An eradicating vaccine for Plasmodium falciparum: Possibility or Pipe-dream?

Balazs Fazekas, University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 0SP

“Every 45 seconds, a child dies of Malaria in Africa”
World Health Organisation - 2010

Balazs Fazekas discusses the challenges facing the development of a malaria vaccine, and the progress so far.


Table of Contents

  1. Introduction
  2. Targeting the pre-erythrocytic antigens using subunit vaccines
    • 2.1. RTS,S vaccine candidate
    • 2.2. Liver-Stage Antigen-1
  3. Targeting blood-stage parasites
    • 3.1. Merozoite Surface Protein-1
    • 3.2. Plasmodium falciparum erythrocyte membrane protein-1
    • 3.3 Targeting the proteins on the red blood cell: Basigin
  4. Targeting the sexual stage and mosquito antigens by ‘altruistic’ vaccination;
    • 4.1. Targeting antigens on the gametocyte
    • 4.2. Targeting mosquito antigens
  5. Whole-cell parasite approaches
    • 5.1. Radiation attenuated sporozoites
    • 5.2. Genetically attenuated parasites
    • 5.3. Infectious sporozoites
  6. Targeting multiple life stages with multi-epitope vaccines
  7. Acquired immunity to malaria, can we ever achieve sterile immunity with a vaccine? If not, do we ever need to?
  8. Integration with other approaches
  9. Conclusion

1. Introduction

Plasmodium falciparum is a deadly parasite protozoon that annually infects an estimated 451 million people worldwide, the majority of the burden falling on the continents Africa and Asia (1). The parasite’s long evolutionary history alongside humans and its extensive genetic diversity pose a major challenge both to our immune system and to the development of a successful vaccine (2). Paradoxically, it has been possible to eliminate the parasite from many parts of the world through the use of effective medical treatments and pest-control schemes (3, 4), however, the complete eradication from the world has remained been a ‘pipe-dream’ for researchers, for the past two generations.

Dramatic improvements in mortality and morbidity rates, as well as the abolition of numerous cerebral and pregnancy-related complications would represent potential benefits of a successful vaccination campaign (5). Multiple candidate vaccines are currently in clinical trials around the world, which target different lifecycle stages of P. falciparum, each with a different design and mechanism of action (6). Despite the extensive efforts in this field progress has been slow. This is due to the fact that the parasite employs various effective techniques to evade the host’s immune responses. This includes a high level of antigenic variation and the infection of erythrocytes which not only lack MHC molecules required for the presentation of parasite peptides to host T-lymphocytes, but also do not possess the nuclear machinery which would allow it to respond to the presence of the parasite.

Furthermore, the parasite induces the formation of parasitophorous vacuoles, which conceal many of the parasitic proteins from host’s immune cells. This article does not aim to cover all malarial vaccines in development. Instead, it will focus on key representatives of the various approaches being undertaken and will evaluate the likelihood of these candidates successfully eradicating P. falciparum in the near future. In addition, progress in understanding the aspects of the natural immune response to P. falciparum will be discussed, since these may shed light on whether it will be possible to eradicate this parasite.





Figure 1 (adapted from Scherf et al (7)). The lifecycle of P.falciparum

Figure 1 shows the lifecycle of P. falciparum. Initially, elongated sporozoites are injected into a human host by a female Anopheles mosquito. The sporozoites then migrate to the liver in the bloodstream, where they enter hepatocytes and develop into schizonts through several rounds of asexual, mitotic replication. After 7 days, the schizonts rupture releasing thousands of merozoites into the bloodstream, which subsequently infect erythrocytes and develop into blood-stage merozoites within them. Every 48-72 hours, the infected erythrocytes rupture releasing the merozoites, and giving rise to the characteristic cyclic paroxysms of fever associated with malaria. Some of the released parasites differentiate into male and female gametocytes, which can then be taken up by the mosquito during a blood-meal. The gametes then fuse in the mid-gut of the mosquito forming an ookinete (zygote) and subsequently an oocyst, after migration across the gut epithelium. Oocysts undergo meiosis to form new sporozoites, which then travel to the salivary glands of the mosquito, from where they can be injected into a human host to reinitiate the cycle.

Each of the lifecycle stages of P. falciparum has been targeted by potential vaccine candidates. Vaccines may be directed against three antigenically distinct stages. These include pre-erythrocytic antigens comprising the sporozoite and liver-stage antigens, the asexual blood-stage antigens formed within the erythrocytes, as well as the mosquito stages of the parasite lifecycle (8). Various types of vaccine constructs including subunit, multi-subunit and whole-cell strategies present the parasite epitopes to the host immune system, with the aim of priming it for subsequent attack. So far, none of the potential vaccines have been successful enough for clinical use. Nevertheless, recently, a subunit vaccine targeting the sporozoite antigens has shown remarkable promise in clinical trials and hence offers a glimmer of hope for the eradication of P. falciparum.

2. Targeting the pre-erythrocytic antigens using subunit vaccines:

The fact that immunisation with irradiated sporozoites can induce sterilising protection in humans has been known since the 1970s (9). This led researchers to investigate whether the protein components of these early, pre-erythrocytic stages could be used to prime the host immune response. The leading subunit vaccine candidate is the RTS,S Malaria Vaccine, which is currently undergoing Phase III clinical trials in sub-Saharan Africa (10). If these are successful, the RTS,S could potentially become the first ever licensed vaccine against P. falciparum.

2.1 RTS,S vaccine candidate:

The RTS,S vaccine targets the circumsporozoite (CS) protein of the parasite; this protein is the main immunodominant antigen covering the entire surface of the sporozoite, forming a key component of its plasma membrane (11) (12). CS protein plays a role in the binding of the sporozoites to hepatocytes via heparin sulphate proteoglycan components and is also expressed in liver-stage parasites (13). As shown in Figure 2, a surprising feature of the CS protein is the existence of four amino acid NANP repeats in its extracellular domain.




Figure 2 (Adapted from Casares et al (10)). The Circumsporozoite protein of P. falciparum and the region of the protein incorporated into the RTS,S vaccine

Figure 2 shows the Circumsporozoite protein of P. falciparum and the region of the protein incorporated into the RTS,S vaccine. The four amino-acid (NANP) repeats, the Th2R and Th3R T- cell epitope sites, as well as the conserved region II near the C-terminal of the protein are included in the vaccine design. For the RTS,S vaccine, the CS protein is fused with Hepatitis B surface Antigen (HBsAg) and this is then expressed in combination with the unfused HBsAg to ensure assembly. Adjuvants such as AS02A or more recently AS01 are also added.

In-vitro experiments carried out by Gysin et al (14), confirmed that the CS protein was an effective target for vaccination. These researchers reported that antibodies raised to synthetic peptides of the repetitive NANP epitope of the CS protein could neutralise the infectivity of sporozoites. Since RTS,S incorporates these repetitive epitopes of the CS protein, it can prime the immune system against CS protein on the surface of the parasite. RTS,S’s protective capabilities have also been extensively tested in-vivo, both in endemic and non-endemic areas. Analysis of the immune response to RTS,S vaccines has been carried out in human clinical trials conducted amongst Gambian adults (15). The results of this trial, depicted in Figure 3, revealed that the vaccine induces a very high immunoglobulin G antibody response to the NANP repeat of the CS protein, much greater than that produced by life-long natural immunity to malaria.





Figure 3 (modified from Bojang et al (15)) demonstrates the changes in the mean anti-CS IgG antibody titres of Gambian men over time after receiving either the RTS,S vaccine or a control Rabies vaccine. Clearly, RTS,S induces a very high antibody response, several times higher than the natural acquired immune response, as revealed by the control subjects receiving the Rabies vaccine. The latter group would have been exposed to similar levels of endemic infection of P. falciparum compared to that of the vaccine cohort. The trial enrolled 306 men.

High levels of anti-CS antibodies induced by the RTS,S have been reported to be associated with protection against malaria in some experimental studies such as the human preliminary trial of RTS,S by Stoute et al (16), and have been used as correlates of protection. This trial reported that in vaccinated individuals, protected subjects showed higher antibody titres. It was also found that RTS,S vaccine had an efficacy of 86%, protecting 6 out of 7 previously non-exposed subjects from infection.

In addition to antibody mediated response elicited by RTS,S, it has also been reported to induce CD4+ and CD8+ T-cells specific for CS protein (Fig.4) (17). These cell-mediated responses are thought to have an important role in the immunity against malaria (18). Confirmational studies are, however, required to ascertain whether there is a positive correlation between T-cell proliferation induced by the RTS,S and protection against P. falciparum.
However, whether RTS,S could potentially become a tool for eradication in endemic areas was more recently questioned by the Phase IIb field trials enrolling over 2000 infants in Mozambique. Alonso et al (19) reported that RTS,S had a protective efficacy of as low as 37% among the vaccinated infants against clinical malaria and 57.7% against cases of severe malaria (Fig. 5). Compared to the higher efficacy reported in the preliminary trials, the subunit vaccine seems to offer the infants relatively minor protection. Nevertheless a follow up study of the trial reported that the partial protection lasted at least 18 months, which is more promising (20).

A major concern for RTS,S in vaccine development is that it targets only one immune-dominant target, the CS protein, which is not actually considered to be an important antigen in naturally acquired immunity to malaria (21). It would also seem paradoxical for P. falciparum to evolve a surface protein that would serve as an eradicating target for protective immunity. Other vaccines targeting sporozoites have showed less success than RTS,S, for example the DNA-MVA-ME-TRAP vaccine, and are reviewed in (22) and (23).

2.2 Liver-stage antigen-1

Since P. falciparum multiplies over 10,000 fold in hepatocytes (24), targeting the liver-stage of the parasite could potentially greatly reduce its burden on the host. Consequently, the cloning of a protein expressed only during the liver-stage of the parasite lifecycle initially brought high hopes for its use as a vaccine candidate (25). Liver-Stage Antigen-1 (LSA-1) shows similarities to other pre-erythrocytic proteins, such as the CS protein, in having repeated epitopes and in being immunogenic in humans, which caused further excitement amongst researchers. However, recent clinical Phase I/II trials using recombinant LSA-1 indicated that despite the strong anti-LSA-1 antibody response and a CD4+ T-cell response that were elicited, the individuals were not protected at all by immunisation with LSA-1 (26). The lack of efficacy was attributed to the particular design of the LSA-1 recombinant used in the trial. As the whole protein was not incorporated into the design, an important B- or T-cell epitope could have been omitted according to the research group. These results suggest that the LSA-1 antigen will not be the key vaccine candidate sought. It may nevertheless play a role in the potential eradication of malaria through its incorporation into multi-component vaccines (see section 6).

3. Targeting Blood-stage parasites:

Vaccine development directed at blood-stage antigens has been a very rapidly changing field, with a wide array of new vaccines investigated. The targeted antigens are usually on the merozoite life-stage form and are only exposed to the host immune system for a very short period, namely, before their rapid invasion of erythrocytes (Fig. 4) (27). This makes the recognition of the merozoites by the immune system even more difficult. Consequently, vaccines that induce a response to these merozoite epitopes need to be very effective; the aim is to produce sustained, high levels of functional antibodies for protection (28).





Fig 4 (Snapshot from Miller et al (29)) shows an electron micrograph image of a merozoite (Mz) invading an erythrocyte (RBC). Two apical organelles, the micronemes (M) and the rhoptries (R) contain the ligands required for binding to receptors on the erythrocyte. Due to the rapidity of the invasion process, the merozoite surface proteins are only exposed for approximately one minute to the immune system. The triangle-shaped arrows indicate the receptor-ligand interactions between the erythrocyte and the merozoite.

3.1 Merozoite surface protein-1:

One of the antigens present on the blood-stage form of the parasite is the Merozoite Surface Protein-1 (MSP-1), a GPI (glycosylphosphatidyl-inositol) -linked protein, reported to be important in the invasion of erythrocytes (31). The empirical evidence supporting the use of MSP-1 in malaria vaccines has come from rodent models, in which the injection of purified MSP-1 protein could confer resistance to subsequent P. yoelii infection (32). Antibodies were shown to be fundamental for inducing a protective effect since passively transferred monoclonal antibodies from protected mice conferred protection to naïve mice (33). The 190kDa MSP-1 pre-protein is proteolytically cleaved to yield several fragments. One of these smaller fragments, a 42kDa protein (MSP1-42), has been extensively investigated for use in vaccines, since it is more easily expressed in the correct conformation in recombinant systems (34). In combination with AS02 adjuvants, MSP1-42 recombinants have undergone a Phase II trial in Kenya. Surprisingly, despite the rise in antibody titres to MSP-1 protein, no protective effect was reported; parasite densities in blood and the number of clinically affected individuals in the vaccinated group were not reduced significantly (35). The reason for the lack of efficacy is not clear; however, the authors speculate that it may have been due to allelic polymorphism of the MSP-1, or the lack of antibody specificity to MSP-1. Either way, these sobering results call into question whether it will ever be possible to eradicate P. falciparum with blood-stage vaccines.

A common approach to improve efficacy has been combination vaccines, which incorporate MSP-1 antigens to generate sustained and protective antibody titres. Combination B vaccine is an example of a recombinant vaccine comprising the MSP-1 and two other merozoite surface proteins, MSP-2 and Ring-infected erythrocyte surface antigen (RESA). Combination B was reported to lower parasite densities by 62% in a Phase I/II study, involving 120 children in Papua New Guinea (36). Its suitability as a vaccine was, however, questioned because of the lack of significant efficacy in the reduction of incidence of clinical episodes. Nonetheless, its dual design has shown that using multiple antigens in combination can at least reduce parasite densities, even if it does not prevent clinical episodes. The fact that MSP-1 continues to be a promising vaccine candidate is shown by recent strategies incorporating MSP-1 into adenoviral vectors as well as Pox vectors (37, 38). It remains to be seen whether these designs will be successful.

3.2. Plasmodium falciparum Erythrocyte Membrane Protein-1:

Like MSP-1, many other blood-stage antigens are polymorphic, which can render the process of vaccine design more difficult. If a polymorphic protein is going to be part of an eradicating vaccine, many of its alleles will need to be included in the design, which may limit practicality and cost-efficiency of vaccine production. One way to deal with the issue of polymorphism seen in many of the blood-stage antigens is to target conserved regions of polymorphic proteins. For example, Baruch et al (39) targeted a functional receptor-binding domain of the Plasmodium falciparum Erythrocyte Membrane Protein-1 (PfEMP-1), a highly polymorphic erythrocyte surface protein. This region has limited variation for maintaining function of binding to CD36 on endothelial cells. It was targeted using a recombinant 179 amino-acid fragment mimicking the cysteine-rich interdomain region, CIDR, of PfEMP-1. The immune systems of some vaccinated monkeys challenged with homologous parasites were able to control the primary infection, as well as the secondary recrudescent parasites with different PfEMP-1 antigens on the erythrocyte surface. This confirmed that cross-reactive antibodies could be generated, despite the extensive polymorphism. (Fig. 5). Indeed PfEMP-1 has 59 variants encoded in the P. falciparum genome (40), the expression of which can be switched frequently to vary the erythrocyte phenotype, and hence its visibility to the immune system (41). Therefore, reducing this arsenal may be a crucial step towards the development of a possible eradicating vaccine.





Figure 5 (adapted from Baruch et al (39)) shows the parasitemia of Aotus monkeys immunised with either P. yoelii mice CS protein (control) or with the PfEMP-1 y179 recombinant protein fragment, following parasite challenge. In A both monkeys were treated with drugs (arrowheads) due to high parasitaemia. In B however, the y179 immunised monkeys showed delayed-onset, lower levels of parasitaemia. Recrudescence, i.e. the secondary infection due to ‘reinfecting’ parasites with different PfEMP-1 surface proteins compared to the primary infection, was also observed in monkey 3 (green) on day 25. This monkey had lower parasitaemia on secondary infection, suggesting a more efficient immune response second time round. Each different colour represents an experimental monkey. In both cases Freunds Adjuvant was the tested adjuvant. Drug treatment due to high parasitaemia is shown by arrows.

Furthermore, another way to deal with the issue of polymorphism is to identify subsets of the genes that are associated with virulence so that these subgroups, containing semi-conserved sequences, can be targets for vaccine design (42).

3.3 Targeting the proteins on the red blood cell: Basigin

Most recently, a group of scientists have provided support for the possibility of targeting proteins that are found on human red blood cells as a potential form of human vaccination (30). They showed that parasite entry could be potently inhibited (by 80-90%) by mutating Basigin, one of the receptors used by P. falciparum for the entry into the red blood cell. Adding excess Basigin or indeed antibodies raised against also drastically reduces the entry of the parasite. This is a very encouraging finding especially because the inhibition was observed with every strain of the malarial parasite which was tested, including those obtained from the field. Therefore, although these are very early findings, Basigin, may yet prove to be a successful vaccine candidate in the near future.

4. Targeting the sexual-stage and mosquito antigens by ‘altruistic’ vaccination:

Thus far, this paper has focussed on vaccinations that provide direct protective effects to the host by priming their immune response to malarial antigens. However, innovative vaccines are now emerging that confer protection to the surrounding community, rather than solely benefiting the vaccinated individual (43). These ‘altruistic’ vaccines block transmission of the parasite by targeting the sexual-stages of their lifecycle. Two approaches may be used to accomplish this. One involves the injection of gametocyte antigens into the human host so that the sexual-stage of the parasite becomes covered with antibodies and will not be able to produce viable oocysts within the mosquito. The other entails inducing the production of antibodies to mosquito antigens within humans; these antibodies are then taken up during a blood-meal by the mosquito and thus interfere with the development of the parasite within the vector (44). It thus offers a potential mechanism to protect against several malaria species.

Transmission-blocking vaccines have many advantages over vaccinations targeting other life-cycle stages. Firstly, the sexual-stage proteins are much less polymorphic than the asexual stage antigens (45). Secondly, since the exposure to sexual-stage antigens in the blood is prolonged, the level of antibody necessary for protection is lower (46). However, ‘altruistic’ vaccines also have to overcome several hurdles before they can become an effective vaccine approach. In contrast with ‘canonical’ malaria vaccines, natural boosting does not occur in response to the antigens solely expressed in the mosquito stages of the lifecycle. Consequently, any induced vaccination response has to be sustained for longer periods of time in the host for it to produce the same effect (43).

4.1. Targeting antigens on the gametocyte:

The targeting of the sexual gametocyte antigens was first investigated by Gwadz (47) who noted that injection of sexual-stage gametocytes into chickens could dramatically reduce the infectivity of the parasite protozoa in the mosquito vector, as measured by the number of oocysts that developed subsequently. Since then, the protein targets on the gametocyte surface have been identified as being the proteins Pfs48/45 and Pfs230 (48), and have been characterised immunologically. Insights into the protein structure have enabled a smaller sub-domain of the Pfs48/45, C10, to be expressed in E. coli (49). Injection of this P. falciparum C10 sub-domain into mice resulted in the production of antibodies that recognised the native Pfs48/45 epitopes of the parasite. These antibodies were able block fertilisation in the mosquito when taken up in the blood-meal, thus confirming its transmission blocking activity (Fig. 6).





Figure 6 (49) shows the transmission-blocking activity of the C10 fragment of the Pfs48/45 protein. In general, as the level of antibodies induced by C10 in mice increases, the mean oocyst production in the mosquito decreases. The level of antibodies induced by C10 was measured by a competition assay. In this assay, the higher the level of C10-induced antibody, the greater the % competition against the constant amount of peroxidase-conjugated anti-epitope I antibodies. Each of the various shapes of the data-points in the diagram represents one experimental mouse and there were 2 control mice receiving phosphate buffered saline (PBS). 10C is a recombinant protein fragment containing 10 cysteine residues; these correspond to the 4 cysteine residues in the middle of the protein and the 6 C-terminal cysteine modules of the Pfs48/45 protein.

However, to date, the most developed transmission blocking target is the Pfs25 protein, a mosquito-stage antigen that is not expressed until after fertilisation (43). Its application as a vaccine candidate has been suggested by both animal and human clinical studies. In a Phase I clinical trial involving 10 humans, Wu et al (50) reported variable transmission-blocking antibody production induced by Pfs25. The transmission-blocking antibody titres, however, were not sustained in most of the vaccinated subjects. The vaccine also resulted in high levels of adverse reactions among the participants of the clinical trial, which would need to be dealt with if it were to become a useful vaccine. Despite the importance of this trial in demonstrating that the transmission blocking principle can be applied to human models of infection, the level of transmission-blocking activity is not yet sufficient for a dramatic impact in endemic areas. Other protein targets need to be found on gametocytes, which may potentially have greater transmission blocking activity.

4.2. Targeting Mosquito Antigens:

The second approach to blocking transmission of P. falciparum is to vaccinate against components within the mosquito itself rather than targeting the sexual-stages of the parasite (44). A promising Anopheles mosquito antigen is the mid-gut carboxypeptidase B, CPBAg1. CPBAg1 is a digestive enzyme that has been reported to be up-regulated by P. falciparum in the mid-gut of the mosquito (51). This suggests that CPBAg1 is essential for the development of the parasite. Consequently, it is no surprise that anti-CPBAg1 containing sera inhibit the development of P. falciparum as measured by the rate of mosquito infection (51). As illustrated in Figure 7, a single injection of recombinant CPBAg1 into mice produced anti-CPBAg1 antibodies that could inhibit transmission of P. berghei by an average of 63%. Moreover, the reproductive capacity of the mosquitoes was also reduced in this experiment as shown by the number of larvae in the first egg batches of the mosquitoes.





Figure 7 ( Adapted from Lavazec et al (51)) illustrates the development of Plasmodium berghei oocysts in Anopheles gambiae mosquitoes fed on control mice and mice immunised with mosquito antigen Carboxypeptidase B. The graph shows that oocyst development is reduced by 63.2% in Anopheles mosquitoes fed on mice immunised with CPBAg1 compared to controls. This inhibition of the oocyst development was measured by calculating the rate of mosquito infection by counting the oocysts in the mosquito 9 days post-feeding. The reduction of the mosquito infection seen in CPBAg1-immunised mice demonstrates the transmission blocking activity of the vaccine. Whether the levels of transmission-blocking are sufficiently high will need to be determined in human models with P. falciparum.

These are very encouraging results demonstrating that antibodies against mosquito epitopes can be effective in reducing parasite transmission, as well as the reproductive capacity of the mosquito. Whether the levels of inhibition are sufficiently high to considerably reduce human transmission in endemic areas is still unknown.

5. Whole-cell parasite approaches:

Targeting one immunodominant antigen clearly has its drawbacks for potential vaccine efficacy, namely that an immune response is only generated against the selected epitopes. In contrast, whole-cell parasite vaccines make use of attenuated whole parasites and thus imitate natural parasite exposure more closely.

5.1. Radiation attenuated sporozoites:

The benefit of using whole-cell stages of the protozoan parasite was demonstrated when it was established by Nussenzweig et al (52) that sterile immunity – i.e., complete protection -can develop in mice following the injection of radiation-attenuated sporozoites. This beneficial trend was also reported in humans in response to both homologous and heterologous strains of P. falciparum (9). The fact that this approach might provide protection against heterologous strains is important because in endemic areas the parasite population is genetically highly diverse (53). Further human clinical trials have investigated whether radiation-attenuated parasites could be employed as a viable vaccine strategy. One such study, by Hoffman et al (54), involved the administration of more than one thousand Anopheline mosquito bites to US Navy volunteers, yielding an almost complete level of protection (93%), which lasted for at least 23-42 weeks (Table 1).





Table 1 (Adapted from Hoffman et al (54)) summarises of all the P. falciparum challenges with 5-14 infected mosquitoes in US Navy soldier volunteers, who were immunised to various degrees with radiation-attenuated sporozoites. Each mosquito bite could deliver approximately 10-100 sporozoites. With higher immunisation loads, better levels of protection can be reached.

The results confirm the high protection elicited by vaccinating with radiation-attenuated parasites. However, a great concern is the excessively large dose of irradiated sporozoites that have to be injected into the host in order to induce protection. Moreover, these sporozoites must be alive, suggesting the need for cryo-preservation. Therefore, it might prove very difficult to store and deliver the vaccine to regions affected by malaria, raising doubts as to whether it would be a generally applicable tool for eradication.

It should be noted that the issue of logistics has been an area of intense research, especially for the company ‘SANARIA’, who have even developed their own first generation vaccine, called PfSPZ and are attempting to commercialise its production (55). PfSPZ are metabolically active, non-replicating sporozoites, which fail to proceed beyond the liver-stage of the lifecycle. Recent animal studies using P. yoelii sporozoites to model P. falciparum infection in mice have suggested that large doses of sporozoites may not be critical for maximal protection. By immunising mice with 3 doses of only 750 attenuated sporozoites Chattopadhyay at al (56) reported a 100% protection in 10 mice. Since fewer parasites are needed, this might simplify the process of vaccine production. Whilst this result is encouraging, it seems that there are still many practical issues which need to be overcome before this strategy can be implemented for vaccination; for example, intramuscular vaccination has not yet been shown to be effective.

5.2. Genetically attenuated Parasites (GAPs):

Attenuation by radiation is a relatively crude method of disabling the parasite, by preventing its progression beyond the hepatocyte stage of the life-cycle. Recently, the sequencing of the genome of P. falciparum by Gardner at al (40), along with development of methods to alter specific genes and their expression, has allowed the identification and disruption of specific crucial genes involved at different stages of the parasite’s lifecycle. Mueller et al (57) genetically modified P. falciparum and used these as protective experimental malaria vaccines. (Fig. 8) Their target for gene disruption, Upregulated in Infective Sporozoites-3, UIS3, is a small trans-membrane protein found in the parasitophorus vacuolar membrane, which is essential for early liver-stage development (58). By disrupting this specific gene, sporozoites were able to infect hepatocytes, but were not able to progress to the blood-stage forms. Immunised mice, receiving 3 rounds of 10,000 sporozoites, turned out to be completely protected.





Figure 8 (Adapted from Mueller et al (57)) shows targeted gene replacement strategy to form a UIS3(-) mutant parasite, which could then be used as a vaccine. The UIS-3 gene is replaced by a Toxoplasma gondii dhfr/ts selectable marker. It is targeted by a EcoRI/HindIII linearised replacement plasmid containing the 5’ and 3’ untranslated regions of the UIS-3. After a double-crossing recombination event the UIS-3 is replaced by the selection marker.

These animal experiments support the notion that it may be possible to develop a genetically attenuated vaccine for humans. In fact, a P. falciparum GAP, the dual mutant p52(-)/p36(-), has now been selected for human Phase I/IIa trials (59). These mutant parasites offer more severe intra-hepatocytic growth defects in comparison to the single gene mutants. The virtual impossibility of mutations that could cause it to revert justifies its testing in humans. Needless to say, in animal studies the p52(-)/p36(-) parasites offered complete protection against P. yoelii challenge with homologous strains, through both injection and mosquito biting (60). In addition, as with radiation-attenuated sporozoites, GAPs are proving to be highly effective at very low doses. This might prove advantageous in vaccine production. For example, recent, unpublished data suggest that 3 doses of a thousand SAP1(- mutant) sporozoites may induce complete protection against sporozoite challenge (61). SAP1 (Sporozoite Asparagine-rich Protein 1) is essential for liver-stage infection of the parasite and also affects the expression of other UIS genes including UIS3 and P52, most likely through post-transcriptional effects. Consequently, its deletion leads to a quasi multi-locus attenuated strain of P. falciparum (62). Nevertheless, this field is still in its early stages and there are some obstacles to overcome. For example, to date little is known about the mechanism of protection by these genetically altered sporozoites and whether it is similar to that elicited by radiation-attenuated sporozoites.

5.3 Infectious Sporozoites:

Yet another approach to vaccine development promises complete protection and possibly even greater vaccine efficacy than that with irradiated and genetically attenuated sporozoites. It involves the administration of infectious P. falciparum sporozoites whilst treating the subject with anti-malarial chloroquine. This approach has been demonstrated to be an effective means of vaccination in mice using Plasmodium berghei (63), as well as in human studies. Roestenberg et al (64) reported a 100% protection of 10 individuals when challenged by homologous strains of P. falciparum after a 3 months immunisation phase (Fig. 9). The higher level of efficacy compared to irradiated parasites may be due to a broader array of antigens being presented to the host immune system, since the chloroquine only inhibits the later stages of the blood-stage parasites (65). Despite its success in this limited sample group, all vaccinees reported adverse effects at least once during their immunisation phase. This finding raises concerns about the possibility of designing a safe, implementable vaccine. Furthermore, in endemic areas, certain strains of P. falciparum are chloroquine resistant making this strategy non-applicable, as yet (66).





Figure 9 (64) illustrates the efficacy of immunising humans with infectious parasites concomitantly with treatment. The graph shows the mean P. falciparum parasite levels in the blood of volunteers (10 in the vaccine group and 5 in the control) following an immunisation programme of 3 injections, as shown by real-time PCR analyses. No blood-stage parasites are recorded in the vaccine group, but there is a cyclical multiplication of blood-stage parasite seen in the controls. 

Nevertheless, recently, vaccine-like effects of infectious sporozoites have been reported whilst using a similar drug, primaquine, which ablates liver-stage parasites. Putrianti et al (67) hypothesize that an immune response to the pre-eythrocytic stages can be elicited by repeated exposure to infected mosquitoes during prophylactic primaquine treatment; if this holds true, prophylactic treatments in endemic areas could both eliminate liver-stage parasites and prime the immune response, which in turn could potentially protect the individual in the long-term, after the treatment.

6. Targeting multiple life-stages, with next-generation, multi-epitope vaccines:

Whole-cell vaccines offer, to this day, the most effective defence against P. falciparum. This protection depends on the presentation of an extensive array of antigens to the host immune system. By identifying immunodominant epitopes on the parasite’s surface, it has been possible to ‘build’ multi-epitope constructs that can induce immune protection (68). A well-known example is the Spf66, a compilation of four immunogenic epitopes, incorporated from both pre-erythrocytic and blood-stages phases of the life-cycle. These are linked to one another through cysteine residues at the N-terminal and C-terminal ends, as shown in Figure 10 (69).





Figure 10 (69) demonstrates the Spf66 costruct; 4 fragments of immunogenic surface proteins from both the pre-erythrocytic and blood-stages of the parasite lifecycle are used within this hybrid molecule. One fragment is the NANP amino acid repeats from the circumsporozoite protein, present in the RTS,S vaccine candidate. The other 3 fragments correspond to merozoite surface-proteins with relative molecular masses of 83K, 55K and 35K. These fragments were shown to induce protection in Aotus triviǫatus monkeys. The synthetic peptides are polymerised through the addition of terminal cysteine amino acids.

Spf66 was reported to confer high efficacy in several field trials around the world including Phase III clinical trials in Columbia, 38.8% (70), and Ecuador, 66.8% (71), with children above the age of 1. However, as part of the Extensive Programme of Immunisation, another trial of Spf66 in Tanzania with 1207 infants (below the age of 1) yielded an efficacy of 2%, highlighting the sobering nature of vaccine trials for malaria (72). The reason for the dramatic failure with infants is not understood but the age-dependent difference in the immunological reactions to Spf66 is the most likely explanation. Very young children showed different immunological responses, rendering them less protected by the vaccine. Nevertheless, this vaccine was a milestone because it highlighted the importance of taking into account the host genotype and its variability when developing vaccines. Murillo et al (73) reported that 73.3% of the non-responders to Spf66 were of the HLA DR4 serotype (and 42% were of the HLA DQw6 type) providing support for the association of certain MHCII alleles of the host, with the level of immune response seen to Spf66. Encouragingly, in the near future, it may become possible to overcome the issue of varying host genotypes by monitoring the frequency of the HLA alleles in the population and only selecting those drugs that are recognised by a large proportion of the varying HLA molecules (74).

Many multi-stage vaccines are currently being evaluated in clinical trials that employ viral vectors. For example, the ‘Naval Medical Research Centre’ is now currently recruiting subjects to investigate the efficacy of the NMRC-M3V-Ad-PfCA vaccine, which is a mixture of two attenuated adenoviral recombinants, one of which encodes the AMA-1 blood-stage antigen and the other the circumsporozoite protein (75). Unpublished preliminary data from Tamminga, claims that the vaccine induced IFN-γ and antibody responses that are dose-responsive in humans (76). These are promising results and the trial is set to finish by July 2011.

7. Acquired Immunity to malaria: Can we ever achieve sterile immunity with a vaccine? If not, do we ever need to?

Much of what we understand about the immunity developed against malaria stems from the observations of Robert Koch, a biologist who worked in malarial endemic areas such as Papua New Guinea, at the beginning of the 20th century (77). On his journey, Koch identified fundamental features of the immunity against malaria and some of his observations raise concerns about the feasibility of constructing an effective eradicating vaccine. Koch in his 4th report to the German Colonial Office highlighted the chronic, asymptomatic nature of malaria infection in endemic areas (78). Since then it has become clear that even the most protected individuals in endemic areas cannot acquire sterile protection from prolonged exposure to the parasite (79). Even those people who have been exposed to malaria their whole lives have a low level of parasitaemia in their bloodstream, known as ‘premunition state’. These individuals may still be at risk of an episode of malaria, albeit at substantially lower risk than non-protected individuals (80). This raises doubts as to whether a single exposure to a vaccine will ever be capable of achieving what a lifetime exposure to the parasite cannot, i.e. reaching sterile immunity to the parasite. Perhaps then, the aim of a vaccination programme should be to attain an asymptomatic premunition state within human hosts rather than sterile protection.

Koch also established that developing immunity to malaria takes years in endemic areas, a period which is now thought to be needed to build up a repertoire of specific antibodies against the various polymorphic epitopes (81). If this is true, a single vaccination that could rapidly confer protection, in theory at least, becomes much more unrealistic. Nevertheless, this ‘slow’ model for the mechanism of acquired immunity has been contested by an alternate view that strain-transcending immunity to malaria can develop more rapidly than previously thought (79). The view that immunity to malaria could develop rapidly initially came from a Neuro-syphilis malaria-therapy programme that began in 1917 and involved the inoculation of the parasite to induce fever as a form of treatment for syphilis (82). It was observed that after 4-10 inoculations with a homologous strain of P. falciparum, a sterile immunity to malaria developed (Fig. 11) (83).





Figure 11 (adapted from Baird (83)) demonstrates the acquired protection to malaria induced by repeated inoculations of homologous strains of P. falciparum. The graph shows that as the number of inoculations of homologous parasites increases the percentage of patients not experiencing fever and parasitaemia increases, whereas the percentage of patients suffering from fever and parasitemia decreases. Inoculations were part of the Malaria Therapy programme.

Although this relatively rapid acquisition of immunity was attributed to using homologous strains of malaria, there was also a rapid development of partial protection to heterologous strains that seemed to have been overlooked by supporters of the ‘slow’ theory of acquired immunity (83). Moreover, in migratory studies it was reported that adults can actually develop a rapid natural immunity to circulating strains of malaria but that children cannot, due to their immature immune systems (84). This point was demonstrated by comparing the immune responses of migrant populations of Java moving to highly endemic areas of Jaya, Indonesia with those of the natives in the highly endemic areas. A new model of acquired immunity has therefore emerged, one that is dependent on age and the degree of exposure.

The view that a brief heavy exposure can protect in an age-dependent fashion, if true, may have both positive and negative predictions for vaccine development. On one hand, in adults it offers hope and suggests that protection may be possible through fewer exposures to the parasite. On the other hand, the age-dependent acquisition implies that it will be difficult to protect children due to their insufficient immune response. Importantly, in endemic areas, the greatest burden of malaria is on the lives of young children (85), and so this is a serious concern. Nevertheless, although the acquisition of premunition protective state may take longer in children, Gupta et al (86) showed that vaccines in this group could nonetheless protect relatively quickly against the most severe lethal forms of malaria. Therefore, vaccination could have a dramatic impact even without parasite eradication.

Koch also highlighted the need for continual exposure to the parasite for effective protection (77). Disruption of continual exposure to P. falciparum can cause a loss of protective immunity as was seen in a Malaria Epidemic in Madagascar in the 1980s, which claimed over 40,000 human lives (87). This requirement for continuous exposure to the parasite for protective immunity raises doubts about whether a single vaccine can ever mimic continual, long-term exposure, or bypass the need for it. This may be a difficult task, because our immune system has failed to do just this for many millennia.

8. Integration with other approaches:

Even if a fully eradicating vaccine cannot be developed, partially effective vaccines could nonetheless reduce the prevalence of the parasite significantly. What’s more, when these are used alongside other existing malaria control strategies, such as mass screening and treatment and the use of insecticide-impregnated bed-nets, they could in combination help to eliminate the parasite. A support for this assumption was provided by a mathematical model by Griffin et al (88), which predicts the outcome of these strategies in Kjenjojo, Uganda. (Fig. 12)





Figure 12 (Adapted from Griffin et al (88)) illustrates a mathematical model of the impact of vaccination and other interventions on the parasite prevalence in Kjenjojo, Uganda. The figure shows that malaria control interventions such as the use of insecticide impregnated nets, mass screening and treatment may have a drastic impact on the prevalence of the parasite. According to the model, in Uganda, if high levels of adherence were to be achieved, elimination of the parasite could potentially be possible through a combination of mass vaccination and mass screening and treatment.

Whether this model is valid and whether it can be applied on a global scale for parasite eradication remains to be seen.

9. Conclusion:

In conclusion, although many different approaches are currently being undertaken to tackle the task of eradicating P. falciparum, they have until now all fallen short of this objective. The lack of success observed in the clinical setting can be attributed to several factors. It has often been the case that animal models showed promising results initially, but when these agents were then applied in the field, they failed to show an equally high level of efficacy in humans. Other considerable challenges that hinder vaccine production include: our incomplete understanding of the mechanisms of action of certain vaccines; logistical difficulties of vaccine production and storage and the unsuitability of existing dosing regimes for potential mass-vaccination programmes. Despite these short-comings and in view of the slow, yet significant progress that has been made in the field thus far, an eradicating vaccine is less of a ‘pipe-dream’ today, than it was 60 years ago, when the vaccination experiments began. It is probably only a matter of time before our knowledge of P. falciparum’s complex structure and our better understanding of the host’s immune response will be used to corner the parasite. It is likely that vaccination will initially be used alongside other malaria control schemes to reduce the parasite prevalence (Fig. 13). In this regard, much hope is placed in the RTS,S vaccine, which is set to complete Phase III clinical trials by the end of 2012. A vaccine that could induce complete protection or one that could totally block transmission will almost certainly require more time to create; a recent promising example includes Basigin, a surface protein on red blood cells, the disruption of which potently reduces parasite entry.

In the future, one strategy that is likely to be important in wiping out the parasite is the combination of existing approaches of vaccination such as the use of RTS,S with the transmission-blocking vaccines. In this way, both individual protection and reduction of transmission within the community could be provided by a ‘dual’ vaccine. In theory, these could be further incorporated into viral delivery platforms, such as the one seen in the NMRC-M3V-Ad-PfCA vaccine; this would allow the induction of a sustained immune response to these antigens, by both the innate and the adaptive immune systems. Evidently, these complex vaccine-designs are still only a ‘pipe-dream’, but at the rate new candidates are being tested, it is not likely to remain like that for long. In addition, our growing understanding of the immune response to malaria is also likely to play an important role in the development of novel vaccine strategies. What we ought to do now is to support the anti-malaria schemes like ‘Bill and Melinda Gates Malaria Foundation’ (89), and hope for a pleasant end to our ‘pipe-dreams’.





Figure 13 shows the integrated approach to potential P. falciparum eradication. The main obstacles encountered by these strategies are shown in red. It shows how vaccination could potentially be integrated with existing malaria control schemes, such as habitat removal schemes etc. Although the aim is to eradicate the parasite, induction of asymptomatic protection to children suffering from severe malaria may be more likely in the near future.

References: 

1. Hay SI, Okiro EA, Gething PW, Patil AP, Tatem AJ, Guerra CA, et al. Estimating the global clinical burden of Plasmodium falciparum Malaria in 2007. PLoS Med. 2010;7(6):e1000290. Epub 2010/06/22. doi: 10.1371/journal.pmed.1000290. PubMed PMID: 20563310; PubMed Central PMCID: PMC2885984.

2. Hughes AL, Verra F. Extensive polymorphism and ancient origin of Plasmodium falciparum. Trends in Parasitology. 2002;18(8):348-51. doi: Doi: 10.1016/s1471-4922(02)02290-0.

3. Zucker JR. Changing patterns of autochthonous malaria transmission in the United States: a review of recent outbreaks. Emerg Infect Dis. 1996;2(1):37-43. Epub 1996/01/01. PubMed PMID: 8964058; PubMed Central PMCID: PMC2639811.

4. Stapleton DH. Lessons of history? Anti-malaria strategies of the International Health Board and the Rockefeller Foundation from the 1920s to the era of DDT. Public Health Rep. 2004;119(2):206-15. Epub 2004/06/15. PubMed PMID: 15192908; PubMed Central PMCID: PMC1497608.

5. Snow RW, Marlies CH, Charles NRJC, Richard. SW. The public health burden of Plasmodium falciparum malaria in Africa: Deriving the
numbers.: Working Paper No. 11, Disease Control Priorities Project. Bethesda, Maryland: Fogarty International Center, National Institutes of Health; 2003.

6. WHO. Date Accessed. Malaria Vaccine Rainbow Tables. http://wwwwhoint/vaccine_research/links/Rainbow/en/indexhtml2010.

7. Scherf A, Lopez-Rubio JJ, Riviere L. Antigenic variation in Plasmodium falciparum. Annu Rev Microbiol. 2008;62:445-70. Epub 2008/09/13. doi: 10.1146/annurev.micro.61.080706.093134. PubMed PMID: 18785843.

8. Richie TL, Saul A. Progress and challenges for malaria vaccines. Nature. 2002;415(6872):694-701. Epub 2002/02/08. doi: 10.1038/415694a415694a [pii]. PubMed PMID: 11832958.

9. Clyde DF, McCarthy VC, Miller RM, Hornick RB. Specificity of protection of man immunized against sporozoite-induced falciparum malaria. The American Journal of the Medical Sciences.1973;266(6).

10. Casares S, Brumeanu TD, Richie TL. The RTS,S malaria vaccine. Vaccine. 2010;28(31):4880-94. doi: 10.1016/j.vaccine.2010.05.033. PubMed PMID: WOS:000280659600007.

11. Herrington DA, Clyde DF, Losonsky G, Cortesia M, Murphy JR, Davis J, et al. Safety and immunogenicity in man of a synthetic peptide malaria vaccine against Plasmodium falciparum sporozoites. Nature. 1987;328(6127):257-9. Epub 1987/07/16. doi: 10.1038/328257a0. PubMed PMID: 2439920.

12. Nussenzweig RS, Nussenzweig V. Antisporozoite Vaccine for Malaria: Experimental Basis and Current Status. Reviews of Infectious Diseases. 1989;11:S579-S85.

13. Kappe SH, Buscaglia CA, Nussenzweig V. Plasmodium sporozoite molecular cell biology. Annu Rev Cell Dev Biol. 2004;20:29-59. Epub 2004/10/12. doi:10.1146/annurev.cellbio.20.011603.150935. PubMed PMID: 15473834.

14. Gysin J, Barnwell J, Schlesinger DH, Nussenzweig V, Nussenzweig RS. Neutralization of the infectivity of sporozoites of Plasmodium knowlesi by antibodies to a synthetic peptide. J Exp Med. 1984;160(3):935-40. Epub 1984/09/01. PubMed PMID: 6470623; PubMed Central PMCID: PMC2187411.

15. Bojang K, Milligan P, Pinder M, Doherty T, Leach A, Ofori-Anyinam O, et al. Five-year safety and immunogenicity of GlaxoSmithKline's candidate malaria vaccine RTS,S/AS02 following administration to semi-immune adult men living in a malaria-endemic region of The Gambia. Hum Vaccin. 2009;5(4):242-7. Epub 2009/03/12. doi: 7050 [pii]. PubMed PMID: 19276646.

16. Stoute JA, Slaoui M, Heppner DG, Momin P, Kester KE, Desmons P, et al. A preliminary evaluation of a recombinant circumsporozoite protein vaccine against Plasmodium falciparum malaria. RTS,S Malaria Vaccine Evaluation Group. N Engl J Med. 1997;336(2):86-91. Epub 1997/01/09. doi: 10.1056/nejm199701093360202. PubMed PMID: 8988885.

17. Sun P, Schwenk R, White K, Stoute JA, Cohen J, Ballou WR, et al. Protective immunity induced with malaria vaccine, RTS,S, is linked to Plasmodium falciparum circumsporozoite protein-specific CD4+ and CD8+ T cells producing IFN-gamma. J Immunol. 2003;171:6961-7.

18. Nardin EH, Nussenzweig RS. T Cell Responses to Pre-Erythrocytic Stages of Malaria: Role in Protection and Vaccine Development Against Pre-Erythrocytic Stages. Annual Review of Immunology. 1993;11(1):687-727. doi: 10.1146/annurev.iy.11.040193.003351.

19. Alonso PL, Sacarlal J, Aponte JJ, Leach A, Macete E, Milman J, et al. Efficacy of the RTS,S/AS02A vaccine against Plasmodium falciparum infection and disease in young African children: randomised controlled trial. Lancet. 2004;364(9443):1411-20. Epub 2004/10/19. doi: S0140673604172231 [pii] 10.1016/s0140-6736(04)17223-1. PubMed PMID: 15488216.

20. Alonso PL, Sacarlal J, Aponte JJ, Leach A, Macete E, Aide P, et al. Duration of protection with RTS,S/AS02A malaria vaccine in prevention of Plasmodium falciparum disease in Mozambican children: single-blind extended follow-up of a randomised controlled trial. Lancet. 2005;366:2012-8.

21. Schofield L. The circumsporozoite protein of Plasmodium: a mechanism of immune evasion by the malaria parasite? Bull World Health Organ. 1990;68 Suppl:66-73. Epub 1990/01/01. PubMed PMID: 1709835; PubMed Central PMCID: PMC2393050.

22. Hill AV. Pre-erythrocytic malaria vaccines: towards greater efficacy. Nat Rev Immunol. 2006;6(1):21-32. Epub 2006/02/24. doi: nri1746 [pii] 10.1038/nri1746. PubMed PMID: 16493425.

23. Moorthy VS, Imoukhuede EB, Milligan P, Bojang K, Keating S, Kaye P, et al. A randomised, double-blind, controlled vaccine efficacy trial of DNA/MVA ME-TRAP against malaria infection in Gambian adults. PLoS Med. 2004;1(2):e33. Epub 2004/11/05. doi: 10.1371/journal.pmed.0010033. PubMed PMID: 15526058; PubMed Central PMCID: PMC524376.

24. Kurtis JD, Hollingdale MR, Luty AJ, Lanar DE, Krzych U, Duffy PE. Pre-erythrocytic immunity to Plasmodium falciparum: the case for an LSA-1 vaccine. Trends Parasitol. 2001;17(5):219-23. Epub 2001/04/27. doi: S0169-4758(00)01862-7 [pii]. PubMed PMID: 11323304.

25. Guerin-Marchand C, Druilhe P, Galey B, Londono A, Patarapotikul J, Beaudoin RL, et al. A liver-stage-specific antigen of Plasmodium falciparum characterized by gene cloning. Nature. 1987;329(6135):164-7.

26. Cummings JF, Spring MD, Schwenk RJ, Ockenhouse CF, Kester KE, Polhemus ME, et al. Recombinant Liver Stage Antigen-1 (LSA-1) formulated with AS01 or AS02 is safe, elicits high titer antibody and induces IFN-[gamma]/IL-2 CD4+ T cells but does not protect against experimental Plasmodium falciparum infection. Vaccine. 2010;28(31):5135-44. doi: DOI: 10.1016/j.vaccine.2009.08.046.

27. Dvorak JA, Miller LH, Whitehouse WC, Shiroishi T. Invasion of Erythrocytes by Malaria Merozoites. Science. 1975;187(4178):748-50.

28. Mahanty S, Saul A, Miller LH. Progress in the development of recombinant and synthetic blood-stage malaria vaccines. Journal of Experimental Biology. 2003;206(21):3781-8.

29. Miller LH, Good MF, Milon G. Malaria Pathogenesis. Science. 1994;264(5167):1878-83.

30. Crosnier C, Bustamante LY, Bartholdson SJ, Bei AK, Theron M, Uchikawa M, et al. Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum. Nature. 2011;advance online publication. doi:http://www.nature.com/nature/journal/vaop/ncurrent/abs/nature10606.html#supplementary-information.

31. Goel VK, Li X, Chen H, Liu SC, Chishti AH, Oh SS. Band 3 is a host receptor binding merozoite surface protein 1 during the Plasmodium falciparum invasion of erythrocytes. Proc Natl Acad Sci U S A. 2003. p. 5164-9.

32. Holder AA, Freeman RR. Immunization against blood-stage rodent malaria using purified parasite antigens. Nature. 1981;294(5839):361-4.

33. Majarian WR, Daly TM, Weidanz WP, Long CA. Passive immunization against murine malaria with an IgG3 monoclonal antibody. The Journal of Immunology. 1984;132(6):3131-7.

34. Holder AA, Sandhu JS, Hillman Y, Davey LS, Nicholls SC, Cooper H, et al. Processing of the precursor to the major merozoite surface antigens of Plasmodium falciparum. Parasitology. 1987;94 ( Pt 2):199-208. Epub 1987/04/01. PubMed PMID: 3295686.

35. Ogutu BR, Apollo OJ, McKinney D, Okoth W, Siangla J, Dubovsky F, et al. Blood Stage Malaria Vaccine Eliciting High Antigen-Specific Antibody Concentrations Confers No Protection to Young Children in Western Kenya. PLoS ONE. 2009;4(3):e4708.

36. Genton B, Betuela I, Felger I, Al-Yaman F, Anders RF, Saul A, et al. A Recombinant Blood-Stage Malaria Vaccine Reduces Plasmodium falciparum Density and Exerts Selective Pressure on Parasite Populations in a Phase 1-2b Trial in Papua New Guinea. The Journal of Infectious Diseases. 2002;185(6):820-7.

37. Bruder JT, Stefaniak ME, Patterson NB, Chen P, Konovalova S, Limbach K, et al. Adenovectors induce functional antibodies capable of potent inhibition of blood stage malaria parasite growth. Vaccine. 2010;28(18):3201-10. doi: 10.1016/j.vaccine.2010.02.024.

38. Jiang G, Shi M, Conteh S, Richie N, Banania G, Geneshan H, et al. Sterile Protection against Plasmodium knowlesi in Rhesus Monkeys from a Malaria Vaccine: Comparison of Heterologous Prime Boost Strategies. PLoS ONE. 2009;4(8):e6559.

39. Baruch DI, Gamain B, Barnwell JW, Sullivan JS, Stowers A, Galland GG, et al. Immunization of Aotus monkeys with a functional domain of the Plasmodium falciparum variant antigen induces protection against a lethal parasite line. Proc Natl Acad Sci U S A. 2002. p. 3860-5.

40. Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW, et al. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature. 2002;419(6906):498-511. Epub 2002/10/09. doi: 10.1038/nature01097nature01097 [pii]. PubMed PMID: 12368864.

41. Roberts DJ, Craig, Berendt AR, Pinches R, Nash G, Marsh K, et al. Rapid switching to multiple antigenic and adhesive phenotypes in malaria. Nature. 1992;357(6380):689-92.

42. Bull PC, Berriman M, Kyes S, Quail MA, Hall N, Kortok MM, et al. Plasmodium falciparum Variant Surface Antigen Expression Patterns during Malaria. PLoS Pathog. 2005;1(3):e26.

43. Saul A. Mosquito stage, transmission blocking vaccines for malaria. Curr Opin Infect Dis. United States2007. p. 476-81.

44. Lavazec C, Bourgouin C. Mosquito-based transmission blocking vaccines for interrupting Plasmodium development. Microbes Infect. France2008. p. 845-9.

45. Escalante AA, Lal AA, Ayala FJ. Genetic Polymorphism and Natural Selection in the Malaria Parasite Plasmodium falciparum. Genetics. 1998;149(1):189-202.

46. Saul A. Kinetic constraints on the development of a malaria vaccine. Parasite Immunology. 1987;9(1):1-9.

47. Gwadz RW. Successful immunization against the sexual stages of Plasmodium gallinaceum. Science (New York, NY). 1976;193(4258):1150-1.

48. Kumar N. Target antigens of malaria transmission blocking immunity exist as a stable membrane bound complex. Parasite Immunology. 1987;9(3):321-35.

49. Outchkourov N, Vermunt A, Jansen J, Kaan A, Roeffen W, Teelen K, et al. Epitope Analysis of the Malaria Surface Antigen Pfs48/45 Identifies a Subdomain That Elicits Transmission Blocking Antibodies. Journal of Biological Chemistry. 2007;282(23):17148-56.

50. Wu Y, Ellis RD, Shaffer D, Fontes E, Malkin EM, Mahanty S, et al. Phase 1 Trial of Malaria Transmission Blocking Vaccine Candidates Pfs25 and Pvs25 Formulated with Montanide ISA 51. PLoS ONE. 2008;3(7):e2636.

51. Lavazec C, Boudin C, Lacroix R, Bonnet S, Diop A, Thiberge S, et al. Carboxypeptidases B of Anopheles gambiae as Targets for a Plasmodium falciparum Transmission-Blocking Vaccine. Infection and Immunity. 2007;75(4):1635-42.

52. Nussenzweig RS, Vanderberg J, Most H, Orton C. Protective immunity produced by the injection of x-irradiated sporozoites of plasmodium berghei. Nature. 1967;216(5111):160-2. Epub 1967/10/14. PubMed PMID: 6057225.

53. Snounou G, Beck HP. The Use of PCR Genotyping in the Assessment of Recrudescence or Reinfection after Antimalarial Drug Treatment. Parasitology Today. 1998;14(11):462-7. doi: 10.1016/s0169-4758(98)01340-4.

54. Hoffman SL, Goh LM, Luke TC, Schneider I, Le TP, Doolan DL, et al. Protection of humans against malaria by immunization with radiation-attenuated Plasmodium falciparum sporozoites. J Infect Dis. 2002;185(8):1155-64. Epub 2002/04/04. doi: JID010922 [pii] 10.1086/339409. PubMed PMID: 11930326.

55. Hoffman SL, Billingsley PF, James E, Richman A, Loyevsky M, Li T, et al. Development of a metabolically active, non-replicating sporozoite vaccine to prevent Plasmodium falciparum malaria. Hum Vaccin. 2010;6(1):97-106. Epub 2009/12/01. doi: 10396 [pii]. PubMed PMID: 19946222.

56. Chattopadhyay R, Conteh S, Li M, James ER, Epstein JE, Hoffman SL. The Effects of radiation on the safety and protective efficacy of an attenuated Plasmodium yoelii sporozoite malaria vaccine. Vaccine. 2009;27(27):3675-80. doi: DOI: 10.1016/j.vaccine.2008.11.073.

57. Mueller A-K, Labaied M, Kappe SHI, Matuschewski K. Genetically modified Plasmodium parasites as a protective experimental malaria vaccine. Nature. 2005;433(7022):164-7.

58. Sharma A, Yogavel M, Akhouri RR, Gill J. Crystal structure of soluble domain of malaria sporozoite protein UIS3 in complex with lipid. J Biol Chem. 2008;283(35):24077-88. Epub 2008/06/26. doi: M801946200 [pii]10.1074/jbc.M801946200. PubMed PMID: 18577521; PubMed Central PMCID: PMC2527117.

59. VanBuskirk KM, O'Neill MT, De La Vega P, Maier AG, Krzych U, Williams J, et al. Preerythrocytic, live-attenuated Plasmodium falciparum vaccine candidates by design. Proc Natl Acad Sci U S A. 2009;106(31):13004-9. Epub 2009/07/25. doi: 0906387106 [pii]10.1073/pnas.0906387106. PubMed PMID: 19625622; PubMed Central PMCID: PMC2714279.

60. Labaied M, Harupa A, Dumpit RF, Coppens I, Mikolajczak SA, Kappe SHI. Plasmodium yoelii Sporozoites with Simultaneous Deletion of P52 and P36 Are Completely Attenuated and Confer Sterile Immunity against Infection. Infection and Immunity. 2007;75(8):3758-68.

61. Vaughan AM, Wang R, Kappe SH. Genetically engineered, attenuated whole-cell vaccine approaches for malaria. Hum Vaccin. 2010;6(1):107-13. Epub 2009/10/20. doi: 9654 [pii]. PubMed PMID: 19838068.

62. Aly ASI, Mikolajczak SA, Rivera HS, Camargo N, Jacobs-Lorena V, Labaied M, et al. Targeted deletion of SAP1 abolishes the expression of infectivity factors necessary for successful malaria parasite liver infection. Molecular Microbiology. 2008;69(1):152-63.

63. Beaudoin RL, Strome CPA, Mitchell F, Tubergen TA. Plasmodium berghei: Immunization of mice against the ANKA strain using the unaltered sporozoite as an antigen. Experimental Parasitology. 1977;42(1):1-5. doi: Doi: 10.1016/0014-4894(77)90054-6.

64. Roestenberg M, McCall M, Hopman J, Wiersma J, Luty AJ, van Gemert GJ, et al. Protection against a malaria challenge by sporozoite inoculation. N Engl J Med. 2009;361(5):468-77. Epub 2009/07/31. doi: 361/5/468 [pii] 10.1056/NEJMoa0805832. PubMed PMID: 19641203.

65. Yayon A, Waa JAV, Yayon M, Geary TG, Jensen JB. Stage-Dependent Effects of Chloroquine on Plasmodium falciparum In Vitro1. Journal of Eukaryotic Microbiology. 1983;30(4):642-7.

66. Trape JF. The public health impact of chloroquine resistance in Africa. American Journal of Tropical Medicine and Hygiene. 2001;64(1_suppl):12-7.

67. Putrianti Elyzana D, Silvie O, Kordes M, Borrmann S, Matuschewski K. Vaccineâ Like Immunity against Malaria by Repeated Causal Prophylactic Treatment of Liver Stage Plasmodium Parasites. The Journal of Infectious Diseases. 2009;199(6):899-903. doi: 10.1086/597121.

68. Doolan DL, Southwood S, Freilich DA, Sidney J, Graber NL, Shatney L, et al. Identification of Plasmodium falciparum antigens by antigenic analysis of genomic and proteomic data. Proceedings of the National Academy of Sciences of the United States of America. 2003;100(17):9952-7.

69. Patarroyo ME, Amador R, Clavijo P, Moreno A, Guzman F, Romero P, et al. A synthetic vaccine protects humans against challenge with asexual blood stages of Plasmodium falciparum malaria. Nature. 1988;332(6160):158-61. Epub 1988/03/10. doi: 10.1038/332158a0. PubMed PMID:2450281.

70.Valero MV, Amador LR, Galindo C, Figueroa J, Bello MS, Murillo LA, et al. Vaccination with SPf66, a chemically synthesised vaccine, against Plasmodium falciparum malaria in Colombia. The Lancet. 1993;341(8847):705-10. doi: 10.1016/0140-6736(93)90483-w.

71. Sempertegui F, Estrella B, Moscoso J, Piedrahita L, Hernandez D, Gaybor J, et al. Safety, immunogenicity and protective effect of the SPf66 malaria synthetic vaccine against Plasmodium falciparum infection in a randomized double-blind placebo-controlled field trial in an endemic area of Ecuador. Vaccine. 1994;12(4):337-42. Epub 1994/03/01. PubMed PMID: 8178556.

72. Acosta CJ, Galindo CM, Schellenberg D, Aponte JJ, Kahigwa E, Urassa H, et al. Evaluation of the SPf66 vaccine for malaria control when delivered through the EPI scheme in Tanzania. Tropical Medicine & International Health. 1999;4(5):368-76.

73. Murillo LA, Rocha CL, Mora AL, Kalil J, Goldenberg AK, Patarroyo ME. Molecular analysis of HLA DR4-beta 1 gene in malaria vaccinees. Typing and subtyping by PCR technique and oligonucleotides. Parasite Immunol. 1991;13(2):201-10. Epub 1991/03/01. PubMed PMID: 2052406.

74. Patarroyo ME, Cifuentes G, Bermúdez A, Patarroyo MA. Strategies for developing multi-epitope, subunit-based, chemically synthesized anti-malarial vaccines. Journal of Cellular and Molecular Medicine. 2008;12(5b):1915-35.

75. Tamminga C. NMRC-M3V-Ad-PfCA Vaccine - Clinical Trial 1. http://clinicaltrials.gov/ct2/show/NCT00392015; 2011.

76. Limbach KJ, Richie TL. Viral vectors in malaria vaccine development. Parasite Immunology. 2009;31(9):501-19.

77. Koch R. Professor Kochs Investigations on Malaria. British Medical Journal; 1900. p. 1183-6.

78. Koch R. Professor Koch's Investigations on Malaria: Fourth Report to the Colonial Department of the German Colonial Office. Br Med J. 1900;1(2061):1597-8. Epub 1900/06/30.PubMed PMID: 20759083; PubMed Central PMCID: PMC2506827.

79. Doolan DL, Dobano C, Baird JK. Acquired immunity to malaria. Clin Microbiol Rev. 2009;22(1):13-36, Table of Contents. Epub 2009/01/13. doi: 22/1/13 [pii]10.1128/cmr.00025-08. PubMed PMID: 19136431; PubMed Central PMCID: PMC2620631.

80. Obi RK, Okangba CC, Nwanebu FC, Ndubuisi UU, Orji NM. Premunition in Plasmodium falciparum malaria. African Journal of Biotechnology. 2010;9(10):1397-401. PubMed PMID: WOS:000275890700001.

81. Bull PC, Lowe BS, Kortok M, Molyneux CS, Newbold CI, Marsh K. Parasite antigens on the infected red cell surface are targets for naturally acquired immunity to malaria. Nat Med.1998;4(3):358-60. Epub 1998/03/21. PubMed PMID: 9500614.

82. Ernest JN. Malaria in Neuro-Syphilis 1923-43. Journal of Mental Science; 1943.

83. Baird JK. Host Age as a determinant of naturally acquired immunity to Plasmodium falciparum. Parasitology Today. 1995;11(3):105-11. doi: 10.1016/0169-4758(95)80167-7.

84. Baird JK, Jones TR, Danudirgo EW, Annis BA, Bangs MJ, Basri H, et al. Age-dependent acquired protection against Plasmodium falciparum in people having two years exposure to hyperendemic malaria. American Journal of Tropical Medicine and Hygiene. 1991;45(1):65-76.

85. Roca-Feltrer A, Carneiro I, Armstrong Schellenberg JRM. Estimates of the burden of malaria morbidity in Africa in children under the age of 5 years. Tropical Medicine & International Health. 2008;13(6):771-83.

86. Gupta S, Snow RW, Donnelly CA, Marsh K, Newbold C. Immunity to non-cerebral severe malaria is acquired after one or two infections. Nat Med. 1999;5(3):340-3.

87. Mouchet J, Laventure S, Blanchy S, Fioramonti R, Rakotonjanabelo A, Rabarison P, et al. [The reconquest of the Madagascar highlands by malaria]. Bull Soc Pathol Exot. 1997;90(3):162-8. Epub 1997/01/01. PubMed PMID: 9410249.

88. Griffin JT, Hollingsworth TD, Okell LC, Churcher TS, White M, Hinsley W, et al. Reducing Plasmodium falciparum Malaria Transmission in Africa: A Model-Based Evaluation of Intervention Strategies. PLoS Med. 2010;7(8):e1000324.

89. Gates B. All Lives Have Equal Value by 'The Bill and Melinda Gates Foundation' http://www.gatesfoundation.org/topics/Pages/malaria.aspx#2010.