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Malaria vaccines:where are we and where are we going?
Review
Malaria vaccines
Malaria vaccines: where are we and where are we going? Shirley A Moore, Emma GE Surgey, and Anthony M Cadwgan Malaria is still killing over one million people each year and its incidence is increasing. The need for an effective vaccine is greater than ever. A major difficulty with vaccine research is that the malaria parasite presents thousands of antigens to the human immune system that vary throughout its life cycle. Identifying those that may prove to be vaccine targets is complicated and time consuming. Most vaccines are targeted at individual stages of the malaria life cycle, although it is likely that only the development of a multistage vaccine will offer complete protection to both visitors to, and residents of, a malaria-endemic area. With the development of a successful vaccine other issues such as cost, distribution, education, and compliance will have to be addressed. This review describes some of the current vaccine candidates for immunising against malaria. Lancet Infect Dis 2002; 2: 737–43
Malaria is caused by infection with a single-cell parasite, plasmodium. Four Plasmodium spp cause malaria in human beings: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae. P falciparum is the most important because it accounts for the majority of infections and causes the most severe symptoms. Human beings are the intermediate (or vertebrate) hosts in the plasmodia life cycle and are usually infected with malaria when bitten by a female anopheles mosquito (the definitive host) carrying the parasite. Although its mode of transmission was known more than 100 years ago,1 malaria remains a serious public-health problem for roughly 40% of the world’s population, killing over one million people each year. Malaria need not be a fatal disease and death is avoided by prompt diagnosis and treatment; however, without this a child may die of acute malaria within 24 h. Transmission can be prevented by the use of bednets, repellents, and insecticides, but, according to WHO, the prevalence of malaria is on the rise. This increasing prevalence is mainly due to drug and insecticide resistance but also in part to social and economic change. The cost of malaria is especially important in developing countries, because the economic effects of preventing and treating malaria are a large burden on the health budget. Due to the lack of cost-effective treatments and the emergence of resistance, a malaria vaccine is likely to be crucial in reducing both the morbidity and the mortality of this disease.
Life cycle of the malaria parasite The malaria parasite life cycle can be divided into two distinct stages, one occurring in the intermediate host and another in the definitive host (figure 1). Plasmodia sporozoites are introduced into the subcutaneous tissue and, less frequently, the bloodstream of the intermediate host via the bite of an
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infected mosquito.2 After just minutes in the bloodstream the sporozoites reach the liver.3,4 It is not certain exactly how the sporozoites enter hepatocytes,1 although it has been suggested that hepatocyte receptors to the major surface protein on the sporozoite, the circumsporozoite protein (CSP), are involved.5 Once inside hepatocytes, the sporozoites develop into schizonts containing thousands of merozoites.1,2 In certain species of malaria the sporozoites may remain as latent hypnozoites in the liver before this stage.1 The merozoites are released into the blood flowing through the sinusoids of the liver after 6–15 days, depending on the Plasmodium spp. The circulating merozoites infect erythrocytes within a few seconds and begin the asexual cycle (figure 2).1 Within the erythrocyte, the parasite passes through the ring and trophozoite stages before the production of the erythrocytic schizont.6 About 20 merozoites are produced from each mature schizont. The merozoites are released after 48 h, with the destruction of the erythrocyte, and are then free to infect further cells.2 Symptoms coincide with the release of merozoites into the blood and the release of cytokines such as tumour necrosis factor (TNF) and interleukin 1 from macrophages in response to the rupture of erythrocytes.4,6,7 The asexual cycle continues until infection is controlled, either by the host’s immune response or chemotherapy, or until the host dies.1 Eventually some merozoites within the erythrocytes differentiate into immature gametocytes. For the sexual reproduction of plasmodia to take place, these gametocytes must be taken up into the alimentary tract of an anopheles mosquito. On biting an infected intermediate host the mosquito ingests blood containing the parasite and the normally asexually dividing bloodstream forms (merozoites) die. The gametocytes are stimulated and the sexual stage of the cycle then continues in the mosquito.1 The gametocytes are released from the ingested erythrocytes and transform into male and female gametes.8 The male form (microgamete) undergoes flagellation and fertilises the female form (macrogamete), forming a zygote. Within 24 h the zygote develops into a worm-like ookinete, which penetrates the midgut wall of the mosquito, forming an oocyst between the midgut epithelium and the basal lamina. Many sporozoites are then formed asexually within the oocyst, which, on reaching maturity, bursts open to release the sporozoites. These sporozoites migrate to the mosquito’s salivary glands where they can be transmitted through a bite into another intermediate host approximately 7–18 days after gametocyte ingestion.1 SAM and EGES are at the University of Aberdeen, Aberdeen, UK; and AMC is at the Aberdeen Royal Infirmary, Aberdeen. Correspondence: Dr Anthony M Cadwgan, Infection Unit, Aberdeen Royal Infirmary, Foresterhill, Aberdeen AB25 2ZN, UK. Tel +44 (0)1224 553705; email [email protected]
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Review
Malaria vaccines
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Figure 1. Life cycle of malaria. (1) Mosquito ingests erythrocytes infected with malaria gametocytes. (2) Gametocytes released from erythrocytes and transform into male and female gametes. (3) Zygote formed from the fertilisation of the female gamete by the male. (4) Zygote develops into ookinete and penetrates the midgut wall of the mosquito. (5) Ookinete differentiates into oocyst. (6) Oocyst ruptures releasing sporozoites that migrate to the mosquito’s salivary glands. (7) Sporozoites introduced into intermediate host through the bite of an infected mosquito. (8) Sporozoites migrate towards the liver. (9) Sporozoites invade hepatocytes. (10) Sporozoites develop into schizonts containing thousands of merozoites. (11) Alternatively, sporozoites may remain latent in the liver as hypnozoites. (12) Merozoites released into the blood to infect circulating erythrocytes. (13) The parasite passes through the ring stage. (14) Next comes the trophozoite stage. (15) Finally the erythrocytic schizont is produced. (16) Merozoites are released, with the destruction of the erythrocytes to begin the cycle again. (17) Alternatively, some merozoites differentiate into gametocytes.
Developing a malaria vaccine Plasmodia are large and complex organisms. Each infection presents thousands of antigens to the human immune system, which differ at each stage of the parasite’s life cycle. Plasmodia’s ability to adapt to the human immune system and misdirect or suppress it presents the main difficulty in finding a successful vaccine.6 Another difficulty is our poor understanding of the natural immune response to malaria, and, therefore, that which should be elicited by a vaccination programme. Multiple infections of malaria (different strains and/or different species) are also possible, so protection against one type does not necessarily protect against another.6 Until now vaccine development has targeted four different stages of the plasmodia life cycle: the sporozoite and liver stages (together known as the pre-erythrocytic stage), the merozoite, or erythrocytic, stage where the parasite invades or grows in the red blood cells, and the sexual stage occurring in the mosquito.9 There is also work under way to produce a vaccine that will contain a range of antigens from all stages of the plasmodium life cycle9 and adjuvants are being investigated to boost a vaccine’s effect. Most research into malaria-vaccine development is directed against P falciparum, the organism responsible for the highest rates of morbidity and mortality of all of the malaria parasites.1 Many people living in areas of high endemicity develop a degree of immunity to plasmodia. These people rarely show
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clinical evidence of infection because they are able to reduce circulating parasite numbers to within the limits tolerated by their immune system.1,10 However, this natural immunity is acquired only gradually after repeated infection11 and babies and young children are still at risk. According to WHO, 700 000 under-5s die from malaria each year. It can take more than 10 years to reach optimum (incomplete) protection, the time taken correlating inversely with the intensity of parasite transmission.12 This timescale supports the hypothesis that the antigens inducing protective immune responses are only weak inducers of immunity. Alternatively, plasmodium antigens may be very polymorphic (vary between strains) or may change with time within strains, therefore requiring acquired immunity to a large number of different epitopes for optimum protection.12 It is thought that children develop resistance to malaria more slowly than adults,13 although it is uncertain whether this slower development can be explained by differences in the immune response between children and adults, or by sufficient exposure to different antigens.12,13 Naturally acquired immunity is normally specific to one species of plasmodia and requires continual “boosting” by regular reinfection.1 Therefore, people who leave an endemic area are at risk of infection with other species of the malaria parasite against which they have no protection. They also risk clinical symptoms of malaria on return to their original
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area due to the loss of (natural) partial immunity. A vaccine targeted at antigens expressed during the erythrocytic stage would mimic the immunity acquired by persistent infection, associated with residence in an endemic area. Some merozoites would persist in causing symptoms but with reduced severity of illness.14 This type of vaccine would be suitable for immunising children living in a malariaendemic area. A vaccine targeting the erythrocytic stages of the parasite would be of little value to the so-called “malaria naive” traveller or newcomer to a malariaendemic area who would require complete protection.1 To achieve this level of protection, the vaccine must target the pre-erythrocytic sporozoite or liver-stage parasites, thus preventing merozoite production and release, the process responsible for clinical disease.4,6,7 A third type of vaccine is aimed at preventing Figure 2. P falciparum parasites in blood. sexual reproduction of plasmodia in the mosquito, therefore preventing the transmission of disease to another Pre-erythrocytic vaccines intermediate host. The “altruistic” transmission-blocking A vaccine that targets the pre-erythrocytic stages of plasmodia vaccines (TBVs) offer no protection from disease for the would be the only vaccine capable of preventing the clinical vaccine recipient, but rather prevent the spread of disease by manifestations of malaria. Such a vaccine would benefit those interrupting the plasmodia life cycle. Malaria is transmitted individuals who had not previously been exposed to the mainly within a few hundred metres from an infected parasite, and would therefore be at greater risk of severe human source; therefore, the use of TBVs within a morbidity and mortality. Due to the increase in travel to community would protect that community.15 TBVs target malaria-endemic areas, and the inconvenience of the current the antigens present on gametes, zygotes, or ookinetes. The prophylactic regimen, development of a pre-erythrocytic production of antibodies that are specific to such antigens is vaccine has proved popular with many researchers.16 The immune response needed to neutralise sporozoites is stimulated in human beings by vaccination with antigenic matter. These antibodies, freely circulating in the blood, are specific and different from that needed to destroy the parasite ingested by the mosquito in the blood meal, and cause in other stages. A successful vaccine would have to neutralise destruction of the developing parasite stages in the each individual sporozoite because each has the potential to become more than 30 000 merozoites.4 This is especially alimentary system of the mosquito.1,15 Immunity to an antigen from one stage of the difficult since the sporozoites only circulate in the blood plasmodium life cycle is confined to that stage11 so a stream for a few minutes. It is, therefore, necessary for a universally effective malaria vaccine would need to include sustained high antibody concentration to be present in the antigens from all stages of the parasite’s life cycle. Effective blood. Once sporozoites are inside hepatocytes they are more vaccine-induced immunity would also have to be faster protected from the body’s immune system and are harder to acting, longer lasting, and protect against more strains than destroy. To eliminate these liver-stage parasites, a cellular acquired immunity. A summary of the different possible immune response, involving both helper and cytotoxic T cells antigens used in candidate vaccines is shown in table 1. is necessary.17 CD8+ and CD4+ T cells recognise parasiteTable 1. Summary of different vaccine candidates and their action Stage of malaria life cycle Pre-erythrocytic stage Sporozoite
Liver schizont Blood stage Merozoite Infected erythrocytes Toxins Mosquito (sexual) stage Gametes Zygotes/ookinetes Blood meal
Target antigen
Immune response
Comments
Whole, irradiated sporozoite Circumsporozoite protein (CSP) derivatives
Humoral Humoral, cellular
Liver stage antigen 1 (LSA1)
Cellular
In-vitro culture very difficult Promising when attached to recombinant microorganisms with “booster” viruses Relatively uniform sequence throughout the different plasmodia strains
Merozoite surface protein 1 (MSP1) Apical membrane antigen 1 (AMA1) Serine repeat antigen (SERA) Glycosylphosphatidylinositol (GPI)
Humoral, cellular Humoral, cellular Humoral Humoral
Pfs230, Pfs48/45 P25, P28 Chitinase
Humoral Humoral Humoral
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Vaccines mimic natural immunity
Initial stages of research “Altruistic” vaccine candidates
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derived peptides presented by MHC class I and II molecules on the surface of plasmodia-infected hepatocytes leading to their destruction.3,4 There is evidence that protective immunity against this stage of malaria can be mediated in vitro through cytotoxic mechanisms targeted against these parasitic peptides.6 Early malaria-vaccine research examined the immune response to irradiated sporozoites. It was discovered that proteins expressed on the surface of the sporozoite induced an immune response giving protection against subsequent sporozoite challenge, preventing the progression of the parasite to the asexual blood stages.18 However, the use of irradiated sporozoites has not been widely pursued due to the difficulty in culturing plasmodia in vitro. Current research into the development of a pre-erythrocytic vaccine involves the use of the sporozoite surface proteins and the manipulation of the immune system to full advantage.
Shortly after the sporozoite invades the liver, a new antigen, liver-stage antigen 1 (LSA1), is synthesised by the parasite. This is increasingly expressed on the surface of the developing merozoite, while the CSP antigens are detected in decreasing amounts.27 LSA1 is proposed as a vaccine because it can be used to develop an immune response to the parasite in the hepatic stage by promoting a CD8+ T-cell response. This response has been shown to be present in those with naturally occurring infection and in patients vaccinated with irradiated sporozoite containing LSA1 antigens.28 Investigations into the effects of expressing LSA1 in Escherichia coli or with hepatitis B surface antigen (HbsAg) are also under way.28 LSA1 has a relatively uniform sequence throughout the strains of plasmodia,27 suggesting a crucial role in the development of the parasite and therefore a promising vaccine target.
Circumsporozoite protein
Anti-merozoite vaccines
Circumsporozoite protein (CSP) is a surface protein synthesised by sporozoites developing in the mosquito salivary gland.19 It is present throughout the pre-erythrocytic stage and presents epitopes stimulating both a humoral and cellular mediated immune response.20 Positive results from studies of the immune response to CSP have led to the proposal that CSP be used to vaccinate against malaria. It has been shown that using specific CSPderived epitopes as a primer, followed by boosting with a vaccinia virus expressing the entire protein, gave significantly improved results over using either fragments of CSP, or the entire CSP as a single vaccine.21 Other studies have highlighted the benefits of modifying CSP, for example using a branched recombinant protein as opposed to the natural linear counterpart,22 or by the use of adjuvants such as alum, or universal T-cell epitopes which decrease genetic restriction (antigens only presented to T cells of a host with a certain MHC haplotype) in the vaccinated recipient by acting as universal T-cell epitopes.16 Research studies have also identified a benefit in attaching plasmodia antigens to recombinant microorganisms or elements derived from them. An example currently in phase II clinical trials is RTS,S/AS02; part of the CSP from P falciparum attached to an entire hepatitis B surface antigen, RTS,S, with the AS02 adjuvant.7 A similar unit given as a vaccine elicited potent humoral and cellular immune responses, offering 41% protection against plasmodia-infected mosquito challenge.23 Murine studies using Plasmodium yoelii CSP expressed in an adenovirus, with a modified vaccinia virus (MVA) booster have shown 100% protection from symptomatic malaria, although these results have yet to be mirrored in human trials.24,25 The T-lymphocyte response to CSP, or part of it, has been shown by Reece et al26 to vary between naturally infected populations. In one group an interleukin 4 response was predominant, whereas in another group the response was mainly interferon ␥. Whilst this may prove to be an insignificant finding, it is more likely that it will offer knowledge needed to target a specific immune response necessary for vaccine success.26
Creating a vaccine to sensitise the immune system to infected erythrocytes is difficult, the main reason being the lack of knowledge about how the immune system deals with the infected cell. Erythrocytes do not express MHC class I or II molecules, limiting T-cell immunity. The immune response is therefore predominantly dependent on antibody-associated processes; neutralising the merozoite, preventing its entry into erythrocytes,6,16 or attacking infected erythrocytes expressing merozoite surface antigens.16,29 Antibody-dependent cell-mediated cytotoxicity (ADCC) and complement lysis are thought to be important in eliminating merozoite-infected erythrocytes.3,30
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Liver-stage antigen 1
Merozoite surface protein 1
The most extensively studied of the merozoite antigens, merozoite surface protein 1 (MSP1) is a glycoprotein found on the merozoite surface. Its function is unclear, although it has been suggested that it may be involved in erythrocyte invasion.16 It has been shown that immunisation with the C terminal of MSP1 protects mice against a normally lethal plasmodia challenge.31 MSP119 (a fragment of MSP1) antigens are known to evoke both a T and B cell proliferation.32,33 Immunisation with parasite-derived MSP1 has shown the best results in model systems; however, recombinant MSP1-based proteins can also offer immunity. The optimum fragment has not yet been confirmed, but there is good evidence for MSP119, possibly alongside other epitopes.34 One advantage of MSP119 over other epitopes is its lack of polymorphism. Individuals previously exposed to P falciparum have been shown to have both antibodies and T cells that recognise MSP1.19,32,33 MSP4 is another merozoite surface protein that has been identified as a possible vaccine candidate. More than 94% of a study population living in an area of Vietnam where malaria is highly endemic had antibodies corresponding to MSP4. A significant antibody response was shown to antigens synthesised in recombinant bacteria, suggesting that this protein also has potential as a vaccine.29
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Apical membrane antigen 1
Pre-fertilisation antigens
Apical membrane antigen 1 (AMA1) is synthesised late in the development of the schizont.29 A vaccine generated using murine Plasmodium chabaudi AMA1 expressed in E coli has been found to be effective in mice, and produced an antibody response, as well as a CD4 T-cell response. Protection was also given by passive transfer of antibodies when this vaccine was tested in rabbits.35
Several antigens have been identified that are expressed either completely or predominantly on the surfaces of both male and female gametes. The most frequently studied of these include Pfs230 and Pfs48/45, against which monoclonal antibodies act to block transmission in vitro.1,15 There is evidence that these proteins may be ligands in the fertilisation process.15 Substantial work, however, is needed before transmission blocking is successful in vivo and the development of these antigens for a vaccine is still in the initial stages of research.1,15,39,40 During a natural infection these pre-fertilisation antigens are also expressed in the intermediate host, which in a vaccinated individual may cause natural boosting of the antibody.1,41,42
Serine repeat antigen
Serine repeat antigen (SERA) is produced by trophozoites and schizonts of P falciparum and P vivax and then secreted into the parasitophorous vacuole. A recombinant section of SERA expressed in yeast with an adjuvant, produced a strong immune response against P falciparum challenge in aotus monkeys.36
Transmission-blocking vaccines TBVs are designed to prevent the development of malaria within the mosquito host by acting against the sexual stages of the parasite. Experiments in avian and other animal malaria systems have shown that immunisation of the intermediate host with antigens of the sexual and midgut stages of malaria parasites (gametes, zygotes, and ookinetes) can effectively block malaria transmission.15 This type of immunity is mediated by antibodies against surface protein antigens on extracellular parasites that have emerged from the erythrocytes in the mid gut of a mosquito after a blood meal. The antibodies are induced by vaccination of the intermediate host and ingested by the mosquito.37 Gametocytes in the erythrocytes of the immunised intermediate host are not affected.15,38 Antibodies against the gametes prevent fertilisation or destroy the gametes or zygotes within 5–10 minutes of entering the mosquito midgut. Antibodies against ookinetes act 12–24 h later to prevent them penetrating the midgut and forming the sporozoites, which could eventually infect another intermediate host.15
Post-fertilisaton antigens
The second class of antigens consists of proteins expressed solely or predominantly on zygotes or ookinetes but which are not present in the intermediate host. Antibody responses cannot, therefore, be boosted after natural infection.1 Two of these proteins are being investigated— namely P25 and P28. These proteins are targets for antibodies that interfere with ookinete maturation and oocyst formation, although the mechanism is unknown.43 Although they do not show synergism,44 P25 and P28 have partly redundant functions, since ookinete/oocyst development in parasites lacking one of these proteins is compromised only slightly.43 The same study suggested that since neither of the molecules is individually essential for survival of the parasite, but the loss of both results in a loss of viability, it would be essential to target both molecules simultaneously in a vaccine.40 Clinical-grade material is available for Pfs25 and phase 1 trials in human volunteers have begun.15,17,40 Chitinase as an antigen
After a blood meal reaches the mosquito midgut, epithelial cells in the midgut secrete the peritrophic matrix (PM), a
Table 2. Summary of antigens in MuStDO multistage vaccine.54 Antigen Pre-erythrocytic antigens 1 CSP 2 SSP2 3 LSA1 4 LSA3 5 Exp1
Location
Details
Sporozoite surface Sporozoite surface protein 2 Liver-stage parasite vacuole Liver-stage parasite vacuole and blood stage merozoite Liver-stage vacuole membrane, host cell cytoplasm
These antigens aim to induce both a humoral and cellular immune response to infected hepatocytes, and to neutralise any free sporozoites in the blood
Erythrocytic antigens 6 MSP142 7 MSP2 8 MSP4 9 MSP5 10 MSP3 11 EBA175 12 RII 13 AMA1 14 SERA 15 RAP2
Merozoite surface Merozoite surface Merozoite surface Merozoite surface Merozoite surface Microneme Microneme Merozoite surface Parasitophosphorus vacuole Rhoptry complex
These vaccine components aim, by a predominantly humoral immune response, to prevent the entry of merozoites into erythrocytes, prevent intra-erythrocytic development, and to inhibit the adherence of infected erythrocytes to vascular endothelium.
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chitin-containing sheath that completely surrounds the bolus of food. To traverse this barrier, the malaria parasite secretes a chitinase.8,45 Chitinase is also secreted by the mosquito midgut epithelium and serves to regulate the properties (thickness and permeability) of the PM. If antibodies against the mosquito and/or malaria chitinase inhibit the chitinase activity and so make the PM much harder for the malaria parasite to traverse, these may be effective in a TBV.8,45 Experiments using these antibodies are in progress.45
The future Until recently, cell-mediated immunity was relatively neglected in terms of malaria-vaccine development. It has been shown that the parasitic antigens that are expressed on the surface of infected erythrocytes also give rise to a CD4+ T-cell cytokine-mediated immunity.46 Research into the effect of antibody-independent cell-mediated immunity has shown the role of T cells in reducing parasitaemia. The study showed that despite the absence of immunoglobulins specific for the strain of P falciparum, a degree of immunity was evident after several low-dose challenges of infection.47 It is generally accepted that the future of malaria vaccination lies in the development of a multistage, multicomponent vaccine. Different strains of plasmodia should be included in a vaccine to help reduce the risk of resistant strains emerging.48–50 A vaccinated individual, on being bitten by an infected mosquito, would initially mount an antibody response against sporozoite surface antigens. Any surviving sporozoites would then invade the hepatocytes against which CD8+ and CD4+ T cells would act to destroy the infected cells. Antibodies to merozoite surface antigens would target merozoites released from liver schizonts that escape destruction. If the parasite is still not completely destroyed and invades erythrocytes, antibodies to erythrocytic surface antigens would neutralise the infected cells. Finally, transmission-blocking antibodies would make sure that if the infection is not completely alleviated, it is not passed on to another mosquito to infect another intermediate host.48 The problem in designing a multicomponent vaccine is determining which antigens to deliver and by what means.51 NYVAC-Pf7
The first multistage multiantigen vaccine candidate for P falciparum was NYVAC-Pf7.52 NYVAC-Pf7 is an attenuated vaccinia virus, genetically engineered to include seven P falciparum genes. These genes include those encoding the pre-erythrocytic antigens CSP and LSA1, the asexual blood stage antigens MSP1, AMA1, and SERA, and a transmission-blocking antigen Pfs25.50–52 Initial work, however, did not produce a vaccine that protected against P falciparum in human volunteers, although detectable immune responses were elicited.53
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Search strategy and selection criteria Data for this review were identified by searches of Medline, WHO documents, documents from the Malaria Vaccine Initiative, and parasitology textbooks. Search terms were “malaria”, “vaccine”, “transmission blocking”, “pre-erythrocytic” and “anti-merozoite”. Only papers written in English were reviewed.
MuStDO
The multi-stage DNA-based malaria vaccine operation (MuStDO) is a collaborative vaccine-development programme involving scientists from several worldwide organisations.54 Current research is directed towards the induction of good immune responses to 15 P falciparum antigens, five pre-erythrocytic, and ten erythrocytic proteins to be used in a multistage, multivalent vaccine.49 Transmission-blocking antigens will also be considered for future MuStDO vaccines (table 2).54 Anti-disease approaches
Two studies have suggested a new approach to antimalarial vaccination. In one study, repeated exposure to low densities of malarial parasites followed by cure with drugs led to raised cell-mediated immunity and clinical resistance to further infection in healthy volunteers.47 In another study a vaccine directed against Glycosylphosphatidylinositol (a malarial toxin) provided protection against cerebral and pulmonary oedema, acidosis, and death in mice infected with malaria.55
Conclusions An effective malaria vaccine may be several years away, but the need for such a vaccine is as great as ever. The success achieved so far in identifying antigens capable of eliciting immune responses to the different stages of the malaria life cycle has taken us one step closer to a successful vaccine. Trials are already under way for some vaccine candidates; for example, DNA-based vaccines using a “prime/boost” approach to maximise T-cell immunogenicity against liverstage parasites have been trialled in Oxford, UK, and The Gambia. These vaccines have shown good tolerability and safety with moderate efficacy in phase 1 trials, with trials continuing.11 Sequencing of the parasite56 and host genome may help in the future development of vaccines, although many sequences from which vaccine candidates have been developed have been known for some time. The licensing of a vaccine, however, is only part of a much bigger picture. Cost is very important; the countries that need this vaccine the most are some of the poorest in the world. Substantial funding will be required to bring the vaccine from trials to a licensed product. Encouragingly, there has been a resurgence in government interest into malaria research, with many of the poorer endemic countries contributing to the cause, despite their limited funds.17 Distribution, education, and compliance are also important issues. If and when a malaria vaccine becomes
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available, it is essential that the means to ensure that the vaccine reaches those who need it are in place. Populations must be made aware of the vaccine’s availability and benefits. Putting these measures into place may be as challenging as developing the actual vaccine. We can only hope that investment into vaccine research continues, ensuring that a vaccine will become a reality in the future. References 1 2 3 4 5 6 7
8 9 10
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17 18 19
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Phillips RS. Current status of malaria and potential for control. Clin Microbiol Rev 2001; 14: 208–26. Miller LH, Baruch DI, Marsh K, Doumbo OK. The pathogenic basis of malaria. Nature 2002; 415: 673–79. Plebanski M, Hill AVS. The immunology of malaria infection. Curr Opin Immunol 2000; 12: 437–41 Good MF, Doolan DL. Immune effector mechanisms in malaria. Curr Opin Immunol 1999; 11: 412–19. Sinnis P, Kim Lee Sim B. Cell invasion by the vertebrate stages of plasmodium. Trends Microbiol 1997; 5: 52–58. Wakelin D. Immunity to parasites: how parasitic infections are controlled, 2nd edn. Cambridge: Cambridge University Press, 1996: 44–69. Bojang KA, Milligan PJM, Pinder M. Efficacy of RTS,S/AS02 malaria vaccine against Plasmodium falciparum infection in semi-immune adult men in The Gambia: a randomised trial. Lancet 2001; 358: 1927–34. Ghosh A, Edwards MJ, Jacobs-Lorena M. The journey of the malaria parasite in the mosquito: hopes for the new century. Parasitol Today 2000; 16: 196–201. Graves P, Gelband H. Vaccines for preventing malaria (Cochrane Review). In: The Cochrane Library, Issue 1, 2002. Oxford: Update Software. Rhee MSM, Akanmori BD, Waterfall M, Riley EM. Changes in cytokine production associated with acquired immunity to Plasmodium falciparum malaria. Clin Exp Immunol 2001; 126: 503–10. Moorthy V, Hill AVS. Malaria vaccines. Br Med Bull 2002; 62: 59–72. Haviid L. Clinical disease, immunity, and protection against Plasmodium falciparum malaria in populations living in endemic areas. Exp Rev Mol Med 1998; June 24. http://www-ermm.cbu.cam.ac.uk/lhc/txt001lhc.htm Baird JK. Host age as a determinant of naturally acquired immunity to Plasmodium falciparum. Parasitol Today 1995; 11: 105–11. Hoffman SL, Rogers WO, Carucci DJ, Venter JC. From genomics to vaccines: malaria as a model system. Nat Med 1998; 4: 1351–53. Carter R. Transmission blocking malaria vaccines. Vaccine 2001; 19: 2309–14. Tsuji M, Rodriguez EG, Nusselweig RS. Progress towards a malarial vaccine: efficient induction of protective anti-malaria immunity. Biol Chem 2001; 382: 553–70. Richie TL, Saul A. Progress and challenges for malaria vaccines. Nature 2002; 415: 694–701. Nussenzweig V, Nussenzweig RS. Rationale for the development of an engineered sporozoite malaria vaccine. Adv Immunol 1989; 45: 283–334. Menard R, Sultan AA, Cortes C, et al. Circumsporozoite protein is required for development of malaria sporozoites in mosquitoes. Nature 1997; 385: 336–40. Nardin E, Zavala F, Nussenzweig V, Nussenzweig RS. Pre-erythrocytic malaria vaccine: mechanisms of protective immunity and human vaccine trials. Parassitologia 1999; 41: 397–402. Oliveira-Ferreira J, Miyahira Y, Layton GT, et al. Immunogenicity of Ty-VLP bearing a CD8+ T cell epitope of the CS protein of P yoelii: enhanced memory response by boosting with recombinant vaccinia virus. Vaccine 2000; 18: 1863–69. Nardin EH, de Oliveira GA, Calvo-Calle JM, Nussenzweig RS. The use of multiple antigen peptides (MAPs) in the analysis and induction of protective immune responses against infectious diseases. Adv Immunol 1995; 60: 105–49.
Useful websites World Health Organization Malaria Foundation International Malaria Vaccine Initiative
Acknowledgments
We acknowledge the contributions of Deborah Kerr, Louise Marshall, Mhairi Grierson, and Robert Laing for their contributions to the initial literature search and review of the manuscript. Conflict of interest
We have no financial or personal relationships with persons or organisations that could influence the content of this review.
23 Kester KE, McKinney DA, Tormeporth N, et al. Efficacy of recombinant circumsporozoite protein vaccine regimes against experimental Plasmodium falciparum malaria. J Infect Dis 2001; 183: 640–47. 24 Gilbert SC, Schneider J, Hannan CM, et al. Enhanced CD8+ T cell immunogenicity and protective efficacy in a mouse malaria model using a recombinant adenoviral vaccine in heterologous prime-boost immunisation regimes. Vaccine 2002; 20: 1039–45. 25 Bruña-Romero O, González-Aseguinolaza G, Hafalla JCR, Tsuji M, Nussenwieg RS. Complete, long-lasting protection against malaria of mice primed and boosted with two distinct viral vectors expressing the same plasmodial antigen. Proc Natl Acad Sci USA 2001; 98: 11491–96. 26 Reece WHH, Plebanski M, Akinwunmi P, et al. Naturally exposed populations differ in their T1 and T2 responses to the circumsporozoite protein of Plasmodium falciparum. Infect Immun 2002; 70: 1468–74. 27 Fidock DA, Gras-Masse H, Lepers JP, et al. Plasmodium falciparum liver stage antigen-1 is well conserved and contains potent B and T cell determinants. J Immunol 1994; 153: 190–204. 28 Kurtis JD, Hollingdale MR, Luty AJF, et al. Preerythrocytic immunity to Plasmodium falciparum: the case for an LSA-1 vaccine. Trends Parasitol 2001; 17: 219–23. 29 Wang L, Richie TL, Stowers A, Nhan DH, Coppel RL. Naturally acquired antibody responses to Plasmodium falciparum merozoite surface protein 4 in a population living in an area of endemicity in Vietnam. Infect Immun 2001; 69: 4390–97. 30 Dubovsky F. Creating a vaccine against malaria. The malaria vaccine initiative, PATH (Program for Appropriate Technology in Health) 2001. http://www.malariavaccines.org/downloads.htm (accessed July 14, 2002). 31 Daly TM, Long CA. Humoral response to a carboxylterminal region of the merozoite surface protein-1 plays a predominant role in controlling blood-stage infection in rodent malaria. J Immunol 1995; 155: 236–43. 32 Shi YP, Sayed U, Qari SH, et al. Natural immune response to the C-terminal 19-kilodalton domain of Plasmodium falciparum merozoite surface protein 1. Infect Immun 1996; 64: 2716–23. 33 Soares IS, Levitus G, Souza JM, Del Portillo HA, Rodrigues MM. Acquired immune responses to the N- and C-terminal regions of Plasmodium vivax merozoite surface protein 1 in individuals exposed to malaria. Infect Immun 1997; 65: 1606–14. 34 Falciparum Malaria MSP-1 Workshop. The Malaria Vaccine Initiative, PATH (Program for Appropriate Technology in Health) 2000. http://www.malariavaccines.org/downloads.htm (accessed July 14, 2002). 35 Anders RF, Crewther PE, Edwards S, et al. Immunisation with recombinant AMA-1 protects mice against infection with Plasmodium chabaudi. Vaccine 1998; 16: 240–47. 36 Inselburg J, Bathurst IC, Kansopon J, et al. Protective immunity induced in Aotus monkeys by a recombinant SERA protein of Plasmodium falciparum: adjuvant effects on induction of protective immunity. Infect Immun 1993; 61: 2041–47. 37 Stowers A, Carter R. Current developments in malaria transmission-blocking vaccines. Expert Opin Biol Ther 2001; 1: 619–28. 38 Vincent AA, Fanning, S, Caira FC, Williamson KC. Immunogenicity of malaria transmission-blocking vaccine candidate y230.CA14 following crosslinking in the presence of tetanus toxoid. Parasite Immunol 1999; 21: 573–81.
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39 Roeffen WFG, Raats JMH, Teelen K, et al. Recombinant human antibodies specific for the Pfs48/45 protein of the malaria parasite Plasmodium falciparum. J Biol Chem 2001; 276: 19807–11. 40 UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) Publication TDR/RBM/MAL/VAC/2000.1. Malaria transmission blocking vaccines: an ideal public good. http://www.who.int/tdr/publications/publications/mal aria_tbv.htm (accessed May 18, 2002). 41 Bustamente PJ, Woodruff DC, OH J, Keister DB, Muratova O, Wiliamson KC. Differential ability of specific regions of Plasmodium falciparum sexual-stage antigen, Pfs230, to induce malaria transmissionblocking immunity. Parasite Immunol 2000; 22: 373–80. 42 Healer J, McGuinness D, Carter R, Riley E. Transmission-blocking immunity to Plasmodium falciparum in malaria-immune individuals is associated with antibodies to the gamete surface protein Pfs230. Parasitology 1999; 119: 425–33. 43 Tomas AM, Margos G, Dimopoulos G, et al. P25 and P28 proteins of the malaria ookinete surface have multiple and partially redundant functions. EMBO J 2001; 20: 3975–83. 44 Hisaeda H, Collins WE, Saul A, Stowers AW. Antibodies to Plasmodium Vivax transmissionblocking vaccine candidate antigens Pvs25 and Pvs28 do not show synergism. Vaccine 2001; 20: 763–70. 45 Shen Z, Jacobs-Lorena M. Characterisation of a novel gut-specific chitinase gene from the human malaria vector Anopheles gambiae. J Biol Chem 1997; 272: 28895–900. 46 Taylor-Robinson AW, Phillips RS, Severn A, Moncada S, Liew FY. The role of Th1 and Th2 cells in a rodent malaria infection. Science 1993; 260: 1931–34. 47 Pombo D, Hirunpetcharat C, Rzepczyk C, et al. Immunity to malaria after ultra-low doses of red blood cells infected with Plasmodium falciparum. Lancet 2002; 360: 610. 48 Doolan DL, Hoffman SL. Multi-gene vaccination against malaria: a multistage, multi-immune response approach. Parasitol Today 1997; 13: 171–78. 49 Doolan DL, Hoffman SL. DNA-based vaccines against malaria: status and promise of the multi-stage malaria DNA vaccine operation. Int J Parasitol 2001; 31: 753–62. 50 Engers HD, Godal T. Malaria vaccine development: current status. Parasitol Today 1998; 14: 56–64. 51 Stanley SL Jr. Malaria vaccines: are seven antigens better than one? Lancet 1998; 352: 1163–64. 52 Tine JA, Lanar DE, Smith DM, et al. NYVAC-Pf7, a poxvirus-vectored, multiantigen, multistage vaccine candidate for Plasmodium falciparum malaria. Infect Immun 1996; 64: 3833–44. 53 Ockenhouse CF, Sun PF, Lanar DE, et al. Phase I/IIa safety, immunogenicity and efficacy of NYVAC-Pf7, a pox-vectored, multiantigen, multistage vaccine candidate for Plasmodium falciparum malaria. J Infect Dis 1998; 177: 1664–73. 54 Kumar S, Epstein JE, Richie TL, et al. A multilateral effort to develop DNA vaccines against falciparum malaria. Trends Parasitol 2002; 18: 129–35. 55 Scofield L, Hewitt MC, Evans K, Siomos MA, Seeberger PH. Synthetic GPI as a candidate antitoxic vaccine in a model of malaria. Nature 2002; 418: 785–89. 56 Carlton JM, Angiuoli SV, Sub BB. Genome sequence and comparative analysis of the model rodent malaria parasite Plasmodium yoelii. Nature 2002; 419: 512–19.
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Malaria vaccines
Malaria vaccines: where are we and where are we going? Shirley A Moore, Emma GE Surgey, and Anthony M Cadwgan Malaria is still killing over one million people each year and its incidence is increasing. The need for an effective vaccine is greater than ever. A major difficulty with vaccine research is that the malaria parasite presents thousands of antigens to the human immune system that vary throughout its life cycle. Identifying those that may prove to be vaccine targets is complicated and time consuming. Most vaccines are targeted at individual stages of the malaria life cycle, although it is likely that only the development of a multistage vaccine will offer complete protection to both visitors to, and residents of, a malaria-endemic area. With the development of a successful vaccine other issues such as cost, distribution, education, and compliance will have to be addressed. This review describes some of the current vaccine candidates for immunising against malaria. Lancet Infect Dis 2002; 2: 737–43
Malaria is caused by infection with a single-cell parasite, plasmodium. Four Plasmodium spp cause malaria in human beings: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae. P falciparum is the most important because it accounts for the majority of infections and causes the most severe symptoms. Human beings are the intermediate (or vertebrate) hosts in the plasmodia life cycle and are usually infected with malaria when bitten by a female anopheles mosquito (the definitive host) carrying the parasite. Although its mode of transmission was known more than 100 years ago,1 malaria remains a serious public-health problem for roughly 40% of the world’s population, killing over one million people each year. Malaria need not be a fatal disease and death is avoided by prompt diagnosis and treatment; however, without this a child may die of acute malaria within 24 h. Transmission can be prevented by the use of bednets, repellents, and insecticides, but, according to WHO, the prevalence of malaria is on the rise. This increasing prevalence is mainly due to drug and insecticide resistance but also in part to social and economic change. The cost of malaria is especially important in developing countries, because the economic effects of preventing and treating malaria are a large burden on the health budget. Due to the lack of cost-effective treatments and the emergence of resistance, a malaria vaccine is likely to be crucial in reducing both the morbidity and the mortality of this disease.
Life cycle of the malaria parasite The malaria parasite life cycle can be divided into two distinct stages, one occurring in the intermediate host and another in the definitive host (figure 1). Plasmodia sporozoites are introduced into the subcutaneous tissue and, less frequently, the bloodstream of the intermediate host via the bite of an
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infected mosquito.2 After just minutes in the bloodstream the sporozoites reach the liver.3,4 It is not certain exactly how the sporozoites enter hepatocytes,1 although it has been suggested that hepatocyte receptors to the major surface protein on the sporozoite, the circumsporozoite protein (CSP), are involved.5 Once inside hepatocytes, the sporozoites develop into schizonts containing thousands of merozoites.1,2 In certain species of malaria the sporozoites may remain as latent hypnozoites in the liver before this stage.1 The merozoites are released into the blood flowing through the sinusoids of the liver after 6–15 days, depending on the Plasmodium spp. The circulating merozoites infect erythrocytes within a few seconds and begin the asexual cycle (figure 2).1 Within the erythrocyte, the parasite passes through the ring and trophozoite stages before the production of the erythrocytic schizont.6 About 20 merozoites are produced from each mature schizont. The merozoites are released after 48 h, with the destruction of the erythrocyte, and are then free to infect further cells.2 Symptoms coincide with the release of merozoites into the blood and the release of cytokines such as tumour necrosis factor (TNF) and interleukin 1 from macrophages in response to the rupture of erythrocytes.4,6,7 The asexual cycle continues until infection is controlled, either by the host’s immune response or chemotherapy, or until the host dies.1 Eventually some merozoites within the erythrocytes differentiate into immature gametocytes. For the sexual reproduction of plasmodia to take place, these gametocytes must be taken up into the alimentary tract of an anopheles mosquito. On biting an infected intermediate host the mosquito ingests blood containing the parasite and the normally asexually dividing bloodstream forms (merozoites) die. The gametocytes are stimulated and the sexual stage of the cycle then continues in the mosquito.1 The gametocytes are released from the ingested erythrocytes and transform into male and female gametes.8 The male form (microgamete) undergoes flagellation and fertilises the female form (macrogamete), forming a zygote. Within 24 h the zygote develops into a worm-like ookinete, which penetrates the midgut wall of the mosquito, forming an oocyst between the midgut epithelium and the basal lamina. Many sporozoites are then formed asexually within the oocyst, which, on reaching maturity, bursts open to release the sporozoites. These sporozoites migrate to the mosquito’s salivary glands where they can be transmitted through a bite into another intermediate host approximately 7–18 days after gametocyte ingestion.1 SAM and EGES are at the University of Aberdeen, Aberdeen, UK; and AMC is at the Aberdeen Royal Infirmary, Aberdeen. Correspondence: Dr Anthony M Cadwgan, Infection Unit, Aberdeen Royal Infirmary, Foresterhill, Aberdeen AB25 2ZN, UK. Tel +44 (0)1224 553705; email [email protected]
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8 7 9 6 11
10
5
12
1
13
4 2
3 14
16 15 17
Figure 1. Life cycle of malaria. (1) Mosquito ingests erythrocytes infected with malaria gametocytes. (2) Gametocytes released from erythrocytes and transform into male and female gametes. (3) Zygote formed from the fertilisation of the female gamete by the male. (4) Zygote develops into ookinete and penetrates the midgut wall of the mosquito. (5) Ookinete differentiates into oocyst. (6) Oocyst ruptures releasing sporozoites that migrate to the mosquito’s salivary glands. (7) Sporozoites introduced into intermediate host through the bite of an infected mosquito. (8) Sporozoites migrate towards the liver. (9) Sporozoites invade hepatocytes. (10) Sporozoites develop into schizonts containing thousands of merozoites. (11) Alternatively, sporozoites may remain latent in the liver as hypnozoites. (12) Merozoites released into the blood to infect circulating erythrocytes. (13) The parasite passes through the ring stage. (14) Next comes the trophozoite stage. (15) Finally the erythrocytic schizont is produced. (16) Merozoites are released, with the destruction of the erythrocytes to begin the cycle again. (17) Alternatively, some merozoites differentiate into gametocytes.
Developing a malaria vaccine Plasmodia are large and complex organisms. Each infection presents thousands of antigens to the human immune system, which differ at each stage of the parasite’s life cycle. Plasmodia’s ability to adapt to the human immune system and misdirect or suppress it presents the main difficulty in finding a successful vaccine.6 Another difficulty is our poor understanding of the natural immune response to malaria, and, therefore, that which should be elicited by a vaccination programme. Multiple infections of malaria (different strains and/or different species) are also possible, so protection against one type does not necessarily protect against another.6 Until now vaccine development has targeted four different stages of the plasmodia life cycle: the sporozoite and liver stages (together known as the pre-erythrocytic stage), the merozoite, or erythrocytic, stage where the parasite invades or grows in the red blood cells, and the sexual stage occurring in the mosquito.9 There is also work under way to produce a vaccine that will contain a range of antigens from all stages of the plasmodium life cycle9 and adjuvants are being investigated to boost a vaccine’s effect. Most research into malaria-vaccine development is directed against P falciparum, the organism responsible for the highest rates of morbidity and mortality of all of the malaria parasites.1 Many people living in areas of high endemicity develop a degree of immunity to plasmodia. These people rarely show
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clinical evidence of infection because they are able to reduce circulating parasite numbers to within the limits tolerated by their immune system.1,10 However, this natural immunity is acquired only gradually after repeated infection11 and babies and young children are still at risk. According to WHO, 700 000 under-5s die from malaria each year. It can take more than 10 years to reach optimum (incomplete) protection, the time taken correlating inversely with the intensity of parasite transmission.12 This timescale supports the hypothesis that the antigens inducing protective immune responses are only weak inducers of immunity. Alternatively, plasmodium antigens may be very polymorphic (vary between strains) or may change with time within strains, therefore requiring acquired immunity to a large number of different epitopes for optimum protection.12 It is thought that children develop resistance to malaria more slowly than adults,13 although it is uncertain whether this slower development can be explained by differences in the immune response between children and adults, or by sufficient exposure to different antigens.12,13 Naturally acquired immunity is normally specific to one species of plasmodia and requires continual “boosting” by regular reinfection.1 Therefore, people who leave an endemic area are at risk of infection with other species of the malaria parasite against which they have no protection. They also risk clinical symptoms of malaria on return to their original
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area due to the loss of (natural) partial immunity. A vaccine targeted at antigens expressed during the erythrocytic stage would mimic the immunity acquired by persistent infection, associated with residence in an endemic area. Some merozoites would persist in causing symptoms but with reduced severity of illness.14 This type of vaccine would be suitable for immunising children living in a malariaendemic area. A vaccine targeting the erythrocytic stages of the parasite would be of little value to the so-called “malaria naive” traveller or newcomer to a malariaendemic area who would require complete protection.1 To achieve this level of protection, the vaccine must target the pre-erythrocytic sporozoite or liver-stage parasites, thus preventing merozoite production and release, the process responsible for clinical disease.4,6,7 A third type of vaccine is aimed at preventing Figure 2. P falciparum parasites in blood. sexual reproduction of plasmodia in the mosquito, therefore preventing the transmission of disease to another Pre-erythrocytic vaccines intermediate host. The “altruistic” transmission-blocking A vaccine that targets the pre-erythrocytic stages of plasmodia vaccines (TBVs) offer no protection from disease for the would be the only vaccine capable of preventing the clinical vaccine recipient, but rather prevent the spread of disease by manifestations of malaria. Such a vaccine would benefit those interrupting the plasmodia life cycle. Malaria is transmitted individuals who had not previously been exposed to the mainly within a few hundred metres from an infected parasite, and would therefore be at greater risk of severe human source; therefore, the use of TBVs within a morbidity and mortality. Due to the increase in travel to community would protect that community.15 TBVs target malaria-endemic areas, and the inconvenience of the current the antigens present on gametes, zygotes, or ookinetes. The prophylactic regimen, development of a pre-erythrocytic production of antibodies that are specific to such antigens is vaccine has proved popular with many researchers.16 The immune response needed to neutralise sporozoites is stimulated in human beings by vaccination with antigenic matter. These antibodies, freely circulating in the blood, are specific and different from that needed to destroy the parasite ingested by the mosquito in the blood meal, and cause in other stages. A successful vaccine would have to neutralise destruction of the developing parasite stages in the each individual sporozoite because each has the potential to become more than 30 000 merozoites.4 This is especially alimentary system of the mosquito.1,15 Immunity to an antigen from one stage of the difficult since the sporozoites only circulate in the blood plasmodium life cycle is confined to that stage11 so a stream for a few minutes. It is, therefore, necessary for a universally effective malaria vaccine would need to include sustained high antibody concentration to be present in the antigens from all stages of the parasite’s life cycle. Effective blood. Once sporozoites are inside hepatocytes they are more vaccine-induced immunity would also have to be faster protected from the body’s immune system and are harder to acting, longer lasting, and protect against more strains than destroy. To eliminate these liver-stage parasites, a cellular acquired immunity. A summary of the different possible immune response, involving both helper and cytotoxic T cells antigens used in candidate vaccines is shown in table 1. is necessary.17 CD8+ and CD4+ T cells recognise parasiteTable 1. Summary of different vaccine candidates and their action Stage of malaria life cycle Pre-erythrocytic stage Sporozoite
Liver schizont Blood stage Merozoite Infected erythrocytes Toxins Mosquito (sexual) stage Gametes Zygotes/ookinetes Blood meal
Target antigen
Immune response
Comments
Whole, irradiated sporozoite Circumsporozoite protein (CSP) derivatives
Humoral Humoral, cellular
Liver stage antigen 1 (LSA1)
Cellular
In-vitro culture very difficult Promising when attached to recombinant microorganisms with “booster” viruses Relatively uniform sequence throughout the different plasmodia strains
Merozoite surface protein 1 (MSP1) Apical membrane antigen 1 (AMA1) Serine repeat antigen (SERA) Glycosylphosphatidylinositol (GPI)
Humoral, cellular Humoral, cellular Humoral Humoral
Pfs230, Pfs48/45 P25, P28 Chitinase
Humoral Humoral Humoral
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Vaccines mimic natural immunity
Initial stages of research “Altruistic” vaccine candidates
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derived peptides presented by MHC class I and II molecules on the surface of plasmodia-infected hepatocytes leading to their destruction.3,4 There is evidence that protective immunity against this stage of malaria can be mediated in vitro through cytotoxic mechanisms targeted against these parasitic peptides.6 Early malaria-vaccine research examined the immune response to irradiated sporozoites. It was discovered that proteins expressed on the surface of the sporozoite induced an immune response giving protection against subsequent sporozoite challenge, preventing the progression of the parasite to the asexual blood stages.18 However, the use of irradiated sporozoites has not been widely pursued due to the difficulty in culturing plasmodia in vitro. Current research into the development of a pre-erythrocytic vaccine involves the use of the sporozoite surface proteins and the manipulation of the immune system to full advantage.
Shortly after the sporozoite invades the liver, a new antigen, liver-stage antigen 1 (LSA1), is synthesised by the parasite. This is increasingly expressed on the surface of the developing merozoite, while the CSP antigens are detected in decreasing amounts.27 LSA1 is proposed as a vaccine because it can be used to develop an immune response to the parasite in the hepatic stage by promoting a CD8+ T-cell response. This response has been shown to be present in those with naturally occurring infection and in patients vaccinated with irradiated sporozoite containing LSA1 antigens.28 Investigations into the effects of expressing LSA1 in Escherichia coli or with hepatitis B surface antigen (HbsAg) are also under way.28 LSA1 has a relatively uniform sequence throughout the strains of plasmodia,27 suggesting a crucial role in the development of the parasite and therefore a promising vaccine target.
Circumsporozoite protein
Anti-merozoite vaccines
Circumsporozoite protein (CSP) is a surface protein synthesised by sporozoites developing in the mosquito salivary gland.19 It is present throughout the pre-erythrocytic stage and presents epitopes stimulating both a humoral and cellular mediated immune response.20 Positive results from studies of the immune response to CSP have led to the proposal that CSP be used to vaccinate against malaria. It has been shown that using specific CSPderived epitopes as a primer, followed by boosting with a vaccinia virus expressing the entire protein, gave significantly improved results over using either fragments of CSP, or the entire CSP as a single vaccine.21 Other studies have highlighted the benefits of modifying CSP, for example using a branched recombinant protein as opposed to the natural linear counterpart,22 or by the use of adjuvants such as alum, or universal T-cell epitopes which decrease genetic restriction (antigens only presented to T cells of a host with a certain MHC haplotype) in the vaccinated recipient by acting as universal T-cell epitopes.16 Research studies have also identified a benefit in attaching plasmodia antigens to recombinant microorganisms or elements derived from them. An example currently in phase II clinical trials is RTS,S/AS02; part of the CSP from P falciparum attached to an entire hepatitis B surface antigen, RTS,S, with the AS02 adjuvant.7 A similar unit given as a vaccine elicited potent humoral and cellular immune responses, offering 41% protection against plasmodia-infected mosquito challenge.23 Murine studies using Plasmodium yoelii CSP expressed in an adenovirus, with a modified vaccinia virus (MVA) booster have shown 100% protection from symptomatic malaria, although these results have yet to be mirrored in human trials.24,25 The T-lymphocyte response to CSP, or part of it, has been shown by Reece et al26 to vary between naturally infected populations. In one group an interleukin 4 response was predominant, whereas in another group the response was mainly interferon ␥. Whilst this may prove to be an insignificant finding, it is more likely that it will offer knowledge needed to target a specific immune response necessary for vaccine success.26
Creating a vaccine to sensitise the immune system to infected erythrocytes is difficult, the main reason being the lack of knowledge about how the immune system deals with the infected cell. Erythrocytes do not express MHC class I or II molecules, limiting T-cell immunity. The immune response is therefore predominantly dependent on antibody-associated processes; neutralising the merozoite, preventing its entry into erythrocytes,6,16 or attacking infected erythrocytes expressing merozoite surface antigens.16,29 Antibody-dependent cell-mediated cytotoxicity (ADCC) and complement lysis are thought to be important in eliminating merozoite-infected erythrocytes.3,30
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Liver-stage antigen 1
Merozoite surface protein 1
The most extensively studied of the merozoite antigens, merozoite surface protein 1 (MSP1) is a glycoprotein found on the merozoite surface. Its function is unclear, although it has been suggested that it may be involved in erythrocyte invasion.16 It has been shown that immunisation with the C terminal of MSP1 protects mice against a normally lethal plasmodia challenge.31 MSP119 (a fragment of MSP1) antigens are known to evoke both a T and B cell proliferation.32,33 Immunisation with parasite-derived MSP1 has shown the best results in model systems; however, recombinant MSP1-based proteins can also offer immunity. The optimum fragment has not yet been confirmed, but there is good evidence for MSP119, possibly alongside other epitopes.34 One advantage of MSP119 over other epitopes is its lack of polymorphism. Individuals previously exposed to P falciparum have been shown to have both antibodies and T cells that recognise MSP1.19,32,33 MSP4 is another merozoite surface protein that has been identified as a possible vaccine candidate. More than 94% of a study population living in an area of Vietnam where malaria is highly endemic had antibodies corresponding to MSP4. A significant antibody response was shown to antigens synthesised in recombinant bacteria, suggesting that this protein also has potential as a vaccine.29
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Apical membrane antigen 1
Pre-fertilisation antigens
Apical membrane antigen 1 (AMA1) is synthesised late in the development of the schizont.29 A vaccine generated using murine Plasmodium chabaudi AMA1 expressed in E coli has been found to be effective in mice, and produced an antibody response, as well as a CD4 T-cell response. Protection was also given by passive transfer of antibodies when this vaccine was tested in rabbits.35
Several antigens have been identified that are expressed either completely or predominantly on the surfaces of both male and female gametes. The most frequently studied of these include Pfs230 and Pfs48/45, against which monoclonal antibodies act to block transmission in vitro.1,15 There is evidence that these proteins may be ligands in the fertilisation process.15 Substantial work, however, is needed before transmission blocking is successful in vivo and the development of these antigens for a vaccine is still in the initial stages of research.1,15,39,40 During a natural infection these pre-fertilisation antigens are also expressed in the intermediate host, which in a vaccinated individual may cause natural boosting of the antibody.1,41,42
Serine repeat antigen
Serine repeat antigen (SERA) is produced by trophozoites and schizonts of P falciparum and P vivax and then secreted into the parasitophorous vacuole. A recombinant section of SERA expressed in yeast with an adjuvant, produced a strong immune response against P falciparum challenge in aotus monkeys.36
Transmission-blocking vaccines TBVs are designed to prevent the development of malaria within the mosquito host by acting against the sexual stages of the parasite. Experiments in avian and other animal malaria systems have shown that immunisation of the intermediate host with antigens of the sexual and midgut stages of malaria parasites (gametes, zygotes, and ookinetes) can effectively block malaria transmission.15 This type of immunity is mediated by antibodies against surface protein antigens on extracellular parasites that have emerged from the erythrocytes in the mid gut of a mosquito after a blood meal. The antibodies are induced by vaccination of the intermediate host and ingested by the mosquito.37 Gametocytes in the erythrocytes of the immunised intermediate host are not affected.15,38 Antibodies against the gametes prevent fertilisation or destroy the gametes or zygotes within 5–10 minutes of entering the mosquito midgut. Antibodies against ookinetes act 12–24 h later to prevent them penetrating the midgut and forming the sporozoites, which could eventually infect another intermediate host.15
Post-fertilisaton antigens
The second class of antigens consists of proteins expressed solely or predominantly on zygotes or ookinetes but which are not present in the intermediate host. Antibody responses cannot, therefore, be boosted after natural infection.1 Two of these proteins are being investigated— namely P25 and P28. These proteins are targets for antibodies that interfere with ookinete maturation and oocyst formation, although the mechanism is unknown.43 Although they do not show synergism,44 P25 and P28 have partly redundant functions, since ookinete/oocyst development in parasites lacking one of these proteins is compromised only slightly.43 The same study suggested that since neither of the molecules is individually essential for survival of the parasite, but the loss of both results in a loss of viability, it would be essential to target both molecules simultaneously in a vaccine.40 Clinical-grade material is available for Pfs25 and phase 1 trials in human volunteers have begun.15,17,40 Chitinase as an antigen
After a blood meal reaches the mosquito midgut, epithelial cells in the midgut secrete the peritrophic matrix (PM), a
Table 2. Summary of antigens in MuStDO multistage vaccine.54 Antigen Pre-erythrocytic antigens 1 CSP 2 SSP2 3 LSA1 4 LSA3 5 Exp1
Location
Details
Sporozoite surface Sporozoite surface protein 2 Liver-stage parasite vacuole Liver-stage parasite vacuole and blood stage merozoite Liver-stage vacuole membrane, host cell cytoplasm
These antigens aim to induce both a humoral and cellular immune response to infected hepatocytes, and to neutralise any free sporozoites in the blood
Erythrocytic antigens 6 MSP142 7 MSP2 8 MSP4 9 MSP5 10 MSP3 11 EBA175 12 RII 13 AMA1 14 SERA 15 RAP2
Merozoite surface Merozoite surface Merozoite surface Merozoite surface Merozoite surface Microneme Microneme Merozoite surface Parasitophosphorus vacuole Rhoptry complex
These vaccine components aim, by a predominantly humoral immune response, to prevent the entry of merozoites into erythrocytes, prevent intra-erythrocytic development, and to inhibit the adherence of infected erythrocytes to vascular endothelium.
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chitin-containing sheath that completely surrounds the bolus of food. To traverse this barrier, the malaria parasite secretes a chitinase.8,45 Chitinase is also secreted by the mosquito midgut epithelium and serves to regulate the properties (thickness and permeability) of the PM. If antibodies against the mosquito and/or malaria chitinase inhibit the chitinase activity and so make the PM much harder for the malaria parasite to traverse, these may be effective in a TBV.8,45 Experiments using these antibodies are in progress.45
The future Until recently, cell-mediated immunity was relatively neglected in terms of malaria-vaccine development. It has been shown that the parasitic antigens that are expressed on the surface of infected erythrocytes also give rise to a CD4+ T-cell cytokine-mediated immunity.46 Research into the effect of antibody-independent cell-mediated immunity has shown the role of T cells in reducing parasitaemia. The study showed that despite the absence of immunoglobulins specific for the strain of P falciparum, a degree of immunity was evident after several low-dose challenges of infection.47 It is generally accepted that the future of malaria vaccination lies in the development of a multistage, multicomponent vaccine. Different strains of plasmodia should be included in a vaccine to help reduce the risk of resistant strains emerging.48–50 A vaccinated individual, on being bitten by an infected mosquito, would initially mount an antibody response against sporozoite surface antigens. Any surviving sporozoites would then invade the hepatocytes against which CD8+ and CD4+ T cells would act to destroy the infected cells. Antibodies to merozoite surface antigens would target merozoites released from liver schizonts that escape destruction. If the parasite is still not completely destroyed and invades erythrocytes, antibodies to erythrocytic surface antigens would neutralise the infected cells. Finally, transmission-blocking antibodies would make sure that if the infection is not completely alleviated, it is not passed on to another mosquito to infect another intermediate host.48 The problem in designing a multicomponent vaccine is determining which antigens to deliver and by what means.51 NYVAC-Pf7
The first multistage multiantigen vaccine candidate for P falciparum was NYVAC-Pf7.52 NYVAC-Pf7 is an attenuated vaccinia virus, genetically engineered to include seven P falciparum genes. These genes include those encoding the pre-erythrocytic antigens CSP and LSA1, the asexual blood stage antigens MSP1, AMA1, and SERA, and a transmission-blocking antigen Pfs25.50–52 Initial work, however, did not produce a vaccine that protected against P falciparum in human volunteers, although detectable immune responses were elicited.53
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Search strategy and selection criteria Data for this review were identified by searches of Medline, WHO documents, documents from the Malaria Vaccine Initiative, and parasitology textbooks. Search terms were “malaria”, “vaccine”, “transmission blocking”, “pre-erythrocytic” and “anti-merozoite”. Only papers written in English were reviewed.
MuStDO
The multi-stage DNA-based malaria vaccine operation (MuStDO) is a collaborative vaccine-development programme involving scientists from several worldwide organisations.54 Current research is directed towards the induction of good immune responses to 15 P falciparum antigens, five pre-erythrocytic, and ten erythrocytic proteins to be used in a multistage, multivalent vaccine.49 Transmission-blocking antigens will also be considered for future MuStDO vaccines (table 2).54 Anti-disease approaches
Two studies have suggested a new approach to antimalarial vaccination. In one study, repeated exposure to low densities of malarial parasites followed by cure with drugs led to raised cell-mediated immunity and clinical resistance to further infection in healthy volunteers.47 In another study a vaccine directed against Glycosylphosphatidylinositol (a malarial toxin) provided protection against cerebral and pulmonary oedema, acidosis, and death in mice infected with malaria.55
Conclusions An effective malaria vaccine may be several years away, but the need for such a vaccine is as great as ever. The success achieved so far in identifying antigens capable of eliciting immune responses to the different stages of the malaria life cycle has taken us one step closer to a successful vaccine. Trials are already under way for some vaccine candidates; for example, DNA-based vaccines using a “prime/boost” approach to maximise T-cell immunogenicity against liverstage parasites have been trialled in Oxford, UK, and The Gambia. These vaccines have shown good tolerability and safety with moderate efficacy in phase 1 trials, with trials continuing.11 Sequencing of the parasite56 and host genome may help in the future development of vaccines, although many sequences from which vaccine candidates have been developed have been known for some time. The licensing of a vaccine, however, is only part of a much bigger picture. Cost is very important; the countries that need this vaccine the most are some of the poorest in the world. Substantial funding will be required to bring the vaccine from trials to a licensed product. Encouragingly, there has been a resurgence in government interest into malaria research, with many of the poorer endemic countries contributing to the cause, despite their limited funds.17 Distribution, education, and compliance are also important issues. If and when a malaria vaccine becomes
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Review
Malaria vaccines
available, it is essential that the means to ensure that the vaccine reaches those who need it are in place. Populations must be made aware of the vaccine’s availability and benefits. Putting these measures into place may be as challenging as developing the actual vaccine. We can only hope that investment into vaccine research continues, ensuring that a vaccine will become a reality in the future. References 1 2 3 4 5 6 7
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Useful websites World Health Organization Malaria Foundation International Malaria Vaccine Initiative
Acknowledgments
We acknowledge the contributions of Deborah Kerr, Louise Marshall, Mhairi Grierson, and Robert Laing for their contributions to the initial literature search and review of the manuscript. Conflict of interest
We have no financial or personal relationships with persons or organisations that could influence the content of this review.
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