A Library of Functional Recombinant Cell-surface and Secreted P. falciparum Merozoite Proteins

Malaria, an infectious disease caused by parasites of the Plasmodium genus, is one of the world''s major public health concerns causing up to a million deaths annually, mostly because of P. falciparum infections. All of the clinical symptoms are associated with the blood stage of the disease, an obligate part of the parasite life cycle, when a form of the parasite called the merozoite recognizes and invades host erythrocytes. During erythrocyte invasion, merozoites are directly exposed to the host humoral immune system making the blood stage of the parasite a conceptually attractive therapeutic target. Progress in the functional and molecular characterization of P. falciparum merozoite proteins, however, has been hampered by the technical challenges associated with expressing these proteins in a biochemically active recombinant form. This challenge is particularly acute for extracellular proteins, which are the likely targets of host antibody responses, because they contain structurally critical post-translational modifications that are not added by some recombinant expression systems. Here, we report the development of a method that uses a mammalian expression system to compile a protein resource containing the entire ectodomains of 42 P. falciparum merozoite secreted and cell surface proteins, many of which have not previously been characterized. Importantly, we are able to recapitulate known biochemical activities by showing that recombinant MSP1-MSP7 and P12-P41 directly interact, and that both recombinant EBA175 and EBA140 can bind human erythrocytes in a sialic acid-dependent manner. Finally, we use sera from malaria-exposed immune adults to profile the relative immunoreactivity of the proteins and show that the majority of the antigens contain conformational (heat-labile) epitopes. We envisage that this resource of recombinant proteins will make a valuable contribution toward a molecular understanding of the blood stage of P. falciparum infections and facilitate the comparative screening of antigens as blood-stage vaccine candidates.Parasites of the Plasmodium genus are the etiological agents responsible for malaria, an infectious disease mostly occurring in developing countries with up to 40% of the world''s population described as being at risk of the disease. Among the Plasmodium species that can affect humans, Plasmodium falciparum is responsible for the highest mortality, causing around one million deaths annually, mostly in children under the age of five (1). The clinical symptoms of malaria occur during the cyclic asexual blood stage of the parasite lifecycle when merozoites, that have invaded and replicated within host erythrocytes, are released into the bloodstream before invading new red blood cells (2). Despite intensive efforts from the research community there is currently no licensed vaccine for malaria. The leading candidate RTS,S/AS01, which targets the pre-erythrocytic stage of the disease and was tested in phase III trials, conferred 30 to 50% protection from clinical malaria, depending on the age group studied (3, 4). This limited efficacy has led to calls for a more effective vaccine and many have suggested that a combinatorial vaccine that additionally targets the blood stage may increase efficacy.A vaccine targeting the proteins expressed on the surface of the blood stage of the parasite is conceptually attractive because merozoites are repeatedly and directly exposed to the human humoral immune system and naturally acquired antibodies against these proteins have been shown to confer at least partial immunity (5–8). Despite this, only a few antigens discovered before the completion of the parasite genome sequence have been assessed in detail (9) and clinical vaccine trials using antigens that target the blood stage have so far shown limited efficacy, mostly caused by antigenic diversity (10). The sequencing of the parasite genome (11) has identified all possible targets but the systematic screening of these new candidates to assess their potential as a vaccine is hampered by the inability to systematically express recombinant Plasmodium proteins in their native conformation (12–15). Likely explanations might be the high (∼80%) A:T content of the P. falciparum genome resulting in low codon usage compatibility in heterologous expression systems, the large size (> 50 kDa) of many proteins, the presence of long stretches of highly repetitive amino acids, and the difficulty in identifying clear structural domains within these proteins using standard prediction computer programs (11). Extracellular proteins, in particular, present an additional challenge because they often have signal peptides and transmembrane regions that can negatively impact expression (16–18) and contain structurally important disulfide bonds. However, unlike most other eukaryotic extracellular proteins, Plasmodium cell surface and secreted proteins are not modified by N-linked glycans because of the absence of the necessary enzymes (19).To express Plasmodium proteins for basic research and vaccine development, a diverse range of expression systems have been tried (12) ranging from bacteria (17, 18), yeast (13), Dictyostelium (20), and plants (21) to mammalian cells (22) and cell-free systems (23–25). To circumvent the problem of codon usage, bacterial (26) and yeast (27) strains with modified tRNA pools have been developed, or sequences of the gene of interest synthesized and codon-optimized to match that of the expression host (28, 29). Although Escherichia coli has been the most popular expression system because of its relative simplicity and cost effectiveness, large-scale production of soluble functional Plasmodium falciparum recombinant proteins remains challenging with success rates ranging from just 6 to 21% (17, 18) and is often hindered by the need for complex refolding procedures. Similarly, attempts have been made to compile large panels of parasite proteins using in vitro translation systems (23, 25, 30, 31). These systems, however, require reducing conditions and are therefore not generally suitable for the systematic expression of extracellular proteins that occupy an oxidizing environment and critically require the formation of disulfide bonds for proper function. As a result, functional analyses of extracellular parasite proteins have often been restricted to smaller subfragments of the proteins that can be expressed in a soluble form rather than the entire extracellular region. Although eukaryotic expression systems are able to add disulfide bonds, they also often inappropriately glycosylate parasite proteins, adding further complication (32). A generic method that would overcome these technical challenges to express, in a systematic way, panels of recombinant Plasmodium proteins that have retained their native function and conformation would therefore be a valuable resource for the molecular investigations of erythrocyte invasion and the development of a blood stage vaccine.To generate a resource of correctly folded recombinant merozoite proteins, we used a mammalian expression system and established the parameters necessary for high-level expression. Using this method, we compiled a panel of 42 proteins that corresponds to the repertoire of abundant cell surface and secreted merozoite proteins of the 3D7 strain of Plasmodium falciparum. Biochemical activity of these proteins was demonstrated by recapitulating known protein interactions and by showing conformation-sensitive immunoreactivity of the recombinant proteins using immune sera.