Malaria is the most widespread deadly parasitic disease, affecting 500 million individuals and causing the death of 1–2 million children under 5 years of age and, among adults, mainly prima gravida women. Vaccines against malaria have been the object of constant endeavor by the scientific community and pharmaceutical industries for over three decades, with little success so far. Hopes for developing a vaccine against malaria stem from several observations or experiments:
• People older than 5 years of age and living in disease-endemic areas are partially or totally protected from clinical malaria
• Transfer of purified immunoglobulins obtained from immune individuals diminishes parasitemia in affected individuals
• Infants are protected during the first 6 months of life, most likely due to the presence of maternal immunoglobulins in their blood
• Radiation-attenuated sporozoites can confer immunity in humans
In the absence of a malaria vaccine, parasites and disease have been contained by several measures:
• Use of pesticides
• Use of impregnated bednets
• Antimalarial drugs
• Environmental management
Additional important correlates of success in the fight against malaria are a better education and economical status of the population under malaria siege.
This series of measures was undertaken at the beginning of the last century in Italy, with subsequent elimination/eradication of malaria in 1962 Citation[1].
The environmental and economic conditions existing in countries currently affected by malaria do not give us too much hope of achieving eradication in the near future, even though the combined measures taken in the last few years in specific geographical areas are certainly very promising in drastically reducing the local malaria burden.
Past vaccine development
In this scenario, vaccines represent another powerful, cost-effective tool with limited temporal interventions. On the other hand, one can ask why a malaria vaccine has not yet been fully developed and implemented. The reasons for this failure are multiple and reside in the complexity of the parasite, which expresses over 5000 proteins during its different life stages, the intricate interplay between parasite biology and host immunity, a lack of adequate resources and a lack of effective global cooperation.
Hope and pessimism concerning malaria vaccine development have been recurrent in the last 30 years. As soon as the sequence of the circumsporozoite (CS) protein was published, a number of laboratories started working on a vaccine primarily based on the simple sequence (NANP)n of the CS repeat region. The results obtained regarding the efficacy of the repeat region of the corresponding Plasmodium berghei protein in protecting mice were an optimistic omen for similar results in humans. Unfortunately, the early clinical assays in humans were very disappointing Citation[2,3]. This fostered new research on the CS protein, and numerous trials were designed to test other CS-based constructs and new adjuvants. In the meantime, Patarroyo and colleagues developed a hybrid vaccine by combining together different short epitopes derived from the CS repeat region and three erythrocytic proteins followed by polymerization of the construct.
In clinical trials, none of the constructs consistently protected volunteers against malaria (initial results obtained by Patarroyo’s team were positive) Citation[4,5], with one exception, called RTS,S. This consists of the repeat and C-terminal regions of the CS protein attached to the S protein of HBV and coexpressed with the same S protein. However, protection with RTS,S was only observed in conjunction with the GlaxoSmithKline proprietary AS02A or closely related adjuvants Citation[6].
Current vaccine development
The RTS,S construct formulated with the adjuvant AS02 gave approximately 50% protection from clinical malaria in a Phase IIa trial. Field trials ensued with an efficacy of 30–60% in a 6–12-month interval following the last injection Citation[7,8]. A large-scale Phase III clinical trial is underway for the RTS,S vaccine. Other antigens were tested with negative results.
Given this not very promising scenario why do we continue to believe that development of a malaria vaccine is possible Citation[9]?
There are several reasons to believe that malaria vaccine development has not been optimal during the last 25 years since the CS protein was cloned.
First, the number of antigens developed was limited and, in general, they represented proteins that happened to be accidentally discovered because of their abundance. Second, they were mainly polymorphic and structurally rather complex. Third, a rigorous down-selection approach was never applied to this limited number of vaccine candidates. Some were forced through different phases of clinical trial because there was nothing else to be tested. In addition, until recently, the biological activities and properties of various candidates were never compared in side-by-side tests.
Future hopes
The publication of the Plasmodium falciparum genome gave the illusion that new antigens would quickly be discovered and developed Citation[10]. A total of 7 years after its publication, only a handful of new candidates for further development have emerged. In general, the established laboratories continued to work with their traditional antigens owing to the time previously invested and the conviction that they were close to a successful product.
On the other hand, meeting the same challenges encountered previously – expression of complex molecules, often of limited solubility; refolding to mimic the native 3D structure; and, finally, the gathering of experimental evidence that the chosen antigen is a promising candidate – still remains time-consuming and expensive Citation[11–13].
We tried to solve these problems by exploiting the wealth of genomic data through the rapid production of antigens, such that a few of the most promising ones could be rapidly selected from a large pool Citation[14,15]. For this purpose, we developed a strategy that uses the concept of structural epitopes. Antibodies are raised either against structural/conformational (discontinuous) or linear (continuous) epitopes Citation[16]. While constructing linear epitopes is rather trivial, mimicking conformation-dependent epitopes by peptides 30–50 residues long, which is suitable for multiple-channel chemical synthesis, represents a major challenge to protein chemists, molecular modelers and immunologists.
Previous attempts were focused on engineering artificial mini proteins that are able to mimic the conformational epitopes of native proteins Citation[17,18]. We applied a ‘bioinformatics’ approach that became possible only after the sequencing of the malaria genome. The first step was to select potential conformational epitopes by using a genome-wide, bioinformatics-based approach. It was based on the following principles: the peptide (potential epitope) should be of small size (<∼30–50 residues), which is appropriate for high-throughput multiple-channel peptide synthesis Citation[1]; it should have a native conformation when isolated from the context of the whole protein Citation[2]; and sequence motifs corresponding to the peptide should be easily detected and abundant in the genome Citation[3].
To this end, two simple protein motifs, the a-helical coiled coil and natively unstructured/random coil domains, were chosen. The a-helical coiled coils share a seven-residue repeat (abcdefg)n, containing nonpolar residues at positions a and d, and generally polar residues elsewhere. The coiled coils of over 20 amino acid residues in length are able to fold into a stable native structure Citation[19]. The natively unstructured regions, in contrast to the a-helical coiled coil motifs, do not fold in any particular unique structure Citation[20] and, as previously mentioned, are the most suitable for mimicking native linear epitopes. If long unstructured fragments (>60 amino acids) are present, they can be synthesized as shorter overlapping peptides without losing recognition by specific antibodies. The sequence features of both types of motifs render their bioinformatics-based identification in genomes easily accomplished. Several hundred putative a-helical coiled coil domains longer than 25 amino acid residues and thousands of natively unstructured regions longer than 50 residues were identified in P. falciparum proteins Citation[21] [Kajava A, Corradin G, Unpublished Data].
We chose to focus on proteins associated with the erythrocytic stage of the parasite for two reasons. First, in our opinion, it is not possible to down-select CD4+ and/or CD+8 T-cell epitopes relevant to the hepatic stage in a rational manner. Second, since protection from clinical malaria is associated with IgG and, in particular, IgG1 and IgG3 responses, measurement of recognition of new antigens by antibodies as a first step is rather trivial and hasty. After this initial step, subsequent rigorous downselection procedures, such as the use of affinity-purified human specific antibodies to demonstrate expression of the epitope in parasites, in vitro determination of biological activity, and determination of the polymorphism of the selected fragments and immunogenicity in animal models, are reasonably rapid.
Thus, starting from a large number of segments identified by bioinformatics and chemically synthesized, the most suitable and promising vaccine candidates could be rapidly identified. In 2 years, the fragment P27A of 104 amino acids derived from the novel protein PFF0165c (sequence 223–326) went from initial identification to planning for a Phase I clinical trial Citation[22]. Given the appropriate financial support, a screen of the whole genome for fragments presenting structural features similar to those described above could be performed in a couple of years.
Two important features are associated with the two structural motifs selected. One is the low extent of polymorphism of putative a-helical coiled coil domains found in field and laboratory parasite strains and in the single-nucleotide polymorphism data available at PlasmoDB Citation[23]. The second is the high solubility in aqueous solutions of all a-helical coiled coil or unstructured peptides synthesized (>250). In addition, homologous a-helical coiled coil sequences are present in the Plasmodium vivax parasite such that the development of a cross-reacting vaccine for both P. falciparum and P. vivax can be envisaged.
Other approaches, such as the use of a wheat germ system to rapidly express P. falciparum recombinant proteins, can easily be incorporated in our strategy Citation[13]. Given the fact that the clinical trials needed to demonstrate protection from malaria involve rather limited number of volunteers and short trial durations (500–1000 children; 1–2 years), we are confident that an efficacious vaccine could be developed in a record time, provided a cooperative global effort is established.
The identification and selection strategy outlined here can be easily adopted and applied to other complex pathogens.
Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.
References
- Snowden F. The Conquest of Malaria: Italy, 1900–1962. Yale University Press, CT, USA (2006).
- Herrington DA, Clyde DF, Losansky G et al. Safety and immunogenicity in man of a synthetic peptide malaria vaccine against Plasmodium falciparum sporozoites. Nature328, 257–259 (1987).
- Ballou WR, Hoffman SL, Sherwood JA et al. Safety and efficacy of a recombinant DNA Plasmodium falciparum sporozoite vaccine. Lancet1, 1277–1281 (1987).
- Valero MV, Amador LR, Galindo C et al. Vaccination with Spf 66 a chemically synthesized vaccine, against Plasmodium falciparum malaria in Colombia. Lancet341, 705–710 (1993).
- Alonso PL, Smith T, Armstrong Schellenberg JQM et al. Randomised trial of efficacy of Spf 66 vaccine against Plasmodium falciparum malaria in children in South Tanzania. Lancet344, 1175–1181 (1994).
- Stoute JA, Slaoui M, Heppner D 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.336, 86–91 (1997).
- Alonso PL, Sacarlal J, Aponte JJ 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. Lancet366, 2012–2018 (2005).
- Aponte JJ, Aide P, Renom M et al. Safety of the RTS,S/AS02D candidate malaria vaccine in infants living in a highly endemic area of Mozambique: a double blind randomised controlled Phase I/IIb trial. Lancet370, 1543–1551 (2007).
- Sherman IW. The Elusive Malaria Vaccine: Miracle or Mirage. ASM Press, Washington, DC, USA (2009).
- Gardner MJ, Hall N, Fung E, White O et al. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature419, 498–511 (2002).
- Aguiar JC, LaBaer J, Blair PL. High-throughput generation of P. falciparum functional molecules by recombinational cloning. Genome Res.14, 2076–2082 (2004).
- Mehlin C, Boni E, Buckner FS. Heterologous expression of proteins from Plasmodium falciparum: results from 1000 genes. Mol. Biochem. Parasitol.148, 144–160 (2006).
- Tsuboi T, Takeo S, Iriko H et al. Wheat germ cell-free system-based production of malaria proteins for discovery of novel vaccine candidates. Infect. Immun.76, 1702–1708 (2008).
- Corradin G, Villard V, Kajava AV. Protein structure based strategies for antigen discovery and vaccine development against malaria and other pathogens. Endocr. Metab. Immune Disord. Drug. Targets7, 259–265 (2007).
- Villard V, Agak GW, Frank G et al. Rapid identification of malaria vaccine candidates based on a-helical coiled coil protein motif. PLoS One2, e645 (2007).
- Barlow Barlow DJ, Edwards MS, Thornton JM. Continuous and discontinuous protein antigenic determinants. Nature322, 747–748 (1986).
- Martin L, Vita C. Engineering novel bioactive mini-proteins from small size natural and de novo designed scaffolds. Curr. Protein Pept. Sci.1, 403–430 (2000).
- Lu SM, Hodges RS. A de novo designed template for generating conformation-specific antibodies that recognize a-helices in proteins. J. Biol. Chem.277, 23515–23524 (2002).
- Harbury PB, Zhang T, Kim PS, Alber T. A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants. Science262, 1401–1407 (1993).
- Wright PE, Dyson HJ. Intrinsically unstructured proteins: re-assessing the protein structure-function paradigm. J. Mol. Biol.293, 321–331 (1999).
- Feng ZP, X. Zhang X, Han P, Arora N, Anders RF, Norton RS. Abundance of intrinsically unstructured proteins in P. falciparum and other apicomplexan parasite proteomes. Mol. Biochem. Parasitol.150, 256–267 (2006).
- Olugbile S, Kulangara C, Bang G et al. Vaccine potentials of an intrinsically unstructured fragment derived from the blood stage associated P. falciparum protein PFF0165c. Infect. Immun.77, 5701–5709 (2009).
- Kulangara C, Kajava AV. Corradin G, Felger I. Sequence conservation in Plasmodium falciparum α-helical coiled coil domains proposed for vaccine development. PLoS One4, e5419 (2009).