Malaria is a potentially fatal, infectious blood-borne disease, caused by the protozoan parasite plasmodium, which is transmitted by female mosquitoes of the genus Anopheles. Plasmodium sporozoites are inoculated into the skin of the vertebrate host during mosquito probing or feeding (Rennenberg et al., 2010). Malaria is an enormous worldwide public health problem found predominantly in tropical and sub-tropical areas, such as Africa, Asia and parts of America, which have the favoured ecological conditions for parasite and vector survival (Stressman, 2010). The success of many anti-malarial drugs has been hindered by widespread resistance, which has provoked further research into the development of new drug combinations. Millions of lives are threatened every year; an effective vaccine is desperately needed (Taylor-Robinson, 1998).
A Brief History of Malaria
Through-out our history, malaria has plagued humans for thousands of years, causing high mortality rates. In ancient literature there are references to intermittent fevers, characteristic of malaria, as far back as 2700 BC in Chinese Literature. Descriptions by Hippocrates in the fourth and fifth centuries BC demonstrate an awareness of such symptoms of those living near stagnant water. Evidence suggests malaria arrived in Europe between the end of the last Ice Age, a period the vector and parasite were most likely absent due to unfavourable ecological conditions, and 500 BC (Sallares, 2004; Cox, 2010).
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As reviewed by Cox, before the discovery of bacteria in 1676 by Antoni van Leeuwenhoek, the consensus was swamp vapours caused malarial fevers. However once microorganisms were identified as possible causes for infections and the germ theory of infection proposed by Louis Pasteur and Robert Koch in 1878-1879, the aetiology was speculated. This escalated in 1880 when Charles Louis Alphonse Laveran discovered the protozoan parasite of malaria through the observation of crescent shaped leucocytes in the blood of malaria patients, which were never present in healthy individuals (Cox, 2010).
With the guidance of Patrick Manson, Ronald Ross demonstrated culicine mosquitoes transmitted the malarial parasite to birds in 1897, which prompted Italian scientists Bastianelli, Bignami and Grassi to provide evidence that human malaria was transmitted similarly but with anopheline mosquitoes (Shortt, 1951). After the discovery of the parasite and vector, research into the life cycle of Plasmodium followed; the pre-erythrocytic phase and erythrocytic phase were soon described and in 1982 the dormant hypnozoites were discovered in Plasmodium species vivax and ovale (Krotoski et al., 1982).
Despite the discovery of insecticides such as DDT and the development of anti-malarial drugs, malaria has failed to be eradicated and remains a serious public health problem. Current widespread resistance to anti-malarial drugs plagues doctors and scientists with difficulties and the complex life cycle of Plasmodium creates immense challenges to develop an effective vaccine (Taylor-Robinson, 1998).
Epidemiology of Malaria
Affecting millions of people annually, malaria is the most prevalent vector-borne infectious diseases, endemic in over one hundred countries across the world and responsible for the highest fatality rates (WHO, 2009; Friesen et al., 2010). According to the World Health Organisation, in 2008 there were an estimated 243 million cases and 863,000 deaths worldwide, the majority of which were present in sub-Saharan Africa. Worldwide, 85% of deaths were among children under five years of age (WHO, 2009).
In malaria endemic areas such as Africa, there is a high incidence of the sickle cell gene, indicating a selective advantage. Sickle cell disease is an inherited autosomal recessive blood disorder characterised by an abnormal form of haemoglobin in the blood, resulting in distorted crescent shaped erythrocytes. Heterozygous individual with the asymptomatic sickle cell trait (SCT), who have a mixture of normal and abnormal haemoglobin, have a distinct resistance against malaria with reduced chances of acquiring the disease (Allison, 1954; Willcox et al., 1983; Aidoo et al., 2002).
As discussed by Stressman, the distribution and prevalence of malaria is dependent upon specific ecological conditions required for vector and parasite survival. Temperature is a primary factor, important for both mosquito and parasite development. As altitude increases there is a reduction in temperature and therefore, little or no malaria is found above 1500m. Mosquitoes inhabit wet environments and thus precipitation is important (Lindsay & Martens, 1998; Stressman, 2010). The number of tourists visiting tropical and sub-tropical countries implicates malaria as one of the most common infections imported in the United Kingdom with 1500 to 2000 cases each year; the majority are caused by plasmodium falciparum species and are acquired through visiting friends or family in a malaria endemic country (Lalloo et al., 2007).
The Life Cycle of Plasmodium
Always on Time
Marked to Standard
Plasmodium is an apicomplexan parasite characterised by an apical complex containing secretory bound rhoptries and micronemes, which are important for motility and invasion (Singh et al., 2010). An actin-myosin motor called the glideosome powers the gliding motility of apicomplexan parasites (Frénal et al., 2010). There are around two hundred species of plasmodium, four of which are well known to cause human malaria: Plasmodium falciparum (P. falciparum), the deadliest form, Plasmodium vivax (P. vivax), Plasmodium ovale (P. ovale) and Plasmodium malariae (P. malariae). Zoonotic forms of Plasmodium can cause infection in humans such as Plasmodium knowlesi, which usually infects monkeys (Rich & Ayala, 2003; Kantele et al., 2008).
The plasmodium life cycle is highly complex with multiple stages involving morphological transformations. The life cycle is similar for all human species, with some variations. Malaria is usually transmitted through the female anopheles mosquitoes, although transmission can occur congenitally or through blood transfusions and organ transplants (Kitchen & Chiodini, 2006; Falade et al., 2007).
Inoculation and the pre-erythrocytic stage PIC FROM Prudêncio M et al, 2006
Motile sporozoites within saliva are injected into the dermis of the vertebrate host through the bite of a female anopheline mosquito infected with malaria (Rennenberg et al., 2010). Small numbers of the parasite are inoculated, despite the mosquito harbouring thousands of sporozoites (Vanderberg, 1977; Rosenberg et al., 1990). The sporozoites do not immediately enter the bloodstream and remain in the inoculation site for many minutes (Sidjanski & Vanderberg, 1997). Sporozoites have a slender morphology and are covered by a protein surface coat containing circumsporozoite protein (CSP) and thrombospondin-related anonymous protein (TRAP), which are significant for motility and invasion (Sultan et al, 1997). Sporozoites rapidly migrate to the liver, where they are arrested in the liver sinusoid by the interaction of sporozoite surface proteins, such as CSP and TRAP, with heparan sulphate proteoglycans (HSPGs) projecting from liver cells. The sporozoites are believed to traverse the sinusoidal endothelial cells by migrating through Kupffer cells, specialised macrophages which line the sinusoid. After crossing the space of Disse they are able to invade hepatocytes (Prudêncio et al., 2006). A sporozoite invades a number of hepatocytes before the formation of a parasitophorous vacuole is formed, an environment the parasite can reside in, in the final hepatocyte (Mota et al, 2001; Mota MM et al, 2002). This migratory to invasive sporozoite switch is poorly understood (Ejigiri & Sinnis, 2009). Attachment to the hepatocyte is suggested to be mediated by CSP, which binds to hepatocyte HSPGs (Pinzon-Ortiz et al., 2001). Research indicates hepatocyte invasion involves the secretion of microneme proteins TRAP and AMA-1 (Silvie et al., 2004). Inside the parasitophorous vacuole the sporozoite differentiates into an exoerythrocytic schizont, which contains 10,000-30,000 merozoites (Singh et al., 2007). Merozoites are released from the hepatocyte once the parasite has induced apoptosis of the cell and initiated detachment. Some merozoites will infect other hepatocytes whilst others leave the liver and circulate in the bloodstream where they can invade erythrocytes (Rennenberg et al., 2010).
P. vivax and p. ovale enter a dormant stage during the pre-erythrocytic stage of their life cycle, forming hypnozoites in the liver, which causes relapses months or even years later (Krotoski et al., 1982; WHO, 2010 International travel and health).
Figure 2. Merozoite invasion of erythrocyte
(Cowman & Crabb, 2006)
Merozoites are transported to the bloodstream in host cell derived vesicles called merosomes, which are important for immune evasion, preventing phagocytosis (Sturm et al., 2006).
Invasion of the erythrocyte is a multi-step process (Figure 2). The merozoite has a surface coat containing proteins such as MSP-1, which make initial contact with erythrocyte (A). Reorientation is necessary for invasion; the apical end must be adjacent to the erythrocyte surface to form a tight junction (B). This contact induces micronemes and rhoptries to release their contents, which induces the invagination of the erythrocyte; the merozoite is engulfed (Preiser et al., 2000). The merozoites shed their coat before infecting the erythrocyte, establishing a parasitophorous vacuole in the cell (Cowman & Crabb, 2006). Morphological changes occur and early trophozoites, called ring forms, develop. The degradation of haemoglobin from the erythrocyte along with other nutrients enables the trophozoites to mature and form a schizont, which asexually produces 16-32 merozoites. The subsequent rupturing of the erythrocyte releases the merozoites, which are then able to infect other erythrocytes. Some merozoites differentiate into male and female sexual forms called gametocytes, which can be taken up by mosquitoes. The erythrocytic cycle takes 48 hours in p. falciparum and p. vivax species (*****Ramasamy, 1998, immune evasion journal).
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When a mosquito takes a blood meal, ingested gametocytes enter the gut and immediately undergo gametogenesis (Billker et al., 1997). As reviewed by Kuehn & Pradel, this is initiated by the transition of vertebrate host to invertebrate vector environment. Together the 5°C reduction in temperature and presence of mosquito derived gametocyte activating factor, identified as xanthurenic acid in 1998, in the mosquito, induce gametogenesis in the mosquito gut (Billker et al., 1998; Garcia et al., 1998; Arai, 2001). Mature gametocytes undergo DNA replication three times before shedding eight motile flagella, called microgametocytes, in a process termed exflagellation. A microgametocyte fertilises a female gametocyte to form a diploid zygote, which undergoes meiosis and differentiates into a motile ookinete. The ookinete penetrates the midgut epithelium and upon arrival at the basement membrane transforms into an oocyst (Hirai et al., 2006). Thousands of sporozoites develop inside the oocyst and after around ten days are released into the hemolymph of the mosquito, where they can migrate to the salivary glands for transmission to humans (Cirimotich, 2010). The mechanism involved in the migration of the sporozoites to the salivary glands of the mosquito remains unknown; however it is believed that the sporozoites recognise the organ via specific receptor-ligand interactions (Ghosh & Jacobs-Lorena, 2009).
Pathology of malaria
Malaria has an incubation period of seven days or more; after which unspecific flu-like symptoms are initially presented in patients, caused by the erythrocytic stage of the plasmodium life cycle. The most common presentation is fever, occurring in conjunction with the erythrocytic cycle. Other symptoms include headache, muscular pain, fatigue, sweating, nausea and diarrhoea (Singh et al., 2010; WHO, 2010 International). Severe malaria, principally caused by p. falciparum, occurs if the parasite is allowed to multiply; according to the WHO one or more of the following clinical manifestations are usually present: "coma (cerebral malaria), metabolic acidosis, severe anaemia, hypoglycaemia, acute renal failure or acute pulmonary oedema." At this stage, the disease is often fatal if left untreated (WHO, 2010 treatment guidelines).
The pro-inflammatory immune response elicited against erythrocytic stages of infection is thought to contribute to the pathology. The cytokine TNF-Î± is suggested to cause many of the clinical symptoms (REF). Due to the critical role the spleen plays in the immune response to the erythrocytic phase of infection, splenomegaly is common in malaria endemic areas, as individuals are constantly exposed to malarial infection (REF).
Severe anaemia is a common feature due to the degradation of haemoglobin by parasites and the destruction of erythrocytes. This results in a limited oxygen supply to organs and tissues and can induce further complications (REF).
Trophozoite and schizont stage parasitised erythrocytes (PRBCs) are able to disappear from the circulation, called sequestration, which prolongs the infection as splenic destruction is avoided. This sequestration, confined to p. falciparum, occurs as PRBCs adhere to the endothelial cells of the microvasculature, termed cytoadherence, which is mediated by proteins inserted onto the surface of the PRBC after invasion (David et al., 1983). P. falciparum red blood cell membrane protein 1 (PfEMP-1), is a major protein mediating cytoadherence, which binds to host receptors such as CD-36, thrombospondin (TSP) and intercellular adhesion molecule 1 (ICAM-1) on endothelial cells (Baruch et al., 1996). PRBCs also adhere to healthy erythrocytes, forming clumps of red blood cells called rosettes (David et al., 1988; Chen et al., 2000). Cytoadherence, autoagglutination of PRBCs and rosette formation contribute to causing acute pathology in various organs due to an impaired oxygen supply, resulting from occlusion of the blood vessel(s) (Chen et al., 2000).
One of the most common and fatal complications of severe p. falciparum infection is cerebral malaria, which is usually characterised by comas (WHO, 2010 treatment). The pathogenesis of cerebral malaria is poorly defined, but is characterised by the sequestration of PRBCs to the endothelial cells of the cerebral blood vessels. The congestion and ultimate occlusion of cerebral microvasulature leads to haemorrhages, coma and often death (Johnson et al., 1993).
Metabolic acidosis is a common clinical manifestation leading to respiratory distress. The lactic acid produced by parasites
Sequestration causes organ dysfunction.
Prophylactic measures can reduce malaria transmission and include wearing protective clothing that covers the body, using mosquito repellent, screening houses and using insecticide treated bed nets (WHO, 2006). Insecticide-impregnated bed nets are particularly effective in the protection from malaria, reducing mortality and morbidity rates (Alonso et al, 1993). Indoor residual spraying of insecticide is another effective method used to kill adult mosquitoes, decreasing malaria transmission (Sharp et al, 2007). Methods such as these are currently in practice across the world to prevent malaria, although mosquito insecticide resistance is an issue. Various other measures, which destroy mosquito larvae and reduce the source, are also in place (WHO, 2006).
Chemoprophylaxis is crucial for individuals travelling to malaria endemic areas and should be used in conjunction with preventative measures. Although there is the possibility of side effects and complete protection is not provided, antimalarial drugs reduce the risk of fatal infection (WHO, 2010 International). Varieties of antimalarial drugs are available, each differ in dosing and are administered in accordance to the area visiting and individual circumstances such as pregnancy and age (Table 1).
Table 1. Drugs used in the prophylaxis of malaria (adapted from Castelli et al, 2010)
All malarious areas
250/100 mg daily orally
Paediatric tablets containing 62.5mg ATV and 25mg of PGN-hydrochloride. Tablet number/die depends on weight.
Not recommended for under 11kg
P. vivax, p. ovale, p. malariae and in chloroquine sensitive p. falciparum malaria areas.
300 mg base/weekly
All malarious areas
Controindicated under 8 years of age
Areas with mefloquine
250 mg base (1 tab)/week
Not recommended under 5 kg
Not recommended in the first trimester of pregnancy
P. vivax and p. ovale malaria
30 mg base daily
0.5 mg/kg base daily up to adult dose
Treatment of Malaria
Early diagnosis is essential to treat malaria effectively; light microscopy or alternatively rapid diagnostic tests are methods used to examine the blood for parasites. Treatment will depend upon a number of factors including the species of malaria, the degree of severity and individual circumstances (WHO, 2010, International Health).
The recommended treatments for uncomplicated p. falciparum malaria are arteminisin based combination therapies (ACTs), using derivatives of arteminisin such as artemether and artesunate in conjunction with other anti-malarial drugs. Recommended ACTs are: artemether-lumefantrine, artesunate-amodiaquine, artesunate-mefloquine and artesunate-sulfadoxine-pyrimethamine. Due to previous widespread resistance to monotherapies such as chloroquine, the WHO strongly recommends against using artemisinin and its derivatives as monotherapies to prevent the emergence of resistant parasites (WHO, 2010 Treatment).
In uncomplicated P. vivax, the dormant stage of the disease must be considered and therefore treatments must destroy hypnozoites to prevent relapses. Chloroquine with primaquine is recommended in areas without chloroquine resistance. In areas with resistance, ACTs are most effective, except artesunate-sulfadoxine-pyrimethamine (WHO, 2010 Treat).
Numerous studies have shown ACTs are safe, well tolerated and the most effective form of treatment in comparison to other anti-malarial drug combinations (Sowunmi et al., 2005; Thapa et al., 2007). The mechanism of ACTs remains ambiguous but research has shown parasites are rapidly cleared and transmission can be prevented, as shown by the resultant reduction of gametocytes and decreased infectivity of gametocytes to mosquitoes. Therefore the spread of resistance could be prevented through the use of ACTs (Price et al., 1996; Obonyo et al., 2003; Sowunmi et al., 2007).
Severe malaria caused by P. falciparum or P. vivax requires urgent diagnosis and treatment. The WHO recommends intravenous or intramuscular artesunate to treat adults. Recommended treatment for children is the same but intravenous or intramuscular quinine are also recommended (WHO, 2010 Treat). In children, both quinine and artesunate are effective with no significant differences. In comparison, artesunate treats severe malaria in adults much more effectively in contrast to quinine, as shown through reduced mortality rates (Dondorp et al., 2005). Artesunate eliminates parasites more rapidly and prevents further pathology through significantly reducing cytoadherence and rosetting of PRBCs in comparison to quinine (Udomsangpetch, 1996).
However, resistance has emerged along the border of Thailand and Cambodia; parasites have become less susceptible to the potent effects of artesunate, characterised by a slower clearance rate of parasites in some patients (Dondorp et al., 2009). Reports of resistance are a cause for concern as currently ACTs are the most effective form of treatment against malaria. Prevention of wide-spread resistance is critical and control measures must be put in place in areas of reduced susceptibility. Another disadvantage of this treatment is expense; artemisinin and its derivatives are more expensive compared to other anti-malarial drugs such as chloroquine and therefore endemic areas in developing countries often cannot afford the most effective form of treatment (Mutabingwa, 2005).
The development of other effective and cheaper anti-malarial drugs is crucial to prevent further fatalities due to resistance or expense.
The Immunological Response to Malaria
Our immune system is comprised of many specialised components, which work collectively to defend the body from harmful foreign bodies. Knowledge of the immune response elicited during malarial infections mainly comes from research using small animal models such as rodents; Plasmodium berghei and Plasmodium yoelii are species of rodent malaria commonly used in studies. Although an immune response is elicited against malaria, in many individuals the parasite is not effectively eliminated, allowing the parasite to multiply and induce clinical symptoms. Due to the morphological transformations occurring, a different group of immune components are stimulated at different stages of the life cycle (REF).
Following immunisation of irradiated sporozoites, sterile protective immunity against malaria can be induced in all models studied, including mice, monkeys and humans (Nussenzweig etÂ al., 1967; Edelman et al., 1993; Doolan & Hoffman, 2000). Rodent models have implicated antibodies as mediators of this protective immunity; Potocnjak et al. found that monoclonal antibodies against plasmodium berghei sporozoites neutralised the parasite, blocking hepatocyte invasion and protecting mice from subsequent infection (Potocnjak et al., 1980). However, as discussed by Good & Doolan, parasite elimination in humans by antibodies is unlikely, as high levels of pre-circulating specific antibody would be required at sporozoite inoculation to prevent hepatocyte infection (Good & Doolan, 1999). In addition, studies have demonstrated that antibodies do not mediate protection and instead cell mediated responses are involved (Belnoue et al., 2004).
Schofield et al. highlighted the significance of a group of T lymphocytes called cytotoxic CD8+ T cells and the cytokine interferon-gamma (IFN-Î³). Mice immunised with attenuated sporozoites were not protected from malarial infection when depleted of CD8+ T cells, and when IFN-Î³ was neutralised mice were no longer immune (Schofield et al, 1987). Other studies have reported similar conclusions, suggesting CD8+ T cells and IFN-Î³ are important mediators of an immune response against pre-erythrocytic stages, as reviewed by Doolan & Martinez-Alier (Doolan & Martinez-Alier, 2006). However little is known of the activation or mechanism of CD8+ T cells in malarial infection. Rodent models have suggested naÃ¯ve CD8+ T cells in the lymph nodes near the site of inoculation or in the liver become activated through coming into contact with antigen presenting cells called dendritic cells (DCs), which prime CD8+ T cells through cross presenting sporozoite antigens such as CSP. DCs internalise, process and present antigens in association with MHC class I molecules to CD8+ T cells. After specific interaction and co-stimulatory molecule signals, CD8+ T cells become activated and migrate to or stay in the liver, where they can eliminate parasitised hepatocytes (Jung et al, 2002; Amino et al., 2006). Usually CD8+ T cells kill via cytotoxic mechanisms; however immunity to P. berghei sporozoites in mice was found to be independent of cytotoxicity molecules fas and perforin, which suggests the cytokine secretion of CD8+ T cells, eliminates parasites (Renglli et al., 1997). Evidence also indicates IL-12 and natural killer (NK) cells are important for CD8+ T cells to carry out effector functions (Doolan & Hoffman, 1999).
CD4+ T cells are essential for CD8+ T cell effector responses and optimal functioning; IL-4 secreting CD4+ T cells are crucial (Carvalho et al., 2002; Doolan & Martinez-Alier, 2006). Furthermore, CD4+ T cells have anti-parasitic functions; CD4+ T cells clones derived from mice immunised with irradiated sporozoites, provided protection against sporozoite infection in malaria-naÃ¯ve mice (Tsuji et al., 1990). Belnoue et al. proved both CD4+ T cells and CD8+ T cells were important to eliminate pre-erythrocytic P. yoelii in mice; protection was mediated by IFN-Î³ production and dependent upon nitric oxide (NO) (Belnoue et al., 2004). The toxic effects of NO, suggest it is a critical mediator of effectively eliminating malaria.
The mechanisms remain undefined; studies have implicated many different immune components, which can singularly or collectively confer protection in rodent models, with parallel studies identifying different critical mediators.
Passive transfer studies provide evidence that antibodies are important in eliminating parasites; antibodies from malaria-immune individuals successfully treated individuals with malaria (Cohen S et al, 1961). Furthermore immunity in individuals living in malaria endemic areas may be mediated by high concentrations of antibody specific for a variety of erythrocyte stage parasitic antigens (Osier et al, 2008). This suggests antibodies play a role and are likely to target merozoite proteins such as MSP-1 to prevent erythrocyte invasion. Antibodies may also target parasitic ligands on the surface of PRBCs. Parasite development could be inhibited through these antibodies or antibodies may opsonise PRBCs for phagocytosis or lysis via the complement system (REF).
The spleen is known to be a primary site of cell mediated immune responses against erythrocytic parasites (REF). Research suggests that DCs internalise parasites, mature and migrate to the spleen, where they can present parasitic antigens in association with MHC class I molecules to naÃ¯ve CD4+ T cells. The subsequent differentiation of CD4+ T cells, through IL-12 secretion from DCs mediates protective immunity against erythrocytic malarial parasites. Th1 cells activate macrophages through the secretion of IFN-Î³ and Th2 cells assist B cell maturation for the production of antibodies (Abs) through IL-4, IL-6 and IL-10 secretion (Taylor-Robinson, 1998; REF).
The production of IL-12 is also believed to activate natural killer (NK) cells, which secrete IFN-gamma. Cytokine secretions from activated cells simulate a positive feedback loop, amplifying the immune response through promoting DC function, CD4+ T cells expansion and NK function.
After activation, macrophages secrete TNF-Î±, a mediator of inflammation, which is believed to participate in the pathogenesis of malaria. Macrophages destroy some PRBCs through phagocytosis and by the release of toxic free radicals such as NO.
Toll like receptors- innate immune system receptors, can recognise parasite
Parasite evasion and the importance of immunology
Plasmodium has evolved a variety of methods to evade detection by the immune system. Antigenic diversity and variation keeps the parasite one step ahead, preventing recognition by T cells and antibodies. Antibodies are unable to recognise intracellular parasites and erythrocytes have little to no MHC molecules, making elimination difficult. Cytoadherence of PRBCs to the endothelial lining and immunosuppression further prevent detection (Hisaeda et al., 2005).
Currently there is limited knowledge of the precise mechanisms that mediate an immune response at different stages of the parasite life cycle in humans. Findings from animal models can be extrapolated to humans although they will not be a precise reflection. Immunological findings and the mechanisms by which the parasite evades the immune system are important in the development of an effective vaccine against malaria.
A safe vaccine, which stimulates an effective immune response against the plasmodium parasite, is urgently needed. Knowledge of the immune components which fight against malarial infection is essential in vaccine development. There are currently a number of vaccine candidates which target a specific stage of the life cycle and some are discussed in this section.
Pre-erythrocytic vaccine candidates
The design of pre-erythrocytic vaccines is to prevent the release of merozoites into the bloodstream through targeting sporozoites or infected hepatocytes (Ballou et al., 2004). The most promising vaccine candidate to date, currently in phase three of clinical trials, is the RTS,S vaccine, which targets CSP, neutralising sporozoites and thus limiting hepatocyte infection (Alonso et al., 2004; Casares et al., 2010). The vaccine is composed of part of the CSP fused to hepatitis B surface antigen (HBsAg); both are expressed in yeast unfused with HBsAg (Bojang et al, 2005). The recombinant protein is then formulated with an adjuvant, which is essential to enhance the immune response (Casares et al., 2010).
Studies have shown the efficacy of RTS,S formulated with adjuvant system AS02, also called SBAS2; the vaccine protected six out of seven malaria-naÃ¯ve volunteers when challenged with P. falciparum infection (Stoute et al., 1997). Furthermore the vaccine is safe, well tolerated and highly immunogenic; Bojang et al found RTS,S/AS02 induced powerful humoral responses and T cell responses in semi-immune adults, which resulted in protection for over six months against natural p. falciparum infection (Bojang et al., 2001). A similar vaccine with a varied adjuvant, RTS,S/AS02A, has also been effective; in African children the vaccine reduced the prevalence of p. falciparum infection and remarkably reduced clinical disease over a six month period (Alonso et al, 2004). Subsequent studies emphasised this and one reported partial protection in African children lasting over 18 months with no signs of diminishing (Alonso et al, 2005). Phase 1 and 2 trials have shown RTS,S/AS02A is safe, well tolerated and highly immunogenic among semi-immune children in malaria endemic areas (Bojang, 2005; Sacarlal et al., 2008). Furthermore, phase 2a trials by Kester et al have implicated a different formulation, RTS,S/AS01B, which conferred protection in malaria-naÃ¯ve adults. This suggested further trial studies should be conducted to compare efficacy of RTS/AS01B with RTS/AS02A, to determine the most effective vaccine formula (Kester et al., 2009).
Other antigens of the erythrocytic stage include TRAP and liver stage antigens 1 and 3, which are further targets for vaccine developments (Girard et al., 2007).
Erythrocytic vaccine candidates
Mediators of erythrocytic invasion found on the surface of merozoites, such as AMA-1 and MSP-1, are key targets for blood stage vaccine developments. Blocking erythrocyte invasion through inducing anti-merozoite antibody responses could prevent infection and destruction of erythrocytes (Ballou et al., 2004; Singh et al., 2010).
AMA-1 is a protein expressed at sporozoite, hepatic and erythrocyte stages of malaria and is therefore an excellent target for the development of an effective vaccine. FMP2.1/AS02A is currently in phase 2 trials, composed of an AMA-1 based protein expressed in Escherichia coli and formulated in adjuvant AS02A (Polhemus et al., 2007; Thera et al., 2010). Studies have demonstrated the recombinant vaccine is safe, well tolerated and highly immunogenic, inducing humoral and cell mediated responses in malaria-naÃ¯ve adults and both children and adults living in malaria endemic areas (Polhemus et al., 2007; Thera et al., 2008; Lyke et al., 2009; Thera et al., 2010). AMA-1 based vaccines with different adjuvants are also being tested.
Other candidates include vaccines containing MSP. FMP1/AS02A contains a portion of MSP-1 and is safe, well tolerated and immunogenic in adults living in malaria endemic areas (Stoute et al., 2007). However in a phase 2b trial, the vaccine failed to provide protection against malaria in African children despite inducing humoral responses (Ogutu et al., 2009). Using animal models, Goodman et al have reported potential erythrocytic stage vaccines after designing vectored p. falciparum MSP-1 based vaccines, which induced antibody and T cell responses (Goodman et al., 2010).
Vaccines combining AMA-1 and MSP-1 are also potential candidates such as the PfCP-2.9 vaccine targeting P. falciparum (Li C et al, 2010). Further developments include other MSP molecules such as MSP-3; phase 1 trials have found MSP3-LSP vaccine to be safe and immunogenic in adults and children (Audran et al., 2005; Sirima et al., 2007; Lusingu et al., 2009).
Sexual stage vaccine candidates
Sexual stage vaccines are designed to target gametes, zygotes and ookinetes to block transmission through preventing the development of sporozoites in the mosquito vector. Trialing proves difficult as sexual stage vaccines protect communities from malaria and so assessing their impact in field studies is challenging. Zygote and ookinete major surface antigens P25 and P28 of P. falciparum (Pfs25 and Pfs28) and P. vivax (Pvs25 and Pvs28) are leading candidates (Carter R et al., 2000; Girard et al., 2007).
Pvs25H is a potential P. vivax vaccine candidate; the recombinant protein expressed in Saccharomyces cerevisiae with adjuvant Alhydrogel, has been shown to induce transmission blocking immunity in humans (Malkin et al., 2005). Wu et al have found that both Pfs25 and Pvs25 recombinant proteins formulated in adjuvant Montanide ISA 51 can induce transmission blocking antibodies. Despite causing unexpected local and systemic reactions, the study implicated that a vaccine is feasible, but further developments using Montanide ISA 51 are unlikely (Wu et al., 2008).
The aim of my education project is to develop an exciting new resource for As Level students, to increase knowledge of the plasmodium life cycle and the immune response elicited against the parasite. Having liaised with the Head of Biology of John Cleveland College, a school in Leicestershire, I have studied the As Level Biology curriculum; students learn of the life cycle of malaria, under a module called Human Health and Disease. Before creating my resource, intense study of this curriculum is important to ensure knowledge provided in the resource is beneficial for the students. Currently, I plan to create a life size board game, resembling the plasmodium life cycle, which includes information cards. The students will resemble the "pawns" and throw a giant dice to move through the game. Critiquing the resource is important and therefore the same assessment questions will be given prior and after use. Assessment results will define the effectiveness of the resource and differences between male and female results will also be looked at. The resource will combine the three learning preferences together; kinaesthetic, visual and auditory to enhance learning, creating a fun but informational experience for sixth form students.