Doctoral thesis from the Department of Immunology,

Wenner-Gren Institute, Stockholm University, Sweden

Immunological characteristics of recombinant fragments of

the Plasmodium falciparum blood-stage antigen

Pf332

Halima A. Balogun

Stockholm 2011

ii

These pictures were taken during inhibition assay with rabbit antibodies; they are infected

and non-infected red cells, stained with acridine orange. The picture above in

collaboration with another infected red cell, formed the parasites

probably because they escaped the antibody pressure (the parasites were exposed to

antibodies for 22 hours). The other pictures below are from parasites that were exposed to

antibodies for 42 hours, well they were not so lucky, and they ended up being the

parasites.

Well, more mysteries lie ahead in order to combat malaria.

The possession of knowledge does not kill the sense of wonder and mystery. There is always more mystery. - Anais Nin

Cover image captured & designed by Halima A. Balogun.

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............ I give all praise to Almighty Allah for giving me the strength, when I thought

this was not going to be possible.

To my precious Mum (Yeye ni Wura)

…………L.A.B (R.I.P)

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The first step towards knowledge is to know that we are ignorant.

- Richard Cecil

i

Abstract

An effective malaria vaccine might help in improving control strategies against malaria,

but the development of an effective vaccine faces challenges, such as the complexity of

interactions between the parasite and its hosts. The asexual blood stage antigen Pf332, a

megadalton protein, is transported across the parasitophorous membrane to the iRBC

membrane skeleton during schizogony, and has potentials as one of the proteins in

understanding the complex host-parasite interactions. The functional role of Pf332 in the

parasite’s life cycle is still not well defined, as different studies have assigned different

roles to this antigen. The interest in Pf332 as a possible target for parasite neutralizing

antibodies, evolved from previous studies demonstrating that Pf332-reactive antibodies

inhibits parasite growth in vitro. The presence of such significant antibodies during

natural P. falciparum infection also indicated that Pf332 has the ability to induce

protective antibodies.

In our first study we identified and characterized the immunogenicity of a C-

terminal region of Pf332. Immunological analyses carried out with this fragment revealed

that rabbit anti-C231 antibodies possess parasite in vitro inhibitory capabilities. In

another study, the functional activity of C231 specific antibodies was further confirmed

with human-affinity purified antibodies, where the antibodies inhibited late stage parasite

development, as evidenced by the presence of abnormal parasites as well as disintegrated

red cell membranes.

Using epidemiological data from a malaria endemic area of Senegal, we examined

the pattern of antibodies reactive to two different regions of Pf332 (C231 and DBL) with

regard to Ig classes and IgG subclasses. With both recombinant antigens, we observed

positive correlations between the IgG (R=0.706, p=<0.0001) and IgM (R= 0.711, p=

<0.0001) antibody levels against C231 and DBL. The distribution of the anti-C231

antibodies in the IgG subclasses, gave similar levels of IgG2 and IgG3. Correlation

studies showed that the levels of anti-C231 antibodies were associated with protection

from clinical malaria, which only reached significance with IgE. In contrast, the group

with high anti-Pf332-DBL-IgG3 was found to be protected from clinical malaria attack.

We hereby conclude that antigen Pf332 contains immunogenic epitopes, and is a

potential target for parasite neutralizing antibodies. The Pf332 protein should thus be

considered as a candidate antigen for inclusion in a subunit P. falciparum malaria

vaccine.

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Content

Abstract ............................................................................................................................... i

Content ............................................................................................................................... ii

List of included papers .................................................................................................... iii

Abbreviations ................................................................................................................... iv

1 Introduction ................................................................................................................. 1

1.1 Overview of malaria ................................................................................................ 1

1.2 Control measures against malaria ............................................................................ 3

1.3 The malaria parasite ................................................................................................. 5

1.4 Clinical disease and manifestations ......................................................................... 8

1.4.1 Disease pathogenesis .................................................................................. 9

2 Malaria and the human immune system ................................................................ 12

2.1 Innate immune response to malaria ....................................................................... 12

2.2 Pre-erythrocytic stage immunity in malaria ........................................................... 15

2.3 Erythrocytic stage immunity in malaria ................................................................. 16

2.3.1 Mode of action of antibodies..................................................................... 18

2.3.2 Immunoglobulins and immunity to malaria .............................................. 19

2.4 Immune evasion by malaria parasite...................................................................... 21

2.5 Malaria vaccines .................................................................................................... 23

2.5.1 Asexual blood stage vaccines.................................................................... 25

3 The infected red cell and its modifications ............................................................. 27

3.1 Antigens of the infected red cell ............................................................................ 30

3.2 Antigen 332 ............................................................................................................ 33

3.2.1 Related background .................................................................................. 33

3.2.2 Immune responses to antigen Pf332 ......................................................... 36

4 The Present Investigation ......................................................................................... 38

4.1 Preface.................................................................................................................... 38

4.2 Objectives .............................................................................................................. 39

4.3 Experimental approach .......................................................................................... 40

4.3.1 Study population ....................................................................................... 40

4.3.2 The Malaria Situation in Senegal ............................................................. 40

4.4 Results and discussion ........................................................................................... 42

4.4.1 Paper I ...................................................................................................... 42

4.4.2 Paper II ..................................................................................................... 43

4.4.3 Paper III & IV ........................................................................................... 45

4.5 Concluding remarks ............................................................................................... 49

4.6 Future perspectives ................................................................................................ 51

4.7 Other Publications .................................................................................................. 52

5 Acknowledgements ................................................................................................... 53

6 References .................................................................................................................. 56

iii

List of included papers

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

text by their roman numerals:

I. Balogun AH, Vasconcelos N-M, Lindberg R, Haeggström M, Moll K,

Chen Q, Wahlgren M and Berzins K. Immunogenicity and antigenic

properties of Pf332-C231, a fragment of a non-repeat region of the

Plasmodium falciparum antigen Pf332. Vaccine. 2009;28(1):90-7.

II. Balogun AH, Awah N, Farouk S, and Berzins K. Pf332-C231 reactive

antibodies affect growth and development of intra-erythrocytic

Plasmodium falciparum parasites. (Submitted under revision, 2011).

III. Israelsson E, Balogun AH, Vasconcelos N-M, Beser J, Roussilhon C,

Rogier C, Trape JF and Berzins K. Antibody responses to a C-terminal

fragment of the Plasmodium falciparum blood-stage antigen Pf332 in

Senegalese individuals naturally primed to the parasite. Clinical and

Experimental Immunology. 2008;152(1):64-71.

IV. Balogun AH, Awah N, Nilsson S, Rousillhon C, Rogier C, Trape JF,

Chen Q and Berzins K. Pattern of antibodies to the Duffy binding like

domain of Plasmodium falciparum antigen Pf332 in Senegalese

individuals. (Manuscript, 2011).

iv

Abbreviations

ABRA Acid-basic repeat antigen

ADCI Antibody-dependent cellular inhibition

AMA Apical-membrane antigen

APC Antigen-presenting cell

CD Cluster of differenciation

DC Dendritic cell

CM Cerebral malaria

DBL Duffy binding-like domain

GLURP Glutamate rich protein

GPI Glycosylphospatidyl inositol

His-tag Histidine tag

HZ Haemozoin

ICAM-1 Intercellular-adhesion molecule-1

IFN Interferon

Ig Immunoglobulin

IL Interleukin

iRBC Infected red-blood cell

MHC Major histocompatibility complex

MyD88 Myeloid differentiation primary response gene 88

NK Natural killer

MSP Merozoite-surface protein

PAM Pregnancy associated malaria

PDC Plasmacytoid-dendritic cell

P. f Plasmodium falciparum

PV Parasitophorous vacoule

RBC Red-blood cell

RESA Ring-infected erythrocyte surface antigen

SERA Serine-repeat antigen

SMA Severe malaria anaemia

TCR T-cell receptor

Th T helper

TLR Toll-like receptor

TNF Tumor necrosis factor

TRAP Thrombospondin-related anonymous protein

Treg Regulatory-T cell

1

1 Introduction

1.1 Overview of malaria

Despite the fact that humans have evolved to co-exist and survive with the malaria

parasite Plasmodium for a long period of time, malaria still remains one of the main three

infectious diseases in Africa (Fig. 1b). Malaria creates a global health concern with about

225 million clinical cases per year worldwide, with the vast majority of cases (about 85

%) in the African region, and approximately 1 million deaths annually (Alonso et al.,

2011). The heaviest burden of malaria lies mainly on non-immune individuals, pregnant

women, and children below the age of five years, residing in Sub Saharan Africa. Disease

manifestations in malaria, such as severe malaria anemia (SMA), pose socioeconomic

challenges and can be life threatening in the case of cerebral malaria (CM).

The malaria situation has aggravated since time immemorial, due to insecticide resistance

of the vector, the anopheline mosquito which transmits the parasite, as well as the

emergence of drug resistance in the parasite. Eradication of malaria has also been

hampered by other factors such as inappropriate public health facilities and bottlenecks in

vaccine development. The human host genetic factors have also been shown to play a

vital role in clearance of infection (Allison et al., 2009; Pasvol 2010; Giha et al., 2010).

The malaria parasite has a complex life cycle during which they infect humans and are

transmitted by anopheline mosquitoes. This has also hampered effective control strategies

and vaccine development. In malaria endemic countries, progress to reduce the burden of

malaria has been achieved to an extent by means of improved health care systems (WHO,

2010). However, malaria still needs great attention in areas of vector control, diagnostics

and treatment as impediments still lie in these fields, not exempting basic research on the

biology of the parasite and especially vaccine development, which may sooner or later be

a long-term solution to this acquainted enemy of man.

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Figure1a: The global distribution of malaria, www.cdc.gov.

Figure 1b: Distribution of malaria in Africa, www.mara.org.za.

3

1.2 Control measures against malaria

Malaria control has been a great challenge to malariologists, it requires an integrated

approach of the various preventive/control measures, which targets reduction of disease

incidence, prevalence, morbidity or mortality to an appreciable level. With the current

tools and state of knowledge at hand, strategies against obliteration of malaria may not be

achievable. One of the reasons is due to the fact that malaria is caused by five

Plasmodium species, which are being transmitted by more than 30 Anopheline mosquito

species (Alonso et al., 2011). These complexities result in diverse disease spectra in

different epidemiological settings. Till date, malaria eradication has utilized various

interventions, including control of the mosquito vector, use of therapeutic drugs and

chemoprophylaxis agents and prompt appropriate case management. Strategies that have

proven to have impact on reducing morbidity and mortality/burden of malaria are the

home management system (Pagnoni, 2009) and improved health care systems (RBM

partnership, 2008). However, eradication of malaria still faces enormous setbacks in most

malaria endemic countries.

In most endemic areas, the disease is treated using anti-malarial drugs, which are

important in early control of the attack, and the administration of these drugs has either a

prophylactic or curative effect. Increasing resistance of P. falciparum malaria to

antimalarial drugs poses a major threat to the global effort to roll back malaria, but now

emphasis is being laid on the use of drugs with a short half-life, which minimizes the risk

of development of resistance (WHO, 2010a). Resistance can be prevented, or its onset

slowed considerably, by combining antimalarials with different mechanisms of action

and ensuring very high cure rates through full adherence to correct dose regimens (WHO,

2010a). Mosquito vector control is an important control approach that should be

integrated with other malaria control measures. Significant effect has been observed with

the use of indoor residual spraying (IRS) and long lasting insectide treated nets (LLINs)

(Enayati & Hemingway, 2010), with these most malaria endemic areas have been able to

reduce vectorial capacity. Nothwithstanding, challenges are still encountered, as regions

with very high transmission areas will not benefit from this control measure, due to

4

increased mosquito resistance to insecticides. The global malaria control strategy adopted

by governments and W.H.O. emphasized the need for prompt and effective diagnosis,

which will aid appropriate treatment. However an effective health care system is very

important in this context, and such systems are required to sustain malaria intervention

programmes in malaria endemic countries.

Vaccines have been projected to be part of the control measures in malaria eradication,

but so far no effective vaccine against this disease has been developed. The reason for

this is entangled among various issues, such as breach in knowledge in the area of

parasite biology, whereby the molecular interactions between parasite, its human hosts

and vector host, has not been properly elucidated (Langhorne et al., 2008; The malERA

Consultative Group on Basic Science and Enabling Technologies, 2011). The complexity

of the parasite’s life cycle (Gardner et al., 2002; Florens et al., 2002) and extensive

antigenic variation (Sherf et al., 2008) also poses setbacks to malaria vaccine

development. Vaccines based on asexual blood-stage antigens (will be discussed in

section 2.5) may be effective at reducing parasite densities and provide protection against

clinical disease. In addition, understanding of the clinically silent stages preceding the

blood stage (sporozoite and hepatocyte stages), may help to improve the induction of

protective immune responses in vaccine development. Both approaches are being

integrated in vaccine development, but Plasmodium species are quite complex. While

working on vaccine development, emphasis on adjuvant development should not be

forgotten, as the unavailability of a wide range of potent adjuvants has been a bottleneck

in development of recombinant protein-based vaccines for malaria.

Amidst the decrease in malaria prevalence in some endemic areas, due to various

expanded control programmes, such as preventive mosquito control with ITN and indoor

insecticide spraying (Kappe et al., 2010). Attemps to eradicate or at least ameliorate the

disease are still hampered, due to resistance of parasites to anti-malarial drugs and of

mosquitoes to insecticides, plus socio-economic turmoil in many countries. For these

reasons, there is still need for additional studies in order to understand the interactions

between the malaria parasite and its host, leading to discovery of potential target antigens

for vaccine development.

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1.3 The malaria parasite

Malaria is caused by a unicellular eukaryotic protozoan parasite belonging to the

kingdom Protista, phylum Apicomplexa, class Sporozoa, order Eucoccidia and genus

Plasmodium. There are 172 species of Plasmodium that infect birds, reptiles, and

mammals, but only five cause malaria in humans, P. falciparum, P. vivax, P. ovale and P.

malariae and P. knowlesi. Anopheles mosquitoes are the vectors for transmission of

malaria. There are 435 known species of Anopheles mosquitoes, out of which 30-40

species can transmit malaria. In sub-Saharan Africa, the three major species that transmit

malaria are: Anopheles gambiae, A. funestus, and A. arabiensis. As a means of survival

the malaria parasite inhabits two natural hosts, the female Anopheles mosquitoes and,

during the other half of its life cycle, the mammalian host (Fig. 2). Described below is the

life cycle of P. falciparum, the most lethal and virulent of the five species mentioned

above, and this will be main species referred to in this thesis.

Transmission of P. falciparum into the host cell: During a blood meal, a malaria-

infected female Anopheles mosquito inoculates sporozoites into the human host, which

then circulate in the blood for minutes, before they attach and invade hepatocytes

(Florens et al., 2002). Within the liver the parasite undergoes the pre-erythrocytic stage in

which it multiplies asexually. Since the parasite is in the liver for at least five days, this is

a good site for attack by the immune cells, such as the T-cells and cytokines. Of note, in

P. vivax and P. ovale a dormant stage hypnozoites can persist in the liver and cause

relapses by invading the bloodstream, weeks or even years later. After this initial

replication in the liver (pre-erythrocytic stage), a single sporozoite gives rise to tens of

thousands of asexual parasites called merozoites (Prudêncio et al., 2006). Around one

week after the initial liver infection, merozoites are released from the infected

hepatocytes into the bloodstream, infecting red blood cells (RBCs). In the RBCs the

parasites undergo asexual multiplication (erythrocytic stages) (Fig. 2), were the ring stage

parasite develops in a parasitophorous vacuole (PV) into trophozoites, the feeding stage

of the parasite, where it digests haemoglobin from the RBC (Florens et al., 2002).

6

The schizont stage is the final stage of asexual development in the human host. At this

stage the asexual daughter cells, called the merozoites, develop, and when mature the

infected RBC (iRBC) ruptures and the newly formed merozoites may infect new RBCs.

Each merozoite can multiply up to 20-fold every 48 h in cycle from erythrocyte invasion,

to erythrocyte rupture, and this cycle is responsible for the clinical manifestations of the

disease.

Transmission of P. falciparum from host cell to mosquito: Some merozoites

differentiate into sexual erythrocytic stages (gametocytes). The transmission of

Plasmodium from the human to mosquitoes requires infectious gametocytes that reside in

the capillaries of the inner organs of the infected human. Gametocytes will only appear 3

to 10 days after the merozoites have entered into the bloodstream. However the length of

time depends on the strain of the Plasmodium (WHO: FAQ about malaria, 2009). Once

the female mosquito bites and sucks the blood from the infected host, the formation of

male (microgametes) and female (macrogametes) gametes are released and fusion takes

place to produce zygotes. The zygotes in turn become motile and elongated (ookinetes),

which invade the midgut wall of the mosquito, where they develop into oocysts. The

oocysts grow, rupture, and release thousands of sporozoites, which become infectious

once they migrate to the mosquito’s salivary gland. The infected mosquito then finds a

host to feed on to get blood, and in the process injects the infectious sporozoites into its

victim (Mikolajczak et al., 2008), and the vicious cycle of infection starts all over again.

7

A

B

C

D

Figure 2: Life cycle of P. falciparum, showing the development of the parasite in the

mosquito and human host. Potential immunological mechanisms at the different stages of

the parasite’s life cycle are also indicated, A-D. (Diagram is adapted and modified from,

Crompton et al., 2010).

8

1.4 Clinical disease and manifestations

The malaria parasite’s life cycle initially involves a clinically silent liver stage (Prudêncio

et al., 2006, Shofield & Hacket 1993), and thereafter the asexual blood stage, where

multiple molecular processes contribute to the remodelling of infected as well as the

uninfected red blood cells, (Maier et al., 2009; Layez et al., 2005). These processes lead

to various clinical outcomes, such as severe malaria (SM), including severe malarial

anaemia (SMA) and cerebral malaria (CM), as well as pregnancy-associated malaria

(PAM). In humans, severe cases of malaria are multifaceted and most likely cannot be

respresented by one single pathogenic scheme, rather by compounding effects of multiple

disorders. Such effects include, destruction of uninfected red blood cells (uRBCs),

microvascular obstruction, immunopathological and inflammatory processes (Mecheri,

2011), and not exempting the age and previous immunological experience of the host

(Schofield & Grau, 2005). In malaria endemic areas, the burden of the disease is mainly

borne by infants and young children leading to a severe outcome of the disese, including

SMA and CM. In areas of lower transmission, primary infections might occur in

adulthood, in which severe disease is manifested. In most cases, transmission dynamics,

host age, host genetics and immunological responses are important determinants of

disease severity (Schofield & Grau 2005).

The traditional presentation of malaria, observed in about 50-70% of malarial cases

consists of paroxysms of fever alternating with periods of fatigue (Zaki & Shanbag,

2011). Signs associated with febrile outbursts include high fever, rigors, sweats and

headaches, myalgia, nausea, diarrhoea, pallor and jaundice (Krause et al., 2007). Other

obvious clinical symptoms include fatigue, respiratory distress, pulmonary edema,

convulsions, circulatory collapse, hemoglobinuria, severe anaemia and impaired

consciousness (Ferreira et al., 2008). However, in different endemic regions malaria can

present itself with unusual manifestations due to development of immunity and

increasing resistance to antimalarial drugs (Zaki & Shanbag, 2011). Severe

manifestations of P. falciparum malaria in hyperendemic areas are defined by the

occurrence of one or more of the symptoms mentioned above. SMA is the earliest most

9

severe and frequent dangerous complication of malaria, and it might be the leading cause

of deaths from malaria worldwide in most hyperendemic areas, resulting from a massive

RBC loss and/or impaired erythropoeisis (Lamikanra et al., 2007). CM, a syndrome of

unarguable coma is often associated with fits and other neurological abnormalities, such

as seizures, increased intramuscular muscle tone, and elevated intracranial pressure

(Kwiatkowski 1994; Mishra & Newton 2009). It is one of the most common and

potentially life-threatening complications of P. falciparum malaria commonly found in

low endemic areas.

1.4.1 Disease pathogenesis

The pathogenesis of severe cases of malaria occurring during the erythrocytic cycle is

multifactorial and intertwined. It involves processes from the early parasite stage (rings)

to the mature stages (trophozoites & schizonts) (Fig 2), interactions with the host, as well

as immunological feedbacks from the host immune system. During P. f. infection, the

asexual cycle, where the release of daughter merozoites occurs every 48 hrs from iRBCs,

is a major contributor to the fever chills observed during manifestation of the disease.

Glycosyl-phosphatidyl inositol (GPI), an anchor molecule of some plasmodial membrane

proteins, is thought to function as a critical toxin, which contributes to severe malarial

pathogenesis by eliciting the production of pro-inflammatory responses by the innate

immune system of mammalian hosts (see section 2.1) (Schofield et al., 1993; Schofield &

Hackett 1993; Tachado et al., 1997; Naik et al., 2000). In cases of SMA, which can be a

chronic complication of malaria, it does not only arise from destruction of iRBCs, but

also from the significant loss of uninfected RBCs and decreased production of RBCs.

Indeed infection by the P. falciparum parasite modifies the uninfected RBCs (Maier et

al., 2009; Layez et al., 2005), as a proportion of uninfected RBCs are “decorated” with

parasite molecules (such as RSP2/RAP 2) released during invasion (Layez et al., 2005;

Pouvelle B, 2000; Awah et al., 2009), and these RBCs are susceptible to splenic

retention, phagocytosis or complement mediated lysis (Layez et al., 2005; Awah et al.,

2009). These immunological reactions likely aggravate RBC loss in malaria patients,

10

leading to severe cases of malaria.

In addition there is induction of an array of bioactive molecules (pro-inflammatory and

anti-inflammatory cytokines) which either upregulate or downregulate pathogenic

processes. Sequestration of iRBCs in the microvasculature of vital organs is one of the

most important factors in pathogenesis of P.f. malaria (Turner et al., 1994), caused

mainly by one of the parasite proteins, Plasmodium falciparum erythrocyte membrane

protein 1 (PfEMP1) (for details see section 3.1). Sequestration is characterized as the

removal of iRBCs from the circulation by binding to the vascular endothelium of the host

cells, mostly in the post capillary venules of the deep tissues. Impaired oxygen delivery

due to occlusion of the blood flow in vessels may result in organ dysfunction.

P. falciparum infection during pregnancy can bring about immense adverse effects such

as maternal anaemia. Low birth weight and infant anaemia, have been a hallmark of

pregnancy-associated malaria and these are believed to be the consequence of

sequestration of the parasites in the intervillous spaces of the placenta using the placental

receptor Chondroitin sulphate A (CSA) (Hviid & Salanti, 2007; Rogerson et al., 2007).

Immense sequestration in the brain is alleged to be the cause of coma in CM (Turner et

al., 1994). In patients with CM, the total parasite biomass is greater than in patients with

uncomplicated or other severe attacks (Buffet et al., 2011). In addition to sequestration

of mature forms of the parasites in the brain leading to organ and tissue dysfunction, there

is recruitment and sequestration of host cells, (such as leukocytes or platelets) which

contribute to the pathogenesis of CM (Wassmer et al., 2003), either through local effects

in brain microvessels or through distant effects mediated by the production of potentially

deleterious mediators, such as pro-inflammatory cytokines and induction of nitric oxide

(NO) (Clark et al., 1992).

Another phenomenon with immense pathological effect is rosetting, described as when

matured trophozoite iRBCs attach to two or more uninfected RBCs to form rosettes, or

the adhesion of several iRBCs and RBCs to each other to form giant rosettes (Wahlgren

11

et al., 1990, Udomsangpetch et al., 1989). Rosetting parasites are associated with severe

clinical disease including CM in the human host (Heddini et al., 2001, Rowe et al., 1995,

Rowe J.A et al., 2002, Treutiger et al., 1992, Carlson et al., 1990). In addition rosetting

may also aid in camouflaging of iRBCs from immune cells, and increase the efficiency of

merozoite invasion, imparted by the proximitity of newly ruptured schizonts to

uninfected red blood cells, this ability has been speculated for antigen Pf332 (Moll et al.,

2007).

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2 Malaria and the human immune system

As mentioned earlier, P. falciparum infection is the major cause of morbidity and

mortality in malaria endemic areas. The erythrocytic stage of the parasite is the main

culprit in malaria pathogenesis, and the main target of immune responses to malaria.

Immunity to malaria is complex, and has been shown to be based on factors such as age,

transmission intensity and continuity of exposure to parasite. As it has been emphasized,

immunity to malaria is partial, as older children and adults living in malaria hyper-

endemic regions can develop malarial disease (Marsh & Kinyanjui, 2006). Newborns do

not contract the disease during the first months of life, owing to the maternal antibodies

that crossed the placenta. However, the role of maternal antibodies in this context has

been questioned (Riley et al., 2000). As the level of the passively acquired antibodies

wane around 6-9 months of age, infants in malaria endemic areas may experience their

first clinical episode at this age (Bruce-Chwatt, 1952). This leaves the young children

(about 1-5 years of age), non-immune individuals and pregnant women with the burden

of the disease. In other words, the acquisition of malaria specific immunity is important

in preventing morbidity and mortality from malaria (Sanni et al., 2003).

Immunity to malaria is based on the different stages (pre-erythrocytic and erythrocytic) of

the parasites life cycle, as each stage induces different mechanisms of immune response

(Fig. 2A-D). In this thesis, we will be addressing one of the mechanisms employed by the

host against the erythrocytic stage. Above all, there is no single measure to predict a

protective immunity to malaria, as it involves an interplay of various arms of the immune

system, including the innate as well as the humoral and cellular arms of the adaptive

system.

2.1 Innate immune response to malaria

Innate immunity to malaria is an immediate inhibitory response to the inception of the

infection, not dependent on any previous encounter with the parasite (Doolan et al.,

2009). This type of response may be based on inherent properties of the host which may

13

help to reduce the parasite multiplication rate and in the end prevents disease severity

(Mohan & Stevenson 1998; Doolan et al., 2009). Pattern recognition receptors (PRR) are

molecules on host cells which the innate system use to detect pathogens, they comprise a

family of type I transmembrane receptors which are characterized by an extracellular

leucine-rich repeat (LRR) domain and intracellular Toll/IL-I receptor (TIR) domain

(Medzhitov, 2001; Janeway & Medzhitov, 2002; Beutler, 2004). One family of pattern

recognition molecules present on or in host cells is the Toll-like receptors (TLRs).

The infected RBC, or parasite products, can interact with several Toll-like receptors

(TLRs) on host cells, especially monocytes/macrophages and dendritic cells, which are

used to recognize and respond to a plasmodial infection The interaction between the

iRBC and most TLRs can orchestrate an early defense, and in most cases dependent on a

crucial adaptor molecule MyD88 (myeloid differentiation primary gene 88) and

activation of nuclear factor kappa β (NF-κ β), which often leads to the production of pro-

inflammatory cytokines (Ropert et al., 2008; Gowda, 2007; Arama et al., 2011).

Cells of the innate immune system contribute to innate immunity against malaria, these

cells include NK, gamma delta (γδ) and NK T cells, macrophages and dendritic cells

(DCs). Polymorphonuclear cells such as neutrophils and eosinophils, soluble factors,

such as interferons and complement factors, are also involved in the innate immune

responses against malaria (Perlmann & Troye-Blomberg 2002). NK cells have been

shown to be the first cells to respond to P. falciparum infection by increasing in number

and the ability to lyse infected RBC in vitro (Orago & Facer 1991). In P. falciparum

infection, direct contact between parasitized RBCs and NK cells leads to IL-12 and 18

production, which further leads to IFN-γ production (Artavanis-Tsakonas et al., 2003).

Thus, the IFN-γ produced by the NK cells activates macrophages to eliminate parasitized

RBCs. During malaria infection, γδ T cells are more expanded in the circulation than

other T cell subsets, and they have been shown to directly inhibit the growth of blood-

stage parasites (Farouk et al., 2004). The role of γδ T cells expressing NK receptors are

dominant source of IFN-γ in response to P. f. infection (D’Ombrian et al., 2007).

14

Evidence for the role of macrophages and DCs in innate immunity is their ability to

phagocytose infected erythrocytes in the absence of cytophilic or opsonizing malaria-

specific antibodies, (Gyan et al., 1994; Serghides et al., 2003), and thereby contributing to

the reduction of initial parasitemia. The GPI molecule interacts with macrophages and

DCs to induce pro-inflammatory cytokines such as TNF- , IL 1 and IL-12 (Schofield &

Hackett, 1993; Naik et al., 2000, Gowda, 2007). It activates DCs, through TLR2 and

TLR4 (Krishnegowda et al., 2005), studies on TLRs and malaria in humans, indicated

that common TLR-4 mutations in African children may increase the risk of severe malaria

(Mockenhaupt et al., 2006). Parasite material such as haemozoin (HZ) has effects on the

immune system as demonstrated by HZ purified from P. falciparum being a ligand for

TLR-9, which may further modulate the function of DCs (Coban et al., 2005;

Pichyangkul et al., 2004). However it has also been demonstrated that the parasite DNA

attached to HZ is responsible for the observervations with DCs (Parroche et al., 2007).

The divergencies observed with the specific ligand for TLR-9, may be due to factors such

as heterogeneity of the DCs, different parasites strains and type of HZ used (Urban &

Todryk, 2006; Bousheri & Cao 2008).

The influence of the genetic make-up of the host on susceptibility to malaria infection in

endemic areas has been established, especially in the genetic red cell disorders, including

sickle cell trait, thalassemia, enzyme deficiencies, ovalocytosis and ABO blood groups

(Miller et al., 1994; Modiano et al., 2001, Aidoo et al., 2002; Williams et al., 2005;

Weatherall et al., 2002; Ayi et al., 2004; Mockenhaupt et al., 2003; Wambua et al., 2006;

Durand & Coetzer 2008). These host factors may confer natural protection against

malaria infection. Other innate factors, such as polymorphisms in ICAM-1, a putative

receptor for erythrocyte binding to the brain endothelium, and a polymorphism in the

promoter region of TNF-α, appear related to the frequency of severe disease (Fernandez-

Reyes et al., 1997, McGuire et al., 1994; Keusap et al., 2010). Innate differences in

host’s control of immune responses and differences in parasite virulence each have their

part in determining the response to infection. Polymorphisms in host genes such as those

encoding IFN-γ, TNF, IL-10 and IL-4 (Bayley et al., 2004, Carpenter et al., 2007, Henri

et al., 2002, Jüliger et al 2002) have been associated with susceptibility to the disease.

15

However, proper balance between the regulation of pro- and anti-inflammatory cytokines

vis-à-vis disease endemicity may be critical in determining the extent of pathology

(Kurtzhals et al., 1999, Sinha et al., 2010, Hafalla et al., 2011). Proper understanding of

the innate immune responses to pathogens is very crucial, because it directs the

development of an efficient adaptive immune system (Janeway & Medzhitov 2002).

2.2 Pre-erythrocytic stage immunity in malaria

Natural immune responses to the pre-erythrocytic stages (sporozoite and liver) of malaria

have been suggested to have limited involvement in malaria immunity (Hoffman 1987,

Hoffman 1996, Owusu Agyei et al., 2001, Marsh & Kinyanjui, 2006, Langhorne et al.,

2008; Shwenk & Richie 2011). This immunity precedes the pathogenic stage and if very

strong and sterilizing may be very important in malaria immunity, one of the challenges

encountered in vaccine development against the liver stage. However, our understanding

on immune effectors underlying an effective and sterilizing pre-erythrocytic immunity is

limited. Antibodies to sporozoites may act through neutralizing opsonization of

sporozoites and/or blocking of the invasion of hepatocytes (Fig 2B), yet they are thought

to have negligible function, probably due to inadequate titers of high affinity antibodies

(Nardin et al., 1999).

The infected hepatocyte is an important target of protective immunity, because the

presence of parasites in this MHC-I-expressing cell may render parasite antigens more

accessible to a cell-mediated immune response (Speake & Duffy 2009). During this liver

stage, effector functions of the CD4+ and CD8

+ T cells are important (Tsuji & Zafala

,2003; Wykes & Good, 2009). There are evidences that IFNγ-secreting T cells against

liver stage antigens (Fig 2C), are associated with reduced malaria incidence (Todryk &

Bejon 2009; Perlaza et al., 2008). However, the definitive roles of CD4+ and CD8

+ T

cells in malaria immunity are yet to be determined (Tsuji, 2010). In addition, studies have

demonstrated that antibodies also contribute to protection against the pre-erythrocytic

stages during malaria infection (Hollingdale et al., 1984; Kebaier et al., 2009; Schwenk

&Richie 2011).

16

2.3 Erythrocytic stage immunity in malaria

Red-cell invasion is a crucial activity in malaria pathogenesis and is an important target

for protective immune responses. Blood-stage immunity can be acquired in individuals

that are repeatedly exposed to the pathogen, and it is substantially mediated by

antibodies, but, protective mechanisms such as, innate immune responses, secretion of

IFN-γ by CD4+ T cells are also utilized (Fig 2D), (Plebanski & Hill 2000). During the

asexual blood stage, CD4+ T cells are pivotal in malaria immunity (Pombo et al., 2002)

as well as in the development and regulation of humoral immune response, as they form

interplay with the B cells. This leads to effective class switching based on the cytokine

milieu and efficient antibody production. It has been shown that CD4+ T cells from

malaria exposed individuals naturally exposed to malaria, respond to blood stage antigens

of P. falciparum by proliferation, production of IFNγ and / or IL-4 secretion in vitro

(Troye-Blomberg et al., 1990). γδT cells, whose activation is initiated by IL-2, IL-4 and

IL-15, have been shown to expand both in mice and humans during malaria infection

(Rzepczyk et al., 1997). These cells also recognize schizont derived–phosphorylated

molecules (Pichyangkul et al., 1997) and produce proinflammatory cytokines.

Pathology of severe disease has been linked to an excessive inflammatory response, and

the ability to regulate these responses is important in immunity against severe malaria

(Artavanis-Tsakonas et al., 2003). The regulatory T cells (T-regs) comprise 5-10% of

CD4 T-cells of the immune system (Riley et al., 2006), these IL-10 and TGF-B

producing cells are involved in immunosuppression (Hansen & Schofield, 2010). It has

been shown that these cells achieve this function due to their abilities to regulate the

magnitude and timing of the cellular immune response, and allowing sequential induction

of appropriate levels of inflammatory- and anti-inflammatory cytokines at key stages of

the infection (Li et al, 1999, 2003, Walther et al., 2005, 2009).

A number of evidence has shown that antibodies are very important in the clearance of

parasite loads in both animal and human blood stage infections (Berzins et al., 1991;

Bolad & Berzins, 2000). In most malaria endemic countries, malaria infection induces

17

humoral immune responses, involving production of predominantly IgM and IgG but also

of other immunoglobulin isotypes. While a majority of this immunoglobulin is a result of

polyclonal B-cell activation, up to 5% or more represent species as well as stage-specific

antibodies, reacting with a wide variety of parasite antigens. The level of total

antimalarial antibodies in most cases increases with age, and is usually taken as a

measure of the length and intensity of exposure, and sometimes may indicate protection

against malaria. The biological properties of antibodies produced in response to malaria

are of particular importance, due to their efficiency in antibody-mediated actions and

ability to confer protection based on the presence of one or combination of isotypes and

subisotypes.

Figure 3: Possible mechanisms for parasite neutralization of P. f. asexual blood stages.

[1] Antibody-mediated phagocytosis of whole infected erythrocytes or merozoites. [2]

Antibody-independent phagocytosis. [3] Complement mediated phagocytosis. [4] Effect

of TNF-α and nitric oxide from activated macrophages on intraerythrocytic (IE) parasite

or merozoites. [5] Antibody-mediated inhibition of merozoite invasion. [6-8] Antibody-

mediated inhibition of IE parasite development; [6] by antibodies binding to antigens on

the surface of the iRBC, [7] by antibodies entering through parasitophorous duct, [8] by

antibodies entering through a leaky RBC membrane. (Adapted from Bolad & Berzins

2000).

18

2.3.1 Mode of action of antibodies

Antibodies specific for malaria can carry out their actions through various mechanisms

directed towards different stages of the parasite in the erythrocytic cycle. Antibodies

against erythrocyte surface-associated proteins may block RBC invasion by blocking

merozoite release from ruptured schizonts, a phenomenon referred to as agglutination

(Green et al., 1981, Wåhlin et al., 1984). Antibodies may also enter the infected RBC

through the leaky membrane at the time close to rupture (Green et al., 1981), or through

the parasitophorous duct, which has been suggested to form a connection for direct access

of serum macromolecules to the parasite (Fig 3) (Pouvelle et.al., 1991; Taraschi 1999).

These actions may interfere with intracellular development of the parasite and essentially

lead to growth inhibition (Ahlborg et al., 1996; Bergmann-Leitner et al., 2006). In

addition, antibodies against antigens expressed during the trophozoite- schizont stages

can block sequestration of iRBC to endothelial cells in internal organs thus enhancing

clearance of parasites by the spleen (David et al., 1983), as well inhibiting rosetting of

RBC to iRBC (Carlson et al., 1990). These actions may help prevent severe malaria

complications. Antibodies to the GPI molecule may neutralize parasite toxins and prevent

the induction of excessive inflammation.

Antibodies may carry out their effector functions in alliance with ancillary cells. They

opsonize iRBC or free merozoites to enhance the phagocytic activity of monocytes

(Bouharoun-Tayoun et al, 1995). The mechanism underlying this effective killing of

parasites is the capture of antibodies on the surface of monocytes through receptors that

bind the Fc part of the antibody, while the Fab part of the antibody molecule is bound to

antigen/s on the surface of merozoites (Bouharoun-Tayoun et al., 1990) or late stage

iRBCs (Gysin et al., 1993). This co-operation between malaria specific antibodies (see

2.3.2) and monocytes via Fcγ receptors could induce cellular functions such as

phagocytosis, antibody dependent cellular inhibition (ADCI), mediated by monocyte-

derived factors (Tebo et al., 2001; Jafarshad et al., 2007).

19

Antibodies against well studied target antigens such as merozoite-surface associated

antigens (MSP1, MSP2), and antigens present in the apical complex organelles of

merozoites (EBA-175 in micronemes, Rhop1-3, RAP 1-3 and AMA 1 in rhoptries) or in

dense granules (Pf155/ RESA) (Wåhlin et al., 1984), may block RBC invasion either by

neutralization of free merozoites and further interfering with the merozoite invasion

process (Berzins & Anders, 1999). Other antibodies to antigens expressed during

trophozoite development (SERA, ABRA, GLURP, PfEMP1, RIFINS, STEVORS and

Pf332) have been demonstrated as efficient inhibitors of P. falciparum growth in vitro

and/ or merozoite invasion (reviewed in Bolad & Berzins, 2000).

Importantly, adequate knowledge of blood stage immunity as well as antigen-specific

responses (an issue addressed in this thesis) is very essential in malaria immunology, as

this will give appropriate information about antigens responsible for protection, and to

further scrutinize these antigens for vaccine development.

2.3.2 Immunoglobulins and immunity to malaria

As it has been emphasized, immunoglobulins (Ig) are crucial in immunity to malaria

especially during the pathogenic blood stage. Antibodies produced against malaria

parasites are of different isotypes, with different functional capabilities regarding being

protective or otherwise.

IgM, is the primary antibody produced on the first encounter with an antigen (Leoratti et

al., 2008), and most puzzling of all isotypes. It has been speculated that these antibodies

are involved in anti-parasite immunity (Baird, 1995), also in anti-toxic immunity which

helps to prevent the clinical appearance of a malaria attack (Bate et al., 1990; Boudin et

al., 1993). Elevated levels of IgM, as compared to controls have been observed in several

studies performed in relation to malaria specific antigens, and has been suggested to be

important in agglutination (Doolan et al., 2009) as well as in neutralizing pathogens,

owing to its polymeric structure (Czajkowsky et al., 2010; Ehrenstein & Notley, 2010 ).

In addition it is a potent complement activator (Czajkowsky & Shao, 2009). In a study it

20

was shown that immune IgM is protective against P. chaubadi infection (Couper et al.,

2005). In contrast, other studies have shown that IgM may not play a significant role in P.

f. infection (Branch et al., 1998), which still leaves us with the impression that there is no

clear role of IgM during malaria infection.

The IgG isotype has been shown to be the mostly produced in humans in response to

pathogens, this isotype consists of four subclasses (IgG 1, 2, 3, 4). IgG and its subclasses

have been demonstrated to be significant towards immunity to malaria. The subclasses

differ in their structure and mediate different immune effector functions (Nimmerjahn&

Ravetch, 2008). The cytophilic ones, IgG1 and IgG3, have been shown to predominate

regarding protective humoral responses to P. f. malaria (Bouharoun –Tayoun & Druilhe

1992, Sarthou et al., 1997, Shi et al., 1999). By acting through various mechanisms

mentioned above (Bouharoun Tayoun et al., 1995; Jafarshad et al., 2007; Tebo et al.,

2001), these IgG subclasses of antibodies have been demonstrated to be very important in

immunity to malaria. IgG3, the most short-lived subclass out of all IgG subclasses, has

been the prevailing isotype in terms of responses associated with protection in malaria

(Garraud et al., 2003a), a finding which we also observed in one of our studies.

The other subclasses, IgG2 and IgG4 are non-cytophilic, and have been speculated to be

non-protective. Although in some malaria endemic areas, elevated levels of IgG2 have

been related to decreased risk of infection, especially in individuals carrying a specific

allelic variant of FcγRIIA, which binds IgG2 (Aucan et al., 2000, Garraud et al., 2003b,

Nasr et al., 2009). Meanwhile, IgG4 is thought to compete with the IgG1 and 3 binding

epitopes, thereby inhibiting the parasite neutralizing effects of cytophilic antibodies

(Nutman, 2001, Hagan, 1991, Garraud et al., 2003b). In malaria immunology, the

dynamics of subclass responses and association with protection is important, and at the

same time vary, especially with specific antigens and this area should be explored

properly in order to understand immunity to malaria, as well guiding vaccine

development (Garraud et al., 2003a; Stanisic et al., 2009).

The role of IgE antibodies in malaria is still vague, as it on one hand has been shown that

malaria specific IgE may be associated with protection (Bereczky et al., 2004, Farouk et

21

al., 2005, Duarte et al., 2007). On the other hand, elevated levels of IgE appear to be

associated with pathogenesis, as indicated in patients with severe and cerebral malaria

(Perlmann et al., 1994; Perlmann et al., 2000; Perlmann et al., 1997; Troye-Blomberg et

al., 1999; Seka-Seka et al., 2004, Leoratti et al., 2008).

Regarding IgA, there is no evidence of specific function regarding malaria. Some studies

have shown high titres of naturally occurring plasmodium-specific IgA in sera (Biswas et

al., 1995) and breast milk (Kassim et al., 2000), however some showed that the

frequency of positivity of IgA antibodies does not have a correlation with the number of

previous malaria episodes (Leoratti et al., 2008).

In malaria immunity, different antigens of P. f. induce different classes and subclasses of

antibodies, and production of some of these immunoglobulins in individuals in relation to

protection exists. The different patterns of antimalarial antibody responses observed have

been suspected to be associated to various factors such as antigen properties, host age,

cumulative exposure (Scopel et al., 2006; Nimmerjahn & Ravetch 2008; Garraud et al.,

2002; Stanisic et al., 2009) and genetics (Duah et al., 2009; Nasr et al., 2009; Ntoumi et

al., 2002; Ntoumi et al., 2005). In addition, the quality of antibodies and proper

balance/proportion is a very crucial factor in protective immunity towards malaria.

2.4 Immune evasion by malaria parasite

The human immune system has evolved in order to eliminate infectious diseases, but the

P. f. parasites, which are obligate parasites of human RBCs, have evolved to a complex

life cycle and have been able to maintain an invasive blood-borne infection in its host. A

complex co-existence has been established between the parasite and its host by evading

human immune responses and occasionally not killing their hosts, making P. falciparum

a cunning evader. The ability to evade the immune system is the bases of the lethality of

P. f. infection when compared to other Plasmodium species, and has made the design of

an effective vaccine amazingly difficult as evasion can occur at the various stages of the

parasites’ life cycle.

22

Suppression of the host’s immunity during the pre-erythrocytic stage of malaria infection

takes place. The sporozoite-infected hepatocytes are targets for CD8+ T cells, thus

leading to immune activation upon recognition of parasite derived peptides (Schofield et

al., 1987, Klotz et al., 1995). To avoid this, the parasite suppresses the activation of the

T-cell immune response due to the presence of multiple tandem repeats (Gilbert et al.,

1998, Plebanski et al., 1999).

The pathogenic erythrocytic stage has an armory of evasive machineries, one of them

being the inability of iRBC to process and present antigens, thereby providing protection

from the host’s immune system. Some P. f.-proteins mimic the immune system by

producing antibodies to non-protective repetitive regions of the antigens, thereby

preventing the normal affinity and isotype maturation of an effective immune response

(Anders, 1986; Ramasamy, 1998). The hemozoin from the late trophozoite/schizont

stages have also been demonstrated to be involved in evasion, as HZ modulates the

maturation of DC (Urban et al., 1999; Guisti et al., 2011), and monocytes (Skorokhod et

al., 2004), which may downregulate the immune response.

In most cases, antigenic variation is employed by P. f. as an avenue to evade the immune

system, based on the presence of immunodominant surface antigens. PfEMP1 has

achieved a form of variability (see section 3.1 below), which in turn has allowed the

parasite to cope with the host immune system. Furthermore, P. falciparum parasites show

a remarkably high degree of polymorphism at the various stages of their life cycle

(Lockyer et al., 1989; Miller, 1993; Fenton et al., 1991; Konate et al., 1999), which has

important implications for the efficacy of parasite neutralizing immune responses.

Antigenic diversity may thus delay acquisition of protective immunity to malaria, the

development of which may thus require repeated exposure to different antigenic types or

strains present in the epidemiological area. The antigenic diversity observed in most

malaria antigens is a reflection of allelic gene polymorphisms, which is caused by

variations in the sequence of short tandem repeats and which frequently constitute

immunodominant regions. The best studied are msp1 and 2 genes regarding allelic

23

polymorphisms (Snounou et al., 1999, Aubouy et al., 2003, Kang et al., 2010).

The spleen is an immunologically active organ, where resident macrophages recognize

and remove RBCs with compromised deformability or altered antigenicity (a form of

innate immunity), which is the case with the P. f. infected red blood cells. Mature stage

parasites express proteins which alter the properties of the host RBC membrane (section

3) and thus promotes adhesion of the iRBCs to ICAM-1 of vascular endothelium using

the antigen PfEMP1 (section 3.1), resulting in sequestration and primarily evasion from

clearance by the spleen.

PfEMP1 and RSP2/RAP2 proteins are not the only culprit antigens involved in

pathogenesis and evasive mechanisms observed in P. f. malaria. A number of proteins

involved in modifications of erythrocyte cytoskeleton and/or iRBC membrane might be

involved (Maier et al., 2009), which will be discussed later in section 3. It is believed that

most of the iRBC proteins act alone or in combination with other proteins to perform

their molecular functions (Tilley et al., 2011). Understanding the roles of these antigens

is important in the field of parasite biology, as is the way they elicit strong

immunological responses, which might be protective or used to artifice the human

immune system. These questions are relevant and represent a key point addressed in this

thesis.

2.5 Malaria vaccines

The aim of vaccination is to generate immune responses with capacity to protect the

recipient against the natural infection. The immune response induced by vaccination

should be of long duration and should have the capacity to be effectively reactivated by a

natural infection. The rationale behind the development of a malaria vaccine is supported

by previous studies, which have shown that anti-sporozoite vaccines based on irradiated

sporozoites could elicit sterile immunity in humans (Schofield et al., 2002), and also that

passive transfer of IgG from immune individuals can provide some protection against

malaria (Cohen et al., 1961, Sabchareon et al., 1991). Attempts to develop a malaria

24

vaccine began in the early twentieth century (Desowitz, 1991), and in spite of advances in

biomedical technology and periodic bouts of unsubstantiated optimism in the field, no

effective vaccine is available for widespread use till date.

Ideally a vaccine should be able to follow the central dogma of immune responses. After

antigen encounter, the CD4+ T cells develop in to T helper (Th) 1 or Th 2 effector cells,

depending on the cytokine milieu. The Th1 effector cells produce proinflammatory

cytokines, such as IFN-γ and TNF-α, and these lead to further activation of the CD8+ T

cells and their cytotoxic effector function. The Th2 cells, on the other hand, produce

interleukins, such as IL-4, 5, 10 and 13, which further stimulate B cells to undergo

activation into effector B cells (plasma cells), which secrete antibodies (IgD, IgM, IgE,

IgA or IgG). Memory cells, which are only about 5-10 % of the lymphocyte population,

are phenotypically different from the naïve cells, and they proliferate and produce

cytokines faster upon activation. Whether the persistence of antigen on follicular

dendritic cells is needed or not for maintenance of immunological memory, is not clear.

Several studies have indicated that memory B and T cells can survive without antigen,

while others have suggested otherwise (Haberman & Sclomchick 2003). However,

cytokines such as IL-15 are crucial for the longevity of memory T cells.

The advent of recombinant DNA technology and protein engineering enabled the

expression of immunogenic proteins in bacterial, (an approach employed in our study)

yeast or mammalian cells. After being cloned in a suitable expression vector, such

expressed antigens are used for studies in vaccine development (Dertzbaugh, 1998; Liu

1998; Babiuk, 1999; Liljeqvist & Ståhl 1999). The basics of this technology is to transfer

a gene encoding an antigen, responsible for inducing good humoral responses sufficient

for protection, to a non-pathogenic expression vehicles (Liljeqvist & Ståhl 1999) ,

thereby making the production of the antigen safer and generally more efficient. There

are several limitations with recombinant proteins; they are generally poor immunogens

when administered alone and are unable to induce effector T–cell responses, such a

CD8+ CTLs, that are necessary for elimination of the intracellular pathogens.

25

As mentioned above (section 2.4), Plasmodium species have evolved multiple

mechanisms of immune evasion at the individual and population levels, including stage

specific antigen expression, allelic diversity, variability within T cell epitope sequences

and antigenic variation. During the course of its complex life cycle, the Plasmodium

parasite expresses different, complex mixtures of antigens. Therefore, a vaccine against a

single stage in the parasite life cycle may need to be 100% effective, because parasites

which progress to the next stage may express a new set of antigens, that may be

unaffected by the vaccine induced response. Most malaria antigens are stage-specific and

therefore there are distinct immune mechanisms operating against the different stages of

the complex life cycle. Vaccines against all stages of the parasite life cycle are important,

however for conciseness of this thesis, we will address only issues around vaccines

against the blood stage cycle.

2.5.1 Asexual blood stage vaccines

Vaccines targeted against antigens expressed by the asexual blood stages of the parasite’s

life cycle are aimed at preventing the complications of the disease, such as cerebral

malaria or anemia. Since these are the stages responsible for pathology of the disease,

antibodies to the target antigens should be able to inhibit parasite sequestration, induce

neutralizing antibodies against parasite derived antigens, or to eliminate/reduce the

parasite load, which might further reduce mortality. Despite encouraging progress, the

lack of immune correlates of protection, and insufficient predictive animal models, as

well as antigenic polymorphisms and strain variability of most asexual blood-stage

antigens, constitute major challenges to the development effort of asexual stage vaccines.

Inhibition of parasite invasion, as often measured in in vitro assays, may not be predictive

of immune status in endemic areas. This assay is termed as one of the prerequisite assays

important for evaluating blood-stage vaccine candidates and for identifying targets of

protective antibodies against malaria (Persson et al., 2006).

26

Till date, a small number of blood-stage antigens expressed on the surface of merozoites

are in clinical development as vaccines (Crompton et al., 2010). These include the apical

membrane antigen 1, AMA1 (Sagara et al., 2009), erythrocyte-binding antigen-175,

EBA-175 (El Sahly et al., 2010), glutamate rich protein, GLURP (Esen et al., 2009,

Hermsen et al., 2007), merozoite surface protein 1, MSP1 (Ogutu et al., 2009), MSP2

(Genton et al., 2002), MSP3 (Esen et al., 2009, Audran et al., 2005, Sirima et al., 2009,

Druilhe et al., 2005), and the serine repeat antigen 5, SERA5 (Horii et al., 2010).

However, in a phase II trial some of these vaccine candidates, AMA1 and MSP1, with

novel adjuvants have failed to demonstrate efficacy in African children (Sagara et al.,

2009, Ogutu et al., 2009).

An important setback in the development of vaccines is that the P. falciparum genome

encodes about 5,300 proteins (Gardner et al., 2002), and identification of potential blood-

stage vaccine candidates capable of inducing effective immune response is quite

challenging. Steps such as high-throughput protein expression systems to construct

microarrays of large numbers of P. f.-proteins are being currently employed to

circumvent this setback (Tsuboi et al., 2008, Doolan et al., 2008, Crompton et al., 2010).

Another major factor preventing the development of an effective malaria vaccine, is

extensive parasite genetic diversity due to selective pressure exerted by the human

immune response, which leads to extremely polymorphic antigens and effective measures

to overcome antigenic polymorphism are important to control this setback (Weedall &

Conway 2010; Takala et al., 2009).

To ensure a high degree of protection from malaria disease, a malaria vaccine may

encompass antigens from different stages of the parasite’s life cycle. If found effective,

such a vaccine will interfere with parasite development within the mosquito and further

reduce parasite transmission (transmission-blocking vaccine), as well as prevent blood

stage infection (pre-erythrocytic vaccine) and target pathogenesis (erythrocytic vaccine).

27

3 The infected red cell and its modifications

Simply, the uninfected erythrocyte is a sack of haemogloblin, inherently adapted to

perform specialized tasks of O2 and CO2 transport. This cell is anucleated and therefore

unable to synthesize new proteins and its inability to perform neither intracellular

trafficking nor antigen presentation. The RBC (diameter ~8 µm) is highly deformable

without undergoing fragmentation, demonstrated by its ability to transit through 1-2 µm

interendothelial slits (An & Mohandas, 2008). The capability of the RBCs membrane to

undergo this deformability is due to the composition of the membrane skeleton, which is

made up of a network of proteins, actin and spectrin (Mohandas & Chasis, 1993; Luna &

Hitt 1992), as well as to their low internal viscosity (Maier et al., 2008).

The P. falciparum parasite finds refuge in the uninfected erythrocyte, a perfect cellular

niche that will accommodate its havoc. As the parasite grows within the erythrocyte it

loses its deformability and becomes spherocytic and more rigid (Cooke et al., 2004).

These properties are thought to contribute to the pathogenesis of malaria, in addition to

vascular adhesion of parasitized erythrocytes. The altered deformability is manifested by

export of proteins into erythrocytes that interact with the host cell cytoskeleton and

inserted into the membrane. Upon initial invasion the parasite is enclosed in a

parasitophorous vacuole (PV) and its own parasite plasma membrane, which separate the

parasite from the RBC (Hanssen et al., 2010). It develops into a ring form, where it starts

to take up small amounts of haemoglobin and nutrients, using its cystostome (Bannister

& Mitchell, 2003), as well as synthesizing molecules specific to this stage (Spielmann &

Beck, 2000).

The ring develops into the trophozoite (~ 20-38 h post infection), the period where the

parasite is metabolically active, growth increases, and major modifications occur

(Bannister, 2003; Hanssen et al., 2010). Most of the cell structures are visible, and

parasite organelles such as the Maurer’s clefts (MC) are observed in the iRBC’s

cytoplasm (Aikawa et al., 1986, Atkinson & Aikawa, 1990). Maurer’s clefts are

flattened cisternae that arise from the PV membrane, bud into the RBC cytoplasm, and

28

become attached to the RBC membrane by specialized tubular structures (Hanssen et al.,

2010). It is a secretory organelle that concentrates proteins for delivery to the host iRBC

membrane (Lanzer et al., 2006; Sam-Yellowe, 2009). This organelle harbours a number

of P. f. proteins and is an intermediate compartment in the trafficking some of the

parasite proteins associated with parasite virulence. Observations have shown that defects

in Maurer’s cleft proteins can cause altered morphology of the exo-membrane system

(Tilley et al., 2011).

As the parasite matures it initiates alterations to its host RBC, which facilitates entry of

nutrients and exit of its byproducts, such as digested haemoglobin and synthesized

proteins. The properties of the RBCs are modified by some of these proteins, which are

further transported across the PV membrane via the MC to different sites in the RBC

cytoplasm and the iRBC membrane (Lanzer et al., 2006; Maier et al., 2009).

The concluding phase of the cycle is the schizont stage (38-48 h p.i.), where the parasite

occupies most of the host cell volume. At this stage the parasite consumes and digests

approximately 80% of the host cell haemoglobin (Loria et al., 1999). The parasite

undergoes a sequence of nuclear divisions, generating 16-20 daughter merozoites (about

1.2 µm long) ready for re-invasion as well as intense synthesis and assembly of

molecules needed for RBC invasion (Bannister & Mitchell, 2003). The individual

merozoites, armed with their own invasion machinery, remain within PV until the iRBC

and the PVM ruptures by a protease-dependent process (Salmon et al., 2001).

29

Figure 4: (a) diagrammatic representation of a P. f.-infected erythrocyte, showing the

organelles, Maier et al., 2008, (b) an expanded view of the membrane skeleton of P. f.-

infected red blood cell, showing the location of Pf332 and other membrane related

proteins (modified from Maier et al., 2009).

a

Spectrin repeat

Glycophorin C

Actin

Band 3

30

3.1 Antigens of the infected red cell

The P. falciparum parasite, might be a small haploid organism, but genomically

complicated, with about 5300 proteins expressed by the organism at the different stages

of its life cycle. The parasite exports an estimated 400 proteins, including kinases,

lipases, adhesions, proteases and chaperone-like proteins, to the host cell during the blood

stage. These proteins mediate a profound remodelling of the iRBC, performing different

functions, from structural and functional changes of the iRBC to parasite growth (Tilley

et al., 2011; Maier et al., 2008; Maier et al., 2009). They are also involved in the

modification of the host’s RBCs, such as increasing the iRBCs rigidity, capability to

adhere to the walls of vascular endothelial cells and ability to vary the antigenic coat of

the iRBC to avoid protective antibodies (Cooke et al., 2004; Rowe et al., 2009), which

eventually promote disease severity due to its pathogenic mechanisms. The mechanism of

export of some P. f. proteins from the parasite into the RBC cytoplasm depends on the

possession of an NH2-terminal motif, termed plasmodium export element (PEXEL) or

vacuolar transport signal (VTS), which is conserved across the Plasmodium genus (Hiller

et al., 2004; Marti et al., 2004), some but not all of the P. f. proteins possess this motif.

Very few of these proteins have been highly studied in relation to understanding the

biology of the parasite, therein, some of the proteins transported to iRBC cytoplasm and

membrane as well as those involved in modification of the iRBC are described briefly.

As earlier mentioned, golgi-like structures such as Maurer’s clefts appear in the

cytoplasm of the iRBC as the parasite matures. Significant resident proteins of the MCs

and PEXEL-negative include, SBP1, MAHRP1, REX1, REX 2. Antigen Pf332 (the

antigen of interest in this thesis) is also involved with the cleft but the exact association

with the MC is still elusive. Although there is a recent data, that demonstrated that Pf332

is associated with the cytoplasmic face of the cleft (Nilsson et al., unpublished).

SBP1: the skeleton–binding protein-1, is one of the first MC proteins to be described. It

is a 48 kDa integral membrane protein that spans the Maurer’s cleft membrane (Blisnick

et al., 2000). The NH2-terminal domain is found within the cleft, whereas the COOH-

31

terminal domain is exposed within the iRBC cytoplasm and interacts with a RBC

membrane skeleton protein, possibly participating in the anchoring of the clefts to the

RBC membrane skeleton (Blisnick et al., 2000). SBP1 has been suggested to be involved

in the translocation of the virulence associated protein PfEMP1 to the surface of infected

red blood cells (Cooke et al., 2006).

MAHRP1: the membrane-associated histidine-rich protein-1 is a 28.9 kDa protein,

which consists of a highly conserved N-terminal region, a putative transmembrane (TM)

domain in its middle region, and a variable histidine-rich domain at its C-terminal end

(Spycher et al., 2003). The N-terminal and the TM domain have been suggested to be

important for proper translocation of this protein towards MCs (Spycher et al., 2006).

This protein is also being speculated to be involved in haemozoin production (Lynn et al.,

1999, Sullivan et al., 1996, Garcia et al., 2009). It is important for the intraerythrocytic

development of the parasite (Garcia et al., 2009) as well as the morphology of the MCs,

as disruption of the MAHRP1 leads to increased fragility of MCs, accumulation of

PfEMP1 in the parasite and PV, and in the end no surface expression of PfEMP1

(Spycher et al., 2008).

REX1 & 2: the ring-exported proteins 1 and 2. REX1 is the largest member of a cluster

of the exported proteins (REX1-4) expressed in the ring-stage parasite (Dixon et al.,

2008). It persists at the Maurer’s cleft throughout the erythrocyte infection. REX1 is

peripherally associated with the cytoplasmic surface of the cleft, this protein plays a role

in controlling the overall architecture of the MCs, as disruption of REX1 causes stacking

of the MC lamellae and reduction in their numbers (Spielmann et al., 2006, Hanssen et

al., 2008). REX2 is a 96 amino acid long protein that contains a N-terminal region and a

TM domain, which are suggested to be involved in the transporting the protein to the

MCs (Spielmann et al., 2006, Haase et al., 2009).

Additional proteins such as, KAHRP and PfEMP3 are PEXEL positive MC proteins

associated with the cytoplasmic face of the Maurer’s clefts. About 16 hours post-invasion

of merozoites, the surface of iRBC expresses knob-like protusions (Gruenberg et al.,

1983), these structures contain the knob-associated histidine-rich protein, KAHRP

(Taylor et al., 1987). KAHRP interacts with spectrin and actin (Kilejian et al., 1991, Oh

32

et al., 2000, Chishti et al., 1992), and is essential for knob formation (Crabb et al., 1997;

Wickham et al., 2001; Rug et al., 2006). PfEMP3, P. f erythrocyte membrane protein 3

(Pasloske et al., 1993, Glenister et al., 2002), is important for trafficking of PfEMP1 to

the erythrocyte surface and, hence, for cytoadherence. The truncation of PfEMP3 affects

the MC morphology (Waterkeyn et al., 2000).

Several proteins synthesized by the intracellular parasite have been shown to be

transported to the erythrocyte surface (Cooke et al., 2004). The most characterized

protein on the surface of iRBC is P. f. erythrocyte membrane protein-1, PfEMP 1, an

adhesive protein encoded by the hypervariable var gene family, which consists of about

60 genes encoding proteins ranging in size from 220-350 kDa (Baruch et al., 1995; Su

1995; Smith et al., 1995). During the ring stage several var genes are expressed but

during the trophozoite only one PfEMP1 is dominant. The expression of PfEMP1 on the

iRBC surface leads to sequestration of matured parasites in the vascular endothelium.

Binding specificities of PfEMP1 variants have been mapped to different adhesion

domains of the proteins and have revealed that DBL1α binds blood group A, CR1 and HS

on both endothelial cells and erythrocytes (Barragan et al., 2000a, 2000b; Rowe et al.,

1997; Chen et al., 2000; Vogt et al., 2003). CIDR1α domain has been shown to bind

CD36 and IgM (Chen et al., 2000, Baruch et al., 1997, Robinson et al., 2003), and the

DBLβ-C2 region binds to CD36 and ICAM-1(Baruch et al., 1996, Chattopadhyay et al.,

2004). Different patterns in disease severity have been observed with different PfEMP1

expressed at the time of infection. The variant associated with severe disease is

Var2CSA, which mediates maternal malaria through sequestration to CSA in the placenta

(Salanti et al., 2004; Dahlbäck et al., 2006).

Other variant protein families trafficked via the Maurer’s clefts include the STEVOR

(subtelomeric variable open reading frame) proteins a family encoded by 27-39 gene

copies (Przyborski et al., 2005). The RIFIN (repetitive interspersed family) proteins are

encoded by the largest multicopy gene family in P. falciparum, comprising about 150-

200 gene copies (Helmby et al., 1993; Kyes et al., 1999; Sam-Yellowe, 2009). The

33

SURFIN proteins are encoded by surf genes, consisting of a family of 10 genes. It is

exported along with RIFIN and PfEMP1 to the MCs (Winter et al., 2005).

Additional proteins of interest include: the ring-infected erythrocyte surface antigen,

Pf155/RESA, which is released from the merozoite dense granules during or shortly after

invasion, and it ends up in the RBC cytoplasm destined to the surface of the infected red

cell (Aikawa et al., 1990; Culvenor et al., 1991). RESA is one of the first proteins

exported across the PV membrane to iRBC membrane, where it interacts with the

spectrin network and appears to suppress further invasion by other merozoites (Foley et

al., 1991; Ruangjirachuporn et al., 1991; Pei et al., 2007). This antigen has been

suggested to be involved in modulating the mechanical properties of ring-stage iRBC,

thereby reducing deformability of early ring infected- host red blood cells (Mills et al.,

2007). The mature-parasite-infected erythrocyte surface antigen, MESA, is a high

molecular-mass protein expressed in trophozoites and schizonts this protein is also

associated with the RBC skeleton (Coppel et al., 1988, Lustigman et al., 1990;

Przyborski et al., 2005).

3.2 Antigen 332

3.2.1 Related background

The P. falciparum antigen 332 (Pf332) identified by Mattei et al., (1989), is so far the

largest of all P. f. proteins (~ 1 megadalton) exported to the infected red blood cell

membrane. The Pf332 gene has a two exon structure and is located in the subtelomeric

region on chromosome 11, and it has been detected in all clinical parasite isolates

examined so far. Subtelomeric genes are prone to frequent gene recombination event, the

Pf332 gene displays a marked sequence variation as demonstrated by restriction fragment

length polymorphism (RFLP) (Mattei & Scherf 1992). Pf332 consists of a DBL (Duffy

binding like) domain at the N terminus, followed by a predicted transmembrane domain

and a large number of highly degenerate Glu-rich repeats (Fig.5) (plasmodb; Moll et al.,

2007; Scherf & Mattei 1992). With an overrepresentation of glutamic acids (30%) and

34

valine (13%) (Hinterberg et al., 1994), Pf332 shares related amino acid repeat sequences

with other Glutamic acid rich antigens such as RESA (Cappai et al., 1989) and Pf11.1

(Scherf et al., 1992), and consequently these proteins show antigenic cross-reactivity

(Ahlborg et al., 1991; Mattei et al., 1992).

During the later stages of the intraerythrocytic parasite development, this protein appears

in vesicle-like structures (MCs) in the iRBCs cytoplasm and later becomes associated

with the iRBC membrane skeleton (Hinterberg et al., 1994; Waller et al., 2010). The

functional role of Pf332 in the parasite’s life cycle has not yet been fully elucidated.

Nevertheless, studies have shown that the antigen could be involved in the trafficking of

the major virulence adhesion protein PfEMP1 to the surface of the iRBC, as seen in a co-

localization study, where Pf332 was transported in vesicles together with RIFIN and

PfEMP-1 (Haeggström et al., 2004). Other findings showed that the N-terminal DBL

region of Pf332 might be involved in re-invasion of red blood cells by facilitating uRBC

adhesion (Moll et al., 2007), and as well as modulating iRBC membrane rigidity and

assisting trafficking of PfEMP1 (Glenister et al., 2009, Hodder et al., 2009). Another

region of Pf332, located in the C- terminus binds to actin, suggesting that Pf332 might be

important in modulating the RBC membrane skeleton (Waller et al., 2010).

Studies on another region located C-terminally has showed that anti-Pf332 antibodies

were involved in growth inhibition of parasites and the iRBC integrity (Paper I, Paper II).

As well, it has been demonstrated that a centrally located region might be a target for

opsonizing antibodies (Gysin et al., 1993; Perraut et al., 1995). The different

observations in predicted functional role of antigen Pf332 is probably due to the large

size of the protein and the fact that the different groups have focused on the different

regions of the protein. These has led to the proposition that different regions of Pf332

may be involved in different functions during the erythrocytic cycle of the parasites life

cycle (Hodder et al., 2009; Glenister et al., 2009; Waller et al., 2010; Paper II). Studies

on Pf332 have been carried out using recombinant fragments of the antigen, and some of

the fragments used in our studies are described below:

35

EB200 is a fragment of antigen Pf332 (Mattei &Scherf 1992). It is about 157 aa long, and

centrally located. It consists of mainly glutamic acid (E) repeats often occurring in pairs.

This fragment has been defined as a target of anti-EB200 opsonizing antibodies in P.

falciparum-exposed Saimiri monkeys (Gysin et al., 1993; Perraut et al., 1995). A study

on sera from Senegalese adults displayed EB200-reactive antibodies of mainly the IgG3

subclass (Perraut et al., 2000) and in another study the levels of anti-EB200 antibodies

were found to be associated with fewer future malaria attacks (Ahlborg et al., 2002).

Pf332-C231 is recombinant fragment located at the C-terminal region of the antigen,

which was characterized in papers I-III in this thesis, and it will be dealt with below in

Section 4.

Pf332-DBL is a region located in the N-terminal region of the antigen. This region has a

Duffy binding like (DBL) domain, which is related to the DBL domain of erythrocyte-

binding-like (EBL) family of proteins (Moll et al., 2007). Studies on this region by Moll

et al., 2007, disclosed that antigen Pf332 may be important for merozoite re-invasion, as

infected RBCs were shown to bind to uninfected RBCs. (Moll et al., 2007).

Immunization studies with animal model revealed that recombinant Pf332-DBL was able

to induce a strong IgG response in the presence of an adjuvant approved for human use

(Du et al., 2010). Recently, reactivity with plasma from individuals living in malaria

endemic regions (Uganda, Burkina Faso and Mali) has been reported (Nilsson et al.,

2011, (In press)). This fragment was used in the Paper IV of this thesis.

Pf332EB”00DBL

predictedTM

nDBL EB200 C231Val-richregion

Glu-rich region

Figure 5: A schematic representation of antigen Pf332.

36

3.2.2 Immune responses to antigen Pf332

As mentioned previously, an effective protective immunity against P. f. malaria in

humans develop gradually overtime after repeated exposure, protecting against

progression to symptomatic and severe illness. The importance and quality of the

response in relation to protective immunity is associated with different classes and

subclasses of antibodies induced by the antigen in naturally exposed individuals. Several

data have indicated that Pf332 might be a target of protective immune response as it has

been demonstrated to be a target for ADCI and growth inhibitory antibodies.

The Pf332-reactive human monoclonal antibody (mAb) 33G2 has been demonstrated to

inhibit both P. falciparum growth and cytoadherence in vitro (Udomsangpetch et al,

1989). Furthermore, various rabbit polyclonal antibodies specific for Pf332 (Ahlborg et

al., 1996; Alhborg et al., 1995; paper I), and human polyclonal IgG antibodies, affinity-

purified on Pf332 fragments, also display similar parasite growth inhibitory capacity

(Ahlborg et al., 1993; paper II). In addition, Pf332-reactive antibodies in humans are

associated with decreased number of malaria incidents (Ahlborg et al., 2002; paper III).

Experimental animal vaccinations with Pf332 have mainly been conducted with EB200

fragment (Mattei & Scherf, 1992). In this primate model, EB200 was identified as a

target of opsonizing antibodies present in hyperimmune sera from P. f.- exposed Saimiri

monkeys (Gysin et al., 1993), and the presence of these antibodies correlated with

protection against disease (Perraut et al., 1995).

Evidence from various sero-epidemiological studies with the C231 fragment of Pf332

(Sudan- Nasr et al., 2007; Giha et al., 2009, 2010a, & 2010b; Nasr et al., 2009; Senegal-

paper I, paper III; Nigeria- Iriemenam et al., 2009, BurkinaFaso, Liberia, Mali- paper

I) have demonstrated that antibody responses to this antigen are present during natural

infections. Recently, immunization studies with an experimental model, showed that

Pf332-DBL generated a strong and significant IgG response (Du et al., 2010), and in

37

addition reactivity with several plasma from different malaria endemic regions have been

demonstrated (Nilsson et al., 2011, In press). From a prospective study using microarray,

Malian sera used were highly reactive with antigen Pf332 (Crompton et al., 2010), further

strengthening the fact that antigen Pf332 might be a target for protective antibody

reponses during natural P. f. infection to malaria.

38

4 The Present Investigation

4.1 Preface

Despite efforts made towards vaccine development, malaria still remains uncontrolled

due to the meager knowledge of the natural protective immunity against this disease.

Induction of an effective immune response to malaria is multifaceted and mechanisms

behind the induction, regulation and maintenance, should be considered on antigen-

specific level. As mentioned earlier, the asexual erythrocytic stages of the malaria

parasite are the major cause of morbidity and mortality, and major focus is based on

identifying and characterizing antigens from the blood stage cycle. As the importance of

antibodies in immunity against malaria has been emphasized, antigens from the blood

stage cycle are expected to induce significant antibodies, which may be functional in

protective immunity. These antigens might in the end be effective for vaccine

development.

This present study is based on Pf332, an antigen derived from the late stage of the

erythrocytic cycle, with focus on the two recombinant fragments Pf332-C231 and Pf332-

DBL, derived from two different regions of this megadalton protein. Being an

uncommonly large protein it will be feasible to work with fragments rather than the large

protein as a whole. The rationale behind Pf332-C231 is based on earlier studies which

focused on the central part of Pf332, EB200. This fragment is quite overrepresented with

glutamic acid repeats and there have been difficulties in defining the actual target for

parasite growth inhibitory antibodies due to its antigenic cross-reactivity with other

malaria antigens. In order to address this problem, it was proposed that a less repetitive

fragment Pf332-C231 located in the C-terminal region of the antigen be investigated.

This C-terminal fragment is well represented with various amino acids and most

importantly has a cysteine, which might be involved in the folding of the protein. Studies

on the second fragment, Pf332-DBL, located at the N-terminus are based on the findings

that antibodies targeted against this region might be effective against parasite re-invasion

(Moll et al., 2007) as well as immunization studies carried out in experimental models

(Du et al., 2010).

39

Within the context of this thesis, we addressed issues such as cloning of recombinant

fragments (Paper I), a molecular technology that has facilitated numerous studies such as

characterization of the antigen, generation of antigen specific antibodies, and the ability

of specific antibodies to be effective in invitro killing of parasites and inhibiting growth

of parasites (Paper I & II). Understanding the humoral response to malarial antigens has

been a major objective in malaria research, initially this centered on the identification of

protective immune responses and the classical serum replacement of Cohen and

McGregor (Cohen et al., 1961; McGregor, 1964). In this thesis we looked at the humoral

response of naturally exposed Senegalese against two different fragments (Pf332-C231 –

Paper III, Pf332-DBL – Paper IV) of antigen Pf332, using the conventional ELISA.

4.2 Objectives

The specific aims of the research work carried out in this thesis were:

To study the immunological capabilities of the fragment Pf332-C231 using an

experimental model (Paper I).

To investigate the immunological capacities and possible mode of action of

affinity purified human antibodies reactive with the Pf332-C231 fragment of

antigen Pf332 (Paper II).

To study the profile of the antibody responses to Pf332-C231 in naturally exposed

individuals living in Senegal (Paper III).

To determine the pattern of immunoglobulin responses to Pf332-DBL fragment of

Pf332 in exposed individuals living in Senegal (Paper IV).

40

4.3 Experimental approach

The materials and methods used, as well as ethical approvals are described in detail in the

respective studies (paper I-IV). A brief description of the study area used in the sero-

epidemiological studies (paper III & IV) is given below.

4.3.1 Study population

Senegal is a country south of the Sénégal River in western Africa. The country is

externally bounded by the Atlantic Ocean to the west, Mauritania to the north, Mali to the

east, and Guinea and Guinea-Bissau to the south; internally it almost completely

surrounds The Gambia (Fig. 6). Senegal covers a land area of almost 197,000 square

kilometres (76,000 sq mi), and has an estimated population of about 14 million. The

climate is tropical with two seasons: the dry season and the rainy season. Dakar, the

capital city of Senegal, is located at the westernmost tip of the country on the Cap-Vert

peninsula. The specific study area in this thesis for Paper III & IV is the village of

Dielmo (Fig. 6). Dielmo is in an area of the Sudan-type, savanna region of Senegal,

located 280 km southeast of Dakar (about 4 hr drive from Dakar) and about 15 km north

of the Gambian border (Trape et al., 1994). Rainfall occurs during a four-month period

(from end of June-mid October). The village is situated on the marshy bank of a small

permanent stream (the Nema) that permits the persistence of anopheline breeding sites

year round (Trape et al., 1994). The inhabitants of Dielmo are settled agricultural workers

(Trape et al., 1994).

4.3.2 The Malaria Situation in Senegal

Malaria in Senegal is defined as endemic/stable with seasonal transmission occurring

from June to November, with almost all cases caused by P. falciparum. There are

significant epidemiological variations from north (hypoendemic) to south

41

(hyperendemic). Malaria has been a venerable public health problem in Senegal. At the

national level, 2 million cases (> 2000 deaths) of malaria were recorded. In Dielmo,

malaria transmission is intense and permanent. The annual entomological inoculation rate

(EIR) recorded in 1996 in Dielmo was 305 P. falciparum infective bites per individual.

The longitudinal epidemiological follow-up of this study area has been described in detail

in (Trape et al., 1994). However, in Senegal, the malaria cases and deaths has declined

markedly between 2007 and 2009, due to the effective malaria control programme

established since 2005(WHO, 2010).

http://www.rollbackmalaria.org/ProgressImpactSeries/report4.html,

http://www.who.int/malaria/publications/country-profiles/profile_sen_en.pdf,

Figure 6: Map of Senegal, showing the study area Dielmo. (Modified from

http://en.wikipedia.org/wiki/Senegal).

Dielmo

42

4.4 Results and discussion

4.4.1 Paper I

The majority of all functional studies on malarial proteins have been initiated by

introducing genes downstream of promoters ported by plasmids, and transfection into E.

coli for recombinant expression. There are however other expression systems such as

baculovirus, yeast, mammalian and insect cells, but the approach which we have used in

our study poses some advantages, such as simplicity and low cost, availability of

compatible molecular tools, absence of post translational modifications, higher yield per

unit biomass. In this paper, we were able to clone the recombinant protein of interest,

using the plasmid vector and expression was carried out in E. coli. In molecular biology,

expression in E. coli has been debarred by the formation of insoluble aggregates of the

recombinant protein being expressed, and to circumvent this problem, we enhanced the

purification of our protein, using a solubility enhancing fusion tag. The 6xHis tag is a

small, poorly immunogenic tag, which does not interfere with the structure or function of

the purified protein.

This study on C231, possesses theoretical and practical advantages over those on EB200

regarding functional and immunological studies, as we observed that the primary

sequence of C231 is more complex as the contribution of each type of amino acid is more

equal, and the C231 sequence also consists of one cysteine residue, which theoretically

may contribute to sequence conservation and formation of a disulphide bond. The

characterization of this recombinant Pf332-C231 fragment is one of the steps that have

facilitated the unravelling of the entangled Pf332. Additional bioinformatic analysis also

revealed that C231 consists of conserved B-cell epitopes.

Regarding the immunogenicity of this protein, Pf332-C231 induced high antibody titers

in rabbits, and these antibodies also recognized the native protein expressed in infected

erythrocytes, as revealed by immunoflourescence and immunoblotting. The functionality

of antibodies induced against the asexual blood stages of P. falciparum are evaluated on

43

the basis of their ability to induce anti-parasite activity. Such antibodies may have

different effector functions, e.g. inhibition of invasion or inhibition of parasite

growth/development, depending on the target antigen. In in vitro experiments carried out

with anti-C231 antibodies we observed inhibition of merozoite invasion, as reflected by

inhibition of parasite development of infected RBCs. Reactivities by ELISA with

recombinant C231 of plasma samples of malaria-immune individuals from Senegal,

Liberia, Burkina Faso and Mali, revealed that the Pf332-C231 region is immunogenic

during natural malaria infections, as revealed by its recognition by antibodies at high

frequency. Thus, the B-cell epitopes of Pf332 are not only limited to the glutamic acid

rich repeats.

As mentioned earlier, antibodies to EB200 displayed cross-reactivity with other malaria

antigens, however in this study we could not totally circumvent this effect, as some

degree of cross-reactivity of rabbit anti-C231 antibodies was observed with RESA.

4.4.2 Paper II

Humans repeatedly exposed to malaria have been shown to be reactive with antigen

Pf332 (Ahlborg et al., 2002; Paper I; Paper III), and Pf332 reactive antibodies have been

demonstrated to inhibit the growth of F32 parasites in vitro (Paper I), although the

mechanisms by which such antibodies inhibit invasion/parasite growth is not explicit. In

this paper, using an in vitro model, we investigated the ability of Pf332-C231 affinity

purified human antibodies to inhibit the growth of F32 parasites, and the likely mode of

action of these specific antibodies was studied.

Using seven sera from malaria exposed Liberian adults, Pf332-C231 reactive-antibodies

were affinity purified using CNBr activated Sepharose. All affinity-purified antibodies

were reactive by ELISA, and positive in immunofluorescence, giving the typical Pf332

dot-like staining pattern. To analyse the functional activities of the C231-specific

antibodies, a parasite growth inhibition assay was performed, and it was found that all

44

C231-specific antibody preparations except one inhibited (> 50%) the growth of

parasites, leading to subsequent reduction in parasitaemia. The specificity of this

inhibition was further established by an antigen reversal assay, were the inhibitory effect

seen was reduced/blocked by the recombinant C231. In this study affinity purified

antibodies were of the IgM and IgG isotypes, but the IgG isotype seemed to be

responsible for the inhibitory effect observed.

In addition to the ability of Pf332-specific antibodies to inhibit parasite growth, external

matured parasites were observed in the form of pyknotic parasites as well as parasites

with anomalous morphologies. The presence of these external late stage parasites may

indicate that Pf332-C231 targets the cytoskeleton of the infected RBC. This observation

is highly important, as Pf332 has been affirmed to be involved in modulating the rigidity

of the cytoskeleton of iRBCs (Glenister et al., 2009, Hodder et al., 2009), specifically by

binding to F-actin through a region in the C-terminal domain (Waller et al., 2010). Thus,

the presence of Pf332-specific antibodies may lead to interference with the cytoskeletal

network, eventually leading to destruction of the iRBCs and exposure of the mature

parasites, which may imply a minimal or no reinvasion.

The effect of anti-C231 antibodies was further examined, when antibody pressure on F32

parasites was removed after 42 h and parasites growth monitored. Initially, the growth of

the antibody treated parasites was retarded, as compared to that of the control parasites,

but the parasite growth was recuperated after 5 invasive cycles. However, there may be a

different situation in vivo, as other immune mechanisms (Bolad & Berzins, 2000) may

aid parasite clearance. Indeed, immunity to malaria is not sterile, and asymptomatic

infections are very common in malaria endemic countries, and it has been mentioned that

antibodies to some intracellular malaria antigens might be important in curbing parasite

load (Crompton et al., 2010). Therefore, the implication of antibody pressure is that it

may give rise to parasite clones with a differential sensitivity to growth inhibitory

antibodies, an observation seen earlier with Pf332-reactive antibodies (Iqbal et al., 1997),

and which may be an avenue for immune evasive mechanisms due to antigenic variation.

45

The ultimate position of Pf332 regarding the infected erythrocyte is still uncertain. Moll

et al., (2007) showed that the antigen is expressed on the surface of infected RBCs, but

another study failed to detect this (Hodder et al., 2009). We performed

immunoflourescence on live infected RBCs, but could not reach any conclusion

regarding the surface exposure of the C-terminal part of Pf332, as we only detected a

very weak staining. Although different fragments were used in the above mentioned

studies, we may speculate that C-terminal region may be more involved in the interaction

with the iRBC membrane skeleton, as suggested by Waller et al., (2010), but may also be

partially on the surface of iRBCs. In addition, recent unpublished data showed that the

Pf332 protein is present on the cytosolic face of the cleft via protein-protein interaction

(Nilsson et al., 2011 (in PhD thesis by S. Nilsson). Obviously, the actual location of

Pf332 regarding surface or internal membrane of the iRBC needs intensive structural

studies. If the final location of Pf332 is not on the iRBC membrane, the mode of entry of

antibodies into the iRBC to effect growth inhibition or parasite killing is still unrequited.

Most likely, antibodies may gain access to intraerythrocytic epitopes by passing through

the leaky membrane during the late parasite stages, close to the time of rupture or via the

parasitophorous duct (Pouvelle et al 1991, Taraschi 1999).

4.4.3 Paper III & IV

To understand the dynamics of the host-parasite balance, its relationship to transmission,

and to identify those factors that contribute to the development of disease, it is essential

to carry out studies of well defined cohorts, in which malaria parameters are recorded

longidutinally. As is well established, antibodies are thought to be the primary immune

effectors in the defense against erythrocytic stage P. falciparum. The level of total anti-

malarial antibodies increases with age, and is usually taken as a measure of the length and

intensity of exposure, and sometimes may indicate protection against malaria.

In Paper III, we performed analyses with recombinant Pf332-C231 as antigen in

comparison to crude malaria extract, and looked at the profile of isotypes and IgG

46

subclasses of antibodies in malaria exposed individuals from Senegal. In general, anti-

C231 reactive antibodies were detected in all sera tested regarding all isotypes (IgG, IgM,

IgE) investigated, and the levels increased significantly with age.

The polarization of antibody responses towards the IgG1 and IgG3 subclasses, which

posess the ability to bind to Fcγ receptors on the surface of monocytes, macrophages, and

neutrophils, is believed to play a key-role in immunity to blood-stage P. falciparum

infection. Such cytophilic antibodies (IgG1 and IgG3) have been demonstrated to mediate

parasite-killing responses, such as opsonization and phagocytosis of extracellular

parasites or parasitized red blood cells, and antibody-dependent cellular inhibition of

intracellular parasites. However, there are studies suggesting a protective role of non-

cytophilic IgG2 in vivo (Aucan et al., 2000; Ntoumi et al., 2005), which may to some

extent be explained by a 131 R/H polymorphism in the Fcγ receptor IIa. As a result, IgG2

is cytophilic in individuals carrying the H131 allele positive FcγIIa gene (Warmerdam et

al., 1991). When analyzing the subclasses of C231 specific IgG antibodies in sera from

individuals naturally primed to P. falciparum (Paper III), we observed a bias towards

IgG2 and IgG3 relative to IgG1. We conclude that the IgG subclass distribution of

naturally acquired antibodies to Pf332-C231 in malaria exposed individuals in Senegal

may be epitope driven. Since polarization towards IgG2 was more evident for anti-C231,

this may be affected by cumulative or current exposure to malaria by the age and FcγRIIa

genotype of the subjects. These findings have clear implications for the rational design

and evaluation of antimalarial vaccines that induce antibody-mediated protection.

However, further studies and analysis should be carried out for possible correlation of

IgG2 and H131 allele of FcγRIIa.

Comparison of serological and clinical data in a previous study with the same Senegalese

donors showed that high levels of IgG antibodies to crude malaria antigen and to EB200

were predictive of fewer future clinical attacks of malaria (Ahlborg et al., 2002). With

C231, antibodies of all isotypes tended to be higher in individuals not experiencing any

malaria attack after 12 months of follow up, which however did not reach statistical

significance in a multivariate analysis.

47

In Paper IV, using the same samples and epidemiological data employed in paper III, we

investigated the antibody reactivity pattern to the N-terminal Duffy binding like domain

of Pf332 (Pf332-DBL). In this study we detected antibodies of all isotypes and IgG

subclasses measured in all age groups, with IgG as the main antibody detected. The effect

of age, as seen with most malaria antigens, was observed mainly with IgG, IgG1 and

IgG3.

Most malaria antigens have been shown to induce antibodies of the cytophilic IgG1 and

IgG3 subclasses, which is based on many factors, such as antigenic composition and age

of donors (Tongren et al., 2006; Matondo Maya et al., 2006). In our study with Pf332-

DBL, IgG1 and IgG3 subclasses predominated the non-cytophilic IgG2 and IgG4

antibodies. When investigating if there were correlations among the levels of antibodies

of the different subclasses detected, we found all had a positive correlation, except for

IgG2, which displayed consistent negative associations.

The knowledge of IgG subclass responses associated with protection against malaria with

specific antigens varies (Stanisic et al., 2009). Thus, we investigated the association of

anti-Pf332-DBL IgG subclasses in relation to number of malaria attacks experienced by

the donors between 6 months and 3 years of follow up. The IgG3 response was found

negatively associated with the number of malaria attacks observed after 2 years of follow

up. This indicates that anti-Pf332-DBL IgG3, amidst other factors, might play a role in

the asymptomatic malaria observed in the Dielmo village during the study period. To

further elucidate the role of IgG3, the donors were divided into two groups based on the

level of IgG3 antibodies induced. It was found that the group with higher levels Pf332-

DBL IgG3 was associated with fewer malaria attacks still after 2 years of follow up, and

this remained significant when all other variables were controlled. In a recent preliminary

microarray analysis, it was observed that the antibody reactivity to Pf332 was

significantly higher in protected individuals (Crompton et al., 2010). The observation

with antibodies to Pf332 might be a reflection of anti-disease immunity whereby anti-

Pf332 antibodies (Pf332-DBL IgG3) might prevent pathological symptoms.

48

Even though we observed positive correlations between the IgG (R=0.706, p=<0.0001)

and IgM (R= 0.711, p= <0.0001) antibody responses against Pf332- C231 (paper III)

and Pf332-DBL (paper IV), the pattern of antibody responses differed, even with the

previously studied EB200 fragment (Ahlborg et al., 2002). These results are indicative of

antigen Pf332 being capable of inducing specific polyclonal antibodies. This is not

unexpected, as these fragments, even if from the same antigen, possess different antigenic

properties, owing to the differences in their amino acid composition. In addition, the size

of Pf332 permits this, as it has been suggested that such a megadalton protein may

engage its different regions in various functions (Hodder et al., 2009; Glenister et al.,

2009; Waller et al., 2010; Paper I).

49

4.5 Concluding remarks

The natural immunity to malaria consists of a complex mixture of diverse immune

responses, some non-protective and some protective or inhibitory. Within the capacity of

our studies, we have shown that the C-terminal C231 fragment of antigen Pf332, an

asexual blood stage antigen is immunogenic. Furthermore we have been able to ascertain

the functional activity of Pf332-specific (Pf332-C231), human antibodies using the in

vitro inhibition assay, a prerequisite for evaluation of blood-stage vaccine candidates and

for identifying targets of protective antibodies against malaria. In addition, the presence

of Pf332 (C231 & DBL) reactive antibodies associated with protection, was found in all

age groups of asymptomatic individuals naturally exposed to P. falciparum infection.

The development of an effective malaria vaccine encompasses a whole array of setbacks.

The complexity of the parasite cycle offers various candidates, and there might be doubts

whether these candidates will induce the right type of immunological response, even if it

does in experimental models and some clinical trials. Furthermore, it might not be able to

induce the long term immunological memory essential for long term protection.

Antibodies to intracellular proteins, such as Pf332, are typically viewed as markers of

past infection and not as evidence of vaccine potential, based on the location of such

antigens. However, it is possible that higher antibody levels against intracellular proteins

among protected individuals, as seen in our studies, could be a predictor of anti-disease

immunity and/or a marker of enhanced parasite killing (Crompton et al., 2010). On this

note, it is assumed that it might be too early to dismiss intracellularly located

immunogenic proteins, such as antigen Pf332, as potential vaccine targets based solely on

their subcellular location, as little is known about the molecular host-malaria parasite

interactions. Based on our findings (Paper I- IV), and other recent studies on Pf332

(Glenister et al., 2009; Moll et al., 2007; Hodder et al., 2009; Du et al., 2010, Crompton

et al., 2010), we propose that Pf332 is a target for parasite neutralizing antibodies, which

emphasizes that Pf332 might be a potential vaccine candidate.

50

d

a b

External and dead parasites

Abnormal disintegrating Schizonts

Parasites with pale iRBCs

c

Figure 7a-c: Schematic representation of the possible mechanism of antibody mediated

inhibition of intraerythrocytic development of parasites by anti-Pf332 antibodies. a) is by

antibodies binding to antigens on the surface of iRBC, b) by antibodies entering through

the leaky membrane, c) antibodies entering through the parasitophorous duct, and d) the

morphological effects of (C231-Pf332) affinity purified human antibodies on F32

parasites, in vitro.

51

4.6 Future perspectives

Amidst all inferences mentioned above, further studies should be carried out addressing

different aspects of antibodies to Pf332.

Antibodies induced by proteins are commonly directed against surface exposed regions

of the protein, and certain epitopes may be immunodominant. One strategy for the

selection of antigenic sequences to be included in a subunit vaccine against P. falciparum

malaria, is to define epitopes seen by antibodies which have the ability to interfere with

parasite development. A Pf332 reactive monoclonal antibody (mAb 33G2), which was

shown to have the capacity to inhibit parasite growth/merozoite invasion in vitro,

harbours an epitope of five amino acids long sequence, VTEEI (Ahlborg et al., 1991). In

line with this, the elucidation of specific B-cell epitopes by analyzing the ability of

peptides to induce antibodies may reveal different epitope specificities regarding Pf332-

C231 and other fragments. Furthermore, production of new human monoclonal

antibodies against epitopes in different regions of Pf332 would be important tools for

dissecting the antigen in specific functional assays.

Further studies on the parasite inhibitory capacity of affinity purified human anti-C231

antibodies from human sera from other malaria endemic areas should be carried out. It

might also be interesting to see the inhibitory effect of affinity purified antibodies of

other Pf332 fragments, using the same samples. Also of importance is to carry out

inhibition assays with other parasite strains as we only used the F32 strain. The parasite

neutralizing capacity of these human antibodies can also be tested with human

monocytes, as this mimics the immunological situation in vivo.

To further elucidate the mechanism of the antibody mediated inhibition of parasite

growth, it may be functional to perform inhibition experiments with Pf332 reactive

antibodies on parasites lacking expression of Pf332, (Hodder et al. 2009; Glenister et al.,

2009), as well as inhibition experiments with mixtures of antibodies to different regions

of Pf332 to see if there will be synergistic effects. Such experiments would also give

information about whether Pf332 is the true target for the inhibitory antibodies.

52

4.7 Other Publications

The following articles are of relative importance but not included within the framework

of the thesis:

Nasr A, Iriemenam NC, Troye-Blomberg M, Giha HA, Balogun HA, Osman OF,

Montgomery SM, ElGhazali G, Berzins K. Fc gamma receptor IIa (CD32)

polymorphism and antibody responses to asexual blood-stage antigens of

Plasmodium falciparum malaria in Sudanese patients. Scand J Immunol. 2007

Jul;66(1):87-96.

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

ElGhazali G, Berzins K. FcgammaRIIa (CD32) polymorphism and anti-malarial

IgG subclass pattern among Fulani and sympatric ethnic groups living in eastern

Sudan. Malar J. 2009 Mar 13;8:43.

Giha HA, Nasr A, Iriemenam NC, Balogun HA, Arnot D, Theander TG, Troye-

Blomberg M, Berzins K, El Ghazali G. Age-dependent association between IgG2

and IgG3 subclasses to Pf332-C231 antigen and protection from malaria, and

induction of protective antibodies by sub-patent malaria infections, in Daraweesh.

Vaccine. 2010 Feb 17;28(7):1732-9. Epub 2009 Dec 28.

Iriemenam NC, Okafor CM, Balogun HA, Ayede I, Omosun Y, Persson

JO, Hagstedt M, Anumudu CI, Nwuba RI, Troye-Blomberg M, Berzins K.

Cytokine profiles and antibody responses to Plasmodium falciparum malaria

infection in individuals living in Ibadan, southwest Nigeria. Afr Health Sci. 2009

Jun;9(2):66-74.

Awah N, Balogun H, Achidi E, Mariuba LA, Nogueira PA, Orlandi P, Troye-

Blomberg M, Gysin J, Berzins K. Antibodies to the Plasmodium falciparum

rhoptry protein RAP-2/RSP-2 in relation to anaemia in Cameroonian children.

Parasite Immunol. 2011 Feb;33(2):104-15.

53

5 Acknowledgements

54

☺

55

☺

56

6 References

Ahlborg N., Berzins K. & Perlmann P. 1991. Definition of the epitope recognized by

the Plasmodium falciparum-reactive human monoclonal antibody 33G2. Mol

Biochem Parasitol 46: 89-95.

Ahlborg N., Flyg B.W., Iqbal J., Perlmann P. & Berzins K. 1993. Epitope specificity

and capacity to inhibit parasite growth in vitro of human antibodies to repeat

sequences of the Plasmodium falciparum antigen Ag332. Parasite Immunol 15: 391-

400.

Ahlborg N., Iqbal J., Bjork L., Ståhl S., Perlmann P. & Berzins K. 1996. Plasmodium

falciparum: differential parasite growth inhibition mediated by antibodies to the

antigens Pf332 and Pf155/RESA. Exp Parasitol 82: 155-163.

Ahlborg N., Iqbal J., Hansson M., Uhlen M., Mattei D., Perlmann P., Ståhl S. &

Berzins K. 1995. Immunogens containing sequences from antigen Pf332 induce

Plasmodium falciparum-reactive antibodies which inhibit parasite growth but not

cytoadherence. Parasite Immunol 17: 341-352.

Aidoo M., Terlouw D.J., Kolczak M.S., McElroy P.D., ter Kuile F.O., Kariuki S.,

Nahlen B.L., Lal A.A. & Udhayakumar V. 2002. Protective effects of the sickle

cell gene against malaria morbidity and mortality. Lancet 359: 1311-1312.

Aikawa M., Torii M., Sjölander A., Berzins K., Perlmann P. & Miller L.H. 1990.

Pf155/RESA antigen is localized in dense granules of Plasmodium falciparum

merozoites. Exp Parasitol 71: 326-329.

Aikawa M., Uni Y., Andrutis A.T. & Howard R.J. 1986. Membrane-associated

electron-dense material of the asexual stages of Plasmodium falciparum: evidence

for movement from the intracellular parasite to the erythrocyte membrane. Am J

Trop Med Hyg 35: 30-36.

Allison A.C. 2009. Genetic control of resistance to human malaria. Curr Opin Immunol

21: 499-505.

Alonso P.L., Brown G., Arevalo-Herrera M., Binka F., Chitnis C., Collins F.,

Doumbo O.K., Greenwood B., Hall B.F., Levine M.M., Mendis K., Newman

R.D., Plowe C.V., Rodriguez M.H., Sinden R., Slutsker L. & Tanner M. 2011. A

research agenda to underpin malaria eradication. PLoS Med 8: e1000406.

An X., Mohandas N. 2008. Disorders of red cell membrane. Br J Haematol 141: 367-

375.

Anders R.F. 1986. Multiple cross-reactivities amongst antigens of Plasmodium

falciparum impair the development of protective immunity against malaria. Parasite

Immunol 8: 529-539.

57

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

& Troye-Blomberg M. 2011. Interethnic Differences in Antigen-Presenting Cell

Activation and TLR Responses in Malian Children duringPlasmodium

falciparum Malaria. PLoS One. 2011; 6: e18319.

Artavanis-Tsakonas K., Eleme K., McQueen K.L., Cheng N.W., Parham P., Davis

D.M. & Riley E.M. 2003. Activation of a subset of human NK cells upon contact

with Plasmodium falciparum-infected erythrocytes. J Immunol 171: 5396-5405.

Artavanis-Tsakonas K., Tongren J.E. & Riley E.M. 2003. The war between the

malaria parasite and the immune system: immunity, immunoregulation and

immunopathology. Clin Exp Immunol 133: 145-152.

Atkinson C.T., Aikawa M. 1990. Ultrastructure of malaria-infected erythrocytes. Blood

Cells 16: 351-368.

Aubouy A., Migot-Nabias F. & Deloron P. 2003. Polymorphism in two merozoite

surface proteins of Plasmodium falciparum isolates from Gabon. Malar J 2: 12.

Aucan C., Traore Y., Tall F., Nacro B., Traore-Leroux T., Fumoux F. & Rihet P.

2000. High immunoglobulin G2 (IgG2) and low IgG4 levels are associated with

human resistance to Plasmodium falciparum malaria. Infect Immun 68: 1252-1258.

Audran R., Cachat M., Lurati F., Soe S., Leroy O., Corradin G., Druilhe P. &

Spertini F. 2005. Phase I malaria vaccine trial with a long synthetic peptide derived

from the merozoite surface protein 3 antigen. Infect Immun 73: 8017-8026.

Awah N.W., Troye-Blomberg M., Berzins K. & Gysin J. 2009. Mechanisms of

malarial anaemia: potential involvement of the Plasmodium falciparum low

molecular weight rhoptry-associated proteins. Acta Trop 112: 295-302.

Ayi K., Turrini F., Piga A. & Arese P. 2004. Enhanced phagocytosis of ring-parasitized

mutant erythrocytes: a common mechanism that may explain protection against

falciparum malaria in sickle trait and beta-thalassemia trait. Blood 104: 3364-3371.

Babiuk L.A. 1999. Broadening the approaches to developing more effective vaccines.

Vaccine 17: 1587-1595.

Baird J.K. 1995. Host age as a determinant of naturally acquired immunity to

Plasmodium falciparum. Parasitol Today 11: 105-111.

Bannister L., Mitchell G. 2003. The ins, outs and roundabouts of malaria. Trends

Parasitol 19: 209-213.

Bannister L.H., Sinden R.E. 1982. New knowledge of parasite morphology. Br Med

Bull 38: 141-145.

58

Barragan A., Fernandez V., Chen Q., von Euler A., Wahlgren M. & Spillmann D.

2000. The duffy-binding-like domain 1 of Plasmodium falciparum erythrocyte

membrane protein 1 (PfEMP1) is a heparan sulfate ligand that requires 12 mers for

binding. Blood 95: 3594-3599.

Barragan A., Kremsner P.G., Wahlgren M. & Carlson J. 2000. Blood group A

antigen is a coreceptor in Plasmodium falciparum rosetting. Infect Immun 68: 2971-

2975.

Bate C.A., Taverne J., Dave A. & Playfair J.H. 1990. Malaria exoantigens induce T-

independent antibody that blocks their ability to induce TNF. Immunology 70: 315-

320.

Bereczky S., Montgomery S.M., Troye-Blomberg M., Rooth I., Shaw M.A. &

Farnert A. 2004. Elevated anti-malarial IgE in asymptomatic individuals is

associated with reduced risk for subsequent clinical malaria. Int J Parasitol 34: 935-

942.

Bergmann-Leitner E.S., Duncan E.H., Mullen G.E., Burge J.R., Khan F., Long

C.A., Angov E. & Lyon J.A. 2006. Critical evaluation of different methods for

measuring the functional activity of antibodies against malaria blood stage antigens.

Am J Trop Med Hyg 75: 437-442.

Berzins K., Anders R. F. 1999. The malaria antigens. In: Wahlgren M, Perlmann P, eds.

Malaria Molecular and Clinical Aspects. 181-216.

Berzins K., Perlmann H., Wåhlin B., Ekre H.P., Hogh B., Petersen E., Wellde B.,

Schoenbechler M., Williams J. & Chulay J. 1991. Passive immunization of Aotus

monkeys with human antibodies to the Plasmodium falciparum antigen

Pf155/RESA. Infect Immun 59: 1500-1506.

Beutler B. 2004. Innate immunity: an overview. Mol Immunol 40: 845-859.

Biswas S., Rao D.N., Roy A., Yadav R.P., Ghosh S.K. & Kabilan L. 1995. Humoral

immune responses to the Plasmodium falciparum antigen Pf155 RESA in adults

with differential clinical conditions from an Indian zone where malaria is endemic.

Southeast Asian J Trop Med Public Health 26: 219-227.

Blisnick T., Morales Betoulle M.E., Barale J.C., Uzureau P., Berry L., Desroses S.,

Fujioka H., Mattei D. & Braun Breton C. 2000. Pfsbp1, a Maurer's cleft

Plasmodium falciparum protein, is associated with the erythrocyte skeleton. Mol

Biochem Parasitol 111: 107-121.

Bolad A., Berzins K. 2000. Antigenic diversity of Plasmodium falciparum and antibody-

mediated parasite neutralization. Scand J Immunol 52: 233-239.

59

Boudin C., Chumpitazi B., Dziegiel M., Peyron F., Picot S., Hogh B. & Ambroise-

Thomas P. 1993. Possible role of specific immunoglobulin M antibodies to

Plasmodium falciparum antigens in immunoprotection of humans living in a

hyperendemic area, Burkina Faso. J Clin Microbiol 31: 636-641.

Bouharoun-Tayoun H., Druilhe P. 1992. Plasmodium falciparum malaria: evidence for

an isotype imbalance which may be responsible for delayed acquisition of protective

immunity. Infect Immun 60: 1473-1481.

Bouharoun-Tayoun H., Oeuvray C., Lunel F. & Druilhe P. 1995. Mechanisms

underlying the monocyte-mediated antibody-dependent killing of Plasmodium

falciparum asexual blood stages. J Exp Med 182: 409-418.

Bousheri S., Cao H. 2008. New insight into the role of dendritic cells in malaria immune

pathogenesis. Trends Parasitol 24: 199-200.

Branch O.H., Udhayakumar V., Hightower A.W., Oloo A.J., Hawley W.A., Nahlen

B.L., Bloland P.B., Kaslow D.C. & Lal A.A. 1998. A longitudinal investigation of

IgG and IgM antibody responses to the merozoite surface protein-1 19-kiloDalton

domain of Plasmodium falciparum in pregnant women and infants: associations with

febrile illness, parasitemia, and anemia. Am J Trop Med Hyg 58: 211-219.

Bruce-Chwatt L.J. 1952. Malaria in African infants and children in Southern Nigeria.

Ann Trop Med Parasitol 46: 173-200.

Buffet P.A., Safeukui I., Deplaine G., Brousse V., Prendki V., Thellier M., Turner

G.D. & Mercereau-Puijalon O. 2011. The pathogenesis of Plasmodium falciparum

malaria in humans: insights from splenic physiology. Blood 117: 381-392.

Cappai R., van Schravendijk M.R., Anders R.F., Peterson M.G., Thomas L.M.,

Cowman A.F. & Kemp D.J. 1989. Expression of the RESA gene in Plasmodium

falciparum isolate FCR3 is prevented by a subtelomeric deletion. Mol Cell Biol 9:

3584-3587.

Carlson J., Helmby H., Hill A.V., Brewster D., Greenwood B.M. & Wahlgren M.

1990. Human cerebral malaria: association with erythrocyte rosetting and lack of

anti-rosetting antibodies. Lancet 336: 1457-1460.

Carlson J., Holmquist G., Taylor D.W., Perlmann P. & Wahlgren M. 1990.

Antibodies to a histidine-rich protein (PfHRP1) disrupt spontaneously formed

Plasmodium falciparum erythrocyte rosettes. Proc Natl Acad Sci U S A 87: 2511-

2515.

Carpenter D., Abushama H., Bereczky S., Farnert A., Rooth I., Troye-Blomberg M.,

Quinnell R.J. & Shaw M.A. 2007. Immunogenetic control of antibody

responsiveness in a malaria endemic area. Hum Immunol 68: 165-169.

60

Chattopadhyay R., Taneja T., Chakrabarti K., Pillai C.R. & Chitnis C.E. 2004.

Molecular analysis of the cytoadherence phenotype of a Plasmodium falciparum

field isolate that binds intercellular adhesion molecule-1. Mol Biochem Parasitol

133: 255-265.

Chen Q., Heddini A., Barragan A., Fernandez V., Pearce S.F. & Wahlgren M. 2000.

The semiconserved head structure of Plasmodium falciparum erythrocyte membrane

protein 1 mediates binding to multiple independent host receptors. J Exp Med 192:

1-10.

Chishti A.H., Andrabi K.I., Derick L.H., Palek J. & Liu S.C. 1992. Isolation of

skeleton-associated knobs from human red blood cells infected with malaria parasite

Plasmodium falciparum. Mol Biochem Parasitol 52: 283-287.

Clark I.A., Cowden W.B. 1992. Roles of TNF in malaria and other parasitic infections.

Immunol Ser 56: 365-407.

Coban C., Ishii K.J., Kawai T., Hemmi H., Sato S., Uematsu S., Yamamoto M.,

Takeuchi O., Itagaki S., Kumar N., Horii T. & Akira S. 2005. Toll-like receptor 9

mediates innate immune activation by the malaria pigment hemozoin. J Exp Med

201: 19-25.

Cohen S., McGregor I.A. & Carrington S. 1961. Gamma-globulin and acquired

immunity to human malaria. Nature 192: 733-737.

Cooke B.M., Buckingham D.W., Glenister F.K., Fernandez K.M., Bannister L.H.,

Marti M., Mohandas N. & Coppel R.L. 2006. A Maurer's cleft-associated protein

is essential for expression of the major malaria virulence antigen on the surface of

infected red blood cells. J Cell Biol 172: 899-908.

Cooke G.S., Aucan C., Walley A.J., Segal S., Greenwood B.M., Kwiatkowski D.P. &

Hill A.V. 2003. Association of Fcgamma receptor IIa (CD32) polymorphism with

severe malaria in West Africa. Am J Trop Med Hyg 69: 565-568.

Coppel R.L., Lustigman S., Murray L. & Anders R.F. 1988. MESA is a Plasmodium

falciparum phosphoprotein associated with the erythrocyte membrane skeleton. Mol

Biochem Parasitol 31: 223-231.

Couper K.N., Phillips R.S., Brombacher F. & Alexander J. 2005. Parasite-specific

IgM plays a significant role in the protective immune response to asexual

erythrocytic stage Plasmodium chabaudi AS infection. Parasite Immunol 27: 171-

180.

Crabb B.S., Cooke B.M. 2004. Molecular approaches to malaria: MAM 2004 and

beyond. Trends Parasitol 20: 547.

61

Crabb B.S., Cooke B.M., Reeder J.C., Waller R.F., Caruana S.R., Davern K.M.,

Wickham M.E., Brown G.V., Coppel R.L. & Cowman A.F. 1997. Targeted gene

disruption shows that knobs enable malaria-infected red cells to cytoadhere under

physiological shear stress. Cell 89: 287-296.

Crompton P.D., Kayala M.A., Traore B., Kayentao K., Ongoiba A., Weiss G.E.,

Molina D.M., Burk C.R., Waisberg M., Jasinskas A., Tan X., Doumbo S.,

Doumtabe D., Kone Y., Narum D.L., Liang X., Doumbo O.K., Miller L.H.,

Doolan D.L., Baldi P., Felgner P.L. & Pierce S.K. 2010. A prospective analysis of

the Ab response to Plasmodium falciparum before and after a malaria season by

protein microarray. Proc Natl Acad Sci U S A 107: 6958-6963.

Culvenor J.G., Day K.P. & Anders R.F. 1991. Plasmodium falciparum ring-infected

erythrocyte surface antigen is released from merozoite dense granules after

erythrocyte invasion. Infect Immun 59: 1183-1187.

Czajkowsky D.M., Salanti A., Ditlev S.B., Shao Z., Ghumra A., Rowe J.A. & Pleass

R.J. 2010. IgM, Fc mu Rs, and malarial immune evasion. J Immunol 184: 4597-

4603.

Czajkowsky D.M., Shao Z. 2009. The human IgM pentamer is a mushroom-shaped

molecule with a flexural bias. Proc Natl Acad Sci U S A 106: 14960-14965.

Dahlback M., Rask T.S., Andersen P.H., Nielsen M.A., Ndam N.T., Resende M.,

Turner L., Deloron P., Hviid L., Lund O., Pedersen A.G., Theander T.G. &

Salanti A. 2006. Epitope mapping and topographic analysis of VAR2CSA DBL3X

involved in P. falciparum placental sequestration. PLoS Pathog 2: e124.

David P.H., Hommel M., Miller L.H., Udeinya I.J. & Oligino L.D. 1983. Parasite

sequestration in Plasmodium falciparum malaria: spleen and antibody modulation of

cytoadherence of infected erythrocytes. Proc Natl Acad Sci U S A 80: 5075-5079.

Dertzbaugh M.T. 1998. Genetically engineered vaccines: an overview. Plasmid 39: 100-

113.

Desowitz R.S. 1991. The malaria Capers.

D'Ombrain M.C., Voss T.S., Maier A.G., Pearce J.A., Hansen D.S., Cowman A.F. &

Schofield L. 2007. Plasmodium falciparum erythrocyte membrane protein-1

specifically suppresses early production of host interferon-gamma. Cell Host

Microbe 2: 130-138.

Doolan D.L., Dobano C. & Baird J.K. 2009. Acquired immunity to malaria. Clin

Microbiol Rev 22: 13-36, Table of Contents.

Du C., Nilsson S., Lu H., Yin J., Jiang N., Wahlgren M. & Chen Q. 2010.

Immunogenicity of the Plasmodium falciparum Pf332-DBL domain in combination

with different adjuvants. Vaccine 28: 4977-4983.

62

Duah N.O., Weiss H.A., Jepson A., Tetteh K.K., Whittle H.C. & Conway D.J. 2009.

Heritability of antibody isotype and subclass responses to Plasmodium falciparum

antigens. PLoS One 4: e7381.

Duarte J., Deshpande P., Guiyedi V., Mecheri S., Fesel C., Cazenave P.A., Mishra

G.C., Kombila M. & Pied S. 2007. Total and functional parasite specific IgE

responses in Plasmodium falciparum-infected patients exhibiting different clinical

status. Malar J 6: 1.

Durand P.M., Coetzer T.L. 2008. Hereditary red cell disorders and malaria resistance.

Haematologica 93: 961-963.

Ehrenstein M.R., Notley C.A. 2010. The importance of natural IgM: scavenger,

protector and regulator. Nat Rev Immunol 10: 778-786.

El Sahly H.M., Patel S.M., Atmar R.L., Lanford T.A., Dube T., Thompson D., Sim

B.K., Long C. & Keitel W.A. 2010. Safety and immunogenicity of a recombinant

nonglycosylated erythrocyte binding antigen 175 Region II malaria vaccine in

healthy adults living in an area where malaria is not endemic. Clin Vaccine Immunol

17: 1552-1559.

Enayati A., Hemingway J. 2010. Malaria management: past, present, and future. Annu

Rev Entomol 55: 569-591.

Esen M., Kremsner P.G., Schleucher R., Gassler M., Imoukhuede E.B., Imbault N.,

Leroy O., Jepsen S., Knudsen B.W., Schumm M., Knobloch J., Theisen M. &

Mordmuller B. 2009. Safety and immunogenicity of GMZ2 - a MSP3-GLURP

fusion protein malaria vaccine candidate. Vaccine 27: 6862-6868.

Farouk S.E., Dolo A., Bereczky S., Kouriba B., Maiga B., Farnert A., Perlmann H.,

Hayano M., Montgomery S.M., Doumbo O.K. & Troye-Blomberg M. 2005.

Different antibody- and cytokine-mediated responses to Plasmodium falciparum

parasite in two sympatric ethnic tribes living in Mali. Microbes Infect 7: 110-117.

Farouk S.E., Mincheva-Nilsson L., Krensky A.M., Dieli F. & Troye-Blomberg M.

2004. Gamma delta T cells inhibit in vitro growth of the asexual blood stages of

Plasmodium falciparum by a granule exocytosis-dependent cytotoxic pathway that

requires granulysin. Eur J Immunol 34: 2248-2256.

Fenton B., Clark J.T., Khan C.M., Robinson J.V., Walliker D., Ridley R., Scaife

J.G. & McBride J.S. 1991. Structural and antigenic polymorphism of the 35- to 48-

kilodalton merozoite surface antigen (MSA-2) of the malaria parasite Plasmodium

falciparum. Mol Cell Biol 11: 963-971.

Fernandez-Reyes D., Craig A.G., Kyes S.A., Peshu N., Snow R.W., Berendt A.R.,

Marsh K. & Newbold C.I. 1997. A high frequency African coding polymorphism

in the N-terminal domain of ICAM-1 predisposing to cerebral malaria in Kenya.

Hum Mol Genet 6: 1357-1360.

63

Ferreira A., Balla J., Jeney V., Balla G. & Soares M.P. 2008. A central role for free

heme in the pathogenesis of severe malaria: the missing link? J Mol Med (Berl) 86:

1097-1111.

Florens L., Washburn M.P., Raine J.D., Anthony R.M., Grainger M., Haynes J.D.,

Moch J.K., Muster N., Sacci J.B., Tabb D.L., Witney A.A., Wolters D., Wu Y.,

Gardner M.J., Holder A.A., Sinden R.E., Yates J.R. & Carucci D.J. 2002. A

proteomic view of the Plasmodium falciparum life cycle. Nature 419: 520-526.

Foley M., Corcoran L., Tilley L. & Anders R. 1994. Plasmodium falciparum: mapping

the membrane-binding domain in the ring-infected erythrocyte surface antigen. Exp

Parasitol 79: 340-350.

Frischknecht F., Lanzer M. 2008. The Plasmodium falciparum Maurer's clefts in 3D.

Mol Microbiol 67: 687-691.

Gardner M.J., Hall N., Fung E., White O., Berriman M., Hyman R.W., Carlton

J.M., Pain A., Nelson K.E., Bowman S., Paulsen I.T., James K., Eisen J.A.,

Rutherford K., Salzberg S.L., Craig A., Kyes S., Chan M.S., Nene V., Shallom

S.J., Suh B., Peterson J., Angiuoli S., Pertea M., Allen J., Selengut J., Haft D.,

Mather M.W., Vaidya A.B., Martin D.M., Fairlamb A.H., Fraunholz M.J., Roos

D.S., Ralph S.A., McFadden G.I., Cummings L.M., Subramanian G.M.,

Mungall C., Venter J.C., Carucci D.J., Hoffman S.L., Newbold C., Davis R.W.,

Fraser C.M. & Barrell B. 2002. Genome sequence of the human malaria parasite

Plasmodium falciparum. Nature 419: 498-511.

Gardner M.J., Shallom S.J., Carlton J.M., Salzberg S.L., Nene V., Shoaibi A.,

Ciecko A., Lynn J., Rizzo M., Weaver B., Jarrahi B., Brenner M., Parvizi B.,

Tallon L., Moazzez A., Granger D., Fujii C., Hansen C., Pederson J., Feldblyum

T., Peterson J., Suh B., Angiuoli S., Pertea M., Allen J., Selengut J., White O.,

Cummings L.M., Smith H.O., Adams M.D., Venter J.C., Carucci D.J., Hoffman

S.L. & Fraser C.M. 2002. Sequence of Plasmodium falciparum chromosomes 2,

10, 11 and 14. Nature 419: 531-534.

Garraud O., Mahanty S. & Perraut R. 2003. Malaria-specific antibody subclasses in

immune individuals: a key source of information for vaccine design. Trends

Immunol 24: 30-35.

Garraud O., Perraut R., Diouf A., Nambei W.S., Tall A., Spiegel A., Longacre S.,

Kaslow D.C., Jouin H., Mattei D., Engler G.M., Nutman T.B., Riley E.M. &

Mercereau-Puijalon O. 2002. Regulation of antigen-specific immunoglobulin G

subclasses in response to conserved and polymorphic Plasmodium falciparum

antigens in an in vitro model. Infect Immun 70: 2820-2827.

Garraud O., Perraut R., Riveau G. & Nutman T.B. 2003. Class and subclass selection

in parasite-specific antibody responses. Trends Parasitol 19: 300-304.

64

Genton B., Corradin G. 2002. Malaria vaccines: from the laboratory to the field. Curr

Drug Targets Immune Endocr Metabol Disord 2: 255-267.

Giha H.A., ElGhazali G., Nasr A., Iriemenam N.C., Berzins K., Troye-Blomberg M.,

Theander T.G. & Arnot D. 2010. Clustering of malaria treatment failure (TF) in

Daraweesh: hints for host genetic susceptibility to TF with emphasis on immune-

modulating SNPs. Infect Genet Evol 10: 481-486.

Giha H.A., Nasr A., Iriemenam N.C., Arnot D., Troye-Blomberg M., Theander T.G.,

Berzins K., ElGhazali G. & Pandey J.P. 2009. Antigen-specific influence of

GM/KM allotypes on IgG isotypes and association of GM allotypes with

susceptibility to Plasmodium falciparum malaria. Malar J 8: 306.

Giha H.A., Nasr A., Iriemenam N.C., Balogun H.A., Arnot D., Theander T.G.,

Troye-Blomberg M., Berzins K. & ElGhazali G. 2010. Age-dependent association

between IgG2 and IgG3 subclasses to Pf332-C231 antigen and protection from

malaria, and induction of protective antibodies by sub-patent malaria infections, in

Daraweesh. Vaccine 28: 1732-1739.

Gilbert S.C., Plebanski M., Gupta S., Morris J., Cox M., Aidoo M., Kwiatkowski D.,

Greenwood B.M., Whittle H.C. & Hill A.V. 1998. Association of malaria parasite

population structure, HLA, and immunological antagonism. Science 279: 1173-1177.

Giusti P., Urban B.C., Frascaroli G., Albrecht L., Tinti A., Troye-Blomberg M. &

Varani S. 2011. Plasmodium falciparum-Infected Erythrocytes and {beta}-Hematin

Induce Partial Maturation of Human Dendritic Cells and Increase Their Migratory

Ability in Response to Lymphoid Chemokines. Infect Immun 79: 2727-2736.

Glenister F.K., Coppel R.L., Cowman A.F., Mohandas N. & Cooke B.M. 2002.

Contribution of parasite proteins to altered mechanical properties of malaria-infected

red blood cells. Blood 99: 1060-1063.

Glenister F.K., Fernandez K.M., Kats L.M., Hanssen E., Mohandas N., Coppel R.L.

& Cooke B.M. 2009. Functional alteration of red blood cells by a Mega-Dalton

protein of Plasmodium falciparum. Blood doi:10.1182/blood-2008-05-157735.

Gowda D. 2007. TLR-mediated cell signaling by malaria GPIs. Trends Parasitol 23:

596-604.

Green T.J., Morhardt M., Brackett R.G. & Jacobs R.L. 1981. Serum inhibition of

merozoite dispersal from Plasmodium falciparum schizonts: indicator of immune

status. Infect Immun 31: 1203-1208.

Gruenberg J., Allred D.R. & Sherman I.W. 1983. Scanning electron microscope-

analysis of the protrusions (knobs) present on the surface of Plasmodium

falciparum-infected erythrocytes. J Cell Biol 97: 795-802.

65

Gyan B., Troye-Blomberg M., Perlmann P. & Bjorkman A. 1994. Human monocytes

cultured with and without interferon-gamma inhibit Plasmodium falciparum parasite

growth in vitro via secretion of reactive nitrogen intermediates. Parasite Immunol

16: 371-375.

Gysin J., Gavoille S., Mattei D., Scherf A., Bonnefoy S., Mercereau-Puijalon O.,

Feldmann T., Kun J., Muller-Hill B. & Pereira da Silva L. 1993. In vitro

phagocytosis inhibition assay for the screening of potential candidate antigens for

sub-unit vaccines against the asexual blood stage of Plasmodium falciparum. J

Immunol Methods 159: 209-219.

Haase S., Herrmann S., Gruring C., Heiber A., Jansen P.W., Langer C., Treeck M.,

Cabrera A., Bruns C., Struck N.S., Kono M., Engelberg K., Ruch U.,

Stunnenberg H.G., Gilberger T.W. & Spielmann T. 2009. Sequence requirements

for the export of the Plasmodium falciparum Maurer's clefts protein REX2. Mol

Microbiol 71: 1003-1017.

Haberman A.M., Shlomchik M.J. 2003. Reassessing the function of immune-complex

retention by follicular dendritic cells. Nat Rev Immunol 3: 757-764.

Haeggström M., Kironde F., Berzins K., Chen Q., Wahlgren M. & Fernandez V.

2004. Common trafficking pathway for variant antigens destined for the surface of

the Plasmodium falciparum-infected erythrocyte. Mol Biochem Parasitol 133: 1-14.

Hafalla J.C., Silvie O. & Matuschewski K. 2011. Cell biology and immunology of

malaria. Immunol Rev 240: 297-316.

Handunnetti S.M., David P.H., Perera K.L. & Mendis K.N. 1989. Uninfected

erythrocytes form "rosettes" around Plasmodium falciparum infected erythrocytes.

Am J Trop Med Hyg 40: 115-118.

Hansen D.S., Schofield L. 2010. Natural regulatory T cells in malaria: host or parasite

allies? PLoS Pathog 6: e1000771.

Hanssen E., Hawthorne P., Dixon M.W., Trenholme K.R., McMillan P.J.,

Spielmann T., Gardiner D.L. & Tilley L. 2008. Targeted mutagenesis of the ring-

exported protein-1 of Plasmodium falciparum disrupts the architecture of Maurer's

cleft organelles. Mol Microbiol 69: 938-953.

Hanssen E., McMillan P.J. & Tilley L. 2010. Cellular architecture of Plasmodium

falciparum-infected erythrocytes. Int J Parasitol 40: 1127-1135.

Heddini A., Pettersson F., Kai O., Shafi J., Obiero J., Chen Q., Barragan A.,

Wahlgren M. & Marsh K. 2001. Fresh isolates from children with severe

Plasmodium falciparum malaria bind to multiple receptors. Infect Immun 69: 5849-

5856.

66

Helmby H., Cavelier L., Pettersson U. & Wahlgren M. 1993. Rosetting Plasmodium

falciparum-infected erythrocytes express unique strain-specific antigens on their

surface. Infect Immun 61: 284-288.

Henri S., Stefani F., Parzy D., Eboumbou C., Dessein A. & Chevillard C. 2002.

Description of three new polymorphisms in the intronic and 3'UTR regions of the

human interferon gamma gene. Genes Immun 3: 1-4.

Hermsen C.C., Verhage D.F., Telgt D.S., Teelen K., Bousema J.T., Roestenberg M.,

Bolad A., Berzins K., Corradin G., Leroy O., Theisen M. & Sauerwein R.W.

2007. Glutamate-rich protein (GLURP) induces antibodies that inhibit in vitro

growth of Plasmodium falciparum in a phase 1 malaria vaccine trial. Vaccine 25:

2930-2940.

Hiller N.L., Bhattacharjee S., van Ooij C., Liolios K., Harrison T., Lopez-Estrano C.

& Haldar K. 2004. A host-targeting signal in virulence proteins reveals a secretome

in malarial infection. Science 306: 1934-1937.

Hinterberg K., Scherf A., Gysin J., Toyoshima T., Aikawa M., Mazie J.C., da Silva

L.P. & Mattei D. 1994. Plasmodium falciparum: the Pf332 antigen is secreted from

the parasite by a brefeldin A-dependent pathway and is translocated to the

erythrocyte membrane via the Maurer's clefts. Exp Parasitol 79: 279-291.

Hodder A.N., Maier A.G., Rug M., Brown M., Hommel M., Pantic I., Puig-de-

Morales-Marinkovic M., Smith B., Triglia T., Beeson J. & Cowman A.F. 2009.

Analysis of structure and function of the giant protein Pf332 in Plasmodium

falciparum. Mol Microbiol 71: 48-65.

Hoffman SL 1996. Malaria Vaccine Development: A MultiImmune Response Approach.

ed.

Hoffman S.L., Oster C.N., Plowe C.V., Woollett G.R., Beier J.C., Chulay J.D., Wirtz

R.A., Hollingdale M.R. & Mugambi M. 1987. Naturally acquired antibodies to

sporozoites do not prevent malaria: vaccine development implications. Science 237:

639-642.

Hollingdale M.R., Nardin E.H., Tharavanij S., Schwartz A.L. & Nussenzweig R.S.

1984. Inhibition of entry of Plasmodium falciparum and P. vivax sporozoites into

cultured cells; an in vitro assay of protective antibodies. J Immunol 132: 909-913.

Horii T., Shirai H., Jie L., Ishii K.J., Palacpac N.Q., Tougan T., Hato M., Ohta N.,

Bobogare A., Arakaki N., Matsumoto Y., Namazue J., Ishikawa T., Ueda S. &

Takahashi M. 2010. Evidences of protection against blood-stage infection of

Plasmodium falciparum by the novel protein vaccine SE36. Parasitol Int 59: 380-

386.

67

Hviid L., Salanti A. 2007. VAR2CSA and protective immunity against pregnancy-

associated Plasmodium falciparum malaria. Parasitology 134: 1871-1876.

Iqbal J., Siripoon N., Snounou V.G., Perlmann P. & Berzins K. 1997. Plasmodium

falciparum: selection of parasite subpopulations with decreased sensitivity for

antibody-mediated growth inhibition in vitro. Parasitology 114 ( Pt 4): 317-324.

Iriemenam N.C., Okafor C.M., Balogun H.A., Ayede I., Omosun Y., Persson J.O.,

Hagstedt M., Anumudu C.I., Nwuba R.I., Troye-Blomberg M. & Berzins K.

2009. Cytokine profiles and antibody responses to Plasmodium falciparum malaria

infection in individuals living in Ibadan, southwest Nigeria. Afr Health Sci 9: 66-74.

Jafarshad A., Dziegiel M.H., Lundquist R., Nielsen L.K., Singh S. & Druilhe P.L.

2007. A novel antibody-dependent cellular cytotoxicity mechanism involved in

defense against malaria requires costimulation of monocytes FcgammaRII and

FcgammaRIII. J Immunol 178: 3099-3106.

Janeway C.A.,Jr, Medzhitov R. 2002. Innate immune recognition. Annu Rev Immunol

20: 197-216.

J-P Bayley, T H M Ottenhoff and C L Verweij 2004. Is there a future for TNF

promoter polymorphisms?. Genes Immun 5: 315-329.

Juliger S., Bongartz M., Luty A.J., Kremsner P.G. & Kun J.F. 2003. Functional

analysis of a promoter variant of the gene encoding the interferon-gamma receptor

chain I. Immunogenetics 54: 675-680.

Kang J.M., Moon S.U., Kim J.Y., Cho S.H., Lin K., Sohn W.M., Kim T.S. & Na

B.K. 2010. Genetic polymorphism of merozoite surface protein-1 and merozoite

surface protein-2 in Plasmodium falciparum field isolates from Myanmar. Malar J 9:

131.

Kappe S.H., Vaughan A.M., Boddey J.A. & Cowman A.F. 2010. That was then but

this is now: malaria research in the time of an eradication agenda. Science 328: 862-

866.

Kassim O.O., Ako-Anai K.A., Torimiro S.E., Hollowell G.P., Okoye V.C. & Martin

S.K. 2000. Inhibitory factors in breastmilk, maternal and infant sera against in vitro

growth of Plasmodium falciparum malaria parasite. J Trop Pediatr 46: 92-96.

Kebaier C., Voza T. & Vanderberg J. 2009. Kinetics of mosquito-injected Plasmodium

sporozoites in mice: fewer sporozoites are injected into sporozoite-immunized mice.

PLoS Pathog 5: e1000399.

Kilejian A., Rashid M.A., Aikawa M., Aji T. & Yang Y.F. 1991. Selective association

of a fragment of the knob protein with spectrin, actin and the red cell membrane.

Mol Biochem Parasitol 44: 175-181.

68

Klotz F.W., Scheller L.F., Seguin M.C., Kumar N., Marletta M.A., Green S.J. &

Azad A.F. 1995. Co-localization of inducible-nitric oxide synthase and Plasmodium

berghei in hepatocytes from rats immunized with irradiated sporozoites. J Immunol

154: 3391-3395.

Konate L., Zwetyenga J., Rogier C., Bischoff E., Fontenille D., Tall A., Spiegel A.,

Trape J.F. & Mercereau-Puijalon O. 1999. Variation of Plasmodium falciparum

msp1 block 2 and msp2 allele prevalence and of infection complexity in two

neighbouring Senegalese villages with different transmission conditions. Trans R

Soc Trop Med Hyg 93 Suppl 1: 21-28.

Krause P.J., Daily J., Telford S.R., Vannier E., Lantos P. & Spielman A. 2007.

Shared features in the pathobiology of babesiosis and malaria. Trends Parasitol 23:

605-610.

Krishnegowda G., Hajjar A.M., Zhu J., Douglass E.J., Uematsu S., Akira S., Woods

A.S. & Gowda D.C. 2005. Induction of proinflammatory responses in macrophages

by the glycosylphosphatidylinositols of Plasmodium falciparum: cell signaling

receptors, glycosylphosphatidylinositol (GPI) structural requirement, and regulation

of GPI activity. J Biol Chem 280: 8606-8616.

Kuesap J., Hirayama K., Kikuchi M., Ruangweerayut R. & Na-Bangchang K. 2010.

Study on association between genetic polymorphisms of haem oxygenase-1, tumour

necrosis factor, cadmium exposure and malaria pathogenicity and severity. Malar J

9: 260.

Kurtzhals J.A., Akanmori B.D., Goka B.Q., Adabayeri V., Nkrumah F.K., Behr C.

& Hviid L. 1999. The cytokine balance in severe malarial anemia. J Infect Dis 180:

1753-1755.

Kwiatkowski D. 1994. Prospects of an anti-disease vaccine. 132-143.

Kyes S.A., Rowe J.A., Kriek N. & Newbold C.I. 1999. Rifins: a second family of

clonally variant proteins expressed on the surface of red cells infected with

Plasmodium falciparum. Proc Natl Acad Sci U S A 96: 9333-9338.

Lamikanra A.A., Brown D., Potocnik A., Casals-Pascual C., Langhorne J. &

Roberts D.J. 2007. Malarial anemia: of mice and men. Blood 110: 18-28.

Langhorne J., Ndungu F.M., Sponaas A.M. & Marsh K. 2008. Immunity to malaria:

more questions than answers. Nat Immunol 9: 725-732.

Lanzer M., Wickert H., Krohne G., Vincensini L. & Braun Breton C. 2006. Maurer's

clefts: a novel multi-functional organelle in the cytoplasm of Plasmodium

falciparum-infected erythrocytes. Int J Parasitol 36: 23-36.

69

Layez C., Nogueira P., Combes V., Costa F.T., Juhan-Vague I., da Silva L.H. &

Gysin J. 2005. Plasmodium falciparum rhoptry protein RSP2 triggers destruction of

the erythroid lineage. Blood 106: 3632-3638.

Leoratti F.M., Durlacher R.R., Lacerda M.V., Alecrim M.G., Ferreira A.W.,

Sanchez M.C. & Moraes S.L. 2008. Pattern of humoral immune response to

Plasmodium falciparum blood stages in individuals presenting different clinical

expressions of malaria. Malar J 7: 186.

Li C., Sanni L.A., Omer F., Riley E. & Langhorne J. 2003. Pathology of Plasmodium

chabaudi chabaudi infection and mortality in interleukin-10-deficient mice are

ameliorated by anti-tumor necrosis factor alpha and exacerbated by anti-

transforming growth factor beta antibodies. Infect Immun 71: 4850-4856.

Liljeqvist S., Ståhl S. 1999. Production of recombinant subunit vaccines: protein

immunogens, live delivery systems and nucleic acid vaccines. J Biotechnol 73: 1-33.

Liu M.A. 1998. Vaccine developments. Nat Med 4: 515-519.

Lockyer MJ, Marsh K, Newbold CI. 1989. Wild isolates of Plasmodium falciparum

show extensive polymorphism in T cell epitopes of the circumsporozoite protein.

Mol Biochem Parasitol 37: 275-280.

Loria P., Miller S., Foley M. & Tilley L. 1999. Inhibition of the peroxidative

degradation of haem as the basis of action of chloroquine and other quinoline

antimalarials. Biochem J 339 ( Pt 2): 363-370.

Luna E.J., Hitt A.L. 1992. Cytoskeleton--plasma membrane interactions. Science 258:

955-964.

Lustigman S., Anders R.F., Brown G.V. & Coppel R.L. 1990. The mature-parasite-

infected erythrocyte surface antigen (MESA) of Plasmodium falciparum associates

with the erythrocyte membrane skeletal protein, band 4.1. Mol Biochem Parasitol

38: 261-270.

MacPherson G.G., Warrell M.J., White N.J., Looareesuwan S. & Warrell D.A.

1985. Human cerebral malaria. A quantitative ultrastructural analysis of parasitized

erythrocyte sequestration. Am J Pathol 119: 385-401.

Maier A.G., Cooke B.M., Cowman A.F. & Tilley L. 2009. Malaria parasite proteins

that remodel the host erythrocyte. Nat Rev Microbiol 7: 341-354.

Maier A.G., Rug M., O'Neill M.T., Brown M., Chakravorty S., Szestak T., Chesson

J., Wu Y., Hughes K., Coppel R.L., Newbold C., Beeson J.G., Craig A., Crabb

B.S. & Cowman A.F. 2008. Exported proteins required for virulence and rigidity of

Plasmodium falciparum-infected human erythrocytes. Cell 134: 48-61.

70

malERA Consultative Group on Basic Science and Enabling Technologies, Baum J.,

Billker O., Bousema T., Dinglasan R., McGovern V., Mota M.M., Mueller I. &

Sinden R. 2011. A research agenda for malaria eradication: basic science and

enabling technologies. PLoS Med 8: e1000399.

malERA Consultative Group on Vaccines, Alonso P.L., Ballou R., Brown G.,

Chitnis C., Loucq C., Moorthy V., Saul A. & Wirth D. 2011. A research agenda

for malaria eradication: vaccines. PLoS Med 8: e1000398.

malERA Consultative Group on Vector Control, Alonso P.L., Besansky N.J., Burkot

T.R., Collins F.H., Hemingway J., James A.A., Lengeler C., Lindsay S., Liu Q.,

Lobo N.F., Mnzava A., Tanner M. & Zwiebel L. 2011. A research agenda for

malaria eradication: vector control. PLoS Med 8: e1000401.

Marsh K., Kinyanjui S. 2006. Immune effector mechanisms in malaria. Parasite

Immunol 28: 51-60.

Marti M., Good R.T., Rug M., Knuepfer E. & Cowman A.F. 2004. Targeting malaria

virulence and remodeling proteins to the host erythrocyte. Science 306: 1930-1933.

Matondo Maya D.W., Mavoungou E., Deloron P., Theisen M. & Ntoumi F. 2006.

Distribution of IgG subclass antibodies specific for Plasmodium falciparum

glutamate-rich-protein molecule in sickle cell trait children with asymptomatic

infections. Exp Parasitol 112: 92-98.

Mattei D., Berzins K., Wahlgren M., Udomsangpetch R., Perlmann P., Griesser

H.W., Scherf A., Muller-Hill B., Bonnefoy S. & Guillotte M. 1989. Cross-reactive

antigenic determinants present on different Plasmodium falciparum blood-stage

antigens. Parasite Immunol 11: 15-29.

Mattei D., Scherf A. 1992. The Pf332 gene of Plasmodium falciparum codes for a giant

protein that is translocated from the parasite to the membrane of infected

erythrocytes. Gene 110: 71-79.

Maude R.J., Pontavornpinyo W., Saralamba S., Aguas R., Yeung S., Dondorp A.M.,

Day N.P., White N.J. & White L.J. 2009. The last man standing is the most

resistant: eliminating artemisinin-resistant malaria in Cambodia. Malar J 8: 31.

McGregor I.A. 1964. Studies in the Acquisition of Immunity of Plasmodium falciparum

Infections in Africa. Trans R Soc Trop Med Hyg 58: 80-92.

McGuire W., Hill A.V., Allsopp C.E., Greenwood B.M. & Kwiatkowski D. 1994.

Variation in the TNF-alpha promoter region associated with susceptibility to cerebral

malaria. Nature 371: 508-510.

Mecheri S. 2011. Contribution of allergic inflammatory response to the pathogenesis of

malaria disease. Biochim Biophys Acta

71

Medzhitov R. 2001. Toll-like receptors and innate immunity. Nat Rev Immunol 1: 135-

145.

Medzhitov R., Janeway C.,Jr 2000. The Toll receptor family and microbial recognition.

Trends Microbiol 8: 452-456.

Mikolajczak S.A., Silva-Rivera H., Peng X., Tarun A.S., Camargo N., Jacobs-

Lorena V., Daly T.M., Bergman L.W., de la Vega P., Williams J., Aly A.S. &

Kappe S.H. 2008. Distinct malaria parasite sporozoites reveal transcriptional

changes that cause differential tissue infection competence in the mosquito vector

and mammalian host. Mol Cell Biol 28: 6196-6207.

Miller L.H. 1994. Impact of malaria on genetic polymorphism and genetic diseases in

Africans and African Americans. Proc Natl Acad Sci U S A 91: 2415-2419.

Miller L.H., Roberts T., Shahabuddin M. & McCutchan T.F. 1993. Analysis of

sequence diversity in the Plasmodium falciparum merozoite surface protein-1 (MSP-

1). Mol Biochem Parasitol 59: 1-14.

Mishra S.K., Newton C.R. 2009. Diagnosis and management of the neurological

complications of falciparum malaria. Nat Rev Neurol 5: 189-198.

Mockenhaupt F.P., Cramer J.P., Hamann L., Stegemann M.S., Eckert J., Oh N.R.,

Otchwemah R.N., Dietz E., Ehrhardt S., Schroder N.W., Bienzle U. &

Schumann R.R. 2006. Toll-like receptor (TLR) polymorphisms in African children:

Common TLR-4 variants predispose to severe malaria. Proc Natl Acad Sci U S A

103: 177-182.

Mockenhaupt F.P., Ehrhardt S., Gellert S., Otchwemah R.N., Dietz E., Anemana

S.D. & Bienzle U. 2004. Alpha(+)-thalassemia protects African children from severe

malaria. Blood 104: 2003-2006.

Modiano D., Luoni G., Sirima B.S., Lanfrancotti A., Petrarca V., Cruciani F.,

Simpore J., Ciminelli B.M., Foglietta E., Grisanti P., Bianco I., Modiano G. &

Coluzzi M. 2001. The lower susceptibility to Plasmodium falciparum malaria of

Fulani of Burkina Faso (west Africa) is associated with low frequencies of classic

malaria-resistance genes. Trans R Soc Trop Med Hyg 95: 149-152.

Mohan K S.M. 1998. Acquired immunity to asexual blood stages; in Sherman IW (ed).

467-493.

Moll K., Chene A., Ribacke U., Kaneko O., Nilsson S., Winter G., Haeggstrom M.,

Pan W., Berzins K., Wahlgren M. & Chen Q. 2007. A novel DBL-domain of the

P. falciparum 332 molecule possibly involved in erythrocyte adhesion. PLoS One 2:

e477.

Naik R.S., Branch O.H., Woods A.S., Vijaykumar M., Perkins D.J., Nahlen B.L.,

Lal A.A., Cotter R.J., Costello C.E., Ockenhouse C.F., Davidson E.A. & Gowda

72

D.C. 2000. Glycosylphosphatidylinositol anchors of Plasmodium falciparum:

molecular characterization and naturally elicited antibody response that may provide

immunity to malaria pathogenesis. J Exp Med 192: 1563-1576.

Nardin E., Zavala F., Nussenzweig V. & Nussenzweig R.S. 1999. Pre-erythrocytic

malaria vaccine: mechanisms of protective immunity and human vaccine trials.

Parassitologia 41: 397-402.

Nasr A., Iriemenam N.C., Giha H.A., Balogun H.A., Anders R.F., Troye-Blomberg

M., ElGhazali G. & Berzins K. 2009. FcgammaRIIa (CD32) polymorphism and

anti-malarial IgG subclass pattern among Fulani and sympatric ethnic groups living

in eastern Sudan. Malar J 8: 43.

Nasr A., Iriemenam N.C., Troye-Blomberg M., Giha H.A., Balogun H.A., Osman

O.F., Montgomery S.M., ElGhazali G. & Berzins K. 2007. Fc gamma receptor IIa

(CD32) polymorphism and antibody responses to asexual blood-stage antigens of

Plasmodium falciparum malaria in Sudanese patients. Scand J Immunol 66: 87-96.

Nilsson S. 2011. Plasmodium falciparum antigen 332 associates with the cytoplasmic

side of Maurer´s clefts via protein-protein interactions. (In Phd thesis, Exported

proteins of the malaria parasite Plasmodium falciparum: Characterization of blood-

stage antigen 332, Karolinska Institutet, ISBN 978-91-7457-365-7)

Nilsson S., Moll K., Angeletti D., Albrecht L., Kursula I., Jiang N., Sun X., Berzins

K., Wahlgren M. & Chen Q. 2011. (In press). Characterization of the Duffy-

binding like domain of Plasmodium falciparum blood stage antigen 332. Malar

Reasearch and Treatment.

Nimmerjahn F., Ravetch J.V. 2008. Analyzing antibody-Fc-receptor interactions.

Methods Mol Biol 415: 151-162.

Ntoumi F., Flori L., Mayengue P.I., Matondo Maya D.W., Issifou S., Deloron P., Lell

B., Kremsner P.G. & Rihet P. 2005. Influence of carriage of hemoglobin AS and

the Fc gamma receptor IIa-R131 allele on levels of immunoglobulin G2 antibodies

to Plasmodium falciparum merozoite antigens in Gabonese children. J Infect Dis

192: 1975-1980.

Ogutu B.R., Apollo O.J., McKinney D., Okoth W., Siangla J., Dubovsky F., Tucker

K., Waitumbi J.N., Diggs C., Wittes J., Malkin E., Leach A., Soisson L.A.,

Milman J.B., Otieno L., Holland C.A., Polhemus M., Remich S.A., Ockenhouse

C.F., Cohen J., Ballou W.R., Martin S.K., Angov E., Stewart V.A., Lyon J.A.,

Heppner D.G., Withers M.R. & MSP-1 Malaria Vaccine Working Group 2009.

Blood stage malaria vaccine eliciting high antigen-specific antibody concentrations

confers no protection to young children in Western Kenya. PLoS One 4: e4708.

Oh S.S., Voigt S., Fisher D., Yi S.J., LeRoy P.J., Derick L.H., Liu S. & Chishti A.H.

2000. Plasmodium falciparum erythrocyte membrane protein 1 is anchored to the

73

actin-spectrin junction and knob-associated histidine-rich protein in the erythrocyte

skeleton. Mol Biochem Parasitol 108: 237-247.

Orago A.S., Facer C.A. 1991. Cytotoxicity of human natural killer (NK) cell subsets for

Plasmodium falciparum erythrocytic schizonts: stimulation by cytokines and

inhibition by neomycin. Clin Exp Immunol 86: 22-29.

Owusu-Agyei S., Koram K.A., Baird J.K., Utz G.C., Binka F.N., Nkrumah F.K.,

Fryauff D.J. & Hoffman S.L. 2001. Incidence of symptomatic and asymptomatic

Plasmodium falciparum infection following curative therapy in adult residents of

northern Ghana. Am J Trop Med Hyg 65: 197-203.

Pachlatko E., Rusch S., Muller A., Hemphill A., Tilley L., Hanssen E. & Beck H.P.

2010. MAHRP2, an exported protein of Plasmodium falciparum, is an essential

component of Maurer's cleft tethers. Mol Microbiol 77: 1136-1152.

Pagnoni F. 2009. Malaria treatment: no place like home. Trends Parasitol 25: 115-119.

Parroche P., Lauw F.N., Goutagny N., Latz E., Monks B.G., Visintin A., Halmen

K.A., Lamphier M., Olivier M., Bartholomeu D.C., Gazzinelli R.T. &

Golenbock D.T. 2007. Malaria hemozoin is immunologically inert but radically

enhances innate responses by presenting malaria DNA to Toll-like receptor 9. Proc

Natl Acad Sci U S A 104: 1919-1924.

Pasloske B.L., Baruch D.I., van Schravendijk M.R., Handunnetti S.M., Aikawa M.,

Fujioka H., Taraschi T.F., Gormley J.A. & Howard R.J. 1993. Cloning and

characterization of a Plasmodium falciparum gene encoding a novel high-molecular

weight host membrane-associated protein, PfEMP3. Mol Biochem Parasitol 59: 59-

72.

Pasvol G. 2010. Protective hemoglobinopathies and Plasmodium falciparum

transmission. Nat Genet 42: 284-285.

Pei X., Guo X., Coppel R., Bhattacharjee S., Haldar K., Gratzer W., Mohandas N.

& An X. 2007. The ring-infected erythrocyte surface antigen (RESA) of

Plasmodium falciparum stabilizes spectrin tetramers and suppresses further invasion.

Blood 110: 1036-1042.

Perlaza B., Valencia A.Z., Zapata C., Castellanos A., Sauzet J., Blanc C., Cohen J.,

Arévalo-Herrera M., Corradin G., Herrera S. & Druilhe P. 2008. Protection

against Plasmodium falciparum challenge induced in Aotus monkeys by liver-stage

antigen-3-derived long synthetic peptides. Eur J Immunol 38: 2610-2615.

Perlmann P., and Troye-Blomberg M. 2002. Malaria and the immune system in

humans. Malaria Immunology 229-237.

Perlmann H., Helmby H., Hagstedt M., Carlson J., Larsson P.H., Troye-Blomberg

M. & Perlmann P. 1994. IgE elevation and IgE anti-malarial antibodies in

74

Plasmodium falciparum malaria: association of high IgE levels with cerebral

malaria. Clin Exp Immunol 97: 284-292.

Perlmann P., Perlmann H., Flyg B.W., Hagstedt M., Elghazali G., Worku S.,

Fernandez V., Rutta A.S. & Troye-Blomberg M. 1997. Immunoglobulin E, a

pathogenic factor in Plasmodium falciparum malaria. Infect Immun 65: 116-121.

Perlmann P., Perlmann H., Looareesuwan S., Krudsood S., Kano S., Matsumoto Y.,

Brittenham G., Troye-Blomberg M. & Aikawa M. 2000. Contrasting functions of

IgG and IgE antimalarial antibodies in uncomplicated and severe Plasmodium

falciparum malaria. Am J Trop Med Hyg 62: 373-377.

Perraut R., Mercereau-Puijalon O., Diouf B., Tall A., Guillotte M., Le Scanf C.,

Trape J.F., Spiegel A. & Garraud O. 2000. Seasonal fluctuation of antibody levels

to Plasmodium falciparum parasitized red blood cell-associated antigens in two

Senegalese villages with different transmission conditions. Am J Trop Med Hyg 62:

746-751.

Perraut R., Mercereau-Puijalon O., Mattei D., Bourreau E., Garraud O.,

Bonnemains B., Pereia de Silva L. & Michel J.C. 1995. Induction of opsonizing

antibodies after injection of recombinant Plasmodium falciparum vaccine candidate

antigens in preimmune Saimiri sciureus monkeys. Infect Immun 63: 554-562.

Persson K.E., Lee C.T., Marsh K. & Beeson J.G. 2006. Development and optimization

of high-throughput methods to measure Plasmodium falciparum-specific growth

inhibitory antibodies. J Clin Microbiol 44: 1665-1673.

Pichyangkul S., Yongvanitchit K., Kum-arb U., Hemmi H., Akira S., Krieg A.M.,

Heppner D.G., Stewart V.A., Hasegawa H., Looareesuwan S., Shanks G.D. &

Miller R.S. 2004. Malaria blood stage parasites activate human plasmacytoid

dendritic cells and murine dendritic cells through a Toll-like receptor 9-dependent

pathway. J Immunol 172: 4926-4933.

plasmodb http://www.plasmodb.org/plasmo/home.jsp.

Plebanski M., Flanagan K.L., Lee E.A., Reece W.H., Hart K., Gelder C., Gillespie

G., Pinder M. & Hill A.V. 1999. Interleukin 10-mediated immunosuppression by a

variant CD4 T cell epitope of Plasmodium falciparum. Immunity 10: 651-660.

Plebanski M., Hill A.V. 2000. The immunology of malaria infection. Curr Opin

Immunol 12: 437-441.

75

Pombo D.J., Lawrence G., Hirunpetcharat C., Rzepczyk C., Bryden M., Cloonan N.,

Anderson K., Mahakunkijcharoen Y., Martin L.B., Wilson D., Elliott S., Elliott

S., Eisen D.P., Weinberg J.B., Saul A. & Good M.F. 2002. Immunity to malaria

after administration of ultra-low doses of red cells infected with Plasmodium

falciparum. Lancet 360: 610-617.

Pouvelle B., Spiegel R., Hsiao L., Howard R.J., Morris R.L., Thomas A.P. &

Taraschi T.F. 1991. Direct access to serum macromolecules by intraerythrocytic

malaria parasites. Nature 353: 73-75.

Prudencio M., Rodriguez A. & Mota M.M. 2006. The silent path to thousands of

merozoites: the Plasmodium liver stage. Nat Rev Microbiol 4: 849-856.

Przyborski J.M., Miller S.K., Pfahler J.M., Henrich P.P., Rohrbach P., Crabb B.S.

& Lanzer M. 2005. Trafficking of STEVOR to the Maurer's clefts in Plasmodium

falciparum-infected erythrocytes. EMBO J 24: 2306-2317.

Ramasamy R. 1998. Molecular basis for evasion of host immunity and pathogenesis in

malaria. Biochim Biophys Acta 1406: 10-27.

Riley E.M., Wagner G.E., Ofori M.F., Wheeler J.G., Akanmori B.D., Tetteh K.,

McGuinness D., Bennett S., Nkrumah F.K., Anders R.F. & Koram K.A. 2000.

Lack of association between maternal antibody and protection of African infants

from malaria infection. Infect Immun 68: 5856-5863.

Riley E.M., Wahl S., Perkins D.J. & Schofield L. 2006. Regulating immunity to

malaria. Parasite Immunol 28: 35-49.

Robinson B.A., Welch T.L. & Smith J.D. 2003. Widespread functional specialization

of Plasmodium falciparum erythrocyte membrane protein 1 family members to bind

CD36 analysed across a parasite genome. Mol Microbiol 47: 1265-1278.

Rogerson S.J., Hviid L., Duffy P.E., Leke R.F. & Taylor D.W. 2007. Malaria in

pregnancy: pathogenesis and immunity. Lancet Infect Dis 7: 105-117.

Roll Back Malaria Patnership 2008. The Global Malaria Action Plan.

Ropert C., Franklin B. S. & Gazzinelli R. T. 2008. Role of TLRs/MyD88 in host

resistance and pathogenesis during protozoan infection: lessons from malaria. Semin

Immunopathol. 30: 41-50.

Rowe A., Obeiro J., Newbold C.I. & Marsh K. 1995. Plasmodium falciparum rosetting

is associated with malaria severity in Kenya. Infect Immun 63: 2323-2326.

Rowe J.A., Claessens A., Corrigan R.A. & Arman M. 2009. Adhesion of Plasmodium

falciparum-infected erythrocytes to human cells: molecular mechanisms and

therapeutic implications. Expert Rev Mol Med 11: e16.

76

Rowe J.A., Moulds J.M., Newbold C.I. & Miller L.H. 1997. P. falciparum rosetting

mediated by a parasite-variant erythrocyte membrane protein and complement-

receptor 1. Nature 388: 292-295.

Rowe J.A., Obiero J., Marsh K. & Raza A. 2002. Short report: Positive correlation

between rosetting and parasitemia in Plasmodium falciparum clinical isolates. Am J

Trop Med Hyg 66: 458-460.

Ruangjirachuporn W., Udomsangpetch R., Carlsson J., Drenckhahn D., Perlmann

P. & Berzins K. 1991. Plasmodium falciparum: analysis of the interaction of

antigen Pf155/RESA with the erythrocyte membrane. Exp Parasitol 73: 62-72.

Rug M., Prescott S.W., Fernandez K.M., Cooke B.M. & Cowman A.F. 2006. The role

of KAHRP domains in knob formation and cytoadherence of P falciparum-infected

human erythrocytes. Blood 108: 370-378.

Rzepczyk C.M., Anderson K., Stamatiou S., Townsend E., Allworth A., McCormack

J. & Whitby M. 1997. Gamma delta T cells: their immunobiology and role in

malaria infections. Int J Parasitol 27: 191-200.

Sabchareon A., Burnouf T., Ouattara D., Attanath P., Bouharoun-Tayoun H.,

Chantavanich P., Foucault C., Chongsuphajaisiddhi T. & Druilhe P. 1991.

Parasitologic and clinical human response to immunoglobulin administration in

falciparum malaria. Am J Trop Med Hyg 45: 297-308.

Sagara I., Ellis R.D., Dicko A., Niambele M.B., Kamate B., Guindo O., Sissoko M.S.,

Fay M.P., Guindo M.A., Kante O., Saye R., Miura K., Long C., Mullen G.E.,

Pierce M., Martin L.B., Rausch K., Dolo A., Diallo D.A., Miller L.H. & Doumbo

O.K. 2009. A randomized and controlled Phase 1 study of the safety and

immunogenicity of the AMA1-C1/Alhydrogel + CPG 7909 vaccine for Plasmodium

falciparum malaria in semi-immune Malian adults. Vaccine 27: 7292-7298.

Salanti A., Dahlback M., Turner L., Nielsen M.A., Barfod L., Magistrado P., Jensen

A.T., Lavstsen T., Ofori M.F., Marsh K., Hviid L. & Theander T.G. 2004.

Evidence for the involvement of VAR2CSA in pregnancy-associated malaria. J Exp

Med 200: 1197-1203.

Salmon B.L., Oksman A. & Goldberg D.E. 2001. Malaria parasite exit from the host

erythrocyte: a two-step process requiring extraerythrocytic proteolysis. Proc Natl

Acad Sci U S A 98: 271-276.

Sam-Yellowe T.Y. 2009. The role of the Maurer's clefts in protein transport in

Plasmodium falciparum. Trends Parasitol 25: 277-284.

Sarthou J.L., Angel G., Aribot G., Rogier C., Dieye A., Toure Balde A., Diatta B.,

Seignot P. & Roussilhon C. 1997. Prognostic value of anti-Plasmodium

falciparum-specific immunoglobulin G3, cytokines, and their soluble receptors in

West African patients with severe malaria. Infect Immun 65: 3271-3276.

77

Scherf A., Lopez-Rubio J.J. & Riviere L. 2008. Antigenic variation in Plasmodium

falciparum. Annu Rev Microbiol 62: 445-470.

Scherf A., Petersen C., Carter R., Alano P., Nelson R., Aikawa M., Mattei D., da

Silva L.P. & Leech J. 1992. Characterization of a Plasmodium falciparium mutant

that has deleted the majority of the gametocyte-specific Pf11-1 locus. Mem Inst

Oswaldo Cruz 87 Suppl 3: 91-94.

Schofield L. 2002. Antidisease vaccines. Chem Immunol 80: 322-342.

Schofield L., Grau G.E. 2005. Immunological processes in malaria pathogenesis. Nat

Rev Immunol 5: 722-735.

Schofield L., Hackett F. 1993. Signal transduction in host cells by a

glycosylphosphatidylinositol toxin of malaria parasites. J Exp Med 177: 145-153.

Schofield L., Vivas L., Hackett F., Gerold P., Schwarz R.T. & Tachado S. 1993.

Neutralizing monoclonal antibodies to glycosylphosphatidylinositol, the dominant

TNF-alpha-inducing toxin of Plasmodium falciparum: prospects for the

immunotherapy of severe malaria. Ann Trop Med Parasitol 87: 617-626.

Schwenk R.J., Richie T.L. 2011. Protective immunity to pre-erythrocytic stage malaria.

Trends Parasitol 27: 306-314.

Scopel K.K., Fontes C.J., Ferreira M.U. & Braga E.M. 2006. Factors associated with

immunoglobulin G subclass polarization in naturally acquired antibodies to

Plasmodium falciparum merozoite surface proteins: a cross-sectional survey in

Brazilian Amazonia. Clin Vaccine Immunol 13: 810-813.

Seka-Seka J., Brouh Y., Yapo-Crezoit A.C. & Atseye N.H. 2004. The role of serum

immunoglobulin E in the pathogenesis of Plasmodium falciparum malaria in Ivorian

children. Scand J Immunol 59: 228-230.

Serghides L., Smith T.G., Patel S.N. & Kain K.C. 2003. CD36 and malaria: friends or

foes? Trends Parasitol 19: 461-469.

Shi Y.P., Udhayakumar V., Oloo A.J., Nahlen B.L. & Lal A.A. 1999. Differential

effect and interaction of monocytes, hyperimmune sera, and immunoglobulin G on

the growth of asexual stage Plasmodium falciparum parasites. Am J Trop Med Hyg

60: 135-141.

Sinha S., Qidwai T., Kanchan K., Jha G.N., Anand P., Pati S.S., Mohanty S., Mishra

S.K., Tyagi P.K., Sharma S.K., Awasthi S., Venkatesh V. & Habib S. 2010.

Distinct cytokine profiles define clinical immune response to falciparum malaria in

regions of high or low disease transmission. Eur Cytokine Netw 21: 232-240.

Sirima S.B., Tiono A.B., Ouedraogo A., Diarra A., Ouedraogo A.L., Yaro J.B.,

Ouedraogo E., Gansane A., Bougouma E.C., Konate A.T., Kabore Y., Traore

78

A., Chilengi R., Soulama I., Luty A.J., Druilhe P., Cousens S. & Nebie I. 2009.

Safety and immunogenicity of the malaria vaccine candidate MSP3 long synthetic

peptide in 12-24 months-old Burkinabe children. PLoS One 4: e7549.

Skorokhod O.A., Alessio M., Mordmuller B., Arese P. & Schwarzer E. 2004.

Hemozoin (malarial pigment) inhibits differentiation and maturation of human

monocyte-derived dendritic cells: a peroxisome proliferator-activated receptor-

gamma-mediated effect. J Immunol 173: 4066-4074.

Snounou G., Zhu X., Siripoon N., Jarra W., Thaithong S., Brown K.N. &

Viriyakosol S. 1999. Biased distribution of msp1 and msp2 allelic variants in

Plasmodium falciparum populations in Thailand. Trans R Soc Trop Med Hyg 93:

369-374.

Speake C., Duffy P.E. 2009. Antigens for pre-erythrocytic malaria vaccines: building on

success. Parasite Immunol 31: 539-546.

Spielmann T., Beck H.P. 2000. Analysis of stage-specific transcription in Plasmodium

falciparum reveals a set of genes exclusively transcribed in ring stage parasites. Mol

Biochem Parasitol 111: 453-458.

Spielmann T., Hawthorne P.L., Dixon M.W., Hannemann M., Klotz K., Kemp D.J.,

Klonis N., Tilley L., Trenholme K.R. & Gardiner D.L. 2006. A cluster of ring

stage-specific genes linked to a locus implicated in cytoadherence in Plasmodium

falciparum codes for PEXEL-negative and PEXEL-positive proteins exported into

the host cell. Mol Biol Cell 17: 3613-3624.

Spycher C., Klonis N., Spielmann T., Kump E., Steiger S., Tilley L. & Beck H.P.

2003. MAHRP-1, a novel Plasmodium falciparum histidine-rich protein, binds

ferriprotoporphyrin IX and localizes to the Maurer's clefts. J Biol Chem 278: 35373-

35383.

Spycher C., Rug M., Klonis N., Ferguson D.J., Cowman A.F., Beck H.P. & Tilley L.

2006. Genesis of and trafficking to the Maurer's clefts of Plasmodium falciparum-

infected erythrocytes. Mol Cell Biol 26: 4074-4085.

Spycher C., Rug M., Pachlatko E., Hanssen E., Ferguson D., Cowman A.F., Tilley L.

& Beck H.P. 2008. The Maurer's cleft protein MAHRP1 is essential for trafficking

of PfEMP1 to the surface of Plasmodium falciparum-infected erythrocytes. Mol

Microbiol 68: 1300-1314.

Stanisic D.I., Richards J.S., McCallum F.J., Michon P., King C.L., Schoepflin S.,

Gilson P.R., Murphy V.J., Anders R.F., Mueller I. & Beeson J.G. 2009.

Immunoglobulin G subclass-specific responses against Plasmodium falciparum

merozoite antigens are associated with control of parasitemia and protection from

symptomatic illness. Infect Immun 77: 1165-1174.

79

Su X.Z., Heatwole V.M., Wertheimer S.P., Guinet F., Herrfeldt J.A., Peterson D.S.,

Ravetch J.A. & Wellems T.E. 1995. The large diverse gene family var encodes

proteins involved in cytoadherence and antigenic variation of Plasmodium

falciparum-infected erythrocytes. Cell 82: 89-100.

Sullivan A.D., Ittarat I. & Meshnick S.R. 1996. Patterns of haemozoin accumulation in

tissue. Parasitology 112 ( Pt 3): 285-294.

Takala S.L., Coulibaly D., Thera M.A., Batchelor A.H., Cummings M.P., Escalante

A.A., Ouattara A., Traore K., Niangaly A., Djimde A.A., Doumbo O.K. &

Plowe C.V. 2009. Extreme polymorphism in a vaccine antigen and risk of clinical

malaria: implications for vaccine development. Sci Transl Med 1: 2ra5.

Takala S.L., Plowe C.V. 2009. Genetic diversity and malaria vaccine design, testing and

efficacy: preventing and overcoming 'vaccine resistant malaria'. Parasite Immunol

31: 560-573.

Taraschi T.F. 1999. Macromolecular transport in malaria-infected erythrocytes. Novartis

Found Symp 226: 114-20; discussion 121-5.

Taylor D.W., Parra M., Chapman G.B., Stearns M.E., Rener J., Aikawa M., Uni S.,

Aley S.B., Panton L.J. & Howard R.J. 1987. Localization of Plasmodium

falciparum histidine-rich protein 1 in the erythrocyte skeleton under knobs. Mol

Biochem Parasitol 25: 165-174.

Tebo A.E., Kremsner P.G. & Luty A.J. 2001. Plasmodium falciparum: a major role for

IgG3 in antibody-dependent monocyte-mediated cellular inhibition of parasite

growth in vitro. Exp Parasitol 98: 20-28.

Tilley L., Dixon M.W. & Kirk K. 2011. The Plasmodium falciparum-infected red blood

cell. Int J Biochem Cell Biol 43: 839-842.

Todryk S., Bejon P. 2009. Malaria vaccine development: Lessons from the field. Eur J

Immunol 39: 2007-2010.

Tongren J.E., Drakeley C.J., McDonald S.L., Reyburn H.G., Manjurano A., Nkya

W.M., Lemnge M.M., Gowda C.D., Todd J.E., Corran P.H. & Riley E.M. 2006.

Target antigen, age, and duration of antigen exposure independently regulate

immunoglobulin G subclass switching in malaria. Infect Immun 74: 257-264.

Trape J.F., Rogier C., Konate L., Diagne N., Bouganali H., Canque B., Legros F.,

Badji A., Ndiaye G. & Ndiaye P. 1994. The Dielmo project: a longitudinal study of

natural malaria infection and the mechanisms of protective immunity in a

community living in a holoendemic area of Senegal. Am J Trop Med Hyg 51: 123-

137.

80

Treutiger C.J., Hedlund I., Helmby H., Carlson J., Jepson A., Twumasi P.,

Kwiatkowski D., Greenwood B.M. & Wahlgren M. 1992. Rosette formation in

Plasmodium falciparum isolates and anti-rosette activity of sera from Gambians with

cerebral or uncomplicated malaria. Am J Trop Med Hyg 46: 503-510.

Troye-Blomberg M., Perlmann P., Mincheva Nilsson L. & Perlmann H. 1999.

Immune regulation of protection and pathogenesis in Plasmodium falciparum

malaria. Parassitologia 41: 131-138.

Troye-Blomberg M., Riley E.M., Kabilan L., Holmberg M., Perlmann H.,

Andersson U., Heusser C.H. & Perlmann P. 1990. Production by activated human

T cells of interleukin 4 but not interferon-gamma is associated with elevated levels

of serum antibodies to activating malaria antigens. Proc Natl Acad Sci U S A 87:

5484-5488.

Tsuji M. 2010. A retrospective evaluation of the role of T cells in the development of

malaria vaccine. Exp Parasitol 126: 421-425.

Tsuji M., Zavala F. 2003. T cells as mediators of protective immunity against liver

stages of Plasmodium. Trends Parasitol 19: 88-93.

Turner G.D., Morrison H., Jones M., Davis T.M., Looareesuwan S., Buley I.D.,

Gatter K.C., Newbold C.I., Pukritayakamee S. & Nagachinta B. 1994. An

immunohistochemical study of the pathology of fatal malaria. Evidence for

widespread endothelial activation and a potential role for intercellular adhesion

molecule-1 in cerebral sequestration. Am J Pathol 145: 1057-1069.

Udomsangpetch R., Aikawa M., Berzins K., Wahlgren M. & Perlmann P. 1989.

Cytoadherence of knobless Plasmodium falciparum-infected erythrocytes and its

inhibition by a human monoclonal antibody. Nature 338: 763-765.

Urban B.C., Ferguson D.J., Pain A., Willcox N., Plebanski M., Austyn J.M. &

Roberts D.J. 1999. Plasmodium falciparum-infected erythrocytes modulate the

maturation of dendritic cells. Nature 400: 73-77.

Urban B.C., Todryk S. 2006. Malaria pigment paralyzes dendritic cells. J Biol 5: 4.

Vogt A.M., Barragan A., Chen Q., Kironde F., Spillmann D. & Wahlgren M. 2003.

Heparan sulfate on endothelial cells mediates the binding of Plasmodium

falciparum-infected erythrocytes via the DBL1alpha domain of PfEMP1. Blood 101:

2405-2411.

Wåhlin B., Wahlgren M., Perlmann H., Berzins K., Bjorkman A., Patarroyo M.E. &

Perlmann P. 1984. Human antibodies to a Mr 155,000 Plasmodium falciparum

antigen efficiently inhibit merozoite invasion. Proc Natl Acad Sci U S A 81: 7912-

7916.

81

Waller K.L., Stubberfield L.M., Dubljevic V., Buckingham D.W., Mohandas N.,

Coppel R.L. & Cooke B.M. 2010. Interaction of the exported malaria protein Pf332

with the red blood cell membrane skeleton. Biochim Biophys Acta 1798: 861-871.

Walther M., Jeffries D., Finney O.C., Njie M., Ebonyi A., Deininger S., Lawrence E.,

Ngwa-Amambua A., Jayasooriya S., Cheeseman I.H., Gomez-Escobar N.,

Okebe J., Conway D.J. & Riley E.M. 2009. Distinct roles for FOXP3 and FOXP3

CD4 T cells in regulating cellular immunity to uncomplicated and severe

Plasmodium falciparum malaria. PLoS Pathog 5: e1000364.

Walther M., Tongren J.E., Andrews L., Korbel D., King E., Fletcher H., Andersen

R.F., Bejon P., Thompson F., Dunachie S.J., Edele F., de Souza J.B., Sinden

R.E., Gilbert S.C., Riley E.M. & Hill A.V. 2005. Upregulation of TGF-beta,

FOXP3, and CD4+CD25+ regulatory T cells correlates with more rapid parasite

growth in human malaria infection. Immunity 23: 287-296.

Wambua S., Mwangi T.W., Kortok M., Uyoga S.M., Macharia A.W., Mwacharo

J.K., Weatherall D.J., Snow R.W., Marsh K. & Williams T.N. 2006. The effect

of alpha+-thalassaemia on the incidence of malaria and other diseases in children

living on the coast of Kenya. PLoS Med 3: e158.

Wassmer S.C., Combes V. & Grau G.E. 2003. Pathophysiology of cerebral malaria:

role of host cells in the modulation of cytoadhesion. Ann N Y Acad Sci 992: 30-38.

Waterkeyn J.G., Wickham M.E., Davern K.M., Cooke B.M., Coppel R.L., Reeder

J.C., Culvenor J.G., Waller R.F. & Cowman A.F. 2000. Targeted mutagenesis of

Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) disrupts

cytoadherence of malaria-infected red blood cells. EMBO J 19: 2813-2823.

Weedall G.D., Conway D.J. 2010. Detecting signatures of balancing selection to

identify targets of anti-parasite immunity. Trends Parasitol 26: 363-369.

Wickham M.E., Rug M., Ralph S.A., Klonis N., McFadden G.I., Tilley L. &

Cowman A.F. 2001. Trafficking and assembly of the cytoadherence complex in

Plasmodium falciparum-infected human erythrocytes. EMBO J 20: 5636-5649.

Williams T.N., Mwangi T.W., Wambua S., Alexander N.D., Kortok M., Snow R.W.

& Marsh K. 2005. Sickle cell trait and the risk of Plasmodium falciparum malaria

and other childhood diseases. J Infect Dis 192: 178-186.

Winter G., Kawai S., Haeggström M., Kaneko O., von Euler A., Kawazu S., Palm

D., Fernandez V. & Wahlgren M. 2005. SURFIN is a polymorphic antigen

expressed on Plasmodium falciparum merozoites and infected erythrocytes. J Exp

Med 201: 1853-1863.

World Health Organization FAQ about malaria 2009.

82

World Health Organization 2010a. Guidelines for the treatment of malaria, (2nd edition

2010).

World Health Organization. 2010. World malaria report.

Wykes M.N., Good M.F. 2009. What have we learnt from mouse models for the study

of malaria? Eur J Immunol 39: 2004-2007.

Zaki S.A., Shanbag P. 2011. Atypical manifestations of malaria. Res Rep Trop Med 9-

22.

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