Download Licentiate thesis from the Department of Immunology,

Licentiate thesis from the Department of Immunology,
Wenner-Gren Institute, Stockholm University, Sweden
The role of antibody mediated parasite neutralization in
protective immunity against malaria
Elisabeth Israelsson
Stockholm 2007
Believe nothing, no matter where you read it,
or who said it, no matter if I have said it, unless
it agrees with your own reason and your own
common sense
– Buddha
Summary
Malaria is the most prevalent infectious disease in the world today, in regard to
morbidity and mortality, and it is mostly affecting sub Saharan Africa. High priority is
put on the development of a vaccine against the malaria parasite Plasmodium falciparum,
which due to its prevalence, virulence and drug resistance is the major cause of the high
mortality. But there are many obstacles left before a rational vaccine can be developed,
the major one being the limited knowledge of how the immune system is clearing a
malaria infection and what protective components are involved in this context. The aim
of the work presented in this thesis is to define the role of antibodies as protective
components in natural Plasmodium falciparum infections, since definition of the isotypes
and specificities of the antibodies involved are essential for designing an effective
vaccine. This investigation is based on ethnic differences in susceptibility to malaria. In
Mali and Burkina Faso the Fulani ethnic group shows a relative resistance to malaria as
compared to other sympatric ethnic groups.
In this thesis I present studies of the antibody responses to Plasmodium
falciparum and other pathogens in sympatric ethnic groups living in Burkina Faso and
Mali. We confirm the previous findings of the different anti-malarial antibody responses
between the sympatric ethnic groups. The anti-malarial IgG responses are dominated by
IgG1 and IgG3, suggesting a role of these subclasses in protection, and we also suggest a
protective role of anti-malarial IgM. However, we could not show any consistent
differences between the ethnic groups for non-malarial antigens, nor for total IgG
antibodies, suggesting the relative resistance to malaria seen in the Fulani to be pathogen
specific and not due to a generally hyper-reactivity in this group. We have also analysed
the impact of the Fcγ receptor IIa R131H polymorphism on IgG subclass pattern and
susceptibility to malaria. Our results show that the IgG subclass pattern is different
between the tribes, Fulani having a higher proportion of malaria specific IgG2 than
Dogon, and we also observed a significant difference in genotype frequency, Dogon
being strongly biased towards a RR or HR genotype, while Fulani present an evenly
distributed genotype frequency. However we could not show any consistent results for
the impact of the genotype on IgG subclass pattern. We suggest, based on our results that
IgG2 is related to protection and also the HH-genotype may be related to protection.
ii
List of Papers
This thesis is based on the following original papers, which will be referred to by their
Roman numerals:
I.
A. Bolad,* S.E. Farouk,* E. Israelsson, A. Dolo, O.K. Doumbo, I. Nebié, B.
Maiga, B. Kouriba, G. Luoni, B.S. Sirima, D. Modiano, K. Berzins, M. TroyeBlomberg. Distinct interethnic differences in IgG class/subclass and IgM
antibody responses to malaria antigens but not in IgG responses to nonmalarial antigens in sympatric tribes living in West Africa
Scand J Immunology 2005 Apr;61(4):380-6
*These authors contributed equally to this paper.
II.
E. Israelsson, A. Lysén, M. Vafa, N. Iriemenam, A. Dolo, B. Maiga, O.K.
Doumbo, M. Troye-Blomberg, K. Berzins. Fcγ receptor IIa polymorphism and
IgG subclass pattern in sympatric ethnic groups in Mali
Manuscript
iii
Table of contents
List of Abbreviations ___________________________________________________ v
Introduction___________________________________________________________ 1
The Immune system ___________________________________________________ 1
Immunoglobulins _____________________________________________________ 3
Fc receptors__________________________________________________________ 5
Malaria _____________________________________________________________ 6
Malaria parasite life cycle _______________________________________________ 7
Malaria disease _______________________________________________________ 8
Immunity to malaria ___________________________________________________ 9
Immune responses to malaria ____________________________________________ 9
The importance of antibodies in protection against malaria ____________________ 11
Polymorphisms in Fcγ receptors and malaria protection ______________________ 13
Ethnic groups in West Africa showing differences in susceptibility to malaria_____ 14
General aim of the study _______________________________________________ 15
Material and Methods used in the study___________________________________ 15
Results and Discussion _________________________________________________ 16
Antibody responses to P. falciparum and other pathogens in ethnic groups living in
sympatry in West Africa (Study I) _______________________________________ 16
Fcγ receptor IIa polymorphism and IgG subclass pattern in sympatric ethnic groups in
Mali (Study 2) _______________________________________________________ 18
Future projects _______________________________________________________ 20
Acknowledgments _____________________________________________________ 22
References ___________________________________________________________ 23
iv
List of Abbreviations
ADCI
Antibody dependent cell mediated inhibition
APC
Antigen presenting cell
CRP
C-reactive protein
DC
Dendritic cell
FcR
Fc receptor
H
Histidine
Ig
Immunoglobulin
IL
Interleukin
IFN
Interferon
iRBC
Infected red blood cell
ITAM
Immunoreceptor Tyrosin-based Activation Motifs
ITIM
Immunoreceptor Tyrosin-based Inhibiting Motifs
MHC
Major histocompatibility complex
MSP
Merozoite surface protein
NA
Neutrophil Antigen
NK cell
Natural Killer cell
PAM
Pregnancy associated malaria
R
Arginine
RBC
Red blood cell
TCR
T cell receptor
Th
T helper
TLR
Toll like receptor
VSA
Variant surface antigen
v
Introduction
The Immune system
The immune system is a highly variable and diverse component in all higher
animals. It has evolved throughout the years, and its complex network of cells and
molecules can distinguish between invading pathogens and the body’s own cells.
Traditionally, the immune responses raised to an invading pathogen are divided into
innate immune responses and adaptive immune responses.
The adaptive immunity is mediated by clonally distributed B and T cells and it
exhibits specificity, diversity and memory. Small differences between pathogens can be
distinguished, and a unique response will be raised against all particular antigens. Once
the antigen has been recognized and responded to, an immunological memory will be
developed, which by the next encounter with the same antigen will yield a faster immune
response. The disadvantage with the adaptive immune system is that the primary
response is delayed, due to the clonal expansion.
The innate immune system is a less specific first line of defence and it involves
anatomical barriers (skin and mucosal surfaces), physiological barriers (temperature, pH
and chemical mediators), endocytic and phagocytic cells (monocytes, macrophages and
neutrophils), and inflammatory responses. It reacts immediately upon stimulation of
pathogens and can also activate and shape the adaptive immune responses. The
stimulation of the innate immune system is mainly mediated through pattern recognition
receptors (PRR), which bind conserved molecular structures found in large groups of
pathogens. These receptors can be secreted, be expressed on cell surfaces or in
intracellular compartments. The Toll-like receptors (TLRs) are one of the most important
PRR families and ten TLRs in humans are known 1, of which TLR2 and TLR4 are the
best characterised.
There are many different cell types involved in different stages of the immune
response. The only cells capable of producing antibodies are the B cells, each B cell
producing a unique specificity of their antibodies. Dendritic cells (DCs) are bridging the
gap between the innate and adaptive immune responses. T cells are divided into αβ T
cells and γδ T cells, depending on the composition of the T cell receptor (TCR). The γδ T
1
cells, representing a relatively small part of the T cell repertoire, recognise non-peptidic
antigens in a major histocompatibility complex (MHC) independent manner 2. The αβ T
cells are further divided into CD4+ T cells, which regulate the cellular and humoral
immune responses, and CD8+ T cells, that show a major cytotoxic activity toward cells
infected with intracellular pathogens. The CD4+ T cells are divided into Th1/Th2 type of
cells depending on the cytokines they produce. Traditionally, Th1 cells had been
described to drive the type-1 pathway, the cellular immunity pathway, to fight viruses
and other types of intracellular pathogens, whereas the type-2 cells has been said to drive
the type-2 pathway, the humoral immunity pathway, by up-regulating antibody
production to fight extra-cellular pathogens 3. A less characterised subset of T cells is the
T-regulatory cells (Treg), which can regulate the responses by CD4+ and CD8+ T cells
and NK cells. Natural killer cells have the ability to react with spontaneous cytotoxicity,
without sensitisation, against a broad range of targets, and they are also one of the key
producer of cytokines that will mediate the immune responses of the other immune cells
4
. NKT cells express a TCR, and are therefore by definition T cells, however it shares
some of the characteristic NK cell markers. In contrast to other T cells, NKT cells do not
interact with MHC class I or II, but do interact with glycolipids presented by CD1d, a
non-classical antigen presenting molecule. They can also up- or down regulate immune
responses by secretion of Th1-, Th2- or regulatory cytokines 5.
A key function for the immune responses, is the antigen recognition and
presentation, the antibodies produced by B cells can bind directly to the naive antigen,
but T cells usually need to get the antigen presented as a peptide bound to a MHC
molecule. Two classes of MHC molecules function in antigen presentation, MHC class I
and MHC class II. The MHC class I molecules are expressed on almost all cells, they
primarily present endogenous antigens (e.g. viral proteins), and it is mainly CD8+ T cells
that recognise the MCH class I, leading to lysis of the cell presenting foreign peptides 6.
The MHC class II is only expressed on the professional antigen presenting cells (APCs),
which comprise B-cells, macrophages and DC. They express the MHC class II molecules
together with co-stimulatory molecules that are necessary for the induction of a proper T
cell response. MHC class II presents peptides from exogenous proteins, and APCs
present almost exclusively to CD4+ T cells 6. The CD4+ T cells provide helper functions
2
to stimulate specific antibody responses by B cells and specific responses by CD8+ T
cells.
Immunoglobulins
The immunoglobulin (Ig) molecule can be found both as membrane bound on Bcells and in a secreted form that is produced by activated B-cells, the plasma cells. When
bound on the surface the Ig functions as a receptor involved in differentiation, activation
and apoptosis, while the secreted form can neutralize foreign antigens and recruit other
effector components 7. The Ig consists of two large polypeptide chains, called heavy
chains, and two shorter, called light chains, paired together in a Y-shape. The open upper
part of the Y is the antigen binding part, and the lower part of the Y is the Fc part, which
is responsible for interaction with receptors and complement 7. There are five different Fc
parts, each corresponding to an Ig isotype, IgM, IgD, IgG, IgA and IgE, all of which can
function both as receptors on the cell surface and in a secreted form 7.
Figure 1: A simplified structure of the Ig molecule. Adapted from Martin (1969) 7
IgM represents about 30% of the total serum immunoglobulins 8, and it is the first
antibody class that encounters a new antigen 8. IgM can be found in two forms;
membrane bound monomeric IgM and secreted pentameric IgM. The pentameric form of
IgM makes it a powerful complement activator and it can up-regulate both primary and
memory responses and increase affinity maturation 9.
3
Out of the total serum immunoglobulins, 0.25% is represented by IgD. It appears
not to cross the placenta, and it has a weak or absent binding to normal lymphocytes,
neutrophils and monocytes 10. Native IgD has little or no capacity to activate complement
effects, whereas aggregated monoclonal IgD induces complement activation 10. IgD is coexpressed with IgM on most peripheral B-cells. IgD is conserved across different species
and it is found in all mammals and avian species, suggesting an evolutionary advantage
10
. The role of IgD is still not completely understood, but it seems to behave like IgM
early in infections. Moreover, IgD concentrations are elevated in chronic infections, but if
these specific IgD antibodies are of any clinical importance is unknown 10.
IgG is dominating the humoral responses in humans, around 75% of the total
immunoglobulin concentration in serum being IgG. Human IgG is divided into four
subclasses, IgG1-IgG4, where IgG1 is the largest subclass (66%), followed by IgG2
(24%), IgG3 (7%) and IgG4 (3%) 11. The different subclasses differ in ability to activate
the complement, IgG3 and IgG1 are the most effective ones, and IgG2 is a weak
activator, whereas IgG4 does not activate complement at all. The role of the different
subclasses varies, IgG2 is the main antibody targeting encapsulated bacteria, IgG1 and 3
are mainly directed against protein antigens and IgG4 is common in chronic exposure to
protein antigens and in allergy. IgG can also be transported through the placenta.
IgA is present in normal human serum at about one fifth of the IgG concentration,
and it is the most abundant antibody in secretions
12
. Mucosal surfaces are the main
source of antigenic material in the body, and in the mucosal tissues the local synthesis of
secretory IgA is dominating that of the other antibody classes. Secretory IgA is present in
all mucosal surfaces, and is therefore an important first line of defence, and it can activate
the complement system and also trigger cell-mediated events
12
. In breast milk and
colostrum, the major immunoglobulin is IgA, providing the child protection against
intestinal pathogens 13.
IgE is the antibody class that is the least abundant in human serum. The effect of
IgE is mainly known in allergy, where IgE mediates the hypersensitivity reactions
responsible for the symptoms of hay fever, asthma, hives, and anaphylactic shock. IgE
can up-regulate carrier-specific antibody responses
9
, both primary and memory
responses 14. Elevated levels of IgE have been shown for many helmintic infections
and also in malaria exposed individuals 18-21.
4
15-17
Fc receptors
One receptor type that is involved in antibody recognition comprises the Fc
receptors (FcR). They recognize and bind to the Fc part of the antibodies and they exist
for all antibody classes, FcγR recognize and bind IgG, FcαR for IgA, FcδR for IgD,
FcµR for IgM and FcεR for IgE. Signalling through the Fc receptors induces many
different actions, depending on the cell carrying the receptor, e.g. phagocytosis and
release of inflammatory components
22
. There are also FcRs responsible for
transportation of antibodies through epithelia, they are the polymeric IgA and IgM
receptors and the neonatal FcR, that mediates antibody transportation through the
placenta from the mother to the child 22. The FcRs capable of cell activation all contain
intracytoplasmic activation motifs, designated immunoreceptor tyrosine-based activation
or inhibiting motifs (ITAMs or ITIMs)
23
(Fig 2). These ITAMs can be of two different
types, multichain or single-chain receptors. The FcRs that lack ITAMs do not trigger cell
activation, the exception is FcγRIIIB, which has no activating effect on its own, but
contributes to cell signalling by associating to other FcRs 22.
Figure 2: Human Fc receptors. Adapted from Pleass and Woof (2001) 23
5
Malaria
Malaria is the most prevalent infectious disease in the world, causing more than
300 million acute clinical cases and approximately 2 million deaths every year. About
90% of the deaths related to malaria occur in sub-Saharan Africa, and the disease is the
leading cause of mortality (20%) in children less than five years of age
24
. Women are
also highly susceptible to so called placental- malaria (or pregnancy associated malaria,
PAM) during their first and second pregnancy, which may lead to death. Moreover,
malaria infections in the mother can lead to spontaneous abortion, neonatal death and low
birth weight of the child. Malaria is increasingly becoming a global problem, natural
disasters, agricultural projects, climate changes and the fact that people travel more are
giving the parasite and the mosquito many chances to spread to non-malaria areas. The
rapid development of drug-resistance amongst the malaria parasites is another problem,
since several of the anti-malarial drugs available are now becoming without effect, and
the drugs that still work are expensive, and many countries can not afford them as a first
line treatment. However, a recent optimistic report from Malawi, presents data suggesting
that the previously widely spread chloroquine resistance among the P. falciparum
parasites in this country, seems to have disappeared, and chloroquine is again an effective
anti-malarial drug
25
. The vector has also been a target for control measures of the
disease, the major ones being insecticide usage on wetlands and insecticide treated bed
nets. However, the mosquito is prone to develop insecticide resistance, leaving only the
insecticide treated bed nets as an effective barrier. The distribution of bed nets is ongoing,
but financial issues have slowed down the progress. Today, a main goal for controlling
malaria infections around the world is the development of a functional vaccine. However,
this is not progressing as fast as needed. The knowledge of how the immune system is
clearing a malaria infection, and what protective components that are involved in parasite
neutralisation is still not defined enough for a rational vaccine design.
6
Malaria parasite life cycle
Malaria is caused by a protozoan parasite of the Plasmodium family. Although
four species infect humans, Plasmodium falciparum, P. vivax, P. malariae and P. ovale,
only P. falciparum results in high mortality as a result of its prevalence, virulence and
drug resistance 26. Transmitted by the female Anopheles mosquito, the sporozoites reach
the liver, where they develop into merozoites. After 1-2 weeks the infected liver cells
rupture, releasing thousands of merozoites, which all can invade red blood cells (RBC),
and develop inside the RBC, to the so called ring-stage, trophozoite stage and schizont
stage. When the schizonts rupture, many merozoites will be released and invade new
RBCs. This is known as the asexual blood stage of the parasites life-cycle. Some
merozoites develop into gametocytes, which are taken up by the anopheline mosquito,
and the parasite starts its sexual cycle inside the mosquito midgut with sporozoites as end
products 26 (Fig 3).
The erythrocytic cycle of P. falciparum has a unique feature, mature trophozoites
and schizonts are sequestred in the peripheral circulation, due to parasite mediated
changes of the surface of the infected RBC (iRBC), causing them to adhere to endothelial
cells (sequestration) and other erythrocytes (rosetting). It is an accepted theory that this
adhesion is an immune escape mechanism of the parasite, and that it also may lead to a
better maturation in the microaerophilic venous atmosphere 27.
7
Figure 3: The life cycle of the Plasmodium parasite. The average number of Plasmodium
parasites at each developmental stage is indicated. Adapted from Brown and Catteruccia
(2006)28
Malaria disease
The clinical symptoms of malaria are only presented during the erythrocytic stage
of the parasites life cycle, and mild or uncomplicated malaria is characterised by fever,
followed by nausea, headache, cough, diarrhoea and muscular pain. Infections by P.
vivax, P. ovale and P. malariae usually give milder malaria as compared to that caused
by P. falciparum. This is due to the ability of P. falciparum to adhere to host endothelium
in combination with the high parasitemia usually seen for P. falciparum. WHO defines
severe malaria as a parasitemic person with one or more of the following symptoms:
prostration (inability to sit up without help), impaired consciousness, respiratory distress
or pulmonary edema, seizures, circulatory collapse, abnormal bleeding, jaundice,
hemoglobinuria or severe anaemia (haemoglobin < 50 g/L or hematocrit < 15%)
8
26
.
Cerebral malaria is caused by the sequestration of P. falciparum infected RBCs in small
blood vessels in the brain, causing blocking of the blood flow, leading to coma or other
neurological phenomena, such as seizures and elevated intracranial pressure.
Immunity to malaria
Unlike other acute infections, malaria does not induce a long lasting
immunological memory, but it rather develops gradually and requires repeated infections
to persist. The developed immunity is not sterile, malaria parasites can still survive in the
host but at such low levels so that the clinical symptoms do not show. The development
of this semi-immunity is influenced by age, genetic background, pregnancy, coinfections
and the nutritional status of the host 29.
Immune responses to malaria
The genetic influences on susceptibility to a malaria infection has been well
studied and, not surprisingly, are many of the well-known malaria resistance genes
related to the structure or function of the RBC, including sickle cell trait, thalassemias,
enzyme deficiencies, ovalocytosis and the ABO blood groups.
In the complex immune response to malaria, both the adaptive and the innate
immune responses are important. Monocytes 30, macrophages 31 and NK cells 32 are able
to kill the parasite in the absence of antibodies, probably involving binding of CD36 to
parasite derived surface molecules on iRBC 33. Human NK cells have also been shown to
rapidly produce interferon (IFN) - γ, a cytokine associated with reduced susceptibility to
malaria
34, 35
infections
36
, suggesting a importance of NK cells early in the blood-stage malaria
. The NKT cells produce large amounts of IFN-γ and IL-4, when activated
through the TCR, and this rapid cytokine output may activate other lymphoid cells 37. It
has also been shown that, in response to a Plasmodium berghei infection, the CD1restricted NKT cells contribute to malarial splenomegaly, which is associated with
expansion of splenic B-cells and enhanced parasite-specific antibody formation 37.
The role of DCs in malaria immunity is still relatively unknown, some studies
show that the maturation of human DCs are suppressed, and that their ability to activate
9
T-cells are reduced by iRBC
38, 39
. However, using animal models, it was demonstrated
that DCs from infected mice are fully functional APCs 40.
During the first few days in a malaria infection, γδ T cells expand and they have
been shown to have the capacity to directly inhibit the parasite growth 41. Both the CD4+
and CD8+ T cells play important roles in immunity to malaria, but at different stages.
During the liver stage CD8+ T cell functions are important 42, and they also contribute to
protection against severe malaria
43, 44
. The CD4+ T cells are crucial in the immunity
against asexual blood stage malaria. They produce cytokines which are involved in the
activation of innate immune responses, and they are also required for the B cell
production of anti-malarial antibodies. The immunity to blood stage malaria is dependent
on the CD4+ T cells, anti-malarial antibodies and B cells
45
. For other protozoan
infections, the Th1/Th2 balance is crucial for the clearance of the parasites 46. In malaria,
many studies, using mouse models, have shown that pro-inflammatory Th1 cytokines are
crucial determinants of the outcome of the malaria disease, C57BL/6 mice, which have a
predominant Th1- immune response, are more susceptible to cerebral malaria, than the
Th2 biased BALB/c, which are resistant to cerebral malaria
47
. And in humans, studies
have shown a possible association between IL-4 and levels of anti-malarial antibodies 48.
T regulatory cells are still under investigation for their role in for malaria immunity 49.
The role of TLRs is still not fully understood, but it is known that they can
recognise malaria parasites or their metabolites. The glycosylphosphatidylinositol (GPI)
anchors of the P. falciparum antigens have been shown to mediate signals, mainly
through TLR2 and to a lesser extent by TLR4 50. Furthermore, hemozoin, a parasite heme
metabolite, is recognised by TLR9
51
. Common polymorphisms in TLR4 may be
associated to the clinical outcome of a malaria infection 52, and polymorphisms in TLR4
and TLR9 have been shown to increase the risk of low birth weight in P. falciparum
infected pregnant women as well as the risk of maternal anemia
53
. However, none of
these polymorphisms were found to affect the prevalence and parasite density of the P.
falciparum infection 53.
10
The importance of antibodies in protection against malaria
Innate immune mechanisms involving mononuclear phagocytes and NK cells play
an important role early in malaria infections. However, the importance of antibodies in
protective immunity against P. falciparum infection was demonstrated by the classical
experiments of Cohen and McGregor 54, in which passive transfer of IgG from adults had
curative effects in children. Furthermore, in Thai individuals, passive transfer of human
IgG with a high content of cytophilic antibodies was associated with protection 55. The in
vitro effect of antibodies on P. falciparum isolates have been studied by different
methods using whole sera or Ig fractions from individuals that have experienced malaria
56
. Antibodies can neutralize the parasite either by inhibition of merozoite invasion, by
neutralization of the free merozoites or by interference with the merozoite invasion
process. Antibodies may also react with parasite-derived antigens expressed on the
surface of infected RBC, thereby inhibiting the intraerythrocytic development of the
parasite
56
. The leaky membrane of infected erythrocytes just prior to merozoite release
may give the antibodies access to the intraerythrocytic parasite, and they may interfere
with merozoite dispersal. Another pathway for antibody attack may be the possible
parasitophorus duct, which forms a connection for direct access of serum
macromolecules to the parasite 56. Studies on antibody-mediated inhibition of the growth
of parasite-isolates from different regions, showed significantly better inhibition by
sera/Ig coming from the same area as the parasites, than by those from remote areas
56
.
However, the growth inhibition is not always the result from the action of antibodies, but
rather from some other, as yet undefined, serum factors
56
. Furthermore, some studies
have shown that certain sera or Ig-fractions may enhance the growth of the parasite,
instead of inhibiting the growth
56
. Even though antibodies can inhibit parasite
invasion/growth on their own, the main effects of the malaria specific antibodies are to
induce antibody dependent cell-mediated inhibition (ADCI)
monocyte-derived mediators
57
and the secretion of
58
. The main players in this type of killing are the Fc
receptors on the surface of the effector cells, which will bind the Fc part of the antibodies,
while the Fab part of the antibody is bound to antigens on the surface of merozoites 55 or
late stage infected RBC 59.
Several studies have shown that high titres of malaria specific IgG are related to
protection from severe malaria and seroepidemiological studies in different endemic
11
areas have demonstrated the association of IgG antibodies of the cytophilic subclasses
IgG3 and IgG1 with protection against P. falciparum malaria
60, 61
. This association is,
however, quite inconsistent when considering antibody responses to single malaria
antigens. IgG3 is the major subclass in responses against P. falciparum antigens showing
a high degree of diversity, e.g. merozoite surface protein 2 (MSP-2)
55
, while responses
against more conserved antigens, e.g. the C-terminal part of MSP-1, are dominated by
IgG1 62. Interestingly, in some populations, IgG2 is related to protection, so it is not clear
what IgG subclass profile that is the most protective.
The most important antigens that are being targeted by the antibodies are mainly
expressed during the merozoite stage or the later trophozoite stage (Fig 4). At the
merozoite stage, the MSP antigens, antigens present in the apical complex organelles of
the merozoites (EBA-175, Rhop 1-3, RAP 1-3 and AMA-1) and Pf155/RESA, all have
been shown to be targets to antibodies with the capacity to inhibit merozoite invasion 63.
Also, the recently identified SURFINs
64
are found in this stage. There are several
antigens synthesised during the trophozoite development (e.g. GLURP, SERA, ABRA,
PfEMP1, Pf332, RIFINs, STEVORs), and antibodies to several of these antigens have
been shown to have a high capacity to inhibit parasite growth or invasion 65-68.
Immunity to malaria is parasite and strain specific, and clonal antigenic variation
is common in P. falciparum 69. The mechanism behind this antigenic variation is still not
clear, but one very likely hypothesis is that there is a frequent ongoing switching of
variant surface antigens (VSA) in the parasite population, and an outgrowth of one of
these subpopulations would occur when antibodies are being raised towards the other
VSA presented. The importance of VSAs in immunity can be shown by correlating the
range of different anti-VSA antibodies to protection 70. The pregnancy associated malaria
is caused by accumulation of iRBCs in the placenta. These parasites express a specific
VSA that binds to chondroitin sulphate A (CSA), and the immune responses that are
induced are sex specific and parity dependent
71
. Some important antigenically variable
antigens are PfEMP1, RIFINs, STEVORs and SURFINs.
12
Figure 4: The location of some of the more important antigens involved in parasite
neutralizing immune responses during the erythrocytic cycle. Adapted from (Bolad and
Berzins 2000) 56
Polymorphisms in Fcγ receptors and malaria protection
In humans there are three families of Fc-receptors binding IgG, FcγRI (CD64), RII (CD32) and -RIII (CD16). FcγRI is a high-affinity receptor that binds monomeric
IgG, FcγRII and -RIII are low-affinity receptors only binding complexed or aggregated
IgG. Polymorphisms in the Fcγ receptors critically affect their binding of different IgG
subclasses. Accumulating evidence suggests a relevance of these polymorphisms for
susceptibility to disease 72. FcγRIIa has two codominantly expressed allotypes, differing
at position 131R/H. FcγRIIa 131H is the only human FcγR that efficiently binds IgG2 73.
FcγRIII has two isoforms, FcγRIIIa exhibits a dimorphism at position 158F/V with
different affinity to IgG1 and IgG3
74
. FcγRIIIb occurs in two allotypes, neutrophil
13
antigen 1 (NA1) and NA2, where the FcγRIIIb-NA2/NA2 genotype has a lower capacity
for phagocytosis 75.
Only a few studies on FcγR polymorphisms in relation to malaria have been
performed, most of them are associating the FcγRIIa 131R/R genotype with protection
against malaria and the FcγRIIa 131H/H genotype with susceptibility to the disease 76. A
study in Western Kenya showed that infants carrying the FcγRIIa 131R/R genotype had a
significantly lower risk for high-density P. falciparum infection than 131R/H genotype
carriers 57. In the same region, the FcγRIIa 131H/H genotype was found associated with
enhanced susceptibility to placental malaria in HIV-positive, but not in HIV-negative
women 77. Furthermore, a study in Thailand showed that the FcγRIIa 131H/H genotype
and the FcγRIIIb NA2 allele were associated with susceptibility to cerebral malaria
while the 158F/V polymorphism in FcγRIIIa had no effect in this respect
78
,
79
. Similarly,
the FcγRIIa 131H/H genotype was significantly associated with susceptibility to severe
malaria in a study performed in The Gambia
80
. A study in Burkina Faso indicates the
impact of the genotype of FcγRIIa on protective immunity; a significant correlation was
demonstrated between the levels of anti-malarial IgG2 antibodies and the incidence of
malaria, in a population where the IgG2 binding FcγRIIa 131H allele was predominant 81.
Transfected phagocytic cells, expressing the FcγRIIa 131R allotype, tended to show
higher phagocytosis of P. falciparum infected erythrocytes following opsonisation with
IgG1-containing sera, in contrast to the 131H allotype, that showed the highest
phagocytosis with IgG3-containing sera 82.
Ethnic groups in West Africa showing differences in susceptibility to malaria
Several studies have demonstrated differences in susceptibility to malaria between
different ethnic groups. In East-Africa, the Fulani showed a higher frequency of
splenomegaly and lower incidences of malaria than other sympatric groups, despite the
same exposure to malaria and no differences in socio-cultural circumstances
83
. This
finding was later confirmed by various studies, showing that the Fulani have a lower
parasite prevalence and density and have a more prominent spleen enlargement compared
14
to other ethnic groups
84, 85
. Moreover, the Fulani have generally higher anti-malarial
antibody responses, covering antigens from both the liver stage 86, 87 and the blood stage
87, 88
, as well as the crude P. falciparum extract
85
. HLA analyses have shown that the
Fulani are genetically distinct from other African tribes
89
, and regarding established
genetic malaria resistance factors, the haemoglobin S and C, α thalassemia, G6PDA and
HLA B, have been shown to occur in a lower frequency in the Fulani than in their
sympatric neighbours
9085
. The proportion of individuals not having any of these
protective alleles was more than 3-fold greater in the Fulani, as compared to the other
ethnic groups
90
. IL-4 levels and polymorphisms have been suggested as contributing
factors to this lower susceptibility in the Fulani 48, 91, but these findings are not enough to
explain this ethnic differences, so many studies are ongoing, covering a wide range of
possible effector functions, e.g. cytokine expression, TLR expression, cell activation and
receptor functions.
General aim of the study
The aim of this study was to define the role of antibodies as protective
components in the complex immune responses to natural P. falciparum infections.
Definition of the isotypes and specificities of antibodies in relation to their anti-parasitic
activities is essential for a rational development of a vaccine to malaria. The differences
in susceptibility to malaria between the sympatric living Fulani and their neighbours, give
a unique opportunity to study the importance of anti-malarial antibodies and their role in
protection against severe outcome of the disease.
Material and Methods used in the study
The methodologies, study areas and study populations of the included studies are
described in detail in the corresponding paper.
15
Results and Discussion
Antibody responses to P. falciparum and other pathogens in ethnic groups living in
sympatry in West Africa (Study I)
The well known relative resistance to malaria seen in the Fulani, as compared to
other sympatric tribes, is related to their higher concentrations of anti-malarial antibodies
88
. The ability of the Fulani to mount stronger immune response has been suggested to be
at least in part genetically regulated 48. However, if these inter-ethnic differences can be
ascribed a generally more activated immune system or specifically enhanced antimalarial immune responses in the Fulani is still unknown.
In this study, we investigated the isotypic distribution of malaria specific
antibodies to crude P. falciparum antigen, the total IgG and IgM concentrations and
concentrations of IgG antibodies, reactive with a panel of non-malarial antigens in Fulani
individuals from Burkina Faso and Mali, and compared them to sympatric individuals
from ethnic groups with a different genetic background, in order to clarify if the relative
resistance seen in Fulani is malaria specific or a general hyperreactivity in this tribe.
Despite a difference in transmission intensity, Fulani from Mali showed similar
levels of P. falciparum specific IgG, IgM and IgG subclasses as the Fulani from Burkina
Faso. Fulani from both Burkina Faso and Mali had higher levels of all malaria-specific
antibodies when compared with those of the respective sympatric tribes. Also, total IgM
levels were shown to be higher in Fulani than in the non-Fulani, but for total IgG we
could not show any difference between the tribes. For the non-malarial antigens included
in the study, some showed the same pattern as for malarial antigen, with Fulani having
higher levels of specific antibodies, while some other antigens showed no such
difference.
The higher levels of anti-malarial IgG and IgM in the Fulani groups, suggest a
role of these antibodies in the lower susceptibility to malaria seen in Fulani. This is in
line with previous studies, suggesting a role of malaria specific IgG and IgM in the
defense against malaria
54, 92
. It has been shown that memory IgM+ B cells can persist
long after the malaria transmission seasons 92, 93, which could be further supported in our
result by the consistently higher total concentrations of total IgM in Fulani as compared
16
to non-Fulani groups. However, ethnic differences in persistence of these IgM+ B
lymphocytes has to be further studied before any conclusion of that kind can be made.
IgG subclass antibodies with specificity to malaria antigens have been shown to
be important in protection against malaria. In particular, antibodies of the IgG1 and IgG3
subclasses have been related to protection 94. The suggested mechanism by which these
subclasses are protective, involves their binding to the Fc receptors on monocytes,
leading to antibody dependent cell mediated inhibition of parasite replication 94, 95. This is
supported by our results, since the two most predominant IgG subclasses in this study are
IgG1 followed by IgG3.
The results for the included non-malarial antigens showed higher levels of IgG
against measles and T. gondii antigens in the Fulani compared to the other ethnic groups.
Only the Malian Fulani showed higher antibody levels against M. tuberculosis (PstS-1)
compared to their sympatric tribe, while no difference was seen in Burkina Faso for this
antigen. For Rubella and H. pylori, no differences between the ethnic groups were seen.
The higher levels of anti-mycobacterial antibodies in Fulani of Mali can be an indication
of a higher prevalence of the disease or more frequent vaccinations in this group as
compared to their neighbouring tribe. The responses to measles and T. gondii in the
Fulani may be explained by a possible cross-reaction with P. falciparum, which has also
been shown to signal through TLR 9
96-99
. Thus, maybe polymorphisms in TLR9
100
can
be a contributing factor for the differences in anti-malaria response seen between Fulani
and their sympatric neighbours.
In conclusion, this study supports the previously reported higher anti-malarial
responses seen in Fulani as compared to sympatric tribes. Also, we show that the
response is dominated by IgG1 and IgG3, suggesting a role of these IgG subclasses in
protection against malaria. We also suggest a protective role of anti-malarial IgM, and the
fact that only some of the non-malarial antigens showed the same inter- ethnic
differences as malaria antigens, indicates that Fulani is not immunological hyper-reactive
to all pathogens.
17
Fcγ receptor IIa polymorphism and IgG subclass pattern in sympatric ethnic groups in
Mali (Study 2)
Several studies report a protective effect of a polymorphism in amino acid 131 in
Fcγ receptor IIa to malaria. The wild type allele, arginine (R), has been shown to be
associated with protection against malaria, while the mutated allele, histidine (H), was
linked to susceptibility
76
. The H allele is the only FcγR that efficiently binds IgG2
73
,
which has been suggested to inhibit the anti-malarial effects of IgG1 and IgG3. There are
a few studies suggesting that IgG2 is important in protection
81, 101
, and this conflict in
results could be due to the FcγRIIa polymorphism.
Since we have suggested that the relative resistance in Fulani against malaria is
not due to a general hyper immunological reactivity, we wanted to confirm these results
by comparing the total IgG subclass levels between Fulani and Dogon in Mali. We also
analysed the malaria specific IgG subclass antibodies in relation to Fcγ receptor IIa
polymorphism in order to investigate if this polymorphism could be a contributing factor
for the protection seen in Fulani.
Our results show that Fulani are less parasitized, have fewer parasites clones and
have higher spleen rate than Dogon. This is in line with what has been previously
reported 83, 85, 87.
For the total IgG subclasses, we could not find any consistent difference between
the two ethnic groups, only IgG4 was slightly increased in Dogon as compared to Fulani.
This confirms our suggestion that the relative resistance in Fulani as compared to
sympatric tribes is pathogen specific. We can also confirm the findings that the Fulani
have higher anti-malarial IgG subclasses than the Dogon, however the pattern of
distribution differed between the two groups. While the Dogon and many of the Fulani
had
a
similar
pattern
to
that
previously
described
in
many
studies
(IgG1>IgG3>IgG2>IgG4), some Fulani individuals showed higher IgG2 levels than
IgG3, making the order IgG1>IgG2>IgG3>IgG4. This difference was more obvious
when looking at the ratios between IgG1:IgG2, the Fulani showing more IgG2 than the
Dogon, the ratios being 17.5:1 for Dogon and 6.3:1 for Fulani. The IgG1 and IgG3
subclasses have been given the most attention as protective antibodies, however there are
18
some reports suggesting IgG2 to be protective
81, 101
and our results confirms these
results, since Fulani are supposed to be relatively more resistant to malaria.
Regarding the Fcγ receptor IIa R131H polymorphism, our results are not
confirming the idea of the R-allele being associated with protection from malaria. We
demonstrated a significant difference between the genotype frequencies in Fulani and
Dogon, with RR homozygotes being more common in Dogon than Fulani, and the HH
genotype occurring at higher frequency in the Fulani. Some studies have suggested the
HH genotype to be mildly associated to protection
81, 102
, and our results confirm these
findings. Interestingly, the frequencies of the genotype distribution are for the Fulani very
similar to what has been reported for Caucasians
103, 104
, this suggests that the allelic
change is not driven by malaria pressure in Fulani, but rather is a reflection of their
genetic background
89
. However, the impact of Fcγ receptor IIa 131 R/H polymorphism
on malaria protection has been shown in many studies, so if the HH genotype can be
shown to be a protective factor, then the relative resistance seen in the Fulani could, at
least in part, be explained by this genetic predisposition. The proposed protection of the
R-allele could come from a difference in IgG subclass distribution as compared to the Hallele, and in Fulani individuals we show higher IgG3 levels among R-allele carrier than
among HH individuals. However, no such trend was seen in the Dogon. Interestingly, for
IgG2 the R-allele seemed to be related to higher IgG2 levels in the Dogon tribe. Since the
receptor is less effective in binding IgG2 when the R-allele is present, it is possible that
more IgG2 will be present in the serum of individuals with the R-allele than those with
the H-allele. However, for the Fulani, the higher IgG2 levels were not found in
individuals with the RR genotype, but rather in those with the H-allele, i.e. the total
opposite result from that found in Dogon. These conflicting results may suggest that this
polymorphism is not of high importance in malaria protection, or that the importance of
this polymorphism lies elsewhere and not in the IgG subclass distribution. Importantly,
although the levels of antibodies to the crude malaria antigen are associated with relative
resistance from malaria, their importance in protective immune mechanisms remains to
be defined. Based on our results, we suggest that the FcγRIIa 131HH genotype is
associated to a lower susceptibility to malaria, and that IgG2 may be important in
susceptibility to malaria. We also suggest, that the FcγRIIa R131H polymorphism could
19
influence the IgG subclass responses, and also be a contributing factor to the lower
susceptibility to malaria seen in the Fulani as compared to their sympatric neighbours.
Future projects
So far, our results have shown a possible relation between FcγRIIa and a lower
susceptibility to malaria. We have also been able to show, that the proposed protected
IgG subclass pattern, maybe should include IgG2. However, the transmission intensity
may influence these findings, so it is important to extend the studies on FcγR and IgG
subclass distribution and their relevance for protection against malaria in other areas with
different malaria transmission intensity. I will also investigate the effect of transmission
intensity on other factors known be related to FcγR, such as C-reactive protein.
C-reactive protein (CRP) is an acute-phase serum protein that belongs to the
pentraxin protein family
105
, and it plays a regulating role in infections and
inflammations. CRP can bind to Fcγ receptor I and II, with an allele-specific binding to
FcγRIIa
106
. Studies on P. falciparum malaria show a relation between high CRP
concentrations and parasite density and severity of the P. falciparum infection
107-110
.
Recently, a three allelic single nucleotide polymorphism in the promoter of CRP (-286
C>T>A) were strongly associated with the plasma concentration of CRP, pre-dominantly
in patients with coronary heart disease (CHD) 111. We are interested in analysing the CRP
levels in Fulani and the sympatric ethnic groups and relate the CRP concentrations to the
genotypes of the tri-allelic polymorphism in the promoter region. Our hypothesis is that
higher concentrations of CRP are a contributing factor to malaria outcome, since CRP
may compete with IgG subclasses in the binding to the FcγIIa receptor. Hence, high CRP
levels may lead to a general activation of the immune system instead of a malaria specific
one. We will also correlate the levels of CRP with the levels of IgG subclasses and
FcγRIIa R131H genotypes. The study population is the same as in study 2.
In order to understand the importance of the different IgG subclass antibodies in
protection, and the impact the FcγRIIa R131H polymorphism has on protection, analyses
on the parasitic growth inhibitory capacity of the IgG subclasses on field isolates in intra-
20
and inter ethnic combination among Fulani and their sympatric neighbours will be done,
in relation to the donor’s FcγRIIa genotype and IgG subclass pattern. Ideally, monocytes
from the participating individuals will be genotyped and used in the assays as effector
cells. If this turns out to be too time consuming, or other practical difficulties makes it
impossible, monocytic cell lines, expressing the three different genotypes, will be used
instead. This study will be performed during the malaria transmission season in 2007 at a
study site still to be confirmed.
21
Acknowledgments
I am sincerely grateful to all the people that have contributed with invaluable help and
support during the work included in this thesis.
Without my supervisor I would be a lot more confused than I am today. Klavs, your
patience and good sense of humor have been really helpful and I can say that you are the
best supervisor any student could have!
Maggan, you are always ready to help me solve my problems and you always have time
for non-scientific chats. You will never be forgotten.
I would also like to thank all my colleagues, both seniors and juniors, the allergy-girls,
TB-boys and the Malaria-crew, for nice times spent in the lab and outside, especially
Halima, Manijeh and Petra.
Mamma och Pappa, tack för att ni lät mig gå mina egna vägar och att ni alltid tagit emot
mig när jag fallit och snabbt fått upp mig på fötter igen.
Stora Syster-yster, du har, trots att jag trillade in i ditt ordnade ensambarnsliv, alltid
stöttat mig och brytt dig om hur jag mår. Jag tycker att du är den bästa systern i världen,
bara så att du vet...
Min älskade Joakim, du gör min vardag till ett spännande äventyr! Du gör gräset grönare
och himlen blåare...
Mina vänner, tack för att ni tvingar mig från labbet ut i den vanliga världen med jämna
mellanrum!
22
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