The development of a vaccine against Plasmodium falciparum

The development of a vaccine

against Plasmodium falciparum
Student number: 06901380
Malaria due to infection with Plasmodium falciparum is a leading cause of
death in children under the age of 5 years. Discuss why there is not
a useful vaccine against malaria when other diseases that used to be
common in this age group (such as measles and polio) have been
successfully controlled by vaccination.
It is estimated that malaria is the ninth leading cause of death in low income
countries, causing between one and three million deaths per annum worldwide
(Guerra et al., 2008, Snow et al., 2005). The majority of these deaths are caused by
Plasmodium falciparum, the most deadly of the four species of malaria affecting
humans (Guerra et al., 2008). Current research efforts are targeted at vaccine
prevention of Plasmodium falciparum malaria. It is not possible to create a vaccine
that mimics natural immunity, as with vaccines for polio and measles, since the
inherent immune response in an immunocompetent host does not provide sterile
immunity. This is a consequence of the complex lifecycle of the malaria parasite
and a number of immune evasion techniques the parasite employs. This essay will
briefly discuss the lifecycle of Plasmodium falciparum, the normal immune
response to infection and the immune evasion techniques the parasite employs.
The potential vaccine targets will then be discussed with reference to the efficacy
of vaccines currently in development and trials.
The life-cycle of Plasmodium falciparum
The lifecycle of the protozoan, Plasmodium falciparum, involves transmission from
human to human via the Anopheles mosquito vector, as illustrated in figure 1. The
salivary glands of an infected Anopheles mosquito contain a large number of
sporozoites, the infective form of the Plasmodium parasite. As the Anopheles
initiates feeding it injects a small amount of saliva containing sporozoites into the
wound. These are rapidly taken up by the liver where they multiply inside
hepatocytes as merozoites (Kumar and Clark, 2002). These two phases are
collectively known as the pre-erythrocytic stage. After several days the hepatocytes
rupture releasing the merozoites into the blood. The merozoites then infect
erythrocytes and replicate in a phase termed the asexual blood-stage. In this stage
the merozoites differentiate first into a trophozoite and then a schizont. At the
schizont stage the Plasmodium undergoes DNA replication followed by cellular
segmentation to form between 8 and 24 new merozoites (Kumar and Clark, 2002).
Eventually the erythrocyte ruptures and the merozoites are released to infect
further erythrocytes. This cycle is known as erythrocytic schizogony and is
responsible for many of the clinical features of malaria. A minority of merozoites
differentiate into gametocytes, the sexual form of the parasite, which are not
released from the erythrocytes but remain intracellular until they are taken up by a
feeding mosquito (Kumar and Clark, 2002). If the gametocytes are successful in
infecting a mosquito they differentiate into gametes which then form zygotes.
These then multiply again, changing from zygote to ookinete, oocyst and then
sporozoites ready for infection of the next human host (Hall et al., 2005).

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Student no.: 06901380

The development of a vaccine against Plasmodium falciparum

The immune response and immune
evasion techniques
The immune response to malaria has a
complexity
which
complements
the
parasites complex lifecycle. Different
stages of the parasite present different
antigens, requiring multiple different
immune responses to eradicate the
infection. However, the malaria protozoan
has a number of highly effective methods
of evading the bodies immune response
(Schofield and Tachado, 1996). Even in
areas where malaria is endemic, immunity
is slowly acquired, unstable and is usually
incomplete (Reeder and Brown, 1996).

Figure 1. The lifecycle of malaria.

Sporozoites are injected via the
Anopheles mosquito and travel to the
liver (A). Replication and release of
merozoites then occurs in hepatocytes
(B). Released merozoites repeatedly
infect erythrocytes (C) by replication
and haemolysis. Some merozoites
differentiate into gametocytes which
are taken up the feeding of a second
Anopheles (D). Finally fertilization and
production of infective sporozoites
occurs within the mosquito (E). Figure
adapted from Richie and Saul (2002).

Identified immune responses against the

pre-erythrocytic
stage
include
the
development of antibodies against the
circumsporozoite protein which block
hepatocyte
invasion
of
sporozoites
(Holder,
1994).
If
the
sporozoites
successfully
invade
hepatocytes
a
cytotoxic T cell response is triggered by
parasitic
protein
derived
peptides
presented on the surface of the infected
hepatocytes (Hill et al., 1992).

The most significant immune response is

targeted at the asexual blood-stage
parasites. Antibodies against surface
proteins of the merozoites which can
prevent erythrocyte invasion have been
demonstrated (Holder, 1996). These
surface antigens include apical membrane
antigen 1 (AMA-1) and merozoites surface
antigens 1 and 2 (MSA-1 and MSA-2).
Once inside the erythrocyte the merozoite
(Richie and Saul, 2002)
is
initially
shielded
from
immune
surveillance mechanisms. However, as the
parasite develops inside the erythrocyte changes occur in the erythrocyte
membrane triggering its sequestration and destruction within the spleen (Foley
and Tilley, 1995). The developing parasite is able to prevent this destruction by
causing adherence of the erythrocyte to the postcapillary venular endothelium
(Brown and Rogerson, 1996, Gruarin et al., 2001). The occurrence of this
sequestration within the brain is the likely pathological mechanism underlying the
development of cerebral malaria (Aikawa et al., 1990). Cytoadhesion is achieved
by the insertion of parasitic adhesion molecules into the erythrocyte cell
membrane such as Plasmodium falciparum erythrocyte membrane protein 1
(PfEMP1). These adhesion molecules also act as foreign antigens with antibodies
developed against them causing opsonisation of the infected erythrocyte and
prevention of cytoadhesion (Reeder and Brown, 1996).

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The development of a vaccine against Plasmodium falciparum

The genome of the parasite has evolved to maximise the array of immunogenic
proteins through two distinct mechanisms; antigenic variation and antigenic
diversity. Antigenic variation is the ability of a single clonal parasite population to
alter the antigens presented to the immune system. The parasite exhibits
polymorphisms in many immunogenic proteins. For example a monoclonal parasite
population contains around 40 alleles for the surface antigen PfEMP1 (Saul, 1999).
Switching of antigenic phenotype occurs via a stochastic process at rates
comparable with the time taken to generate a high-titre antibody response (Saul,
1999). High rates of antigenic phenotype switching may allow immune mediated
negative selection pressures to permit replication of parasites which subsequently
express unrecognised antigens and thereby determine this rate of switching
(Reeder and Brown, 1996). More explicitly; the rate at which novel antigens are
displayed by the parasite may be determined by the rate at which the immune
system responds to the previously displayed antigens (Saul, 1999). Antigen
variation permits chronic infection with successive generations of parasites
unimpeded by the antibodies generated to combat their predecessors.
By contrast with antigenic variation, antigenic diversity is defined as the
expression of different alleles in different Plasmodium populations. A number of
different forms of the MSA-1 antigen have been identified in Plasmodium
falciparum and their corresponding genes sequenced (Kemp, 1992). These genes
(shown in figure 2) show a level of amino acid sequence homology as low as 10% in
some regions (Tanabe et al., 1987). Mechanisms by which intragenic
recombination occurs at the meiosis stage of parasite reproduction have been
suggested (Kemp, 1992) and may provide a method to facilitate the transformation
of the antigenic repertoire over time. The MSA-2 gene has also been reported to
have a number of different alleles (Kemp, 1992).
Currently it is unclear how natural immunity to malaria develops. The primary
controversy is whether immunity is achieved by generating response to a finite
number of antigenic variations or by generating a response to a single poorly
immunogenic epitope such as a conserved domain of PfEMP (Reeder and Brown,
1996). If immunity is developed through recognition of the diverse repertoire of
antigens, a polyclonal reservoir of responding B cells would be required. If
immunity is conferred by response to a single epitope it may be possible to induce
this response through vaccination with similar epitope of increased immungenicity.
Alternatively if immunity is achieved by developing a polyclonal reservoir of
Figure 2. A schematic of MSA-1
genes
from
Plasmodium
falciparum. Regions 1 and 17 are
involved in membrane transport
and attachment and are highly
conserved.
The
remaining
variable
and
semiconserved
regions are combined in various
ways
providing
antigenic
diversity. Figure adapted from
Kemp (1992)

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The development of a vaccine against Plasmodium falciparum

responding B cells then a vaccine may need to present multiple immune targets,
thus replicating this polyclonal response.
Vaccine targets
As the antigens presented to the immune system are different at each stage of the
malaria life cycle, current vaccines are designed only to target a single parasite
stage. Pre-erythrocytic stage vaccines target surface antigens on the merozoites in
order to prevent hepatocyte invasion or trigger the destruction of parasites within
infected hepatocytes via T-cell recognition of surface antigens. However these
vaccines offer no protection against parasites which emerge from the preerythrocytic stage and are subsequently able to undergo asexual replication
unimpeded (Cheng et al., 1997). Asexual stage vaccines either target merozoite
surface antigens and prevent erythrocyte invasion or infect erythrocyte surface
molecules to enhance destruction of infected cells. A final type of vaccine is aimed
at preventing sporogenic development within the mosquito vector via the
production of antibodies against the mosquito stages of the parasite which would
be transmitted to the mosquito with the gametocytes during feeding. These are
termed transmission blocking vaccines.
Pre-erythrocytic stage vaccines
Sterile immunity to Plasmodium falciparum has been achieved by exposure to
around 1000-3000 mosquitoes containing irradiated sporozoites (Hoffman et al.,
2002). However this method of vaccination is highly impractical and the current
focus is directed towards recombinant and peptide vaccines (Targett, 2005). There
a currently a wide range of vaccine technologies under investigation including the
use of plasmid DNA, virosomes and synthetic polypeptides but only one construct
has shown significant promise in clinical trials, the RTS,S vaccine (Targett, 2005).
This is perhaps the most promising malaria vaccine to date. The RTS,S vaccine is a
recombinant protein comprised of the Plasmodium falciparum circumsporozoite
protein and the hepatitis B surface antigen molecule. It has shown considerable
promise in clinical trials including significant protection against infection and
clinical disease in children (Bojang et al., 2001). A phase II randomised control
trial in Mozambique demonstrated a 58% decrease in severe malaria episodes in
children six months after vaccination with RTS,S (Alonso et al., 2004).
Asexual stage vaccines
Most potential vaccines against the blood stage of malaria target molecules which
are involved with the invasion of erythrocytes. Unfortunately these invasion
pathways are redundant in Plasmodium falciparum requiring blockade of multiple
targets simultaneously. Some promising results have been demonstrated in
vaccines targeting MSA-1, MSA-2 and MSA-3 (Druilhe et al., 2005, Genton et al.,
2002). These results are, however, strain specific as a result of the antigenic
diversity of these targets. Other vaccine targets include the parasite molecules on
the erythrocyte surface. As these molecules express high levels of antigenic
variation, vaccine development will be challenging. However one surface antigen
VAR2CSA is responsible for erythrocyte sequestration in the placenta and the
cause of severe malaria sequelae in pregnancy (Duffy, 2007). This provides the
possibility of developing a vaccine beneficial in pregnancy (Duffy, 2007).
Transmission blocking vaccines
These vaccines would not provide protection for malaria to the recipient but if
successfully developed could form part of a malaria eradication program. Mosquito
stage antigens are not exposed to the human immune system and therefore have

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The development of a vaccine against Plasmodium falciparum

not developed a high level of antigenic diversity. However some identified
gametocyte antigens have been found to have variants and some are strain specific
(Kaslow, 1993). Nevertheless animal models of transmission blocking vaccines
have shown some success (Saul, 2007).
Combination vaccines
It is likely that combination vaccines will offer the highest level of protection
against malaria. The combination of a pre-erythrocytic vaccine with an asexual
stage vaccine will prevent the problem of parasitic break through that occurs when
a pre-erythrocytic vaccine is used alone. Incorporation of a transmission blocking
vaccine may reduce the probability of emergence of vaccine resistant clones by
limiting malaria spread from vaccinated individuals.
Combination of vaccines which target the same parasite stage through different
antigens is also likely to have beneficial outcomes: If the individual component
vaccines fail to provide sterile immunity such a vaccine may have increased
potency. The existence of a variety of HLA (human leukocyte antigen) types within
the human population result in varying responsiveness to current vaccines (Nardin
et al., 2000), combination of different target vaccines will increase the chance of
generating immunity. A combination vaccine of this type is would also reduce the
probability of vaccine resistance emerging.
Although the benefits of combination vaccines appear evident the cost of vaccine
production rises rapidly with increased complexity and may prove to render such
vaccines impractical. For this reason optimisation of individual vaccines should be
the principal consideration of current research.
Conclusion
Although an effective and practical malaria vaccine appears to be close many
obstacles remain. It is only through an understanding of the molecular and genetic
mechanism that underpin the immune evasion of the malaria parasite and the
human host interaction with these mechanisms that we can hope to develop such a
vaccine. It is likely that a combination vaccine, targeting different parasite stages,
will provide the greatest level of protection against clinical malaria and
development of vaccine resistant malaria.

Word count: 1993

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The development of a vaccine against Plasmodium falciparum

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Student no.: 06901380