One of the major bottlenecks in malaria research has been the difficulty in recombinant protein expression. Here, we report the application of the wheat germ cell-free system for the successful production of malaria proteins. For proof of principle, the Pfs25, PfCSP, and PfAMA1 proteins were chosen. These genes contain very high A/T sequences and are also difficult to express as recombinant proteins. In our wheat germ cell-free system, native and codon-optimized versions of the Pfs25 genes produced equal amounts of proteins. PfCSP and PfAMA1 genes without any codon optimization were also expressed. The products were soluble, with yields between 50 and 200 g/ml of the translation mixture, indicating that the cell-free system can be used to produce malaria proteins without any prior optimization of their biased codon usage. Biochemical and immunocytochemical analyses of antibodies raised in mice against each protein revealed that every antibody retained its high specificity to the parasite protein in question. The development of parasites in mosquitoes fed patient blood carrying Plasmodium falciparum gametocytes and supplemented with our mouse anti-Pfs25 sera was strongly inhibited, indicating that both Pfs25-3D7/WG and Pfs25-TBV/WG retained their immunogenicity. Lastly, we carried out a parallel expression assay of proteins of blood-stage P. falciparum. The PCR products of 124 P. falciparum genes chosen from the available database were used directly in a small-scale format of transcription and translation reactions. Autoradiogram testing revealed the production of 93 proteins. The application of this new cell-free system-based protocol for the discovery of malaria vaccine candidates will be discussed.Plasmodium falciparum is the protozoan responsible for the widespread return of malaria to tropical countries, particularly in Africa. This reemergence is generally credited to two causes: the development of multidrug-resistant parasites and the development of insecticide-resistant mosquitoes (10). Through decades of work, scientists have learned that vaccination could be a potent curative, but efforts to develop a successful vaccine have not yet succeeded (25). One of the bottlenecks in vaccine development is at the malaria protein production step and is mainly due to the lack of a methodology to enable preparation of quality proteins in an efficient manner. P. falciparum genes have a very high A/T content (average, 76% per gene), and a number of them encode repeated stretches of amino acid sequences (8); these features have been proposed as the major factors limiting P. falciparum protein expression in cell-based systems. Moreover, the presence of glycosylation machinery in eukaryotic cell-based systems can produce inappropriately glycosylated recombinant malaria proteins, resulting in incorrect immune responses (9,21,26). In fact, the three pioneering genome-wide studies on the production of P. falciparum proteins in cell-based systems faced serious problems. For instance, Aguiar et al. (1) were able to obtain exp...

Abbreviations: aa, amino acid(s); Ab, antibody; AMA1, apical membrane antigen 1; GST, Glutathione S transferase; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RON, rhoptry neck protein. AbstractErythrocyte invasion is an essential step in the establishment of host infection by malaria parasites, and is a major target of intervention strategies that attempt to control the disease.Recent proteome analysis of the closely-related apicomplexan parasite, Toxoplasma gondii, falciparum, suggesting that co-operative function of the rhoptry and microneme proteins is a common mechanism in apicomplexan parasites during host cell invasion. PfRON2 possesses a region displaying homology with the rhoptry body protein PfRhopH1/Clag, a component of the RhopH complex. However, here we present co-immunoprecipitation studies which suggest that PfRON2 is not a component of the RhopH complex and has an independent role. Nucleotide polymorphism analysis suggested that PfRON2 was under diversifying selective pressure. This evidence suggests that RON2 appears to have a fundamental role in host cell invasion by apicomplexan parasites, and is a potential target for malaria intervention strategies.

The major virulence determinant of the rodent malaria parasite, Plasmodium yoelii, has remained unresolved since the discovery of the lethal line in the 1970s. Because virulence in this parasite correlates with the ability to invade different types of erythrocytes, we evaluated the potential role of the parasite erythrocyte binding ligand, PyEBL. We found 1 amino acid substitution in a domain responsible for intracellular trafficking between the lethal and nonlethal parasite lines and, furthermore, that the intracellular localization of PyEBL was distinct between these lines. Genetic modification showed that this substitution was responsible not only for PyEBL localization but also the erythrocyte-type invasion preference of the parasite and subsequently its virulence in mice. This previously unrecognized mechanism for altering an invasion phenotype indicates that subtle alterations of a malaria parasite ligand can dramatically affect host-pathogen interactions and malaria virulence.dense granule ͉ invasion ͉ malaria ͉ microneme ͉ transfection T he rodent malaria parasite Plasmodium yoelii yoelii has been widely studied to understand the interactions between the malaria parasite and the host cell (1). The nonlethal 17X line mainly infects young erythrocytes (reticulocytes), whereas the lethal 17XL and YM lines infect a wide range of erythrocytes. These lines have previously been studied to identify the genetic determinants of virulence (2, 3). These differences in erythrocyte invasion preference suggest the possible involvement of a parasite ligand that recognizes erythrocyte surface receptors; however, the actual molecular basis of the observed invasion preference differences remains unclear.Erythrocyte invasion by the malaria merozoite is a multistep process, initiated by reversible binding to the erythrocyte surface, followed by the establishment of a tight junction between the apical end of the merozoite and erythrocyte surface and the subsequent movement of the merozoite into the nascent parasitophorous vacuole. Each step involves specific interactions between parasite ligands and erythrocyte receptors. Among the ligands of malaria parasites, the best characterized is a type I integral transmembrane protein encoded by the ebl (erythrocytebinding-like) gene family. Upon release from the micronemes, EBL proteins recognize erythrocyte receptors and initiate the formation of the tight junction. The importance of EBL in malaria virulence is exemplified in the human malaria parasite Plasmodium vivax, which uses an EBL orthologue, PvDBP, to recognize the Duffy antigen on the erythrocyte surface. Because the parasite is apparently unable to use an alternative invasion pathway, individuals in whom the Duffy antigen is not expressed on the erythrocyte surface are completely resistant to P. vivax (4,5). Because of this dramatic association between the disruption of a host-pathogen interaction and protection against a malaria parasite, PvDBP and the Plasmodium falciparum EBL orthologue, EBA-175, have been targeted for ...

One of the solutions for reducing the global mortality and morbidity due to malaria is multivalent vaccines comprising antigens of several life cycle stages of the malarial parasite. Hence, there is a need for supplementing the current set of malaria vaccine candidate antigens. Here, we aimed to characterize glycosylphosphatidylinositol (GPI)-anchored micronemal antigen (GAMA) encoded by the PF08_0008 gene in Plasmodium falciparum. Antibodies were raised against recombinant GAMA synthesized by using a wheat germ cell-free system. Immunoelectron microscopy demonstrated for the first time that GAMA is a microneme protein of the merozoite. Erythrocyte binding assays revealed that GAMA possesses an erythrocyte binding epitope in the C-terminal region and it binds a nonsialylated protein receptor on human erythrocytes. Growth inhibition assays revealed that anti-GAMA antibodies can inhibit P. falciparum invasion in a dose-dependent manner and GAMA plays a role in the sialic acid (SA)-independent invasion pathway. Anti-GAMA antibodies in combination with anti-erythrocyte binding antigen 175 exhibited a significantly higher level of invasion inhibition, supporting the rationale that targeting of both SA-dependent and SA-independent ligands/pathways is better than targeting either of them alone. Human sera collected from areas of malaria endemicity in Mali and Thailand recognized GAMA. Since GAMA in P. falciparum is refractory to gene knockout attempts, it is essential to parasite invasion. Overall, our study indicates that GAMA is a novel blood-stage vaccine candidate antigen.

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