charles arama thesis

Charles Arama Page i

Doctoral thesis from the Department of Immunology, Wenner-Gren Institute, Stockholm

University, Sweden

Novel immunization strategies and interethnic differences in

response to malaria infection

Charles Arama

Stockholm 2012

Charles Arama Page ii

All previously published papers were reproduced with permission from the publishers

© Charles Arama, Stockholm Sweden 2012

ISBN 978-91-7447-450-3

Printed by Universitetsservice AB, Stockholm 2012

Distributed by Stockholm University Library

Charles Arama Page iii

To the memory of my father, Antandou Jean Arama

(25th Anniversary of his death)

Charles Arama Page iv

SUMMARY

A better understanding of the innate immunity, specifically the role of antigen-presenting cells (APCs) in host resistance to malaria is essential to unravel the complex interactions between the host and the parasite. Such knowledge would improve the design of malaria vaccines. A protective malaria vaccine should induce both high levels of neutralizing antibodies and strong T-cell responses. The Plasmodium falciparum circumsporozoite protein (CSp) is a leading pre-erythrocytic vaccine candidate. However, the relative short-lasting immunity conferred by CSp remains the major issue. Mycobacterium bovis Bacillus Calmette-Guérin (BCG) has excellent adjuvant activity and has been utilized as a vector to deliver vaccine candidate antigens. We assessed the immunogenicity of a recombinant BCG-expressing CSp (BCG-CS) as a malaria vaccine candidate by in vitro and in vivo experiments. Immunization of BALB/c mice with BCG-CS augmented the numbers of dendritic cells (DCs) in draining lymph nodes and in the spleen. The activation markers MHC-class-II, CD40, CD80, and CD86 on DCs were significantly upregulated by BCG-CS as compared to wild-type BCG (wt-BCG). In vitro stimulation of bone marrow-derived DCs and macrophages with BCG-CS induced IL-12 and TNF-α production. BCG-CS induced higher phagocytic activity in macrophages as compared to wt-BCG. In addition, BCG-CS induced CSp-specific antibodies and IFN-γ-producing memory cells. Taken together, our results demonstrate that BCG-CS is highly efficient in activating innate immune responses and that it could effectively prime the adaptive immune system. Heterologous prime–boost approaches using vectors are optimal strategies to improve a broad and prolonged immunogenicity of malaria vaccines. We have demonstrated in BALB/c mice that priming with a replication-defective human adenovirus serotype 35 (Ad35) vector encoding CSp (Ad35-CS), followed by boosting with BCG-CS, maintained antibody responses and significantly increased levels of long-lived plasma cells (LLPC) and IFN-γ-producing cells in response to CSp peptides. The increased number of IFN-γ-producing cells induced by the combination of Ad35-CS/BCG-CS and the sustained type 1 antibody profile, together with high levels of LLPCs, may be essential for the development of long-term protective immunity against liver-stage parasites. Fulani and Dogon, two sympatric ethnic groups living in northeastern Mali, are characterized by a marked difference in the susceptibility to P. falciparum malaria. We investigated whether APCs obtained from Fulani and Dogon children exhibited differences in terms of activation status and toll-like receptor (TLR) responses during malaria infection. We observed decreased activation of APCs and markedly suppressed TLR responses in Dogon children as compared to Fulani. These findings suggest that APCs and TLR signaling may be of importance for the protective immunity against malaria observed in the Fulani.

In conclusion, the results presented in this thesis provide new insights that could facilitate a rational design of novel vaccines against malaria, which still remains one of the most devastating tropical diseases. Furthermore, the results elicit some immunological bases of the APC activation underlying the differences in host susceptibility to malaria infections.

Charles Arama Page v

LIST OF PAPERS

This thesis is based on the following papers, which are referred to by their Roman numerals in

the text:

I. Arama C, Waseem S, Fernández C, Assefaw-Redda Y, You L, Rodriguez A,

Radošević K, Goudsmit J, Kaufmann SH, Reece ST, Troye-Blomberg M. A recombinant

Bacillus Calmette-Guérin construct expressing the Plasmodium falciparum circumsporozoite

protein enhances dendritic cell activation and primes for circumsporozoite-specific memory

cells in BALB/c mice. Vaccine 2011; Oct 5. [Epub ahead of print]

II. Arama C, Assefaw-Redda Y, Rodriguez A, Fernández C, Corradin G, Kaufmann SH,

Reece ST, Troye-Blomberg M. Heterologous prime–boost regimen adenovector 35-

circumsporozoite protein vaccine/recombinant Bacillus Calmette-Guérin expressing the

Plasmodium falciparum circumsporozoite induces enhanced long-term memory immunity in

BALB/c mice (submitted manuscript).

III. Arama C*, Giusti P*, Boström S, Dara V, Traore B, Dolo A, Doumbo O, Varani S,

Troye-Blomberg M. Interethnic differences in antigen-presenting cell activation and TLR

responses in Malian children during Plasmodium falciparum malaria. PLoS One. 2011; 6(3):

e18319. * These authors contributed equally to this work.

Charles Arama Page vi

TABLE OF CONTENTS

SUMMARY .................................................................................................................................................................. iv LIST OF PAPERS ..........................................................................................................................................................v ABBREVIATIONS ..................................................................................................................................................... vii I. BACKGROUND .........................................................................................................................................................1 1.1. Introduction to malaria .............................................................................................................................................1 1.2. Parasite life cycle .....................................................................................................................................................2 1.3. The disease ...............................................................................................................................................................6 1.4. Malaria immunology ................................................................................................................................................7 1.5. Effector cells of the innate immune system .............................................................................................................8 1.5.1. Granulocytes .........................................................................................................................................................8 1.5.2. Monocytes-macrophages ......................................................................................................................................9 1.5.3. Dendritic cells .....................................................................................................................................................11 1.5.4. Natural killer cells ...............................................................................................................................................12 1.5.5. Natural killer T cells ...........................................................................................................................................13 1.6.1. Cell-mediated immunity .....................................................................................................................................15 1.6.2. Antibody-mediated immune responses ...............................................................................................................17 II. RELATED BACKGROUND ..................................................................................................................................20 2.1. Ethnic differences in West Africa and clinical implications in malaria ................................................................20 2.2. Activation of DCs by microbial components and induction of protective immunity ............................................21 2.3. Repertoire of PRRs on DC subsets ........................................................................................................................22 2.3.1. Toll-like receptors on DCs ..................................................................................................................................22 2.3.2. C-type lectin receptors on DCs ...........................................................................................................................24 2.3.3. Nucleotide oligomerization domain-like receptors on DCs ................................................................................25 2.4. Vaccines against malaria ........................................................................................................................................26 2.4.1. Transmission-blocking vaccines .........................................................................................................................27 2.4.2. Pre-erythrocytic vaccines ....................................................................................................................................27 2.4.3. Blood-stage vaccines ..........................................................................................................................................29 2.5. Design of vaccines through targeting DCs in vivo .................................................................................................30 2.6. Adjuvants and delivery systems for malaria vaccines ...........................................................................................32 2.7. The prime–boost strategy: tools to improve immunogenicity of malaria vaccines ...............................................33 III. PRESENT STUDIES ..............................................................................................................................................35 3.1. General aims ..........................................................................................................................................................35 3.2. Specific aims ..........................................................................................................................................................36 3.3. Materials and methods ...........................................................................................................................................36 3.4. Results and discussion ...........................................................................................................................................37 3.5. Overall Conclusions ...............................................................................................................................................46 3.6. Future investigations ..............................................................................................................................................47 IV. Acknowledgements .................................................................................................................................................49 V. References ................................................................................................................................................................52 VI. ANNEXES ..............................................................................................................................................................86

Charles Arama Page vii

ABBREVIATIONS

ADCI Antibody-dependent cellular inhibition ACT Artemisinin-based combination therapy APC Antigen-presenting cells BCG Bacillus Calmette-Guérin BDCA Blood dendritic cell antigen BMM Bone marrow-derived macrophage BMmDC Bone marrow-derived myeloid dendritic cells BMpDC Bone marrow-derived plasmacytoid dendritic cells CD Cluster of differentiation CMI Cell-mediated immunity CLR C-type lectin receptors CSp Circumsporozoite protein CTL Cytotoxic T lymphocyte DC Dendritic cells DC-SIGN DC-specific intercellular adhesion molecule-3 (ICAM-3)-grabbing non-integrin EIR Entomologic inoculation rate GPI Glycosylphosphatidylinositol anchors GM-CSF Granulocyte macrophage colony stimulating factor Hz Hemozoin HLA Human-leukocyte antigen IFN Interferon Ig Immunoglobulin IL Interleukin IRF Interferon regulatory factor ITAM Immunoreceptor tyrosine-based activation motif ITIM Immunoreceptor tyrosine-based inhibitory motif LPS Lipopolysaccharide LSA Liver stage antigen

MCP Monocyte chemoattractant protein M-CSF Macrophage colony stimulating factor mDC myeloid dendritic cell MHC Major histocompatibility complex MIP Macrophage-inflammatory protein MSP Merozoite surface antigen MyD88 Myeloid differentiation primary response gene 88 NF-kb Nuclear factor kappa beta NK cells Natural-killer cells NKT cells Natural-killer T cells NLR Nucleotide oligomerization domain-like receptors NO Nitric oxide PAMPs Pathogen-associated molecular patterns PBMC Peripheral mononuclear cells pDC plasmacytoid dendritic cell PFEMP P. falciparum erythrocyte membrane protein polyI:C Polyinosinic: polycytidylic acid PRR Pattern-recognition receptors RAP Rhoptry-associated protein ROS Reactive oxygen species TCR T-cell receptor TGF Transforming growth factor Th Helper-T cell TLR Toll-like receptor TNF Tumour necrosis factor TRAF TNF receptor associated factor TRAP Thrombospondin-related anonymous protein TRIF TIR-domain containing adapter-inducing interferon-β WHO World health organization

Charles Arama Page 1

I. BACKGROUND

1.1. Introduction to malaria

Malaria remains one of the most devastating infectious diseases threatening people living in

the tropical part of the world, especially young children and pregnant women. Human malaria

is caused by five different species of Plasmodium parasites: P. falciparum, P. vivax, P. ovale,

P. malariae and P. knowlesi [1]. Malaria is a mosquito-borne disease and hence it can be

controlled at the level of both humans and mosquitoes. Despite major efforts to control the

disease, approximately 781 000 deaths occurred in 2009 in children under the age of five in

sub-Saharan Africa [2]. The control of this scourge requires a multifaceted approach, including

insecticides, chemotherapy and development of cheap, affordable and efficacious malaria

vaccines. Because of the large gap in our understanding of P. falciparum infection biology it is

difficult to identify the right vaccine candidate among the thousands of potential P. falciparum

antigens constitutes [3]. This gap can be filled, but only with adequate research support and the

commitment of experts in all facets of P. falciparum immunobiology. While significant

advances have been made in vector control strategies and the effectiveness of artemisinin-

based combination therapy (ACT), a licensed malaria vaccine is still not available. In addition,

current malaria control and elimination programs face notable heterogeneity of transmission

dynamics of malaria in endemic areas, including differences in parasite, vector, human, social

and environmental factors.

To date, vaccination remains the most effective method of preventing infectious diseases and

represents the most relevant contribution of immunology to human health. The phenomenal

success of vaccines against polio, smallpox, measles, diphtheria, tetanus, rabies etc.,


demonstrates the potential of this approach in reducing the global burden of infectious diseases

and complete eradication of smallpox in humans [4]. However, despite these remarkable

successes, there are major logistical and technical challenges that still remain to be solved.

There is an urgent need to develop efficient malaria vaccines that might hopefully be an

important tool for the malaria elimination and eradication programs.

1.2. Parasite life cycle

Malaria parasite development requires mosquitoes and human hosts to complete its life cycle.

The parasite undergoes several developmental stages on its way to cause disease in humans.

During its two-host life cycle, P. falciparum undergoes ten morphological transitions in five

different host tissues [5]. The cycle in humans includes:

The pre-erythrocytic stage, which is the first stage of infection in humans where sporozoites

are inoculated to infect the hepatocytes.

The erythrocytic stage, which is the asexual reproduction of the parasite in the blood that

causes the clinical symptoms of the disease.

The gametocyte stage, which enables the sexual reproduction of the parasites in the mosquito

and further transmission.

The pre-erythrocytic stage begins when an infected female Anopheles mosquito inoculates

sporozoites into the skin or into the blood stream of humans during a blood meal. Sporozoites

circulate transiently in the blood stream before invading hepatocytes, where an asexual cycle

occurs. It is estimated that mosquitoes transmit fewer than 100 sporozoites per bite [6, 7].

Recent studies have shown that sporozoites can remain for up to 6 hours at the site of injection


[8], and that one-third of those leaving the injection site may enter the draining lymph nodes

via the lymphatic vessels [9]. The capacity of each of these sporozoites to yield an asexual

erythrocytic-stage infection is low. In humans, at least bites of five P. falciparum-infected

mosquitoes are necessary to assure that 100% of the volunteers get infected [10]. When

sporozoites reach the liver parenchyma, they continue to migrate through several hepatocytes

before they finally infect one. This migration seems to be beneficial for malaria infections in at

least two different ways: by activating sporozoites for infection and by increasing the

susceptibility of host hepatocytes [11]. After a week, the rupture of merosomes within the lung

microvasculature [12] releases thousands of infectious merozoites into the bloodstream, where

they invade circulating erythrocytes and initiate the clinically important intra-erythrocytic

cycle of the asexual replication during the following 48 hours. This stage is responsible for all

the clinical symptoms associated with the disease. After 24-32 hours, when young parasites

mature from rings to the trophozoite stages, infected erythrocytes adhere to endothelial cells in

the microcirculation of various organs (sequestration) causing cerebral malaria if sequestrated

in the brain. The trophozoites mature into schizonts, which finally rupture and release 16-32

daughter merozoites that invade fresh red-blood cells to perpetuate the asexual life cycle. As a

survival strategy, blood-stage parasites have been shown to infect, survive, and replicate within

CD317+ dendritic cells (DCs) and small numbers of these cells release parasites infectious for

erythrocytes in vivo in a murine model [13].

Some of the parasites inside erythrocytes differentiate into male and female gametocytes. It

remains poorly understood which factors stimulate gametogenesis [14]. However,

environmental factors, drugs and innate immune factors have been reported to influence

gametogenesis [15, 16]. Without treatment, most patients with P. falciparum malaria will


develop gametocytemia within 10 to 40 days after the onset of the parasitemia [17]. Upon

ingestion by a feeding female mosquito, the male and female gametes undergo fertilization in

the mosquito midgut to form a zygote and subsequently a motile ookinete. The ookinetes

penetrate the midgut epithelial cells and rest between the midgut epithelial cells and the basal

lamina to form oocysts. The oocysts undergo a complex asexual development phase, which

eventually generates infective sporozoites that can be introduced into the human host at the

next blood meal, thereby perpetuating the continuation of the parasite life cycle.


Figure 1: P. falciparum life cycle (J Clin Invest. 2010; 120 (12): 4168–4178, reprinted with

permission from the ASCI).


1.3. The disease

Clinical manifestations of P. falciparum infection are induced by the asexual stages of the

parasite that develop in the red-blood cells. Uncomplicated malaria is defined as the absence of

symptoms of severe malaria. The main symptoms often encountered in malaria are fever,

headache, fatigue, abdominal discomfort muscle and joint aches, chills, perspiration anorexia

and vomiting.

In non-immune individuals, the clinical manifestation is more patent and a minority of these

can become severe and life threatening. Ranges of discrete and overlapping disease syndromes

are manifested with complex etiologies. Those dying of malaria can have complications in

single-organ, multiple-organ or systemic involvement [18]. The patterns of the syndromes

depend markedly on the age and on previous immunological experience of the host [19]. In

areas of high malaria transmission, infants and young children are the most vulnerable. They

are more susceptible to life-threatening disease, which consists of metabolic acidosis (which

leads to respiratory distress), cerebral malaria (CM) and severe malarial anemia (SMA).

However, in areas of lower transmission, primary infections might occur in adulthood in which

severe disease often involves additional disturbances, such as renal failure, pulmonary edema,

shock, jaundice, hemoglobinuria, high fever and hyperparasitemia [20]. Thus, transmission

dynamics and host age are important determinants of disease severity, together with host

genetics and immunological responses.


1.4. Malaria immunology

In general, residents of malaria-endemic areas eventually develop some immunity to the

disease. This type of immunity is slow to acquire and requires repeated parasite exposure to be

maintained [21, 22]. The immune responses induced in humans by malaria parasites are

complex and vary depending on the level of endemicity, epidemiological and genetic factors,

host age, parasite stages and parasite species [23-25].

In humans, the immune system is generally divided into innate and adaptive responses that

cooperate together in order to combat pathogens. The innate immune system is rapid and

employs cells that directly sense the environment by germ line-encoded pattern recognition

receptors (PRRs). Cells that belong to the innate immune system include mainly

monocytes/macrophages, DCs, granulocytes and natural killer (NK)- and γδ T-cells. These

cells initiate the adaptive immune response, for example via the release of cytokines and

antigen presentations. In contrast, the adaptive immune response settles slowly, involves gene

rearrangement and clonal selection of T- and B-cell receptors that allows specificity and the

development of immunological memory. During a malaria infection, innate and acquired

mechanisms are induced against both liver and erythrocytic stages and their involvement in

protection and pathogenesis will be highlighted. Today, advances in immunological methods

and flow cytometry allow for different approaches to study malaria immunology in detail.

However, the role of the different cell populations and understanding their relevance in vivo

are often difficult to elucidate. The functional roles of various immune mechanisms are usually

based on an accumulation of data obtained from different approaches using in vitro and in vivo

studies, animal models, immuno-epidemiological and genetic studies.


Experimental models, such as various combinations of rodent Plasmodium species and inbred

mouse strains, have been utilized to mimic human malaria infections. However, no single

rodent model replicates all of the features of human malaria in terms of either pathology or

immune responses [26]. It is also important to consider that there are some differences between

the human and mouse immune system, including functional differences in NK cells, DCs, and

γδ T-cell subsets [27], which are key players in the innate immune response. In this regard,

primate models that are phylogenetically closer to humans may represent an interesting

alternative to rodent models.

1.5. Effector cells of the innate immune system

1.5.1. Granulocytes

Granulocytes (or polymorphonuclear leukocytes) are circulating leukocytes, which are

characterized by the presence of granules in their cytoplasm. Granulocytes express receptors to

which the Fc part of immunoglobulin (Ig) can bind. The role of granulocytes during malaria

infection remains elusive. The neutrophils are the most investigated so far. It has been shown

that neutrophils are able to phagocytize P. falciparum merozoites, and that opsonins such as

complement and antibodies present in sera from malaria-immune individuals were required in

this process [28, 29]. Moreover, cytokines such as tumor-necrosis factor alpha (TNF-α),

interferon gamma (IFN-γ), granulocyte-macrophage colony stimulating factor (GM-CSF) and

interleukin (IL)-1β, significantly induce the capacity of neutrophils to phagocytize merozoites

[30]. It has also been shown that neutrophils can inhibit the multiplication of the parasite

probably via anti-microbial components [31].


1.5.2. Monocytes-macrophages

Monocytes originate in the bone marrow and are released into the peripheral circulation. They

constitute 5-10% of the leukocytes in human peripheral blood and are morphologically and

functionally heterogeneous [32]. The initial marker for the identification of monocytes was the

expression of CD14. The combination of CD14 and CD16 allows the identification of two

subsets of monocytes: CD14++CD16- cells and CD14+CD16+ cells. Functionally, the

CD14+CD16+ cells appear to produce more pro-inflammatory cytokines and have higher

antigen presentation capacity than the CD14++CD16- monocytes [33]. Monocytes are important

effector cells in innate immunity. After their release from the bone marrow, monocytes

circulate in the blood before they enter tissues where they differentiate into macrophages.

Macrophages are generally grouped in subpopulations with different functions depending on

the mode of activation. The classically activated macrophages (M1) develop in response to

IFN-γ and microbial products. They are identified through their increased endocytic functions,

ability to present antigens and produce pro-inflammatory cytokines [34]. The alternatively

activated macrophages (M2) are further subdivided into three subsets according to their

functions. M2a macrophages are activated by anti-inflammatory cytokines, like IL-4 and IL-

13. The function of M2a cells includes secretion of chemokines that recruit eosinophils,

basophils and T cells to the site of infection and promote anti-inflammatory immune responses

[34]. M2b cells are induced upon stimulation with lipopolysaccharide (LPS) or IL-1β. These

cells promote the induction of type 2 adaptive immune responses, which result in activation of

T helper (Th)-2 cells producing anti-inflammatory cytokines. Finally, the M2c macrophages

are activated by IL-10, transforming growth factor (TGF)-β or glucocorticoids, and their


functions include down regulation of pro-inflammatory cytokines and increased scavenging

activity [35].

Many studies indicate that monocytes/macrophages play an important role in both cell-

mediated and humoral mechanisms against malaria infection [36, 37]. In fact,

monocytes/macrophages secrete soluble factors such as pro-inflammatory cytokines and

release nitric oxide (NO) or reactive oxygen species (ROS). It has been shown that the malaria

glycosylphosphatidylinositol (GPI) anchor activates monocytes to release TNF-α and IL-6 [38]

by triggering toll-like receptors (TLR) 2 and TLR4 [39]. Recent findings have provided a role

for malaria derived GPI also in TLR-mediated cell signaling in macrophages [40, 41].

In contrast, other data have also shown that monocyte/macrophage functions appear to be

impaired during malaria infection. In fact, hemozoin, which is a by-product generated after

hemoglobin degradation by the parasite impairs both antigen presentation and

immunomodulatory functions of the monocytes/macrophages [42, 43]. Other studies have also

shown that pigmented monocytes are negative correlates of protection against severe and

complicated malaria in Ugandan children [44].

All together, these results suggest that monocytes/macrophages play a key role in the early

clearance of the parasite. In addition, during the later phases when the acquired immunity has

been initiated, monocytes/macrophages may contribute to the clearance of parasites by the

antibody-dependent cellular inhibition (ADCI) mechanism [45].


1.5.3. Dendritic cells

Dendritic cells are professional APCs that respond to pathogen-associated molecular patterns

(PAMPs). They represent the first line of defense in the immunological battlefront in tissues.

DCs originate from the bone marrow and have a common progenitor cell, generating two main

lineages, the myeloid and lymphoid subsets [46, 47]. Myeloid cells expand into two major

lineages, macrophages and DCs. These cells leave the bone marrow as precursor cells and

populate the tissues. In secondary lymphoid organs, such as the spleen, the Fms-related

tyrosine kinase 3 (Flt3) receptor regulates plasmacytoid (p) DC and myeloid (m) DC

generation from bone marrow progenitors [48]. It may also induce progenitor-cell

differentiation providing a constant low self-sustaining population of DCs [49, 50]. In addition,

inflammatory DCs are sometimes recruited from monocytes during the course of the

inflammatory process both in the tissues and also directly from the blood [51, 52].

Lymphoid-lineage DCs derive from bone marrow-derived lymphoid precursors and colonize

the primary and secondary lymphoid tissues (thymus, spleen, liver and lymph nodes) and

reside there. DCs have a more defined migratory role in transporting antigen to the secondary

lymphoid tissues than macrophages that may live and die in situ after recruitment to the

tissues. In humans, it has been suggested that this difference in behavior between macrophages

and DCs is related to the expression of cell surface adhesion receptors such as CD312 [53].

DCs are fundamental for initiating immune responses by educating B and T cells, the effector

cells of the adaptive immunity. The existence of different DC subsets and their plasticity are

prominent determinants to the complexity of the DC system. Indeed, the different subsets are

distributed in peripheral tissues and in the blood and display different microbial receptors,


surface molecules and cytokine secretion, all of which influence the immunological response.

In response to inflammatory stimuli, DCs mature and are able to initiate T-cell responses. A

hallmark of DC maturation is down-regulation of endocytosis, which is assumed to restrict the

ability of mature DCs to capture and present antigens encountered after the initial stimulus

[54]. The maturation of DCs is a process in which morphological changes occur on their

surfaces such as expression of activation markers and chemokine receptors.

1.5.4. Natural killer cells

NK cells are described as a type of lymphocytes, which are mainly found in the peripheral

blood, the spleen and in the bone marrow. They constitute an important component of the

innate immune system. Although it is well documented that NK cells play a major role in

killing cells infected by viruses and in the rejection of tumors, their role in protective immunity

against malaria infection remains only partially elucidated. During human malaria infection,

activation of NK cells in vivo has been inferred from the observation that NK-cell activity was

increased in malaria-infected children and that this activity was correlated with parasitemia and

plasma levels of IFN-γ [55]. Another study has shown that NK cells are the first cells to

respond by producing IFN-γ after in vitro exposure of human peripheral blood mononuclear

cells (PBMCs) to P. falciparum infected red-blood cells (iRBC) [56]. In contrast, others have

shown that NK cell secretion of IFN-γ in response to iRBC is not detected in all malaria-naive

or previously exposed donors [57]. These authors showed that NK cells comprise only a small

proportion of the in vitro IFN-γ-producing cells, and that the depletion of NK cells does not

affect the IFN-γ production following 24 hours exposure to live late stage iRBC [57]. Instead,

the predominant source of innate IFN-γ was attributed to γδ T-cells expressing NK receptors

[57]. Moreover, the same group has shown in another study in Papua New Guinea that high


levels of ex vivo IFN-γ production were associated with protection from high parasite densities

and clinical disease [58]. The release of IFN-γ by NK cells seems to require both a direct

contact between iRBC and NK cells, and on the presence of accessory cells (monocytes and

myeloid DCs), while TGF-β suppressed NK-cell activation [59]. Altogether, these data

highlight an important role of NK cells in the early immune response against Plasmodium

parasites.

1.5.5. Natural killer T cells

Natural killer T (NKT) cells are a recently described subset of innate immune cells that have

features of both T and NK cells [60]. Their ability to rapidly secrete large amount of cytokines

upon activation allows them to bridge the innate and the adaptive immune response [61]. NKT-

cell activation leads to downstream recruitment and activation of DCs, NK cells, CD4+ and

CD8+ T cells. There is accumulating evidence that NKT cells play a role in protective

immunity against malaria. One study showed that the humoral immune response against the

circumsporozoite protein (CSp) was strongly diminished in CD1d-deficient mice compared to

wild-type mice [62]. Since the CD1d is a lineage commitment marker of NKT cells, mice

lacking this marker have a deficiency in NKT cells. The study further demonstrated that GPI

purified from the asexual blood stages of P. falciparum was able to stimulate murine CD4+

NKT cells in vitro, suggesting that CD4+ NKT cells can act as helper-T cells to facilitate the

production of anti-CSp antibody by B cells in vivo [62]. The role of NKT cells as helper T cells

in the regulation of anti-malarial humoral responses in vivo was later confirmed by two

independent studies [63, 64]. Overall, NKT cells do not seem to have a clear physiological role

against pre-erythrocytic stages. However, once the artificial ligand α-galactosylceramide (α-

GalCer) activates NKT cells, they display an inhibitory effect against the development of liver


stages of malaria in vivo [65]. This anti-plasmodial activity of α-GalCer-activated NKT cells

seems to be mediated by IFN-γ, since the activity was abolished in mice lacking IFN-γ or IFN-

γ receptors.

1.5.6. Gammadelta T cells

Gammadelta (γδ) T cells are defined as lymphocyte subsets that have several innate cell-like

features, which allow their early activation following recognition of conserved stress-induced

ligands. The γδT cells interact with a wide range of cell types, including innate immune cells,

T cells and B cells, as well as non-immune tissue cells. Such interactions might trigger

regulatory and cytotoxic responses of γδT cells. They also induce innate and adaptive

immunity, and can promote tissue repair and wound healing [66]. They share common

characteristics with both NK and NKT cells, such as the capacity to secrete high amount of

pro-inflammatory cytokines and the cytolytic activity against both tumors and pathogens.

Increasingly, it is accepted that γδT cells form part of the protective immune response directed

against P. falciparum. It has been shown that γδT cells inhibit the growth of the asexual blood

stages of P. falciparum by the granule exocytosis-dependent cytotoxic mechanism [67, 68].

Moreover, studies have shown that P. falciparum infection induces expansion of γδT cells to

reach up to 30% of circulating T cells in humans [69-71], suggesting their potent role in the

clearance of the parasites [68].


1.6. Adaptive immunity

The adaptive immune system develops as a specific response to pathogens. Both humoral and

cellular components of the adaptive branches contribute to the protective immune responses.

1.6.1. Cell-mediated immunity

In the context of cell-mediated immunity (CMI), cytotoxic T cell (CTL) responses are

mediated by CD8+ T cells, while the T helper (Th) cell responses mainly involve CD4+ T cells.

In general, CD8+ T cells respond to peptide fragments presented in association with class I

major histocompatibility complex (MHC) molecules, while CD4+ T cells recognize peptides in

association with MHC class II molecules present on APCs. Based to the cytokine profile

produced by CD4+ T cells, they can be subdivided into various subsets of Th cells such as the

Th1 and Th2 types [72]. The Th1 cells are specialized producers of pro-inflammatory

cytokines such as IFN-γ and IL-2, while the Th2 cells produce anti-inflammatory cytokines

like IL-4 and IL-13, which regulate specific antibody responses. CMI induced by malaria

infections, may protect against both pre-erythrocytic and erythrocytic stages. In addition to

these cells, the role of Th17 cells and regulatory T cells (Tregs) remains an interesting research

topic with regard to protection against malaria.

The central role of CD8+ T cells in pre-erythrocytic immunity is more and more investigated

[73]. CD8+ T cells play an essential effector role and contribute to protection against severe

malaria, as shown in murine experimental models [74, 75]. Three major subsets of antigen-

encountered CD8+ T cells have been identified based on the expression of CD62L and CD127:

(i) central memory T cells (TCM) are defined as CD62L+ and CD127+; (ii) effector memory T

cells (TEM) which are CD62L¯ CD127+; (iii) effector T cells (TE) are negative for both markers


[76]. The ability of each subset to protect is dependent on the functional properties and the

type of infection [77]. In contrast, there are only few studies investigating the role of CD8+ T

cells in human malaria. The pioneering study demonstrating a protective role of CD8+ T cells

against human malaria was performed by Hill and colleagues, who showed that the human-

leukocyte antigen (HLA)-B53 MHC class I allele was associated with protection from severe

forms of P. falciparum infection in Gambian children [78]. They also identified HLA-B53-

restricted CD8+ T cells, which recognize a conserved nonamer peptide from the liver-stage

antigen (LSA)-1 among malaria-immune Africans. The HLA-B53-restricted CD8+ T cells

against several epitopes of LSA-3 and the exported protein 1 (Exp-1) were also identified to be

associated with protection against rodent malaria [79]. Interestingly, it was shown that the

HLA-A2-restricted CD8+ T-cell clone could be activated upon recognizing human hepatocytes

infected with a recombinant Vaccinia virus expressing the P. falciparum CSp [80].

Furthermore, individuals that were able to mount a significant CD8+ T-cell response against

irradiated sporozoites (IrSp) were protected against a parasite challenge [81]. However, more

efforts are needed to identify key antigens that can elicit protective CD8+ T-cell responses in

humans and thus provide more evidence to support a protective role for CD8+ T cells against

human malaria.

The regulatory and effector functions of CD4+ T cells are well established in both experimental

and human malaria [21, 82, 83]. In earlier studies, vaccinees receiving P. falciparum IrSp were

protected from malaria infection and elicited CSp-specific CD4+ T cells exhibiting a cytolytic

activity [84]. When CD4+ T-cell clones were isolated from volunteers immunized with a

synthetic peptide containing a universal CD4+ T-cell epitope of the CSp, it was found that Th1-

type CD4+ T-cell clones were more predominant and produced high levels of IFN-γ [85].


Others have shown that the protective immunity induced by RTS,S, the leading malaria

vaccine candidate, is linked to P. falciparum CSp-specific IFN-γ producing CD4+ and CD8+ T

cells [86]. After an experimental infection with P. falciparum, volunteers were protected

against malaria challenge with infected mosquitoes [87]. The proportion of T cells (mostly

CD4+ CD45RO+ memory T cells) producing multiple cytokines in response to iRBC was

significantly higher in the vaccinees. Interestingly, such IFN-γ, IL-2 and TNF-α

multifunctional Th1 cells have been defined as a promising immunological marker of

protection in other infectious diseases [88, 89]. Overall, a better understanding of the induction

of the CMI will contribute tremendously to the development of an effective and safe malaria

vaccine.

1.6.2. Antibody-mediated immune responses

B cells are considered to be the main player in humoral immunity and they are the only cells

capable of producing antibodies. B cells constitute 15% of peripheral blood lymphocytes. They

differentiate from hematopoietic stem cells in the bone marrow. In the secondary lymphoid

organs such as the spleen, germinal centers are important sites where B-cell responses are

initiated and the activated B cells proliferate. In response to antigens, three important B-cell

differentiation events take place within the germinal center: affinity maturation,

immunoglobulin isotype-switching and formation of memory B cells that can rapidly

differentiate into plasma cells on re-exposure to antigen. The plasma cells migrate into the

bone marrow where they reside and produce antibodies.

In residents of endemic areas, malaria infection induces strong humoral immune responses,

involving production of predominantly IgM and IgG as well as the other immunoglobulin


isotypes [21]. Several studies support a role for antibodies in the development of immunity to

the Plasmodium parasite in humans: (i) Passive transfer of IgG from immune serum into non-

immune humans can prevent infection in the recipient [90, 91]; (ii) Infants are protected

against malaria during the first few months of life by maternally transferred antibodies [92-94];

(iii) In longitudinal studies, the prevalence and levels of antibodies against defined malarial

antigens have been associated with resistance against disease [95-97]; (iv) Several

investigations have found an association between antibodies against ring-infected erythrocyte

surface antigen (Pf155/RESA) and low levels of parasitemia [98-101], and an association

between antibodies directed against the merozoite surface protein-1 (MSP-1) and parasite

clearance [96, 98, 102]. Other studies have reported on positive associations between low

levels of parasitemia and antibodies specific for the sporozoite CSp [103], MSP-1 [104], MSP-

2 [105], GPI [106], the rhoptry-associated protein 1 (RAP-1) [107, 108] and the variant antigen

P. falciparum erythrocyte membrane protein-1 (PfEMP-1) [109].

The main effector mechanisms of antibody-mediated immunity are: opsonization,

neutralization or blocking, activation of complement and antibody-mediated cell cytotoxicity

(ADCC). Antimalarial IgG subclasses play an important role in malaria immunity; in particular

cytophilic IgG1 and IgG3 have been related to protection [110, 111]. The suggested

mechanism by which these cytophilic antibodies are protective, involves their binding to the Fc

receptors on monocytes, leading to ADCI of parasite replication [45]. However, some reports

suggest a protective role of antimalarial IgG2, in which high level of IgG2 and low level of

IgG4 have been associated with human resistance to P. falciparum malaria [112]. A more

recent study has shown that FcγRIIa R131H genotype may influence the IgG subclass


responses related to protection against malaria and that H-allele carriers had higher anti-

malarial IgG2 levels in the Fulani [113].

Overall, data from field studies support an association between antibody responses and

immunity to malaria. However, it is important to consider that antibody responses may be

influenced by factors such as age, previous exposure to parasite, transmission rate, host genetic

factors, as well as the statistical power of the study. Furthermore, it is likely that multiple and

diverse immune mechanisms are required for full protection.


II. RELATED BACKGROUND

2.1. Ethnic differences in West Africa and clinical implications in malaria

The Fulani from Sudan and West Africa constitute a particularly interesting ethnic group

because of their lower susceptibility to P. falciparum infections as compared to other

sympatric populations, the Dogon in Mali [114] and the Mossi and Rimaibé in Burkina Faso

[115, 116]. These sympatric groups live under the same conditions of meso-endemic (Mali)

and hyperendemic (Burkina Faso) transmission in the Sudanese-savannah area. Numerous

studies have shown obvious interethnic differences in the susceptibility and immune responses

to malaria, the Fulani are showing lower infection and disease rates and stronger humoral

responses to various P. falciparum antigens than other sympatric ethnic groups [117, 118].

More recently, genetic polymorphism studies have shown that the IL4- 590 T allele is present

in 75% of the Fulani, twice the frequency compared to their neighbors, raising the possibility

that in this ethnic group it marks out a protective haplotype [119, 120]. The association

between the polymorphism and elevated serum antibody levels in the Fulani would be

consistent with this. However, the lack of association in non-Fulani indicates that the

polymorphism is not functional by its self in this context [121]. The higher immune reactivity

to malaria in the Fulani further supports the existence in this ethnic group of unknown

immunogenetic factors, probably involved in the regulation of humoral and/or other immune

responses. These differences should be considered in the development of anti-malaria vaccines

and in the evaluation and application of malaria control strategies, such as drug treatment.

Also, the possibilities of combining inter- and intra-ethnic comparisons to characterize critical

determinants of malaria immunity in natural settings will be more useful in the control of this


infection. It is known that protective immunity against malaria depends on the magnitude and

the quality of certain components of the innate immune system needed for initiation of the

adaptive arm. However, we are far from having a clear understanding of the mechanism for

this relative resistance. There are many basic aspects of the P. falciparum infection

immunobiology that are poorly understood and many others that have not been investigated

yet, limiting our knowledge on the mechanisms involved in the acquisition of protective

immunity to malaria.

2.2. Activation of DCs by microbial components and induction of protective

immunity

Pathogen-associated molecular patterns are recognized by DCs through at least three PRR

families of molecules: the Toll-like receptors, the cell surface C-type lectin receptors (CLRs)

and the intracytoplasmic nucleotide oligomerization domain (NOD)-like receptors (NLRs)

[122]. The activation of DCs can occur directly by microbes through their PRRs or indirectly

by capture of apoptotic/necrotic products of other cells dying in response to microbial

exposure. In addition, microbes can also induce certain types of cells, such as epithelial cells,

fibroblasts and other cells of the innate immune system to secrete cytokines capable of

activating DCs. The process of DC maturation comprises:

(a) changes in morphology such as the loss of adhesive structures, cytoskeleton reorganization

and the acquisition of high cellular motility [54]

(b) loss of endocytic/phagocytic receptors


(c) secretion of chemokines according to the type of immune cells that are needed for such

responses [123]

(d) up-regulation of costimulatory molecules such as CD40, CD80 and CD86 [124]

(e) translocation of MHC class II compartments to the cell surface [125, 126]

(f) secretion of cytokines that differentiate and shape the effector immune responses [127].

Immature DCs sense different microbial stimuli and convey this information to cells of the

adaptive immune system [128]. Thus, the type of adaptive immune response induced is highly

dependent on the nature of the activating stimuli that they receive from DCs.

2.3. Repertoire of PRRs on DC subsets

2.3.1. Toll-like receptors on DCs

TLRs are expressed on a wide repertoire of cells including DCs. Until now, ten TLRs have

been described in humans and thirteen TLRs have been identified in mice [129]. TLRs are

divided into subfamilies such as the subfamily of TLR1, TLR2, TLR4 and TLR6 that

recognizes mainly lipidic molecules, whereas TLR3, TLR7, TLR8 and TLR9 recognize nucleic

acids and related structures [130]. TLR1, TLR2, TLR4, TLR5 and TLR6 are expressed on the

cell surface, whereas TLR3, TLR7, TLR8 and TLR9 are expressed in intracellular

endolysosomal compartments. Such specific localization might provide insight into the

mechanism by which DCs avoid spontaneous activation by self nucleic acids [131, 132]. The

differential expression of TLRs by distinct DC subsets might shape the type of the immune

response and subsequently its regulation. This also allows DC subsets to respond differently to

microbes. Thus, in humans, blood pDCs express TLR1, TLR6, TLR7, TLR9 and TLR10, while


mDCs express TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 and TLR10 [133, 134].

The dermal interstitial DCs (intDCs) express TLR2, TLR4 and TLR5 that recognize bacterial

PAMPs [135]. For that reason, dermal intDCs are considered as a DC subset specialized for

bacterial recognition in the skin.

Importantly, certain differences are found between human and murine DC subsets in terms of

their TLR expression and some aspects of the innate systems [27]. In the mouse, TLR9 is

expressed both on mDCs and pDCs [136], while TLR9 is only expressed in human pDCs

[133].

The recognition of different PAMPs delivers distinct molecular signals, thereby resulting into

distinct types of DC maturation and therefore distinct immune responses [137]. For example,

TLR3 stimulation by polyinosinic: polycytidylic acid (polyI: C) induces DCs to produce TNF-

α; TLR2 stimulation by peptidoglycan increases IL-10 secretion [138]. Likewise, Escherichia

coli LPS induces IL-12 secretion through TLR4 sensing and promotes a Th1 type of response.

In contrast, Porphyromonas gingivalis LPS activates DCs through TLR2 to secrete IL-10,

which eventually polarizes towards a Th2-type of response [139]. For a better coordination of

the immune response, TLRs cooperate to increase DC activation, thereby ensuring efficient

control of the different PAMPs. For example, synergistic action of TLR3/TLR4 and

TLR7/TLR8 or TLR9 stimulates DCs to produce IL-12p70 and triggers a Th1 type-polarizing

program in DCs [134].


2.3.2. C-type lectin receptors on DCs

DCs express constitutively on their surface a large group of CLRs that can bind the

carbohydrate moiety of glycoproteins [140, 141]. Like TLRs, distinct DC subsets express

different CLRs. Thus, blood DC antigen 2 (BDCA2) is specific for pDCs [142],

langerin/CD207 is mainly expressed on Langerhans cells (LCs) [143], and DC-specific

intercellular adhesion molecule-3 (ICAM-3)-grabbing non-integrin/CD209 (DC-SIGN) is

preferentially present on intDCs and macrophages [144].

CLRs operate as anchors for several pathogens including viruses, bacteria, parasites and fungi

that allow their internalization. In addition to this function, CLRs appear to behave as adhesion

molecules between DCs and other cell types. The interaction between several CLRs and their

ligands is still unclear. Some CLRs contain motifs in their cytoplasmic domains and deliver

activation/suppression signals [145, 146]. This means that the sensing of CLRs depends on the

type of the signal-transducing pattern associated to this specific CLR. For instance, DC-SIGN

[143], the asialoglycoprotein receptor (ASGPR) [147] and dectin-1 [148] are found to contain

an immunoreceptor tyrosine-based activation motif (ITAM), which allows activation signals to

DCs. In contrast, the dendritic-cell immunoreceptor (DCIR) [149] and the myeloid inhibitory

C-type lectin receptor (MICL) [150] carry an immunoreceptor tyrosine-based inhibitory motif

(ITIM), therefore predicting an inhibitory function. Similar to TLR expression, CLR

expression differs between humans and mice [146]. Multiple DCIRs (DCIR1-4) have been

identified in the mouse, while only one single DCIR has so far been found in humans [122].

This is one of the reasons to that it can be difficult to extrapolate results obtained from mouse

studies to humans.


2.3.3. Nucleotide oligomerization domain-like receptors on DCs

NOD-like receptors constitute a specific group of intracellular proteins that play a critical role

in the regulation of the host’s innate immune response. NLRs act by triggering the downstream

nuclear factor-κB and the mitogen-activated protein kinase signaling pathways [151]. NLRs

control the activation of inflammatory caspases, which are proteins involved in apoptosis, a

process of programmed cell death that occurs in multicellular organisms. In humans, the NLR

family consists of 23 members, the majority of which belonging to two large protein

subgroups: NODs (NOD1-5) and NALPs (NACHT, leucine-rich repeat and pyrin-domain-

containing proteins 1-14) [122]. These molecules are mainly involved in host-pathogen

interactions and in inflammatory responses.

The expression of the majority of NLR members in human DCs is poorly understood. Some

studies have shown that NOD1 and 2 are expressed in the cytosol of macrophages and DCs

[152]. Recently, one study showed that NOD2 stimulation induces autophagy in DCs [153].

The distribution profiles of NALP1 and NALP3 vary in different DC subsets. All DC subsets

express NALP1 and NALP3, but the highest levels of NALP1 are found in LCs [154].


2.4. Vaccines against malaria

The purpose of vaccination strategies is to induce immunological responses in order to attack

and disrupt the replicative amplification of the malaria parasite. The development of a vaccine

against malaria is an active research area that encounters enormous challenges. As the parasite

proceeds from a sporozoite through the liver stage to the replicative cycling of the blood stage,

it undergoes morphological changes and displays antigenic variation. These features allow the

parasite to evade protective immune responses. As a result, the acquisition of long-term sterile

immunity, which is often associated with recovery from many other infectious diseases, does

not occur in malaria-infected individuals. Despite these difficulties, the most compelling

evidence that vaccination against malaria is feasible has come from laboratory studies in which

attenuated sporozoites induced sterile protective immunity in rodents, monkeys and human

subjects [155].

An ideal malaria vaccine requires three essential features:

(i) multiple components that will induce an effective immune response to the different

stages of the malaria infection (sporozoites, infected hepatocytes, the asexual and

sexual stages)

(ii) multiple epitopes that are restricted to presentation by different MHC molecules in

order to overcome genetic diversities and antigenic variation

(iii) multi-immunogenicity inducing more than one type of immune response, including

cell-mediated and humoral components. Such a multicomponent vaccine would

increase the probability of a more sustainable and effective host response [156].


Malaria vaccine research pursues intensive investigations on three groups of vaccine

candidates targeting different stages in the parasite's life cycle: (i) transmission-blocking

vaccines; (ii) pre-erythrocytic vaccines; (iii) blood-stage vaccines (Figure 2).

2.4.1. Transmission-blocking vaccines

Transmission-blocking vaccines target antigens on gametes, zygotes and ookinetes in order to

prevent parasite development in the mosquito midgut [157]. The aim of these vaccines is to

induce antibodies against the sexual stage antigens in order to prevent the development of

infectious sporozoites in the salivary glands of the Anopheles mosquitoes [157]. Importantly,

transmission-blocking vaccines would not protect the recipient from contracting malaria, but

could be helpful in preventing the spread of the disease. These vaccines are intended to protect

entire/selected communities from infection. The leading vaccine candidates from this subgroup

contain the P. falciparum ookinete surface antigens Pfs25 [158] and Pfs28 or their P. vivax

homologues Pvs25 and Pvs28 [159, 160]. Transmission-blocking vaccines could be important

tools for malaria elimination.

2.4.2. Pre-erythrocytic vaccines

Liver-stage vaccines are designed to prevent malaria in the human host. But because of the

high rate of replication of the sporozoites one single parasite may be sufficient for the infection

to proceed to the blood stage. Therefore a liver stage vaccine must be 100% effective in order

to protect humans with no natural immunity. Such vaccines include those containing whole

killed sporozoites and those based on antigenic portions of the circumsporozoite proteins

[161]. So far, the most advanced vaccine candidate is RTS,S, which consists of a truncated

circumsporozoite protein of P. falciparum directly fused to the hepatitis B surface (S) antigen.


This vaccine has shown good safety with 30-50% protection in human field trials in Africa

[162]. Since the protection was relatively short-lasting, one of the challenges is now how to

improve the protection by manipulating the immune response of the host. The mechanism by

which RTS;S confers protection against blood-stage disease remains poorly understood. It

seems that RTS,S induces protection against clinical malaria by temporarily reducing the

number of merozoites emerging from the liver. This may allow prolonged exposure to

subclinical levels of asexual blood-stage parasites, therefore boosting the naturally acquired

blood-stage immunity [163]. Recently a novel antigen, the “Cell-traversal protein for ookinetes

and sporozoites” (CelTOS) was identified as an essential protein for the traversal of the

malaria parasite in both the mammalian and the insect host [164]. In a murine model, the

CelTOS antigen is a potentially interesting pre-erythrocytic vaccine candidate, which induces

complete sterile protection against sporozoite challenge [165].

The case for a whole sporozoite vaccine has its origins in the early finding that mice could be

protected by inoculation of P. berghei irradiated sporozoites by the bite of mosquitoes [166].

Today, the whole-organism vaccine gains a new interest thanks to the recent highly successful

human trial, using experimental sporozoite inoculation under chloroquine prophylaxis [87].

Moreover, four of the six volunteers mounted sterile protection against a re-challenge infection

two years later [167]. Sporozoites attenuated by targeted gene disruption are being increasingly

evaluated as whole parasite vaccines. Among the whole sporozoite vaccines, the choice is in

favor to the late liver stage-arresting genetically attenuated parasites. In fact, recent data have

clearly demonstrated that the late liver stage-arresting genetically attenuated parasite engenders

high levels of cross-stage and cross-species protection and complete protection when

administered by intradermal or subcutaneous routes [168]. This avoids the potentially


problematic irradiation step in the manufacturing process, but other challenges of

manufacturing and delivering a viable cryopreserved whole parasite vaccine remain [169].

Therefore, increased research into other potential malaria vaccine antigens and strategies for

their delivery is essential.

2.4.3. Blood-stage vaccines

Blood-stage vaccines are designed to elicit two main types of mechanisms: anti-invasion and

anti-disease [170]. The principle behind these strategies is that if a vaccine could block the

invasion of the erythrocytes by merozoites, it would prevent malarial disease. Today, certain

blood-stage antigens are in clinical trials. These are mainly apical membrane antigen 1

(AMA1) [171], erythrocyte-binding antigen–175 (EBA-175) [172], glutamate-rich protein

(GLURP) [173, 174], merozoite surface protein 1 (MSP1) [175], MSP2 [176], MSP3 [177] and

serine repeat antigen 5 (SERA5) [178]. They are highly expressed on the surface of the

merozoite. AMA1 and MSP1 did not demonstrate efficacy in African children [171, 175],

probably due to their highly polymorphic structures [179]. Efforts to enhance the vaccine

efficacy of AMA1 and MSP1 with novel adjuvants [180, 181], using viral vector prime-boost

strategies [182], or by combining AMA1 and MSP1 [183] are under investigations. The

extensive parasite genetic diversity and the selective pressure exerted on the host´s immune

response remain to be major factors to consider in the development of effective blood-stage

vaccines [184].


Figure 2: The schematic representation with emphasis on targets for the three

types of malaria vaccines.

2.5. Design of vaccines through targeting DCs in vivo

The acquisition of knowledge about DC biology in vivo in humans and in murine experimental

models will hopefully lead to the development of a new generation of vaccines against

infectious diseases such as an effective vaccine against malaria [185]. However, novel

strategies have been proposed to directly target the antigens to DCs in vivo. Various DC

surface molecules have been considered as targets, as they are involved in the internalization of


antigens for further processing and presentation on both MHC class I and class II molecules

[126]. In mice, some studies have shown that targeting the antigens in the absence of DC

activation results in the induction of tolerance [186, 187]. On the contrary, targeting the

antigen to DCs via the DEC-205 receptor resulted in the generation of improved immune

responses against a variety of antigens [188]. The expression of different targets on murine DC

subsets implies various functional outcomes. In particular, the mouse CD8+DEC205+ DC

subset is specialized in MHC class I presentation, whereas the CD8-DCIR2+ DC subset is

specialized in MHC class II presentation [189]. Moreover, these subsets of DCs use different

mechanisms for CD4+ T-cell priming. The DEC205+DCIR2-CD8+ DCs prime T-cells to

produce IFN-γ in an IL-12-independent manner, while the DEC205-DCIR2+CD8- DCs prime T

cells in an IL-12-dependent manner [190]. On mouse DCs, other surface molecules have been

extensively studied as targets for vaccination purposes including a CLR that binds to heat

shock proteins (HSP) 70 [191], the mannose receptor, the FcγRII/III [126] as well as the CD40

molecule [192]. Regarding human DCs, the functional biology of these cells remains to be

explored. However, some studies have been conducted involving anti-DC-SIGN coupled to the

key-hole limpet hemocyanin (KLH) [193], anti-DEC205 with the gag protein [194] and the

anti-mannose receptor with human chorionic gonadotropin hormone [195]. Considerable

efforts are needed in the field of DC targeting that may hopefully result in potential vaccines

against infectious diseases.


2.6. Adjuvants and delivery systems for malaria vaccines

A range of adjuvant formulations and viral/bacterial vectors is available for use in combination

with antigen candidates. An ideal adjuvant is a component that enhances the potency, longevity

and quality of specific immune response to antigens, with minimal toxicity to the recipient.

Adjuvants have been divided into two major groups according to their component sources,

physiochemical properties or mechanisms of action, namely: (i) immunostimulants such as

TLR ligands, cytokines, saponins and bacterial exotoxins that directly act on the immune

system to increase responses to antigens and (ii) vehicles, such as mineral salts, emulsions,

liposomes, virosomes and biodegradable polymer microspheres that present vaccine antigens

[196].

Effective protection against different pathogens requires distinct types of immune responses

and therefore the role of adjuvants is of great importance. Indeed, the nature of the adjuvant

can determine the specific type of immune response, which may skew towards CTL responses,

antibody responses or particular types of Th responses. Although the concept of adjuvants was

known since the early vaccination trials, it is only recently that their mechanism of action is

getting understood [197]. The adjuvants that have been approved for human clinical trials or

tested in malaria vaccines include alum, saponins (Quil-A, ISCOM, QS-21, AS02 and AS01),

montanides (ISA51, ISA720), the oil-in-water emulsion MF59™, the monophosphoryl lipid A

(MPL®), virus-like particle, virosomes and cholera toxin. Today, more and more

immunostimulatory oligonucleotides (CpG motifs), imidazoquinolines (imiquimod,

resiquimod) and others TLRs ligands are used as adjuvants due to their properties to induce

Th1 type immunity and CTL responses [198]. Moreover, some adjuvants such as virosomes,


liposomes and immunostimulating complexes (ISCOMs) seem to promote priming of CD8+ T

cells and antigen cross-presentation.

The M. bovis Bacillus Calmette-Guérin (BCG) has been used for a century as a tuberculosis

vaccine. The long-lasting adjuvant property and the intrinsic immunostimulatory activity of

BCG form the basis of its registration as a treatment for bladder carcinoma [199]. Previous

studies have shown that vaccination with recombinant BCG-based vaccine modalities results in

strong humoral and cell-mediated responses, elicited by the host to a variety of foreign

antigens inserted into and expressed by the recombinant vector [200]. These studies highlight

BCG as a very promising vaccine vector and provide a rationale to utilize it for malaria

vaccine development. Usually, multi-antigen vaccines are better favored as compared to

single-antigen vaccines, since high and broad immune coverage inhibits immune escape of

viruses and parasites. Therefore, the capacity of a recombinant BCG expressing CSp to induce

anti-CSp immunity is discussed in detail in paper I.

2.7. The prime–boost strategy: tools to improve immunogenicity of malaria

vaccines

Today, a novel strategy involving priming with DNA vaccines, such as adenovirus and

boosting with fowlpoxvirus (FPV) or modified vaccinia virus Ankara strain (MVA), has

resulted in the generation of good protection against infectious agents that currently pose great

problems for vaccine development. Several features of the vectors used in prime–boost

vaccination underlie their capacity to induce enhanced immune responses, particularly CMI.

The bacterial plasmid backbone of the DNA vector itself contains short, dinucleotide

sequences that strongly stimulate IL-12 production and may favor the development of CMI


against encoded vaccine antigens. Another critical element in the ability of DNA vaccines to

prime T cells for greatly enhanced secondary responses might be their persistent expression of

immunogenic proteins in vivo. This also promotes the phenomenon of affinity maturation of B-

cell responses, with steadily increasing antibody affinities arising after multiple exposures to

antigen. Furthermore, studies have also demonstrated that T cells may display higher average

affinities for MHC–peptide molecules after multiple exposures to antigen [201, 202].

Concerning malaria vaccines, major efforts are currently being made at developing optimal

heterologous prime-boosting systems. This issue has been thoroughly addressed in paper II.


III. PRESENT STUDIES

3.1. General aims

It is now generally accepted that the immunological control of P. falciparum infection will

require a vaccine that induces both high levels of neutralizing antibodies as well as a strong T-

cell response. Based on their ability to elicit strong antigen specific humoral and cellular host

responses, recombinant BCG and recombinant adenovirus vectors were identified as the best

antigen delivery systems. In the first part of this thesis, we assess the immunogenicity of the P.

falciparum circumsporozoite protein (CSp) expressed by the two delivery systems,

recombinant Bacillus Calmette-Guérin construct (BCG-CS) and Adenovector 35 (Ad35-CS) in

a murine model.

In malaria endemic areas, clinical immunity to P. falciparum malaria develops gradually over

years of repeated exposure. As previously mentioned, it is well established that the Fulani are

less susceptible and mount a relatively stronger immune response to malaria than their

sympatric neighboring ethnic groups, the Dogon. We hypothesize that the innate immune

responses might contribute as a driving force by constantly promoting the activation of the

adaptive immunity. In the second part of this thesis, we characterize the behavior of APCs and

their TLR-induced responses in naturally exposed children from ethnic groups with different

susceptibility to P. falciparum infection.


3.2. Specific aims

The specific aims of this thesis were to investigate:

the capacity of BCG-CS to induce activation of APCs in BALB/c mice and to prime the

acquired immunity (Paper I)

the quantity and quality of the immune responses induced by heterologous prime-boost

Ad35-CS/BCG-CS immunization in BALB/c mice (Paper II)

the behavior of APCs and their TLR-induced responses in naturally exposed children

from ethic groups with different susceptibility to P. falciparum infection (Paper III).

3.3. Material and methods

The material and methods that we used in our different studies are addressed in detail in each

paper.


3.4. Results and discussion

Paper I.

The P. falciparum antigen finally selected for incorporation into the vaccine vectors in our

study is the CSp. The rationale for choosing this antigen is based on the fact that upon entry of

P. falciparum into the vaccinated host, antibodies against CSp will neutralize sporozoites.

However, since the exposure of the sporozoites in the bloodstream is transient, CSp-specific T

cells would subsequently kill sporozoite-infected liver cells and prevent parasite propagation in

the liver.

We assessed the capacity of a recombinant BCG (BCG-CS) to induce activation of innate

immune responses in immunized BALB/c mice. Our data showed that the BCG-CS was highly

efficient in inducing innate immune responses by activating DCs to produce pro-inflammatory

cytokines, and to up-regulate costimulatory molecules both in vitro and in vivo. The uptake of

BCG-CS into bone marrow-derived macrophages (BMM) in vitro was slightly better than that

seen of wt-BCG. We also found that the BCG-CS induced significant production of TNF-α in

BMM as compared with wt-BCG. The role of pro-inflammatory cytokines such as TNF-α is

crucial in the inhibition of parasite growth in hepatocytes as well as in erythrocytes [203].

Furthermore, TNF-α induces production of ROS and NO in macrophages, which results in the

elimination of the pathogens [204]. It is well established in a murine model that early pro-

inflammatory responses are associated with protection against malaria [26].

Interleukin (IL)-12 is believed to be influential in protective immunity against malaria [205].

IL-12 is a multifunctional cytokine, acting as a key regulator of cell-mediated immune

responses through the differentiation of naïve CD4+ T cells into Th1 type cells producing IFN-


γ [206]. We found that BCG-CS immunization induced IL-12p70 production in bone marrow-

derived DCs (BMDCs). The fact that BMDCs obtained from BCG-CS-infected BALB/c mice

released significant levels of IL-12p70, suggests that this malaria vaccine candidate might be

potent to initiate the adaptive immune response via priming of naïve T cells.

Our results also showed that the BCG-CS induced the production of CSp reactive antibodies as

well as CS-specific IFN-γ production. Our findings are in agreement with a previous study that

showed high production of IFN-γ by spleen cells stimulated with rBCG expressing P. yoelii

MSP-1 [200]. Taken together, our data support the fact that BCG-CS could be a good malaria

vaccine candidate against the pre-erythrocytic stages, since it activates the innate immune

system, which then in turn promotes the CS-specific protective acquired immune system.

Antibodies specific to CSp have been shown to be involved in the neutralization of sporozoites

in the bloodstream [207, 208]. In addition, our data showed that BCG-CS induced CSp-specific

T-cell responses by producing IFN-γ, which is crucial for eliciting liver stage malaria specific

T-cell responses [82].

During malaria infections the innate immune system stimulates and instructs the adaptive

immune system. It does so by secreting appropriate cytokines and chemokines and by

expressing costimulatory molecules, such as CD40, CD80 and CD86 on the surface of APCs.

The most important APCs involved in this process are DCs who fight against infection in all

tissues [209]. Together, both the innate and the adaptive immunity function to provide an

optimal host defense and DCs are the key cells that link the innate and the adaptive immune

responses by inducing the activation of T and B cells. Upon immunization via the

subcutaneous (s.c.) route, DCs present at or recruited to the site of vaccination phagocytize

pathogens. They migrate into the draining lymph nodes, thereby playing important roles in


immune activation and dissemination of pathogens [210]. Results from our study also

confirmed that the population of DCs was expanded in the draining lymph nodes and the

spleen. Furthermore, the costimulatory molecules CD40, CD80 and CD86 were significantly

upregulated on the surface of the DCs recovered from the spleens of BCG-CS-immunized

mice. We observed that the peak of DC expansion was achieved at day 2 post-vaccination in

the spleen. This suggests that the BCG-CS efficiently induces the activation of an innate

immune response.

In order to mount an efficient protective immune response, it is crucial that the BCG is able to

grow in the lungs, and that it can disseminate into other organs such as the spleen [211, 212].

We investigated if there were differences between the BCG-CS and the wild type (wt)-BCG in

terms of uptake by macrophages and dissemination into different organs in vivo. In agreement

with earlier findings [212], we observed an increased capacity of BCG-CS to grow in the lungs

as compared to the wt-BCG after intranasal (i.n.) immunization of BALB/c mice. Taken

together, our results suggest that the use of recombinant BCG as vehicle for P. falciparum CSp

delivery might not require an adjuvant and thus could be an excellent system to be used safely

in humans.

Paper II.

In this paper we investigated the capacity of a heterologous prime-boost regimen, Ad35-

CS/BCG-CS, to induce specific antibody responses against P. falciparum circumsporozoite as

well as generation of long-lived plasma cells in BALB/c mice. Our main findings are that the

heterologous prime-boost regimen was highly efficient in inducing CSp-specific memory

immunity. This CSp-specific memory immunity was shown to be mediated by high levels of


IFN-γ-producing cells and by the induction of stronger long-lived plasma cell (LLPC)

responses against CSp.

For liver-stage malaria vaccines, CD8+ and CD4+ T-cell responses against intrahepatic parasite

antigens are crucial, and these can be induced by either experimental immunization or natural

infection [213]. Parasite-derived peptide epitopes are presented on the surface of infected

hepatocytes, which can be recognized by CD8+ T cells in association with MHC class I

molecules. This is likely the mechanism that is involved for the heterologous prime–boost

regimen Ad35-CS/BCG-CS. CD4+ T cells are known to play a role in liver-stage immunity,

since it has been shown that professional APCs are able to induce activation of Th1 responses

via cross-presentation of sporozoite antigens [214]. In addition, upon activation of T cells, a

cytokine cascade involving both IFN-γ and IL-12 is evoked, inducing intracellular killing of

the parasite through NO [215]. Our findings support the previous suggestion that IFN-γ is a

good surrogate marker of P. falciparum pre-erythrocytic stage protective immunity [216].

Although antibodies are considered to provide a key effector mechanism for blood-stage

immunity against malaria, in particular antibodies directed against conformational epitopes,

more evidence is needed to prove the involvement of humoral immunity in the maintenance of

protection against the liver-stage parasites. In this context, the ability of a vaccine to confer a

long-term immunity depends on both memory B cells and LLPCs [217, 218]. Our data clearly

show that sequential immunization with different delivery systems, i.e. the heterologous

prime–boost regimen Ad35-CS/BCG-CS, induced significantly stronger CSp-specific antibody

responses, and that the cytophilic antibodies such as IgG2a mainly mediated these responses.

Furthermore, we found that the heterologous prime–boost regimen Ad35-CS/BCG-CS induced

higher numbers of LLPCs as compared to the homologous prime–boost regimen BCG-


CS/BCG-CS, suggesting the maintenance of immunological memory to hepatic-stage parasites.

The essential role of LLPCs and memory B cells against Plasmodium has been demonstrated in

rodent models [219] and in people living in endemic areas [220, 221]. However, in individuals

who are chronically exposed to P. falciparum, atypical memory B cells are greatly expanded,

although it is uncertain if they are beneficial to the host or play a role in immune evasion of the

parasite [222].

In general, the development and delivery of an efficacious malaria vaccine remains a critical

tool for sustainable malaria control, and how protective responses are induced, regulated and

maintained following sporozoite inoculation are key considerations for malaria vaccine

development [12]. To tackle some of these issues, heterologous prime-boost immunization

approaches have been used as strategies for antigens delivery systems [182]. In this context,

immunogenicity and efficacy can be improved. Thus, the heterologous prime–boost regimens

utilizing distinct vectors are suggested as an optimal strategy to target the desired type of

protective immunity, and there are increased efforts to develop specific vectors not only to

generate specific CTLs and/or antibody responses, but also to promote T-cell help [223]. The

concept of heterologous prime-boosting arose when evidence showed that an immunization

with plasmid DNA followed by a viral vector, both encoding the same antigen, was more

potent for generating immune responses and provided better protective immunity to malarial

challenge than either the DNA or viral vector alone or administrating in opposite order [224].

In this study, we attempted to improve the immunogenicity of CSp-based malaria vaccine by

exploiting this concept.


Paper III.

In this study, we investigated whether APCs obtained from the two ethnic groups, Fulani and

Dogon differ in their activation profile and their cytokine responses upon TLR stimulation.

The major findings obtained from this study are:

(i) Specific subsets of DCs from the Fulani children were more activated and less

frequent in the periphery during P. falciparum infection, as compared to what was

seen for the Dogon children.

(ii) Differences in circulating monocyte frequency and activation were observed

between the Fulani and Dogon children and intra-ethnically between infected and

non-infected children.

(iii) P. falciparum infection induced suppression of TLR-stimulated cytokine responses

in infected Dogon but not in infected Fulani children.

(iv) P. falciparum infection induced a decrease in the TNF-α/IL-10 ratio of CpGA and

imiquimod stimulated responses of PBMCs from Dogon children but not those of

the Fulani.

In the Dogon children, P. falciparum infection induced an increased frequency of BDCA-3+

DCs and pDCs and decreased expression of costimulatory molecules. Conversely, P.

falciparum induced a significantly decreased frequency of BDCA-3+ DCs, pDCs, and up-

regulation of the activation markers HLADR and CD86 on circulating PBMCs of Fulani

children. These findings imply that the Fulani and Dogon children harbored distinct innate

immune responses against P. falciparum infection, as reflected by differences in the activation


of their APCs. This is in line with previous findings that the malaria parasites or the products

derived from their metabolism may impair the differentiation and function of mDCs [43, 225].

In addition, the HLA-DR levels are decreased on circulating DCs of P. falciparum-infected

children as compared to healthy controls [226]. Higher numbers of circulating BDCA-3+ DCs

have been detected in blood of severely infected children, combined with reduced stimulatory

ability, indicating that malaria may impair DC functionality and disclosing a modulatory role

for the BDCA-3+ DC subset in immunity [227, 228].

To gain a better understanding of the effect of P. falciparum infection on the behavior of

APCs, we performed ex vivo stimulation of PBMCs using different TLR stimuli and assessed

cytokine responses. The results obtained in the present study showed that the production of

pro-inflammatory cytokines was suppressed in infected Dogon children, while PBMCs of the

infected Fulani children were still able to produce significant levels of inflammatory cytokines.

This finding is consistent with previous observations, demonstrating that the Fulani harbored

higher proportions of malaria specific IFN-γ-producing cells compared to the Dogon [118].

More recently, investigations have shown that mononuclear cells from Fulani individuals

elicited over 10-fold stronger IFN-γ production following a 24-hour in vitro co-incubation with

asexual parasites than cells from the sympatric Dogon individuals. These responses were

highly specific for P. falciparum, since it was not seen in response to a panel of other human

pathogens [229]. In support of the latter finding, we also found interethnic differences in IFN-

γ, IFN-α, TNF-α, IL1-β, IL-6 and IL-10 between infected Dogon and Fulani children. The

Fulani children secreted higher levels of those cytokines upon TLR4, TLR7 and TLR9

stimulations. It is known that early production of IFN-γ is crucial for the control of parasitemia

during malaria infection. Furthermore, it was recently shown that the interferon regulatory


factor (IRF) polymorphisms correlated well with the ability to control parasitemia but not the

incidence of severe malaria in Burkina Faso [230, 231].

Recent studies suggest that TLR polymorphisms have been associated with an altered risk of

disease or disease severity in numerous infections [232-234]. However, only a few studies tried

to investigate the genetic polymorphisms of some relevant TLR involved in malaria [235-237].

In vitro evidence suggests that TLR2, TLR4 and TLR9 are important for the recognition and

signaling in response to P. falciparum ligands. Murine studies have shown that TLR2 and

TLR4 recognition of P. falciparum GPI are associated with an increased production of TNF-α

[238]. In addition, TLR9 signaling in response to hemozoin (a by-product of host hemoglobin

digestion by malaria parasites) upregulates TNF-α, IL-12p40, monocyte chemoattractant

protein (MCP)-1 and IL-6 production by DCs [239]. In humans, certain TLR9 single

nucleotide polymorphisms (SNPs) have been associated with altered IFN-γ levels in children

with cerebral malaria [240]. Understanding the polymorphism of these innate receptors and

their potential role in susceptibility to P. falciparum infection may provide insight into the

development of new drugs and vaccines against malaria.

The frequencies of both classical and inflammatory monocytes were increased both in Dogon

and Fulani children upon P. falciparum infection. These results are in accordance with

previous findings that a higher frequency of inflammatory monocytes was found in peripheral

blood of patients with acute malaria [241]. We also found that P. falciparum infection

suppressed monocyte function, as reflected by a reduced expression of activation markers on

classical and inflammatory monocytes both in Fulani and Dogon children. This might suggest

that P. falciparum alters the functionality of these cells in both ethnic groups. However,


interethnic differences were observed during malaria infection, as classical monocytes were

significantly more activated in infected Fulani than in infected Dogon children.

Taken together, our findings suggest that the prevalence and the activation status of APCs, as

well as the TLR-induced cytokine responses differ between Fulani and Dogon children in the

course of a P. falciparum infection. It seems likely that the parasites induce suppression of the

APC function in the Dogon children, resulting in a deficient and delayed activation of the

acquired immune responses, while the Fulani children elicit an early innate immune response

that might contribute to the protection against malaria infection.


3.5. Overall Conclusions

The results presented in this thesis provide further insight into innate immune responses

induced by the putative BCG-CS malaria vaccine candidate construct to activate APCs in

BALB/c mice. The BCG-CS was potent to prime the adaptive immune response by inducing

CSp-specific IFN-γ production in T cells and CSp-specific antibody responses. Furthermore,

we have demonstrated that BCG-CS is driving the adaptive immune response toward a Th1

type response, which reflects an increase in the IgG2a/IgG1 ratio in sera of mice that received

the homologous prime–boost BCG-CS/BCG-CS regimen. Therefore, the accumulated evidence

presented here strengthens the use of BCG as a vehicle and adjuvant for the induction of

protective immunity in malaria vaccine development.

The development of novel malaria vaccines that promote effective cellular immunity is

required for the control of malaria for which classical humoral-based vaccines have been

ineffective. Prime–boosting has emerged as a powerful approach for establishing cellular

immunity, and our results have demonstrated the efficacy of prime–boost vaccines in

generating protective immunity in animal models. The use of the heterologous prime–boost

concept and the insights gained from the preclinical vector vaccines point towards the potential

of different vectors in developing effective malaria vaccines, as well as for illuminating the

incredible complexities of the effectors of memory and protective immunity. Further

development of these vaccine strategies depends on advances in our basic understanding of the

mechanisms how to better target APCs that promote the induction of a long-term protective

immunological memory.


Furthermore, our study reveals some immunological bases for the differences in host

susceptibility to malaria infections induced by the activation of APCs. The observation that the

innate immune system is primed differently in Fulani and Dogon children during P. falciparum

infection could not have been predicted. In the Dogon children, most immune responses appear

to be strongly suppressed, thus leading to an immune state that is often described as

“immunoparalysis”. By the time a patient with P. falciparum infection is diagnosed, the innate

immune system is markedly immunosuppressed. Hence, the Dogon children who probably

represent how most people would respond to malaria are more vulnerable to the occurrence of

the clinical symptoms. Just the opposite appears to occur during acute malaria in the Fulani

children. Even though they were also infected, they elicited efficient innate immune responses,

which can potentially hamper the appearance of clinical symptoms. Clearly, a better

understanding of the innate immune responses is critical for the rational design of novel

therapies and malaria vaccines. Such effective vaccines would not only prevent a great number

of cases in the short term, but will probably also be necessary for the optimal control or

elimination of the disease in the long term.

3.6. Future investigations

With regard to the findings from Paper I and II, we will continue to investigate the efficacy of

rBCG by carrying out a P. falciparum sporozoite challenge study. To further characterize this

rBCG, we will evaluate the immune responses in immunodeficient mice such as MyD88 and

TLR2 knockout mice since the TLR2 signaling pathway might be involved in its recognition.

TLR2 has been suggested as an important PRR, which is involved in the induction of

protective immunity against mycobacterial infection. Therefore, the investigation of the TLR2

signaling pathway will hopefully elucidate the underlying recognition mechanism of the rBCG.


In order to better understand the importance of the different TLR-induced cytokine responses

in protection against malaria it is useful to investigate TLR4 and TLR9 polymorphisms in the

Fulani and the Dogon and to evaluate the risk factors involved in the outcome of the disease in

relation to these polymorphisms. It seems that TLR4-mediated responses to malaria in vivo and

TLR4 polymorphisms are associated with disease manifestation. Since migration of APCs to

lymphoid organs is driven by chemokines, investigations of chemokines and their receptors

during P. falciparum infection remains an exciting area of research in malaria immunology in

general and in particular between these two ethnic groups. This may lead to a better

understanding of the underlying mechanisms of protection against malaria.


IV. ACKNOWLEDGEMENTS

This work, carried out at the Department of Immunology, Stockholm University was supported

by grants from the European and Developing Countries Clinical Partnership (EDCTP CG-TA

05 40204_003), the Swedish Agency for Research and Development with Developing

Countries (Sida/SAREC), the BioMalPar European Network of Excellence (LSHP-CT-2004-

503578), EVIMALAR, from the European Community's Seventh Framework Program

(FP7/2007-2013 under grant agreement N° 242095) and from the European Community's Sixth

Framework Program (FP6/2007-037494, PRIBOMAL project).

I am thankful to all the people that have contributed with invaluable help and support during my PhD

study, be it academically, socially and spiritually.

I would like to acknowledge:

My supervisor Marita Troye-Blomberg, thank you for your strong support and for giving me the

opportunity to learn from you. Tack Marita (my “Swedish mother” a name given by my wife) for accepting

me in your team.

The seniors in the departments: Klavs Berzins, Carmen Fernández, Eva Severinson and Eva Sverremark-

Ekström. Thank you for your training in many aspects of immunology and for scientific interactions.

Stefania Varani, I am thankful for accepting to co-supervise the field study and my 20 points project

work. It was for me a real pleasure to work with you.


Mes superviseurs sur le terrain: Ogobara Doumbo et Amagana Dolo. Vous m´aviez apporté votre soutien

sans faille pour la réalisation de mes travaux au MRTC et sur les sites d´études. Recevez ma profonde

gratitude.

Au populations Dogon et Peulh de Manteourou, Binedama et environs. Merci pour votre participation

aux activités de recherche sur le paludisme.

Pablo Giusti my co-author and “my junior supervisor”, thanks for being such a nice friend to work with,

and for all the numerous discussions we had about research and life. I also enjoyed the nice summer time

with your parents in the archipelago.

Yohannes Assefaw-Redda and family, thank you for the academic and social activities we shared during

my study.

John Arko-Mensah, Salah Eldin Farouk, Nnaemeka Iriemenam Manijeh Vafa Homann, Amre Nasr, Lisa

Israelsson, Shanie Saghafian-Hedengren Halima Balogun and Family, Nancy Awah, Muhammad J

Rahman, Sabina Dahlström, Shahid Waseem, Sriwipa Chuangchaiya, Samad Ibitokou, Liya You,

Richards Bola, Bakary Maiga and Aminatou Koné: thanks for your friendship and help in academic and

social aspects during PhD journey.

My colleagues and friends at the department, Stephanie Boström, Ioana Bujila, Olga D Chuquimia Flores,

Maria Johansson, Ebba Sohlberg, Natalija Gerasimcik, Yeneneh Haleselassie, Sophia Björkander,

Jacqueline, Ulrika Holmlund and Dagbjort Petursdottir, thanks for providing the friendship and nice

atmosphere needed for research.

Margareta Hagstedt, Hedvig Perlmann and Ann Sjölund, I sincerely thank you for all technical issues and

for all your help during these years.


Gelana Yadeta and Anna-Leena Jarva, I am grateful for all administrative matters, and for organizing

some of the best social events at the department.

The animal facility staff, I thank you for the technical support.

The members of the PRIBOMAL project consortium from the Max Plank Institute for Infection Biology

(Berlin Germany) and from Crucell company (Crucell Holland B.V. Leiden The Netherland): I would like

to thank you for your invaluable collaboration and critical discussions.

Father Dominik Terstriep and the Catholic University Chaplaincy students, I am grateful for the spiritual

and social activities we shared.

A ma mère, mes sœurs et frères merci pour le soutien familial.

Enfin, à Assa Marthe et Erè Célestin, merci pour cette chaleur familiale et cette joie de sentir aimé. Je suis

reconnaissant pour tous vos sacrifices dans cette aventure. Que le Seigneur nous accorde la force et la joie

de nous supporter mutuellement. "Amba be wi tama".

To everyone whose name I missed in the acknowledgment, it does not mean that I think about you less, I

am grateful that you were part of my PhD journey.

Tack så mycket.


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VI. ANNEXES

charles arama thesis

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