Targeting different stages of malaria has attracted interest for many years, as people living in endemic countries acquire immunity to stages of the malarial parasite following repeated exposure. After extensive research, the three stages of the life cycle of the malarial parasite (the pre-erythrocytic, erythrocytic and transmission stages) were identified as targets of the immune system. RTS,S against the pre-erythrocytic stage is currently the most successful vaccine candidate and it is hoped that in the future the property of mounting an immune response to the malarial parasite will be exploited further to develop an effective Malaria vaccine
Introduction - remember to justify
Malaria causes widespread death particularly in tropical countries. In humans, malaria is caused by four species of Plasmodium: Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax. Of the four species, the most common cause of infection is by P.falciparum, which is responsible for the death of one million African children annually. The Malarial life cycle consists of three stages: the pre-erythrocytic stage, the erythrocytic stage and the transmission stage (fig. 1) Each of these are potential targets for a vaccine, by using the host's own immune response as it has been found that protection against malaria was conferred by the passive transfer of hyperimmune immunoglobulins from humans immune to malaria into humans that were not. (Sabchareon et al., 1991). The development of a vaccine must therefore use a sub-unit approach, by targeting particular stages of the infection, as opposed to a whole organism approach. The major advance in achieving a malarial vaccine occurred in 1967, when mice were immunised by injecting X-irradiated sporozoites intravenously obtained from the salivary glands of the mosquito, P. berghei, with the result that there was almost total protection against subsequent sporozoite challenges. However, the protection achieved from the irradiated sporozoites was limited to the sporozoite stage of infection, and as such did not work against erythrocytic stages. (Nussenzweig et al., 1967). There has been further progress at developing a vaccine targeted at each of the three stages of the life cycle of the malarial parasite, using the host's immune response. So far, prevention of contracting malaria is the only viable option to limit the number of cases of malaria. Prevention measures include: mosquito control programs, repellents, and chemoprophylaxis. Antimalarial drugs are being used in anticipation of disease for travellers to endemic malaria regions (Lin et al., 2006) but as of yet, an effective vaccine has not been developed. There is an increasing incidence of parasite resistance to the current anti-malarial drugs, and a large proportion of the population cannot afford such drugs and preventative measures. Despite the fact that there are logistical problems in delivering an effective vaccine to endemic countries, continuing research into the immune system's response to the malarial parasite at different stages of development is being undertaken, with the hope that in the future, an effective vaccine will be developed.
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Fig 1)The three stages in the life cycle of the malarial parasite
The first stages are pre-erythrocytic stages consisting of the sporozoites that are injected by the mosquito into the skin, and the liver stage whereby each sporozoite undergoes development into schizonts. These rupture to give rise to as many as 30,000 uninucleate merozoites. The second stage is the Erythrocytic (red blood cell) stage, whereby the merozoites infect the erythrocytes of the host. After this, a dormant version of the parasite waits to be ingested by another mosquito in order to be passed onto another host. This third stage of infection is the Transmission or sexual stage. Gametocytes are taken up by an anopheline mosquito, and, following fertilisation, zygotes and ookinetes are produced.
(Source: Ménard et al., 2005)
It is important to define what an adjuvant is as this has proved significant in developing an effective vaccine. Adjuvants are substances that help to elicit a strong, long-lasting immunity, and they have been used in Malaria vaccines to up-regulate some cytokines, as well as helping to deposit the antigen to the target site. (Cox and Coulter., 1997).The major adjuvant system that is used in prospective vaccines, is the AS02A adjuvant system, as it helps in generating both a strong cell-mediated and humoral immunity, to interfere with various stages of the parasite life cycle.
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Targeting the pre-erythrocytic stage (sporozoite and liver stage)
A major target of vaccine development has been against the sporozoite stage of infection of the malarial parasite, as this stage initiates the parasitic life cycle that results in the manifestation of disease. A key observation that it would be possible to develop a malaria vaccine occurred when irradiated sporozoites induced protective immunity in both humans and animals (Nussenzweig et al., 1967). This illustrated that the sporozoite stage of infection could be targeted by the host's own immune responses which would ultimately result in the attack of the parasite. Furthermore, the sporozoites, when injected by the mosquito into the skin, are free-living. This means that the host's immune system responds to the sporozoite stage of infection by the production of monoclonal antibodies (which are antibodies that have the same affinity for the malarial parasite antigens) with the result that protective immunity is achieved. (Cochrane et al., 1982).
Efforts have been made to target the Circumsporozoite (CS) proteins. CS proteins are stage and species-specific polypeptides that cover the surface of the sporozoite, and investigations have been made into how they are shed when they are cross-linked by antibodies. In 1982, progress was made as specific monoclonal antibodies 3D6 (an IgG1 antibody with a κ light chain against the P.falciparum sporozoite) and 2F2 (an IgG2b antibody with a κ light chain against the P.vivax sporozoite) were identified as specific monoclonal antibodies that could be directed against the CS protein of the malarial parasite (Nardin et al., 1982).
The hepatocyte stage of infection of malaria presents another important target for a vaccine. Researchers believe that there are CD8+ T cells that are specific for MHC class I molecules found on the surface of infected hepatocytes. These cytotoxic T cells are able to react in an antigen specific manner against liver stage malaria, thus implicating the need for inducing cellular immunity (involving the activation of antigen-specific cytotoxic T-lymphocytes) as well as humoral immunity (involving antibodies and complement). This was illustrated in 1988, when Weiss et al found that depletion of CD8+ T cells in sporozoite-immunised mice abolished protection against the sporozoite stage of infection. This was because the infected hepatocytes only expressed MHC class I molecules on their surface, and thus, could be recognised by CD8+ T cells, which could then interact in a cell specific manner to cause the destruction of the infected hepatocytes (Weiss et al., 1988).
CD4+ T cells do not appear to be important in the destruction of the malarial parasite, but they do play a role in helping recruit other effectors to fight infection. Thus, their role should be considered when deciding on how to establish an appropriate vaccine, as epitopes recognised by the CD4+ T cells would have to be included in a vaccine if it were to succeed. CD4+ T cells been illustrated to play an important role in inducing protective immunity with irradiated sporozoites. Anti - CD4 monoclonal antibody (mAb) treated mice were illustrated to be less protected against small numbers of sporozoites (Weiss et al.,1993) Moreover, in many other diseases it has been illustrated that anti - CD4 mAb treated mice have slower development of CD8 effector cells. However, in malaria, there is only a 48 hour period before development of the blood stage. Thus, although it has not been experimentally proven, it is believed that CD4 cells may ensure the CD8 cells are able to elicit a quicker response to the malarial parasite, meaning there is less chance of development into the erythrocytic stage of infection (Weiss et al., 1993).
Implications for future medicine against the pre-erythrocytic stages of infection
Targeting the CS protein has received great interest in vaccine development as the host antibody response is against the CS protein that covers the surface of the sporozoite. (Nussenzweig et al., 1984). The CSP protein also elicits a cellular response that allows infected hepatocytes to be destroyed.
After Dame et al (Dame et al., 1984) cloned and sequenced the P.falciparum CS gene, the prospect of producing a pre-erythrocytic vaccine to target the immune response of an individual was enhanced. The fact that the repeat region of the CS protein of P.falciparum is conserved in geographic isolates (Zavala et al., 1985) also proved promising. This made way for the development of the singular CS protein vaccine. Despite the fact that this induced antisporozoite antibodies, the CS protein on its own in a vaccine overall had poor immunogenicity (Hoffman et al., 1987).
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Recently the RTS,S/AS02A vaccine was developed by GlaxoSmithKline in collaboration with the Malaria Vaccine Initiative (MVI) Programme for Appropriate Technology in Health (PATH), which illustrates much promise for a successful vaccine against the pre-erythrocytic stages of Malaria, as it aims to overcome the problems of poor immunogenicity of the singular CS protein by fusing the protein with a surface antigen from Hepatitis B, to create enhanced immunogenicity. It was found that the RTS, S/AS02A vaccine was able to reduce the risk of clinical malaria by 35% and was able to decrease the number of cases of severe malaria over a period of 18 months (Alonso et al., 2005). Furthermore, trials indicate that the RTS,S/AS02A vaccine could potentially have several effects, which helps to increase its efficacy. The RTS,S/AS02A vaccine was designed with the outcome of initiating several areas of the immune system, and in trials this also appeared to be the case. The vaccine increased the numbers of antibody levels in some subjects, as a response to the circumsporozoite protein, however, in other subjects, methods of cell mediated killing by CD8 +responses resulted in the destruction of the antigen in the liver (Stoute et al., 1998), perhaps with the help of CD4+ T cells, to help amplify the role of the CD8+ T cells.
As the erythrocytic stage is responsible for the symptoms of disease, a vaccine at this stage would limit the number of cases of disease, particularly as a back-up mechanism if pre-erythrocytic vaccines were not 100% effective. As the erythrocytes lack MHC molecules on their surface, cellular immunity involving the MHC cannot be used as a mechanism of killing the malarial parasite. Thus, the type of immune response involved in dealing with the malarial parasite is antibody mediated.
P. falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by var genes, on the exterior surface of the infected erythrocyte, ensures that the parasite can bind to multiple receptors on the endothelial cells lining the capillary walls. (fig 2)
Fig 2) Infected erythrocytes are covered by proteins of the PfEMP1 family. These are able to bind to various receptors of the endothelial cells lining the capillary walls The PfEMP1 spans Knob-like projections that are found on the surface of infected erythrocytes, that allow attachment between the infected erythrocyte and the endothelial cell. (Macpherson et al., 1985) The PfEMP1 molecules, despite having variable regions, also contain conserved regions which could be future targets for a vaccine. (Source: Borst and Genest., 2006)
Naturally acquired immunity to malaria in individuals who live in regions where malaria is endemic is brought about by the immune system producing monoclonal antibodies to PfEMP (Bull et al., 1998). These antibodies are able to prevent the cytoadherence of the infected erythrocytes to the endothelial cells (Smith et al., 2000) particularly in the brain, which is important in preventing cerebral malaria.
Moreover, P.vivax, Apical Membrane Antigen (AMA) has been studied. AMA-1 has been implicated to play an important role in the invasion of erythrocytes (Triglia et al., 2000) and in individuals with a naturally acquired immunity to malaria, the AMA-1induces the long-lasting specific production of IgG-1 (Morais et al., 2006).
Implications for vaccine development
Rosette formation is when uninfected erythrocytes agglutinate around parasitized erythrocytes, which often causes mild cases of malaria, but can be reversed by immune serum (David et al., 1988). The Duffy binding-like 1 alpha (DBL1α) domain is expressed in PfEMP1, and has been found to mediate rosette-formation (Chen et al., 1998). Immunisation using the DBL1α used alone, or in combination with other duffy like-binding domains, was found to elicit protection against PfEMP1 sequestration, by means of antibody production, in both rats and monkeys, against heterologous parasite strains (Moll et al., 2007). However, as this was recent work, further studies would need to be carried out, particularly on humans, to see if using the DBLα domain would prove effective in protecting against severe malaria in humans.
The most recent advance in malaria vaccine development that proves the most promising is the AMA1-based malaria vaccine FMP2.1. This vaccine has been shown to stimulate the production of IgG. Where AMA-1 was formulated in the adjuvant system AS02A, there was the induction of the IgG antibody to a great degree. When 3 doses of the FMP2.1/AS02A vaccine were implemented, there was the production of 100-fold increases in the level of IgG antibody, specific for the AMA1 antigen. It has thus been implied that it may be possible in the future to develop a vaccine that could incorporate AMA-1 with RTS,S in an effective adjuvant system, such as AS02A to be administered as a multi-stage vaccine, or to use AMA-1 as a disease blocking vaccine. (Thera et al., 2010).
The transmission stage of malaria is the final target for a malaria vaccine. Targeting a vaccine to this stage would reduce the burden of disease in many of the world's countries where malaria is a persistent problem, by eliciting immunity to block transmission of the parasite from the mosquito to the vertebrate host. Furthermore, it is vital to target the transmission-blocking stage with a vaccine, in combination with vaccines to other stages, as this would help to prevent the parasite becoming resistant to vaccines for the other stages of development.
The immune system responds to the sexual stage of the malarial parasite mainly by the production of antibodies against surface antigens of the parasite which include gametes, zygotes and ookinetes (Mendis et al., 1990). Firstly the host immune system acts against gametes by the production of antibodies, against antigens on the surface of the gametes (known as 'pre-fertilisation' antigens'). A few hours later, antibodies that can neutralise and act cytotoxically are directed against the mature ookinetes, that are found in the mid-gut of the mosquito, and are known as 'post-fertilisation' antigens
after the malarial parasite has ingested a blood meal. It has been found that a major target of the immune system is against two major classes of antigens found on the surface of gametes, zygotes and ookinetes. The first of these is are known as Pfs48/45 and Pfs230, and these are expressed on the surface of male and female gametes of P.falciparum. Monoclonal antibodies have been found to target against these antigens (Carter et al., 1990) with the result that the effectivity of parasites to mosquitoes is eliminated.
The second set of antigens that are targeted by the immune system are known as Pfs25 and Pfs28, which are found on the surface of zygotes and mature ookinetes. These act against conformational epitopes on the surface of the mature sexual stages in the mosquito mid-gut in a complement-dependent manner. These post-fertilisation antigens act in a complement dependent manner, to kill the malarial parasite, and it was found that IgG1 was elicited in large numbers when Rhesus Monkeys were immunised with the Pfs25 antigen (Kumar 2007).
Implications for future vaccine development - requires frequent boosting
Vaccine development against the transmission stage of malaria has been difficult due to the fact that the pre-fertilisation antigen, Pfs 230 was found to be too large to express in its full length. Furthermore, Pfs230 in smaller fragments lacked enough immunogenicity to mount an effective immune response to malaria. The most prospective candidates for a malaria vaccine to this stage have been the post-fertilisation antigens, Pfs25 and Pfs28. Pfs25, in particular, has received a lot of attention, and has been developed in a vaccine in clinical trials. It was found that Pfs25 was effective in Rhesus macaques and mice, by using DNA plasmid as a vector of the vaccine (Kumar 2007). The result was a high immunogenicity in the laboratory animals. However, problems have been encountered when developing the vaccine for use in humans, due to the fact that DNA plasmids are less immunogenic in larger animals, and, although electroporation using the DNA plasmids, has resulted in enhancement of the effectiveness of the antigens, there is still need for further testing of the Pfs25 antigen in a vaccine to determine the correct dosage and complete effectiveness of this antigen in killing of the malarial parasite (LeBlanc et al., 2002).
Conclusions and future directions in vaccine development.
Vaccination is key, along with preventative measures to control the fatalities that malaria causes. Over the years, further advances have been made, by studying how the immune system responds to the malarial parasite in the different stages of development, often using people in endemic countries, that show an acquired immunity to the parasite, in order to achieve the goal of an effective vaccine. The aim is to develop a vaccine that will dramatically reduce the number of cases of malaria, by targeting each individual stage (the pre-erythrocytic, erythrocytic, and transmission stages) separately. The most promising vaccine candidates have included, in particular: RTS,S for the pre-erythrocytic stage, AMA-1 for the erythrocytic stage and a vaccine encorporating Pfs25 against the transmission stages of malaria, often involving the well-known adjuvant system, AS02A, to amplify the immune response. However, despite the fact that it is thought that a malaria vaccine will be developed in the near future, with WHO estimating a vaccine to be in place by 2015 (Greenwood 2008), there are still major problems to overcome. Firstly, multiple vaccines would be needed, to target each individual stage separately, which would be expensive, which would possibly mean that people living where malaria is most prevalent (ie in countries such as Africa) may not be able to afford the cost of the treatment. In conjunction with this, regular boosting would also be required, as the malarial parasite occurs in many strains and species, and one vaccine against one particular stage or species may not necessarily work against every strain and species. Finally, logistical problems, such as transportation of the required treatment would limit the effectiveness that the vaccines could have. Thus, although there is the great possibility that in the future a successful malaria vaccine targeting the immune system could be developed, there are further obstacles that need to be overcome.