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Cover illustration: © Palle Ryde Figure 1, 2, 11 © Palle Ryde All previously published papers were reproduced with permission from the publishers. Distributed by Stockholm University Library Printed in Sweden by Universitetsservice AB, Stockholm 2009 © Nnaemeka Chukwukaelo Iriemenam, Stockholm 2009 ISBN 978-91-7155-894-7

iii

‘‘If I haven’t seen further, it is by standing in the footprints of giants’’ Ketil Malde

To my parents, Jonathan Iriemenam Okonkwo and Monica Chinwa Okonkwo And to my lovely wife, Chizube Iriemenam and my wonderful baby

Chikamdabere Iriemenam

iv

‘‘Try not to become a man of success but rather to become a man of value’’ Albert Einstein

Above the cover The picture depicts a cartoon of a plausible mechanism by which antibodies mediate their parasite clearance of infected erythrocytes by binding to free merozoites or to antigens on the surface of infected erythrocytes.

v

SUMMARY Immunity to asexual blood-stage of Plasmodium falciparum malaria is believed to be associated with protective antibodies of certain immunoglobulin classes and subclasses. This thesis addressed the importance of antibodies in relation to malaria infection and their effective interactions with Fc gamma receptor IIa (FcγRIIa) polymorphisms in individuals residing where malaria is endemic. Our data indicate that the frequency of FcγRIIa-R/R131 genotype was significantly higher in Sudanese patients with severe malaria when compared to patients with mild malaria, while the FcγRIIa-H/H131 genotype showed a significant association with mild malaria. The levels of IgG1 and IgG3 subclass antibodies were statistically significantly higher in the mild malaria patients when compared with the severe malaria patients. A reduced risk of severe malaria with IgG3 antibodies in combination with H/H131 genotype was also observed. Our data suggest the importance of cytophilic antibodies in parasite-neutralising immune mechanisms and also signify that the H/H131 genotype, which binds IgG3 more efficiently, is less likely to be associated with severe malaria while R/R131 genotype was associated with severe malaria in this population. The Fulani ethnic group in West Africa has been shown to be relatively resistant to malaria. We investigated the possible impact of FcγRIIa polymorphisms in the ethnic differences in malaria susceptibility seen between Fulani and non-Fulani in Mali and Sudan, and also analysed their malaria reactive IgG subclass profiles. The H131 allele was associated with the Fulani ethnic group, and the R131 allele with the Dogon in Mali and the FcγRIIa-H/H131 genotype and H131 allele were also found to be significantly more prevalent in the Fulani of Sudan as compared to the non-Fulani ethnic groups. The Fulani had higher serum levels of IgG1-3, with higher proportion of IgG2 than the Dogon in Mali while IgG1 and IgG3 were relatively lower among the Fulani in Sudan with a higher proportion of IgG2 than the non-Fulani. The higher IgG2 antibody levels seen in the Fulani individuals may be due to the relatively high prevalence for the H/H131 genotype in this population. Most clinico-epidemiology studies have been in areas with holo- and hyper-malaria endemicity. We have analysed antibody responses to a panel of six blood-stage antigens in relation to clinical malaria outcome in a hospital-based study in Sudan. The pattern of the IgG subclasses towards the different antigens varied but the IgG1 and IgG3 subclasses were the most prominent. Our results revealed a linear association with anti-AMA-1 IgG1 antibodies and reduced risk of severe malaria while a non-linear relationship with IgG3 antibodies was observed for MSP-2, MSP-3 and GLURP. In the combined final model, the highest levels of IgG1 subclass antibodies to AMA-1, GLURP-R0, and the highest levels of IgG3 subclass antibodies reactive to 3D7 MSP-2 were independently associated with a reduced risk of clinical malaria. The results further indicated that for most of the antigens, IgG3 antibodies, with their shorter half-life, may be more efficient than IgG1 in limiting parasitaemia in individuals with clinical malaria, probably due to its structurally biological function. Taken together, these data suggest the importance of the studied merozoite antigens as targets for parasite neutralising antibody responses of the IgG1 and IgG3 subclasses. Furthermore, our results suggest a possible association between FcγRIIa-R/H131 and anti-malarial antibody responses in the clinical outcome of malaria.

vi

TABLE OF CONTENTS SUMMARY.......................................................................................................................V TABLE OF CONTENTS................................................................................................VI LIST OF INCLUDED PAPERS.................................................................................VIII LIST OF ABBREVIATIONS.........................................................................................IX INTRODUCTION.............................................................................................................1 THE IMMUNE SYSTEM...............................................................................................1 Innate Immunity...........................................................................................................1 Cells involved in innate immunity.............................................................................3 Adaptive (Specific) Immunity........................................................................................6 T lymphocytes..............................................................................................................7 Antigen presentation....................................................................................................8 B lymphocytes.............................................................................................................8 Plasma cells................................................................................................................10 Cytokines...................................................................................................................10 Immunoglobulins..........................................................................................................11 Immunoglobulin classes.............................................................................................13 Effector functions mediated by antibodies................................................................16 Fc receptors.................................................................................................................17 FcγRIIa.......................................................................................................................19 MALARIA.....................................................................................................................20 The life cycle of Plasmodium malaria.......................................................................21 Clinical features of malaria.......................................................................................22 Malaria endemicity....................................................................................................26 MALARIA IMMUNOLOGY.......................................................................................29 The pre-erythrocytic stage.........................................................................................29 The erythrocytic stage...............................................................................................29 The importance of antibodies in P. falciparum immunity.........................................31 Target blood-stage antigens.....................................................................................33 B-cell memory in malaria..........................................................................................35 The haemoglobinopathies..........................................................................................36 Haemoglobin variant...............................................................................................37 The ABO blood group system...................................................................................39 RELATED BACKGROUND.........................................................................................40 Epidemiology of malaria in stable and unstable malaria transmissions........................40 Ethnicity.........................................................................................................................41 Antibody reactivities to blood-stage antigens in relation to malaria disease.................42 FcγRIIa and malaria.......................................................................................................43 THE PRESENT STUDY.................................................................................................45 AIM OF THE THESIS..................................................................................................45 SUDAN..........................................................................................................................46 Country profile...........................................................................................................46 Daraweesh project......................................................................................................46 MALI.............................................................................................................................48 Country profile...........................................................................................................48

vii

Study area...................................................................................................................48 METHODOLOGY........................................................................................................50 Ethical permissions....................................................................................................51 RESULTS AND DISCUSSION....................................................................................52 FcγRIIa polymorphism and antibody responses in clinical malaria outcome...........52 FcγRIIa polymorphism and IgG subclasses in relation to malaria............................54 FcγRIIa polymorphism on the IgG subclass patterns of antibodies...........................55 IgG subclass responses to different asexual blood-stage malaria antigens................57 CONCLUDING REMARKS AND FUTURE PERSPECTIVES................................61 ACKNOWLEDGMENTS...............................................................................................64 REFERENCES.................................................................................................................68

viii

LIST OF INCLUDED PAPERS This doctoral thesis is based on the following original papers, which are referred to by their Roman numerals in the text:

I. Nasr A*, Iriemenam NC*, Troye-Blomberg M, Giha HA, Balogun HA, Osman OF, Montgomery SM, ElGhazali G, Berzins K. Fc gamma receptor IIa (CD32) polymorphism and antibody responses to asexual blood-stage antigens of Plasmodium falciparum malaria in Sudanese patients. Scand J Immunol 2007; 66:87-96.

*The authors A. Nasr and N. C. Iriemenam contributed equally to this article.

II. Israelsson E, Vafa M, Bakary M, Lysén A, Iriemenam NC, Dolo A, Doumbo

OK, Troye-Blomberg M, Berzins K. Differences in Fcγ receptor IIa genotypes and IgG subclass pattern of anti-malarial antibodies between sympatric ethnic groups in Mali. Malar J 2008; 7:175.

III. Nasr A, Iriemenam NC, Giha HA, Balogun HA, Anders RF, Troye-Blomberg M,

ElGhazali G, Berzins K. FcγRIIa (CD32) polymorphism and anti-malarial IgG subclass pattern among Fulani and their sympatric tribes in eastern Sudan. Malar J 2009; 8:43.

IV. Iriemenam NC, Khirelsied AH, Nasr A, ElGhazali G, Giha HA, Elhassan TM,

Agab-Aldour AA, Montgomery SM, Anders RF, Theisen M, Troye-Blomberg M, Elbashir MI, Berzins K. Antibody responses to a panel of Plasmodium falciparum malaria blood-stage antigens in relation to clinical disease outcome in Sudan. Vaccine 2009; 27:62-71.

ix

LIST OF ABBREVIATIONS

ADCC Antibody-dependent cell cytoxicity ADCI Antibody-dependent cell-mediated inhibition APC Antigen presenting cell BCR B-cell receptor CLRs C-type lectin-like receptor CNS Central nervous system CR1 Complement receptor 1 CRP C-reactive protein CSA Chondroitin sulphate A CTL Cytotoxic T-cell DBL Duffy-Binding-like DC Dendritic cell DN Double negative DP Double positive EIR Entomological inoculation rate FcγR Fc gamma receptors H Histidine IFN Interferon IL Interleukin ITAM Immunoreceptor-tyrosine-based activation motif ITIM Immunoreceptor-tyrosine-based inhibitory motif KIR Killer-cell immunoglobulin-like receptors MBL Mannose-binding lectin MHC Major histocompatible complex NK cell Natural-killer NKT cell Natural-killer T cell NO Nitric oxide NOD Nucleotide binding and oligomerization domains PAM Pregnancy-associated malaria PAMPs Pathogen-associated molecular patterns PCR Polymerase-chain reaction PfEMP1 Plasmodium falciparum erythrocyte membrane protein 1 PPRs Pattern recognition receptors R Arginine RBC Red blood cell RIG-1 Retinoid acid-inducible gene-1 SNP Single-nucleated polymorphism SRs Scavenger receptors TCR T-cell receptor Th T helper cell TLRs Toll-like receptors TNF Tumour necrotic factor Treg Regulatory T cells VSA Variant surface antigen

Antibody responses and Fc gamma receptor IIa polymorphism in relation to Plasmodium falciparum malaria

Nnaemeka Chukwukaelo Iriemenam, 2009

1

INTRODUCTION

The immune system

The immune system is an organisation of cells and molecules with specialized roles in

defending against infection [1]. Its complex structure helps to detect a wide variety of

infectious agents, (viruses, bacteria, fungi, parasites) and distinguishes them from the

organism’s own healthy cells and tissues. The immune system can be classified into two

fundamental different types, the innate (natural) and adaptive (acquired) immune system

[1, 2].

Innate immunity

The innate immune system provides the frontline host defence to any microbial invader,

in a non-specific manner. It provides the immediate host defence against infection and is

found in all classes on plants and animals. It comprises four types of defensive barriers:

anatomic (skin, mucous membrane), physiologic (temperature, low pH, chemical

mediators), phagocytic (internalization), and inflammatory (tissue damage). Microbial

invaders/intruders that manage to escape these mechanisms, encounter a number of innate

cellular mediators with variant host defensive capabilities like tissue macrophages,

neutrophils, natural-killer cells (NK), and dendritic cells (DC) [3]. The innate immunity

signals plays a critical role in initiating and instructing the development of adaptive

effector mechanisms when it fails to successfully eliminate the pathogen and this process

requires several days to produce armed effector cells.



2

The innate immune system recognises pathogen through an array of soluble and

membrane bound sensors called pattern-recognition receptors (PRRs) that bind pathogen-

associated molecular patterns (PAMPs), found in a broad type of organisms [4].

Scavenger receptors (SRs) represent a large family of structurally unrelated distinct gene

products, expressed by myeloid cells, selected endothelial cells and some epithelial cells

and recognise different ligands including microbial pathogens as well as endogenous and

modified host-derived molecules [5, 6]. SRs are classified into eight different classes

(Class A-H) [6]. The nucleotide binding and oligomerization domains (NOD)-like

receptors (NLRs), which are intracellular, cytoplasmic sensors; and the retinoid acid-

inducible gene-1 (RIG-1)-like receptors (RLRs), which are cytosolic helicases are

additional classes of PRRs [7]. Other members of PRRs family include C-type lectin-like

receptor (CLRs), cytoplasmic double-stranded RNA [8], cytoplasmic-dsDNA receptor

[9], C-reactive protein (CRP), mannose-binding lectin (MBL) and complement proteins.

Of the PRRs, Toll-like receptors (TLRs) are the most studied. TLRs are evolutionary

conserved proteins that have a highly conserved intracellular Toll/IL-1R domain involved

in protein-protein interactions and signalling activation [10]. In humans, 11 TLRs have

been identified and based on their cellular localization, they are classified into two major

groups: TLR 3, 7, 8, and 9 are expressed in endosomes, while TLR 1, 2, 4, 5, and 6 are

present on the surface of many different cells [11]. Upon activation, these sensors trigger

response pathways to form an alarm system critical to the activation of myeloid lineage

cells (macrophages and DCs) that phagocytose and degrade pathogens, migrate to

secondary lymphoid tissue and present antigen to T cells [12]. TLRs control DC



3

functions, activate signals involved in the initiation of the adaptive immune response

[13], and thus may contribute directly to the perpetuation and activation of long term T-

cell memory [14]. B-cell intrinsic TLR signals are not required for antibody production or

maintenance [15]. Members of the TLR family recognise bacteria, viruses, fungi and

protozoa; NLRs detect bacteria, and RLRs detect viruses. The interplay between these

families ensures the efficient co-ordination of innate immune responses, through either

synergistic or co-operative signalling [16].

Cells involved in innate immunity

Mammalian species possess linked cluster of genes, the major histocompatible complex

(MHC), which play a role in inter-cellular recognition and discrimination of self and non-

self. MHCs are glycoproteins found on the surface of cells and are categorized into three

classes; Class I, II and III. Class I is expressed on the surface of all nucleated cells, while

Class II is expressed by professional antigen-presenting cells (APCs) [macrophages, DCs,

B cells], and Class III encodes molecules critical in immune function.

Monocytes and macrophages belong to the mononuclear phagocyte system. They are

phagocytes whose functions are non-specific. They help to phagocytose (engulf and

digest) debris and pathogens and stimulate lymphocytes to respond to the pathogen.

Monocytes are known to originate from the bone marrow through the common myeloid

progenitor (shared with neutrophils) and are released into the peripheral blood, where

they circulate for several days before entering the tissues and differentiate into tissue

macrophages, contributing to local trophy interactions and tissue homeostasis [17].



4

Monocytes also have the capacity to differentiate into DCs and therefore play an

important role in innate and adaptive immunity, due to their microbicidal potential,

capacity to stimulate CD4+ and CD8+ T-cell responses and ability to regulate

immunoglobulin production by B cells [18]. Monocytes express PRR such as TLRs, and

Fc gamma receptors (FcγR) I-III and may contribute to the shaping of memory-T cell

responses [19]. The heterogeneity of macrophages reflects their specialized functions in

different anatomical locations, including the following; alveolar macrophages in the

lungs, macrophages in the central nervous system (CNS) [17], splenic macrophages,

tangible-body macrophages [20], Kupffer cells present in the liver, mesangial cells in the

kidney, etc. Activated macrophages have more effective phagocytic activities because

they express higher levels of MHC Class II molecules and the co-stimulatory B7

membrane molecule thereby allowing them to function more effectively as APCs.

DCs are the most effective APCs because they express high level of MHC class II

molecules and co-stimulatory molecules. They are able to activate naïve T cells and are

critical for the development of the adaptive immune system [21]. DCs are sentinels, able

to capture, process and present antigens and to migrate to lymphoid tissues to select rare,

antigen-reactive T-cell clones [22]. Two DCs subsets, in humans have been described;

myeloid DC which originates from the monocytes arising from the myeloid pathway, and

the plasmacytoid DC which originates from the lymphoid pathway. These subsets differ

slightly in their expression of TLRs but are both effective in the stimulation of naïve T

cells [13, 23]. A new novel subset of cytotoxic DCs has recently been described, with



5

functions of NK cells and DC cells, called killer DC [24, 25], that is believed to

participate in the control of tumour progression [25, 26].

NK cells are large granular lymphocytes with natural cytotoxicity that constitute a major

part of the innate immune system. They kill target cell through the release of cytoplasmic

granules of proteins (perforin and granzyme) that cause the target cell to die through

apoptosis or necrosis [27]. Unlike cytotoxic-T lymphocytes, which need to be activated

before granules appear, NK cells are constitutively cytotoxic. NK cells discriminate

between target cells and healthy self cells by a variety of cell surface activating and

inhibitory receptors and their interactions with DC cells shape innate and adaptive

immunity [28]. They express the surface markers CD16 (FcγRIII) and CD56 in humans,

and NK1.1/NK1.2 in certain strains of mice, enabling them to detect antibody-coated

target cells and to exert antibody-dependent cell cytoxicity (ADCC) [27]. Activating

receptors detect the presence of ligands on cells in distress by NKG2D (humans) as well

as RAE1 (mouse) while they use the inhibitory receptors (killer-cell immunoglobulin-like

receptors – KIR) to gauge the absence of self molecules on susceptible target cells [29].

By interacting with MHC Class 1 molecules that are expressed by most healthy cells,

inhibitory MHC Class 1 receptors provide a way for NK cells to ensure tolerance to self

while allowing toxicity towards stressed cells [27]. Two major subsets of NK cells are

distinguished based on their low and high expression of CD56 (CD56bright and CD56dim

respectively). About 90% of human NK cells are CD56dim/CD56bright, found in the blood,

spleen and display stronger cytotoxic functions while the remaining 10% are

CD56bright/CD56dim, found in the lymph nodes, with less cytotoxicty [30].



6

Adaptive (Specific) Immunity

Unlike the innate immune response, the adaptive immune response provides the immune

system with the ability to recognize and remember specific pathogens, and to mount

stronger attacks each time the pathogen is encountered. This is done through four

different attributes; antigenic specificity (distinguishes between different antigens),

diversity (recognizes different structures on foreign antigens), immunologic memory

(second encounter of the same antigen produces a heightened reactivity) and self/non-self

recognition (distinguishes between self and non-self). Adaptive immune responses are

mediated by lymphocytes. Lymphocytes are one of the many types of white blood cells

produced in the bone marrow by the process of haematopoiesis. These cells recognize

individual pathogens specifically and possess memory.

Two major populations of lymphocytes are known – T lymphocytes (T cells) and B

lymphocytes (B cells). T cells and B cells recognize antigens through the specificity of

their antigen receptors, T- and B-cell receptors (TCR and BCR) respectively. B cells

respond to pathogens by producing large quantities of antibodies which then can

neutralize foreign objects like bacteria, viruses, etc. In response to pathogens some T

cells, called helper-T cells produce cytokines that direct the immune response while other

T cells, called cytotoxic-T cells, produce toxic granules that induce the death of pathogen

infected cells. Following activation, B cells and T cells leave a lasting legacy of the

antigens they have encountered, in the form of memory cells. Through the lifetime of an

animal, these memory cells will remember each specific pathogen encountered, and are

able to mount a strong response if the pathogen is detected again.



7

T lymphocytes

T cells are generated in the thymus throughout adult life [31] and play central role in cell-

mediated immunity. Two lineages of T cells, αβ and γδ, generated in thymus are defined

by their expression of αβ- and γδ- TCR complexes [32]. The most immature subsets lack

the expression of the CD4 and CD8 molecule, thus called double negative (DN). Later,

the αβ lineage progresses to a CD4 CD8 double positive (DP) stage, during which they

undergo further positive and negative selection to generate MHC-restricted and self-

tolerant CD4 and CD8 single positive T cells [33]. The resulting mature γδ T cells, and

αβ T cells comprise; the CD8+ cytotoxic, CD4+ helper and regulatory lineages that form

the basis of cellular immunity [32]. CD4+ T cells regulate cellular and humoral immunity

while CD8+ T cells show cytotoxic activity towards intracellular pathogens. Both CD4+

and CD8+ T cells develop into memory-T cells which increase the specificity of the

adaptive immune responses. CD4+ T cells can differentiate into Th1, Th2, Th17 and Treg

subsets depending on the nature of the cytokine milieu [34].

Th1 cells drive the type-1 pathway, the cellular pathway, mediated by interferon gamma

(IFN-γ) and fight against intracellular pathogens, while Th2 cells drive the type-2

pathway, the humoral immunity pathway, mediated by interleukin-4 (IL-4), IL-5, IL-13

and helps in antibody production and fights against extracellular pathogens [35]. Th17

cells are characterized by their production of IL-17 and may have evolved for host

protection against microbes that Th1 or Th2 immunity are not well suited for, such as

extracellular bacteria and fungi. The effector cytokines produced by Th17 cells are

IL17A, IL17F, TNF-α, IL-22, IL-26 and IFN-γ [36]. Tregs are crucial for the



8

maintenance of immunological tolerance. Naturally occurring Tregs,

CD4+CD25+FoxP3+, arise in the thymus, while the induced Tregs (Tr1 and Tr3) may

originate during an immune response. Natural-killer T cells (NKT cells) share similar

characteristics with NK cell markers by the expression of αβ-TCR. Unlike the T cells

that recognize antigens presented by MHC molecules, NKT cells recognize glycolipid

antigen presented by CD1d. Upon activation, NKT cells can function as either Th or CTL

cells.

Antigen presentation

Antigen processing and presentation are major requirements of the immune response. B

cells can bind directly to the naïve antigen while T cells identify and destroy infected

cells through recognition of foreign determinants associated to self components. These

self components are MHC molecules expressed on the surface of APC and encoded by

the MHC-gene located on chromosome 6 in humans. MHC class I molecules bind to

peptidic fragments which are generated in the cytosol of the cell (endogenous processing

pathway), e.g. viral-infected cell, while MHC class II molecules bind peptidic fragments

that have been internalised by phagocytosis (exogenous processing pathway) and

degraded in the endocytic compartment.

B lymphocytes

B cells are lymphocytes that play a major role in the humoral immune response. Their

primary function is the production of antibodies against antigens, to function as APC and

develop into memory-B cells after antigen stimulation. Many B cells are produced in the



9

bone marrow throughout life. B-cell development occurs through several stages:

generation of mature immunocompetent B cells (stage of maturation), activation of

mature B cells (stage of antigen interaction), and differentiation of activated B cells into

memory-B cells and plasma cells (Figure 1). B-cell activation often proceeds through two

different routes: thymus-dependent antigens that require direct contact with Th cells, eg

proteins while thymus-independent antigens can activate B cells in the absence of Th

cells, eg lipopolysaccharide, peptidoglycan.

Figure 1. B-cell development. During antigen-independent maturation phase, immunocompetent B cells express membrane IgM and IgD. When activated by Th cells, B cells proliferate and differentiate into high-affinity mIg memory-B cells and plasma cells, which may express different isotypes due to class switching.



10

Plasma cells

Conventional models postulate that the maintenance of serum antibodies requires the

continuous proliferation and differentiation of memory-B cells into antibody-secreting

plasma cells [37-39]. Plasma cells typically represent less than 1% of the cells in the

lymphoid organs, yet they are responsible for all antibodies in circulation [40]. An early

study [41] had suggested that some plasma cells have a half-life of only a few days [42-

43] to at most few weeks [44]. However, accumulating evidence, from this last decade,

suggest that plasma cells have the potential to be very long lived, maintaining persisting

antibody production in the event of re-infection [45-47]. The bone marrow is believed to

be a major site of long-term antibody production [47]. Two populations of plasma cells

have been described; long-lived and short-lived [48]. Long-lived plasma cells are a

central part of immune memory, as these cells are largely responsible for the long term

continuous secretion of antibodies. The possible explanation of plasma cell survival in

vivo could be that antibody levels are maintained by the continuous development of

short-lived plasma cells from memory cells, or that plasma cells themselves are long-

lived and maintained independently of memory-B cells and antigen [40].

Cytokines

Cytokines are low molecular weight regulatory polypeptides secreted by white blood

cells and various other cells in response to a number of stimuli. Cytokines bind to specific

receptors on the membrane of target cells triggering signal-transduction pathways that

alter gene expression in the target cell. Cytokines can bind to receptors on the membrane

of the same cell that is producing them (autocrine actions), bind to receptors on the target



11

cell in immediacy (paracrine action), or bind to target cells in distant (endocrine action).

Cytokines are involved in the regulation of the innate and adaptive immunity and

depending on the effect of the immune response; cytokines can be classified as Th1 (pro-

inflammatory) or Th2 (anti-inflammatory) cytokines. The cytokines secreted by the Th1

subset act primarily in cell-mediated responses, whereas those secreted by the Th2 subset

function mostly in B-cell activation and humoral responses.

Immunoglobulins

Immunoglobulins (Ig) are antigen-binding proteins present on the B-cell membrane and

secreted by plasma cells. When bound on the cell surface, the Ig functions as a receptor

involved in differentiation, activation and apoptosis, while the secreted form can

neutralize foreign antigens and recruit other effector components [49]. The Ig molecule

consists of two identical light (L) chains and two identical heavy (H) chains associated in

the form of a Y-shape (Figure 2). It consists of the antigen-binding site (Fab) and the

crystallisable fragment (Fc). Each light chain is bound to a heavy chain by disulfide

bonds and noncovalent interactions, forming the antigen-binding sites. The two heavy

chains are linked to each other by disulfide bonds to form the Ig molecule. The tail of the

Fc part interacts with receptors present on neutrophils, monocytes, macrophages, etc, and

complements. Based on the structure of their heavy chain constant regions, Igs are

classed into classes which may further be divided into subclasses. There are five classes

of heavy chains, δ, µ, γ, α and ε which form the five antibody isotypes IgD, IgM, IgG,

IgA and IgE. The different isotypes perform different functions, and help in directing the

appropriate immune response.



12

Figure 2. Structure of the Ig molecule and the five different Ig isotypes.

The antibody interacts with antigenic determinants, or epitopes, via antigen-combining

sites present in the hyper-variable regions of the Ig molecule. The antigenic determinants

on Ig molecules fall into three categories: isotypic, allotypic, and idiotypic determinants.

Isotypes are antigenic determinants that characterize classes and subclasses of heavy-

chains and types and subtypes of light-chains. They are constant-region determinants that

distinguish each Ig class and subclass within a species. Allotypes are antigenic

determinants specified by allelic forms of the Ig. Allotypes are determined by the amino

acid sequence and corresponding three-dimensional structure of the constant region of the

Ig molecule and reflect genetic differences between members of the same species. This

signifies that not all members of the same species will have any particular allotype.

Idiotypes are unique antigenic determinants present on individual antibody molecules or

on molecules of identical specificity. Idiotypes are determined by the amino acid



13

sequence and corresponding three-dimensional structure of the variable region of the Ig

molecule. It reflects the antigen-binding specificity of any particular antibody molecule.

The individual determinant is called indiotope, and the sum of the individual idiotopes is

the idiotype.

Immunoglobulin classes

Five classes of Ig are described based on their ability to carry out various effector

functions, their average serum concentrations, and their half-life.

IgM predominates in early immune responses, and can exist as monomeric IgM

(expressed as a membrane-bound antibody on the B-cell) or pentameric IgM together

with a J chain. IgM is found in the blood and lymph fluid and is the first antibody

produced in a primary response to an antigen by the neonate, naïve B cells, and during a

primary immune response. Because of its high valency, pentameric IgM is more effective

than the other classes in neutralizing pathogens, agglutination and activation of

complement [50]. IgM does not cross the placenta.

IgG is the most abundant class in serum (75% of serum Ig), important in clearing antigen,

and is the only class that crosses the placenta (Table 1). Maternal IgG, from the placental

transport, protects the child during its early months of life. IgG fixes complement and

binding of IgG to Fc receptors on phagocytic cells (monocytes, macrophages) results in

their opsonisation. The IgG subclasses in humans differ in the number of disulfide bonds

and length of the hinge region, and are named IgG1, IgG2, IgG3 and IgG4. IgG1 makes



14

up most of the total IgG (66%), followed by IgG2 (24%), IgG3 (7%) and IgG4 (3%) [51].

IgG1, IgG3 and IgG4 cross the placenta and play an important role in protecting the new

born from infection. IgG3, however, is the dominant complement activator followed by

IgG1 and IgG2. One prominent feature of IgG3 is its 11 interchain disulfide bonds. IgG4

does not activate complement. Furthermore, IgG1 and IgG3 bind Fc receptors on

phagocytic cells with high affinity, thus mediate opsonisation while IgG4 has an

intermediate affinity, and IgG2 low affinity (Table 1).

Table 1. Selected functional properties of human immunoglobulins

+ to +++, increasing activity. – No activity. ? Questionable

IgA is predominant in external secretions; breast milk, mucous, saliva, tears,

genitourinary and digestive tract. It is important in mucosal immunity. In humans, two

subclasses are known; IgA1 and IgA2. IgA1 is monomeric while IgA2 can polymerize



15

into multimers. While serum IgA is predominantly monomeric in nature, secretory IgA is

polymeric, exists as a dimer or tetramer linked by disulfide bonds to the J chain and

secretory components. Secretory IgA is found on mucosal surfaces (lining of the

respiratory, gastrointestinal, genitourinary tracts) and can be considered an important first

line of defence against invading pathogens [52]. Human colostrum and milk contain a

very high concentration of secretory IgA and therefore, mother’s milk (which contains

secretory IgA against a wide range of microbial antigens) is able to protect the child

against mucosal pathogens [52].

IgD and IgE are the least abundant in the serum. IgD, as IgM, is the major membrane-

bound antibody on mature B cells. IgD does not bind complement and its exact role in

serum is uncertain.

IgE mediates mast-cell degranulation. It binds tightly to Fc receptors on basophils and

mast cells, and as a result, is involved in allergic reactions. IgE mediates the immediate

hypersensitivity reactions that are responsible for the symptoms of hay fever, allergy,

hives, asthma, and anaphylactic shock. IgE also plays a role in parasitic diseases.

Eosinophils have Fc receptors for IgE and binding of eosinophils to IgE-coated helminths

results in killing of the parasite. Mast cells are needed to clear intestinal helminthic

infections [53] and, when infected, IgE-deficient mice have higher burdens of

Schistosoma mansoni [54]. Elevation of P. falciparum-specific and total IgE in severe

malaria disease had been reported [55-56] while P. falciparum-specific IgE response also

seems to contribute to the control of parasites [57-58].



16

Effector functions mediated by antibodies

Antibodies, generally, do not kill pathogens by binding to them. They recognize

pathogens and invoke responses (effector functions) that remove or kill pathogens. The

variable region of the molecule (Fab) helps to bind to antigens while the H chain region

(Fc) is responsible for its interaction with other cells that results in the effector functions

of the humoral response. Antigen binding by the antibodies is the primary function of

antibodies. The binding of Ig to various cell types (phagocytic cells, lymphocytes), which

have Ig receptors can activate the cells to perform effector functions. There are four

major effector functions that enable antibodies to remove or kill pathogens:

Opsonisation promotes antigen phagocytosis by monocytes, macrophages and

neutrophils. Neutralization or blocking – antibodies can prevent pathogen adherence.

Blocking of viral and bacterial adherence to mucous membrane is the major defensive

role of IgA in secretions. Complement activation – the important by-product of the

complement system is a protein fragment called c3b, which binds non-specifically to cell-

and antibody-complexes near the site at which the complement was activated. The

collaboration between antibody and the complement system is important to the

inactivation and removal of antigens and in the killing of pathogens. Antibody-

dependent cell mediated cytoxicity (ADCC) – linking of antibody bound to target cells

with the Fc receptors of a number of cell types particularly NK cells, can direct the

cytotoxic activities of the effector cell against the target cell.



17

Fc receptors

BCR and TCR are the receptors involved in antigen recognition by cells of the immune

system. Fc receptors (FcR) bind the Fc part of the antibodies [59] and thus play a role in

immune regulation as they serve to link antibody-mediated immune responses with

cellular effector functions. FcR exist in every Ig class, FcγR bind IgG, FcαR bind IgA,

FcεR bind IgE, FcµR bind IgM, and FcδR bind IgD [59]. There are two different classes

of FcγRs, categorized according to the receptors’ affinity for specific IgG subclasses and

the type of signalling pathway they trigger [60-61]. The activating receptor contains

intracytoplasmic activation motifs called immunoreceptor-tyrosine-based activation motif

(ITAM) involved in triggering activating signalling while the inhibitory receptor

transmits their signals through an immunoreceptor-tyrosine-based inhibitory motif

(ITIM) involved in down-regulating signalling [61]. Innate immune effector cells

(monocytes, macrophages, DCs, basophils and mast cells) express both activating and

inhibitory FcγRs. Activating FcR trigger innate immune functions resulting in the release

of pro-inflammatory cytokines and an increased phagocytic capacity; in contrast anti-

inflammatory cytokines downregulate the activating FcR expression by increasing

FcγRIIb, and thus creating a balance between the activating and inhibiting receptors [62].

Since FcR are important players in regulating immunity, they are at the forefront of

immunological research [63] and are new targets for new immunotherapeutic design [64].

In all mammalian species studied to date, four different classes of Fc receptors have been

defined known as FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16) and FcγRIV (Figure 3)

the latter only found in mice [61]. The genes encoding the Fc-binding chains of each of



18

the FcγRs are located on chromosome 1q21-24. FcγRI is expressed on monocytes,

macrophages, neutrophils and has the highest affinity for IgG and is the only FcR that

binds monomeric IgG. FcγRII and FcγRIII have low intrinsic affinity for monomeric IgG,

binds IgG in the form of immune complexes and due to their polymorphisms; they

increase the IgG binding affinity. Both FcγRII and FcγRIII interact with human IgG1 and

IgG3, whereas FcγRII is the sole FcγR class capable of binding human IgG2 complexes

[65]. FcγRIIIb exhibits the genetic polymorphisms FcγRIIIb-NA1/NA2, coded for

different capacities of IgG binding and phagocytosis [66]. Both FcγRII and FcγRIII have

been demonstrated on neutrophils, eosinophils, monocytes and macrophages. Signalling

through FcγRIII on NK cells and monocytes triggers ADCC and the production of IFN-γ

[67].

Figure 3. The family of Fc receptors for IgG. Both human and mice FcRs can be distinguished by their affinity for antibody Fc fragment and the signalling pathways induced. Adapted from Nimmerjahn and Ravetch [61].



19

FcγRIIa

The human FcγRII is a 40 kDa glycoprotein expressed on monocytes, macrophages,

basophils, endothelial cells in placenta, platelets, and B cells and initiates phagocytosis,

ADCC and cellular activation [66]. At least six human FcγRII isoforms have been

identified which are encoded by a total of three genes, FcγRIIa, FcγRIIb, and FcγRIIc,

located at chromosome 1q23 [66]. The FcγRIIa gene has two single-nucleated

polymorphisms (SNPs) at position 27 and 131 on the amino acid sequence.

The SNP in the FcγRIIa gene (G494A) (rs1801274) is of interest because of its functional

consequences. A SNP at amino acid position 131 of the mature protein results in either

histidine (His131) or arginine (Arg131). These alleles are codominantly expressed;

therefore, an individual might phenotypically express RR, HR, or HH. This change alters

the ability of the receptor to bind certain IgG subclasses. FcγRIIa-H131 has a higher

affinity for human IgG3 and is the only human FcγR that efficiently binds IgG2, whereas

FcγRIIa-R131 binds IgG2 weakly. The clinical importance of FcγRIIa polymorphism has

been evaluated for encapsulated bacterial infections, which showed an association

between FcγRIIa-His/His131 and protection, whereas the poorly IgG2-binding allotype

FcγRIIa-Arg/Arg131 is associated with increased susceptibility to bacteraemia [68-69].

However, FcγRIIa polymorphism could constitute additional factor that might influence

the clinical manifestation of systemic lupus erythematosus [70-71]. Furthermore, IgG

may act via the activating FcγRIIa to regulate the CD1 expression profile of DCs [72].



20

Malaria

The name malaria is derived from the Italian ‘mal’aria’, which means ‘bad air’ from the

early association of the disease with marshy surroundings [73]. Malaria is the most

widespread and persistent parasitic disease, which affects human populations throughout

the world and especially in tropical and sub-tropical countries. Each year, an estimated

300-500 million clinical cases occur, and between 1-3 million deaths, primarily among

children and pregnant women [74-75]. Every 30 second, a child dies of malaria

somewhere in the world [76], resulting in a daily loss of more than 2000 young lives

worldwide [77].

Malaria is caused by the intracellular parasitic protozoa of the genus Plasmodium and is

transmitted by the bite of an infected female Anopheles species mosquito [78]. Infection

can also occur through blood transfusion or congenital transmission. There are four

species of malaria parasites that infect humans: P. falciparum, P. vivax, P. ovale and P.

malariae. Recently, the discovery of P. knowlesi malaria in humans [79] suggests a

probable fifth human malaria parasite [80-82]. Accumulating evidence (from Malaysia,

Singapore and Philippines) suggest a potential public health importance due to

misdiagnosis of P. knowlesi malaria, short-term travel, immigration and zoonotic

transmission [82-88]. Further evidence shows that P. knowlesi infections in humans are

widely distributed in Malaysian Borneo and not newly emergent [89]. The most severe

form of human malaria is caused by P. falciparum. Malaria is a devastating infectious

disease that not only affects the health system, but also slows the rate of long-term

economic growth and development. The emergence of drug-resistant strains of the



21

parasite has exacerbated the situation, and global climate change, disintegration of health

services; human migration and population displacement have also contributed. Recently,

evidence of artemisinin-resistant malaria report from Western Cambodia confirm fears

that artemisinin is losing its effectiveness in the area [90-91].

The life cycle of Plasmodium malaria

The malaria parasite life cycle is complex involving the sexual stage and the asexual

stages both in the mosquito and vertebrate host respectively. At the time the female

infected mosquito takes a blood meal, sporozoites are injected into the blood stream

(Figure 4). Sporozoites migrate to the liver within 2-30 minutes, invade hepatocytes and

multiply into exo-erythrocytic merozoites. Each sporozoite gives rise to thousands of

merozoites which are released into the blood stream at hepatocyte rupture. The released

merozoites invade other erythrocytes, starting a new cycle of schizogony. After several

cycles, some merozoites develop into gametocytes, the sexual stages, which cause no

symptoms but are infective to the mosquitoes [78]. Cyclical fevers are the hallmark of

malaria, and typically occur shortly before or at the time of red blood cell (RBC) lysis, as

schizonts rupture to release new infectious merozoites. This occurs every 48h in P. vivax,

P. ovale and P. falciparum and every 72h in P. malariae infections. Intense fever is

accompanied by nausea, headaches and muscular pain, amongst other symptoms. In

patients infected with P. vivax and P. ovale, relapse may recur months to years after

initial infection. This is caused by re-activation of the silent liver-stage form of the

parasite (hypnozoites).



22

Figure 4. Life cycle of the malaria parasite. Adapted from Lozano and Patarroyo [92].

Clinical features of malaria

The erythrocytic cycle represents the only stage of the malaria life cycle that is

responsible for the clinical manifestation of malaria. Clinical manifestation of malaria can

be summed into mild (uncomplicated) and severe (complicated) malaria according to the

World Health Organisation set criteria (WHO) [93-94]. Mild malaria is defined with

headache, cough, diarrhoea, fever, muscular pain, nausea and no signs of severe

manifestation. Renal failure, hypoglycaemia, hepatic dysfunction, severe malarial

anaemia (haemoglobin <5 g/dL or haematocrit <15%), pulmonary oedema, convulsions



23

and shock are complications in severe malaria (Table 2). Cerebral malaria is a frequent

presentation of severe P. falciparum infection and has been attributed in part of the

unique ability of the parasite to alter the surface of infected red blood cell (iRBC) so that

they can bind to endothelial surfaces causing obstruction of cerebral blood flow, leading

to coma or other neurological phenomena, such as seizures and elevated intracranial

pressure [95]. Observations suggest that pro-inflammatory cytokines such as tumour

necrosis factor α (TNF-α) and nitric oxide (NO) induced by parasite material may also

contribute to the pathogenesis of cerebral malaria [96-97].



24

Table 2. Clinical features of severe malaria. Manifestation Clinical features Cerebral malaria Unarousable coma which persist for about 30 minutes

not attributable to any other cause of infection. A coma score for children is Blantyre coma score and it ranges from 0 to 5; 2 or less indicates unarousable coma. Glasgow coma score is used to measure coma in adults which ranges from 3 to 14; 9 or less indicates unarousable coma.

Severe malarial anaemia Haemoglobin <5.0 g/dL or haematocrit <15% in the

presence of malaria parasite count >10, 000/µl Prostration Inability to sit up unsupported Impaired consciousness Assessed by Blantyre and Glasgow coma scales for

children and adults respectively Respiratory distress Sustained nasal flaring, intercostal indrawing

(recession), deep breathing Multiple convulsions Seizures (2 or more within 24 hour period) Circulatory collapse Core-to-skin temperature differences >10oC. Systolic

blood pressure <50 mmHg in patients 1-5 years, <70 mmHg in patients >5 years.

Pulmonary oedema Diagnosis by chest X-ray Abnormal bleeding Abnormal bleeding from gums, nose, gastrointestinal

tract or venepuncture sites. Jaundice Sclera examination and mucosal surfaces of the mouth Haemoglobinuria Dark red or black urine. The absence of microscopic

haematuria suggests haemoglobinuria or myoglobinuria Hypoglycaemia Defined as whole blood glucose <2.2 mmol/L (<40

mg/dL) Acidosis Plasma bicarbonate concentration <15 mmol/L or base

excess >-10 Hyperparasitaemia Peripheral parasitaemia of 4% or more in non-immune

individuals Renal impairment Urine output of <12 mL/kg/24 h Adapted from WHO [93-94].



25

Immunity to malaria is relatively slow to develop and incomplete, host-age dependent,

can be lost failure to lack of continuous exposure, especially when the immune adults

leave malaria endemic regions. Immunity to severe diseases (acquired much faster) is

complete after only a few infections for children living in areas of high malaria

transmission but not for those living in areas of low malaria transmission [98] suggesting

that in regions of hyper- and holoendemic malaria transmission, adults after years of

repetitive exposure, reach a state of relative resistance and individuals develop effective

immunity that control parasitaemia and prevents severe and life-threatening

complications [99-104] (Figure 5). This nonsterile immunity with low parasitaemia

usually witnessed with no symptoms has been termed premunition [105-106] and is host-

age dependent.

Figure 5. Population indices of immunity in an endemic setting of P. falciparum malaria. Adapted from Langhorne et al., [102].



26

In holoendemic areas, a small proportion of infections progresses to severe malaria (life-

threatening complications like severe malarial anaemia and cerebral malaria) while a

larger population become ill with fever when infected, but eventually recovers [107]

(Figure 6). The presence of P. falciparum parasitaemia at any given time therefore may

not correlate well with clinical disease in areas of holoendemicity as children may

harbour high parasite loads with no malarial symptoms [104].

Figure 6. Progression of malaria disease in a malaria-endemic region. Adapted from MalariaGEN [107].

Malaria endemicity

Malaria endemicity is classified on the basis of frequency of infection of individuals with

the parasite (parasite rate) and the frequency of enlarged spleen in children (spleen rate)

[108]. Four groups have been identified namely:



27

• Holoendemic – these areas/regions have intense all-year round malaria

transmission. The parasite rate in the one-year group is over 75% during the peak

transmission season. The spleen rate in adults is either high or low and the

population is relatively immune. Parasite density decreases with increase in age.

• Hyperendemic – these areas/regions have seasonal transmission. The parasite rate

and spleen rate in 2-9 years is between 51-75% during the transmission season.

The population’s immunity does not confer adequate immunity irrespective of the

age.

• Mesoedemic – these areas/regions have some malaria transmission. Occasional

epidemics could constitute a public burden due to low immunity acquisition. The

parasite rate and spleen rate in 2-9 years is 11-50% during transmission seasons.

• Hypoendemic – these areas/regions have limited or little transmission. The

population have little or no immunity to malarial disease. The parasite rate and

spleen rate in 2-9 years is <10%.

Malaria is also commonly classified epidemiologically as stables or unstable (Figure 7)

[109-110]. Stable malaria has the characteristics seen in holo-and hyperendemic areas

while unstable malaria has the characteristics seen in meso-and hypoendemic areas. In

stable malaria the level of immunity is high after childhood and a form of equilibrium is

maintained throughout life if the individual do not live outside the region. In unstable

malaria, equilibrium is not attained in the population and epidemics therefore occur when

mosquito breeding becomes seasonally prolific.



28

Figure 7. P. falciparum malaria risk defined by annual parasite incidence with temperature and aridity. Dark grey areas represents stable transmission, middle grey areas (unstable transmission) while light grey represents no risk. The data shown are age-standardized (PfPR2–10) and shown as a continuum from 0–100%. Adapted from Tatem et al. and Guerra et al. [110-111].



29

Malaria Immunology

The exact mechanism by which immunity to malaria is achieved remains a subject of

discussion. The complex life cycle of Plasmodium sp that involves two different hosts

and different stages makes it more complicated. However, it is believed that both cell-

mediated and humoral immune responses are involved.

The pre-erythrocytic stage

Immunity to pre-erythrocytic stages is believed to be mediated by both antibodies and

cellular mechanisms [112]. Antibodies directed against sporozoite surface proteins could

potentially protect either through opsonisation leading to clearance before reaching the

hepatocyte, or through interfering with the process of hepatocyte invasion [113]. Both

CD8+ T cells and CD4+ T cells are involved in protective immunity against malaria but in

different stages. In rodent models, CD8+ T cells have been implicated as the principal

effector cells, and IFN-γ as a critical effector molecule. IL-4 secreting CD4+ T cells are

required for induction of the CD8+ T-cell responses, and Th1 CD4+ T cells provide help

for optimal CD8+ T-cell effector activity. γδT cells, NK cells and NKT cells also play a

role [114].

The erythrocytic stage

CD4+ T cells are crucial in immunity against asexual stage, producing cytokines involved

in the activation of innate immunity and help activated B cells to produce antibodies

essential for parasite clearance. Although blood-stage protection is mediated by

antibodies other protective mechanisms are also used including innate immunity, IFN-γ



30

and CD4+ T cells [115-116]. IFN-γ produced by CD4+ T cells to specific blood-stage

antigens has been shown to be associated with protection against re-infection [117].

CD4+ T cells secretion of IFN-γ might also help to induce cytophilic IgG blood-stage-

specific antibodies and assist in the clearance of infected RBCs [118]. The absence of

MHC molecules on RBC indicates the importance of humoral immune responses against

the erythrocytic stages. The role of B cells in immunity against malaria was established in

mice lacking B cells, which were unable to eliminate blood stage malaria infections [119]

while one study showed that B cells are not required for the control of early acute

parasitaemias, but appear to be important for the final clearance of the infection, probably

by producing specific antibodies against the parasite [120].

Invasion of RBCs by merozoites is a key step in the establishment of malaria infection

and therefore is likely to be an important target of protective immune responses [113].

The process involves a complex cascade of events involving interactions between the

RBC and parasite proteins (Figure 8) including merozoite surface proteins (MSP-1 to 11)

[92, 121-126], RBC surface (PfEMP1, Pf332, PfAARP1, RIFINS, SURFINS) [122, 124,

127-129], parasitophorous vacuole (GLURP) [122, 124, 127, 130], EBA and AMA-1

[122, 124, 127, 131], etc. Given the short time that many of these proteins are exposed to

the host immune system, it is likely that antibodies are the main stay of immunity against

merozoites [113].



31

Figure 8. The erythrocytic cycle with some merozoite representation describing the main proteins expressed. Adapted from Bolad and Berzins [124] and Patarroyo [121].

The importance of antibodies in P. falciparum malaria immunity

Not only are antibodies markers of exposure but also they are effectors of protection. The

classic example of the importance of antibodies in protective immunity against P.

falciparum infection was established by Cohen et al. [132] in which passive transfer of

immunoglobulins by the intramuscular route contributed to the control of parasite density

and protection against P. falciparum clinical malaria. In a similar study, gamma globulin

from adult West Africans was administered to East African subjects with severe P.

falciparum infections which led to abrupt reduction in parasite density and recovery from

clinical illness [133]. In Thai patients, the protective effect of African IgG antibodies

against P. falciparum malaria was also shown by passive transfer [134] and antibodies

from immune Africans were able to control asexual blood parasites in Saimiri monkeys

SURFINS



32

[135]. To date, the exact mechanisms by which malaria specific antibodies interfere with

the development and/or multiplication of the asexual stages of P. falciparum in protection

is still unclear, and the actual target antigens of protective antibodies remain uncertain.

Several seroepidemiological studies in different malaria endemic areas have supported

and also confirmed the association of IgG antibodies especially of the cytophilic

subclasses (IgG1 and IgG3) in protection [136-141]. Accumulating results from different

populations have also suggested a protective role of IgG2 antibodies in malaria infection

probably through its binding to the FcγRIIa-H131 [142-144]. Uncertainty remains as to

which IgG subclass antibody is more important in protective immunity against malaria.

However, in clinically protected individuals, the cytophilic antibodies were more

pronounced [136] and binding of antibodies to infected RBCs results in their opsonisation

facilitating their phagocytosis by monocytes/macrophages [145-147]. A number of

potential mechanisms has been suggested by which antibodies mediate parasite

neutralisation [122-124] including complement-mediated lysis of infected RBC,

preventing processing of invasion proteins or blocking their erythrocyte-binding sites

[113]. Inhibitory antibodies are thought to act by inhibiting erythrocyte invasion through

targeting merozoite surface antigens [148]. Children can acquire growth-inhibitory

antibodies at young age [149], and a correlation of plasma antibody-mediated growth

inhibition of blood stage P. falciparum with age has been shown [150]. The major

mechanism for antibody-mediated parasite neutralisation is thought to involve monocytes

or other leukocytes as effector cells [118, 124, 151], through antibody-dependent cell-

mediated inhibition (ADCI). In corporation with monocytes, the soluble factors (TNF-α,



33

NO) are released by the monocytes upon uptake of opsonised merozoites. But antibodies

by themselves neutralise parasites by interference with the merozoite invasion [152].

Target blood-stage antigens

The clinical symptoms of malaria manifest only during the blood-stage and much

attention has been given to the parasite molecules that interact with the host cells during

RBC invasion as potential targets of host immune responses (reviewed by Berzins and

Anders [122]). The important antigens of parasite neutralising antibodies are expressed

during the merozoite and trophozoite stages (Figure 8) [153-154] that play a role in RBC

invasion and are thought to be targets of immunity (Table 3). While antibodies to AMA-

1, Pf155/RESA and Pf332 inhibit merozoite invasion and parasite growth on their own

[131, 155-156], antibodies to MSP-3 and GLURP appears to be dependent on their

interaction with Fcγ receptors on monocytes or other effector cells [157-158].

Table 3. Selected asexual blood-stage targets of P. falciparum malaria

Adapted from Bolad and Berzins [124] and Taylor-Robinson [154].



34

P. falciparum, the most virulent of the four malaria parasites of humans, adheres to a

molecule called chondroitin sulphate A (CSA) on the surface of syncytiotrophoblasts

[159-160], and women with higher levels of anti-adhesion antibodies against CSA-

binding parasites are protected from malaria infection during pregnancy [159-162] by

blocking parasite adhesion to placental CSA [163]. One important feature of P.

falciparum malaria is the switching of its variant surface antigens (VSA) [164]. The

mechanism of this is still not known but believed to be one way the parasite avoid the

immune response. The importance of VSA in naturally acquired protective immunity to

P. falciparum malaria has been shown and parasites causing clinical disease express

VSA to which the patient has no pre-existing antibody response. Thus, the immune

system responds to a clinical disease episode by mounting an antibody response with

specificity for the VSA expressed by the parasites causing it [165].

Antibodies specific for the VSA are of critical importance to protection [166] and VSA

expressed by parasites causing pregnancy-associated malaria (PMA), characterised by the

accumulation of iRBC in the placenta, are fundamentally different from all other VSA

(reviewed by Hviid [167]). These parasites express a P. falciparum erythrocyte

membrane protein 1 (PfEMP1) VSAPAM that binds to CSA. The identification of a

distinct PfEMP1 variant (VAR2CSA) as the dominant variant surface antigen and shown

to be a clinically important target for protective immune response to PAM raises interest

in developing preventive strategies [168]. VAR2CSA is a large polymorphic protein (350

kD) consisting of six Duffy-Binding-like (DBL) domains and several inter-domain

regions, expressed on the surface of infected erythrocyte on CSA from infected placentas



35

[168-169]. In areas of intense malaria transmission, antibody levels to VSACSA increase

with gravidity, block adhesion of iRBC to CSA [170] and protect against low-birth

weight and maternal anaemia while in regions of low endemicity, the antibody levels did

not appear to confer protection against placental malaria infection [171]. However,

despite the structure and interclonal conservation of VAR2CSA among other PfEMP1,

and with constraints on recombinant protein production, significant challenges still exist

concerning the development of a VAR2CSA-based vaccine [172]. Defining smaller parts

of the molecule which will induce antibodies against DBL domain may inhibit CSA

binding of the parasite [173].

B-cell memory in malaria

The short-lived nature of immune responses to P. falciparum blood-stage antigens in

malaria endemic regions questions the durability of memory-B cell [174]. A previous

study suggested that B-cell memory is a potential determinant of levels of antibodies to

malarial antigens in many individuals [175] and a relationship between memory-B cell

frequencies and serum antibody levels to P. falciparum extract had been reported [176].

A prior study in children indicates that antibodies against merozoite antigens have very

short half-live [177] and individuals with malaria have profound disturbances in B-cell

homeostasis [178]. Furthermore, a recent study denotes that children most likely have

short-lived plasma cells, thus, experience a rapid decline in antibody levels while older

children have longer-lasting antibody responses that depend on long-lived plasma cells

[179]. When malaria remerged in Madagascar after a long period of complete control,

people present 30 years earlier during malaria exposure were much more resistant to

clinical disease than the children [180]. Data from mice malaria study suggests that B-cell



36

response to a second infection is more efficient than the primary response, generating

long-lived plasma cells making the second encounter with parasite more efficient [181].

Acquired immunity to falciparum malaria is believed to be lost without repeated exposure

[102, 104]. However, repeated exposure to malaria may not result in long-lasting B-cell

memory [175]; perhaps it may result in specific memory-B cells being converted into

effector cells and the persistence of effector mechanisms [182]. The ability of the

immune environment to support long-lived plasma cells may gradually increase with age,

such that older individuals have higher proportion of long-lived plasma cells, enabling

antibodies to be continually produced without restimulation by persistent antigen [179].

Obviously, more studies of memory-B cell responses to human malaria are needed to

elucidate the inadequate understanding of naturally acquired immunity [102, 104].

The haemoglobinopathies

The haemoglobinopathies, disorders of haemoglobin structure production, were the first

genetically determined conditions to be implicated in malaria protection [183]. The

haemoglobinopathies can be divided into two categories: those associated with the

production of structurally variant forms of haemoglobin (HbS, C, E), and those caused by

the reduced production of normal α-and β-globin – the α-thalassaemias and β-

thalassaemias respectively [184-185]. The clinical effects of these conditions are highly

variable.



37

Haemoglobin variant

Haemoglobin S

Sickle-cell disease is a blood disorder characterized by red-blood cells that assume an

abnormal, rigid, sickle shape. Mutation of the β-chain of the globin due to single base

pair gene and its substitution of a single amino acid (Glu→Val) results in reduced

solubility of the deoxyhaemoglobin molecule and erythrocytes assume irregular shapes

[186]. The homozygous (HbSS) are associated with severe sequelae and reduced life

expectancy while the heterozygous have a protective advantage over clinical malaria

episodes [187-190]. The mechanism whereby HbAS mediates protection remains unclear

though studies have described a reduced parasite growth and density [191], enhanced

phagocytosis of ring-parasitized mutant erythrocytes [192], an impaired cytoadherence of

falciparum-infected erythrocytes containing sickle haemoglobin [193] and higher titres of

IgG antibodies [194].

Haemoglobin C

Haemoglobin C is abnormal haemoglobin with substitution of a lysine residue for

glutamic acid residue at the 6th position of the β-globin chain. This mutated form reduces

the normal plasticity of host erythrocytes causing a haemoglobinopathy. Although HbAC

is asymptomatic, HbCC causes mild haemolysis, splenomegaly, or gallstones [195].

Recent studies appear to show selected advantage of homozygous HbCC [195-197].

Modiano et al. showed that the protective effect of HbAC was only 30% as compared to

>90% in HbCC [197]. The extent to which HbC reduces the risk of severe malaria and of

its variable symptoms in areas of different endemicity is yet to be established. However,



38

it is believed that regarding HbC, there is reduced ability of P. falciparum parasites to

grow and multiply in variant RBCs [198] and HbC-containing red-blood cells have

modified displays of malaria surface proteins that reduce parasite adhesiveness which

could reduce the risk of severe disease [199].

Haemoglobin E

Haemoglobin E, resulting from a single point mutation (Glu→Lys) at codon 26 of the -

globin gene, is common in Southeast Asia. Like HbC, the homozygous HbE is relatively

benign. A genetic analysis from Thai population revealed that HbE β26 Glu→lys, the

most frequent variant in Southeast Asia, has reached its current frequency in less than

5000 years [200]. Chotivanich et al. cultured P. falciparum parasites in media containing

various mixtures of normal and variant RBCs at relatively high parasite densities

designed to reflect progression to heavy parasitaemias and severe infection. Only 25% of

HbAE were susceptible to invasion suggesting the possibility that HbAE might protect

against severe malaria by limiting the ability of infections to achieve higher parasite

densities [201].

The thalassaemias

The thalassaemias are classified according to which chain of the haemoglobin molecule is

affected. In α thalassaemias, production of the α globin chain is affected, while in β

thalassaemia production of the β globin chain is affected. Thalassaemia produces a

deficiency of α or β globin. β globin chains are encoded by a single gene on chromosome

11 while α globin chains are encoded by two closely linked genes on chromosome 16.



39

Few studies suggest a protective advantage of α thalassaemia over mild clinical disease

[202-203]. Unlike HbAS, which protects against all forms of disease, the effect of α

thalassaemia appears to be relatively specific for severe malarial anaemia [203-204]. α

thalassaemia is associated with complement receptor 1 (CR1) production [205], and

could protect against malaria anaemia via reduction in CR1-mediated rosetting.

The ABO blood group system

The ABO blood group system is the most mysterious, genetic polymorphism in humans

[206]. Relationship between ABO blood groups and susceptibility to malaria is

controversial due to contradictory results [206-207]. However, parasitized erythrocytes

form rosettes more readily with RBCs of A, B or AB groups than with O group [208-209]

and are associated with severity of clinical disease [210] with the development of

cerebral malaria [211]. Furthermore, recent data reveals that blood group O protects

against severe P. falciparum malaria through the mechanism of reduced rosetting [212].



40

Related background

Epidemiology of malaria in stable and unstable malaria transmissions

The transmission pattern of malaria normally governs the development of clinical

immunity to malaria. In stable malaria endemic area, a high entomological inoculation

rate (EIR) is often associated with increase in the incidence of parasitaemia and fever

[213], and parasitaemia is most common in young children, with the incidence of

parasitaemia decreasing steadily with age. Adults, however, have latent parasitaemia but

hardly develop clinical malaria. In unstable transmission settings, malaria transmission is

very seasonal and fluctuating, while in stable transmission settings it is stable over the

seasons or years. Children less than 5 years and especially primigravidae are known to be

more at risk to malaria disease due to inadequate acquisition of immunity. In stable

endemic areas, the most frequent manifestation of malaria is severe malarial anaemia

[214] which peaks at 6-12 months after trans-placental maternal immunity has faded

while cerebral malaria (the most fatal complication) peaks at 2-3 years, and by 5 years,

malaria morbidity declines [215-216]. In areas of unstable malaria transmission, like in

Sudan, the whole population is highly vulnerable. The mean age for severe malarial

anaemia is 5 years, cerebral malaria is about 14 years and the risk of clinical attack is

twice as high in the 5-20 age group and declines markedly after the age of 20 years [217-

219]. This decline has been suggested to be due to development of partial immunity after

having experienced a number of both symptomatic and asymptomatic infections [218].



41

Ethnicity

Differences in susceptibility to malaria between ethnic groups suggest that host genetic

factors might have a role in resistance/susceptibility to malaria. Different studies have

reported differences in susceptibility to malaria between ethnic groups living in sympatry.

In Malaysia, the Orang Asli (aborigine) showed a lower susceptibility to malaria as

compared to other ethnic groups from the same region [220]. In Nepal, decreased malaria

morbidity in the Tharu people have been reported when compared to their sympatric

populations [221]. In Gambia, the Fulani showed lower incidence of malaria and higher

frequency of splenomegaly when compared to other sympatric groups albeit no apparent

socio-cultural differences and same exposure to malaria [222]. Other studies also

established lower parasite density and prevalence of malaria in the Fulani group [223-

224]. Studies from Burkina Faso, Mali and Sudan have also confirmed significant inter-

ethnic variation in susceptibility to malaria with the Fulani tribe developing less frequent

clinical malaria when compared to other ethnic groups living in sympatry (Mossi and

Rimaibé in Burkina Faso, Dogon in Mali and Masaleit in Sudan) [225-227].

In general, the Fulani have higher anti-malarial antibodies against liver-stage antigens

[228-229], blood-stage antigens [229-230] as well as crude P. falciparum blood-stage

extract [226, 231-232]. Increased numbers of specific IL-4- and IFN-gamma-producing

cells in the Fulani’s have also been suggested as contributing factor to the lower

susceptibility [232]. The mechanism behind the lower susceptibility to malaria by the

Fulani group remains unknown and studies continue to elucidate the unknown immuno-

genetic markers. A study from Sudan revealed a higher frequency of HbAS in non-Fulani



42

(Masaleit) than the Fulani [233], while another study suggests that the higher resistance

to malaria of the Fulani tribe could be derived from the functional deficit T regulatory

cells [234]. However, the low proportion of individuals with known genetic malaria

resistant factors like HbS, HbC, α−thalassaemia, G6PDA, HLAB*5301 in Fulani group

suggests lack of association with classic malaria-resistance genes and protection against

malaria in this group [235].

Antibody reactivities to blood-stage antigens in relation to malaria disease

Several asexual blood-stage antigens have been identified as possible vaccine candidates

(Figure 8) and their recognition in immune sera have been documented. A number of

sero-epidemiological studies has reported differences in IgG subclass patterns to different

asexual blood-stage antigens. MSP-2 is believed to induce very strong IgG3 responses

[236-238] in contrast to MSP-119 which induces IgG1 or mixed IgG1/IgG3 response

[239]. The most abundant IgG subclass responses to MSP-3 were IgG3, followed by

IgG1 and the presence of IgG3 antibodies to MSP-3 is associated with reduced risk of

clinical malaria [240-241], and long-lasting natural protection against malaria [242].

AMA-1 was recognised by predominantly IgG1 subclass antibodies in young children

and adults [243], and high antibody levels (IgG1 and IgG3) against GLURP were

associated with reduction in clinical malaria episode [241, 244-246].

Studies on naturally acquired antibody-mediated immunity to clinical malaria have

largely focused on the presence of antibody responses to individual antigens and their

associations with decreased morbidity. In a cross-sectional study, at peak and end of



43

malaria transmission from two villages with different levels of malaria transmission,

children who were high antibody responders to the selected malaria antigens were at

lower risk of clinical malaria than their counterparts classified as non-responders [247].

In a population of adult individuals living in hyperendemic area of Senegal, no

differences were seen between IgG1 and IgG3 subclasses before the rainy season, but

there were a significant decrease in specific IgG after the rainy season that affected IgG3

more than IgG1 [248]. A recent study on breadth (number of important targets to which

antibodies were made) and magnitude (antibody level measured in a random serum

sample) suggest that immunity to malaria may result from high titres of antibodies to

multiple antigenic targets [249] which was further suggested in another study [250].

However, the fine specificity or function of antibody responses is believed to be more

critical for the protection than the magnitude of antibody responses [251].

FcγRIIa and malaria

The importance of FcγRIIa dimorphism in relation to malaria, which indicates R/H131

polymorphism in regulating the clinical outcome of the disease, has been suggested but

shown with conflicting observations. In the Gambia, the HH131 genotype was

significantly associated with susceptibility to severe malaria while the presence of the

R131 allele, rather than the RR131 genotype, appeared to be an important factor

protecting from malaria disease [252]. Omi et al. found that the FcγRIIa-H/H131

genotype in combination with the FcγRIIIb-NA2 allele were associated with

susceptibility to cerebral malaria in Thai patients [253]. In Kenyan infants, FcγRIIa-

R/R131 carriers were found to be at lower risk to succumb to severe disease than the



44

FcγRIIa-R/H131 carriers [254]. In a cross-sectional study in western Kenya,

homozygotes for the R131 alleles were protected against high-density parasitaemia while

the H131 alleles were mildly protective and the FcγRIIa-131 polymorphism did not

protect against malaria anaemia [255]. Conversely, recent studies have suggested a

protective role of the HH131 genotype rather than the RR131 genotype in clinical malaria

from different malaria endemic regions (Burkina Faso, India and Brazil) probably

through its interaction with IgG2 [142-144].



45

The present study

Aim of the thesis

This study addresses the importance of antibodies in relation to malaria infection and

their effective interactions with FcγRIIa in both symptomatic and asymptomatic

individuals. Papers I and IV focused on the pattern of antibody responses in clinical

malaria while papers II and III centred on FcγRIIa polymorphisms in sympatric ethnic

groups.

The specific objectives were:

• To investigate a possible association between FcγRIIa-R/H131 polymorphism and

anti-malarial antibody responses and clinical outcome of P. falciparum malaria

among Sudanese patients.

• To study the interethnic differences in malaria susceptibility seen between the

Fulani and Dogon in Mali if it could be attributed to their FcγRIIa-R/H131

polymorphism and IgG subclass pattern of anti-malarial antibodies.

• To examine the influence of FcγRIIa-R/H131 polymorphism and IgG subclass

pattern of antibodies among sympatric Fulani and non-Fulani in a dry savannah

area of sub-Saharan Sudan.

• To analyse immunoglobulin G profiles of six leading blood-stage vaccine

candidate antigens in relation to clinical malaria outcome.



46

Sudan

Country profile

Sudan is the largest and one of the most diverse countries in Africa, home to deserts,

mountain ranges, swamps and rain forests. Sudan has a population of 39.4 million with

an area of 2.5 million sq km (966,757 sq miles). The northern part has a desert climate

and rainfall is very infrequent, daytime temperatures are very high. The rainy season

varies between regions; rare in the north and frequent in the south. The human

development report for Sudan is 0.526, which gives the country a rank of 147th out of

177 countries [256]. Male life expectancy is 55.4 years while female is 57.8 years and

adult literacy is 50% [257]. Sudanese children suffer an under-five mortality rate of 112

deaths per 1,000 live births, an infant mortality rate of 81 deaths per 1,000 live births and

a maternal mortality ratio of 1,107 deaths per 100,000 live births [258]. In Sudan, malaria

transmission is very unstable, seasonal and the duration varies between north and south

Sudan (Figure 9). Malaria transmission is low in the north while it is moderate with

seasonal epidemic outbreaks in the south.

Daraweesh project

The Daraweesh project started as a collaboration between the Universities of Khartoum

(Sudan), Edinburgh (UK), Copenhagen (Denmark), and Brigham Young University

(USA) [217]. Daraweesh is situated on the Sudanese savannah, at the altitude of 603 m

above sea level, 16 km from Gedaref town, 80 km from the Ethiopian border and is a

region characterized by seasonal and unstable malaria transmission. The cost-cutting

measure is built on agriculture, and is mostly inhabited by a population of Fulani group



47

originating from Burkina Faso whose relations settled in Sudan over 100 years ago [217].

Daraweesh is characterized by wet and dry periods. The rainy season is short followed by

a long dry season extending over 9 months. Malaria incidences during the dry season are

very rare, while during the wet seasons, 20-40% of the population suffer at least 1

malaria attack. The risk of clinical malaria is much higher in ages 5-20 years than

individuals more than 30 years of age though all age-groups are affected. Daraweesh is a

hypo-endemic malaria area. More details of the demographic and social descriptions of

the study area, epidemiological settings can be found in previous publications [217-219,

259].

Figure 9: Distribution of endemic malaria and duration of transmission season in Sudan. The areas where the studies were conducted are marked with a red circle. Adapted from MARA/ARMA (www.mara.org.za).



48

Mali

Country profile

Mali is the seventh largest country in Africa, bordering Algeria on the north, Niger on the

east, Burkina Faso and the Côte d'Ivoire on the south, Guinea on the south-west, and

Senegal and Mauritania on the west. Mali has a population of 12 million with an area of

1,240,000 km². Mali is one of the world’s poorest nations rated 173 out of the 177

countries. The under 5 mortality rate is 219 per 1, 000 live births, maternal mortality ratio

is 1200 deaths per 100, 000 live births and the infant mortality rate is 121 deaths per 1,

000 live births. The human development report for Mali is 0.391, which gives the country

a rank of 168th out of 179 countries with data [260]. Male life expectancy is 45 years

while female is 48 years and adult literacy is 24% [261]. Mali experiences three types of

malaria transmission each year: (i) 6 months of seasonal transmission in the south; (ii) 3

months of transmission in the Sahelian area; and (iii) irregular transmission with

epidemics in the north (Figure 10).

Study area

The study area is located in the Mopti area about 850 km northeast of Bamako, the

capital of Mali. Details of the study area, epidemiological settings can be found in

previous publications [226, 231-232]. The study took place in four rural villages

(Mantéourou, Naye, Binédama, and Anakédié) where Fulani and Dogon ethnic groups

live in sympatry. The Mantéourou and Naye are divided into two subdivisions,

Mantéourou Peulh/Mantéourou Dogon and Naye Peulh/Naye Dogon-Dinsogou. The two

subdivisions are separated by 300–500 meters and inhabited either by Fulani or Dogon.



49

The other two villages, Binédama and Anakédié, are exclusively populated by either

Fulani or Dogon, respectively, and are located approximately 1 km apart. Malaria

transmission is meso-endemic, with P. falciparum as the main cause of infection.

Transmission is seasonal and EIR of 0.38 infective bites per person per night has been

previously reported [226].

Figure 10: Distribution of endemic malaria and duration of transmission season in Mali. The area where the study was conducted is marked with a red circle. Adapted from MARA/ARMA (www.mara.org.za).



50

Methodology

The materials and methods, briefly described below, are properly described accordingly

in the respective papers.

Paper I

A total of 256 individuals, comprising 115 patients with severe malaria, 85 with mild

malaria and 56 malaria-free controls, were enrolled in a prospective clinical study over

two successive malarial transmission seasons. Human genomic DNA was purified using a

commercial DNA purification kit QIAmp® DNA blood mini kit and polymerase-chain

reaction (PCR) was performed for FcγRIIa-R/H131 polymorphism. Plasma samples were

tested for malaria-specific IgE, IgM, IgG and IgG subclasses using ELISA.

Paper II

Randomly selected asymptomatic Fulani and Dogon (164 respectively) were included in

this study. Human genomic DNA was extracted from filter papers using Chelex-100 and

PCR was performed for FcγRIIa-R/H131 polymorphism. ELISA was used to determine

total IgG subclasses and malaria-specific IgG subclasses.

Paper III

Two hundred and fifty one Fulani and 101 non-Fulani were genotyped for FcγRIIa-

R/H131 polymorphism. Plasma antibodies to four malaria merozoites (AMA-1, MSP-

23D7 and FC27, Pf332-C231) were measured using ELISA.



51

Paper IV

Five hundred and fourty five individuals, aged 6 months to 65 years, were consecutively

enrolled in the study. Plasma levels of IgG and IgG subclases against six malaria antigens

(AMA-1, 3D7 MSP-2, FC27 MSP-2, MSP-3, GLURP-R0 and GLURP-R2) were

determined by ELISA.

Ethical permissions

The studies received ethical clearance from the ethical committees at the Faculty of

Medicine, University of Khartoum, Sudan, and the National Research Directorate of the

Sudanese Federal Ministry of Health, ethical committee of the Faculty of Medicine and

Pharmacy of Mali and the ethical committee of Karolinska Institute, Sweden.



52

Results and discussion

FcγRIIa polymorphism and antibody responses in clinical malaria outcome (Paper I)

Fc receptors are expressed on the surface of all types of cells of the immune system. They

bind the Fc portion of Ig, thereby bridging specific antigen recognition by antibodies with

cellular effector mechanisms. FcγRIIa (the low affinity receptor), one of the three

receptors for human IgG, is believed to play a major role in eliciting monocyte- and

macrophage-mediated effector responses against blood-stage malaria parasites. The

importance of FcγRIIa dimorphism in relation to malaria, which indicates R/H131

polymorphism in regulating the clinical outcome of the disease, has been shown with

conflicting observations (reviewed by Braga et al. [262]).

In this study, we investigated a possible association between the FcγRIIa-R/H131

polymorphism and anti-malarial antibody responses with clinical outcome of P.

falciparum malaria among Sudanese patients. Our data showed that the frequency of

FcγRIIa-R/R131 genotype was significantly associated with severe malaria when

compared with patients with mild malaria. Furthermore, the frequency of FcγRIIa-

H/H131 genotype was statistically significantly associated with mild malaria, suggesting

that the FcγRIIa-H/H131 genotype might be associated with a reduced risk of severe

malaria, or possibly have a protective role against severe malaria. Recent data from

different endemic regions have also confirmed this result [142-144]. However, our data

are in contrast to those of earlier studies [252-254] which showed an association between

FcγRIIa-H/H131 genotype and susceptibility to malaria. These discrepant results may

most likely be explained by genetic differences between the populations studied, as well



53

as differences in gene–environment interactions and differences in patterns of malaria

transmission in the different study areas.

Cytophilic antibodies (IgG1 and IgG3) are believed to be influential in protective

immunity against malaria while non-cytophilic antibodies (IgG2 and IgG4) compete

against the available epitopes recognised by the cytophilics [136, 145]. Evidence showed

that in clinically protected individuals, cytophilic antibodies are the most dominant [136].

Sero-epidemiological studies from hyper- and holoendemic regions have also confirmed

this association [136-138]. Our data, from a meso-endemic setting, are in line with

previous data [136-141] regarding the association between cytophilic antibodies and

protection against natural exposure to P. falciparum malaria. While the FcγRIIa-R131

molecule shows a similar binding of the cytophilic subclasses [263], the H131 receptor

which binds IgG3 more efficiently, is the only FcγR that binds IgG2 efficiently [65, 263-

264]. We showed that IgG3 antibodies were more associated with reduced risk of severe

malaria in individuals with H/H131 genotype than in individuals with R/R131 genotype.

We also found an association with low level of IgG2, although it was weak taking into

account confounding factors such as sex and age. The importance of FcγRIIa in parasite

neutralizing events mediated by monocytes was indicated by Tebo et al. using THP-1

cells transfected with the 131H or 131R allele of the receptor [265]. The cells expressing

the 131R allele showed enhanced phagocytosis of P. falciparum infected erythrocytes

with sera containing high levels of IgG1 antibodies, while phagocytosis by cells

expressing the 131H receptor showed preference for sera containing high IgG3 antibody

levels. In antibody-dependent cell-mediated inhibition of P. falciparum growth in vitro,



54

the optimal parasite neutralizing activity by human monocytes was dependent on the

cooperation between FcγRIIa and FcγRIII receptors and only cytophilic IgGs were

potent, in agreement with immunoepidemiological findings [266].

Taken together, our study signified that the H/H131 genotype, which binds IgG3 more

efficiently, is less likely to be associated with severe malaria, while the R/R131 genotype

was associated with severe malaria in this population. The ability to develop clinical

immunity, however, appears to correlate much stronger with cytophilic antibodies

suggesting that these antibodies may play significant roles in parasite-neutralizing

immune mechanisms.

FcγRIIa polymorphism and IgG subclasses in relation to malaria (Paper II)

Studies have reported differences in susceptibility to malaria between ethnic groups

living in sympatry [220-227]. In Gambia, the Fulani showed lower incidence of malaria

and higher frequency of splenomegaly rates [222]. Studies in Burkina Faso extended the

results implying that Fulani are less parasitized, less affected by malaria and have higher

anti-malarial immune responses than the sympatric Mossi and Rimaibé ethnic groups

[229]. A study of the Fulani ethnic group in Mali also has confirmed this similar result

[226] and this relative resistance seems to be pathogen related and not a general hyper-

reactive immune system [231]. It was expected that the conventional haemoglobinopathy

resistance factors will be high in Fulani but the reverse was found [235]. In view of these

differences, we investigated the possible impact of FcγRIIa polymorphism in the ethnic



55

differences in malaria susceptibility seen between Fulani and Dogon in Mali and also

analysed both their total and malaria reactive IgG subclass profiles.

Our data showed a statistically significant difference with regard to the frequency of

homozygotes carrying the R/R131 genotype, which dominated in the Dogon group, while

the homozygotes R/R131 and H/H131 genotypes were evenly distributed in the Fulani

group. The Fulani also showed significantly higher levels of anti-malarial IgG1, IgG2 and

IgG3 antibody subclasses when compared with the Dogon. However, in some Fulani

individuals, a higher proportion of IgG2 over IgG3 was seen. In the Fulani, the frequency

of the H/H131 genotype is common and H-allele carriers had higher anti-malarial IgG2

levels than R/R homozygotes. In line with this finding, the Fulani ethnic group in Sudan

have been shown to have higher frequencies of both H/H131 genotype and H131 allele

than the sympatric Masaleit [233], and other studies suggested that the H/H131 genotype

might contribute to this relative protection from malaria [142-144].

In conclusion, our results suggest that the FcγRIIa-H131 allele may be associated with

the lower susceptibility to malaria seen in the Fulani group, and that anti-malarial IgG2

antibodies may be important in this context.

FcγRIIa polymorphism on the IgG subclass patterns of antibodies (Paper III)

In view of understanding the differences seen in Fulani when compared with other ethnic

groups, we analysed the influence of FcγRIIa-R/H131 polymorphism on the IgG subclass

patterns of antibodies to four malaria antigens (AMA-1, 3D7 MSP-2, FC27 MSP-2 and



56

Pf332-C231) in the Fulani and their sympatric non-Fulani ethnic groups (Masaleit, Hausa

and Four) in eastern Sudan. Our results show that FcγRIIa-H/H131 genotype and H131

allele were significantly more prevalent in the Fulani as compared to the non-Fulani

ethnic groups. In contrast to our previous findings in Burkina Faso and Mali, the Fulani

in Sudan showed lower anti-malarial IgG1 and IgG3 antibody levels, but higher levels of

IgG2 subclass antibodies. These discrepant results seen in Sudan may most likely be

explained by differences in patterns of malaria transmission in the different study areas

(Burkina Faso, Mali and Sudan).

Sudan is a country characterized by hypo/meso-endemicity, with seasonal and unstable

malaria transmission, unlike Burkina Faso and Mali, characterized by hyper/holo-

endemic malaria transmission. In general, the serum levels of IgG1 and IgG3 subclass

antibodies were found to be at higher levels than IgG2 and IgG4 antibodies in both ethnic

groups. However, the antibody levels varied between the different antigens. In line with

our previous finding in Mali (Paper II), the Fulani showed a higher frequency of the

H131 allele and higher levels of anti-malarial IgG2 antibodies than the sympatric non-

Fulani ethnic groups.

In the presence of the FcγRIIa-H131 receptor, IgG2 antibody is essential for effective

IgG2-mediated cellular activation. We found that higher levels of IgG2 and IgG3

antibodies were associated with the H/H genotype, while higher levels of IgG1 antibodies

were associated with the R/R131 genotype. However, the higher IgG2 antibody levels

seen in these Fulani individuals may be due to the relatively high prevalence for the



57

H/H131 genotype in this population. Additional studies are further needed to explicate

this.

In brief, we showed that the FcγRIIa-H/H131 genotype and H131 allele is at higher

frequency in the Fulani ethnic group and the H/H131 genotype was consistently

associated with higher levels of anti-malarial IgG2 and IgG3 antibodies, while the

R/R131 genotype was associated with higher levels of IgG1 antibodies.

IgG subclass responses to different asexual blood-stage malaria antigens (Paper IV)

Evidence shows that antibodies to blood-stage antigens of P. falciparum play a role in

protection from malaria, although the precise targets and mechanisms mediating

immunity remain unclear. In this study, we have analysed antibody responses to a panel

of six blood-stage antigens in relation to clinical malaria outcome in a hospital-based

study in Sudan. We focused our analysis on asexual blood-stage antigens namely; AMA-

1, 3D7 and FC27 alleles of MSP-2, MSP-3, R0 and R2 regions of GLURP based on their

association with protection in both pre-clinical and clinical studies. Our results show that

the pattern of the IgG subclasses for the different antigens varied but IgG1 and IgG3

antibody subclasses were the most prominent. AMA-1 antibodies were predominately of

the IgG1 subclass while both alleles of MSP-2, MSP-3 were biased for IgG3. GLURP

showed a bias for the IgG3 subclass especially in individuals above 5 years of age. The

dominance of IgG3 antibodies in individuals above 5 years was observed with MSP-2,

MSP-3 and GLURP.



58

To demonstrate the characteristics of our associations, we divided the distributions into

groups by ranking them into fifths of the distribution. All measures were coded as series

of binary dummy variable, including age. In other to maximise power, we examined the

linear association by modelling the antibody distributions as ordinal variables based on

the fifths of the distributions. Our results revealed a linear association with anti-AMA-1

IgG1 antibodies and the linearity was more pronounced in the combined final model.

However, in individuals above 5 years, a non-linear (inverted U-shaped) relationship with

IgG3 antibodies was observed for MSP-2, MSP-3 and GLURP. Furthermore, in the

combined final model, the highest levels of IgG1 subclass antibodies to AMA-1,

GLURP-R0, and the highest levels of IgG3 subclass antibodies reactive to 3D7 MSP-2

were independently associated with a reduced risk of clinical malaria both before and

after adjustment for confounding factors. For reasons not fully understood, the different

merozoite antigens induced different relative levels of IgG1 and IgG3 antibodies.

Probably, the intrinsic properties of the antigen itself and/or age (in the context of our

results) may play a major role in determining the subclass of the antibody response.

Antibodies have the capacity to bind antigens through its Fab part, but its effector role is

mediated by the antibody’s Fc portion [267-268]. IgG1 and IgG3 subclass antibodies are

the key components of FcγR-mediated effector responses, and they have the ability to

neutralise pathogens at ports of entry into the body [145]. It has been suggested that IgG3

is more efficient at mediating these processes [266, 269]. Malaria specific IgG1 and IgG3

isotypes are transferred to the offspring more often and more efficiently than IgG2 and

IgG4 [270]. Furthermore, individuals with no detectable level of malaria specific IgG3



59

antibodies had bleak prognostic chance of recovery from severe malaria [271] and in

patients with the most severe form of malaria, a very low level of antibodies were

detected [272]. Evidence also shows a protective role of high levels of IgG3 antibodies in

uninfected primigravidae without placental malaria infection at enrolment and delivery

[273]. Antibodies to merozoite antigens are thought to function in vivo by inhibition of

merozoite invasion of erythrocytes, opsonisation of merozoites for phagocytosis, ADCI

[118, 122, 124, 274-275]. The subclass of antibodies produced against antigens is

probably very important as IgG subclasses differ in their structures and mediate different

immune effector functions [61, 276]. High titres of antibodies to the combinations of

merozoite antigens were reported to be associated with protection from clinical malaria

[249] and enhance the likelihood of therapeutic success in malaria patients [277].

Furthermore, IgG subclass specific responses against merozoite antigens are associated

with protection from symptomatic malaria and high-density parasitaemia [278], while

anti-VSA antibodies were associated with acquiring asymptomatic infection rather than

febrile malaria [279]. However, while antibodies to AMA-1 may directly inhibit the

merozoite invasion of erythrocytes [131], parasite neutralization by antibodies to MSP-3

and GLURP is dependent on their interaction with Fcγ receptors on monocytes or other

effector cells [157-158, 241, 274]. Recently, antibodies to another merozoite antigen

(MSP-1) were also able to inhibit in vitro growth of P. falciparum only in cooperation

with human monocytes [280].

In conclusion, this study provides further evidence of the potential importance of the

studied merozoite antigens as targets for parasite neutralising antibody responses of the



60

IgG1 and IgG3 subclasses. The results further indicated that for most of the antigens,

IgG3 antibodies, with their shorter half-life, may be more efficient than IgG1 in limiting

parasitaemia in individuals with clinical malaria, probably due to its structurally

biological function.



61

Concluding remarks and future perspectives

This study aimed at elucidating some probable mechanisms by which antibodies mediate

their effective functions in malaria infection. We have studied their interactions with

FcγRIIa genotype in both malaria patients and asymptomatic individuals (individuals

with or without malaria parasites but no malaria) from different endemic settings.

The data from this thesis concludes that FcγRIIa-R/H131 polymorphism may play a role

in regulating the clinical outcome of malaria disease. In the case of severe malaria, we

found that the R/R131 genotype was significantly associated with susceptibility to severe

malaria, while the presence of H131 allele and H/H131 genotype, which binds IgG3

antibodies more efficiently, appeared to be less likely to be associated with severe

malaria. The results also indicated that the Fulani ethnic group, who are less susceptible

to malaria, have higher spleen rate, lower parasite prevalence, have a higher frequency of

the FcγRIIa-H/H131-and H131 allele carriers.

Binding of antibodies to infected RBCs is believed to evoke their opsonisation involving

mainly the cytophilic antibodies (Figure 11). IgG1 and IgG3 antibodies, however, have

high affinity for Fc receptors on phagocytic cells. Our data indicated that the levels of

IgG1 and IgG3 antibodies were statistically significantly higher in mild (uncomplicated)

malaria patients when compared with the severe malaria patients. Our results also showed

that Fulani in Mali had significantly higher levels of IgG1 and IgG3 subclass antibodies.

However, the Fulani in Sudan had lower levels of IgG1 and IgG3 antibodies which could

be attributed to differences in malaria endemicity. Interestingly, both the Fulani in Mali



62

and Sudan had significant higher levels of IgG2 antibodies when compared with their

sympatric groups. In individuals carrying H/H131, these results suggest that the

protective role of IgG2 and IgG3 antibodies in malaria infection might be through the

activation of FcγRIIa-H131 (Figure 11). Furthermore, our data showed that the higher

IgG2 subclass antibody levels seen in Fulani group may be due to the relatively high

prevalence of H/H131 in this ethnic group.

High titres of antibodies to the combination of merozoite antigens are thought to be

associated with protection from clinical malaria. However, the magnitude of anti-malarial

responses may not necessarily be the key element involved in protection but the antibody

specificity and biological function. To our studied antigens (paper IV), we found a bias

towards IgG3 antibodies which dominated in individuals above 5 years of age. Although

IgG3 has a very short half-life, continuous exposure to malaria infection (especially in

adults) may maintain its circulation leading to complement-mediated lysis of infected

RBC.

Future prospects would include longitudinal studies, follow-up studies and case-control

studies to evaluate the influence of FcγRs during malaria infections. Consecutive

antibody responses during these studies might also facilitate the understanding of its

functional mechanisms in malaria outcome. Functional experiments with FcγR allotypes

on the circulating levels of antibody subclasses would be interesting to investigate in

view of elucidating mechanisms of susceptibility/resistance to malaria.



63

Figure 11. Probable mechanisms of antibody-mediated clearance of infected erythrocytes. (a) Antibodies can bind to free merozoites or bind to antigens on the surface of infected erythrocytes, thereby inhibit merozoite entry into the erythrocyte by complement-mediated lysis; (b) Antibodies can bind to monocytes/macrophages and optimise parasite killing through the release of soluble factors (TNF-α, NO) upon uptake of opsonised merozoites; (c) IgG2 can participate in parasite clearance in individuals with higher frequency of FcγRIIa-H131 that bind IgG2 along with IgG3 subclass antibodies.



64

Acknowledgements

The journey has been tough, knotty and strenuous. For years, I wondered how it would

come to an end with little or no resources to help maximise my potentials. I would like to

express my heartfelt gratitude to everyone who, one way or the other, have contributed

immeasurably in making this thesis a reality.

Klavs Berzins, my supervisor, for the opportunity to work with you and your advice,

constructive criticisms and suggestions. Thank you for sharing your vast knowledge with

me, nurturing my ideas and interests, and the interesting discussions we always have.

Marita Troye-Blomberg, the Head of Department and my second supervisor, for the

conducive intellectual micro-environment existing at the department. Tack så mycket!

The seniors at the department; Eva Severinson, Carmen Fernández, and Eva

Sverremark-Ekström. Thank you for the favourable environment for scientific

interactions.

I am indebted to my parents, Jonathan Iriemenam Okonkwo and Monica Chinwa

Okonkwo who fought very hard in making sure that every child of the family received

the priceless legacy – education. I am very proud of you all!!! My younger brothers,

Chukwudi, Onyekachukwu, Ginikanwa, Amaobichukwu and my only sister Adaobi,

for your continued support, encouragement, kind-heartedness and prayers. I cannot thank

you enough! Many thanks to Osita Chukwuma, Elonna Okonkwo, Nwanneka



65

Umeigbo, Richard Uche and Immaculata Uche for your love, understanding, support,

advice and encouragement. Ka onye nwe anyị bi n’igwe gọzie ụnụ niile mmaji pụrụ

mmaji!

Dr. Grace Adeoye who first introduced me to the field of Parasitology and Immunology

and Dr. Olubunmi Otubanjo who fostered the interest I have in medical research.

Professor Adetayo Fagbenro-Beyioku and Dr. Wellington Oyibo for the invaluable

support, advice, encouragement and help anytime it is needed.

Hedvig Perlmann, Margareta Hagstedt (Maggan) and Ann Sjölund for your

technical support whenever I had difficulties with practical issues. I am very grateful.

Gelana Yadeta and Anna-Leena Jarva for your superb administrative services that

helps the department to run smoothly without hitches. Keep it up!

I also appreciate the technical support I received from my friends Amre Nasr, Halima

Balogun, Dr. Salah Farouk, and Dr. Gehad ElGhazali.

Adeola Aderounmu who first introduced me to my supervisor and helped me at the

initial stage of my arrival in Sweden. Thank you so much for your wonderful support and

encouragement! It is very much appreciated.



66

I am very grateful to the patients, Daraweesh community, volunteers and participants in

the study areas for their support. This study is a success because of you. The staff at New

Halfa Teaching Hospital, Halfa Sugar Factory, Gedaref Teaching Hospital, El-Obied

Teaching Hospital, are appreciated for their excellent assistance in the field and

laboratory.

Past and present colleagues at the Department of Immunology – Magdi M. Ali, John

Arko-Mensah, Ahmed Bolad, Salah Eldin Farouk, Jacob Minang, Alice Nyakeriga,

Valentina Mangano, Anna Tjärnlund, Khaleda Qazi Rahman, Manijeh Vafa

Homann, Nina-Maria Vasconcelos, Yvonne Sundström, Nora Bachmayer, Petra

Amoudruz, Amre Nasr, Elisabeth Israelsson, Halima Balogun, Nancy Awah, Pablo

Giusti, Jubayer Rahman, Ylva Sjögren, Stefania Varani, Shanie Saghafian-

Hedengren, Olivia Simone, Charles Arama, Maria Johansson, Luis Fernando

Tordoya, Bakary Maiga, Judith Anchang, Ebba Sohlberg, Christian Okafor, Olga

Flores, Jacqueline Calla, Gishanti Arambepola, Nicky Thrupp, Stephanie Boström

and Shahid Waseem for making the department a pleasant place for research. Thank you

for your friendship.

To all my wonderful friends in Sweden; Newlife, Newlife International Cell Group,

Newlife Worship Team, African Ministry, African Gospel Mass Choir (All Nations),

Benjamin Benson, Margaret Benson, John van Dinther, Dr. Diddy Antai, Samuel

Nweze, Mukasa’s family, Jeffrey Idumu, Evelyn Nde, Daniel Ekerieke, Jae Sung

Kim, Gawa Bidla, Adeola Aderounmu, Amani Kheir, Ernest Ampadu, Saliu



67

Amedu, Abbas Owoade, Ronke Adedeji, Divine Eke, Yetunde Amao, Ademola

Johnson and Ikechukwu Ohajekwe. Your friendship is highly treasured!

Finally, I cannot describe the joy and peace I have with my family. Chizube and

Chikamdabere, your smiles, encouragement, support and love keep me strong each day.

Thank you so much for your perfect understanding! Ọ bụ ụnụ n’enye m obi ụtọ ụbọchị ọ

bụla.

These studies were funded by ICSC World Laboratory, UNESCO-IUMS-SGM, Swedish Institute,

SIDA/SAREC and BioMalPar European Network of Excellence.



68

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