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MSc Dissertation

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Introduction
Malaria is the most important parasitic disease of man. There are 300-500 million new cases and 1.5-3 million deaths annually. This makes it about 3,000 lives lost every day
(1).

Plasmodium falciparum is the major contributor

in the above statistics. It causes the most devastating symptoms of the disease and due to its epidemiological and clinical significance it has been extensively studied. The P. falciparum genome has been mapped
(17)

and is

available at PlasmoDB (http://plasmodb.org/restricted/GridddPf.shtml). The parasite causes its symptoms during its multiplication in the red blood cells. The cycles of replication and RBC destruction go on until the host becomes immuno-competent to ward off the infection. Chemotherapeutic prophylaxis and treatments combined with chemical prophylaxis and other vector eradication strategies have proven effective in control of the disease in recent years, (117) but the drug resistant strains mortality and morbidity levels constant. Human hosts react to the antigens present on the surface of the parasite. As the blood stage of the life cycle is the longest in terms of exposure to the immune cells, recent research is putting a lot of emphasis on the merozoite stage to find a cure in the form of both drugs and vaccines. Among the antigens available for vaccine development, merozoite surface proteins (MSPs) are considered to be the most important blood stage antigens.
(9) (49)

have emerged keeping the

Several clinical trials have been performed to assess the usefulness of vaccine preparations containing one or more merozoite surface proteins with mixed results.
(90, 91, 96, 105)

Many other strategies have been proposed to

further develop the efficacy of the vaccines (83, 84) and MSPs have emerged as prime vaccine candidate for an ideal malarial vaccine.

Plasmodium Surface Proteins and their Role in the life cycle

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The study of electron microscopic structure of different stages of P. falciparum has given a good insight into the pathological processes which take place during the course of infection
(2).

Recently, three-dimensional (3-D)

organization of the merozoite, ring, trophozoite and schizont stages of the parasite has been determined (figure-1). These studies have revealed new information about the pathogenesis and the involvement of different parasitic organelles in invasion and host-immune evasion (3).

Fig. 1: P. falciparum Merozoite: 3 dimensional organization. Inset: relative sizes of merozoite and red blood cells. Source: L. H. Bannister et al. 2000 Parasitology today.16 (10) 427-433

One of the major steps in understanding the parasite is the discovery of surface proteins at different stages of its life cycle. Circumsporozoit proteins (CSP) and Merozoit surface proteins (MSP) are the most extensively studied among them. Although much more information is required to have a definite idea of their function in host invasion, but vital clues have been found which have helped in ascertaining previously unknown processes. CSPs have been found to contain an amino acid sequence thought to be involved in mediating recognition of sulphated polysaccharides on the surface of a liver cell, whereas merozoite surface protein-1 may be involved in the initial recognition of red blood cells; this protein undergoes a complex series of modifications in the time between its synthesis as a precursor molecule and successful

Lutaf Islam

MSc Dissertation
(19, 20, 21)

3 Other merozoite proteins located at the apical

erythrocyte invasion. proteins (4).

end of the parasite have been identified as erythrocyte or reticulocyte binding

Life cycle inside the human host Malaria is caused by the bite of female anopheline mosquitoes. The sporozoite form of Plasmodium enters the blood stream and migrates to the liver. This migration can be slowed down because of the acquired immunity conferred in previous infections (10). The amazing immune evasion mechanism of the parasite kicks in when it comes in contact with Kupffer cells in the liver. The Circumsporozoit protein (CSP) and thrombospondin-related-adhesiveprotein (TRAP) present on the surface of the sporozoites interact with the proteoglycans on the surface of Kupffer cells, invade them and stay inside a vacuole and eventually exit the cells unharmed(11). After this the sporozoites gain entry into the hepatocytes. In the liver cell, the parasite develops into a spherical, multinucleate liver-stage schizont which contains 2,000 to 40,000 uninucleate merozoites. This process of enormous amplification is called exoerythrocytic schizogony, and after 43 hours post-invasion, the asexually reproduced Merozoites are released into the blood-stream in their thousands.
(12)

The Merozoite Stage Merozoites have a dense surface coat of proteins most of which is the merozoite surface protein (MSP)-1, which it uses to make initial contact with a red cell. The exact manner in which the parasite attaches itself and then enters the erythrocyte is studied in P. knowlesi
(see figure 2). (16)

The different

organelles involved in this process are micronemes and rhoptries, which are present at the apical end of the merozoite. Rhoptry-associated protein (RAP1/2), membrane antigen (AMA-1), (14) and Rhop-1-3 complex, (15) interact

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with each other and the erythrocyte surface to facilitate the reorientation and entry.

Figure 2 : A schema showing the sequence of red cell invasion as seen in P. knowlesi merozoites: A–D. Merozoite maturation (A) and release (B) from the schizont stage, initial attachment (C) and reorientation with apical attachment (D). E– G. Details of merozoite invasion showing apical attachment and early rhoptry discharge (E) secretion of microneme and rhoptry contents and movement of the merozoite into the invasion pit (F) and finally the complete enclosure of the merozoite in the parasitophorous vacuole and exocytosis of the dense granules from the merozoite surface. (Source: Peter Preiser et al. Microbes and Infection. 2(12):1461-1477)

Merozoites use several receptors when they invade human red blood cells. Recently, a merozoite erythrocyte-binding protein, EBA-140, has been identified that specifically binds to glycophorin C on red blood cells.
(13)

EBA-

175 has also been identified as a protein very active in erythrocyte binding process via glycophorin A. After invasion of a red cell, the merozoite replicates to produce as many as 32 invasive merozoites. However, upon rupture of an infected host cell, only those merozoites that can quickly invade a suitable erythrocyte will survive.
(16)

These proteins which identify the

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glycophorins on erythrocytes are crucial in the survival strategy of the parasite. It is very likely that a balance is maintained in order to make the invasion progress steady but not too aggressive.

Merozoite Surface Proteins
A recent study has established that there are three distinct types of proteins active on the merozoite surface during the intraerythrocytic development cycle (IDC).
(18)

Two of these have been briefly mentioned above; namely rhoptry

and erythrocyte binding antigen families. The third major family categorized in this study is of Merozoit Surface Proteins (MSPs). The precursor to the major merozoite surface proteins was described first in 1984.
(19)

This precursor MSP-1 is present in the form of a complex of

fragments derived by proteolytic processing. Originally, it is a 190 k-Da precursor protein which is fixed at the parasite membrane via a glycosylphosphatidyl-inositol (GPI) anchor. This protein undergoes cleavage into four major fragments during a first step which takes place at the time of merozoite maturation, and into five fragments in a second processing step, which takes place right before the invasion of the host cell. The C-terminal processed product of MSP-1 interacts with the band-3 integral proteins on RBC surface. And experiments have shown a major band 3 binding site located within the 19-kDa domain of MSP-1.
(20)

This is further

reinforced by the fact that disrupting the 19-kDa C-terminal region of the MSP1 gene is lethal to blood-stage P. falciparum. (21) This 19 k-Da fragment is the only part of the complex carried inside the host cells. This fragment carries two Epidermal Growth factor (EGF) like domains, which are very immunogenic. (29) MSP-2 is a 46–53 k-Da integral membrane glycoprotein antigen of Plasmodium falciparum merozoites which was identified soon after MSP-1.
(22)

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It is subjected to significantly less processing then MSP-1 but has glycosylphosphatidylinositol (GPI) anchor.
(23)

It is believed to play important

role in the invasion process of erythrocyte stage. This was shown by in-vitro inhibition of erythrocyte invasion, by use of antibodies directed against the protein.
(27)

MSP-2 shows allelic dimorphism, consisting of two major allelic It is postulated that this

families (C27 family and the 3D7/IC-1 family), but this dimorphism is not related to the invasion process directly.
(28)

dimorphism may be involved in protein-protein interactions on merozoite surface, e.g., trafficking of other surface proteins. MSP-3 is a secreted polymorphic antigen associated with merozoites (SPAM) which was recently discovered. It is a highly polymorphic 48 k-Da antigen present on the merozoite surface
(24)

which is secreted on the parasite surface

during schizont stage and undergoes proteolytic processing. After the schizont ruptures, most of this processed protein is shed where as some smaller fragments stay on the membrane. Just like other proteins of the family, MSP-3 shows a considerable amount of antigenic diversity. due to tandem blocks of heptad repeats present in the gene.
(26) (25)

This is

There are

four allelic variants, and the general sequence of the protein is conserved in all the polymorphs. The main reason for the antigenic diversity of the protein is due to the alanine-heptad repeat polymorphisms. (25) MSP-4 has been identified recently, containing an N-terminal signal sequence and a hydrophobic C-terminal sequence in the protein with an EGF like domain.
(5)

The MSP-4 gene is present on chromosome 2 in tandem with
(41)

MSP-2 and MSP-5 genes.

The protein contains a GPI anchoring signal

near the C-terminus, an epidermal growth factor-like domain just next to the GPI attachment site, and a hydrophobic secretory signal motif at the Nterminal. There is very limited polymorphism present on the antigen and antibodies against it give protective immunity. This limited diversity in structure can be of great use in multi-component vaccine development. (48) MSP-5 is transcribed in asexual stages and is a 40 k-Da protein. It is found adjacent to MSP-2 and MSP-4 genes on chromosome 2. It is very similar to

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MSP-2 as both contain hydrophobic signal sequences, GPI anchors, and single EGF-like domain at their carboxyl termini. But the remainder of their protein coding regions are quite dissimilar. Immunofluorescence assays show that it is located on the merozoite surface. (41) MSP-6 is a precursor merozoite surface protein comprising of about 371 amino acids. After processing it releases a 36 k-Da fragment which is found covalently bound to MSP-1 fragments on the merozoite surface. After the second proteolytic cleavage in MSP-1, all the proteins on it are shed apart from the 19 k-Da fragment. In the discarded complex, the 36 k-Da fragment belongs to MSP-6. The sequence of this fragment is very similar to MSP-3 C terminal region. And it can also be specified as a secreted surface antigen.
(46)

No sequence diversity has been found on MSP-6 which suggests a possible role in immune evasion mechanism of the parasite similar to that suggested for MSP-1. (47) MSP-7 is described as approximately 135 amino acid polypeptide, which is processed in very similar fashion as MSP-6. A 22 k-Da fragment is found noncovalently attached to the MSP-1 complex which is shed from merozoite surface prior to invasion of erythrocytes. It is also thought that a similar proteolytic enzyme is responsible for the cleavage of both MSP-6 and MSP-7 precursor proteins
(51).

This 22 k-Da fragment was already known to be a part
(52)

of the MSP-1 complex shed from merozoite surface.

But now it is

established that it is derived from MSP-7 as a result of proteolytic processing and is attached to the MSP-1 complex in the same way is MSP-6 36 k-Da fragment. Evidence also suggests that this 22 k-Da fragment undergoes more processing and is the precursor of a 19 k-Da fragment found in the shed complex. (51) MSP-8 is located on chromosome 5. This protein has a predicted mass of about 69.4 k-Da. And like MSP-1 19k-Da fragment, it also carries two EGF like domains. There is also evidence that this protein undergoes same kind of proteolytic processing like MSP-1. The protein is present on the surface of the parasite during the asexual stage and is found on the merozoites prior to the

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erythrocyte invasion. Evidence also suggests that only smaller fragments are left on the merozoite during the ring stage. sequences with MSP-1 and 2.
(30)

The EGF like domains on the

protein are highly conserved and there is little homology in non-EGF
(30)

Protein sequence and structural studies

have shown that there are similarities in amino acid sequences of MSP-8 and MSP-1. This is further confirmed in in-vitro erythrocyte binding assays. The assays show that EGF like domains are less immunogenic and bind to erythrocytes just like MSP-1 EGF domains, where as N-terminal which is immunogenic does not play any role in the binding process. This suggests an evasion strategy as well as the role of MSP-8 in erythrocyte invasion. (31) Another protein p101 or acidic–basic repeat antigen (ABRA) is also located at the merozoite surface and in the parasitophorous vacuole.(42) A recent research has found that very similar orthologous proteins are present on other Plasmodium species and they have been classified as MSP-9. Hence it is suggested that P. falciparum ABRA be known as MSP-9 in future. highly conserved and binds to Band 3 receptors on erythrocytes. analysis has revealed other RBC binding sites on the protein. the near future. MSP-10, a predicted 80 k-Da protein (524 amino acids) has also been described recently. Just like MSP-1 and MSP-8, this protein is also present in all asexual stages of parasite life cycle and undergoes proteolytic cleavage. It has a GPI anchor and two EGF like domains, suggesting similar invasive function like that of MSP-1 and MSP-8. The EGF motif is highly conserved and antibodies for EGF motif are very specific. Maximum concentration of the protein is found at the merozoite apex which is highly suggestive of an active role in erythrocyte invasion. The proteolytic processing is also another common feature with MSP-1 and MSP-8. A 36 k-Da protein with C-terminal peptide is isolated with two EGF like domains. Although none of the fragments
(43)

This

protein is 101 k-Da in size. The cysteine rich N terminus of the protein is
(44)

There is

some evidence to suggest some protease like activity too. And in vitro
(45)

There is a

strong likelihood that we will see a more detailed picture of MSP-9 functions in

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of MSP-10 is believed to be carried inside. There is no evidence that this protein is present during the ring stage (post invasion). (32)

Functions of Merozoite Surface Proteins.
To date only 10 MSPs have been described; some are better characterized than the others. There can be more members of this family still not known, but we can safely assume that the structural similarities and the functional relationships between MSPs will be better understood through intensive research on the proteomics and functional genomics of the known proteins. The presence of particular common structural features like EGF like domains, GPI anchors and PEST sequences on certain MSPs indicate a collective function for the whole family of proteins. A comparative approach towards understanding the possible functions of these structures has given us useful insight into the nature of these proteins and their interactions.

Epidermal Growth Factor like Domains:
The Presence of EGF like motifs on many MSPs suggests activity on macromolecular level, such as proteolysis and other protein-protein interactions, including receptor binding.
(33)

The EGF motifs of 19 k-Da

fragment of MSP-1 interact with receptors on erythrocyte surface. We already have evidence suggesting a close interaction between Band 3 receptors on erythrocyte surface and MSP-1. process.
(20)

Recent studies on P. vivax MSP-1 have

provided evidence that the EGF motifs are directly involved in binding
(56)

Presence of homologous EGF domains on MSP-4, MSP-5 and

MSP-8 also indicates that this binding occurs at multiple points and all interactions might have some specific function individually or together.

Glycosylphosphatidylinositol (GPI) anchor:

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MSP-1, 2, 4, and 5 contain a GPI anchor site. The presence of this structure in P. falciparum indicates a variety of possible functions as described below. Possible Functions of GPI anchor: The GPI anchor is believed to take part in cell adhesion as well as enzymatic catalysis.
(34)

The free GPI lipids from Plasmodium spp. are found to be

bioactive and can elevate expression of host adhesion molecules, such as ICAM-1, V-CAM-1, and E-selectin in human umbilical vein endothelial cells. (37) The presence of GPI anchors in MSP molecules suggests many different possible functions for the whole protein family. GPI anchors are known to be involved in a variety of process, all of which can be of some importance in the blood stage plasmodia. As GPI lack the transmembrane domain, they are able to show lateral mobility.
(36)

This mobility of proteins on the merozoite

surface gives a distinct advantage to the parasite to freely attach and manoeuvre on RBC surface and is certainly of help in facilitating a quick entry before the immune recognition. There is also evidence suggesting a rapid proteolytic cleavage process, which helps in shedding the GPI linked proteins; another characteristic which is of great help in immune evasion. Retention of one GPI anchored MSP fragment (19 k-Da of MSP-1) suggests that it is used for other processes, like potocytosis
(35),

a form of endocytosis. Once the

merozoite is inside the host cell, it needs to change the RBC environment to grow and multiply. The GPI anchor is most likely to function in active transfer of molecules and ions across the parasite membrane during this stage. GPI anchor and Malaria Pathogenesis. The pathological symptoms of malaria may also be explained by the presence of the GPI anchors on the parasite surface.
(61)

In experimental studies it has

been established that GPI raises the levels of TNF, Interleukin 1 in the macrophages and regulates the glucose metabolism in adipocytes. The GPI anchor moiety of P. falciparum has been classified as highly toxic component of the parasite.
(58)

These cytokines cause high fevers and the glucose

metabolism regulation gives hypoglycaemic symptoms in laboratory animals.

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As mentioned above, GPI anchor causes the production of cytokines I-CAM1, V-CAM-1 and E-selecting.
(37)

This is accomplished by Protein Tyrosine
(59)

Kinase (PTK) and Protein Kinase C (PKC) signalling pathways.

The

expression of these cytokines promotes cell adhesion and lymphocyte migration. This starts a cascade of events leading to fever and clot formation in case of cerebral malaria. The insulin mimicking capability of GPI moieties
(60)

is responsible for the hypoglycaemia seen in patients.

(61)

So far it is

unclear as to which receptors the GPI anchors interact with and this still needs further thorough investigation. As far as the pathological significance goes, we can utilize the knowledge so far to study the anti-GPI antibodies to treat the symptoms of the disease or the disease itself. Recently, the first synthetic malaria GPI anchor was produced.
(62)

Further advances in this

direction will give us a better picture of Plasmodium GPI structures and this can be used to develop new drugs targeting the parasite in a better, more effective way. Possible Role for GPI anchors in Malaria Vaccines: So far, the GPI motifs of Plasmodium species have been tested in vaccines. The data suggests that some GPI anchors maybe useful in producing a better immune response when used with other candidate antigens, the cellular level.
(63)

while the

others may cause reduced antibody titres due to their bioactive functions at
(64)

This leaves us with another area where further

information on the GPI anchor motifs on MSPs can be of potential use in vaccine development.

PEST sequences and Proteolysis:
Another interesting find in recent years is the presence of PEST (enriched in proline, glutamic acid, serine, and threonine) sequences in MSP-1, MSP-2 and MSP-5 genes.
(38)

These PEST sequences of amino acids are part of a After the

protease, Calpain. There are PEST +ive sequences on the erythrocyte surface, like Band 3 integral proteins which attach to MSP-1.
(20)

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invasion, the RBC membrane in contact with the merozoite becomes devoid of Band 3 receptors.
(39)

This indicates that the MSP-1 fragment is responsible
(38)

for the cell adhesion reaction assisted by PEST sequence.

After the cell

adhesion, next step is the growth and multiplication of the parasite inside the erythrocyte. This is facilitated by bringing about changes in the permeability of host cell membrane. An increase in the influx of extracellular Ca2+ into infected erythrocytes is evident at this stage of parasite development, which is exclusively found in the parasite compartment. The Ca2+ is essential for the growth of rapidly multiplying parasite. (40) Direct Role of MSPs in Erythrocyte Invasion: The major role in invasion of the erythrocytes is accomplished by proteins secreted at the apical end of the merozoite and the organelles i.e., rhoptry, dense granules and micronemes. The initial attachment and the reorientation of the parasite and its subsequent entry take around 20 seconds
(13).

But the

initial attachment is accomplished by MSP-1. The fact that MSP-1 complex is formed and shed during this short time leads us to believe that apart from initial attachment there are more specialized functions for the whole MSP family which are yet not fully known. The interactions of the antigens on merozoite surface, with the RBC receptors are broadly specified as sialic acid dependent and non-sialic acid dependent pathways. It was initially thought that P. falciparum depends on sialic acid pathways to enter the erythrocytes. Erythrocyte binding antigen (EBA)-175 is known to bind with Glycophorin-A. Another similar protein EBA-140, binds with Glycophorin-C.
(13, 16)

Field At

isolates of P. falciparum exhibit sialic acid independent invasion too.

(78)

least one of the merozoite surface proteins, MSP-1 takes the sialic acid pathway. In laboratory conditions, MSP-1 binds freely to spectrin on the erythrocyte surface.
(79)

The fact that certain elements of MSPs are also

involved in erythrocyte binding and antibodies against EGF motifs have a blocking effect on the invasion process leads us to believe that this multifaceted approach is essential in the overall progress of the merozoites into the next stage.
(20, 56)

MSP-8 also attaches with the erythrocyte membrane by

high binding capacity structures in its sequence. The binding capacity is said

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to match or even exceed MSP-1 and there is a fair amount of proofs suggesting that MSP-8 takes the sialic acid route of invasion. affinity for Band 3 receptors on erythrocytes.
(31)

Recently

characterized MSP-9 (ABRA) protein is also observed to have particular
(44)

This indicates that MSPs

definitely have an active role in the invasion process. Exact manner in which this process works is still being investigated and the outcome of future research in this area will be of valuable use in understanding the invasion process. This will open up further possibilities in better drug and vaccine designs. Proteolytic Processing: Proteolytic processing of the proteins involved in the invasion has a fundamental role in almost all the known MSPs. The role of cysteine protease Calpain has been described above. It is now known that the same serine protease pfSUB-1 is active in the processing of two major surface proteins. P. falciparum apical membrane antigen-1 (pfAMA-1) and MSP-1 are shed from the merozoite surface as a result of proteolytic cleavage at the residues adjacent to GPI anchor motif.
(53)

Only such merozoites are successful in
(54)

invading the erythrocytes in which the MSP-1 complex has been shed.

The other major serine protease active during pre-erythrocytic stage is pfSUB2. It is also implicated to be a major player in the processing of MSP-1 complex.
(55)

The proteases involved in the MSP processing can be useful

targets for new drugs. (116)

Immune Evasion
Evading the immune system is another aspect of function and pathology related to the merozoite surface protein structure and processing during the invasion. A lot still remains unclear but we have evidence which suggests an active role of MSP molecules in evading the human immune response. For example, MSP-1 C- terminal elicits an antibody response which effectively inhibits the erythrocyte invasion. This is accomplished by blocking the second

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round of proteolytic processing which culminates in eventual shedding of the MSP-1 complex and parasite entry into the host cell.
(54, 47)

Antibodies Thus

produced against the amino terminal of the MHC-1 protein inhibit the binding of anti-MSP-1 (19 k-Da) antibodies to the C-terminal of the fragment.
(66)

different sequences on the MSP produce a conflicting immune response resulting in successful immune evasion by the parasite. MSP-2 has been implicated in another form of immune evasion. Experiments have shown that more than one merozoite can invade an erythrocyte which are linked via a bivalent antibody produced against MSP-2. in its highly variable sequence. The presence of variable blocks of sequences on MSP-1 and MSP-2 genes also indicates a possible immune evasion. The phenomenon of clone
(108) (114)

So MSP-2 is most probably

involved in promoting multiple invasions by using the immunogenic epitopes

fluctuation has been implicated in multi-strain infections.

As immunity to

the parasite is mainly based on strain-specific response, the appearance of diverse sub-populations due to genetic polymorphisms causes the failure of immune system to identify a definite set of antigens to act upon. The genetic diversity of merozoite surface proteins may have been a result of rapid evolution of the parasite in recent times. (112)

Genetics and Polymorphisms
All the plasmodia stay inside the host for a long time. They have intracellular and extracellular phases in a variety of environments and they are always exposed to the host immune system. In order to survive, like other parasites P. falciparum relies on its antigenic diversity and variation between strains, stages of the lifecycle and stages of infection. Among all the antigens, the MSPs show considerable amount of variations as described below. MSP-1 is the most polymorphic of all the proteins in the family. Initial studies on MSP-1 were done to classify the polymorphic regions on this gene. One

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research partitioned the MSP-1 gene into 17 compartments based on the degree of polymorphism or conservation exhibited. partitions, block 2 highly repetitive tripeptide domain. This block
(110)

Among these intragenic

locus on chromosome 3 shows a high polymorphic and shows

recombinations, a unique method not found in other blocks. In addition to this domain the rest of the gene shows extensive polymorphisms as well. (65, 110) MSP-2 is located on chromosome 2 and has a highly variable sequence. The single nucleotide polymorphisms found in this gene are more common in MSP-2 genes than any other on chromosome 2
(67).

This high level of

sequence variations has led to the division of MSP-2 protein into two distinct allelic families. Both the alleles have highly conserved N and C terminal regions, but vary greatly in the regions in between.
(28, 69)

Both the allelic

families have areas of high numbers of genetic repeats. Such repeats are implicated in generation of immunodominant parts of the antigens. A hypothesis suggests that gene duplication events, followed by point mutations and deletions have led to the diversity in MSP-2 gene. (111) MSPs-3, 4, 5 and 6 show very little variation in their structures.

(5, 25, 46, 48, 68)

The very limited variations in the structure of MSP-4 are mainly due to aminoacid deletions and substitutions. The gene shows very little polymorphism. and MSP-2. possible
(25)

MSP-4 does not show much variation its sequence as compared to MSP-1
(5)

The latest experiments conducted to look for sequence in multi-component vaccines. MSP-5, located on

variations found MSP-4 to be least polymorphic of all the MSPs, suggesting a use
(48)

chromosome 2 shows little sequence diversity among various geographical isolates of P. falciparum. It shows less sequence variations than MSP-4. MSP-5 might be the least polymorphic of all the MSPs.
(68)

MSP-6 is located

on Chromosome 11. The coding sequence consists of a single exon. It does not show much sequence diversity as compared to MSP-1 and MSP-2. (46) Chromosome 2 carries MSP-2, MSP-4 and MSP-5 in head to tail configuration. All three genes are expressed at the same stage of the lifecycle i.e. blood stage.
(57)

The unique location of these three genes with high

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similarity in their structures, EGF like domains and GPI anchors, suggests that these proteins evolved through the phenomenon of gene duplication. (68) The polymorphisms and allelic variations found in MSPs have been a result of selection pressure from the host immune system. It is very likely that children and adults are infected by different variants of the parasite. This explains the high mortality rate among the children. A study has shown high prevalence of a more lethal genotype (FC27) in children suffering from cerebral malaria. These children were found to have low antibody titre against the MSP-2. Whereas the patients from the same age group and same area, who were infected with 3D7 genotype had a better prognosis. They also had higher antibody titre against MSP-2.
(73)

In other studies these genetic variations

have been found to have an effect on the pathogenicity of the parasite. A study showed that parasites carrying the FC27-like genotype for MSP-2 were twice as likely to be found in symptomatic malaria cases as in asymptomatic controls. (72) As MSP-2 is one of the more extensively studied antigens, due to its significance as a vaccine candidate, we have enough data suggesting that the genetic diversity seen on this particular locus has been a result of positive selection pressure. Same can hold true for other groups of antigens being investigated. On the other hand the parasite seems to have conserved the loci which may be involved in immune evasion process. In general, it is observed that P. falciparum shows both allelic polymorphism as shown in MSP-1 and MSP-2 and antigenic variation. Antigenic variation is thought to be caused by gene switching.
(112)

Thus we can find sibling parasites in the same population

showing antigenic diversity; another problem which has to be addressed while developing vaccines.

Vaccine Development

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Although the primary strategy against malaria is still the use of drugs and chemical prophylaxis, but attempts are underway to bring in the first comprehensive antimalarial vaccine. The alarming increase in drug resistance in malaria parasites in last two decades means that development of an effective malaria vaccine is of vital importance. Blood stage vaccines are being tested at different levels all over the world. Almost all of them are using one or more Merozoite surface Proteins as the candidate antigens. As mentioned above, the merozoite is literally coated by MSPs prior to its entry into the erythrocytes. We have also seen that most of these proteins carry immunogenic sequences which make the blood stage vaccines as a realistic option. The first ever malaria vaccination was done by X-irradiation of mosquitoes and then letting them feed on human volunteers. One out of three volunteers acquired strain specific immunity against the disease. (71) This immunity was achieved only with 1000 mosquito bites and obviously has its own serious limitations. Vaccine development in post-genomic era is a whole new science. We have a lot more options to play with while developing vaccines. A historic glimpse into the attempts on malaria vaccine development will show us that the complexity of the protozoan parasites and their ever deceptive polymorphisms have never allowed us to have a chance against them on immunological level. The first attempt to develop a subunit malaria vaccine was done by using the Circumsporozoit Proteins. It was thought that challenging the parasite as soon as it enters the human host was easier. In-vivo immunological experiments on natural and synthetic CSPs were conducted with promising results.(7) But human volunteer studies did not show much potential for a vaccine.(8) Although the genetic sequence of P. falciparum has been published, but the experimental limitations are hindering the progress on understanding the morphology and function of CSPs. (9) As the functional analysis of the proteins

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is fundamental in determining their use in a vaccine, we will have to wait until technology allows us to understand the proteomics of plasmodium antigens in a better way. Merozoite Surface Proteins as Vaccine Candidate Antigens Among the merozoite surface proteins, MSP-1 is the strongest vaccine candidate antigen. It has been extensively studied and we know that antibodies against the 19 k-Da fragment elicit a reasonable antibody response. (29) Experimental immunity was achieved by recombinant MSP-1 19 k-Da C-terminal proteins carrying EGF motifs.
(74)

A strong T helper cell

response is needed to achieve good antibody titres against the merozoite antigens. The 19 k-Da fragment being more stable molecule among the rest, does not induce the Th cell response required. This stability is due to disulfide bonds which resist proteolysis which is integral process in MHC class II antigen processing pathways. (75) In comparison to the T helper cell response to 19 k-Da fragment, experiments have show that the 42 k-Da fragment of MSP-1 gives a better T helper cell response.
(76)

Only recently, immunogenicity of 42 k-Da fragment was tested

on monkeys and it showed promising results. The antibody response was better and it was declared safe for testing in phase I and II studies. Furthermore, there was some evidence that this protein may provide with cross strain immunity.
(77)

The role of MSP-1 in immune evasion and the fact

that many pathways are likely to lead to invasion, restricts the use of the antigen as a sole vaccine component. High levels of polymorphisms are found on MSP-2. It was thought that it was a sign of evasion strategy. But now there is evidence suggesting that the variant epitopes on the proteins can be divided into serogroups whish show extensive serological cross-reactivity. These epitopes are immunodominant, and so can be manipulated to produce a broad spectrum immune response.
(85)

Recombinant and synthetic peptide vaccines based on immunogenic loci

of MSP-2 have been proposed based on experimental data. Immunological

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studies conducted in malaria endemic areas have shown a strong relationship between acquired natural immunity and MSP-2. This immunity is allele family specific
(86)

and the highly polymorphic MSP-2 can get past the defence in

subsequent re-infections. Recombinant and Synthetic Peptide based Vaccines Synthetic and recombinant peptides from immunogenic regions of the antigens can be manipulated in-vitro. The antigenic epitopes from different proteins can be fused together in an expression vector. This technique was applied in preparation of multi-peptide based vaccine. Antigenic regions from MSP-1 and MSP-2 were isolated and incorporated with two immune stimulating epitopes (IL-1 and tetanus toxin) through an appropriate expression vector. The protein produced was administered in various laboratory animal species and results showed a remarkable antibody response against the individual peptides as well as the whole parasites.
(87)

Another chimeric vaccine containing the C-terminal epitope of 19 k-Da fragment of MSP-1 and C-terminal epitope of apical membrane antigen was shown to give high level of antibody production and these antibodies inhibit the parasite growth in vitro. As the domains are found conserved among different lines of the parasite, this response was measured to be of equal magnitude in various P. falciparum lines tested in vitro. It is thought that the disulfide bond dependent configuration on the antibodies produced cause the inhibitory effect.
(89)

A three component vaccine has been developed and
(91)

tested on human volunteers in Papua New Guinea.

It consists of

components from MSP-1, MSP-2 and Ring Infected Erythrocyte Surface Antigen (RESA). In earlier research, the Ring-infected Erythrocyte Surface Antigen (RESA), was found to be transferred from inside merozoites to the erythrocyte surface at about the time of merozoite invasion, and it was deduced that the presence of immunogenic repeat sequences are of great significance in vaccine development.(6) The vaccine was safe and induced good cellular response against the antigens. There was no marked increase in humoral response which is required to clear the parasites from the blood.
(91)

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MSP-3 is also known to be a part of antibody mediated cellular inhibition (ADCI) mechanism. Antigenicity and functional assays identified a 70-amino acid conserved domain of MSP3 C terminal half which produces good immune response in mice.
(84)

This domain can also be incorporated in a

multivalent vaccine. Previously, a hybrid protein construct composed of MSP3 and glutamine rich protein (GLURP) was shown to elicit a strong MSP-3 specific response in the experimental animals.
(88)

In individuals with acquired

immunity against P. falciparum, an antibody specific to MSP-3 has been discovered. This antibody has been found to be involved in ADCI, by activating monocytes against the merozoites. It is now established that the target region for the antibody is a non-polymorphic site on MSP-3. (24) Limited diversity of MSP-4 and MSP-5 make them ideal candidates for vaccines. The protein structure is surprisingly similar to certain motifs on MSP-2. We do not have much data to support that peptide based vaccine of these antigens can be feasible. But experimental data in mice suggests a great value of these antigens in vaccine development. MSP-4/5 of P. yeolii is the homologue of P. falciparum MSP-4 and MSP-5. The P. yeolii MSP-4/5 gene was expressed in E. coli and the recombinant protein was used to vaccinate mice. The mice showed a strong antibody response to the vaccine and were immune to subsequent P. yeolii infections.
(93, 82, 83) (95)

The DNA based

vaccines do present a better use for MSP-4 and MSP-5 in foreseeable future. The hurdle which is causing a lag in the rapid development of malaria vaccines seems to be our inability to determine the antigenecity of the candidate proteins. Although work is underway on the P. falciparum trascriptome, we still need better tools to understand the structural and functional characteristic of each protein. A high throughput expression system for all the known and predicted proteins on P. falciparum genome is needed.
(9)

Similarly, the peptides which become candidates for recombinant peptide

vaccines have to be processed by the right MHC molecules in order to produce adequate level of immunity. Keeping in mind that both MHC and

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antigen are highly polymorphic, designing vaccines is a complex and never ending challenge.

DNA Vaccine Technology and MSPs
DNA based vaccines should be able to reduce the problems we face in designing a comprehensive vaccine. DNA plasmid fragments encoding a protein can be expressed in host cells. Such proteins when expressed produce a Th1 cells mediated response, which is aimed at intracellular pathogens. But there is also evidence that these vaccines can produce a Th2 cells initiated humoral response resulting in production of antibodies.
(100)

In

case of complex parasites like plasmodium, A CD8+ cell response against the expressed protein processed by MHC class II can produce immunity. This was proposed as a strategy to develop a DNA vaccine against malaria.
(90)

In

case of intra-hepatocytic and intra-erythrocytic antigens in such a vaccine, the immune response initiated via MHC class I causes the cytotoxic T cell to cause the death and lysis of infected cells, but for the blood stage parasites a strong CD4+ T cell response is required to produce antibodies. And a strong antibody response has been shown to clear the asexual stage parasite from the experimental animals. So far the chimeric or peptide based vaccines have failed to produce a strong antibody response in the human volunteer trials.
(91)

We know that in order to remove the circulating parasites, antibody

dependent immunity must be produced. These antibodies can trigger phagocytosis. Another possible mechanism can be of opsonization by FcγRII and subsequent killing by cytokines released by monocytes. The antibodies implicated in merozoite binding and phagocytosis are IgG3 and IgG1 type. Whereas the IgG2 type antibodies are thought to be involved in cytotoxic response. (94) All the MSPs yet discovered have immunogenic structures which can be of use in a multi-epitope vaccine. As opposed to conventional vaccines, DNA vaccine components will need modifications to improve their immune

Lutaf Islam responses.

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(99)

22

The A+T rich composition of Plasmodium genome makes it
(102)

difficult to express in mammalian cells.

Additionally, the protein product

needs to be folded and presented to the immune system of the host in such a manner that both humoral and cellular arms can be manipulated sufficiently. Immunization of mice with DNA vaccines encoding the C and N termini of Plasmodium spp. MSP-1 provided partial protection against sporozoite challenge and resulted in boosting of antibody titres after challenge.
(80)

Another plasmid DNA vaccine encoding the C-terminal sequence of 42 k-Da fragment of MSP-1 was tested on monkeys. The results showed a high T cell response and when boosted by recombinant MSP-1 19k-Da protein the antibody levels were enhanced. Adding the sequence for an adjuvant in the plasmid also resulted in a better antibody response.
(92)

The experiments on

non-human primates and their results show that DNA vaccine consisting of one or more sequences from antigens coupled with a boost component can be designed for human testing. Taking it step further, multi-antigen vaccines based on DNA sequences were tried with successful CD8+ cell response. (81) MSP4/5 of P. chabaudi adami which is the murine homologue of P. falciparum MSP-4 and MSP-5 has been tested as a DNA vaccine on laboratory mice. The data shows that although the antibody production is dependent of the route of administration and the vector used, but the vaccine confers immunity against lethal infection especially if administered in a prime/boost sequence.
(93)

The DNA vaccine technology offers a potentially affordable solution for mass production of malaria vaccines. It is the design of the vaccine and its components which need further research and investigation. At this point in time no single vaccine candidate can be relied upon for complete protection against the parasite and our understanding of the complexities of parasite antigenic structure has indicated that a vaccine has to be based on many epitopes from each stage specific protein. As for the blood stage epitopes are concerned, just one component will not be good enough. Experiments of a multi-epitope DNA vaccine on rhesus monkeys have confirmed that MSP-1 19 k-Da component does not produce protective immunity. Although memory T-

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cell response was raised after repeated doses, the vaccine failed to stop the development of parasitemia.
(98)

This leads us to believe that in addition to

MSP-1, more immunogenic segments of MSP class of proteins must be added in DNA vaccines to enhance their effectiveness. A DNA vaccine consisting of sequences from seven different antigens from different stages of P. falciparum lifecycle was tested on human volunteers in 1998. The DNA construct was vectored in attenuated pox virus and administered to volunteers at different intervals. Just one of the volunteers developed immunity and the others showed immune responses of various degrees. The antibody response was not strong, but there was evidence of cell mediated immune response in all the volunteers.
(105)

This study has

underlined the importance of a proper design for a malaria vaccine. The reasons why it failed to produce desired results are still not fully understood, but we know that a number of strategies can be employed to boost the effectiveness of such preparations. Prime boost vaccine strategy has been tried for malaria vaccines with some very positive results. The problem of inadequate cell mediated immunity in general and humoral immunity in particular has been solved in other pathogens by the use of DNA vaccine in specially designed constructs and vectors. Vectors like fowlpox virus (FPV) and modified vaccinia virus Ankara strain (MVA) are ideal for DNA vaccine delivery. Still the DNA vaccines solely based on viral vectors are not always successful. The prime boost strategy consists of a “priming” dose of DNA vaccine which is boosted by the same or similar DNA construct via FPV or MVA. Results so far have shown excellent humoral and cellular response in case of Influenza virus and HIV in experimental animals. Similar results were obtained by prime boost vaccination in mice against sporozoite challenge. (97) Until now, the trials conducted on humans have shown that multi-epitope DNA construct vaccines can delay the intrahepatic phase of the parasite by almost 48 hours. A vaccine plasmid containing TRAP (found on sporozoite surface) sequence and other immunogenic epitopes, was administered to human

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volunteers followed by a boost by the same sequences through a different vector (MVA). The results obtained showed that hepatic stage to blood stage transition (parsitaemia) was delayed by up to 48 hours. (96) It is postulated that this time can be used to build up immunity against the released merozoites from liver by administering appropriate blood stage vaccines. As naturally acquired immunity in malaria endemic areas is against the blood stage antigens, we know that MSPs and other merozoite proteins will form the essential components of such vaccines. Only further experiments will tell whether this strategy is applicable or not, it may be the right direction for vaccine development for now. Multi-Stage DNA-based Malaria vaccine Operation (MuStDO) As the parasite has both intracellular and extracellular phases inside the human host, it is important that the multivalent vaccine induces both cell mediated and humoral immune responses. A collaborative effort called MultiStage DNA-based Malaria vaccine Operation (MuStDO) is underway. The MuStDO operation has proposed a multi-stage, multi-antigen malaria vaccine based on DNA Plasmid immunization technology. The proposed vaccine includes the sequences from sporozoite, merozoite and sexual stage antigens. The DNA based vaccine will be able to prime the naturally acquired immunity in populations. Apart from the liver stage components (CSP) the other major components are the MSPs. It is proposed that the 42 k-Da fragment of MSP-1, MSP-2, MSP-3 and MSP-5 should be included in the MuStDO vaccine and tested at phase I and II trials. (82) The MuStDO programme will consist of two components. The first component of the vaccine is specifically aimed at hepatic (Sporozoite) stage of the plasmodium lifecycle. A multivalent DNA vaccine (MuStDO 5) will be designed to induce CD8+ T cell response against the intracellular liver stage of the lifecycle. There are a number of strong candidate antigens from P. falciparum sporozoites which have been shown to induce a good immune response in DNA vaccine form.
(101)

It is expected that this component of MuStDO vaccine

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programme will successfully inhibit the release of infectious merozoites into the circulation. (82, 83) A Comprehensive Blood Stage Vaccine and Suggested Improvements The second component of the MuStDO programme will contain a number of blood stage antigens. The MSP antigens are aimed at producing the immunity to clear the parasitemia which will be much milder after the first phase of the vaccine. Other improvements suggested for MuStDO vaccine programme is addition of Codon optimization, use of adjuvants and Prime/Boost immunization. (82) Codon optimization means that parasite genes are not transcribed fully because the DNA fragment from P. falciparum is rich in A+T sequences which is very different from the human genome sequence. To solve this problem mammalian codons were inserted in the plasmids containing MSP-1 (42 kDa). The protein production in mice cell lines increased 10-100 folds and the vaccinated mice needed far less dosage of the vaccine to produce a better antibody response.
(102)

Similar technique can be employed in the design of

human vaccines for better results. The use of adjuvants in conjunction with DNA vaccine is a proven way of optimizing the immune response. Adjuvants can be chemical or genetic; i.e. a stimulatory gene. Genetic adjuvants are usually cytokine genes, which provide general immune stimulation and can also bias the immune response toward a Th1 or Th2 type. But it is debatable whether adjuvants like cytokine genes play any role in long term immunity.
(103)

It is suggested that the
(82)

Cytokine adjuvants should be added to the vaccine to optimize their effect. requirement.

A Th1 or Th2 bias can also be directed at different targets according to

The best enhancement in malaria vaccine protocol can be Prime/Boost immunization strategy. A multi-component plasmid based DNA vaccine containing both pre-erythrocyte and erythrocyte stage antigens was

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administered in rhesus monkeys and it was boosted by the same genes inserted in attenuated vaccinia virus. Results showed that the immunized animals resolved their parasitemia while control subjects did not. A strong IFNγ response was measured in the immunized animals. humans. Other factors which may increase the efficacy of the vaccine are the route of administration and delivery system, modifications in the vector DNA backbone, modifications in the insert sequence and targeted delivery etc. (118) The MuStDO vaccine programme will go through the trials and setting any hopes for a quick “miracle” vaccine is unrealistic. We have to bear in mind the fact that DNA vaccine technology is still in its infant stages. We have yet to see a successful gene based vaccine in humans.
(99) (104)

This showed

that prime/boost regimen coupled with cytokine adjuvants can be tested on

A lot needs to be learnt

about the safety and effectiveness of such preparations. With an anti-GM attitude among the educated public, we have to prove through time consuming testing that a malaria DNA vaccine if effective does not carry any side effects. Whether we can find a preventative vaccine breakthrough or not, we still have the option for working on therapeutic vaccines based on blood stage antigens. Prospects of therapeutic malaria vaccines and MSPs: GPI anchor motif is termed as the major malarial toxin involved in dramatic manifestation of the symptoms which include pyrexia, metabolic acidosis, hypoglycaemia, seizures, comma and cerebral oedema etc.
(59, 106)

The toxic

effect of the GPI moiety has been discussed above. We also know that a number of MSPs carry GPI anchors as their integral structural domains. As MSPs are expressed during the blood stage of the parasite life cycle, the GPI domains exhibit their toxicity at this point. The toxicity leads to release of proinflammatory cytokines
(59),

leading to a cascade of events which contribute to

the overall clinical picture of malaria. A vaccine targeting the GPI anchor would lead to suppressing of its pathological activity. A vaccine based on synthetic peptide structures derived from the GPI motif was tested on rodent

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malaria model. The vaccine substantially reduced malarial acidosis, pulmonary oedema, cerebral syndrome and fatality in the subjects
(107).

The

antibodies produced against the GPI anchor motifs are thought to block the binding of the GPI glycans to cell surface receptors. The receptors, not classified yet, may be the start point for cytokine production events.
(106)

Data

collected from malaria endemic areas confirms that anti-GPI antibodies are present in immune individuals. In addition to this, the asymptomatic individuals show high antibody levels against GPI protein. disease in near future. Emergence of more virulent strains: A possible hazard. A challenge that scientist will have to face is to develop a comprehensive vaccine against the blood stage of the parasite. As such vaccines will be aimed at blocking the toxic effects and growth rate of the parasite, thus providing a selection pressure which will result in evolution of a much more virulent parasite population. As parasites have been evolving under the selection pressure as a result of host immune response, they are capable of maintaining the populations even in the presence of incomplete vaccines. This was proven in a vaccine trial study done in Papua New Guinea in 2002. A combined vaccine consisting of peptides from MSP-1 and MSP-2 was administered to human volunteers. Although the infection by the vaccine allelic forms of parasites was markedly reduced in the vaccinated individuals, the morbidity level was the same owing to the infection by the other allelic form.
(70) (113)

A GPI anchor

vaccine, either DNA or peptide based, can bring us closer to eradicating the

Keeping in mind the immense nature of antigen diversity and

polymorphisms, designing a vaccine of such quality will not be easy. If compared to the projected effect of a vaccine against the pathogen infection, which will not result in increased virulence, we have to be very careful and precise in our approach towards designs, clinical trials and administration of blood stage vaccines.
(50)

So an ideal blood stage vaccine should include the

peptides representative of all the major allelic forms during the entire blood stage of the parasite. (70, 117) But with the available knowledge a safe approach

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would be to design a vaccine with both transmission blocking and infection blocking components. Further research in proteomics and recent advances in the area of recombinant vaccine technology has opened up many options, some of which can be of particular use in developing an anti malarial vaccine. Improved expression of parasite antigens in the cell lines and other expression vectors by use of high quality synthetic techniques
(109)

means that we are exploring

the antigens related to immunity in greater detail. The odds of a malaria vaccine causing increased virulence should be minimum in this post-genomic age. Conclusion: The functional studies on MSPs need further improvements. We know that they play an active role in the invasion of erythrocytes. Gradually a vague picture is emerging giving us hints of the structural and functional correlation between MSPs and the molecules of human host cells. New drug targets are being identified which can offer us more effective treatment in coming years.
(116)

The role of merozoite surface proteins in vaccine development is of primary significance. The experiments and trials so far conducted have shown that neither sporozoite antigen based vaccines nor sexual stage antigen based vaccines alone can give complete immunity against malarial infection. The blood stage of the life cycle of parasite is where the parasite can be most vulnerable to an immune attack for a number of reasons; a) owing to the immunogenic structures present on its surface b) The continued exposure of these antigens to the immune cells c) the ability of the host to ward off the challenge in endemic areas due to the immune clearance of the blood stage parasite d) the ability of the natural immune response to boost itself after consecutive infections. Keeping the above factors in mind, we can manipulate the antigen structures and genetic sequences in-vitro to generate more immunogenic vaccine components. The antigens from other stages of the lifecycle can be employed to reinforce the whole vaccine regimen. It seems

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very likely that within the next few years we will have transmission (host to vector) blocking
(96) (115)

and infection (vector to host to blood) blocking vaccines

available.

There are a number of other candidate antigens from the

merozoite stage which can be useful in a multi-epitope design of a blood stage vaccine. MSPs will be the primary components of such vaccine, but we will have to surmount the challenges of incomplete or inadequate immune responses, parasite evolution and polymorphisms and appropriate vaccine delivery in a huge population of prospective recipients.

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