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.
ii
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
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