human genetic factors involved in immunity to198584/fulltext01.pdf · human genetic factors...

Doctoral thesis from the Department of Immunology,

The Wenner-Gren Institute, Stockholm University, Sweden

Human genetic factors involved in immunity to

Plasmodium falciparum infection

Manijeh Vafa Homann

Stockholm 2008

Human genetic factors involved in immunity to Plasmodium falciparum infection

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All previously published papers were reproduced with permission from the publishers. © Manijeh Vafa Homann, Stockholm 2008 ISBN 978-91-7155-614-1 Cover illustration: Palle Ryde Printed in Sweden by Universitetsservice AB, Stockholm 2008 Distributer: Stockholm University Library

Manijeh Vafa Homann iii

To my parents, Mohammad Esmael Vafa and Sara Madadi

And to my husband, Peter Homann


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To be conscious that you are ignorant is a great step of knowledge.

Aristotle (384-322 BC)

Manijeh Vafa Homann v

SUMMARY

Development of clinical immunity can be influenced by host genetic factors. Some genetic disorders in erythrocytes have been shown to confer protection against malaria. Inter-ethnic differences in malaria susceptibility offer an approach to identify genes involved in immunity to malaria. The Fulani ethnic group, living in several African countries, has been shown to be more immunologically responsive to, and less frequently infected by Plasmodium falciparum, than their sympatric ethnic groups. Fewer malaria attacks and higher spleen rates have also been seen in the Fulani compared to the non-Fulani. IL-4 and IL-10 are two cytokines with crucial roles in the regulation of both inflammatory- and antibody responses. IL-4 -590 C/T and IL-10 -1087 A/G single nucleotide polymorphisms (SNP) have been shown to result in variable production of the respective cytokine.

This study investigates; the associations between the above mentioned polymorphisms and malariometric indexes in the Fulani and the Dogon in Mali; the correlations between malaria-specific IgG, IgG1-4, IgE, as well as total IgE and parasite prevalence, multiplicity of malaria infection as well as splenomegaly; and the impact of IL-4 -590 variants on the levels of the studied antibodies.

The allele and genotype frequencies of both studied SNPs differed significantly between the two groups. The Fulani IL-4 T allele carriers had a significantly higher infection prevalence compared with those carrying the CC genotype. No correlation between anti-malarial antibody levels and parasite prevalence was seen in any of the communities. In the Fulani, the increase in total IgE levels was related to the presence of infection. Malaria-specific IgG4 levels were negatively correlated to the number of clones within the Fulani. The Fulani IL-4 T allele carriers had higher total and malaria-specific IgE levels, compared to the CC genotype carriers.

Taken together, these results suggest that the amount of antibodies may not be the key element in the protection against malaria. Rather, specificity, function or affinity might be of more importance in this context. IgG4 might be involved in protection against malaria. The impact of IL-4 -590 variants on the antibody levels may be affected by other genetic/epigenetic/epistatic or environmental factors. The role of IgE in malaria remains to be clarified. Multiplicity of infection (MOI) is the number of various P. falciparum strains co-infecting a single individual. MOI has been shown to be influenced by age, seasonal variation and transmission intensity. Both negative and positive effects of MOI on the risk of clinical malaria have been reported. We investigated the impact of erythrocyte variants on the MOI, the correlation between MOI and malaria morbidity, and association between MOI and age, parasite density and aslo seasonal variation. MOI increased after the transmission season in all subjects, except in α-thalassaemic and in G6PD-mutated children. MOI was positively correlated to parasitemia, and did not vary over age (in the range of 2 to 10 years). No relation between MOI and clinical attack was noted.

These data suggest that α-thalassaemia may protect against infection by certain parasite strains. G6PD-mutated individuals may resist against increase in MOI after the transmission season due to rapid clearance of infection at an early stage. Furthermore HbAs and the ABO system may have an impact on protection against severe disease, but do not seem to affect MOI in asymptomatic individuals. Further studies are required to confirm the age-independency of MOI and to better clarify the relations between MOI and clinical attacks.


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ORIGINAL ARTICLES

This doctoral thesis is based on the following original papers, which in the text, are referred to

by their roman numerals.

I. Manijeh Vafa, Bakary Maiga, Klavs Berzins, Masashi Hayano, Sandor Bereczky, Amagana Dolo, Modibo Daou, Charles Arama, Bourema Kouriba, Anna Färnert, Ogobara K. Doumbo and Marita Troye-Blomberg, Associations between the IL-4 -590 T allele and Plasmodium falciparum infection prevalence in asymptomatic Fulani of Mali. Microbes Infect. 2007; 9: 1043-8

II. Manijeh Vafa, Elisabeth Israelsson, Bakary Maiga, Amagana Dolo, Ogobara K. Doumbo and Marita Troye-Blomberg. Relationship between immunoglobulin isotype response to Plasmodium falciparum blood stage antigens and parasitological indexes as well as splenomegaly in sympatric ethnic groups living in Mali. Acta Tropica, under revision

III. Manijeh Vafa, Elisabeth Israelsson, Bakary Maiga, Amagana Dolo, Ogobara K. Doumbo and Marita Troye-Blomberg. Impact of the IL-4 -590 C/T transition on the levels of Plasmodium falciparum specific IgE, IgG, IgG subclasses and total IgE in two sympatric ethnic groups living in Mali. Manuscript

IV. Manijeh Vafa, Marita Troye-Blomberg, Judith Anchang, Andre Garcia and Florence Migot-Nobias, Multiplicity of Plasmodium falciparum infection in asymptomatic children in Senegal: relation to transmission, age and erythrocyte variants. Malaria Journal 2008; 7:17

Manijeh Vafa Homann vii

TABLE OF CONTENTS

SUMMARY ................................................................................................................................................................................... V ORIGINAL ARTICLES ............................................................................................................................................................ VI ABBREVIATIONS ....................................................................................................................................................................... 1 INTRODUCTION ......................................................................................................................................................................... 3

THE IMMUNE SYSTEM ............................................................................................................................................. 3 Innate immunity ................................................................................................................................................ 3

Cells of the innate immune system ................................................................................................................................ 4 Acquired immunity ............................................................................................................................................ 5

T lymphocytes ................................................................................................................................................................ 5 B lymphocytes ................................................................................................................................................................ 8 Antibodies and Ig class switching .................................................................................................................................. 9 FcRs .............................................................................................................................................................................. 11

Cytokines ......................................................................................................................................................... 11 IFN-γ ............................................................................................................................................................................. 12 IL-4 ............................................................................................................................................................................... 12 IL-10 ............................................................................................................................................................................. 13

MALARIA .............................................................................................................................................................. 13 Life cycle of malaria parasite ......................................................................................................................... 14 Clinical features of malaria infection ............................................................................................................ 15

IMMUNE RESPONSE TO MALARIA .......................................................................................................................... 16 Clinical immunity ............................................................................................................................................ 16 Innate immunity to blood stages ..................................................................................................................... 16 Acquired immunity to blood stages ................................................................................................................ 18

Role of antibodies in P. falciparum immunity ............................................................................................................ 18 CD4+ T lymphocytes .................................................................................................................................................... 19

Cytokines ......................................................................................................................................................... 20 RELATED BACKGROUND ..................................................................................................................................................... 22

HUMAN GENETIC FACTORS AND MALARIA IMMUNITY ......................................................................................... 22 RBC variants ................................................................................................................................................... 22

The ABO blood group system ..................................................................................................................................... 22 The haemoglobinopathies ............................................................................................................................................ 23 G6PD deficiency .......................................................................................................................................................... 25 Others ............................................................................................................................................................................ 25

Human leukocyte antigen (HLA) .................................................................................................................... 25 Ethnicity .......................................................................................................................................................... 26 FcγR gene polymorphisms .............................................................................................................................. 27 Cytokine gene polymorphisms ........................................................................................................................ 28

GENETIC DIVERSITY OF MALARIA PARASITE ........................................................................................................ 28 Multiplicity of malaria infection ..................................................................................................................... 29 MSP2 ............................................................................................................................................................... 30

PRESENT STUDY ...................................................................................................................................................................... 31 AIM OF STUDY ...................................................................................................................................................... 31 METHODOLOGY .................................................................................................................................................... 32 RESULTS AND DISCUSSIONS .................................................................................................................................. 33

Paper I ............................................................................................................................................................. 33 Paper II ........................................................................................................................................................... 34 Paper III .......................................................................................................................................................... 36 Paper IV .......................................................................................................................................................... 38

CONCLUDING REMARKS AND FUTURE PERSPECTIVES .......................................................................................... 40 ACKNOWLEDGMENT ............................................................................................................................................................. 42 REFERENCES ............................................................................................................................................................................ 44


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Manijeh Vafa Homann 1

ABBREVIATIONS

ADCC Antibody-dependent cellular cytotoxicity ADCI Antibody-dependent cellular inhibition APC Antigen-presenting cell BCR B-cell receptor CM Cerebral malaria CR1 Complement receptor 1 CSR Class switching recombination CTLA4 Cytotoxic T lymphocyte-associated antigen-4 DC Dendritic cell DP Double positive ELISA Enzyme-linked immunosorbent assay FcR Fc receptor Foxp3 Forkhead box P3 G6PD Glucose-6-phosphate dehydrogenase GPI Glycosylphosphatidylinositol Hb Haemoglobin HC Heavy chain HLA Human leukocyte antigen IFN Interferon Ig Immunoglobulin IL Interleukin iRBC Infected-Red-Blood Cell IRF-1 Interferon-regulatory factor-1 LC Light chain MHC Major-histocompatibility complex MOI Multiplicity of infection MSA Merozoite-surface antigen MSP Merozoite-surface protein NK Natural killer NKR NK receptors NO Nitric oxide PAMPs Pathogen-associated molecular pattern PCR Polymerase-chain reaction PfEMP1 P. falciparum-erythrocyte membrane protein 1 PfRBCs P. falciparum-infected red-blood cells PRRs Pattern-recognition receptors RBCs Red-blood cells SNP Single-nucleotide polymorphism STAT Signal transduction and activator of transcription Tc T cytotoxic TCR T-cell receptor TGF Transforming-growth factor Th T helper TLR Toll-like receptor TNF Tumor-necrosis factor Treg Regulatory T-cell


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INTRODUCTION

The immune system

The immune system consists of a substantial number of components and

mechanisms by which humans are protected from invaders. It detects a wide variety of

foreign agents and discriminates them from self components. The immune system is branched

into two arms, the innate and the acquired immune system.

Innate immunity

The innate immune system provides an immediate defence against infections

and has been found in almost all multicellular plants and animals. The innate immune system

consists of physical, chemical and cellular barriers. The invaders that manage to pass the non-

specific host anatomical barriers, encounter a number of cells, with variable capacities in

defence mechanisms, such as neutrophils, macrophages, dendritic cells (DCs) and natural

killer cells (NK).

The innate immune system detects infections through an array of soluble and

membrane bound sensors called pattern-recognition receptors (PRRs). PRRs recognize

conserved microbial components, which are usually crucial for microbial survival, known as

pathogen-associated molecular patterns (PAMPs). The scavenger receptors (SRs) are

members of the PRRs family, present on macrophages, DCs and certain epithelial cells. SRs

not only recognize different microbial ligands, but also detect endogenous and modified host-

derived molecules [1]. Scavenger receptors are structurally diverse and accordingly

categorized into eight classes (A to H) [2]. Pathogens evading from extra-cellular recognition,

face another line of detection in the host cytosol. Nucleotide binding and oligomerization

domains (NOD)-like receptors (NLRs) and retionid acid-inducable gene I (RIG-I)-like

receptors are two recently identified cytosolic PRR that recognize PAMPs in the intracellular

compartments [3]. Toll-like receptors (TLRs) are the most studied PRRs. They recognize a

wide range of microbial motifs at the cell surface or within the endosomes. TLRs are highly

expressed by macrophages, DCs and B cells. Hence they also play an important role in the

induction of the adaptive immune response. Eleven TLRs have been identified in humans


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until now. Based on the cellular localization, TLRs are classified into two groups. TLR 3, 7, 8

and 9 are expressed in endosomes, while the second group, including TLR1, 2, 4, 5 and 6, are

present on the surface of many different cells [4, 5]. Complement proteins, mannose-binding

lectin (MBL) and C-reactive protein (CRP) are other members of the PRR family. Once PRRs

detect PAMPs, multiple pro-inflammatory signalling pathways are activated and an effective

response against the intruder is mounted [3].

Cells of the innate immune system

Macrophages and DCs take up, degrade and present antigens, associated with

major histocompatibility (MHC) molecules, on their surface to activate the cells of the

acquired immune system. Thus these cells are also called antigen presenting cells (APCs).

MHC molecules are glycoproteins found on the surface of cells and are categorized into class

I and II. Class I MHC is expressed by almost all nucleated cells, while MHC class II is

expressed by professional antigen-presenting cells (i.e. macrophages, DCs and B cells).

DCs are leukocytes specialized in presenting antigens. They can activate naïve T

and B cells, and thus link the innate and the adaptive immune responses. Immature DCs are

widely scattered in peripheral tissues, at the entrance sites of pathogens, and are very efficient

in engulfing antigens. Following the ingestion of antigen, DCs travel to lymph nodes, where

they present antigens to T cells. Meanwhile, they mature, and their ability to take up antigens

is reduced. Instead they get more efficient in presentation of antigens through the up-

regulation of MHC class II and co-stimulatory CD80 and CD86 molecule expression [6].

Macrophages also express more MHC class II molecules, after activation, but in contrast to

DCs, exhibit a greater phagocytic ability.

NK cells are large, granular lymphocytes, widely distributed in the body, and

can be recruited to the inflammation sites in different tissues. They are the first line of defence

against various viral infections. NK cells are capable to induce the death of self cells, which

have been modified upon intracellular infections and malignant transformation. NK cells

discriminate between target and non-target cells using a set of activating (e.g. NKG2D,

NKp30, NKp 44, NKp 46 and CD16) or inhibitory receptors, such as KIR-inhibitory receptors

(KIR stands for killer-cell immunoglobulin-like receptor) [7]. Inhibitory receptors recognize


MHC class I molecules, as a marker of self cells, and inhibit the activation of NK cells. The

expression of MHC class I molecule is usually down-regulated in infected or transformed

cells. Thereby, interaction of activating receptors with the ligands on infected or transformed

cells, in the absence of MHC class I, leads to NK cells activation. Activated NK cells release

perforin and granzymes containing cytotoxic granules, which induce programmed cell death

in the target cells [8]. NK cells are identified as CD3-CD56+ lymphocytes. Human NK cells

consist of two subsets, CD56bright and CD56dim NK cells. The latter subset is mainly found in

the blood and spleen and display stronger cytotoxic effector functions than CD56bright cells,

which are more abundant in the lymph nodes [7]. NK cells can rapidly get activated and

produce pro-inflammatory cytokines, in particular interferon (IFN)-γ. Therefore, they can

influence the adaptive immune response by inducing T helper (Th) 1 type of responses [9].

Acquired immunity

While the innate immune system recognizes patterns shared by groups of non-

self molecules, T- and B lymphocytes more specifically identify foreign molecules through

the specificity of their antigen receptors, T- and B-cell receptors (TCR and BCR). The TCR is

a heterodimer of either αβ or γδ chains, which recognizes antigen and is responsible for the

specificity of the T cell. This heterodimer is associated with CD3, a multicomponent signal-

transducing complex. CD3 triggers intracellular signalling, induced by binding of the antigen

to the TCR. In contrast to the BCR, which can identify various cell-surface or soluble

antigens without prior modifications, the TCR recognizes antigen-derived peptides in

association with MHC molecules, presented on the surface of other cells [10].

T lymphocytes

T lymphocytes are derived from bone-marrow-resident stem cells, and develop

into self-tolerant, functional T cells through a multistage differentiation process in the thymus.

These stages are distinguished by the expression of the key surface molecules, CD4 and CD8.

The very first immature cells express none of these molecules and therefore are called double

negative (DN). At this stage, αβ and γδ lineages are generated based on the expression of the

αβ or γδ subunits of the TCR complex. The αβ lineage differentiates into a CD4CD8 double

positive (DP) stage. Further positive and negative selections occur that lead to deletion of non


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functional and autoreactive TCRs. Eventually, MHC-restricted and self-tolerant CD4 and

CD8 single positive (SP) T cells are generated and leave the thymus. Thus, mature αβ T

lymphocytes comprise of CD8+ T cytotoxic (Tc), CD4+ helper- and regulatory T cell lineages

[11].

Despite the expression of antigen-receptors, γδ T cells display innate

characteristics and form an independent population of lymphocytes [12]. γδ T cells, in most

instances, do not detect peptide antigens presented on the surface of APCs, as αβ T cells do.

Instead they recognize alkalamines, nonpeptideprenyl pyrophosphates and phosphorylated

uridine and thymidine compounds [13].

CD8+ T lymphocytes participate in responses to infections, through their

cytotoxicity and cytokine production. They can produce IFN-γ, tumor necrosis factor (TNF)-

α, interleukin (IL)-2, IL-4, IL-5, IL-10 and transforming growth factor (TGF)-β. Based on

their migration capacity and cytokine production, CD8+ T cells are named Tc1 and Tc2. CD8+

T lymphocytes exert their cytotoxicity through two mechanisms. They can induce apoptosis

in target cells by the expression of FasL/CD95L, that makes the target cell to terminate the

expression of the receptor Fas/CD95, and thereby initiating apoptosis. They also can kill the

target cell via exocytosis of lytic granules that forms pores in the membrane of the target cell,

which then allows the lytic granules to enter the cell and to induce apoptosis. CD8+ T cells

recognize antigens presented by MHC class I, and need help from DCs and CD4+ T cells to be

fully activated. However, the exact mechanism behind this has not yet been fully understood.

It has been proposed that DCs present antigen to CD4+ T cells in associations with MHC class

II. CD4+ T cells differentiate, proliferate and up-regulate the expression of the CD40L which

interacts with CD40 molecules on DCs. This enables DC to activate and transform CD8+ T

cells to effector cells that identify antigens in association with MHC class I molecules. A

suppressive CD8+ T cell subset, with regulatory function has also been reported [14].

Upon activation, after interaction of TCR with antigen-MHC, naïve CD4+ T

cells can differentiate into Th1, Th2, Th17 or regulatory T cell (Treg), according to cytokine

milieu (Fig 1). Once differentiated, each lineage is characterized by its own cytokine profile.

In the presence of IL-12 and through the activation of signal transduction and activator of

transcription (STAT)-4 and T-bet, CD4+ T cells differentiate into a Th1 lineage that produces


IFN-γ, a key cytokine in response to intracellular infections. Naïve CD4+ T cells become Th2,

when IL-4 is present and this process is regulated by STAT-6 and GATA-3. Th2 cells

produce IL-4, a cytokine with an important role in the host defence against helminth

infections. IL-23 synergizes with IL-1 and IL-6, inducing Th17 differentiation, a process

regulated by STAT-3 and retinoic acid-related orphan receptor (RORγt) transcription factors.

Th17 cells secrete the pro-inflammatory cytokine IL-17, and have been proposed to

participate in responses against extra-cellular bacteria and fungi, and also to mediate

autoimmune diseases. TGF-β1 upregulates forkhead box P3 (Foxp3) expression and induces

the differentiation of naïve CD4+ T cells to become Treg, which produce TGF-β1 [15]. This

process is regulated by STAT-5. CD4+ T cell subsets can regulate each other via changes in

the cytokine milieu.

Figure 1: Modified from Chen and O’Shea [15]

Treg cells are named after their functional ability to regulate/suppress immune

responses, and can develop in the thymus (natural Treg or nTreg) or be induced in the

periphery (inducible or iTreg). Foxp3, IL-2 production and CD25 (IL-2 receptor α chain)


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expression are essential for Treg development, and their function and survival. Naturally

occurring CD4+CD25+ Treg cells are key elements of self-tolerance maintenance. In the

presence of TGF-β, uncommitted CD4+ T cells in periphery can convert into iTreg and release

the regulatory cytokine IL-10 (Tr1 subset) or TGF-β (Th3 subset). In vitro studies have shown

that suppression of CD4+CD25- effector T cells, by Treg cells, depends on cell-cell contact

rather than soluble mediators. Membrane bound TGF-β, the cytotoxic T lymphocyte-

associated antigen-4 (CTLA-4) and the glucocorticoid-induced tumour necrosis factor

(GITR), are suggested to be important in this contact. Tregs have been shown to directly kill

activated CD4+ and CD8+ T cells in a perforin- or granzyme-dependent manner. It has been

proposed that upon cell-cell contact, Tregs modulate APCs function and make them unable to

activate effector T cells. Moreover, Tregs mediate immune-response suppression via the

induction of naïve T-cell differentiation into regulatory cells rather than pathogen-specific

effector cells. This phenomenon is called “infectious tolerance” and was suggested to be

mediated by IL-10 or TGF-β [16].

Another subset of T lymphocytes, which does not fit well in acquired immunity

is the NKT cell subset. These cells are derived from DP thymocytes, and are mainly present in

the bone marrow and liver, but can also be found in other lymphoid compartments. NKT cells

share characteristics of both NK and T cells [17]. NKT cells get activated upon interaction of

their TCR with glycolipids associated with the MHC class I-like molecule CD1d, which is

expressed by APCs. NKT cells are known to be major producers of IFN-γ, a crucial cytokine

for early activation of macrophages [18].

B lymphocytes

B-cell development from hematopoietic cells occurs before birth in the foetal

liver and subsequently in the bone marrow. In the bone marrow, B cells develop from pro-B

to pre-B cells and eventually to membrane-bound IgM-expressing immature B cells. Naïve

mature B cells leave the bone marrow and travel to secondary lymphoid tissues, where they

become activated. Following activation, B cells become either memory or plasma cells.

Memory B cells are more abundant than naïve resting B cells and can be rapidly re-stimulated

by antigens. They often harbour membrane-bound IgG and IgA. Plasma cells are antibody-


producing cells. They are large cells, enriched in endoplasmic reticulum, ribosome and golgi.

Plasma cells do not express membrane Ig and do not divide [19].

Antibodies and Ig class switching

The BCR is present on the surface of B cells, and is a complex of

immunoglobulin (Ig) associated with two accessory proteins, called Igα and Igβ. The BCR

identifies and binds the antigen, and Igα and Igβ trigger intra-cellular signalling induced by

binding of the antigen [10].

Antibodies are expressed and secreted by activated B lymphocytes. Antibodies

are glycoproteins with a common structure, but differing in size, charge and amino acid

sequence. The basic structure consists of two identical light chains (LC) which are bound to

two identical heavy chains (HC) via disulfide bonds and non-covalent interactions. The

amino-terminal portion of the chains, called variable (V) region, vary amongst different Igs

and determine the antibody specificities. The carboxyl-terminal portions of the chains

constitute the constant (C) region and are rather conserved. According to the sequence

patterns of the constant region, the heavy chain is grouped into five classes: μ, δ, γ, ε and α,

each named an isotype, and the light chain is found in two isoforms, κ and λ. In the process of

Ig synthesis, rearrangements in gene segments (VHDJH in HC and VLJL in LC) and somatic

mutations produce variations in amino acid sequences in the amino-terminal portions of the

chains. This generates 106 to 107 different variable regions, which reflects the generation of

a repertoire of Igs with 106 to 107 various specificities. Based on the heavy chain, antibodies

are also classified into five classes, IgM, IgD, IgG, IgE and IgA. In humans, minor variations

in amino acid sequences of the α and the γ heavy chains result in further classification of IgA

and IgG into two (IgA1 and IgA2) and four (IgG1-4) subclasses, respectively.

Rearrangement in the Ig heavy chain occurs during B cell development from

pro-B to pre-B cell. In the pre-B cell, the membrane bound μ chain and the surrogate light

chain (a complex of two proteins which are non-covalently bound to form a light chain-like

structure) associate with the Igα/Igβ heterodimer to build the pre-BCR. The light-chain gene

is then rearranged, and intact IgM is displayed on the immature B cells. In an antigen-

independent manner, naïve mature B cells begin to splice the variable region genes with the δ


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chain constant region and express IgD (in addition to IgM) on their surface to form fully

mature naïve B cells.

All Igs consist of two regions, the Fab and the Fc. The Fab contains the variable

region of the Ig, differs from one antibody to the other, and is the part of the antibody that

identifies and binds antigenic epitopes. The Fc fragment is conserved in each class and

subclass of the antibodies and mediates effector functions specific to that class.

A set of antibodies of different classes can be produced in response to a

particular antigen as a result of class switching recombination (CSR). In the IgH locus, the

genes for the constant regions are positioned in the following order, Cμ, Cδ, Cγ, Cα and Cε.

In CSR, one of these C regions recombines to the rearranged variable (VDJ) region to

generate the respective isotype of the antibody. The first antibody produced after exposure to

antigen is mainly of the IgM class. By substituting the CH regions of IgM with those of IgG,

IgA and IgE, CSR modulates the effector functions of antibodies without changing their

specificity. Antibody-class switching is mainly regulated by extra-cellular signals, in

particular cytokines. [19-23].

Antibody-mediated effector functions

Antibodies recognize and bind to antigens, but they can not remove or kill the

pathogen by themselves. Antibodies interact with other proteins or cells to perform the

effector function of a humoral response. Binding of antibody-antigen complexes to the Fc

receptors (FcR) on the surface of phagocytic cells, promotes the phagocytosis of the antigen

(opsonization).

In humans, IgM, IgG1, IgG2 and IgG3 activate the classical pathway of

complement. IgA1, IgA2 and IgE can activate the alternative pathway of complement.

Complement refers to a set of serum glycoproteins which interact and cooperate with other

immune molecules or cells to remove the antigen or kill the pathogen.

IgG also mediates antibody-dependent cellular cytotoxicity (ADCC). ADCC is

the initiation of cytotoxic activities of the effector cells against the pathogen, upon binding of

antibody in complex with antigen, to the FcR on a number of immune cells, particularly NK

cells. No effector function has been identified for IgD [19, 20, 24].


FcRs

Antibody-mediated responses are regulated by different FcRs. Unlike, CH

regions of IgM and IgD, CH regions of IgG, IgE and IgA can bind to their respective receptors

(Fcγ, Fcε and Fcα) on innate immune cells and promote their pro-inflammatory and killing

functions. This links the specificity of adaptive responses to potent effector functions of the

innate immune response. Co-expression of activating and inhibitory FcRs on innate and

adaptive immune cells regulates the immune response and thus avoids tissue damage induced

by exaggerated immune responses. In addition to immune cells, FcRs are expressed on

endothelial cells, mesangial cells and osteoclasts [22, 25]. B cells only express inhibitory

FcRs (FcγRIIB) [26].

Cytokines

Cytokines are low molecular weight polypeptides produced by different cells,

whereby cells communicate with each other. Cytokines are released in response to a variety of

inflammatory stimuli such as infectious agents and their products, or in response to other

cytokines. In general, a cascade of cytokines is produced by different cell types, eventually

ending with the final immune-effector function. The majority of cytokines are transiently

secreted and generally have a short half-life. Accordingly, cytokines are mostly produced and

act locally, either on the same cell that produced them (autocrine effect) or on the adjacent

cells (paracrine effect). A minority of cytokines enters the circulation and acts on distant

tissues (endocrine or hormonal effect).

According to their role in inflammation, cytokines are classified into pro- and

anti-inflammatory cytokines. Cells at the site of inflammation such as DCs, macrophages and

mast cells release a panel of pro-inflammatory cytokines, including IL-12, IL-18, IL-8, TNF-

α and IFN-γ, which causes the inflammation. Anti-inflammatory cytokines, like IL-4, IL-10

and IL-13, are able to down-regulate the production of pro-inflammatory cytokines. The

balance between pro- and anti-inflammatory cytokines during the course of an inflammation

determines the outcome of the inflammatory response. Importantly, the early inflammatory

cytokine production also influences the subsequent development of the adaptive immune

response [27, 28].

Cytokines are also involved in the regulation of the adaptive immunity. They

play an important role in activating and regulating lymphocytes [27]. Cytokines are important


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in the regulation of humoral response and antibody-class switching. For example in humans,

IgG4 and IgE production is induced by IL-4 and IL-13. IL-4-induced IgE production is

inhibited by IL-10, IL-12, IFN-α, IFN-γ and TGF-β. Production of IgM, IgG1, IgG3 and IgA

is enhanced by IL-10. TGF-β induces isotype switching to IgA, and IgG2 production is

induced by IFN-γ [19, 21].

In addition to the regulation of the innate and the adaptive immune responses,

cytokines also regulate cell growth and differentiation, and influence embryo implantation

and foetal development [27, 28].

Below, cytokines of interest in recent studies are described more in detail.

IFN-γ

IFN-γ is crucial in both innate and adaptive immune responses. It plays a critical

role in immunity against intracellular pathogens and in tumor control. IFN-γ is mainly

produced by NK, NKT, Th1 and CD8+ T lymphocytes. The constitutive expression of IFN-γ

mRNA by NK- and NKT cells allows them to mount a rapid IFN-γ response against

infections. CD4+- and CD8+ T cells produce little IFN-γ shortly after initial activation. IFN-γ

induces expression of MHC class I and II molecules on APCs, and consequently increases

their antigen-presentation capacity. IFN-γ enhances the phagocytic capacity of macrophages.

IFN-γ also induces the production of pro-inflammatory cytokines and antimicrobial products

such as nitric oxide (NO) in macrophages. In neutrophils and monocytes, IFN-γ stimulates the

expression of high-affinity FcγRs. IFN-γ is a growth-promoting factor for T lymphocytes,

whereas, IFN-γ inhibits the IL-4-induced B-cell growth. IFN-γ amplifies the recruitment of

lymphocytes into tissues and prolongs their activation. The excessive production of IFN-γ has

been associated with a number of autoimmune and inflammatory diseases. IFN-γ production

by NK cells is inhibited by TGF-β [8, 29].

IL-4

IL-4 is mainly produced by activated Th2 cells [28]. Mast cells also secrete IL-

4, upon engagement of their FcεR or FcγR [30]. IL-4 induces proliferation and differentiation


of activated B cells, and enhances the expression of MHC class II and the IgE low affinity

receptor (CD23) on resting B cells [31]. In activated B cells, IL-4 induces IgG4 production.

IL-4 also induces isotype switching from IgM to IgE. IL-4-induced antibody-class switching

is antagonized by IFN-γ [32, 33]. IL-4 stimulates NK cells, CD4+ T- and CD8+ T-cell

proliferation [34]. IL-4 suppresses the production of IL-1β, TNF-α and IL-8 [28].

IL-10

A wide variety of cells, such as activated monocytes, macrophages, mast cells,

epithelial cells, B cells, CD4+- and CD8+ T cells, produce IL-10. Also a variety of blood cells

are affected by the inhibitory or stimulatory functions of IL-10. IL-10 promotes B-cell

proliferation, survival and differentiation into Ig-secreting plasma cells. In addition, IL-10

displays inhibitory effects on various blood cells. The inhibitory effect of IL-10 is believed to

be due to the induction of regulatory T cells. IL-10 inhibits IL-1, IL-6, TNF-α, IL-12 and

subsequently IFN-γ production. IL-10 also down-regulates macrophages, NK cells and Th1

cells. IL-4- and IFN-γ-induced expression of MHC class II-antigen is suppressed by IL-10.

Thus, IL-10 is an important regulatory cytokine [16, 19, 35-38].

Malaria

The term malaria originates from Italian ‘mal aria’, which means ‘bad air’, for

the incidence of the disease in marshy areas [39]. The malaria parasite has infected humans

for 50,000-100,000 years [40] and remains a major cause of morbidity and mortality in the

developing word, particularly in sub-Saharan Africa. About half a billion episodes of clinical

malaria is estimated to occur each year, which results in deaths of more than one million

children in sub-Saharan Africa alone [41].

Malaria is a vector-borne infectious disease caused by an intracellular protozoan

parasite of the genus Plasmodium. More than 100 species of Plasmodium parasites are

known, of which only four types can infect humans: P. falciparum, P. vivax, P. ovale and P.

malariae. Among these four species, P. vivax and P. falciparum are the most prevalent


14

species and P. falciparum is the main cause of severe and fatal malaria [42]. Plasmodium

parasites are transmitted to the vertebrate host by the female Anopheles mosquitoes.

Life cycle of malaria parasite

The life cycle of the malaria parasite is complex, includes sexual and asexual

developmental stages and requires both mosquito and vertebrate hosts to be completed.

The infected Anopheles mosquitoes carry Plasmodium sporozoites in their salivary glands.

When an infected mosquito takes a blood meal, it injects 5 to 20 infective sporozoites into the

bloodstream of its victim (Fig. 2).

Figure 2: Plasmodium falciparum life cycle

Sporozoites migrate to the liver within 30 minutes and invade hepatocytes, where they

multiply asexually for a period of 9-16 days. This division is called schizogony and generates

merozoite forms of the parasite. Each sporozoite gives rise to thousands of merozoites which

http://en.wikipedia.org/wiki/Image:MalariacycleBig.jpg


are released into the bloodstream when the infected hepatocyte ruptures. Released merozoites

invade erythrocytes. Once inside the erythrocyte, the merozoite enlarges to a uni-nucleated

cell named trophozoite. Trophozoites undergo asexual divisions and produce schizonts with

multiple nuclei. The schizonts then segment to generate 15 to 30 merozoites which are

released into the blood, ready to invade new erythrocytes. Several such cycles occur and

periodic, synchronized release of merozoites and toxins into the blood, results in malaria

associated waves of fever (within 48–72 hours, depending on the species of Plasmodium).

A small proportion of merozoites differentiate into micro- and

macrogametocytes (male and female, respectively). A mosquito taking a blood meal on an

infected individual may ingest these gametocytes. In the mosquito gut, the gametocytes form

male and female gametes, which fertilize to form zygotes. These zygotes further differentiate,

migrate through the mosquito gut wall, and undergo repeated divisions to produce large

number of sporozoites. The infective sporozoites travel to the salivary gland and the life cycle

is completed.

Clinical features of malaria infection

Infections with malaria parasites have a wide variety of symptoms, ranging from

asymptomatic carriage of infection to severe malaria or even death. A large number of

individuals living in malaria endemic areas carries parasites without presenting signs of

clinical malaria. Clinical malaria can be categorized into mild (uncomplicated) or severe

(complicated) malaria. In general, malaria is a curable disease if diagnosed and treated

quickly and properly.

A combination of fever, chills, sweats, headaches, nausea, malaise, cough,

diarrhoea and muscular pain is presented in uncomplicated forms of malaria disease.

Uncomplicated malaria can be caused by all four Plasmodium species, whereas P. falciparum

is responsible for severe pathology and almost all deaths.

Sequestration, a characteristic feature of P. falciparum infection, happens when

P. falciparum infected red blood cells (iRBC) adhere to uninfected RBCs, endothelial cells

and in some cases to placental cells (Fig. 1). Sequestration of iRBCs in various organs such as

brain, kidney, placenta and lung may lead to serious organ failures or abnormalities in the

patients’ blood and consequently to severe pathology [43]. Cerebral malaria (CM), severe


16

anaemia, hemoglobinuria, hypoglycaemia, renal failure, pulmonary oedema, abnormalities in

blood coagulation and thrombocytopenia, convulsion and shock are some complications of

severe malaria.

Immune response to malaria

The immune response to the malaria parasite, as the parasite’s life cycle, is

complex and depends on the parasite species and on the stage of infection [44]. The infection

starts when the sporozoites enter the human body. At this level protective immune responses

to the parasite rely on antibodies against antigens on the surface of sporozoite, like the

circumsporozoite protein (CSP). During the liver stage, when the parasite is inside the

hepatocyte, a predominant cellular immune response is required. Later on, when the infection

involves erythrocytes, protective immunity depends mainly on antibody responses.

Merozoite-derived antigens expressed on the surface of the infected RBCs, merozoite surface

proteins/antigens (MSP/MSA) and parasite antigens released into circulation, following

erythrocyte rupture, are the major targets of antibody response in this stage [45].

Clinical immunity

Individuals residing in malaria endemic areas experience repeated infections and

acquire immunity to clinical malaria, termed “clinical immunity”, which prevents the disease

despite the persistence of infection [46].

Clinical symptoms of malaria are related to the blood stage of infection. Thus, it

is important to understand the immune response during this stage. The present study has

mainly focused on the blood stage of the malaria infection. Hence, immune responses to

asexual blood-stage of malaria parasite, specifically P. falciparum, are overviewed in this

thesis.

Innate immunity to blood stages

Following the initial rise in parasitemia in blood, innate type of immune

responses control parasite expansion, but this is usually not enough to eliminate the infection.


Macrophages, DCs, NK cells and γδ T cells are effector cells involved in the innate immune

responses to the malaria parasite [47].

PRRs mediate direct interaction between innate immune cells and the parasite.

To date, TLR2, 4 and 9 have been shown to recognize malarial antigens in humans. The P.

falciparum glycosyl-phosphatidylinositol (GPI) interacts with the TLR2/TLR1 complex, with

a minor contribution of TLR4, and induces cytokine production in macrophages. Hemozoin, a

product of haemoglobin digestion by P. falciparum in RBC, was surprisingly identified as a

non-DNA ligand for TLR9. TLR9 was earlier described as a receptor for CpG-containing

DNA. More recent studies have suggested that hemozoin presents DNA to TLR9, while

hemozoin by itself, is not able to induce innate immune responses [5]. Plasmodium profilin-

like proteins have been shown to activate the innate immune system in mice through TLR11

[48]. Of other PRRs involved in recognition of P. falciparum-derived antigens, the scavenger

receptor CD36 can be mentioned. The P. falciparum erythrocyte membrane protein-1

(PfEMP-1) expressed on the surface of iRBC is the ligand for CD36 [4]. Binding of PfEMP-1

to CD36 enables macrophages to attach to, and engulf iRBCs in the absence of cytophilic or

opsonizing malaria-specific antibodies. This suggests an important role for macrophages in

innate immunity to malaria [49].

Besides ingesting iRBCs, macrophages can take the parasites out of iRBCs and

let the parasite-free RBCs back to circulation [50, 51]. Macrophages can also kill/inhibit in

vitro growth of the asexual blood stage through the release of NO and H2O2 and also mediate

antibody-dependent cellular inhibition (ADCI) [52, 53].

Activation of DCs is one of the first events in the innate response to malaria.

Malaria parasites interact with DCs to modulate their functions. Although studies have

revealed a role of DCs in immune responses to many intracellular pathogens, not much is

known about the role of DCs in malaria immunity. The first study on the role of DCs in

malaria, showed that P. falciparum infected red blood cells (PfRBC) adhere to DCs, inhibit

the maturation of DCs [54], and subsequently reduce their capacity to activate T cells [55].

PfRBCs were later shown to interact with the CD36 and CD51 molecules on DCs [54, 56].

Upon this interaction, DCs upregulate the expression of the co-stimulatory molecules CD40,

CD80 and CD86, and instead of initiating a pro-inflammatory response, produce IL-10 and


18

elicit an anti-inflammatory response [57]. However, studies using experimental animals have

shown that DCs from infected mice are fully functional APCs [58].

γδT-cells have been reported to expand during the first few days of P.

falciparum infection [59]. P. falciparum–derived phosphorylated non-peptide antigens have

been identified as ligands for human γδT-cells [60]. In vitro studies have shown that Pf-

activated γδT-cells produce large amounts of IFN-γ [61, 62] and inhibit parasite growth [63].

Extracellular merozoites or late schizonts have been suggested to be targets for γδT-cells,

since γδT-cells have been shown to inhibit growth of the asexual blood stages in vitro at the

time of reinvasion [64]. γδT-cells kill the parasite by exocytosis of granulysin and perforin,

following direct contact with iRBCs [65].

NK cells can lyse PfRBC without prior sensitization. Studies by Artavanis-

Tsakonas et al. have suggested that NK cells are the earliest source of pro-inflammatory

cytokine IFN-γ [66], which is essential to control malaria parasitemia [67, 68], and that NK

cells produce IFN-γ following a direct interaction with PfRBC [69]. However, recent studies

have revealed that the innate IFN-γ production was mainly (in 93.3% of the analysed

individuals) derived from γδT-cells. The majority of these γδT-cells expressed NK receptors

(NKR). The overall IFN-γ levels produced by NK-like γδT-cells were positively associated

with the expression of CD94 on these cells [70].

Acquired immunity to blood stages

Role of antibodies in P. falciparum immunity

Erythrocytes do not express the classical MHC class I and II molecules.

Therefore, antibodies and B-cells are crucial in clinical immunity. Reduction of parasitaemia

and clinical disease after passive transfer of gamma-globulins from immune donors to malaria

patients first suggested a protective role for IgG antibodies [71]. Antibodies are generally

accepted as indicators of infection.

Malaria protective antibodies are mainly restricted to certain classes and

subclasses. Analyses of anti-malarial IgG subclass profiles, in humans, have suggested that


IgG1 and IgG3 (called cytophilic antibodies) provide protection, whereas IgG2 and IgG4 do

not. However, IgG2 has recently been demonstrated to be associated with malaria protection

in individuals carrying a certain polymorphism in the FcγRII gene [72]. IgG4 is thought to

counteract with anti-parasitic functions of IgG1 and IgG3 through inhibiting their binding to

the antigens [73]. While IgG has been correlated to protection against malaria disease,

antibodies of the IgE isotype have been linked to severe pathology. Higher levels of total and

anti-malarial IgE were detected in patients suffering from cerebral malaria than individuals

with uncomplicated malaria [74]. However, recent studies in Tanzania and Senegal have

suggested a protective role for IgE against malaria, where elevated levels of P. falciparum-

specific IgE were shown to reduce the risk of subsequent malaria attack [75, 76]. Thus, the

role of IgE (total and malaria specific) in the pathogenesis/immunity to malaria is still

unclear.

Malaria-specific antibodies exert their protective roles through mediating a

number of anti-parasitic effector functions including: inhibition of cytoadherence and thus

preventing sequestration of iRBCs and instead letting them be removed by spleen [77];

inhibition of RBC invasion [78] and antibody-dependent cytotoxicity [79]. Anti-malarial

antibodies are involved in the ADCI mechanism where the parasite growth is inhibited by

antibodies bound to Fc receptors on phagocytes [80]. Antibodies may also facilitate parasite

clearance by opsonization and phagocytosis by macrophages [81].

CD4+ T lymphocytes

CD4+ T cells are important in the development and regulation of the immune

response to malaria infection. Murine experimental systems have shown that Th1-produced

cytokines, such as IFN-γ, initiate inflammatory responses needed to control infection at early

stage. Cytokines produced by Th2 lymphocytes, e.g. IL-4 and IL-10, not only help B cells to

produce antibodies, but also suppress Th1 responses, which if left uncontrolled, can lead to

immunopathology [82, 83]. While Th1/Th2 type of responses seem to be of importance in

certain murine experimental systems, the situation is less clear in humans.

Another subset of CD4+ T lymphocytes involved in malaria blood stage immune

response is Treg cells. A direct evidence for the role of Tregs in the outcome of malaria

infection comes from studies in mice. The significant delay in parasite growth in CD4+ CD25+

depleted naïve mice, infected with P. berghei NK65, revealed that effector responses are


20

enhanced in the absence of Tregs. Depletion of CD4+ CD25+ T cells in P. yoelii 17XL

infected mice, was also shown to result in amplified T-cell responses to parasite antigens and

control the normally lethal infection [84]. Moreover, studies in P. yoelii 17XL susceptible

(BALB/c) and resistant (DBA/2) mice have indicated an increase in the Treg population early

after infection, in BALB/c but not DBA/2 mice, suggesting that the suppression of the Th1

response during early infection probably results in enhanced parasitemias and fatal disease in

BALB/c mice [85]. Similarly, in vivo studies in humans have shown that blood-stage

infection rapidly induces Tregs which in turn results in a burst of TGF-β production. This

leads to reduced pro-inflammatory cytokine production and antigen-specific immune

responses and consequently gives rise to higher rates of parasite growth [86]. Optimally, it

looks as if Treg activation and/or Treg-derived cytokine (IL-10 and TGF-β) production are

needed to be triggered at the time that pro-inflammatory cytokines have brought parasite

replication under control.

Cytokines

Besides the ability of individuals to control parasite multiplication, the term

“clinical immunity” also refers to the ability of individuals to control the inflammatory-

cytokine response induced against the parasite [87]. In mice it seems as if an early pulse of

pro-inflammatory cytokines is essential to limit the initial parasite replication [88]. An early

IFN-γ response is crucial for the resolution of the primary infection [89]. Elevated plasma

levels of IFN-γ have also been linked to resistance to re-infection by P. falciparum [90].

TNF-α synergises with IFN-γ to induce production of NO and thus parasite killing [91].

A pro-inflammatory response is essential for parasite clearance. However,

excessive production of these cytokines may lead to malaria pathogenesis. Malaria patients

have been shown to have higher plasma levels of IFN-γ as compared to asymptomatic

individuals, suggesting a correlation between IFN-γ levels and malaria disease. Elevated IFN-

γ levels have also been associated with the onset of CM [92]. Circulating levels of TNF-α

have been correlated with the risk of death from CM [93].

Anti-inflammatory cytokines have also been shown to play a critical role in

malaria pathogenesis. An infection by P. chabaudi chabaudi AS has been shown to be more

lethal in IL-10-deficient mice than in their wild type counterparts [94]. Similarly, in humans,

lower IL-10 levels were found in fatal cases than survivors [95]. African children suffering


from anaemia have been reported to have low serum levels of IL-10 [96]. In anaemic children,

the ratio of IL-10/TNF-α concentrations was linked to the severity of anaemia [97].

Thus, a delicate balance in levels and timing of pro- and anti-inflammatory

responses may determine the degree of parasitemia, severity and outcome of the disease.


22

RELATED BACKGROUND

Human genetic factors and malaria immunity

Development of clinical immunity may be influenced by age, pregnancy,

nutritional status, co-infections with other pathogens and host genetic factors [98]. The

importance of human genetic factors in immunity to malaria has long been suggested by the

high frequency of some genetic disorders in malaria endemic areas, which despite general

disadvantages, confer protection against malaria.

RBC variants

RBCs are crucial in the life cycle of the malaria parasite. The parasite uses the

RBC as shelter and food resources. Moreover, the interaction between infected RBCs and

uninfected RBCs and other tissues results in pathogenesis of the disease. Therefore, changes

in RBC structure or function may interfere with RBC invasion by the parasite and/or parasite

growth and multiplication inside RBCs, and consequently affect the outcome of the disease.

Following, some RBC variants important in malaria immunity and included in this study are

listed.

The ABO blood group system

Polymorphisms in carbohydrate structure of glycoproteins and glycolipids on

the surface of erythrocytes generate the ABO system, which is the most medically important

RBC polymorphism. Studies on associations between the ABO system and malaria, have

reported contradictory results. Some have shown a correlation between the ABO system and

severity of the disease, while others reported no correlations between malaria disease and the

ABO blood groups [99, 100]. However, blood groups A, B and AB have been shown to form

rosettes (binding of iRBCs to uninfected ones and to endothelial cells, which is linked to

severe pathology caused by P. falciparum), more readily than blood group O [101]. Thus,

individuals with blood group A, B and AB are more likely to develop CM compared to


individuals with blood group O [102]. In malaria endemic areas, the ratio of blood group O to

non-O is greater than 1, whereas this ratio is smaller than 1 in other populations. This has

been suggested to be the result from the selection of blood group O by P. falciparum

infections. However, no study has linked the ABO system to prevalence of infection,

frequency of repeated malaria episodes or anti-malaria antibody levels [99, 100].

The haemoglobinopathies

Haemoglobinopathies, disorders of haemoglobin (Hb) structure and production,

are the most common human genetic disorders. Hb is the iron-containing protein found in

RBCs that transports oxygen from the lungs to the rest of the body. Hb molecules consist of

four polypeptide chains named globins (usually 2 alpha and 2 beta chains) and an iron-

containing molecule, termed haem, attached to each chain [103].

The haemoglobinopathies are classified into two categories. One caused by

variations in Hb structure such as HbS, C and E, and the other results from variable

production of normal α- or β-globin, causing α- and β- thalassemia, respectively [104]. The

high prevalence of these disorders in malaria-endemic areas, are considered as being the result

of selection under malaria pressure [105].

Sickle cell disease

Sickle cell disease results from a point mutation in the β-globin subunit of

haemoglobin. Haemoglobin S is formed when two wild-type α-globin subunits are associated

with two mutant β-globins. Normal RBCs contain HbA and are quite elastic, which enables

the cells to deform in order to pass through capillaries. HbS carrying RBCs are distorted, tend

to lose their elasticity, and have shorter life time as compared to the normal ones. The

homozygous form of HbS (HbSS) causes a severe disease and is lethal at early childhood, in

the absence of adequate treatment. The heterozygous state (HbAS), also called sickle-cell

trait, has long been linked to protection against severe malaria [106, 107].

A number of mechanisms, by which sickle-cell disease may mediate resistance

to malaria, has been proposed in several studies [108]. In vitro studies have indicated:

enhanced phagocytosis of ring-stage parasitized HbAS erythrocytes [109], reduced


24

erythrocyte invasion and impaired intra-erythrocytic parasite growth in AS-RBCs, as

compared to infected normal erythrocytes [110, 111]. HbAS parasitized erythrocytes also tend

to sickle [110], which may enhance removal of premature infected erythrocytes in the spleen

[110, 112]. Enhanced immune responses to parasitized HbAS erythrocytes have also been

reported [113, 114]. A recent study has shown significantly reduced binding of P. falciparum

infected AS erythrocytes to micro-vascular-endothelial cells and blood monocytes as

compared to infected normal RBCs [115]. This may take part in protection to severe malaria

conferred by the sickle cell trait. HbAS has not been shown to influence asymptomatic

parasitaemia, however, it protects against severe and fatal malaria [107]. Thus, the precise

mechanism whereby sickle cell disease protects against malaria is yet unclear.

Alpha-thalassemia

The haemoglobin-alpha-chain gene is located on chromosome 16. Normally,

there are 2 alpha-chain genes on each chromosome, 4 in total. Alpha-chain-protein production

is evenly divided among the four genes. Alpha-thalassemia occurs when one or more genes

are mutated or fail to work. The severity and type of disease depend on the number of affected

genes, ranging from 4 genes deleted, to one deleted gene. The first defect is fatal prior to or

shortly after birth, whereas the latter form is clinically silent (α+) [103]. Depending on the

size of the deletion, α+ is categorized into different types [116]. In Africa, -α3.7 is the most

prevalent α+–thalassemia [105].

Several studies have indicated that α-thalassemia confers significant protection

against severe and fatal malaria [117, 118]. It has also been related to protection against mild

malaria [118, 119]. However, α-thalassemia does not protect against asymptomatic

parasiteamia [104], and does not influence parasite densities in individuals that go on to

develop the disease [117-119]. Studies by Carlson et al. [120] proposed that α-thalassemic

individuals are less likely to develop severe malaria, probably due to impaired ability of α-

thalassemic RBCs to form rosettes. It has been shown that α-thalassemia gives rise to reduced

expression of the red-cell complement receptor 1 (CR1), an essential receptor for rosetting

[121]. Therefore, impaired CR1-mediated rosetting may confer resistance to severe malaria

seen in α-thalassemic individuals.


G6PD deficiency

Glucose-6-phosphate dehydrogenase (G6PD) is a housekeeping enzyme that

functions as an antioxidant, and thus protects the cell from oxidative damage. Although the

lack of this enzyme in RBCs is fatal, mutant forms with defects in function are common. The

G6PD gene is located on the long arm of the X chromosome. Four hundred and forty two

G6PD variants have been identified. More than 130 variants were found at the DNA level that

lead to impaired enzyme activity [122].

Malaria parasites break down haemoglobin inside the RBC. This gives rise to

products such as oxidized iron, which are toxic to the parasite. Any defects in G6PD function,

which is important for cells to overcome the oxidative stress, is lethal for the parasite and can

thus confer resistance to malaria infections [122]. The geographic distribution of G6PD

variants matches with the historical distribution of malaria disease [123, 124]. The mutant

G6PD A- with 10-50 percent of normal enzyme activity, is the most common G6PD

deficiency allele in sub-Saharan Africa [125-127]. In two large case-control studies, Ruwende

et al. have shown that G6PD A- causes a 46% reduction in the risk of developing severe

malaria in heterozygous females, and 58% in hemizygous males [125]. Studies by Roth et al.

revealed that parasitemia levels in cultures with G6PD-deficient RBCs (from hemizygous

males and heterozygous females) were three times lower than those cultured in normal blood

(blood from individuals with normal RBC) [128]. It has also been shown that ring-stage

infected G6PD deficient RBCs are phagocytosed 2.3 times more intensely than infected

normal RBCs [129]. However, the actual mechanism(s) whereby G6PD deficiencies protect

against malaria is still not well understood.

Others

Duffy and Gerbich blood-group antigen deficiencies, HbC, HbE, β-thalassemia,

CR1 polymorphism and Southeast Asian Ovalocytosis (SAO) are other examples of RBC

variants, involved in malaria resistance [104].

Human leukocyte antigen (HLA)

The MHC gene loci in humans are referred to the HLA complex and are highly

polymorphic. There are evidences that specific HLA alleles might be correlated to


26

susceptibility/resistance to malaria disease. For instance, studies in The Gambia have

correlated an HLA class I antigen, HLA-B*5301, and an HLA class II haplotype, HLA-

DRB1*1302-DQB1*0501, with resistance to severe malaria [130]. HLA-DQB1*0501 has

been linked to the reduced risk of re-infection and anaemia in Gabonese children [131], where

DRB1*04 and DPB1*1701 were related to severe disease [132]. A case-control study in

Ghana has revealed an association between HLA-DRB1*04 and severe malaria [133].

Ethnicity

A recent study in Kenya has estimated that almost 25% of the variation in

malaria incidence is a consequence of host genetic factors. This ratio was far beyond the

effects expected for the haemoglobinopathies alone. The HbS gene, which is the strongest

known resistance genetic factor, constitutes only 2% of the total variation. This suggests that a

large number of protective genes exits in the population, each of which taking a small part in

the protection [134].

Ethnic differences in susceptibility to malaria offer an important approach to

identify immune genes behind the resistance or susceptibility to malaria. Studies in Mali

[135], Burkina Faso [136]and Sudan [137] have revealed a significant inter-ethnic variation in

susceptibility to malaria. In spite of exposure to the same parasite and living under the same

transmission intensity, the Fulani ethnic group less frequently develop clinical malaria, as

compared to their sympatric ethnic groups (the Dogon in Mali, the Mossi and Rimaibe in

Burkina Faso and the Masaleit in Sudan). The Fulani have also been reported to have higher

anti-malarial-immune reactivity and lower asymptomatic infection prevalence than their

neighbours [135, 136, 138-142]. This relative resistance to malaria has been shown not to be

correlated to classical malaria-resistance genes, since higher frequencies of α-thalassaemia,

G6PD, HbC and HLA-B*5301 were found in the Mossi and Rimaibe as compared to the

Fulani. Similar frequencies of HbS were observed amongst three sympatric groups [143].

Similarly in Mali, HbC was more frequent in the Dogon group than in the Fulani, and HbS

showed a low similar frequency in both communities [135]. A recent study in Sudan has

revealed a significantly higher frequency of HbAS in non-Fulani (Masaleit) compared to the

Fulani [144]. Higher frequencies of spleen enlargement have been reported in the Fulani than

non-Fulani groups [135, 137, 145-147].


A recent study in Burkina Faso has revealed enhanced expression of both Th1-

(IL-18 and IFN-γ) and Th2 (IL-4 and GATA 3) related genes, in peripheral blood

mononuclear cells (PBMC) from the Fulani compared to the Mossi. However, the Fulani had

a reduced expression of Treg determinant genes (Foxp3 and CTLA4), suggesting that the

defect in functional Treg in the Fulani could reflect their higher resistance to malaria [148].

These data are in agreement with previous reports from Mali, showing a more efficient Th1

and Th2 response towards P. falciparum antigens in the Fulani [141]. Thus, it is obvious that

many different factors are involved in the protection against malaria, and these factors might

differ in different populations.

FcγR gene polymorphisms

Three families of human FcγRs have been identified, FcγRI (CD64), FcγRII

(CD32) and FcγRIII (CD16). Each FcγR family constitutes of several isoforms. A-, B- and C

isoforms have been described for FcγR- I and II, whereas FcγRIII includes only two isoforms,

A and B. The FcγR isoforms are expressed on a variety of cell types and vary in their binding

affinity to different IgG subclasses.

A number of studies has related some FcγRs gene polymorphisms (for instance

FcγRIIA-G494A [149-151], FcγRIIB-T695C [152] and FcγRIIIB-NA1/NA2 [149]) to

susceptibility to and/or severity of malaria disease. The G494A SNP in the FcγR IIA gene

gives rise to amino acid exchange from arginine (R) to histidine (H) at position 131. FcγR

IIA-131H is the only human FcγR which efficiently binds the IgG2 subclass [153]. The H

allele has been correlated to both protection and susceptibility to malaria in different studies

(reviwed by Braga et al. [154]). Studies in Sudan [144] and Mali [Israelsson et al.,

unpublished] have shown significantly higher frequencies of the H allele and the H/H

genotype in the Fulani as compared to the non-Fulani. However, whether or not the FcγR IIA-

131H allele plays any role in reducing the susceptibility to malaria disease in the Fulani, is not

clear yet.


28

Cytokine gene polymorphisms

Accumulating evidence suggests that inter-individual variations in the

production of cytokines involved in malaria immunity may influence the course of infection,

and determine the outcome of the disease. It is also evident that the ability to produce variable

amounts of cytokines can be genetically regulated. A number of polymorphisms in cytokine-

related genes that modifies cytokine expression and/or function has been identified. For

example, replacement of a C by a T at -590 position in the IL-4 gene promoter has been

described to result in enhanced IL-4 production [155]. Similarly, a G to A substitution in the

IL-10 gene promoter, position -1087, has been identified and related to elevated levels of IL-

10 production [156].

Quite a few studies have tried to relate polymorphisms in cytokine-related genes

to various infections/diseases, including malaria. For instance, enhanced levels of TNF-α in

cerebral malaria patients have been shown to be a consequence of a variation in the TNF-α

promoter in these individuals [157-159]. In Thai malaria patients, the IL-13 -1055 T allele has

been shown to protect against severe malaria [160]. The IL-4 -590 T allele has been correlated

to elevated IgE levels in asthmatic children [155]. This allele has also been associated with

elevated total IgE levels in children suffering from severe malaria. However, the frequency of

the T allele did not differ between severe and mild malaria cases [161, 162]. In Burkina Faso,

the IL-4 -590 T allele was associated with increased malaria-specific IgG levels within the

Fulani individuals but not in non-Fulani [163]. Polymorphisms in interferon-regulatory factor

(IRF)-1 have recently been linked to the control of P. falciparum infection in the same study

area [164]. However, none of the studied polymorphisms has yet been suggested as a

determinant of susceptibility/resistance to malaria infection/disease.

Genetic diversity of malaria parasite

The success of P. falciparum in evading host immunity, particularly in avoiding

antibody-mediated immunity against parasite-derived surface proteins, is attributed to the

antigenic diversity in the parasite population as a whole, as well as antigenic variation in the

individual parasite. The malaria parasite is able to switch the expression of surface antigens

and, thus, change the antigenicity during the course of infection [165]. The existence of

phenotypically variable strains of the parasite in endemic areas enables the P. falciparum


parasite to re-infect previously exposed individuals. This leads to strain and variant specific

immunity [166, 167]. Human experimental malaria studies have shown that a primary

infection with a certain strain generates protective immunity against the infecting strain but

not against infections by other strains [168]. This has been suggested to be the reason why

long term exposure (years), to a pool of variable parasite strains, is required for the

development of naturally acquired immunity to the Plasmodium parasite [169]. Thus, the

acquired immunity has been suggested to develop after infection by multiple parasite strains.

Multiplicity of malaria infection

MOI is the number of various P. falciparum strains simultaneously infecting a

single person. Individuals residing in malaria endemic areas usually carry multiple parasite

strains. MOI can be an indicator of transmission intensity and immune status. MOI has been

shown to be well related to transmission intensity [170, 171]. The presence of diverse

infections in asymptomatic inhabitants in endemic areas is suggested to reflect acquired

immunity [172], and thus may affect the outcome of the infection. Some studies have

indicated that the increase in MOI decreases the risk of developing clinical malaria [173-175],

whereas others indicated a positive link between malaria disease and MOI [176, 177]. Patients

suffering from cerebral malaria have been shown to have higher MOI than mild malaria

patients. However, a slight increase in clone numbers was found in healthy controls compared

to patients with mild malaria [178]. In contrast to this, a recent study in Nigeria has revealed

that children with single strain infection are at the higher risk to develop cerebral malaria as

compared to those with multiple infections [179]. Thus, the impact of MOI on the

development of malaria immunity is still unclear.

MOI is suggested to vary over age, and this variation is highly influenced by

transmission intensity [170, 180-182]. MOI seems to increase as immunity develops [172].

The age of acquiring natural immunity differs according to transmission intensity [183]. In

areas with intensive malaria transmission, immunity is acquired at younger ages compared to

less intense transmission areas [183]. However, the relation between infection by multiple

parasite strains and malaria disease is still unclear.

The number of msp2 variants is widely used as a marker to determine MOI

[184].


30

MSP2

MSP2, also called MSA2, is a 45 kDa polymorphic protein anchored in the

merozoite membrane. The msp2 gene is a single-copy gene and consists of five blocks,

encoding conserved, dimorphic and polymorphic sequences (Fig. 3). Blocks 1 and 5 encode

highly conserved N (43 residues) and C (74 residues) termini, respectively. Blocks 2 and 4 are

dimorphic and classified into two families, the 3D7 and FC27 (also known as type A and type

B, respectively). The central region, block 3, consists of a series of variable repeat motifs,

which vary in number, length and sequence among isolates. Individual MSP2 alleles are

defined by the central repeats [185-187]. MSP-2 is involved in RBC invasion by merozoites

[188].

Figure 3: Modified from Damon Eisen [189]

Conserved regions

Family specific non-repetetive regions

Allelic specific repetetive regions

FC27

3D7


PRESENT STUDY

Aim of study

The overall aim of this study was to investigate possible associations between

human genetic factors and malariometric indexes, with the following specific objectives:

• To investigate correlations between IL-4 -590 C/T and IL-10 -1087 A/G SNPs, and

inter-ethnic differences in malariometric indexes observed between the Fulani and the

Dogon, living in Mali.

• To correlate the levels of malaria-specific IgG, IgG1-4, IgE, as well as total IgE, to

prevalence and multiplicity of asymptomatic infection and splenomegaly in the Fulani,

and to compare the results to those observed in the Dogon.

• To investigate the influence of IL-4 -590 C/T SNP on anti-malarial IgG, IgG1-4, IgE

and total IgE levels in the Fulani and the Dogon.

• To study: the impact of erythrocyte variants on MOI; correlation between MOI and

the risk of clinical malaria; and associations between MOI and age, parasite density

and seasonal variation, in asymptomatic children in Senegal.


32

Methodology

Materials and methods, briefly mentioned below, are described in details in the

respective papers (manuscripts).

Paper I

Age-matched asymptomatic individuals of the Fulani (214) and the Dogon

(212), were included in this study. Finger-prick blood was collected on filter papers. Human

and parasite DNA were extracted using the Chelex- and TE buffer-based methods,

respectively. Polymerase-chain reaction (PCR) was performed for both human (IL-4 T/C and

IL-10 A/G SNPs) and parasite (msp2/3D7 and msp2/FC27) genotyping.

Paper II

Parasite DNA was extracted from blood on filter papers using Chelex-100.

Number of concurrent parasite clones was defined by msp2 genotyping using PCR. ELISA

was performed to determine malaria-specific IgG, IgG1-4, IgE as well as total IgE antibody

levels.

Paper III

Human genomic DNA was extracted from blood on filter papers using the

Chelex-100 and genotyped for the IL-4 T/C SNP by PCR. Antibody levels were determined

using ELISA.

Paper IV

Asymptomatic children (2 -10 years old), living in Senegal, were included in

this study. Parasite densities were measured in June 2002 and January 2003. An active survey

of malaria attacks was conducted during the transmission period in 2002 for a sub-group of

154 children. ABO blood groups were determined by serology, and the other red-blood-cell

polymorphisms were detected using genomic DNA. Parasite DNA was extracted from blood

on filter papers and analysed for the number of msp2 (FC27 and 3D7) clones per infection by

PCR.


Results and discussions

Paper I

IL-4 and IL-10 are two crucial cytokines in immune responses in general and

anti-malarial responses in particular [190-193]. IL-4-590 T/C and IL-10 -1087 A/G

polymorphisms have been shown to result in variable production of these cytokines [155,

156]. We investigated the associations between the above mentioned SNPs and malariometric

indexes amongst sympatric ethnic groups living in Mali, the Fulani and the Dogon.

We observed that the allele and genotype frequencies of the IL-4 -590 T/C and

IL-10 -1087 A/G SNPs varied significantly between the Fulani and the Dogon, indicating that

these ethnic groups had distinct genetic backgrounds. This was consistent with studies in

Burkina Faso, where HLA class I markers in the Fulani were found to be genetically very

distant from their neighbours [194]. The Fulani from Mali (present study) and Burkina Faso

[163] represented similar allele and genotype frequencies of IL-4 -590 T/C, supporting the

same origin for the Fulani in the two countries [194].

In Burkina Faso, the Fulani T allele carriers had higher anti-malarial IgG levels

compared to the CC genotype carriers. This was not seen in the non-Fulani groups living in

the same area, suggesting that the IL-4 -590 polymorphism is not functional, but may be

linked to other factor(s), which can be the real determinant of resistance to malaria [163]. We

found that the IL-4 -590 T allele was correlated to the presence of P. falciparum infection in

the Fulani group but not in the Dogon. This finding suggests that the persistence of infection

in the T allele carriers gives rise to constitutive anti-malarial antibody production.

Pronounced differences in the IL-4 -590 allele and genotype frequencies were

observed between the non-Fulani groups living in the two countries (recent study and [163]).

The Dogon carried a T/C allelic ratio of 82/18, which was very different from those reported

for Mossi and Rimaibe (18/82 and 25/75, respectively). These data suggest that unknown

factor(s) linked to this position, may be polymorphic in non-Fulani from different areas, and

that those factors have been selected differently under variable selection pressures, including

malaria.

Within the Fulani, the T allele carriers had higher infection prevalence than

those carrying the CC genotype. Although parasite densities did not vary significantly

between the two groups, the Fulani CC genotype carriers had lower parasite loads compared


34

to the Dogon carrying the same genotype. This suggests that the Fulani CC genotype carriers

may succeed better in clearing the infection, compared to the T allele carriers, and to the

Dogon CC genotype carriers. However, to draw a firm conclusion regarding the correlations

between IL-4 genotypes and parasitemia, parasite counts need to be more accurately

determined in consecutive samplings.

In line with former studies [135, 141, 145, 146], we noted lower frequencies of

infection and higher spleen rates in the Fulani group than in the Dogon. Within the Fulani,

spleen enlargement was associated with the presence of P. falciparum infection. This was not

the case in the Dogon group. Our results contradict those reported by Bereczky et al. [195],

where splenomegaly was associated with P. falciparum infections within the Dogon but not

the Fulani. These data underline that more standardized analyses are needed to evaluate host-

parasite interactions.

No significant inter- and intra-ethnic differences, in the studied malariometric

indexes, between different IL-10 genotypes were seen in this study.

All together, these data suggest that unknown factor(s) is probably linked to the

IL-4 -590 C and is polymorphic in the non-Fulani. This factor(s) might be involved in the

control of infection and thus, avoiding the clinical presentation. However, further studies are

required to better clarify the contribution of IL-4 and particularly the studied SNP in malaria

immunity.

Paper II

Antibodies play important roles in clearance of blood-stage parasites [196]. In

this study, we assessed correlations between anti-malarial antibody levels and malariometric

characteristics in asymptomatic individuals of the Fulani and the Dogon.

Higher anti-malarial IgG, IgG1-3, IgE and total IgE levels were detected within

the Fulani as compared to the Dogon. However, no significant correlations between antibody

levels and parasite prevalence, number of infective clones and spleen enlargement were noted.

The higher spleen- and lower infection rates, seen in the Fulani compared to

their neighbours, have been proposed to be the result from their enhanced anti-malarial

reactivity. Interestingly, our results revealed no significant correlation between malaria-

specific antibody levels and the presence of P. falciparum infection. This suggests that the

relative resistance to malaria infection seen in the Fulani group is not mainly dependent on the


magnitude of anti-malarial humoral response. In support of this idea, studies in Sudan have

shown that the Fulani had lower malaria-specific IgG1 and almost equal IgG and IgG3 levels

compared to their neighbours, the Masalite (Nasr et al., unpublished).

In this study, we noted that amongst the Fulani individuals, the increase in total

IgE levels was weakly related to the presence of malaria parasites. In malaria patients,

elevated total IgE levels have been associated with the development of CM [74]. Other

studies have shown that unlike malaria-specific IgE [75, 76], total IgE levels did not influence

the risk of subsequent clinical attacks [75]. Thus, the actual consequence of the increase in

total IgE levels during malaria infection within the Fulani remains to be clarified.

Correlations between the studied antibody levels and clone numbers were then

analysed. An increase in Log10 values of malaria-specific IgG4 levels within the Fulani was

linked to a decrease in clone numbers. ADCI, which is suggested to be the most efficient anti-

malarial mechanisms mediated by protective antibodies, is believed not to be mediated by

IgG4 antibodies [72]. However, IgG4 might still display other anti-parasitic functions, such as

inhibition of cytoadherence or inhibition of RBC invasion by the parasite. IgG4, raised

against parasite antigens involved in cytoadherence, may help parasite clearance in the spleen

through inhibiting sequestration of iRBCs. By binding to parasite antigens involved in

parasite invasion, IgG4 by itself could interrupt invasion of new RBCs. Thus, the specificity,

function and/or affinity of an antibody might be more important than the amount of the

antibody. We observed no significant differences in IgG4 levels between the two studied

groups. In the Fulani, IgG4 levels were negatively associated with the number of clones.

Taken together, our results suggest that IgG4, in the two groups, may be raised against

different parasite epitopes or have different affinities, and thus mediate various anti-parasitic

functions.

Spleen enlargement is mostly seen in individuals at the age of 15 and younger

(children). Thus, the relation between antibody levels and splenomegaly was evaluated in the

whole study population and separately in children. A weak correlation between elevated IgG

and IgG3 levels and splenomegaly was noted in the Fulani group (in all ages included and in

children only). Despite higher levels of IgG1 compared to IgG3, splenomegaly was not linked

to IgG1 levels. This may be reflected by different specificities of IgG1 and IgG3. That is,

IgG3 may identify antigens which may lead to less sequestration and thus, to more efficient

parasite clearance by the spleen. However, our observation might also be the result from

overestimation of significances due to not correcting the P values for multiple analyses.


36

Trends for correlations between IgG2 levels and splenomegaly, and multiplicity

of infection within the Dogon were noted in this study. These results could not be explained

by the frequency of the FcγRIIA-131H variant, which enables IgG2 to act as cytophilic

antibody in parallel with IgG1 and IgG3 [73], since the H allele is less frequent than the R

allele in the Dogon (Israelsson et al., unpublished). This emphasizes the need for further

studies to investigate the actual role of IgG2 in responses to malaria.

All together, our results suggest that the stronger anti-malarial humoral response

seen in the Fulani is not responsible for the differences in malariometric indexes observed

between the Fulani and the non-Fulani ethnic groups. A rapid, efficient and well-regulated

innate immune response, which controls parasite counts before the acquired immune response

has developed, may be the reason for the relative resistance to malaria seen in the Fulani.

Paper III

The impact of IL-4 -590 T/C SNP on the level of malaria-specific IgG, IgG1-4,

IgE and total IgE levels, in asymptomatic individuals of the Fulani and the Dogon, was

analysed in this study. We noted that amongst the Fulani, the increases in total and anti-

malarial IgE levels were associated with the IL-4 T allele after adjustment for age and sex. No

correlation between IL-4 -590 variants and other studied antibodies were seen in any of the

studied groups.

IgE has been suggested to be involved in both pathogenesis and protection

against malaria disease. Studies have shown higher levels of total and malaria-specific IgE in

patients suffering from severe malaria compared to those with uncomplicated malaria [74,

197]. However, in asymptomatic individuals, enhanced levels of anti-malarial IgE decreased

the risk of developing clinical symptoms [75, 76]. This suggests that the role of IgE in malaria

is still unclear. In our study, we noted that total and malaria-specific IgE levels were higher in

the T allele carriers compared to the CC genotype, only within the Fulani. Our former studies

have shown a higher frequency of P. falciparum infection in the Fulani T allele carriers than

those with the CC genotype (Paper I), and that the elevated levels of total IgE were correlated

to the presence of infection (Paper II). Others have shown that the T allele is correlated to

elevated levels of total IgE amongst children with severe malaria whereas the frequencies of

the T allele did not differ significantly between severe and mild malaria cases [161, 162].

Therefore, whether the increase in the levels of IgE, seen in the T allele carriers in this study,


is a direct consequence of the impact of IL-4 -590 variants on the antibody-class switching, or

if it is due to the higher infection frequencies observed in the T allele carriers, is not clear and

remains to be investigated.

All together, these data suggest that IL-4 and IgE might influence the outcome

of the disease, in concert with other factors (genetic, epigenetic, epistatic or environmental). A

recent study in Burkina Faso has revealed associations between a number of SNPs in the IRF-

1 gene and carriage of P. falciparum infection in both the Fulani and the Mossi. However,

different patterns of polymorphisms were seen in the two groups, probably reflecting their

different haplotypic architecture [164]. IRF-1 elicits the expression of IL-12p 40 [198, 199]

and is critical for production of IL-12 [200], and subsequently for initiation of the Th1 type of

responses [201, 202]. Thus, IRF-1 can modulate the balance between Th1 and Th2 type of

responses, which is important for the IgE response in malaria [141].

Studies in Burkina Faso have revealed an association between the IL-4 -590 T

allele and elevated levels of anti-malarial IgG, in the Fulani group [163]. In contrast to this,

we noted that IgG levels did not vary significantly between IL-4 genotypes in any of the

studied groups. Production of IL-4 and any other factor(s) involved in the regulation of

antibody responses could also be affected by co-infections with other parasitic or bacterial

pathogens or time after last exposure to malaria. This idea is supported by findings from other

studies, where reverse or no relations between IL-4 -590 variants and IgG levels were seen

[203, 204], indicating that the impact of IL-4 variants on IgG production, in turn, is

influenced/regulated by other factors.

Correlations between IL-4 -590 variants on the level of IgG subclasses have not

been studied previously. IL-4 and IL-13 have been shown to promote IgG4 production [32,

33]. In this study, a trend for an increase in IgG4 levels in the T allele carriers was observed

only within the Fulani individuals. However, analysis of IgG4 responses is difficult due to the

generally low levels of these antibodies. Therefore, the impact of IL-4 -590 variants on IgG4

responses needs to be more analysed including larger study groups.

No significant variations in levels of malaria-specific IgG1-3 between IL-4 -590

genotypes were seen in any of the studied groups.

Taken together, our data suggest that IL-4 may play other roles in malaria

immunity in addition to the regulation of antibody responses. The implication of the IL-4 -590

T allele in susceptibility and/or resistance to malaria infection/disease within the Fulani


38

remains to be addressed in follow-up and case-control studies. Furthermore, the actual role of

IgE in protection and pathogenesis of malaria remains to be uncovered.

Paper IV

This study examined the impact of erythrocyte variants on MOI. Relations

between MOI and age, seasonal variations as well as malaria morbidity were also analysed.

MOI was measured before and after transmission season in asymptomatic children, in the age

range of 2 to 10 years, living in a malaria mesoendemic area in Senegal.

As expected, parasite density and MOI increased after the transmission season.

MOI was positively correlated to parasite density, which is in concordance with data reported

by others showing that with higher parasite counts, it is more probable to detect concurrent

clones [205, 206].

The impact of different RBC variants on MOI has been limitedly studied. We

noted that the general increase in MOI after the transmission season was not seen in α-

thalassaemic and G6PD mutated children. Alpha-thalassaemia alters the membrane properties

of RBCs [207, 208], which may affect their invasion by merozoites. MSP2 is a polymorphic

antigen involved in invasion [188]. Thus, the altered membrane of α-thalassaemic RBCs may

impair the invasion by certain MSP2 variants and consequently protect against infection by

certain parasite strains. However, other studies have shown no impact of α-thalassaemia on

parasite prevalence, density and diversity in symptomatic individuals [209]. A reason for this

contradiction might be due to that we studied asymptomatic subjects, whereas the former

study included symptomatic cases. It is known that α -thalassaemia does not affect parasite

loads in individuals who develop clinical disease [118, 119, 209]. The differences in sample

sizes in the two studies could also be considered as a reason for this discrepancy. These data

suggest that α-thalassaemia may protect against infection by certain parasite strains, but does

not protect against clinical malaria in ongoing infections.

Infected G6PD-deficient RBCs have been shown to be more efficiently

phagocytosed than infected normal RBCs [129]. In this study, MOI did not increase after the

transmission season in the G6PD-mutated children, which might be due to rapid parasite

clearance in these children as compared to those carrying the normal G6PD gene. This effect

was stronger in G6PD-mutated girls carrying the HbAS gene. However HbAS alone did not


affect MOI. G6PD deficiency did not influence MOI in male subjects, which might be due to

the small size of this group.

In the present study, the ABO blood system did not show any influence on MOI

at any time points. The ABO system has been implicated in the outcome of the disease, where

blood group O is less likely to develop CM as compared to A, B and AB carriers [101].

However, the ABO system did not seem to affect parasite density and diversity in

asymptomatic individuals.

Sickle cell trait did not influence MOI in our study. Former studies have

reported none [210] or reverse [211] relations between HbAS and MOI. The reason for these

discrepancies is not known, but they might be due to differences in the ages of the studied

populations and/or variations in transmission intensities between the study areas.

MOI has been shown to be age-dependent in areas with intense transmission,

and to be independent of age in a malaria mesoendemic area [210, 212]. In line with the latter

report, MOI was not influenced by age in the present study. However, these findings need to

be confirmed in a study population with a broader age range.

A clinical survey was performed including 154 children to evaluate correlations

between MOI and clinical attacks. Forty five percent of these children had at least one clinical

attack during the survey. No significant association between clinical malaria and MOI was

noted, at any time points. Both reverse [173-175] and positive [176, 177] correlations between

MOI and malaria morbidity have been reported. Our finding does not agree with any of those

reports, which may partly be due to different genotyping methods used in these studies.

Differences in sensitivity and accuracy of genotyping protocols have been shown to give rise

to variations in results from different laboratories [206, 210, 213, 214]. Moreover, in the

present study, sampling was not done at the time of clinical presentation. This may also be a

reason why MOI was not related to morbidity. In any case, these results emphasize that

malaria morbidity is not only affected by parasitological indexes, but could also be influenced

by host genetic factors and/or transmission levels.

Taken together, methodology should be considered when comparing results

from different studies on MOI. The results from PCR may vary if different protocols and

reagents are used or even may differ from hand to hand [206]. PCR may underestimate the

MOI. Importantly, PCR is unable to differenciate between virulent and avirulent strains. Thus,

to draw a firm conclusion from studies on MOI in different areas, a more standardized

methodology is required.


40

CONCLUDING REMARKS AND FUTURE PERSPECTIVES

We conclude from our studies that the Fulani and the Dogon are genetically

markedly different, using IL-4 -590 and IL-10 -1087 variants, as markers. The IL-4 genotype

pattern was similar in the Fulani from different areas, whereas it varied significantly between

non-Fulani groups living in different countries. The IL-4 T allele was previously suggested to

be linked to unknown factor(s), which could be the real determinant of resistance to malaria

[163]. Our results suggest that the unknown factor is probably linked to the C allele and is

polymorphic in non-Fulani. This factor might be involved in a better parasite clearance.

This study concludes that the magnitude of anti-malarial IgG response may not

be as important as it is thought to be. Instead, other features of antibodies such as specificity,

function and/or affinity may be more crucial in protective response to malaria. The

implications of IgE antibodies in malaria protection and pathogenesis remain to be better

clarified. IgG4 may not mediate ADCI, but might still have some roles in malaria protection,

through mediating anti-parasitic functions different from those mediated by IgG1 and IgG3.

Our results also indicate that the relative resistance to malaria infection seen in the Fulani

does not rely mainly on their augmented anti-malarial antibody response, as it has been

believed to be. Rather, other factors such as a rapid and well-regulated innate response, which

can control the infection and consequently avoid the clinical presentations, might also take

part in this relative resistance to malaria.

Studies on the associations between IL-4 -590 variants and IgE have indicated

that relationship between IL-4 and IgE might be affected by other genetic/epigenetic/epistatic

or environmental factors. Therefore, the outcome of the disease could be reflected by the

action of these factors together with IL-4 and IgE. The production of IL-4 and other

cytokines/factors, in turn, could be affected by the time after last exposure to malaria or by

co-infection with other pathogens which might explain the contradictory results of the impact

of IL-4 -590 variants on IgG levels observed by a number of studies. All together, studies on

IL-4 (genotype/levels/-producing cells) in Mali and Burkina Faso suggest that the

contribution of IL-4 in inter-ethnic differences in malaria susceptibility is probably not

through its role in antibody regulation, rather owing to its implication in other aspects of

immune responses, e.g. induction of NK cell proliferation which help in parasite killing

and/or suppression of anti-inflammatory cytokine productions which avoids immuno-

pathogenesis.


The last study concludes that in asymptomatic children; α-thalassaemic RBCs

may resist against infection by certain parasite strains; protection conferred by G6PD

deficiency might be due to rapid parasite clearance at early stage of infection; the ABO blood

system and HbAS do not affect the MOI. The multiplicity of infection was correlated to

parasite density in a malaria mesoendemic area, but did not vary over the age, in the range of

2 to 10 years. The risk of clinical attack was not affected by MOI. However, these data need

to be confirmed using different genotyping methods and including larger size of the study

group with broader age range.

To address the actual implications of IL-4 -590 variants in malaria (especially

inter-ethnic variations in malaria susceptibility), a follow-up study of a population with

known IL-4 -590 genotypes of the Fulani and the Dogon, joined with a case-control study, is

required. Consecutive determination of antibody levels together with parasitological data,

during these studies, might help to clarify the impact of IL-4 -590 variants on antibody

responses.

Most importantly, studying other genetic factors adjacent to IL-4 gene on

chromosome 5q may help in identifying the actual factor(s), involved in resistance/protection

to malaria infection. Sequencing of this region in individuals of the two groups would be of a

great help in this regard.


42

ACKNOWLEDGMENT

I left my family and home with the wish to continue my studies and become a researcher. It

was a long, tough journey to bring the wish close to reality; a long way that I would never

have been able to make on my own. I would, therefore, like to express my sincere gratitude to

all those who have helped and supported me during these years. In particular, I would like to

thank: Professor Marita Troye-Blomberg; for accepting me into your group, for your

support and guidance, for being patient with my endless questions, and for always being calm

and caring. I learnt so much from you. Present and former seniors at the Immunology

Department; Professor Klavs Berzins, Professor Carmen Fernandez, Professor Eva

Severinson, Docent Eva Sverremark Ekström and Docent Manuchehr Abedi-Valugerdi;

for being excellent teachers, for your critique and advice, and above all for providing a

family-like environment at the department. Margareta Hagstedt (Maggan) and Ann

Sjölund; for your excellent technical support and for your incredible speed in responding to

any call for help. Gelana Yadeta and Anna-Leena Jarva; for excellent administration,

always having a smile, and for your readiness to assist. Present and former students at the

Immunology Department, for making the department a nice and friendly place to work, co-

authors, for your contributions, friends, in and out of university, for the very beautiful time

we shared and the Nadjafi family in Stockholm, for giving me the warmth and feelings of

home and family whilst in cold Stockholm.

I would also like to thank my dear siblings, Aboutaleb, Mahmood, Kobra, Azra, Mehri and

Monireh, and their families; your love and prayers have been my best company all these

years. What would my life mean without you guys?! Mehri, you are the best of friends and

sister, ever. You were available anytime I needed you, and filled every moment of my

loneliness after I left home; despite miles of physical distance our hearts and minds have been

tightly tied together moment by moment, I love you and miss you very much.

My love, my friend, my husband, Peter Homann; where would I have been if not for you

going through the whole emotional rollercoaster with me during the past months; for your


morning encouraging cuddles and evening relaxing hugs, for believing in me and bringing my

self-confidence back when I had none left.

And for my incomparable mother and father, Mohammad E. Vafa and Sara Madadi; words

are not enough to express my love and gratitude to you. Whatever I have, whatever I am,

whatever I get, it is all because of you. This thesis is in fact yours.


44

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