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The completion of the genome sequence for Plasmodium falciparum, the species responsible for most malaria human deaths, has the potential to reveal hundreds of new drug targets and proteins involved in pathogenesis. However, only approximately 35% of the genes code for proteins with an identifiable function. The absence of routine genetic tools for studying Plasmodium parasites suggests that this number is unlikely to change quickly if conventional serial methods are used to characterize encoded proteins. Here, we use a high-density oligonucleotide array to generate expression profiles of human and mosquito stages of the malaria parasite's life cycle. Genes with highly correlated levels and temporal patterns of expression were often involved in similar functions or cellular processes.

The completion of the Plasmodium falciparum clone 3D7 genome provides a basis on which to conduct comparative proteomics studies of this human pathogen. Here, we applied a high-throughput proteomics approach to identify new potential drug and vaccine targets and to better understand the biology of this complex protozoan parasite. We characterized four stages of the parasite life cycle (sporozoites, merozoites, trophozoites and gametocytes) by multidimensional protein identification technology. Functional profiling of over 2,400 proteins agreed with the physiology of each stage. Unexpectedly, the antigenically variant proteins of var and rif genes, defined as molecules on the surface of infected erythrocytes, were also largely expressed in sporozoites. The detection of chromosomal clusters encoding co-expressed proteins suggested a potential mechanism for controlling gene expression.

The gene for the circumsporozoite (CS) protein of Plasmodium falciparum has been cloned and its nucleotide sequence determined. The gene encodes a protein of 412 amino acids as deduced from the nucleotide sequence. The protein contains 41 tandem repeats of a tetrapeptide, 37 of which are Asn-Ala-Asn-Pro and four of which are Asn-Val-Asp-Pro. Monoclonal antibodies against the CS protein of Plasmodium falciparum were inhibited from binding to the protein by synthetic peptides of the repeat sequence. The CS protein of Plasmodium falciparum and the CS protein of a simian malaria parasite, Plasmodium knowlesi, have two regions of homology, one of which is present on either side of the repeat. One region contains 12 of 13 identical amino acids. Within the nucleotide sequence of this region, 25 of 27 nucleotides are conserved. The conservation of these regions in parasites widely separated in evolution suggests that they may have a function such as binding to liver cells and may represent an invariant target for immunity.

The commitment of Plasmodium merozoites to invade red blood cells (RBCs) is marked by the formation of a junction between the merozoite and the RBC and the coordinated induction of the parasitophorous vacuole. Despite its importance, the molecular events underlying the parasite's commitment to invasion are not well understood. Here we show that the interaction of two parasite proteins, RON2 and AMA1, known to be critical for invasion, is essential to trigger junction formation. Using antibodies (Abs) that bind near the hydrophobic pocket of AMA1 and AMA1 mutated in the pocket, we identified RON2's binding site on AMA1. Abs specific for the AMA1 pocket blocked junction formation and the induction of the parasitophorous vacuole. We also identified the critical residues in the RON2 peptide (previously shown to bind AMA1) that are required for binding to the AMA1 pocket, namely, two conserved, disulfide-linked cysteines. The RON2 peptide blocked junction formation but, unlike the AMA1-specific Ab, did not block formation of the parasitophorous vacuole, indicating that formation of the junction and parasitophorous vacuole are molecularly distinct steps in the invasion process. Collectively, these results identify the binding of RON2 to the hydrophobic pocket of AMA1 as the step that commits Plasmodium merozoites to RBC invasion and point to RON2 as a potential vaccine candidate.AMA1-RON2 | malaria | moving junction A picomplexa are a group of parasitic protozoa, including the human malaria parasite Plasmodium falciparum (Pf) and Toxoplasma gondii (Tg), that use apical secretory organelles to invade host cells. Invasion of Plasmodium spp. merozoites into erythrocytes begins with an initial weak attachment of the merozoite to the red blood cell (RBC) surface through yet-unidentified parasite receptor-RBC ligand interactions, followed by a reorientation that ultimately brings the apical end of the merozoite into close apposition with the RBC surface (1, 2). The merozoite then triggers the formation of a junction with the erythrocyte that by electron microscopy appears as a dense area below the erythrocyte membrane at the site of the merozoite's apposed apical end. In addition, the merozoite secretes its rhoptry contents into the RBC that may facilitate the invasion of the merozoite (2-4). The merozoite subsequently moves through the junction as it pulls itself into the RBC through connections between parasite surface proteins and its actin-myosin motor (5). Hence, the formation of the junction and its connection with the molecular motor through the cytoplasmic tail of parasite receptors is critical for invasion (6, 7). Formation of the parasitophorous vacuole, created by the inward flow of the RBC membrane (8-10), occurs coordinately with the entry of the parasite into the RBC (4). At the end of invasion, the electron-dense junction becomes part of the parasitophorous vacuole that surrounds the newly invaded parasite (2). In cytochalasin-treated merozoites where actin polymerization is disrupted, the parasites apically ...

Intermittent episodes of febrile illness are the most benign and recognized symptom of infection with malaria parasites, although the effects on parasite survival and virulence remain unclear. In this study, we identified the molecular factors altered in response to febrile temperature by measuring differential expression levels of individual genes using high-density oligonucleotide microarray technology and by performing biological assays in asexual-stage Plasmodium falciparum parasite cultures incubated at 37°C and 41°C (an elevated temperature that is equivalent to malaria-induced febrile illness in the host). Elevated temperature had a profound influence on expression of individual genes; 336 of approximately 5,300 genes (6.3% of the genome) had altered expression profiles. Of these, 163 genes (49%) were upregulated by twofold or greater, and 173 genes (51%) were downregulated by twofold or greater. In-depth sensitive sequence profile analysis revealed that febrile temperature-induced responses caused significant alterations in the major parasite biologic networks and pathways and that these changes are well coordinated and intricately linked. One of the most notable transcriptional changes occurs in genes encoding proteins containing the predicted Pexel motifs that are exported into the host cytoplasm or inserted into the host cell membrane and are likely to be associated with erythrocyte remodeling and parasite sequestration functions. Using our sensitive computational analysis, we were also able to assign biochemical or biologic functional predictions for at least 100 distinct genes previously annotated as "hypothetical." We find that cultivation of P. falciparum parasites at 41°C leads to parasite death in a time-dependent manner. The presence of the "crisis forms" and the terminal deoxynucleotidyltransferasemediated dUTP-biotin nick end labeling-positive parasites following heat treatment strongly support the notion that an apoptosis-like cell death mechanism might be induced in response to febrile temperatures. These studies enhance the possibility of designing vaccines and drugs on the basis of disruption in molecules and pathways of parasite survival and virulence activated in response to febrile temperatures.

Abstract. Plasmodium falciparum malaria parasites invade human erythrocytes by means of a parasite receptor for erythrocytes, the 175-kD erythrocyte binding antigen . Similar to invasion efficiency, binding requires N-acetylneuraminic acid (Neu5Ac) on human erythrocytes, specifically the glycophorins. EBA-175 bound to erythrocytes with receptor-like specificity and was saturable . The specificity of EBA-175 binding was studied to determine if its binding is influenced either by simple electrostatic interaction with the negatively charged Neu5Ac (on the erythrocyte surface) ; or if Neu5Ac indirectly affected the conformation of an unknown ligand, or if Neu5Ac itself in specific linkage and carbohydrate composition was the primary ligand for EBA175 as demonstrated for hemagglutinins of influenza viruses . Most Neu5Ac on human erythrocytes is linked to galactose by a2-3 and a2-6 linkages on glycophorin A . Soluble Neu5Ac by itself in solution did not competitively inhibit the binding of F invasion of malaria parasites into human erythrocytes were prevented, the malaria life cycle would be interrupted and disease prevented . Thus, considerable effort has gone into understanding the molecular basis of parasite invasion into erythrocytes . Invasion requires recognition by the parasite of the appropriate host cell during one or more steps thatinclude merozoite attachment, apical reorientation, apical junction formation, release of the contents of apical organelles, and entry into the erythrocyte (16, 21). The glycophorins, major sialoglycoproteins present on the erythrocyte surface, appear to be responsible for the sialic aciddependent invasion into erythrocytes by Plasmodium falciparum malaria merozoites (10,11,16,18,29,30,(33)(34)(35)(36) Similarly, enzymatic modification of erythrocyte surface molecules by either trypsin or neuraminidase, treatments that have a direct effect on the integrity of the glycophorins, render the erythrocyte resistant to invasion (4,16,18,33) .In parallel experiments, erythrocytes with modifications or deficiencies in the glycophorins also had marked reductions in the binding of the 175-kD erythrocyte binding anti-

We report the first safety and immunogenicity trial of the Plasmodium falciparum vaccine candidate FMP2.1/AS02A, a recombinant E. coli-expressed protein based upon the apical membrane antigen-1 (AMA-1) of the 3D7 clone formulated with the AS02A adjuvant. We conducted an open-label, staggered-start, dose-escalating Phase I trial in 23 malaria-naïve volunteers who received 8, 20 or 40 g of FMP2.1 in a fixed volume of 0.5 mL of AS02A on a 0, 1, and 2 month schedule. Nineteen of 23 volunteers received all three scheduled immunizations. The most frequent solicited local and systemic adverse events associated with immunization were injection site pain (68%) and headache (29%). There were no significant laboratory abnormalities or vaccine-related serious adverse events. All volunteers seroconverted after second immunization as determined by ELISA. Immune sera recognized sporozoites and merozoites by immunofluorescence assay (IFA), and exhibited both growth inhibition and processing inhibition activity against homologous (3D7) asexual stage parasites. Post-immunization, peripheral blood mononuculear cells exhibited FMP2.1-specific lymphoproliferation and IFN-␥ and IL-5 ELISPOT assay responses. This is the first PfAMA-1-based vaccine shown to elicit both potent humoral and cellular immunity in humans. Encouraged by the potential of FMP1/AS02A to target host immunity against PfAMA-1 that is known to be expressed by sporozoite, hepatic and erythrocytic stages, we have initiated field trials of FMP2.1/AS02A in an endemic population in the Republic of Mali.