Universit of Kha U ty artoum

Faculty of Medic y cine

Departmen of Bioche nt emistry

The Imm muno-Pathogenesis of Severe Plasmodi e ium falcipa arum Malaria in S M Sudanese C Children

By

Atif Has ssan Khire elsied

BSc (A Alexandria U University)., MSc (U of K). ,

A Thesis Submitted in Fulfillment of th Requirem he ments for th Degree of he f PhD in Medical Biochemistry July 2008. upervisor. Su Pr rofessor. Mustafa Idris Elbashir s MD, PhD Biochemi D. istry.

Title:

The Immuno-pathogenesis of Plasmodium falciparum Malaria in Sudanese Children:

Location:

Department of Biochemistry. Faculty of Medicine, University of Khartoum P. O. Box 102, Khartoum, Sudan.

Supervisor:

Mustafa Idris Elbashir MD, PhD, Professor of Biochemistry Faculty of Medicine, University of Khartoum.

Table of Content

Tableofcontents..............................................................................................................................3 Dedication........................................................................................................................................6 Acknowledgement............................................................................................................................7 Listofabbreviations.........................................................................................................................8 Abstract.......................................................................................................................................... 0 1 Listoffigures.................................................................................................................................. 1 1 Listoftables.................................................................................................................................... 1 1

ChapterOne:IntroductiontoMalariaImmunityandPathogenesis................................... 2 1 Theglobalburdenanddistributionofmalaria.............................................................................. 3 1 Thebiologyofmalaria.................................................................................................................... 5 1 ThelifecycleofPlasmodiumfalciparum........................................................................................ 5 1 Themalariadisease........................................................................................................................ 8 1 Uncomplicatedmalaria.................................................................................................................. 8 1 Severemalariacomplications......................................................................................................... 8 1 Cerebralmalaria............................................................................................................................. 9 1 Severemalarialanemia.................................................................................................................. 9 1 ImmunitytoMalaria...................................................................................................................... 0 2 Theinnateimmuneresponsestomalaria..................................................................................... 0 2 2 Adaptiveimmuneresponsestomalaria........................................................................................ 2 Antisporozoiteimmunity ............................................................................................................. 2 . 2 Immunitytoliverstagesofmalariaparasite................................................................................. 3 2 Immunitytobloodstagesofmalariaparasite.............................................................................. 3 2 AntibodymediatedimmunitytobloodstagesofP.falciparum.................................................. 4 2 CellmediatedimmunitytobloodstagesofP.falciparum............................................................ 5 2 TheImmunoPathogenesisofSevereMalaria............................................................................. 6 2 Thepathogenesisofcerebralmalaria........................................................................................... 6 2

Thepathogenesisofseveremalariaanemia................................................................................. 8 2 Theroleofcytokinesinimmunopathogenesisofmalaria.......................................................... 9 2 Interferon(IFN)........................................................................................................................ 0 3 Tumornecrosisfactor(TNF).................................................................................................... 0 3 Lymphotoxin(LT)........................................................................................................................ 1 3 Interleukin1(IL1).......................................................................................................................... 1 3 Interleukin4(IL4).......................................................................................................................... 2 3 3 Interleukin6(IL6).......................................................................................................................... 2 Interleukin8(IL8)andchemokines ............................................................................................. 3 . 3 Interleukin10(IL10)..................................................................................................................... 3 3 Interleukin12(IL12)...................................................................................................................... 5 3 Interleukin18(IL18)...................................................................................................................... 5 3 GranulocyteMacrophageColonyStimulatingFactor(GMCSF).................................................... 5 3 TransformingGrowthFactor(TGF)......................................................................................... 6 3 3 Cytokinegenepolymorphismsandmalariaprotectionandpathogenesis.................................... 6 Interleukin10genepolymorphisms............................................................................................. 7 3 ThePresentStudy......................................................................................................................... 9 3 Rationale........................................................................................................................................ 9 3 StudyI............................................................................................................................................. 9 3 StudyII............................................................................................................................................ 1 4 Thespecificobjectives................................................................................................................... 3 4 ChapterTwo:MaterialandMethods..................................................................................44 Thestudyareaandpopulation...................................................................................................... 4 4 Microscopyfordiagnosisandestimationofparasitedensity ...................................................... 7 . 4 ParasiteDNAextraction,andestimationofP.falciparummultiplicityofinfection(MOI)............ 7 4 Preparationofthecrudeparasiteantigensanddeterminationofspecificantibodylevels......... 8 4 5 ExtractionofhumanDNAandgenotypeofinterleukin10(1082A/G)SNP................................ 0 Determinationofcirculatinglevelsofinterleukin10 .................................................................. 1 . 5 Statisticalanalyses......................................................................................................................... 2 5

ChapterThree:Results............................................................................................................ 4 5 StudyI............................................................................................................................................ 4 5 Thedemographicandclinicalfeaturesofthestudypopulation .................................................. 4 . 5 CorrelationofIgGantibodieswithage,parasitedensity,MOIandmsp2genotypesin thestudypopulation..................................................................................................................... 6 5 5 ComparisonofIgGantibodysubclasslevelsbetweenstudygroups............................................ 6

StudyII........................................................................................................................................... 1 6 Thedemographicandmalariologicalindicesofthestudygroups................................................ 1 6 Thecirculatinglevelsofinterleukin10inthedifferentstudygroups........................................... 1 6 Thefrequencyofallelesandgenotypesininterleukin10(1082A/G)singlenucleotide polymorphismamongclinicalgroups. .......................................................................................... 2 . 6 ChapterFour:DiscussionandConclusions.................................................................................. 6 6 StudyI............................................................................................................................................. 8 6 StudyII........................................................................................................................................... 1 7 Conclusions.................................................................................................................................... 4 7 Perspectives................................................................................................................................... 4 7 References..................................................................................................................................... 5 7 Appendices................................................................................................................................... 9 9

Dedication

To the memory of my father, the great teacher who taught me the love and respect of knowledge, to my mother, my wife and children and to all sisters and brothers, without their patience, understanding, support and most of all love, the completion of this work would not have been possible.

Acknowledgements
I am very much indebted to my supervisor, Professor Mustafa Idris Elbashir, The Dean

Faculty of Medicine University of Khartoum. His wide knowledge and way of thinking have been of great value for me since I knew him fifteen years ago. His understanding, encouraging and personal guidance have provided a good basis for the present thesis. I
would like to express my deep and sincere gratitude to Dr Hayder Geha whose advice, stimulating suggestions and encouragement helped me in all the time of research and writing of this thesis. I have furthermore to thank Professor Marita Troye-Blomberge of the Department of Immunology, Wenner-Gren Institute, Stockholm University who gave me the possibility to to do the necessary research work and to use departmental resources to complete this thesis. I want to thank my colleagues in the Department of Biochemistry, University of Khartoum and the Department of Immunology, Wenner-Gren Institute, Stockholm University, for the invaluable discussions and immense technical assistance. I am especially obliged to Dr. Thorya Moh Elhassan for her great help in difficult times, and to Nnaemeka C. Iriemenam and Amre Nasr

for the invaluable assistance. I would like also to thank all patients, their guardians/families for
their cooperation and the staff at El-Gedarif and El-Obied hospitals for their collaboration and help. I express my gratitude to field team, Adil, Faiz, Ihsan and Mustafa for the great help and assistance,

This study was partially supported by a MIM/WHO/TDR grant to Malaria Immunology and Pathogenesis Consortium in Africa (MIMPAC); (Grant ID A10622), and the International University of Africa, Khartoum, Sudan.

List of abbreviations ADCI AMA-1 APCs CM CSP DNA EDTA ELISA GLURP GM-CSF GPI ICAM-1 IFN- IgG IL iRBCs LT- MHC MIF MOI MSP NK NKT PBS PCR PECAM pRBC rs SM SMA SNP TGF- Th TNF : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : Antibody-dependent cellular inhibition Apical membrane antigen-1 Antigen presenting cells Cerebral malaria Circumsporozoite protein Deoxyribonucleic acid Ethylene diamine tetraacetic acid Enzyme-linked immunosorbent assay Glutamate-rich protein Granulocyte-macrophage colony stimulating factor Glycosylphosphatidylinositol Intracellular adhesion molecule-1 Interferon gamma Immunoglobulin-G Interleukin Infected red blood cells Lymphotoxin- Major histocompatibility complexes Macrophage migration inhibitory factor Multiplicity of infection Merozoite surface protein Natural killer cells Natural Killer T cells. Phosphate buffer saline Polymerase chain reaction Platelet-endothelial cell adhesion molecule Parasitized red blood cells Reference sequence Severe malaria Severe malarial anemia. Single nucleotide polymorphism Transforming Growth Factor- T helper types of CD4+ T cells Tumor necrosis factor 8

TRAP UM VSA WHO

: : : :

Thrombospondin related adhesive protein Uncomplicated malaria Variant surface antigens World Health Organization

Abstract Malaria causes considerable morbidity and mortality among young children in malaria endemic areas worldwide. Anti-malarial antibodies are crucial determinants of Plasmodium falciparum infection outcome. In this study the levels of circulating immunoglobulin-G antibodies specific to crude Plasmodium falciparum antigens were determined by an indirect enzyme-linked immunosorbent assay in a hospital based casecontrol study, of children with varying malaria severity living in the rural area of Gedarif eastern Sudan and El-Obied west of Sudan. The aims were to investigate if immunoglobulin-G subclasses correlated with the clinical presentation of the disease and other malariometric indices among children exposed to seasonal and unstable Plasmodium falciparum transmission. The levels of circulating interleukin-10 and the genotypes of the single nucleotide polymorphism at position -1082 of IL-10 gene were also investigated in relation to malaria severity. The results revealed that individuals with severe malaria had significantly lower prevalence and levels of IgG1 and IgG4 as compared to those with uncomplicated malaria, suggesting a protective role for these molecules against severe malaria. The levels of IL-10 were lower in severe malaria patients as compared to those who presented with uncomplicated malaria, but no significant association was found between the specific alleles or genotypes in the -1082 A/G polymorphic site and any particular form of clinical malaria. The present data argue for protective roles for IgG1 and IgG4 antibodies and IL-10 against severe malaria, but find no evidence supporting significant effect of the different alleles or genotypes at position (-1082A/G) of the IL-10 gene on protection from SM. 10

 :
. . . - . -01 )-2801( .

-1 4 -01 . -01 , )-2801( -01 . 1 4 -01 )-2801( -01 .

11

List of Figures
1. Figure 1.1 2. Figure 1.2 3. Figure 3.1. The Global Distribution of Malaria Transmission Risk. The life cycle of Plasmodium falciparum.

Page
15 18

General comparisons of IgG subclass levels between the study groups. Comparisons of IgG levels between the patient groups. Levels of circulating IL10 in the study groups of malaria patients and healthy controls.

59 60

4. Figure 3.2. 5.

Figure 3.3.

67

List of Tables 1. Table 3.1. The demographic and malariological indices of the study groups Antimalarial immunoglobulin-G subclass sera reactivity and risk of severe malaria. Demographic features of study II population The Hardy-Weinberg Equilibrium for the interleukin-10 (-1082 A/G) SNP in the study groups Estimation of odds ratios, assuming different modes of A/G alleles inheritance. 66 56

2. Table 3.2.

61 64

3. Table 3.3. 4. Table 3.4 5. Table 3.5

65.

12

Chapter one

Introduction to Malaria Immunity and Pathogenesis

13

1.1.

Introduction to malaria:

1.1.1. The global burden and distribution of malaria. Malaria is by far the worlds most important parasitic disease with a staggering global impact [1]. Current estimates indicate that, malaria threatens the lives of 3.2 billion, causes between 350-500 million clinical episode and kills more than one million people each year [2, 3]. Most morbidity and mortality occur among the vulnerable children and women at child bearing age in the poorest communities of sub-Saharan Africa (Fig 1.1) [4]. Conventional approaches used to assess the impact of malaria seem inadequate, and the reported figures of incidence, morbidity, and mortality are likely to be largely underestimated [5]. Recent innovative approaches, exploiting satellite imagery and climatic modeling of malaria transmission levels, further support this view [6-9]. In addition to its direct heavy toll on health, malaria has immense socio-economic impact, and it has long been recognized that malarious communities are impoverished communities. This is clearly evidenced by the fact that, malaria endemic countries are the least developed ones with the lowest rates of economic growth [10]. The disease is responsible for around 1.3 % reduction in the overall annual economic growth rates in countries with high malaria endemicity. Malaria impedes economic through its effects on worker productivity, school performance and medical costs [10-13]. The global distribution and clinical patterns of malaria are widely variable and are largely determined by the local levels of parasite transmission which are influenced by several biological and environmental factors [7, 14]. Genetic characteristics of the host, the parasite, and the vector as well as the environmental factors including socioeconomic, and the malaria control measures contribute differently to the disease distribution. 14

Conventionally, levels of transmission are measured by estimating the entomological inoculation rate [15-18]. Alternatively, the epidemiology of malaria in a specific geographical area can be defined in terms of spleen enlargement and parasite rates among children [19]. More conveniently, the pattern of malaria transmission can be described as stable or unstable based on the fluctauation in the annual transmission. However, local epidemiologic features of malaria transmission may be drastically altered by environmental and/or social disturbances causing frequent epidemics.

Figure. 1.1 The Global Distribution of Malaria Transmission Risk; Malaria is endemic in regions of 107 contries, mainly in sub-Saharan Africa.
Source: The world malaria report 2005; http://www.rbm.who.int/wmr2005/html/map1.htm.[20]

15

1.1.2. The biology of malaria. Malaria is a mosquito-borne disease that affects several vertebrate species including rodents, reptiles, birds and primates. The infection is transmitted by the bite of an infectious female Anopheles mosquito carrying a protozoan parasite of the genus Plasmodium. There are over 175 species of plasmodia currently recognised, many of these are known to cause malaria in non-human vertebrates [21]. However, humans malaria can solely be caused by four species of Plasmodium: P. vivax, P. ovale, P. malariae, and P. falciparum. Among these, P. falciparum is the most virulent and is responsible for almost all cases of severe disease and deaths. P. falciparum is the most common parasite in tropical Africa, and exists in all malaria endemic regions worldwide, while other species are variably distributed in different geographical locations [22, 23]. More than four hundred species of Anopheles were identified, but only seventy of these are known to naturally transmit malaria to various vertebrates. [24, 25]. Human malaria parasites are transmitted by a few Anopheles species, of which, An. gambiae, An. arabiensis, and An. funestus are the most prevalent in Africa whereas other species such as An. albimanus, An. culicifacies, An. dirus, An. anthropophagus are found in other parts of the world [26].

1.1.3. Plasmodium falciparum life-cycle. Plasmodium falciparum has a remarkably complex life cycle (Figure 1.2). The infection begins when sporozoites are released into the blood stream by a female Anopheles mosquito. The inoculated sporozoites circulate briefly before they invade the liver parenchymal cells where they undergo asexual replicative phase called exo-erythrocytic

16

schizogony. This liver stage of parasite lasts for approximately a week before releasing merozoites which invade red blood cells (RBCs). Erythrocyte invasion is crucial for parasite survival, it provides a rich source of nutrients in a niche (the parasitophorous) that is largely protected from host immune defense. The erythrocytic schizogony involves rapid successive mitotic divisions and generate thousands of merozoites which are released to re-invade new erythrocytes. It is this stage that is responsible for the acute clinical episodes of malaria. After a period of 7 to 15 days of asexual replicative cycles in RBCs, a small proportion of the merozoites undergo transformation into male and female gametocytes, the micro- and macro-gametocytes respectively. The half-life of the mature gametocyte in the blood is about 2.4 days during which it could be taken up by a feeding Anopheles mosquito. A few minutes after ingestion by the mosquito, the micro-gametocytes exflagellate in the insect midgut, each forms eight micro-gametes which move to fertilize the female macro-gametes. Eighteen to 24 hours after fertilization, the zygote elongates into an ookinete which traverses the membranes and the epithelial cells of the mid-gut and transforms into oocyst. The oocyst enlarges progressively as the nucleus divides repeatedly to form sporozoites. Mature sporozoites exit the oocyst to the coelomic cavity and migrate to salivary glands awaiting transfer to a new victim [27-29].

17

Fig 1.2. The life cycle of Plasmodium falciparum.

Modified from the Center of Biologics Evaluation and Research, US, Food and Drug Administration: www.fda.gov/Cber/blood/malaria071206sk.htm. Accessed on the 3rd of June 2008.

An infectious mosquito injects sporozoites into the blood stream of the human host, which then circulate with the blood to the liver. An exo-erythrocytic stage in the liver leads to release of merozoites into the blood stream. The merozoites invade the red blood cells and undergo an asexual replicative cycle (the erythrocytic schizogony) to produce multiple merozoites capable of re-invading new red blood cells. Occasionally, erythrocytic trophozoites transform into gametocytes which are released into the blood stream and are eventually taken up by the mosquito, and then undergo the sexual stage of development (sporogenic cycle).

18

1.1.4. The malaria disease. 1.1.4.1. Uncomplicated malaria.

The clinical presentation of P. falciparum malaria is quite variable and generally mimics that of many other diseases caused by bacteria, rickettsia, and viruses. Initially malaria can have a gradual course of onset with non-specific symptoms including slight fever, anorexia, malaise and headache [30, 31]. The classical manifestations of the acute P. falciparum include fever with a feeling of coldness and chills followed by profuse sweating. Generalized symptoms such as backache, myalgia, aching joints, and gastrointestinal symptoms, i.e., anorexia, nausea, vomiting, abdominal pain, and mild diarrhea may also be found. Unless the malaria infection in young children and nonimmuned individuals is diagnosed early and promptly treated, the condition may progressively deteriorate to a severe life-threatening disease with complex multi-system dysfunction [32].
.

1.1.4.2.

Severe complications of malaria.

Severe malaria (SM) is a complex multi-system disorder presenting with a considerably variable range of clinical manifestations [33]. In children and non-immune adults, many of severe complications of P. falciparum may occur in combination or as isolated manifestations. Such complications include cerebral malaria (CM), severe malarial anemia (SMA), respiratory distress, renal failure, and serious metabolic derangements including hypoglycemia and lactic acidemia. However, CM and SMA are the leading causes of malaria-related deaths in Africa. [31, 34-36].

19

1.1.4.2.1. Cerebral Malaria (CM). Cerebral malaria (CM) is a syndrome of impaired consciousness associated with a P. falciparum malaria infection. Clinically, the term is loosely used to describe those at high risk of death due to SM. However, standardized criteria defining CM have been established by a panel of WHO experts, accordingly, the strict research definition of CM; is a condition of unarousable coma for more than 30 minutes after a generalized convulsion, confirmed by P. falciparum infection and exclusion of other causes of coma [37-41]. In malaria endemic areas, CM accounts for up to 10% of all hospitalized cases of P. falciparum malaria, and for more than 80% of fatal cases. It occurs predominantly in patients with little or no parasite specific immunity, i.e., children under five years age in endemic areas or individuals coming from malaria-free areas.

1.1.4.2.2. Severe malarial anemia (SMA). Severe malarial anemia (SMA) is defined as a haemoglobin concentration 50g/L (or a haematocrit 15%) in a patient with a P. falciparum parasitaemia with normocytic indices. The prevalence of SMA has increased during the last decade as a result of spread of chloroquine-resistant P. falciparum across the world [42-44]. SMA has a profound effects on communities in malaria endemic regions [45], anemia is associated with an increased risk of death [32], impaired cognitive and motor development [46-48], retarded growth [49], diminished immune protection [50], and reduced physical work capacity [51].

20

1.2.

Immunity to malaria.

The immune system plays an important role in the host defense against infection with P. falciparum, and in limiting the extent of infection once it occurs. Although most

individuals living in malaria endemic areas are equally exposed to infectious bites of the Anopheles mosquitoes, a large proportion of the infected individuals either spontaneously resolve the infection, or remain asymptomatic i.e. harboring some parasitemia without significant clinical disease. A small proportion of infected individuals, mostly young children, develop clinical symptoms of malaria, and only less than 5% of these clinical cases proceed into severe life-threatening complications [52]. The clinical pattern of malaria is markedly influenced by the age-dependent acquisition of anti-malaria immunity [53]. Different components of the immune system including both antibody-dependent and cell-mediated mechanisms operate at different stages of the parasites life cycle (figure 1.2) [54]. However, protective immunity to malaria takes years of exposure to infection before it develops significantly [55], and even with repeated infections, this immunity is rarely sterile, and is species-, variant-, strain-, and stage-specific [56, 57]. This inefficient and slow acquisition of anti-malaria immunity is attributed to the P. falciparum virulence and variability [54].

1.2.1. The innate immune responses to malaria. Early interactions between sporozoites and host cells produce no significant systemic immune response and disease. Only merozoites are the stages that elicit strong immunoinflammatory responses. Although not fully understood, several humoral components of innate immunity comprising the complement proteins [58], C-reactive proteins [59-61]

21

and mannose binding lectins [62-64] were implicated in protection from malaria. However, the principal innate effector mechanisms against malaria parasites are mediated by cellular components including monocytes-macrophages, natural killer cells, natural killer T cells and / T cells [65]. The activation of monocytes-macrophages is one of the first events in the innate resistance to P. falciparum infection. Circulating monocytes and tissue resident macrophages in the liver, spleen, and other tissues are directly involved in phagocytosis and clearance of the infected red blood cells (iRBCs) [66, 67]. Malarial antigens such as the hemozoin pigment and/or the membrane-anchored glycosylphosphatidylinsitol, phagocytosed by the macrophages or acting through surface receptors, activate these cells to produce a series of pro-inflammatory mediators and cytokines. The inflammatory agents produced may have a direct anti-parasite activity or contribute to the activation of other effector immune mechanisms. However, these cells are also implicated in pathogenesis of severe malaria [68, 69]. The natural killer (NK) cells interact with iRBCs and liberate pro-inflammatory cytokines, particularly IFN- [70-74]. In non-immune donors, NK cells can kill plasmodia-infected erythrocytes in the absence of opsonizing antibodies through direct cell-cell interaction [72-76]. Early production of IFN- by NK cells stimulated by IL-12 during malaria infection is associated with a better prognosis of the disease [70, 72, 77]. Additionally, the interaction of NK with iRBCs induces production of the chemotactant IL-8 [78], suggesting a role for NK cells in recruitment and activation of other leukocytes during malaria infection.

22

Natural killer T cells (NKTs) are a specialized subset of lymphocytes that share properties of both T cells and natural killer (NK) cells. They differ from conventional T cells in that; they express the distinct NK.1.1 marker and possess a unique single invariant V14+ antigen receptor that recognizes lipids and glycolipids presented by CD1d molecules, rather than peptide-MHC complexes [79-81]. Experimental models have indicated a role for NKTs in conferring protection against murine malaria, and NKT cells were linked to malaria-associated splenomegaly and expansion of the splenic B cell pool forming the parasite-specific antibody. [82-85]. The gamma/delta (/) T cells constitute a minor subset of T lymphocytes characterized by an antigen receptor formed of and chains rather than the common and chains that make up the antigen receptors on the conventional T cells [86]. Peripheral / T cells are strongly up-regulated during malaria, and they respond to malaria antigens by proliferation and lymphokine production [87-89]. Activated / T cells but not / T cells from malaria-nave donors were shown to inhibit parasite replication in eryhrocytes in vitro, indicating an important innate protective a role against malaria [90].

1.1.1. Adaptive immune responses to malaria. 1.1.1.1. Anti-sporozoite immunity.

Protective immunity against malaria can be induced in animal models and humans following immunization with irradiated sporozoites.[91-93]. Although it confers complete resistance to subsequent sporozoite challenges, such immunity is stage-specific and does not resist challenges with blood stage Plasmodium. Anti-sporozoite antibodies are the principal effectors that promote phagocytosis and block hepatocytes invasion [94].

23

Such antibodies are mainly directed against the most abundant sporozoite surface proteins, the circumsporozoite protein (CSP) and the thrombospondin related adhesive protein (TRAP) as well as several other sporozoite and liver stage antigens. Most adults in malarious areas were found to possess high titer of antibodies to the CSP of P. falciparum [95], and the immune sera obtained from sporozoite-immunized human volunteers, and naturally infected individuals, were found to block the invasion of P. falciparum sporozoite into hepatocytes [96].

1.1.1.2.

Immunity to liver stages of malaria parasite.

Several cellular mechanisms are implicated in generating protective immunity against the liver stages of Plasmodium [97-101]. T cells were shown to be involved in the generation of protective immunity in rodents immunized with irradiated sporozoites.[102, 103], and the passive transfer of CD8+ T cells and CD4+ T cells clones derived from sporozoite immunized mice was found to confer protection [104-107]. Cytotoxic T lymphocyte response to CS proteins was observed in field studies and believed to protect against preerythrocytic stages of malaria [108].

1.1.1.3.

Immunity to blood stages of malaria parasite.

As previously noted, invasion of red cells is the key step in malaria infection and is the cause of clinical disease, hence erythrocytic stages of P. falciparum are likely to be the most important targets for protective immune responses. Protective immunity against erythrocytic stages involves both antibody-dependent and cell-mediated immunity [109, 110].

24

1.1.1.3.1.

Antibody-mediated immunity to blood stages of P. falciparum.

Passive transfer of anti-Plasmodium specific antibodies to non-immune mice, and treatment of P. falciparum infected patients with immunoglobulins extracted from naturally immune individuals, result in reduction of parasitemia and clinical symptoms [111-115]. Moreover, studies in gene-targeted, and B-cell deficient mice demonstrated that B cells are essential for the resolution of blood stage infection of murine malaria, thus, protection from malaria was thought to be mainly an antibody-mediated type of response [116, 117]. In people living in malaria endemic areas, exposure to repeated infections with different variants of P. falciparum induces production of polyclonal antibodies, predominantly IgM, IgG isotypes [118-122], and the acquired protective immunity to malaria correlates with the individual age and the level of antibodies against the asexual blood stage antigens of the parasite [14, 123-125]. Such epidemiologic observations explain the slow acquisition of protective immunity to malaria during the first years of life in parallel with the expansion of the antibody repertoire against variant surface antigens (VSA). Accordingly, a clinical episode in a child living in a malaria endemic area represents an infection with a parasite expressing VSA not recognized by the existing repertoire of antibodies.[121, 126-129]. Early studies have clearly demonstrated the predominance of immunoglobulin-G (IgG) in resolution of the blood stage infection, and several antibody-mediated mechanisms of parasite growth inhibition and killing have clearly been elucidated [130-132]. IgG antibodies directed against merozoite surface proteins enhance various effector mechanisms including; opsonization, phagocytosis [132-134], complement-mediated

25

damage of merozoites [135], blockage of RBC invasion [136, 137], and antibodydependent cellular inhibition (ADCI) of parasite growth [132, 138].

1.1.1.3.2. Cell-mediated immunity to blood stages of P. falciparum. The first observations showing the importance of T lymphocytes in recovery from malaria infections were made by Brown and co-workers in their experiment with thymectomized rats [139]. Later, it was shown that B cell deficient mice can be immunized by natural infection with Plasmodium, and that adoptive transfer of CD4+ Tcell clones could confer protection against malaria [140-142]. However, because erythrocytes do not express MHC molecules, it seems unlikely that T cells could have a direct cytotoxic effects on infected erthrocytes. Alternatively, it was suggested that T cells respond to malaria antigens by cytokines secretion. In consistence with this view, it was shown that stimulation of T cells by malarial antigens presented by antigen presenting cells (APCs) results in release of various cytokines, and experiments with gene-knocked-out mice, have shown that, immune responses to blood stages of Plasmodium are mostly mediated by IFN--secreting T lymphocytes [99, 116, 143]. Both Th1 and Th2 types of CD4+ T cells were implicated in malaria protection and pathogenesis in mice. It has been shown that; CD4+ T cells of the Th1 type which produce IL-2 and IFN-, are important during the acute phase of murine infection with Plasmodium chabaudi chabaudi AS whereas Th2 type of these cells is required during later phases to provide help to B-cells through IL-4 secretion.[144, 145]. The secretion of IFN- by T lymphocytes is believed to induce the cytophilic IgG blood-stage-specific antibodies and assist in antibody-dependent cellular inhibitory mechanisms [138]. Additionally, the CD4+ Th1 cells can kill parasites in the absence of antibodies through 26

monocyte-macrophage activation with the subsequent generation of reactive oxygen and nitrogen intermediates [146, 147]. In humans, CD4+ T cells were shown to inhibit parasite growth in vitro [148], and the production of IFN- by CD4+ T cells in response to specific erythrocytic antigens was associated with protection from malaria in field studies in Africa [149, 150]. In the other hand, the CD8+ T cells seem to play no significant role in protection against blood stage malaria in mice. Consistent with this, depletion of CD8+ T cells during primary blood stage infection did not diminish the ability of the mice to resolve infection. [151, 152]. However, experiments with mice treated with anti-CD8+ mAb showed significantly higher levels of parasitemia than untreated control mice, suggesting some role for CD8+ cells in containing the blood stages of murine malaria [153]. However, in humans, CD8+ T cells may have indirect regulatory effects on the immune responses to blood stage infection during acute malaria [154, 155].

1.2.

The immuno-pathogenesis of severe malaria. Malaria is a complicated syndrome and the clinical outcome of infection with P. falciparum is determined by several factors. Such factors include the intensity of parasite transmission, the parasite virulence, together with numerous host genetic, nutritional, and immunological characteristics [35, 52, 156-165]. However, the mechanisms leading to severe life-threatening complications of malaria are not fully understood [52, 166].

1.2.1. The pathogenesis of cerebral malaria. The mechanisms underlying the pathogenesis of CM remain controversial, and several hypotheses were set to explain the cause of this deadly syndrome [167-170]. The

27

mechanical

(sequestration)

hypothesis

assumes

that

P.

falciparum-parasitized

erythrocytes bind to the post capillary venules, causing obstruction of blood flow, and thereby decrease tissue perfusion and impair wastes disposal leading to brain pathology [169, 171]. Histological examination of brain sections from fatal cases of CM revealed sequestration of iRBCs in multiple organs and tissues [172]. However, due to lack of sequestration in some patients with cerebral symptoms, and the fact that most patients with CM fully recover without evidence of ischaemic damage, the mechanical hypothesis cannot adequately explain the cause of CM. Alternatively, it has been suggested that; malaria toxins bind to and stimulate monocytes to secrete pro-inflammatory cytokines [156, 173, 174]. Although, the cytokines themselves are not harmful, they may induce production of nitric oxide (NO) [175-178] which diffuses through the blood-brain barrier and interferes with the synaptic function as do general anesthetics, leading to a state of reduced consciousness [179]. Several malarial antigens, such as the membrane-anchored glycosylphosphatidylinositol (GPI) and the by-product of parasite metabolism the hemozoin, can cause cytokines release from various host immune cells [180]. The released cytokines up-regulate the expression of various endothelial adhesion receptors such as ICAM-1, PECAM-1/CD31, and thus enhance cytoadherence of iRBCs and sequestartion [179, 181]. Sequestered parasites in the brain vasculature may locally release more toxins and induce the production of inflammatory mediators that disrupt the brain function and metabolism leading to the CM. Most recently van der Heyde et al., suggested a unified hypothesis to explain the etiology of CM [170]. They attributed CM to a combination of parasite-host specific interactions that promote iRBCs adhesion and sequestration in the microvasculature of various organs

28

and tissues causing the induction and release of cytokines and several other bioactive molecules which trigger excessive immuno-inflammatory responses that disturb the homeostasis [170, 182].

1.2.2. The pathogenesis of severe malaria anemia. Severe malaria anemia (SMA) is the most common complication of malaria in holoendemic areas and contributes substantially to the mortality and morbidity from this disease. The pathogenesis of SMA is multi-factorial, and is only partially understood. It involves both accelerated clearance of erythrocytes from the circulation and suppression of hematopoeisis [164, 183, 184]. During acute P. falciparum infections, parasite replication results in an increased rate of RBCs destruction, but this cannot solely account for the marked reduction in hemoglobin levels observed in patients with SMA. In contrast, children with SMA were shown to have lower peripheral parasite densities than parasitemic children without anemia [185, 186] Moreover, an increased rate of clearance of uninfected erythrocytes associated with low reticulocyte levels was also observed in these patients suggesting involvement of inflammatory processes in the pathogenesis of SMA [187-194]. The analysis of microarrays profile gene expression in a murine model of malaria has shown repression of erythroid-associated transcripts during plasmodial infection, providing additional confirmation for a role of the inflammatory mediators in the etiology of SMA [195]. High circulating levels of the proinflammatory cytokines TNF- and IFN- and low levels of the anti-inflammatory IL-10 were associated with SMA, supporting the hypothesis that specific cytokine disbalance may lead to SMA [196198]. Moreover, impairment of IL-12 response was shown to contribute to the development of SMA [199, 200], and low plasma levels of IL-12 have repeatedly been 29

reported in patients with SMA [201-204]. Macrophage migration inhibitory factor (MIF) has also been suggested to play an important role in the pathogenesis of malarial anemia [68], and elevated plasma MIF concentrations were associated with suppression of erythropoiesis and enhanced severity of anemia in murine models [68, 69]. However, MIF production by monocytes in children with acute malaria was shown to decline progressively with increasing anemia severity (167).

1.2.3. The role of cytokines in immuno-pathogenesis of malaria. The importance of cytokines in protection from and pathogenesis of infectious diseases is well established, and altered production of cytokines is widely held as a mechanism responsible for a variety of immuno-inflammatory diseases [33, 205-207]. The first speculations that malaria parasites elicit a systemic inflammatory response that causes disease appeared early in 1940s [208, 209]. The striking similarities between the patho-physiology of acute P. falciparum malaria and septic diseases together with the detection of elevated levels of circulating inflammatory mediators in animals receiving endotoxin and in animals infected with malaria parasites led to the notion that; macrophage derived factors may be responsible, at least in part, for the pathology observed in malaria [164]. Pro-inflammatory cytokines are now recognized causal mediators of malaria fever and low levels of anti-inflammatory cytokines such as IL-10 and transforming growth (TGF-) and/or increased ratios of pro-inflammatory to antiinflammatory factor- cytokines have repeatedly been associated with increased risk of high fever and severe malaria [196, 198, 202, 210-213] 1.2.3.1. Interferon- (IFN-).

30

There is a growing consensus that IFN- plays a central role in protection from malaria [71, 72, 147, 149, 173, 214-216]. IFN- induces the effector mechanisms essential for control of both pre-erythrocytic and blood-stage malaria infections [99]. Treatment of mice with exogenous IFN- delayed the onset of parasitemia, decreased the parasitemia and increased mice survival [173, 216]. Conversely, treatment of Plasmodia-infected mice with neutralizing monoclonal antibodies to IFN- exacerbated the course of infection [217, 218]. Moreover, studies with IFN- knocked-out mice confirmed the critical role of IFN- in the protective immunity to malaria [219, 220]. In clinical studies, elevated levels of serum IFN- were found in patients with acute malaria [221-226], and individuals heterozygotic for an IFN-R1 polymorphism had a lower incidence of CM and death in Gambia [227].

1.2.3.2.

Tumor necrosis factor (TNF-).

Tumor necrosis factor (TNF-) plays pivotal roles in many inflammatory processes, and is implicated in pathogenesis of various acute and chronic illnesses, encompassing both infectious and non-infectious diseases [164, 228-230]. Ample evidence demonstrated the importance of TNF- in the immune response to malaria infection [33, 175, 222, 231238], and several antigens of P. falciparum blood stage parasites as well as hemozoin granules induced TNF- production in peripheral blood monocytes in vitro. The glycosylphosphatidylinositol (GPI) is the major malaria toxin responsible for the TNF- release and induction of fever [180]. Raised levels of plasma TNF- were found in the general population during the malaria transmission season, and were associated with acute and severe malaria [233, 239-245]. TNF- upregulates the ligands that bind iRBCs

31

and promotes sequestration in the brain capillaries, a process that is central to pathogenesis of CM [246, 247]. In addition, hyperlactaemia [248], hypoglycaemia [222] and fever [241] in malaria patients have all been correlated with elevated levels of serum TNF-. However, the failure of TNF- neutralizing agents to decrease the incidence of clinical CM and the disappointing outcome of trials with pentoxifylline and steroid therapy for CM raise doubts about the role of TNF- in the pathogenesis of CM [249, 250]. Moreover, there is an emerging view claiming that lymphotoxin-, and not TNF-, may be the principal mediator of human CM [238, 251].

1.2.3.3.

Lymphotoxin (LT-).

There is evidence indicating a distinctive role for LT in malaria [238, 251-253]. Like TNF- , LT- has been detected in human malarial serum, and was shown to induce high levels of IL-6 and causes hypoglycemia in a mouse model [253]. LT- but not TNF- was found necessary for IFN- production during malaria infection [251].

1.2.3.4.

Interleukin-1 (IL-1).

Interleukin-1 (IL-1) is a family of pro-inflammatory cytokines which comprises IL-1, IL-1, and the IL-1 Receptor antagonist (IL-1RA) [254]. Production of IL-1 by macrophages, monocytes and dendritic cells enhances T cell-dependent antibody production and increases the expression of adhesion molecules on endothelial cells to enable transmigration of leukocytes to sites of infection [254-256]. Both IL-1 and IL-1 are potent pyrogens that are implicated in the causation of malaria fever. Administration of recombinant IL-1 reduced parasitemia and protected mice from lethal cerebral malaria,

32

and elevated serum levels of IL-1 were found in malaria patients in a holoendemic area [257].

1.2.3.5.

Interleukin-4 (IL-4).

Interleukin-4 is a pleiotropic cytokine that plays a critical role in both the proliferation of lymphocytes and regulation of lymphokine production [160, 258]. IL-4 was shown to inhibit Th1 cell differentiation and to induce a reversion of developing Th1 cells to the Th2 lineage through inhibition of IFN- [259]. Early addition of IL-4 to cultures of human CD4+ T cells was shown to impair IL-2 and IFN- synthesis by these cells suggesting an immune-suppressive effect [260]. However, addition of exogenous IL-4 to in vitro cultures is required for the differentiation of naive CD4+ T cells into IL-4secreting Th2 cells [261-264], indicating a critical role in the development of Th2-type immune responses and possibly in the regulation of immunoglobulin switching to IgE and IgG isotypes [265-267]. Thus, IL-4-producing T cells were suggested to induce the production of malaria specific IgG and elevated levels of IL-4 were detected in parasitemic individuals living in malaria endemic areas [257]. It has also been shown that; IL-4 receptor expression on CD8+ T cells is required for the development of protective memory responses against liver stages of malaria parasites [268].

1.2.3.6.

Interleukin-6 (IL-6).

Interleukin-6 is one of the most important mediators of fever and is required for the activation, proliferation, and survival of T cells and dendritic cells [269-275]. In malaria, IL-6 was shown to mediate the anti-parasitic activity of IL-1 on the liver stages of malaria parasite [276], and patients with malaria fever were found to have 33

elevated serum levels of IL-6 [240, 245, 277-279]. Interestingly, higher serum levels of IL-6 were found in severe malaria than in uncomplicated malaria patients [242, 280, 281] and raised levels of this cytokine were associated with hyperparasitemia, jaundice, and shock in Vietnamese patients [282].

1.2.3.7.

Interleukin-8 (IL-8) and chemokines.

The role of IL-8 in malaria pathogenesis is not clearly understood and conflicting results were reported [245, 283-285]. However, elevated serum levels of IL-8 have been noted during acute malarial attack [283, 284].

1.2.3.8.

Interleukin-10 (IL-10).

Interleukin-10 (IL-10) was initially identifed as a cytokine synthesis inhibitory factor (CSIF) produced by mouse Th2 cells and inhibits Th1 cells cytokine production [286]. It is primarily produced by cells of the monocyte/macrophage lineage and to a lesser extent by activated T and B cells [287-289]. IL-10 has extensively diverse effects mediated through specific cell surface receptor complex (IL-10R and IL-10R) expressed on most hemopoietic cell types. It acts as a suppressor of immune responses, principally through down-regulating the expression of the MHC class II, the costimulatory molecules and cytokines genes in the antigen presenting cells [289-292]. For, example, IL-10 is known to inhibit the secretion of IFN- by Th1 cells [286, 293], and of TNF- and MIF by monocytes and macrophages [290, 294], and it modifies the expression of chemokine receptors [295]. In various experimental models, induction or administration of IL-10 suppressed the antigen-specific T-cell proliferation and contributed to the establishment of persistent infection by a number of pathogens including HIV [296] Mycobacterium 34

tuberculosis [297], Listeria monocytogenes [298], Trypanosoma cruzi [299]. However, although generally precieved as an anti-inflammatory cytokine, IL-10 could have quite opposite effects on some specific models [300, 301]. For instance, IL-10 was found to stimulate the expression of MHC-II molecules and the production of immunoglobulins in B cells [302], and to augment the proliferation and cytotoxicity of NK cells when combined with IL-18 [303]. Increasing evidence demonstrates the role of IL-10 in malaria protection and pathogenesis [210, 226, 245, 304-307]. Murine and in vitro studies have demonstrated IL-10s ability to inhibit TNF- production in response to malarial antigens [221], and high levels early during malaria infection were found to inhbit Th1 type of immune responses in both mice and humans. These raised levels were also associated with less effective clearance of Plasmodial parasites and subsequently the development of severe malarial complications, particularly SMA [210, 245, 305, 308, 309]. In contrast, IL-10 has been suggested to play a protective role against experimental cerebral malaria [307, 310], and clinical studies associated insufficient IL-10 with acute and severe malaria [196, 198, 282]. Moreover, it was demonstrated that, in individuals with SMA, plasma levels of TNF- tend to exceed those of IL-10, and low ratios of IL-10:TNF were found to be a risk factor for both CM and SMA, whereas higher ratios were more frequent among hyperparasitemic individuals [311]. Interestingly, low circulating IL-10 associated with high pro-inflammatory cytokines levels was noted in comatose patients with CM [282], suggesting protective effect for IL-10 against SM. Therefore, while it may be detrimental by decreasing cellular immune responses to parasite, IL-10 may equally be beneficial in preventing excessive inflammation that underlies the SM. It is currently

35

assumed that not only the levels but also timely production of IL-10 is crucial for controlling the parasite growth and replication as well as for preventing excessive inflammatory responses to P. falciparum.

1.2.3.9.

Interleukin-12 (IL-12) .

Interleukin-12 plays an important regulatory role in both the cell-mediated [201-203, 213, 312-315] and antibody-mediated [316] immune responses to malaria. IL-12 has been inversely associated with disease severity in experimental and human malaria. Administration of IL-12 decreased mortality and reduced parasitemia in mice through a mechanism which appears to be critically dependent on IFN- [201, 203, 315, 317, 318]

1.2.3.10.

Interleukin-18 (IL-18). have been found in adult patients with uncomplicated P.

Elevated levels of IL-18

falciparum malaria, and these levels were found to decrease with recovery from disease [319]. Higher levels of IL-18 were found in children with mild malaria than in children with a severe malaria, suggesting a protective effect against severe malaria.[202, 320]). Impaired IL-18 response was shown to contribute to the development of SMA in areas of holoendemicity for malaria [204].

1.2.3.11.

Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF).

Granulocyte-macrophage colony stimulating factor (GM-CSF) gene-knocked out mice were found to be more susceptible to infection with P. chabaudi AS than wild-type mice [321], indicating a protective role against murine malaria. GM-CSF was shown to act

36

synergistically with TNF- in priming neutrophils for enhanced phagocytosis and killing of asexual stages of P. falciparum and to up-regulate expression of cytokines, complement proteins and Fc receptors [322]. Incorporation of a plasmid encoding murine GM-CSF in a DNA vaccine against the CSP of P. yoelii enhanced the efficacy of the vaccine [323]. However, elevated serum levels of GM-CSF were recorded in severe human P. falciparum malaria [223, 324].

1.2.3.12.

Transforming Growth Factor- ( TGF-).

Transforming growth factor- (TGF-) plays a key role in limiting malaria pathology, and reduced levels of TGF- were associated with severe malaria [211, 325, 326]. Increased mortality was observed among IL-10/ mice when infected with the normally non lethal P. chabaudi [265], and treatment of P. berghei infected mice with anti-TGF- antibodies increased the parasite virulence, suggesting a protective effect for TGF- against severe disease [326, 327]. Conversely, treatment of these mice with recombinant TGF- resulted in upregulation of IL-10 and down-regulation of TNF- and extended survival time [326, 327]. However, induction, or administration of TGF- during early, acute blood-stage infection suppresses protective proinflammatory cytokine responses, leading to unrestrained parasite proliferation and increased disease severity [328-330]. 1.2.4. Cytokine gene polymorphisms and malaria protection and pathogenesis. The search for genetic variation associated with disease is an exciting area of medical investigations that has rapidly expanded during the last decades and contributed to better understanding of mechanisms of protection and pathogenesis of many diseases. Genetic markers in cytokine genes are becoming widely used in studies of immune-mediated

37

disease, and it is becoming apparent that inherited differences in cytokine genes contribute to phenotypic variations, risks of disease and responses to the environmental stimuli including infectious challenges [331-340]. Interindividual differences in cytokine profiles appear to be due mainly to allelic polymorphism within regulatory regions of the cytokine genes. The biallelic mutations, the single nucleotide polymorphisms (SNP), are the most common source of variations in the human genome [333]. Analyses of interindividual differences and genetic polymorphisms in association with malaria protection and pathogenesis may provide insight into new strategies for fighting malaria. [341, 342]. Examples of the clearly demonstrated associations of malaria risk with cytokine gene polymorphisms include two substitutions at the positions -308 [343, 344] and -376 upstream of the transcriptional start of the TNF- gene [244]. Both polymorphisms were identified as independent risk factors for cerebral malaria in African populations[243, 244, 345]. A long list of reports associating certain SNPs in different cytokines genes with malaria is currently available. However, the following discussion is confined to polymorphisms in IL-10 which is investigated in the current study.

1.2.4.1.

Interleukin-10 gene polymorphisms.

The IL-10 gene promoter is highly polymorphic, it contains two informative microsatellites, IL10.G and IL10.R and eleven single nucleotide polymorphisms (SNPs), of which the three proximal ones located at positions (-1082 G/A [rs 1800896], -819 C//T [rs 5289772] and -592 C//T [rs5289771]) are the most frequent points of mutations [337, 346-355]. Differences in the genotypes and allele frequencies in these SNPs were

38

observed between different populations. These differences may influence the outcomes of certain infections in different populations [356, 357] Interestingly, the -1082G allele was associated with higher production of IL-10 compared to the A allele both in vitro and in vivo [347, 358, 359], whereas polymorphisms at positions -819 and -592 appear to have no influence on IL-10 production [347]. Several studies have demonstrated the relevance of these SNPs to susceptibility and/or severity of a number of diseases including the inflammatory bowel disease [360-362], psoriasis [363-365], primary Sjogrens syndrome [366], rheumatoid arthritis [358, 367], myocardial infarction [368], coeliac disease [369] and systemic lupus erythematosus [353, 370]. Similarly, several infectious diseases were associated with certain alleles in these SNPs. Genetic predisposition to high IL-10 expression has been reported to be associated with a higher rate of mortality in meningococcal disease [334, 371]. Chronically infected hepatitis C patients who are genetically predisposed to high IL-10 production were reportedly less likely to benefit from IFN- therapy [372]. Moreover, associations have been reported between IL-10 polymorphisms and Epstein Barr virus infection [373], leprosy [374] severe malaria [375], and recurrence of hepatitis C in liver transplant patients [376]. Of particular significance, is the A/G transition at position -1082 which has been associated with rheumatoid arthritis [367], systemic lupus erythematosus [370], psoriasis [365], coeliac disease [369], graft-versus host disease [377], and the severity of P. falciparum malaria in African children [375].

1.3.

The Present Study:

39

This thesis is based primarily on results obtained in two studies which will be referred to as (Study I and II). The studies were carried out as a part of the severe malaria project in the Malaria Research Laboratory, Department of Biochemistry, Faculty of Medicine, University of Khartoum, Sudan, in collaboration with the Department of Immunology, Wenner-Gren Institute, Stockholm University, Sweden. Manuscripts of intended publications originating from these studies, together with reports of other relevant work to which the author contributed during the study period are annexed to the thesis.

1.4.

Rationale. Study I. Natural antibody-mediated immune responses are essential for acquisition of protective immunity against malaria infection and disease in endemic areas [378-380]. Passive transfer of malaria specific immunoglobulins has been shown to reduce parasitemia and to improve the clinical condition of malaria patients [381, 382]. Early studies have clearly demonstrated the predominance of immunoglobulin-G (IgG) in resolution of the blood stage infection, and several antibody-mediated mechanisms of parasite growth inhibition and killing have clearly been elucidated [130-132]. Identification of potential targets and mechanisms of antibody-mediated protective immunity is central for rational vaccine development [383, 384] Generally, natural IgG responses in human comprise four distinct antibody subclasses with varying structural and functional properties. Previous studies which mostly investigated antibody response to either individual or limited panels of malarial antigens, have somehow associated levels of certain subclasses of IgG antibodies with some

40

parameters of protection from malaria as reviewed by Garraud et al.[385]. However, only a few field studies have investigated the role of IgG subclasses in protection against SM. For example, low levels of serum IgG3 against crude extract of P. falciparum blood-stage are associated with poor prognosis of SM, and increased IgG3 is associated with reduced risk of malaria attack in Senegal [308, 386]. In a prospective case-control study of Kenyan children, the mean levels of IgG class and subclass antibodies against crude P. falciparum antigen were comparable between children who developed SM and those who remained healthy for two consecutive transmission seasons [387]. However, in the same study, high levels of IgG1 in relation to IgG2 and IgG4 antibodies were associated with protection, whereas high levels of IgG2 in relation to IgG1 and IgG3 were associated with risk of SM [387]. In contrast, levels of IgG subclasses to P. falciparum were found to be the same in protected and non protected subjects in Madagascar [388]. Interestingly, among IgG class and subclass antibodies directed against four vaccine candidate antigens; the apical membrane antigen-1 (AMA-1), the merozoite surface protein-119 (MSP-119), MSP-3, and the glutamate-rich protein (GLURP), only levels of total IgG, IgG3 and IgG4 antibodies against GLURP and IgG1 antibodies against AMA1 were associated with reduced risk of malaria incidence in Burkina Faso [389]. In addition to the intrinsic properties of antigens, the association between IgG antibody levels and protection from malaria may vary with the age and the genetic constitution of the host, as well as the parasite density and strain. Thus, in spite of the convincing evidence for an important role of the cytophilic IgG1 and IgG3 in parasite killing, the role of IgG antibody subclasses in protection from SM complications is not clearly understood. In particular, the relevance of the quantitative variation of these antibodies and protection

41

from SM in areas of seasonal parasite transmission remains to be clearly defined. Here, we undertook analyses of levels of circulating anti-malarial IgG subclasses directed against crude malaria antigen in order to determine whether particular IgG subclasses are associated with protection from SM, and to find out how such associations might be influenced by the host age, parasite density and multiplicity of infection (MOI).

Study II. Interleukin-10 (IL-10) is an important immuno-suppressive cytokine produced by several cell types including macrophages, B and T lymphocytes and dendritic cells. The main biological function of IL-10 seems to be the restriction of inflammatory responses and the regulation of proliferation and differentiation of immune cells [300, 390]. Interleukin-10 has repetitively been associated in various ways with severe complications of P. falciparum malaria. For example, animal models, although not perfectly reproducing the human disease, have provided evidence for an important role of IL-10 in the pathogenesis of CM [163, 251, 391]. IL-10, principally from splenic T cells, was shown to play a crucial role in protection of C57BL/6 mice against the neurological syndrome associated with P. berghei ANKA infection [310]. This has further been substantiated by the demonstration of IL-10s ability to inhibit IFN- production and to protect against CM [307], and by the observation of enhanced malaria disease of P. chabaudi chabaudi infection in IL-10 knocked-out mice [307, 392, 393]. Therefore, inadequate production or defective respond to IL-10 were suggested as possible pathogenic mechanisms of severe malaria [196, 198, 282, 304]. However, the role of circulating levels of IL-10 in protection from or pathogenesis of human malaria is not

42

well understood. Several reports have associated elevated circulating levels of IL-10 with acute malaria [210, 226, 394], severe malarial anemia (SMA) [212], and CM [210, 245]. Moreover, high IL-10 levels during the early phase of infection were associated with less effective clearance of parasites and hyperparasitemia [305, 309]. Conversely, other authors have associated low levels of IL-10 with CM in deeply comatose patients [282], as well as with SMA [196, 395]. Quantitative differences in IL-10 production may partially be attributed to heritable genetic factors[334, 396]. Several nucleotide polymorphisms (SNPs) identified in the IL10 gene promoter were associated with differences in rates of the cytokine production [348]. The relevance of these SNPs to susceptibility to and/or severity of inflammatory and infectious diseases has been demonstrated [373-375]. For example, the A/G transition at position -1082 (rs 1800896) has been associated with either protection against or enhanced severity of several inflammatory and infectious diseases including EpsteinBarr virus infection [373], inflammatory bowel disease and ulcerative colitis [397], Juvenile rheumatoid arthritis [358] and chronic hepatitis C virus infection [372]. Here, I investigated whether a particular allele or genotype at position -1082 is associated with protection from or susceptibility to SM malaria, and whether this is related to the circulating levels of IL-10 in different forms of clinical malaria among Sudanese children living in areas of unstable seasonal malaria transmission.

1.5.

The General Objectives:

43

The overall objective of these studies was to investigate the role of the anti-malaria immunoglobulin-G subclasses and interleukin-10 in the immune-pathogenesis of malaria.

1.5.1. The specific objectives 1.5.1.1. Study I.

1. To measure circulating levels of anti-malarial immunoglobulin-G (IgG) isotypes in order to examine how these levels relate to malaria protection and pathogenesis. 2. To determine the multiplicity of infection (MOI) by Plasmodium falciparum in patients with severe and uncomplicated malaria, and to assess the effect of MOI in the acquisition of the antibody mediated immunity to malaria.

1.5.1.2.

Study II. 1. To measure the circulating levels of IL-10 in order to determine if these levels correlate with a particular genetic allele, and how they affect the outcome of malaria infection. 2. To determine the genotypes and the allele frequencies of the single nucleotide polymorphism (SNP) at position -1082 [rs 1800896], in order to find out how they relate to protection from severe malaria.

44

Chapter Two

Material and Methods

45

2. 2.1.

Material and Methods:

The study area and population.

These studies were carried out over a period from September to December 2003, in ElGedarif Hospital and during the same period in 2006 in El-Obied Teaching Hospital. ElGedarif town (350 to 400 thousand inhabitants) is situated on the Sudanese Savanna, at an altitude of about 600 m above sea level, and is located 350 km southeast of the capital, Khartoum. The epidemiology of febrile uncomplicated malaria episodes in the area has been reported before [398]. Malaria in this area is highly seasonal, and follows the annual June-October rains; the seasonal incidence of malaria varies considerably from year to year [399]. Anopheles arabiensis is the sole malaria vector in the area. The entomological inoculation rate in Gedarif was found too low and difficult to measure by conventional methods in the surrounding villages [400]. El-Obied, the capital of the state of North Kordofan, is 400 km south-west of Khartoum. North Kordofan State lies in arid semi-desert area, and has a population of around 2.6 million, mainly sedentary farmers and nomad livestock breeders. The rainy season extends from June to mid-September with an annual rainfall of less than 400 millimetres, allowing grazing and seasonal rain-fed agriculture. The malaria epidemiology and clinical features in this area have not been investigated and little information is available about the disease prevalence and incidence. The patients attending the out-patient clinic of El-Gedarif Hospital and the Paediatric Unit of El-Obied Teaching Hospital during the mentioned periods were carefully screened for the signs of malaria. A patient was defined to have malaria if he/she complained of fever, had body temperature exceeding 37.5oC with asexual malaria 46

parasites detected in the blood. Patients who met the criteria for severe and uncomplicated malaria as described by the World Health Organisation (WHO) [24] were selected for study. Patients classified with cerebral malaria were in unarousable coma which persisted for at least 30 minutes after a seizure while patients with severe malarial anaemia had normocytic anemia with haematocrit <15% or haemoglobin <5 g/dl in the presence of microscopically detectable parasitaemia. Uncomplicated malaria cases were described as having a positive blood smear, fever, headache, and no signs of severe manifestation. All patients were promptly treated free of charge according to the hospital treatment protocols. The study was approved by the research committee of the Faculty of Medicine, University of Khartoum, and the National Research Directorate of the Sudan's Federal Ministry of Health. Informed consents were obtained from the patient and/or their guardian before enrollment. Unrestricted immediate access to health care was granted for all patients regardless of their acceptance or refusal of enrolment in the study. Comprehensive demographic and clinical data were recorded in the study questionnaire. All participants were subjected to a series of clinical investigations including haemoglobin (Hb%), blood glucose level, and a complete haemogram. Thin and thick blood smears were prepared to investigate parasitemia, species and density. Samples of venous blood were collected in EDTA tubes before commencing treatment. Approximately, 200 l of blood were blotted onto a Whattman-1 filter paper, air-dried and stored in sealed plastic bags for later use for extraction of DNA. The remaining blood samples were immediately centrifuged and the plasma aliquots were collected into 2 ml eppendorff tubes and stored at -80 C, the RBCs-containing pellets were

47

appropriately treated and cryopreserved for further parasite analyses.

2.2.

Microscopy for diagnosis and estimation of parasite density.

Preliminary diagnosis of malaria was made by quick screen of Giemsa-stained thick peripheral blood films. Standard techniques were used to confirm diagnosis and normocytemia as well as to estimate parasite density by two independent microscopists in duplicates of the thick and thin films. A slide was deemed negative after screening 100 consecutive oil immersion fields of a light microscope (Olympus, Japan; Binocular microscope Model- CH-2; using 100x objective and l0x eye pieces). The parasite density per microliter (L) of blood was calculated from the number of parasitized red blood cells (pRBC) per 200 white blood cells (WBC), assuming an average normal WBC count of 8000/L.

2.3.

Parasite DNA extraction, and estimation of P. falciparum multiplicity of infection (MOI).

Parasite DNA was extracted from filter-papers as described elsewhere [401] with slight modifications. Briefly, three small discs each of an area of 6 mm3 were cut off the bloodblotted filter paper and soaked in 1 ml 0.5 % Saponin in phosphate buffer saline (PBS) and incubated overnight at 4 C. The discs were subsequently washed for 30 minutes with PBS and boiled in 200 l of 5 % Chelex-100 in water for 15 minutes. Samples were vortex-mixed and centrifuged for 2 minutes at 10,000 rpm, the supernatants were collected and stored at -20 C until use for DNA genotyping. The multiplicity of infection is the approximate number of parasite variants specified by distinct alleles of the highly polymorphic MSP-2 gene [402]. Nested polymerase chain 48

reaction was used to amplify specific target sequences in MSP-2 as described elsewhere [403]. Briefly, in an Eppendorf thermal cycler (Mastercycler 5330), the PCR reactions were carried out in a primary amplification program that involved DNA denaturation at 95 C for 1 min, followed by 30 cycles of 98 C/10 sec, 61 C/20 sec/ 72 C/10 sec, and final extension for 5 min. The reaction mixture consisted of 5 l of iProofTM high fidelity master mix (BIO-RAD Laboratories, Hercules, CA), 2.5 l of the DNA extract, 0.5 M of primer pairs and 2 l of sterile distilled water. The outer primers corresponding to the conserved regions of block 3 of msp-2 are detailed elsewhere [404]. The second amplification round was similarly carried out, but with 1 l of the initial PCR products in two separate tubes using allele-specific primers corresponding to the two major MSP-2 families (IC/3D7 and FC27) [405]. The final PCR products were then subjected to electrophoresis on 1.5 % agarose gel loaded with ethidium bromide. Gels were illuminated with GelDoc-It TS 2UV transilluminator (BioDoc-ItTM imaging system, Upland, CA. USA) and photographed to determine number and size of DNA fragments presumed to represent different parasite variants. The least number of fragments of both alleles per sample was used as a proxy for MOI.

2.4.

Preparation of the crude parasite antigens and determination of P. falciparum -specific antibody levels.

Plasmodium falciparum parasites were maintained in continuous in vitro cultures in human erythrocytes (group O+) in RPMI 1640 + L-glutamine-NaHCO3 (GIBCO N.Y. USA) with 0.5 % albumax supplemented with Hepes, NaHCO3, gentamycin and hypoxanthine as described by Trager and Jensen [406]. The parasite cultures were

49

synchronised with 5 % D (-) sorbitol treatment in distilled water and adjusted to 1 % parasitemia of mostly early trophozoites (rings) in 5-6 % hematocrit. Sonicates of late stage infected erythrocytes, enriched by 60 % Percoll gradient centrifugation were used as antigens as previously described [407]. The protein content of the crude extract was estimated by Bradford method [408], and the extract was aliquoted into separate sterile screw capped tubes and stored at 80 C until use.
Malaria specific total IgG and the subclasses, IgG1, IgG2, IgG3, and IgG4 were measured

by an indirect ELISA, using crude antigen extract of the F32 isolate of P. falciparum. Briefly, flat-bottomed microtiter plates (High Binding from Costar, Cat. No. 3590 Corning, NY 14831, USA) were coated with 50 l/well of crude parasite antigen at 10 g/ml in sodium bicarbonate coating buffer and incubated at 4 C overnight. The plates were then blocked with 100 l/well of sodium carbonate buffer (pH 9.6) containing 0.5 % bovine serum albumin (BSA) for 2 h at 37 C. Thereafter, the plates were washed with PBS supplemented with Tween-20 plus 0.15 % Kathon (Mabtech, Nacka, Sweden) in a microplate washer (Skan washer 300, CA, USA). After washing, 50 l/well of serum samples diluted 1:400 (IgG1-4) in incubation buffer (PBS + 0.5 % BSA) were added in duplicates and incubated for 1 h at 37 C. Sera of different clinical groups were blindly allocated into plates to reduce inter-plate error. Plates were washed as above, and bound IgG subclasses were detected with the respective mouse anti-human IgG1 1:1000 (SkyBio, Bedfordshire, UK), IgG2 1:3000 (PharMingen, Erembodegem, Belgium), IgG3 1:1000 (Caltag Laboratories, Paisley, UK), and IgG4 1:2000 (Sigma, St. Louis, USA) and incubated for 1 h at 37 C. The plates were washed again and 50 l of conjugated streptavidinALP (Mabtech) diluted 1:2000 50

in incubation buffer were added and incubated for 1 h at 37 C. The plates were washed once more and developed by the addition of 50 l/well of p-nitrophenyl phosphate (Sigma-Aldrich Chemie, Steinheim, Germany). The optical densities of plates were read at OD405 with a VmaxTM microplate reader (Molecular Devices Corporation, Menlo Park, USA). Serial standard concentrations (ranging from 0.003 to 0.3 g/ml) of IgG subclasses from Biogenesis (U.K) were used for the calibrations, and plasma samples from previously known malaria positive subjects from the Fulani tribe in Mali were used as positive controls. Sera from a few malaria unexposed Swedish blood donors were used as negative controls to assess cross-reactivity and to determine the baseline levels. The mean levels of IgG antibodies detected in these negative control sera were used as baselines above which a test sample is considered positive for the particular antibody.

2.5.

Extraction of human DNA and genotype of interleukin-10 (-1082A/G) SNP:

Extraction of human DNA from filter-paper samples has been carried out by boiling as described above for parasite DNA extraction. The -1082 A/G genotype of IL-10 was determined by analyses of fragment length polymorphisms as described elsewhere [356]. Briefly, extracted DNA was amplified by PCR using sequence-specific primers (Forward; 5-AAG GCA ACA CTA CTA AGG CTT CCT T-3, and Reverse; 5-TAA ATA TCC TCA AAG TTC C-3). The PCR reaction mixture included 5 l of 2X PCR Master Mix (ABgene, Epsom, Surrey, UK), 0.25 l of each primer, 2 l distilled water and 3 l of genomic DNA extract. The reaction was carried out in Eppendorf Mastercycle (Eppendorf AG, Hamburg, Germany) with a program of initial denaturation at 95C/5min followed by 35 cycles of 95C/30 sec, 55C /30 sec, 72C/60 sec and final extension at 51

72C/5 min. The PCR product was then treated with the endonuclease EcoN1 (# R0521L BioLab, New England) according to manufacturer instructions, and incubated at 37 C for 2 hours. EcoN1 cleavage site is the underlined C in the forward primer above. The digested product was visualized on 1.5 % agarose gel with ethidium bromide, the length and number of fragments were used to identify genotypes; where the homozygous GG yields two fragments (280 and 310 bp), the heterozygous AG yields four fragments (310, 280, 252, and 28 bp) and the homozygous AA yields three (310, 252 and 28 bp) fragments.

2.6.

Determination of circulating levels of interleukin-10 .

Circulating levels of IL-10 were determined by direct ELISA. Flat-bottomed microtiter plates (High Binding from Costar, Cat. No. 3590 Corning, NY 14831, USA) were coated with 50 l/well of anti-human IL-10 monoclonal antibody 9D7 (Mabtech AB, Nacka, Sweden) diluted to 2 g/ml in phosphate buffered saline (PBS) (pH of 7.4), and incubated overnight at 4 C. The plates were then washed twice with PBS and blocked with 100 l/well of 0.1 % bovine serum albumin (BSA) in PBS for two hours at room temperature. The plates were then washed with PBS with Tween-20 plus 0.15 % Kathon in a microplate washer (Skan washer 300, Skatron Instruments, Norway). Hundred l/well of sera samples [diluted 1:100 in 0.15 M Tris-Hanks] were added to 20 l/well of Mabtech ELISA diluent and incubated at room temperature for 2 hours and then washed again as above. Then 50 l/well of biotin-conjugated monoclonal antibody ( Mabtech 12G8 [diluted 1:1000 in Mabtech ELISA diluent]) was added and incubated for 1 h at room temperature. The plates were then washed as above followed by addition of 50 l of 52

StreptavidinALP from Mabtech diluted 1:1000 in incubation buffer with 1% BSA and incubated for 1 h at room temperature. The plates were washed, developed and read using Molecular Devices (VmaxTM Kinetic microplate reader Menlo Park, USA) at 405 nm. Serial standard concentrations of (10 to 3.160 pg/ml were used for calibration, the curve fit was log-log = {log10(Y) = A + B * log10(X)}; where A = -01.59 is the log10 Y-intercept of the line of log10 (X) = 0, and B = 0.592 is the line slope. The correlation coefficient was 0.999.

2.7.

Statistical analyses.

The data were analyzed with SPSS software version 16.0.1 (SPSS Inc., Chicago, IL, USA). Logistic regression analyses were used to assess differences in frequencies and proportions and to estimate malaria risk. Scale variables which are not normally distributed were log10 transformed, and differences of means were investigated by t-test and one way analysis of variance. Fishers exact test for contingency tables was used to test for significance in proportions of categorical data between the clinical malaria groups when the expected value was less than 5.

53

Chapter Three

The results

3. The results:

3.1.

Study I. 54

3.1.1. The demographic and clinical features of the study population. The total number of individuals enrolled from El-Gedarif was 177; of them 37 were patients with cerebral malaria (CM), 44 presented with severe malarial anemia (SMA), 55 were patients with UM (UM) and 41 were malaria free donors (MF). The demographic and clinical features and parasitological indices of the study population are given in Table 3.1. Six patients with SMA were excluded from the current analysis because of

insufficient plasma samples or incomplete set of data. Four of those who had CM were mature adults (age range 25-55 years), while all those who had SMA were young children mostly under five years. Individuals presenting with SMA had lower mean age (P = 0.002) as compared to those who had CM. Three patients (two children and one adult) representing 8% of those who presented with CM died within 24 hour after admission into hospital. Whereas parasite densities were comparable between CM and SMA, both groups had significantly higher parasitemia (P = 0.009 and 0.017 respectively) than UM. Neither the MOI nor an individual msp-2 genotype was found significantly different between SM and UM, or between the CM and SMA.

55

Table 3.1.

The demographic and malariological indices of the study groups. Study Groups Cerebral Malaria 37 (14/23) 5.0 (4.0 7.8) 4.4 (0.58) * 1.7 (0.2) 2.1 (0.2) 2.0 (1.2) * Severe Malarial Anemia 38 (12/26) 2.75 (1.5 4.0) * 4.4 (0.48) 1.4 (0.2) 1.8 (0.2) 1.5 (0.9) Uncomplicated Malaria 55 (25/30) 5.0 (3.0 7.0) 4.1 (0.54) 1.6 (0.2) 2.2 (0.2) 2.2 (1.2) Malaria Free Donors 41 (12/29) 5.0 (2.0 - 6.8) -----

Number (female/male) Age years (median, 25 and 75 percentiles) Log10 parasite count. Number of IC/3D7 alleles Number of FC27 alleles Multiplicity of infection (MOI)

Except for age, all values are given as mean (SD), and * significant values are discussed in the text.

56

3.1.2. Correlation of IgG antibodies with age, parasite density, MOI and msp-2 genotypes in the study population. Pearsons correlation analysis showed significant positive association between age and total IgG (r = 0.266, P = 0.001), IgG1 (r = 0.299, P < 0.001), IgG2 (r = 0.459, P < 0.001) and IgG3 (r = 0.350, P < 0.001) but not with IgG4 (r = 0.132, P = 0.089) in all study groups. In an age adjusted analysis, the parasite density correlated significantly inversely with total IgG (r = -0.260, P = 0.006), IgG1 (r = -0.287, P < 0.002), IgG2 (r = -0.204, P = 0.033) and IgG3 (r = -0.205, P = 0.031), but did not correlate with IgG4 (r = -0.06, P = 0.53). In contrast, no significant correlation could be found between any Ab levels and neither MOI nor the number of individual msp-2 genotypes (IC-1 or FC27). The correlation between the levels of individual IgG subclasses and the total IgG concentrations was significant and supports the accuracy of the results (not listed).

3.1.3. Comparison of IgG antibody subclass levels between study groups. The overall comparison of levels of total and subclass IgG antibodies as shown in figures 3.1 and 3.2 revealed higher levels of IgG1 and IgG3 subclasses in the whole study population regardless of the clinical group. The levels of these two antibody subclasses were highly variable both between and within study groups. The pair-wise comparisons of mean antibody levels between the study groups revealed higher levels of total IgG (P < 0.001), IgG1 (P = 0.001) and IgG3 (P < 0.001) but not IgG2 and IgG4 in all malaria patients (SM and UM) compared to the MF individuals. Further age-adjusted analyses splitting malaria patients into SM and UM revealed that there were lower levels of total IgG (P = 0.001), IgG1 (P = 0.004) and IgG4 (P = 0.023), but not IgG2 or IgG3 in the SM compared to UM. Re-analysis of data splitting the SM group into CM and SMA showed comparable levels of total 57

IgG (P = 0.15), IgG2 (P = 0.17), IgG3 (P = 0.30) and IgG4 (P = 0.43) but not of the IgG1 (P = 0.034) subclass in SMA and CM. However, the difference in IgG1 levels between these groups disappeared after adjustment for age (P = 0.33). The SMA patients had lower levels of total IgG, (P = 0.002), IgG1 (P = 0.004) and IgG4 (P = 0.031) compared to those with UM, whereas the levels of all IgG subclasses did not differ significantly between CM and UM. Noteworthy, the differences between the groups of SMA and UM remained significant in the levels of total IgG (P = 0.009), IgG1 (P = 0.03) and IgG4 (P = 0.019) after adjustment for both age and parasite density. Association of risk of SM and levels of total IgG, IgG1 and IgG4 was carried out by calculating the odds ratio of the prevalence of negative versus positive sera for the specific antibody among the SM compared to UM as shown in Table 3.2. There was significantly lower prevalence of IgG1 (P = 0.01) and IgG4 (P = 0.002) reactive sera among the group of SM compared to UM. However, there were no significant differences in the prevalence of total IgG, IgG2 and IgG3 between these two groups.

58

Figure 3.1.

General comparisons of IgG total and subclass levels within and between

different study groups of severe malaria (SM), uncomplicated malaria (UM) and malaria free donors (MF).

59

Figure 3.2.

Comparisons of IgG total and subclass levels within and between groups of

cerebral malaria (CM), severe malarial anemia (SMA) and uncomplicated malaria (UM).

60

Table 3.2. Antibody IgG1

Antimalarial immunoglobulin-G subclass sera reactivity and risk of severe malaria. Reactivity* Severe Uncomplicated Total OR 95 % CI Negative Positive malaria 23 (31.1 %) 51 (68.9 %) 27 (36.5 %) 47 (63.5 %) 1 (1.4 %) 72 (98.6 %) 46 (63 %) 27 (37 %) 1 (1.4 %) 71 (98.6 %) malaria 6 (10.9 %) 49 (89.1 %) 16 (11.1 %) 38 (88.9 %) 3 (5.5 %) 52 (94.5 %) 19 (35.2 %) 35 (64.8 %) 0 (0.0%) 51 (100 %) 29 (22.5 %) 100 (77.5 %) 43 (33.6 %) 85 (66.4 %) 4 (3.1 %) 124 (96.9 %) 65 (51.2 %) 62 (48.8 %) 1 (0.8 %) 122 (99.2 %) 3.68 0.27 1.36 0.73 0.24 4.15 3.14 0.32 1.73 0.58 1.38 9.54 0.11 0.71 0.65 2.87 0.35 1.55 0.03 1.75 0.57 29.66 1.51 6.51 0.15 0.66 0.15 19.79 0.05 6.68

P value 0.010

IgG2

Negative Positive

0.453

IgG3

Negative Positive

0.314

IgG4

Negative Positive

0.002

Total IgG

Negative# Positive

0.418

* Antibody level greater than the cut-off point established as the average level measured in sera from malaria unexposed donors was considered positive. OR = Odds ratios and CI represents Confidence Interval. # Continuity correction was made to eliminate the zero count of negative IgG total sera among persons with uncomplicated malaria, (1/(R+1) and (R/(R+1) were respectively added to counts of SM and UM in total IgG, where R = the total number of SM/ total number of UM = 72/51.

61

3.2.

Study II.

3.2.1. The demographic and malariological indices of the study groups. The total number of individuals investigated in this study was 352 including 171 individuals from the samples collected in El-Gaderif during season 2003 and the rest were from El-Obied and the villages around the city during 2006. Those who were enrolled from El-Obied included 4 patients with CM, 23 with SMA, 82 with UM, and 72 were MF. The sex frequency, age and clinical classification of study II population are shown in Table 3.3. For the overall study population, there are no significant differences in age and sex frequencies between the different clinical groups.

3.2.2. The circulating levels of interleukin-10 in the different study groups. The distribution of the levels of IL-10 was positively skewed, thus scores of IL-10 concentration were log transformed to improve normality. One way analysis of variance (ANOVA) revealed significant differences in the circulating IL-10 between the different clinical groups (F = 7.1, P < 0.001) (Figure 3.3). Tukeys post hoc analysis, showed significantly lower concentrations of IL-10 in CM patients as compared with the MF donors (P = 0.001) or with UM patients (P < 0.001). Although not significant, patients with SMA have had lower IL-10 levels compared to those with UM or the MF donors separately, however, SMA patients had slightly higher levels of IL-10 than patients with CM. Interestingly, there was an inverse correlation between IL-10 levels and the degree of fever indicated by the body temperature (1-tailed P = 0.009) (data not shown). No correlation was observed between IL-10 levels and the patient age or parasitemia separately.

62

2.1.1. The frequencies of alleles and genotypes in interleukin-10 (-1082 A/G) single nucleotide polymorphism. Of the total number of samples compiled from the two sites, we could extract sufficient DNA and genotyped the SNP (-1082 A/G) for 226 samples. The Hardy-Weinberg distribution of alleles and genotypes among different clinical groups and the estimation of risk of severe malaria incurred by carriage of a specific allele or genotype of the -1082 SNP are given in Tables 3.4 and 3.5. The allele distribution among the patients of CM but not SMA or UM deviated significantly from Hardy-Weinberg Equilibrium (HWE). The possible gene-phenotype interactions and outcomes of either allelic or genotypic constitution on malaria outcome are given in Table 3.5. Because it is inapplicable in this situation, we excluded the multiplicative genetic model assuming a risk weight (r) for the heterozygous and (r2) for homozygous candidate allele [409]. The alternative additive model assuming a risk weight (r) for the heterozygous and (2r) for homozygous, were used to test the hypothesis that carrying the G allele influences the risk of CM. Further analysis of the latest model assuming dominant or recessive modes of the allele inheritance was performed by pooling the genotypes into different models (Table 3.5).

63

Table 3.3.
Clinical groups Number (Female + Male). Age in years, mean (SD). (CM)

Demographic features of study II population

(SMA) 61 (22 + 39) 3.28 (2.63) (UM) 137 (56 + 81 M) 4.40 (2.89) (MF) 113 (47 + 66) 4.27 (2.40) Total 352 (141 + 211 ) 4.21 (2.75)

41 (16 + 25) 4.86 (2.41)

Table 3.4. The Hardy-Weinberg Equilibrium for the interleukin-10 (-1082 A/G) SNP in the study groups . Patients groups IL-10 genotype Allele frequency (A:G) %
AA AG GG Total

P values *.

64

Cerebral Malaria . Severe Malarial Anemia . Uncomplicated Malaria. Malaria free donors. Total study population

15 18 42 07 82

8 20 54 08 90

12 14 25 03 54

35 52 121 18 226

54.3 : 45.7 53.8 : 46.2 56.2 : 43.8 61.1 : 38.9 56.2 : 43.8

0.006. 0.264 0.616 0.961 0.016

* Chi Square test with 2 df, P value less than 0.05 indicates inconsistency with Hardy-Weinberg Equilibrium.

65

Table 3.5.
Allele/Genotype 1. Co-dominant inheritance A G Total 2.

Estimation of odds ratios, assuming different modes of A/G alleles inheritance.

SM (n) 83 71 154 30 47 77 53 24 UM (n) 137 99 236 47 71 118 90 28 Total 220 170 390 77 118 195 143 52 OR (95.0% C.I.) 1.18 (0.79 to 1.78) 0.84 (0.56 to 1.27) 1.04 (0.58 to 1.87) 0.96 (0.54 to 1.74) 4.67 (2.69 to 8.09) 0.69 (0.36 to 1.31) 0.32 0.90 P value * 0.42

Dominant G allele inheritance. AA AG + GG Total

3. Recessive G allele inheritance. AA +AG GG

Total 77 118 195 * Pearson Chi-Square test comparing the frequencies among patients with severe malaria (SM) and uncomplicated malaria (UM) revealed no significant risk SM being associated with any of the alleles or genotypes of -1082 A/G single nucleotide polymorphism in IL-10.

66

12

10

Serum levels of IL-10 (pg/ml)

CM

SMA

UM

MF

Patients groups

Figure 3.3

Levels of circulating IL10 in different clinical groups of malaria patients and

healthy controls, box and error bars show the median and 95% CI of circulating IL-10 levels in the cerebral malaria (CM), severe malaria anemia (SMA), uncomplicated malaria (UM) and malaria free donors (MF).

67

Chapter Four

Discussion and conclusions

68

4.1 Discussion. 4.1.1. Study I. In study I, as previously noted in responses to many malaria antigens investigated elsewhere [410-412], the relative abundance of IgG subclasses was profoundly altered, being largely dominated by IgG1 and IgG3. There were higher levels of IgG1 and IgG3 compared to IgG2 and IgG4 in all individuals investigated regardless of the clinical status. Except for IgG2, there were prominent increases in levels of all IgG antibodies in infected individuals compared to the MF donors. The rise in levels of IgG antibodies in the infected individuals is obvious as current parasitemia induces the antibody production. However, the similar levels of IgG2 in the patients and the malaria free individuals cannot be explained as a response to the current infection, rather it may indicate the intensity of P. falciparum transmission during the specified period. Worth mentioning; we collected the samples towards the end of the rainy season after a notably intense transmission season. In spite of the altered natural balance of IgG subclasses serum levels, and apart from the notable differences between the study groups, we could still detect a clear age-dependent increase in the levels of all IgG subclasses except IgG4. This age associated elevation of IgG levels concurs with the well known age-dependent evolution of protection from malaria [306, 378]. However, the lack of association between IgG4 and age in this analysis might partly be explained by the relatively young age of the children investigated hence the limited exposure to parasite. Interestingly, the IgG4 antibodies which are known to be induced by chronic infections [413, 414], might reflect the development of immune memory as a result of cumulative exposure to different parasite strains including the relatively common variants presumed to cause severe malaria. Accordingly, the elevated levels of IgG4 detected in infected individuals as compared

69

to the MF donors, are clearly associated with the current infection. However, this response might have been induced by Th-2 polarized T-cell responses. This is supported by reports demonstrating the induction of IgG4 by interleukin-4 (IL-4) [415, 416]. The effect of parasite density on the levels of anti-malaria IgG antibodies might not be straight forward due to variation in the parasite antigenic characteristics and sequestration in micro capillaries. However, in this study, we observed significant negative associations between the parasite density and the levels of all IgG antibodies except IgG4. All of these associations were age-independent, with IgG1 exhibiting the stronger association as compared to IgG2 and IgG3. The inverse associations of IgG1 and IgG3 with the parasite density are in line with the well established anti-parasite effects of these anti-malarial antibodies. However, the association between IgG2 and parasite densities reveals an unusual anti-parasite effect for this subclass. This is interesting, because unlike the primarily cytophilic IgG1 and IgG3, IgG2 antibodies do not bind the common Fc receptor (FcR) types that mediate the antibody-dependent cellular cytotoxicity. Thus, the observed anti-parasite effect of IgG2 may be mediated through a mutated type of FcRII which was shown to be common in this population [417]. The specific role of IgG4 antibodies in malaria protection and/or pathogenesis is not well understood. Only a weak non significant negative association of IgG4 with parasite density was observed, indicating little if any anti-parasite activity in this subclass. However, the increased risk of SM associated with the low prevalence of IgG1 and IgG4 antibodies in this study suggests a protective role for these two subclasses against SM. Pair-wise comparisons of the mean levels of IgG antibodies between the groups of SM, UM, and MF, and subsequently after splitting the group of SM into CM and SMA provided further support for different levels of IgG1 and IgG4 in SM and UM. While levels of all IgG antibodies are comparable between CM and SA, the combined SM group had significantly lower levels of total IgG, IgG1 and IgG4 than 70

those with UM. Although all these differences persisted after adjustment for age, only the difference in total IgG and IgG4 remained significant after inclusion of both age and the parasite density as covariates indicating a marked effect of parasite density on production of IgG1 but not total IgG and IgG4. Taken together, the associations of the low prevalence and levels of IgG1 and IgG4 antibodies with SM indicate important protective effect for these subclasses against SM, and whereas the protective effect of IgG1 may largely be due to its known antiparasite action, the presumed protective role of IgG4 against SM may be attributed to the clearly evidenced anti-inflammatory effect of IgG antibodies [418, 419]. Although the antiinflammatory role of IgG4 is proved in its clinical use for the treatment of autoimmune diseases, it remains to be demonstrated whether antimalarial IgG4 undergo Fab arm exchange that provides the basis for the anti-inflammatory activity attributed to these antibodies [419]. Several lines of evidence argue for a possible role for this anti-inflammatory activity of IgG4 in protection from severe complications of malaria. For instance, the protection from SM conferred by infection with helminthes [420, 421]. The increased levels of IgG4 associated with helminthes infection might be the underlying cause of the observed protection from clinical malaria. Moreover, IgG4 antibodies are poorly recognized by Fc receptors, thus , do not stimulate inflammation or activate the complement system, rather they were shown to inhibit the complement activation [422]. The mechanisms by which IgG4 mediate anti-severe malaria effects is not understood. However, unlike other antibodies IgG4 exist in vivo as monovalent, bispecific molecules, and are also capable of Fc-Fc interaction with other IgG antibodies [423]. Accordingly, we speculate that, IgG4 may directly block the binding of the cytophilic-antibodycontaining immune complexes to the effector cells. Alternatively, IgG4 may cause the formation of small, non precipitating immune complexes that do not induce inflammatory responses. In conclusion, the results of study I are not only revealing significant difference in IgG1 and 71

IgG4 levels and prevalence between SM and UM but also indicate an important anti-severe malaria protective effect. Thus, it is obvious that factors other than age and parasite density are involved in determining the serum levels of the anti-malaria IgG2 and IgG4 antibodies. In addition to the intensity of P. falciparum transmission other factors such as the number of previous clinical episodes, the genetic background of the host and the parasite strains might also influence the mechanisms regulating serum levels of these antibody subclasses.

4.2. Study II. In humans, the role of IL-10 in malaria is less well defined. It seems to be down-regulating the immunopathology underlying complicated malaria. High circulating levels of IL-10 have repeatedly been associated with SM in Thailand [226], Guinea [281], Mali [245] and Senegal [308]. Interestingly, in contrast to these, our current data showed low levels of circulating IL-10 in both groups of severe malaria (CM and SAM) as compared to those with UM or to HCs. The decrease in IL-10 levels was particularly prominent in the patients of CM. This observation is so far consistent with the suggested protective role of IL-10 previously demonstrated in mice models [307, 392, 424] and accords with a similar finding reported in deeply comatose adult Vietnamese patients with CM [282]. However, it contradicts many previous reports of elevated IL-10 during CM. This contradiction can be justified on the basis of different methodology and inclusion criteria of participants in these studies as well as variation in pattern of malaria transmission in the different epidemiological settings of these studies. Moreover, the immune competency, and human genetic variation in the studied populations might have also contributed to these contradicting results. According to these data, it could be speculated that CM might have arisen as a result of

72

uncontrolled pro-inflammatory responses, and that CM patients would have defective IL-10 production. The link between the circulating IL-10 levels and SMA is not well understood. Previous studies have linked SMA with relatively low levels of IL-10 in African children [196, 198, 425]. However, this is contradicted by other reports of high IL-10 during SMA [212]. Since many previous reports have associated SMA with increased levels of proinflammatory cytokines such as TNF- and IFN- which were suggested to enhance immune mediated destruction of both infected and non-infected RBCs and to suppress the erythropoiesis, it is likely that elevated levels of IL-10 through down-regulating inflammatory processes play a protective rather than deleterious role in SMA. However, this speculation does not go with the recent finding that high IL-10 is associated with inefficient clearance of P. falciparum which could as well lead to persistent parasitemia and severe malaria [305, 309]. Yet, it can still be argued that the role of IL-10 in the immuno-pathogenesis of SMA could be of dual nature, and that the temporal and balanced production of IL-10 rather than the absolute levels of this cytokine are the crucial factors in determining the outcome of malaria infection. Investigation of how the allelic variants and genotypes of the -1082 A/G SNP of IL-10 relate to the clinical forms of malaria revealed no significant association between the risk of severe malaria and any allele or genotypes of this specific SNP (Tables 3.4 and 3.5). Interestingly, tests for the Hardy-Weinberg Equilibrium (HWE) revealed significant deviation from the expected frequencies among the patients with CM (P<0.006) while the distribution of geneotypes was conforming with HWE among the other patients who presented with SMA or UM as well as the few MF donors who were analyzed. Logistic regression analysis of the allele frequencies within clinical groups (Table 3.5) revealed no significant association between any of the alleles and a particular form of clinical 73

presentations, but a non-significant trend of decreased homozygous GG genotypes can be observed among the all patient groups. However, due to lack of information about the frequency of these specific alleles and genotypes among the Sudanese population, it is not possible to draw conclusion about how this SNP relates to malaria pathogenesis. Taking into account the previous reports of higher productivity of IL-10 by the homozygous GG [347, 358, 396, 426, 427], it was interesting to assess how the G allele relates to the circulating levels of IL-10 in the different clinical groups. Comparison of mean levels of IL-10 between the different genotypes across different clinical groups revealed no significant differences between the carriers of either allele (data not shown). However, the homozygous GG individuals had slightly higher levels of IL-10 among the patients of UM, but surprisingly, lower levels in patients of CM. In general it could also be suggested that genetic background and haplotypic patterns rather than individual SNPs may be the cause of variable IL-10 production and eventually protection from and/or susceptibility to severe malarial diseases in different humans [375].

74

4.3. Conclusions. The present data reveal significant differences in the levels of IgG1 and IgG4 among between patients who presented with severe malaria as compared to those with uncomplicated malaria, indicating a protective effect for these antibody subclasess against severe malaria.

The multiplicity of infection (MOI) defined as the number of different Plasmodium falciparum clones showed no significant effect on the circulating levels of IgG antibodies, and the number of parasite clones were similar in the different patient groups.

The IL-10 levels in children with CM were significantly lower than in those with uncomplicated malaria, and IL-10 was particularly lower in comatose patients as compared to other forms of clinical malaria, indicating a protective role for IL-10 against severe malaria.

There was no significant association between the different alleles in the IL-10 gene SNP at position -1082 and the various clinical forms of malaria, and no specific allele or genotype was associated with serum levels of IL-10.

4.4. Perspectives. Future plans include investigating: Genetic factors determing susceptibility/resistance to severe malaria, particularly severe malarial anemia among sympatric tribes (Fulani, Nuba and Hawzma) living in Kordofan region in the middle west of Sudan.

75

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17.

18.

19. 20. 21.

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