Licentiate thesis from the Department of Immunology,

Wenner-Gren Institute, Stockholm University, Sweden

Human genetic factors in relation to Plasmodium

falciparum infection

Manijeh Vafa

Stockholm 2007

Summary

The first study aimed at investigating if there were any correlations between the

IL-10 -1087 A/G and IL-4 -590 C/T polymorphisms and inter-ethnic differences in

susceptibility to malaria observed between two sympatric ethnic tribes, the Fulani and the

Dogon, living in Mali. The genotypes were correlated to both ethnicity and malariometric

indexes. A statistically significant inter-ethnic difference in the allele frequency and genotype

inheritance pattern for both loci was noted. The prevalence of infection was associated with

the IL-4 -590 T allele within the Fulani. Inter-ethnic differences in spleen rates were seen

between the T allele carriers (TT and CT) of both groups. The Fulani CC genotype carriers

had significantly lower parasite densities than those carrying the same genotype in the Dogon.

No significant differences in parasite prevalence, density and diversity between different IL-4

-590 C/T genotypes were found within the Dogon. No associations between the IL-10

genotypes and P. falciparum prevalence, density and diversity were observed in any of the

listed communities. These results suggest an association between; the IL-4 -590 T allele and

parasite prevalence and the CC genotype and control of parasitaemia within the Fulani.

Individuals living in malaria endemic areas generally harbour multiple parasite

strains. Multiplicity of infection (MOI) is known as an indicator of immune status. However,

whether high or low MOI are good or bad for the development of immunity to malaria is still

not clear. Our second study aimed to examine the MOI in asymptomatic children aged 2-10

years and to relate it to the transmission levels, clinical attacks, erythrocyte variants and other

parasitological indexes. MOI was defined as the number of concurrent Plasmodium

falciparum clones per individual using PCR method. Sixty four and 87 percent of PCR

positive cases had multiple infections before and after the transmission season, respectively.

MOI was not age-dependent. A significant association between MOI and parasite density was

observed. No correlation between MOI and malaria attacks was noted. The ABO system and

HbAS did not affect MOI at any time points, indicating that these factors are involved in the

severity of the disease but do not affect asymptomatic infections. α-thalassaemic children did

not show an increase in MOI at the end of the transmission season. This might suggest a role

for α-thalassaemia in protection against certain parasite strains. G6PD deficiency also

protected against an increase in MOI after the transmission season, probably through

clearance of the malaria parasite at early stages of infection.

ii

اى نام تو بهترين سرآغاز بى نام تو نامه آى آنم باز

مددى ينم محمد اسماعيل وفا و سارا تقديم به عزيزتر

To my dearest, Mohammad E. Vafa and Sara Madadi

iii

List of papers

This thesis is based on the following original papers, which will be 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. Submitted.

II. Manijeh Vafa, Marita Troye-Blomberg, Judith Anchang, Andre Garcia and

Florence Migot-Nobias, Multiplicity of Plasmodium falciparum infection in

asymptomatic children in a malaria mesoendemic area in Senegal; its relation to

transmission, age and erythrocyte variants. Preliminary manuscript.

iv

TABLE OF CONTENTS

SUMMARY .......................................................................................................................................................... II

LIST OF PAPERS .............................................................................................................................................. IV

ABBREVIATIONS................................................................................................................................................1

INTRODUCTION..................................................................................................................................................2

GLOBAL INCIDENCE OF MALARIA........................................................................................................................ 2 MALARIA PARASITE ............................................................................................................................................ 2

Parasite life cycle .......................................................................................................................................... 2 MALARIA DISEASE .............................................................................................................................................. 3 IMMUNITY TO MALARIA ...................................................................................................................................... 4

The RBC polymorphisms ............................................................................................................................... 4 Blood group antigens.................................................................................................................................................. 5 The haemoglobinopathies........................................................................................................................................... 5 Enzyme deficiencies................................................................................................................................................... 6

Innate immunity ............................................................................................................................................. 7 Acquired immunity......................................................................................................................................... 8

Cell-mediated immunity ............................................................................................................................................. 8 Antibody-mediated immunity..................................................................................................................................... 9

Cytokines ..................................................................................................................................................... 10 IFN-γ ........................................................................................................................................................................ 10 TNF-α ...................................................................................................................................................................... 11 IL-4........................................................................................................................................................................... 11 TGF-β....................................................................................................................................................................... 12 IL-10......................................................................................................................................................................... 12

Regulation of immune response to malaria ................................................................................................. 13

RELATED BACKGROUND..............................................................................................................................14

HUMAN GENETIC FACTORS................................................................................................................................ 14 PARASITE GENETIC DIVERSITY .......................................................................................................................... 14

PRESENT STUDY...............................................................................................................................................16

OBJECTIVES ...................................................................................................................................................... 16 METHODOLOGY ................................................................................................................................................ 17 RESULTS AND DISCUSSIONS.............................................................................................................................. 18

CONCLUDING REMARKS AND FUTURE PERSPECTIVES.....................................................................21

ACKNOWLEDGMENT .....................................................................................................................................22

REFERENCES.....................................................................................................................................................23

Abbreviations

APC Antigen-presenting cell

CR1 Complement receptor 1

CTL Cytotoxic-T lymphocyte

DC Dendritic cell

G6PD Glucose-6-phosphate dehydrogenase

GPI Glycosylphosphatidylinositol

Hb Haemoglobin

IFN Interferon

Ig Immunoglobulin

IL Interleukin

IPP Isopentenylpyrophosphate

iRBC Infected-Red-Blood Cell

MHC Major-histocompatability complex

MOI Multiplicity of infection

MSP Merozoite-surface protein

NK Natural killer

PCR Polymerase-chain reaction

RBCs Red-blood cells

SNP Single-nucleotide polymorphism

TCR T-cell receptor

TGF Transforming-growth factor

Th T helper

TLR Toll-like receptor

TNF Tumor-necrosis factor

Treg Regulatory T cell

1

Introduction

Global incidence of malaria

Malaria is one of the most important causes of child mortality worldwide. About

half a billion episodes of clinical malaria is estimated to occur each year by Plasmodium

falciparum infection which results in deaths of more than one million children in sub-Saharan

Africa alone 1.

Malaria parasite

Malaria is caused by an obligate, intracellular protozoan parasite of the genus

Plasmodium, transmitted to the vertebrate host by the female Anophele mosquitoes. Of the

four species that infect humans (P. falciparum, P. vivax, P. ovale and P. malariae), P.

falciparum is responsible for virtually all deaths.

Parasite life cycle

The malaria parasite has a complex life cycle, including sexual and asexual

developmental stages in the mosquito vector and in the human host, respectively. Malaria

infection in humans begins when an infected female Anopheles mosquito takes a blood meal

and injects 5 to 20 infective sporozoites into the peripheral circulation (Fig. 1). Within

minutes (about 30 min) the sporozoites invade hepatocytes in the liver and undergo asexual

multiplication for a period of 5 to 10 days, producing liver schizontes. When the infected

hepatocyte ruptures, thousands of merozoites are released into the bloodstream that invade red

blood cells (RBCs), where they complete another round of asexual multiplication within 48–

72 hours depending on the species of Plasmodium. Infected RBC (iRBC) ruptures and

releases 15 to 30 merozoites ready to invade new RBCs and carry on the cycle. This

synchronous release of merozoites is thought to be responsible for the periodic fevers

associated with malaria.

Some invading merozoites do not divide, but differentiate into male and female

sexual forms or gametocytes which are ingested by a mosquito when she takes a blood meal.

Gametocytes mature into male and female gametes in the gut of mosquito and fertilise to form

2

zygotes. These zygotes further differentiate, migrate through the mosquito gut wall and divide

to form thousands of sporozoites.

The infective sporozoites move to the salivary gland, where they await injection

into another human host, thus completing the life cycle.

Figure 1: Adapted from TR Jones and SL Hoffman 2.

Malaria disease

Infection with P. falciparum has a wide spectrum of manifestations classified as

asymptomatic infection or clinical malaria. In malaria endemic areas, a significant proportion

of individuals harbour parasites without presenting signs of clinical malaria. Clinical

manifestations of malaria range from mild uncomplicated to severe malaria. Fever

accompanied by nausea, headache, malaise, cough, diarrhoea and muscular pain are

characterized as uncomplicated malaria.

3

While uncomplicated malaria is caused by all four Plasmodium infections,

complicated malaria is restricted to P. falciparum. Severe anaemia, cerebral malaria, renal

failure, hypoglycaemia, pulmonary oedema, convulsion and shock are the common

complications of severe malaria.

Immunity to malaria

Immunity to malaria is complex and is both stage and species specific. The

liver-stage specific antigens have been suggested to be processed and presented on the surface

of infected hepatocytes in combination with MHC class I and then presented to T cells. This

presentation results in killing of infected cells by cytotoxic T lymphocytes (CTLs) or through

IFN-γ produced by NK and CD4+ T cells which triggers a cascade of immune reactions that

ultimately leads to the death of the infected cells. During a blood stage infection, parasite

antigens released into the bloodstream at the time of erythrocyte rupture, or merozoite-derived

antigens expressed on the surface of infected RBCs are the targets of immune response,

particularly antibody response 3. Since my studies focus mainly on the blood stage of malaria

infection, only factors, cells and components involved in immunity against this stage are

overviewed in this thesis.

The generation and maintenance of clinical immunity to malaria requires

repeated infections over the lifetime. Individuals living in malaria endemic areas eventually

develop clinical immunity to P. falciparum which is usually defined as the ability to control

parasite replication to a level at which they fail to reach the critical threshold needed for the

induction of clinical symptoms.

The development of clinical immunity is influenced by age, genetic background

of host, pregnancy, nutritional status and co infections 4. Both innate and adaptive immune

effector mechanisms are involved in immunity to malaria. However, in most instances the

immune response fails to eliminate the infection completely.

The RBC polymorphisms

The human red blood cell is a central element in malaria life cycle. It provides

the parasite with both food and shelter. Moreover, the pathogenic features of the disease are

consequences of the interaction between infected RBCs, uninfected RBCs and other tissues.

4

Therefore, it is not surprising that many of the known malaria-protective polymorphisms are

related to genes which affect the structure or function of RBCs.

Blood group antigens

Blood groups A and B are more likely to form rosettes 5, i.e. binding of

parasitized red blood cells to uninfected ones which results in severe pathology. Thus,

individuals having these blood groups are more prone to experience cerebral malaria as

compared to individuals carrying blood group O 6. In the case of P. vivax, the RBC Duffy

antigen is important in the merozoite invasion into RBC. P. vivax merozoites are completely

dependent on the Duffy/DARC chemokine receptor for RBC invasion 7. RBCs with Duffy

negative phenotype are resistant to invasion by P. vivax merozoites 8, 9.

The haemoglobinopathies

The haemoglobinopathies, conditions affecting the structure and synthesis of

haemoglobin, are classified into two categories. The first group results from the production of

structurally variable forms of haemoglobin (including haemoglobin S, C and E). The second

group is caused by the reduced production of normal α- or β-globin, resulting in α- and β-

thalassemia, respectively. The clinical effects of these conditions are highly variable

(Reviewed by Thomas N Williams) 10. Haemoglobinopathy provides up to 90% protection

against death due to malaria 11.

Haemoglobin S (HbS), known as sickle cell disease, in the homozygous state

(HbSS) causes severe sequel and is lethal in young children. The sickle cell trait (HbAS)

confers a strong protection against P. falciparum malaria as shown in several studies

conducted in various countries 11-13. Despite this the precise mechanisms behind the

protection remain incompletely understood. To some extent it relates to particular physical or

biochemical properties of HbAS red blood cells. Reduced parasite growth 14, 15 and enhanced

removal of parasitized cells through innate 16 or acquired 17, 18 immune responses, have also

been proposed to be the mechanisms involved in this protection. Although HbAS protects

against severe and lethal malaria, it does not affect asymptomatic parasitaemia 19.

5

In the homozygous form, haemoglobin C (HbCC) and E (HbEE) have mild

clinical effects including haemolysis and splenomegaly. The distributions of these

polymorphisms in malaria-endemic areas support their malaria-protective effects 10. The

protective effect of HbC is probably due to impaired growth and multiplication of P.

falciparum in these RBCs 20. In vitro studies showed that RBCs from subjects with HbE trait

are relatively resistant to invasion by P. falciparum merozoites 21. HbE was associated with

reduced malaria severity in adults admitted to hospital in Thailand 22.

It is evident that α-thalassemia protects from both severe and fatal malaria 23, 24.

Few data also suggest a protective role for it against mild malaria 24, 25. However, α-

thalassemia does not protect against asymptomatic parasiteamia 10 and has no effect on

parasite densities in individuals that go on to develop the disease 23-25. In 1994, Carlson et al. 26 proposed that the impaired ability of red blood cells to form rosettes is the major

mechanism by which α-thalassemia protects against severe malaria. α-thalassemia has also

been associated with the reduced expression of the red cell complement receptor 1 (CR1), an

essential receptor for rosetting 27. Therefore, α-thalassemia may protect against severe malaria

through impaired CR1-mediated resetting.

Enzyme deficiencies

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common

enzymopathy in humans. High prevalence of this X-linked disorder in malaria endemic

communities suggests its natural selection by malaria 28, 29. The most common G6PD

deficiency allele in sub-Saharan Africa is G6PD A-, which confers about 50% reduction in

the risk of developing severe malaria in both heterozygous females and hemizygous males 30.

However, the mechanisms behind the protection are still uncertain; Cappadoro et al. showed

that it probably involves early phagocytosis of iRBCs. Ring stage infected G6PD deficient

RBCs are phagocytosed 2.3 times more intensely than infected normal RBCs 31.

Gerbich blood group antigen negativity, CR1 polymorphism and Southeast

Asian Ovalocytosis (SAO) are additional examples of malaria resistance genes which involve

structure or function of RBCs.

6

Innate immunity

Macrophages, DCs, NK, γδ and NKT cells are important effectors of innate

immunity against malaria.

The importance of macrophages in innate immunity to malaria is due to their

ability to phagocytose infected erythrocytes in the absence of cytophilic or opsonizing

malaria-specific antibodies. Studies by Serghides et al. indicated that CD36 is involved in this

opsonin-independant phagocytosis of P. falciparum-infected erythrocytes by monocytes from

non-immune individuals 32.

Dendritic cells are important in amplifying the innate immune response, in

particular, in stimulating NK cells activation 33, 34. Although studies have revealed a role of

DCs in immune response to many intracellular pathogens, relatively little is known

concerning the role of DCs in immunity to malaria. Urban et al. have shown that P.

falciparum infected red blood cells suppressed the maturation of DCs 35 and reduced their

ability to activate T cells 36. However, studies using experimental animals have shown that

DCs from infected mice are fully functional APCs 37.

Rapid induction of IFN-γ is essential to control malaria parasitemia in mice 38-40,

and NK cells play a crucial role in this early response 38, 41. Similarly, in humans the ability to

mount a robust IFN-γ response is associated with reduced susceptibility to infection 42, 43.

Artavanis-Tsakonas et al. showed that IFN-γ is produced by human NK cells in response to P.

falciparum-infected RBC within 24 hours 44, suggesting a key role for NK cells in the initial

control of blood-stage malaria infections.

Polyclonal expansion of γδT-cells during the first few days of P. falciparum

infection has been reported 45. Phosphorylated non-peptide antigens, such as IPP, synthesized

by P. falciparum have been identified as ligands for human γδT-cells 46. P. falciparum –

activated γδT-cells produce large amount of IFN-γ 47, 48 and inhibits parasite growth in vitro 49. Parasite growth inhibition exerted by γδT-cells has been shown to occur at the time of

reinvasion, suggesting that extracellular merozoites or late schizonts are the targets for γδT-

cells 50. Studies by Farouk et al. revealed that γδT-cells require contact with iRBCs to exert

their P. falciparum inhibition/killing activities and that γδT-cells kill the parasite via a

granule-exocytosis cytotoxic pathway involving granulysin 51. A granule-exocytosis cytotoxic

7

pathway is a way in which the killing of target cell is mediated by the release of cytolytic

molecules such as perforin and granulysin released from cytotoxic granules in the cytolytic

cells. γδT-cells have also been correlated to the pathogenesis of cerebral malaria in P. berghei

ANKA infected mice 52.

NKT cells are mainly involved in pre-erythrocytic anti-malarial immunity. Upon

activation through TCR, NKT cells produce high levels of IFN-γ and IL-4 53. This capacity

may contribute them to exert anti-malarial immunity during blood stage where IFN-γ and IL-4

are involved in parasite clearance.

Toll-like receptors (TLR) are membrane glycoproteins which recognize

components and structures of pathogenic and non-pathogenic microoorganisms. They are

involved in the induction of innate immune response to microbial ligands 54. Of the 13 TLR

known in mammalian so far 55, TLR2, TLR4 56, 57 and TLR9 58 have been shown to recognize

malarial antigens. Hemozoin, a hydrophobic heme polymer, a result from digestion of

haemoglobin by P. falciparum, is released into the bloodstream at the time of RBC rupture.

Hemozoin has been shown to interact with TLR9 58. P. falciparum glycosyl-

phosphatidylinositols (GPIs) bind to TLR2 and to some extent to TLR4 in both mouse and

human macrophages giving rise to TNF-α secretion by these cells 56, 57 which in turn starts

inflammatory reactions (see cytokines).

Acquired immunity

Acquired immunity to malaria involves both cell- and antibody-mediated immunity.

Cell-mediated immunity

CD4+ T cells play a crucial role in immunity against the asexual blood stages of

the malaria parasite. CD4+ T cells produce cytokines which amplify innate immune response

and down-regulate this response later on to avoid immunopathology. CD4+ T cells are also

required to help B cells to produce anti-malarial antibodies which are key components for

parasite clearance. Protective immunity to blood stage of malaria infection is dependent on

CD4+ T cells together with antibodies and B cells (Reviewed in reference 59).

8

Naïve CD4+ T cells can differentiate into two distinct subsets during an immune

response, T helper (Th) 1 and Th 2, with different profiles of cytokine production. Th 1 cells

secrete IFN-γ and are responsible for cell-mediated immunity, whereas Th 2 cells produce IL-

4 and regulate humoral immunity. Cytokines produced by each of these subsets inhibit the

induction of response mediated by the reciprocal subset 60. IFN-γ production by Th1 cells

initiates inflammatory reactions needed for parasite clearance which in excess can lead to

immunopathology 3. Th 2 cells control B-cell growth and induce isotype switching from IgM

and IgG to IgE 61.

Antibody-mediated immunity Antibodies and B cells are critical in immunity against malaria. They play

crucial role during blood stage of infection. B cell-deficient mice infected with Plasmodium

chabaudi chabaudi AS have been shown to fail to eliminate the infection and instead

developed chronic parasitaemias 62.

A protective role for antibodies has long been suggested by passive transfer of

IgG from immune donors which gave rise to reduction of parasitaemia and clinical disease 63.

P. falciparum infected Thai patients treated with IgG purifed from sera of African immune

subjects controlled parasitaemias and reduced clinical symptoms, indicating that protective

antibodies are raised against conserved antigens shared by P. falciparum parasites worldwide 64, 65.

An infection with P. falciparum parasite induces elevated levels of both

polyclonal 66 and anti-malaria specific antibodies. It is evident that antibodies are important in

blood stage parasite clearance 67. Protective anti-malarial antibodies belong to certain Ig

classes and subclasses, with IgG being the most important isotype in this respect. Cytophilic

IgG1 and IgG3 are considered as protective subclasses, whereas IgG2 and IgG4 are known as

non-protective ones. However, IgG2 may also be associated with protection under certain

conditions 68. While IgG has generally been associated with protection against malaria

infection, antibodies of the IgE isotype seem to be involved in both protection and

pathogenesis of malaria. Elevated levels of total and anti-malarial IgE in Plasmodium infected

humans and experimental models have been reported 69, 70. Total and anti-malarial IgE have

been shown to be higher in cerebral malaria patients than those with uncomplicated malaria 69,

71. However, high levels of anti-P. falciparum IgE have also been associated with a reduced

risk of acute malaria in a malaria endemic area in Tanzania 72.

9

Malaria-specific antibodies mediate 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 73; inhibition of RBC invasion 74 and antibody-dependent

cytotoxicity 75. Anti-malarial antibodies are involved in the antibody-dependent cell-mediated

inhibition (ADCI) mechanism where the parasite growth in inhibited by antibodies bound to

Fc receptors on phagocytes 64. Antibodies may also facilitate parasite clearance by

opsonization and phagocytosis by macrophages 76.

Cytokines

Cytokines induced in response to a malaria infection may play both protective

and pathogenic roles. An early pulse of pro-inflammatory cytokines such as TNF-α, IFN-γ,

IL-12 and others may mediate protective immunity by inducing parasite killing, while late

pro-inflammatory responses might contribute to excessive inflammatory reactions and

pathology. Inflammatory response may be down-regulated by anti-inflammatory cytokines,

including IL-4, IL-10 and TGF-β. The timing of the anti-inflammatory response seems to be

critical. Thus high levels of anti-inflammatory cytokines early during infection are generally

believed to suppress mechanisms involved in the resolution of infection, while low levels later

during infection lead to a failure to control the inflammatory cytokine cascade with

subsequent development of severe pathology. The balance between pro- and anti-

inflammatory responses may determine the outcome of the disease.

A large group of cytokines are involved in immunity and pathogenesis of

malaria infection hence only cytokines of our interest and those mentioned in this thesis are

more introduced here.

IFN-γ Interferon γ is a key inducer of the immune effector mechanisms which are

critical to control both pre-erythrocytic and blood-stage of malaria infection 77, 78. IFN-γ, a

macrophage-activating factor, is produced by CD8+ and CD4+ T cell as well as NK cells in

response to the malaria parasite. IFN-γ production by T cells may also induce production of

the cytophilic IgG1 and IgG3 antibodies in humans 3. Serum IFN-γ levels have been

associated with resistance to P. falciparum reinfection 79. Studies in mice have revealed a role

10

for IFN-γ in both protection and pathogenesis of malaria. Although IFN-γ is essential for

control of parasitaemias, elevated levels of IFN-γ are also correlated with the onset of cerebral

malaria (CM). Similarly, in humans, hyperparasitaemia has been shown to be associated with

lower frequencies of IFN-γ producing CD4+ T cells in Gabonese patients suffering from acute

P.falciparum malaria. On the other hand, higher plasma levels of IFN-γ were found in

symptomatic cases than individuals without clinical pathology, indicating a correlation

between disease severity and corresponding IFN-γ levels (reviewed in reference 80).

TNF-α Plasmodium-infected erythrocytes and parasite products including parasite

pigments and GPI induce TNF-α production in macrophages. TNF-α can increase the

expression of Fc-receptors on monocytes resulting in increased phagocytic capacity of these

cells. TNF-α has a regulatory effect on IL-12 production by macrophages. Thus TNF-α is an

important co-factor for IL-12 to induce IFN-γ production in NK cells (Reviewed by

Malaguarnera et al.) 3.

TNF-α has been associated with fever resolution and parasite clearance 81, 82.

TNF-α synergises with IFN-γ to induce production of nitric oxide (NO) and thus parasite

killing 83. However, risk of death from cerebral malaria correlates with circulating levels of

TNF-α 84. It has also been shown that high plasma ratios of TNF-α to IL-10 are linked to

severe anaemia 85.

IL-4 Interleukin (IL)-4 is produced by Th2 and activated mast cells. IL-4 activates

CTL, NK cells and macrophages. Together with Th2 cells, IL-4 is important in regulating the

antibody response induced by the Plasmodium parasite 3. IL-4 production by CD4+ T cell

from malaria exposed donors has been associated with elevated levels of serum antibodies

specific for the antigen used to stimulate T cells in vitro 86. IL-4 has also been shown to

suppress IFN-γ production 87 and macrophage-mediated killing of P. falciparum parasite 88.

IL-4 is involved in the antibody isotype switching from IgM and IgG to IgE 89.

The ratio of mitogen-induced IL-4/IFN-γ producing cells has been correlated with plasma

levels of anti-P.falciparum IgE antibodies 90.

11

TGF-β TGF-β, produced by a wide range of cells such as macrophages, NK, T and B

cells, has a pivotal role in the control of transition of a Th1 response towards a Th2 response.

TGF-β suppresses TNF-α production by macrophages as well as TNF-α and IFN-γ

production by NK cells. TGF-β up-regulates IL-10 production. In low concentrations, TGF-β

stimulates secretion of Ig subclasses by B cells 91, while at higher concentrations antibody

production is inhibited 92.

In mouse models, at the time of peak parasitaemia, a strong TGF-β response was

associated with resolution of infection and abrogated mortality. Administration of neutralizing

antibody to TGF-β led to 100% mortality in BALB/c mice infected with P. chabaudi

chabaudi A/J 93. However, a very early (within 24 hours) burst of TGF-β production leads to

a more rapid parasite growth and early death from anaemia. Similar effects were observed

after administration of exogenous TGF-β (Reviewed by Artavanis-Tsakonas et al.) 80.

IL-10 IL-10 is produced by monocytes, T and B cells. IL-10 induces B-cell

proliferation and antibody production. Hence IL-10 plays an important role in the

development of immune response against blood stage of malaria infections. IL-10 inhibits

IFN-γ, TNF-α and IL-6 production and thus inhibits the cascade of pro-inflammatory

reactions and their pathogenic consequences 3. Thus, IL-10 plays a key role in the regulation

of the immune response 94.

P. chabaudi chabaudi AS infection has been shown to be more lethal in IL-10-

deficient mice than in their wild type counterparts 95. Low serum levels of IL-10 have been

reported in African children with anaemia 96. The ratio of IL-10/TNF-α concentrations

determines the severity of anaemia in infected children 85.

High ratios of IFN-γ, TNF-α and IL-12 to TGF-β or IL-10 are associated with

decreased risk of malaria infection but increased risk of clinical disease in those who get

infected 43.

12

Regulation of immune response to malaria

The regulation of anti-malarial specific immune responses is as important as the induction of

immune responses that determines the outcome of the infection. Accumulating evidence

suggest a central role for regulatory T cells (Treg) in the regulation of immune-mediated

pathology of malaria. Treg comprise 5-10% of peripheral CD4+ T cells in humans and are

characterized by the expression of the IL-2Rα chain (CD25). Treg mediate the suppression of

T-cell activities by inhibiting the IL-2 production and thereby inhibition of the

lymphoproliferation, cytotoxicity and cytokine secretion. Treg is not antigen specific and can

suppress cells of any TCR specificity. The cytotoxic T lymphocyte Ag-4 (CTLA-4), Foxp3,

TLR4, CD25, IL-10 and TGF-β are potential mediators of Treg activities. Antigen-specific,

CD25- and Foxp3- T cells can acquire the ability to express Foxp3 and thus differentiate into

inducible regulatory cells. TGF-β is known as a physiological inducer of Foxp3. Peripheral

CD4+ T cells can be converted to CD4+ CD25+ regulatory T cells by TGF-β and mediate their

effects through the release of IL-10 (Tr1 and CD8+ Treg) or TGF-β (Th3 and CD8+ Treg).

The role of IL-10 and TGF-β has been demonstrated in the regulation of immune response to

malaria (see cytokines). A direct evidence for the role of Treg in the outcome of malaria

infection comes from studies in mice. CD4+ CD25+ depletion of naïve mice, infected with P.

berghei NK65 led to a significant delay in parasite growth, indicating the enhanced effector

responses in the absence of Treg. So far there is no report on the role of Treg on malaria-

induced mortality. Also in P. yoelli 17XL infected mice the T-cell responses to parasite

antigens were amplified after depletion of CD4+ CD25+ T cells, and a normally lethal

infection was controlled. 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 (Reviewed by Riley et

al.) 97.

13

Related background

Human genetic factors

A range of genetic factors might explain the inter-individual variation in

susceptibility/resistance to malaria. Ethnic differences in susceptibility to malaria have been

evident during the last decade. Identifying the genes involved in host-parasite relationship,

and uncovering how they affect this interaction, offer approaches to the prevention and

treatment of malaria. One way to achieve this goal is to uncover the mechanisms involved in

protection offered by known malaria resistance genes such as sickle cell trait (HbAS), α-

thalassaemia, G6PD deficiency and others which are selected in malaria endemic areas.

Genetic comparisons of populations living in similar environmental conditions and infection

but differing in susceptibility to malaria are other approaches to define the genetic variations

making some individuals vulnerable and others resistant to malaria.

Studies on susceptibility to malaria in sympatric ethnic groups in Mali 98 and

Burkina Faso have indicated that despite exposure to the same parasite and living under the

same transmission intensity, the Fulani tribe is less susceptible to malaria infection as defined

by fewer malaria attacks, lower parasite densities and higher anti-malarial antibody levels as

compared to their neighbouring ethnic tribes, the Mossi and Rimaibe in Burkina Faso and the

Dogon in Mali 99-102. Known anti-malarial-protective genetic disorders; α-thalassaemia,

G6PD and HbC, were found to be more frequent in Mossi and Rimaibe as compared to Fulani

tribe, while similar frequencies of HbS were noted amongst all tribes 103. Studies in Mali also

showed higher frequency of HbC in the Dogon group than the Fulani and a similar, low HbS

prevalence in both tribes 98.

Parasite genetic diversity

The Plasmodium parasite has the ability to change the expression of several

surface proteins and consequently alter the antigen profile which the parasite exposes to the

host immune system. The extensive repertoires of hyper variable gene families encoding the

merozoite surface proteins, enables the Plasmodium parasite to evade the host immune

response, particularly to avoid antibody-mediated immunity against parasite-derived surface

proteins. This results in strain specific immune response that may explain the need of long

14

term exposure (10-15 years) to a pool of variable parasite strains for naturally acquired

immunity to the Plasmodium parasite to develop. In experimental malaria in humans, a

primary infection by certain strain resulted in protective immunity against the same strain but

not against infection by other strains 104.

The Merozoite surface protein 2 (MSP2) also called the merozoite surface

antigen 2 (MSA2), encoded by a single-copy gene, is located on the surface of the merozoite

and is considered as a blood stage vaccine candidate. The location on the merozoite surface

and the fact that a specific monoclonal anti-MSP2 antibody can inhibit the parasite growth 105

are evidences that MSP2 is a target of host protective immune responses. Anti-MSP2

antibodies have been detected in sera from 90% of individuals living in a malaria endemic

area 106. MSP2 consists of highly conserved N (43 residues) and C (74 residues) termini

flanking a central variable region (Fig. 2). This central region consists of a series of variable

repeat motifs flanked by dimorphic sequences of one of the two major types, the 3D7 and

FC27 (also known as type A and type B, respectively). The central repeats vary in number,

length and sequence among isolates. Individual MSP2 alleles are defined by the central

repeats 107-109.

FC27

3D7

Conserved regions

Family specific non-repetetive regions

Allelic specific repetetive regions

Figure 2: Modified from Damon Eisen 110

15

Present study

Objectives

Study I

Ethnicity has a great impact on susceptibility/resistance to malaria. Studies in

Burkina Faso 99-102 and Mali 98 have shown that the Fulani tribe are less susceptible against

malaria as defined by having less malaria attacks, less P. falciparum prevalence and higher

anti-malarial antibodies than their sympatric ethnic groups. IL-4 and IL-10 are two cytokines

with critical roles in response to blood stages of the malaria parasite. Cytokine-gene

polymorphisms have been shown to influence susceptibility/resistance to a broad range of

diseases. Here we investigated the associations between the IL-4 -590 T/C and the IL-10 -

1087 A/G polymorphisms and inter-ethnic differences in malariometric indexes observed

between the Fulani and their neighbouring tribe, the Dogon.

Study II

Individuals living in malaria endemic areas generally harbour multiple

Plasmodium falciparum strains. Multiplicity of malaria infection (MOI) is an indicator of

transmission intensity and immune status. MOI has been shown to influence the risk of

clinical attack and to be affected by age, seasonal variation and transmission intensity.

Erythrocyte polymorphisms are known to confer protection against severe malaria. Studies on

the impact of RBC variants on MOI are limited. This study examined MOI in asymptomatic

children in a malaria mesoendemic area and its relationship with age, transmission, malaria

morbidity, erythrocyte variants and other parasitological indexes.

16

Methodology

Materials and methods used in this work are summarised in the text below but

described in details in the respective manuscripts.

Study I

This study included 426 age-matched asymptomatic individuals of the Fulani

(214) and the Dogon (212) sympatric tribes living in the Mopti area, Mali. Thick blood films

were prepared to determine parasite density. Spleen enlargement was graded according to the

Hackett score (1-4). Finger-prick blood was collected on filter papers. Human and parasite

DNA were extracted using the Chelex- and TE buffer-based methods, respectively. PCR was

performed for both human (IL-4 T/C and IL-10 A/G SNPs) and parasite (msp2/3D7 and

msp2/FC27) genotyping. IL-10 and IL-4 genotypes were related to P. falciparum prevalence,

density and diversity, as well as to spleen rate.

Study II

A cohort of 372 asymptomatic subjects (2 -10 years old), living in the Niakhar

area, where malaria is mesoendemic, was included in this study. Parasite densities were

measured in June and in December 2002. An active survey of malaria attacks was conducted

during the transmission period of 2002 for the a sub-group of 154 children.

Venous blood was collected in June 2002. 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. Multiplicity of infection was correlated to age, seasonal

variation, erythrocyte variants and other parasitological indexes.

17

Results and Discussions

Study I

Studies performed in Burkina Faso have suggested a distinctly differing genetic

background between the Fulani and their sympatric ethnic groups, the Mossi and Rimaibe,

using HLA markers 111. In accord to this, we observed a significant difference in IL-4 T/C and

IL-10 A/G allele and genotype frequencies between the Fulani and their neighbours, the

Dogon, living in Mali. Using the IL-4 -590 T/C SNP as a marker, we found that the Fulani

from Mali and Burkina Faso 112 represented almost the same allele and genotype frequencies,

while the non-Fulani from the two countries showed very different distribution of the

mentioned SNPs.

Previous studies on inter-ethnic differences in anti-malarial responses have

shown that the Fulani have higher spleen rate than their neighbours 113, 114, are less infected 98,

101, 102and more immunologicaly responsive to malaria infection 99, 100. We observed higher

numbers of the Fulani individuals presenting enlarged spleen than the Dogon. This was in

contrast to other parasitological indexes (prevalence, density and diversity) where there were

no statistically significant differences between the two groups. Spleen enlargement was

associated with the presence of P. falciparum infection only in the Fulani, suggesting a

different role of spleen in response to malaria infection amongst these communities.

IL-4 and IL-10 have been shown to play roles in parasite clearance 115. Thus,

polymorphisms in their genes which can lead to variable production of the respective

cytokines 116, 117 could affect the parasite counts and other related malariometric indexes.

Infection prevalence was higher in Fulani individuals carrying the IL-4 T allele than the CC

genotype carriers. The IL-4 T allele has been associated with higher anti-malarial antibody

levels in the Fulani. This might indicate that the presence of low grade infections in T allele

carriers may induce a constitutive production of antibodies. However, further studies are

needed to confirm this. The Fulani IL-4 CC genotype carriers had significantly lower parasite

counts as compared to Dogon individuals carrying the same genotype.

In a study performed in Burkina Faso 112, the allele and genotype frequency of

IL-4 -590 T/C in the Fulani and two sympatric tribes (Mossi and Rimaibe) was determined

and related to anti-malarial antibody levels. It was found that the IL-4 T allele was related to

higher anti-malarial antibodies only in the Fulani and not in the other two tribes. This was

interpreted as the IL-4 -590 SNP was not functional and probably linked to an unknown

18

factor(s) which is the actual element regulating susceptibly/resistance to malaria. A

pronounced difference in IL-4 T/C allele frequency was noted between non-Fulani from Mali

(present study) and Burkina Faso 112. Here we observed a significantly lower parasite density

amongst the CC genotypes in the Fulani as compared to Dogon individuals having the same

genotype. All together, these data suggest that the resistance factor(s) may be linked to the C

allele and might be polymorphic in the non-Fulani. The malaria transmission intensity varies

between the study areas in Burkina Faso and Mali. This may contribute to the variable

selection of certain genetic traits involved in immune response to malaria.

When correlating malariametric indexes to the IL-10 -1087 A/G genotypes, we

did not observe any significant differences in malariometric indexes between the different

genotype carriers in any of the studied groups.

Study II

Multiplicity of infection (MOI) is the average numbers of different P.

falciparum strains simultaneously infecting a single host. MOI can be determined using the

polymorphic genes as markers. Genotyping of the highly polymorphic merozoite surface

protein 2 (msp2) gene is the standard to define MOI 118.

MOI increases as the transmission level increases 119, 120. In line with this, we

found higher MOI at the end of the transmission season as compared to dry season.

Previous studies have suggested that high parasite counts increases the

probability of detecting higher number of different parasite strains co-infecting an individual 121, 122. As expected we noted a positive correlation between MOI and parasite density.

Individuals living in malaria endemic areas eventually develop immunity to

malaria. The age at which the clinical immunity is acquired, depends on the level of exposure

and thus differs according to the transmission intensity. The peak MOI is usually seen at this

age 123. Studies in holoendemic areas have suggested that MOI is age-dependent 119, 123-126. In

contrast to this and in line with studies conducted in a mesoendemic area in Senegal 127, we

found that MOI was not being influenced by age. In areas with intense transmission, anti-

malaria specific immunity is achieved faster and at younger ages 128, whereas in areas with

less intense malaria transmission the age of peak MOI (development of immunity) is higher.

Hence, the lack of age-dependence of MOI observed here could perhaps be because of the

low transmission intensity in our study area.

Some studies have suggested that a high MOI protects against malaria 129-131,

while others have correlated a high MOI to a higher risk to succumb to clinical malaria 132, 133.

19

Our observation contradicts with both these suggestions, as we did not find any relation

between MOI and malaria morbidity. These discrepancies may in part be due to the different

genotyping protocols (with variable sensitivity and accuracy) used in the different studies.

RBC variants have long been known to confer protection against severe malaria.

The influences of these genetic disorders on the MOI have so far not been well investigated.

Here we reported that the MOI did not increase in α-thalassaemic children, indicating that

MOI is influenced by α-thalassaemia. Mockenhaupt et al. have shown that α-globin genotype

did not affect the MOI in symptomatic subjects 134. This suggests that α-thalassaemic RBCs

may protect against invasion by certain parasite strains but when infection is established, α-

thalassaemia does not protect against malaria disease.

G6PD deficiency showed a similar effect on MOI as α-thalassaemia. G6PD

deficient iRBC are known to be removed from circulation at early stage of infection 31. This

results in a better control of parasite growth which might lead to a lower increase in MOI at

the end of transmission season. This suggests that the protective effects on MOI seen in G6PD

deficient- and α-thalassaemic children act through different mechanisms.

Blood group O has been known to protect against cerebral malaria 5. However,

no reports are available on the influence of the ABO blood group system on MOI. No impact

of the different blood groups on MOI was noted in this study. This indicates that ABO

antigens may be involved in clinical outcome of malaria but do not affect the MOI.

Our data on the impact of sickle cell trait on MOI are in line with those reported

by Konate et al. 135. Both studies did not observe any effect of this trait on the MOI. This

contradicts with reports from Gabon 136, where asymptomatic AS subjects had higher MOI

than individuals with normal Hb. This suggests that the effects of HbAS on MOI might be

influenced by other factors such as transmission intensity or age of the study groups.

20

Concluding remarks and future perspectives Study I

In conclusion, our results revealed that; the Fulani and the Dogon are genetically

distinct, spleen enlargement was associated with the presence of parasite only within the

Fulani, the Fulani CC genotype carriers had significantly lower parasite density as compared

to the Dogon individuals carrying the same genotype, IL-4 -590 T allele was correlated with

higher parasite prevalence within the Fulani tribe, the difference in spleen rate amongst the

groups was seen only between the IL-4 T allele carriers of the two communities, IL-10 -1087

A/G SNP was not associated with malariometric indexes in any of the tribes.

To better clarify the association between the IL-4 -590 T/C SNP and

malariometric indexes and also to confirm its role in susceptibility/resistance to malaria, we

will determine the allele and genotype distribution amongst symptomatic and asymptomatic

subjects; relate it to clinical presentations, parasitological indexes, splenomegaly and anti-

malarial IgG antibodies. Samples were collected at the end of rainy season year 2005.

Study II

Taken together our data revealed that MOI; was not age-dependent, was correlated with

parasite density, increased after the transmission season, was not influenced by ABO system

and HbAS. However, both α-thalassaemia and G6PD deficiency affected the MOI.

To further investigate the associations between MOI and development of

clinical immunity, we will relate the MOI to the level of anti-MSP2 antibody (total IgG and

IgG subclasses) before and after the transmission season in the same study population.

21

Acknowledgment

I would like to express my sincere gratitude to:

Professor Marita Troye-Blomberg for all your supports, always having time for me

and being patient with my non-stop confusions and questions.

All seniors and colleagues at the department especially Margareta Hagstedt and Gelana

Yadet for always being ready to help.

Former and present PhD students especially; Nina-Maria, Salah, Anna-Karin and

Petra for nice time we shared in and out of university.

Shanie, Camilla, Lisa and Halima for being very good friends.

Mohammad E. Vafa and Sara Madadi; my first and best teachers, the most precious

treasures one could ever have. Without your love and support I would be nothing.

Mehri Vafa and Ali Nadjafi, for simply being the best friends, and being there anytime

I need you.

All my dear siblings, for being with me through all my way, despite miles of distance.

Shiva, for all tears and smiles we shared during years.

با ارزشترين , اولين و بهترين معلمينم, والدين عزيزم. محمد اسماعيل وفا و سارا مددىبا قدر دانى از

.همه وجود من از عشق و حمايت شما ست. سرمايه ام

همراه من بوده وارهمحبتشان هم, مكانىبا تشكر فراوان از همراهى خانواده عزيزم آه عليرغم فاصله

. وهست

به اول سخن داديم دستكاه به آخر قدم نيز بنماى را

22

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