Malaria Vaccine Development: A Case for Attenuated Whole-Cell Pre-Erythrocytic Vaccines for Plasmodium falciparum
Introduction: An unmet need
It is hard to imagine a person alive today who is not aware of the vast prevalence of malaria as well as the crippling rates of mortality and morbidity that are associated with its presence. According to the World Health Organization (WHO) there were 247 million cases of malaria in 2006 that caused nearly one million deaths.1 In countries with high disease rates, malaria can cut economic growth rates by as much as 1.3%.1 Malaria is a preventable and curable disease making it primarily a disease of poverty. There are multiple drugs on the market that can eliminate the parasite from the human host; however, they are expensive and in short supply in sub-Saharan countries that are the most burdened by the disease. Current prevention strategies include indoor residual spraying and long-lasting insecticide-treated bed nets.2 While drugs and prevention strategies have proven successful in reducing disease burden in some countries, few will argue that current strategies in these fields are sufficient. Vast amounts of money today is being directed to malaria vaccine development programs: arguably the most promising public health measure for the control and eventual eradication of malaria.
Many organizations and government-funded programs are involved in the development of vaccines. The big funding organizations are the Bill and Melinda Gates foundation, the Clinton foundation, and the Global Fund to Fight AIDS, Tuberculosis, and Malaria. Research is spear headed by US Military Malaria Vaccine Program, the Seattle Biomedical Research Institute, GlaxoSmithKline (GSK), and the PATH Malaria Vaccine Initiative.
Background: P. falciparum and vaccines
Necessary to the understanding of the current debates regarding malaria vaccine strategies is a basic understanding of the parasite and its effect on humans. Currently there are five know species of malaria that infect humans: Plasmodium falciparum, the most deadly cause of malaria, Plasmodium vivax, the most prevalent cause of malaria, Plasmodium ovale, Plasmodium knowlesi, and Plasmodium malariae.3 Because of its heightened lethal effects, P. falciparum is the overwhelming focus of vaccine development efforts. The most deadly complication of falciparum infection is cerebral malaria, which results in a coma due to tissue hypoxia in the central nervous system.3 P. falciparum parasites insert electron-dense knobs into the surface of the membrane of the red blood cells they infect; these knobs cause adhesion to the vascular walls causing hypoxia and infarction.4 These knobs can also cause adhesion between multiple erythrocytes causing “rosetting” or clumping; this is also responsible for the complicating effects of falciparum.4
When researchers consider vaccine strategies, one criterion assessed is the optimal site of intervention in the life cycle of P. falciparum. Sexual reproduction occurs in mosquitoes of the Anopheles genus, which then transmit infective sporozoites to the human host when taking a blood meal. It has been estimated that 100-300 sporozoites are transferred to the host when the mosquito is feeding.5 Sporozoites are then carried through the blood stream to the liver where they infect hepatocytes and develop into merozoites. This entire process takes five to seven days. Vaccines that stop the malarial life cycle before the development of merozoites are known as pre-erythrocytic vaccines. Upon rupture of the hepatocyte, tens of thousands of merozoites emerge and begin the erythrocytic cycle—the periodic infection and rupture of erythrocytes. This is the stage that causes the symptoms associated with malaria. Vaccines that interrupt proliferation of the virus in the erythrocytic stage are known as blood-stage vaccines. Some merozoites develop into gametophytes that are then taken up by the mosquito when it feeds. Vaccines that prevent the uptake or development of gametocytes are known as transmission blocking vaccines (TBVs). The gametocytes mature and sexually combine in the mosquito eventually resulting in the formation of infective sporozoites.3
Secondly, modern vaccine approaches provide another point of differentiation between possible vaccine strategies. In the literature, the biggest division is between scientists advocating the use attenuated whole parasites and those advocating the use recombinant antigens in order to induce a host immune system response.6 Whole attenuated parasites are living non-replicating forms of the parasite that no longer have the infective characteristics of the wild type parasite. One strategy for attenuation is radiation and another is genetic engineering (the select knockout of relevant genes). Recombinant antigen vaccines use proteins expressed by the parasite (often on the surface) that trigger immune response simulating infection by the whole parasite. Recombinant antigen vaccines are more selective than whole parasite vaccines.6 They can be synthesized with multiple antigens and they are always formulated with an adjuvant. Adjuvants have only been recently understood. They function as secondary signals that greatly amplify the host immune response by activating different classes of immune cells and processes.7 Toll-like receptor (TLR) agonists are a type of adjuvant that can increase antigen-specific immune responses by as much as 5 times to 500 times.8 Other less favored approaches are DNA and viral-vectored introductions. The approaches are more complicated and have so far received the least amount of attention from the research community. With the basic outline of possible malarial vaccine strategies, let us now look at vaccines in development.
Recombinant Antigen Vaccines: Strength and weaknesses
Based on the two criteria mentioned above, most vaccines for malaria will fit into one of five vaccine categories: pre-erythrocytic whole-organism, pre-erythrocytic recombinant antigen, blood-stage whole-organism, blood-stage recombinant antigen, transmission blocking recombinant antigen (No transmission blocking whole-organism vaccines were encountered in the literature). Because of the limited scope of this paper I will focus on the pre-erythrocytic vaccines and blood-stage recombinant vaccines. These approaches prove to be the most promising. The vaccine strategy with the most potential is the pre-erythrocytic attenuated whole-organism model and this will be discussed last.
Pre-erythrocytic recombinant antigen (PERA) vaccines have been moderately successful. One vaccine in particular, RTS,S/AS02, is currently in its Phase III clinical trial: this makes it the most clinically advanced malaria vaccine in development.9 RTS,S is derived from the circumsporozoite protein (CSP) that is expressed on the surface of sporozoites and infected hepatic cells. The vaccine consists of the C-terminus of the CSP fused to the hepatitis B surface antigen.9 The RTS,S antigen is formulated with adjuvant AS02, developed by GSK. “The objective [of adjuvants] is to induce tailored immune responses directed against the pathogen” and AS02 has proved to be very effective at increasing host immune response.8 RTS,S was developed over twenty years ago; consequently, it has been put through a variety of trials: it has “reduced the risk of clinical episodes of malaria in young children by 53 percent over an eight-month follow-up period, and provided 65 percent protection against risk of infection in infants over a six-month follow-up period.”10 In a randomized phase IIb clinical trial in 2022 Mozambican children the prevalence of falciparum was 29% lower in the RTS,S/AS02 cohort after 21 months.11 Although RTS,S is effective, it is by no means comprehensive. Because it is a recombinant antigen vaccine, RTS,S can be relatively cheaply synthesized. Current estimates state that the vaccine will be available for use in children aged 5 years to 17 months in 2012.10
These aspects of RTS,S make it valuable weapon in the fight against malaria, but as can be gleaned from the data presented above, it has limited effectiveness. One reason for incomplete coverage by RTS,S originates from the very specific immune antibody response it generates. There are many surface proteins on sporozoites and infected hepatic cells, and these may be variably expressed. Furthermore, recombinant antigen vaccines are developed based upon a single halpotype, and there may be as many 105 different haplotypes for the gene encoding CSP.12 Mostly, sporozoite antigen diversity is determined by location and physical barriers to gene flow, but there is plenty of overlap (over 21 haplotypes exist in The Gambia alone).12 In the same article (12) one finds a very disconcerting result: “The majority of haplotypes upon which current vaccines are based were found to be present at extremely low frequencies in the global parasite population.” The implications of this are ominous, especially when using focused antibody generators like RTS,S/AS02.
RTS,S/AS02 is the main PERA vaccine in development today. An extensive search through the literature resulted in a select few of other PERA vaccine candidates, but development had been terminated due to poor performance in trials. While results from RTS,S/AS02 trials are significant, it is not potent enough to make possible the eradication of malaria. Other viable vaccine approaches are improving rapidly and getting ready to compete with the RTS,S/AS02 vaccine.
The majority of blood-stage recombinant antigen (BSRA) vaccines are derived from merozoite surface proteins (MSPs), apical membrane antigens (AMAs), and to a lesser degree various surface antigens (VSAs) as well as many others.6 There are no standout candidates in this category. Current efforts are focused on finding the most effective combination of antigens: for example, one combination that has been tested involves MSP-1 and AMA-1. Antibodies of these antigens have been proven to inhibit the invasion of erythrocytes in vitro.9 One group at the Walter Reed Army Institute of Research is developing a multi-antigen, multi-stage vaccine that aims prevent infection and also limit disease if malaria does develop.14 In the proposed vaccine, antigens derived from pre-erythrocytic CSP and liver stage antigen-1 (LSA-1) induce immunity, and antigens derived from blood stage proteins MSP-1 and AMA-1 will serve to limit disease.14 This novel combination will center around RTS,S/AS02, but the added antigens will theoretically increase its scope an efficacy. More testing is needed regarding the interaction of antibody responses when multiple antigens are present, especially ones that are not normally present at the same time during the P. falciparum life cycle.
Like PERA vaccines, BSRA vaccines are more effective when combined with an adjuvant. A unique adjuvant has been described for use with blood-stage P. falciparum vaccinations that is worth noting here because of its specificity and its simplicity to synthesize. The presence of an adjuvant component Hemozoin (HZ) has been discovered and confirmed in blood stage parasites.13 HZ is a malarial heme-detoxification byproduct, and it is able to bind strongly and specifically to toll-like receptor 9 (TLR9) and thereby activate macrophages and dendritic cells. 13 Synthetic Hz (sHZ) is as potent an adjuvant and wild type HZ making sHZ and ideal candidate for development as an adjuvant. Even with a highly potent adjuvant effect inducer, BSRA vaccines face major challenges.
More so than PERA vaccines, BSRA vaccines face the daunting challenge of overcoming antigenic diversity. According to the findings of Barry et al, the diversity of merozoite antigen expression does not vary geographically. Rather, it is hypothesized that the immune response of the host is responsible for the antigenic diversity.12 This opinion is shared Polley and Conway. In 2001 they conducted a study analyzing the gene encoding for AMA-1 and discovered a high degree of polymorphism in a single endemic population. They concluded that naturally acquired protective immune responses in humans were responsible for the selective maintenance of multiple alleles within the population.15 This indicates that antigenic diversity evolved to evade host immune responses. Designing a vaccine that addresses the unusually high amounts of antigenic diversity within a single P. falciparum antigen will take a large amount of additional research. Furthermore, some antigens exist that “are encoded by large multi-gene families and parasites can switch the expression of different genes to facilitate immune evasion.”6 An example of such a antigen is the VSA PfEMPI, which is expressed on the surface of erythrocytes; these are the proteins that form the electron rich “knobs” discussed in the background section.4
Pre-Erythrocytic Whole-Organism Vaccines: the gold standard
The strength of the whole-organism approach lies is the fact that all possible antigens are presented to the immune system so the full spectrum of antibodies can be produced. A test carried out in humans proved that attenuated sporozoites confer immunity for heterologous strains of P. falciparum originating from geographically distinct locations.16 Reasons that research focuses on pre-erythrocytic organisms rather than blood-stage organisms or gametophytes are as follows. First, in the pre-erythocytic stage the number of infected hepatocytes is very low relative to the number of infected erythrocytes in blood stage18: 100-300 infected hepatocytes5 relative to tens of thousands infectious merozoites that emerge from one hepatocyte17 and infect red blood cells. Another reason the pre-erythrocytic stage is targeted is the parasite is not transmittable during this stage. By intervening before erythrocytic infection, individuals are unable to transmit the parasite and they do not experience any symptoms of malaria. Thirdly, it is believed that pre-erythrocytic stages do not exhibit significant antigenic variation17—the major drawback of the blood-stage vaccine approach.
The concept of using live attenuated sporozoites as a vaccine for Plasmodium first arose during the 1970s.16 The recent surge of interest in whole-organism sporozoite vaccines was initiated by Stephen Hoffman in 2002. Hoffman now runs a biotechnology company called Sanaria that is working to scale up production procedures for the isolation of radiation-attenuated Plasmodium falciparum sporozoites from mosquito salivary glands.
Although sporozoites can be attenuated by genetic engineering or chemical treatment, the process approaching clinical trials most rapidly relies on radiation-induced attenuation. There has been success in a human trial where irradiated mosquitoes bit volunteers more than 1,000 times and this conferred almost complete protection to subsequent challenges (94% were protected) up to 42 weeks.16 This method of vaccination has an unbeatable efficacy: the main draw back in vaccine development is the production of a pure, safe, and effective product. The current method employed is the manual dissection of mosquito salivary glands; with a team of six, Sanaria is able to produce 500 doses of vaccine per hour.16 The vaccine, known as Sanaria PfSPZ Vaccine, is currently in clinical trials following FDA approval: the current trial is a dose escalation study.16 One complication of using an attenuated whole organism vaccine is the transport to and storage at immunization sites as the live cells need to remain cryogenically frozen. Without proper transportation infrastructure, rural and poor populations will be unable to use the vaccine. Sanaria believes that distribution in the liquid nitrogen vapor phase eliminates transportation difficulties because there is no need for refrigerated trucks or electricity at the storage site.16 Sanaria PfSPZ Vaccine is exceedingly promising. The effort now is focused on engineering a suitable production facility—to me that is as possible, if not more probable, then comprehending the complexities of antigenic variation and interaction in Plasmodium falciparum. As gene deletion technology catches up with radiation attenuation, Sanaria is prepared to take on its production as well.16
Currently there are a handful of P. falciparum sporozoite genes where deletion has resulted in successful termination of parasite proliferation during the liver stage. Overviewed by Vaughan et al, the gene knockouts are successful at the following loci: jointly at p52 and p36, sap1, jointly at uis3 and uis4, and fabb/f.17 P36/p52 and sap1 cause termination at earlier stages in liver development.17 Questions about the advantages of having longer liver stage development remain as it allows the host immune system exposure to a greater number of antigens but also has a high risk of progressing to blood-stage malaria.
Ahmed et al showed the power of sap1 deletion in a rodent model using Plasmodium yoelii. Sap1 plays an essential role in establishing infection in the liver; its specific role is believed to be post-transcriptional gene expression control.19 The study shows that sap1 is a very safe choice for vaccine development: even at exorbitantly high concentrations of intravenously delivered sap1- sporozoites, no infection proceeded past the liver stage.19 Lastly, the deletion of sap1 does not interfere with the expression of important membrane proteins (such as CSP) that function as antigens stimulating immune response and sterile protection.19 There is a sap1 analogue in P. falciparum and both forms have similar expression patterns, however genetic attenuation of the sap1 gene has not been attempted in P. falciparum.
P. falciparum has been successfully genetically engineered to express deletions at the p36 and p52 loci by VanBuskirk et al. Parasites that harbor deletions are known as genetically attenuated parasites or GAPs. Dual deletion of the p36 and p52 loci caused complete parasite developmental arrest during the liver stages.18 The p36-/p52- GAPs were introduced to human hepatocytes in vitro as well as humanized livers of mice in vivo. Both experiments resulted in consistent termination of parasite proliferation in the liver stages: after four days there was no sign of Plasmodium in the humanized mice liver cells.18 The study presented here confirms the safety of genetic attenuation as a procedure for creating whole-organism vaccines. The next step in the development of GAP vaccines is the production of a master cell bank that can support quantities of vaccine necessary for FDA sanctioned trials.
What I have argued in this paper is that the pre-erythrocytic stage is the most promising for vaccine intervention, and that the preferable method of vaccination is the use of attenuated whole-organisms, specifically sporozoites.
Attempted intervention during the erythrocytic cycle has many obstacles. Dramatic variation of the various antigens targeted for vaccine development severely limits the reach of a vaccine. Also, during this stage of the infection hundreds of thousand of merozoites can be found circulating through the blood. Complete elimination of merozoites is a lofty expectation. Furthermore, transmission is harder to control once the infection reaches the blood.
Transmission blocking vaccines seem to have been abandoned. The vast majority of deaths caused by malaria occur in children under five years old. Children usually die of malarial complications that arise because of a relative lack of exposure to the P. falciparum parasite. With this in mind, a transmission blocking vaccine will not save the lives of those the malarial vaccine campaigns are aiming to save. It allows the development of the infection and this can be lethal in P. falciparum naēve children. Perhaps an altruistic vaccine will be favored in the future when drugs to treat P. falciparum malaria become more available to children living in endemic areas.
In the malarial vaccine development community there is a race between supporters of recombinant antigen vaccines and those of attenuated whole-organism vaccines. Supporters of recombinant antigen vaccines are very close to seeing the first malaria vaccine approved for general use. The biggest set back to antigen specific vaccine development is not due to production compexities—authors stress the cost-effectiveness of this approach—but rather a need for more detailed understanding of antigen variation. The whole organism approach lacks the productive capacity as of now, but they are in possession of a far more powerful and reliable vaccine. I trust that engineers the world over are up to the challenge of designing production and purification processes that will dramatically increase the availability of these incredibly valuable vaccines.
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2 Bill & Melinda Gates Foundation. “Malaria Strategy Overview.” <http://www.gatesfoundation.org/global-health/Documents/malaria-strategy.pdf>
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13 Coban, Cevayir et al. “Immunogenicity of Whole-Parasite Vaccines against Plasmodium falciparum Involves Malarial Hemozoin and Host TLR9.” Cell Host & Microbe 7 (2010): 50-61. Web. 12 Feb. 2010.
14 Heppner, D. Gray et al “Towards an RTS,S-based, multi-stage, multi-antigen vaccine against falciparum malaria: progress at the Walter Reed Army Institute of Research.” Vaccine 23 (2005): 2243-2250. Web. 25 Feb. 2010.
15 Polley, Spencer and David Conway. “Strong Diversifying Selection on Domains of the Plasmodium falciparum Apical Membrane Antigen 1 Gene.” Genetics 158 (2001): 1502-1512. Web. 20 Feb. 2010.
16 Hoffman, Stephen et al. “Development of a metabolically active, non-replicating sporozoite vaccine to prevent Plasmodium falciparum malaria.” Human Vaccines 6.1 (2010): 97-106. Web. 20 Feb. 2010.
17 Vaughan, Ashley M et al. “Genetically engineered, attenuated whole-cell vaccine approaches for malaria.” Human Vaccines 6.1 (2010): 107-113. Web. 24 Feb. 2010
18 VanBuskirk, Kelley M et al. “Preerythrocytic, live-attenuated Plasmodium falciparum vaccine candidates by design.” PNAS 106.31 (2009): 13004-9. Web. 20 Feb. 2010.
19 Aly, Ahmed S I et al. “Targeted deletion of SAP1 abolishes the expression of infectivity factors necessary for successful malaria parasite liver infection.” Molecular Microbiology 69.1 (2008): 152-163. Web. 20 Feb. 2010.