanaians. The prevalence of asymptomatic parasitaemia was significantly higher (P< 0.001) in the children than in the adults. No other Plasmodium species were encountered. 4.2 Lymphoproliferative responses The proliferative responses to the MSA-2 peptides (GI, G2C and G4) and positive control antigens (PPD and TT) are summarized in Figures 1-5 and Table 3a. As indicated, PBMC from Ghanaian adults gave higher responses to all the MSA-2 peptides than those from Ghanaian children and Danish adults (Figures 1-3). On the other hand the lymphoproliferative responses to the control antigens were high in PBMC of all the three groups of donors studied (Figures 4 and 5). Using the criteria for a positive proliferative response as defined in Materials and Methods the frequency of responding cultures was found to be significantly higher (P < 0.03) in Ghanaian adults compared to Ghanaian children or Danish adults for all the MSA-2 peptides tested (Tables 3a and 3b). Approximately 30% of PBMC cultures from Ghanaian adults responded to each of the peptides while only 1 of 21 samples from Ghanaian children and 1 of 18 from Danish adults responded to GI and G2C, respectively (Table 3a). The proportion of cultures responding to PPD and TT did not differ significantly (P = 0.2) between Ghanaian adults, Ghanaian children, and Danish adults (Table 3a). 51 University of Ghana http://ugspace.ug.edu.gh TABLE 2 PREVALENCE OF P. FALCIPARUM PARASITAEMIA AMONG GHANAIAN ADULTS AND CHILDREN Ghanaian Ghanaian Prevalence ratio adults children (95% CI)+ P value' Prevalence of parasitaemia 8/69 21/43 0.14 (0.05-0.59) 0.00001 (11.6%) (48.8%) * Prevalences of P. falciparum parasitaemia were compared by Chi-square for unpaired data. + Prevalence ratio (95 % confidence interval) was calculated as odds ratio for prevalence in adults vs. children. 52 University of Ghana http://ugspace.ug.edu.gh G 1 Figure 1. Proliferative response (increment value) to MSA- 2 peptide GI in peripheral blood mononuclear cells (PBMC) from Ghanaian adults and children and non-exposed Danish adults. Each data point represents the median of triplicate cultures from one individual. 53 University of Ghana http://ugspace.ug.edu.gh G2C >9 E CL o c o -*-» a i— 9 8 E O. O E O IS 4 o CD O CL o„o„o 8oo o8 o o A o o 0-0 — 8-8-8— A — / „oo o o o o w o o o o * c 0.05) between the three groups for responses to either PPD or TT. 58 University of Ghana http://ugspace.ug.edu.gh 4.3 Cytokine production Nine (32%), 8 (29%), and 10 (36%) of the cultures from 28 Ghanaian adults produced IFN-y in response to GI, G2C and G4, respectively, whereas none of the cultures from 7 Danish adults did (Figure 6 ). This difference was not statistically significant (P > 0.05). However, PBMC from a very significantly higher (P < 0.001) proportion of Ghanaians (61%) than Danes (0%) produced IFN-y in response to at least one of the peptides . After re-stimulation with ionomycin and PMA IL-4 was detectable in MSA-2 peptide stimulated cultures of both Ghanaians and Danes (Figure 7). However, using the criteria for a positive IL-4 response, 3 (10.7%), 1 (3.6%) and 1 (3.6%) of the samples from 28 Ghanaian adults but none of those from the 7 Danes were positive for IL-4 in response to GI, G2C and G4, respectively. None of the PBMC samples from Ghanaian adults responded to more than one peptide antigen. 59 University of Ghana http://ugspace.ug.edu.gh 1 1 0 E E Dcn 1 1 1 G1 G2C G4 G 1 G2C G4 Ghanaians Danes Pept ide Figure 6. Interferon- 7 production (increment value) induced by MSA-2 peptides GI, G2C and G4 in PBMC from Ghanaian adults and non-exposed Danish adults. Supernatants were pooled from triplicate cultures and the mean of two measurements recorded. 60 University of Ghana http://ugspace.ug.edu.gh G h a n a i a n s Da n es Pept ide Figure 7. Interleukin-4 production (increment value) induced by MSA-2 peptides GI, G2C, and G4 in PBMC from Ghanaian adults and non-exposed Danish adults. IL-4 was measured in antigen stimulated cultures boosted with ionomycin and PMA for the last 24 h of culture (see text). 61 University of Ghana http://ugspace.ug.edu.gh Using the control antigens PPD and TT it is shown in Tables 4 and 5 that PBMC from both Ghanaian and Danish adults could be stimulated to produce IFN- 7 and/or IL-4. As shown, there were no statistical differences (P > 0.15) between the proportions of PBMC cultures producing IFN- 7 or IL-4 in response to either of the antigens. 4.3.1 Pattern of IFN- 7 and IL-4 production Figure 8 shows corresponding measurements of IFN- 7 and IL-4 in PBMC from Ghanaian adults stimulated with GI, G2C, or G4. Only one of the responding cultures produced both IFN- 7 and IL-4. 4.4 Overall response of PBMC to any of the MSA-2 peptides Eighty two percent of PBMC cultures from Ghanaian adults and 14 % of the cultures from Danes responded to MSA-2 peptides in at least one T-cell assay (Table 6 ). This overall response to the peptides was significantly higher (P = 0.002) for the Ghanaian adults. Similarly, the responses to only the GI and G4 peptides were each significantly higher for the Ghanaians. The responses to G2C were, however, not significantly different (P = 0.1) between the Ghanaians and the Danes. 4.5 Determination of the effects of freezing and storage method on proliferation and IFN-7 assays Figure 9 and 10 show corresponding measurements of proliferation and IFN- 7 production in response to TT stimulation in fresh and frozen PBMC from Danes. A strong positive correlation was found between the fresh and frozen 62 University of Ghana http://ugspace.ug.edu.gh samples in both the proliferation assay (rank correlation coefficient (Rs) = 0.937, P < 0.005) and cytokine production (Rs = 0.760, P = 0.005). 63 University of Ghana http://ugspace.ug.edu.gh o U O O 1 0 0 cn a. ( X ) Figure 8 . IL-4 vs INF- 7 production inSuced^y peptides GI, G2C and G4 in PBMC from Ghanaian adults. The cytokines were measured in parallel cultures with (IL-4) and without (IFN-7 ) boosting with ionomycin and PMA (see text). For every donor corresponding values of IFN- 7 and IL-4 production has been shown for each of the peptides. 64 University of Ghana http://ugspace.ug.edu.gh E Q. O v—/ C o +-> o L_ 0> "Lo_ Q. jn "© u c