J. David Haynes

A proteomic view of the Plasmodium falciparum life cycle.

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.

Binding of Plasmodium merozoite proteins RON2 and AMA1 triggers commitment to invasion

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.

Protection of Aotus Monkeys by Plasmodium falciparum EBA-175 Region II DNA Prime—Protein Boost Immunization Regimen

Aotus monkeys received 4 doses of Plasmodium falciparum EBA-175 region II vaccine as plasmid DNA (Dv-Dv) or recombinant protein in adjuvant (Pv-Pv) or as 3 doses of DNA and 1 dose of protein (Dv-Pv). After 3 doses, antibody titers were approximately 10(4) in DNA-immunized monkeys and 10(6) in protein-immunized monkeys. A fourth dose did not significantly boost antibody responses in the Dv-Dv only or Pv-Pv only groups, but titers were boosted to approximately 10(6) in monkeys in the Dv-Pv group. Four weeks after the last immunization, the animals were challenged with 10(4) P. falciparum-parasitized erythrocytes. Peak levels of parasitemia were lower in the 16 monkeys that received region II-containing plasmids or proteins than in the 16 controls (geometric mean: 194,178 and 410,110 parasites/microL, respectively; P=.013, Students t test). Three of 4 monkeys in the Dv-Pv group did not require treatment. These data demonstrate that immunization with EBA-175 region II induces a significant antiparasite effect in vivo.

Plasmodium falciparum: assessment of in vitro growth by [3H]hypoxanthine incorporation.

To evaluate rapidly Plasmodium falciparum growth in Vitro, [3H]hypoxanthine was added to parasite microcultures and radioisotope incorporation was measured. When culture parameters were carefully controlled, [3H]hypoxanthine incorporation was proportional to the number of parasitized erythrocytes present. Factors affecting [3H]hypoxanthine incorporation included initial parasitemia, duration of culture, duration of radioisotope pulse, parasite stage, concentration of uninfected erythrocytes, the use of serum or plasma to supplement growth, and the concentration of a variety of purines in the culture medium. The method described can be used to measure inhibition of P. falciparum growth by immune serum and has previously been used to study antimalarial drug activity in vitro.

Purification, Characterization, and Immunogenicity of the Refolded Ectodomain of the Plasmodium falciparum Apical Membrane Antigen 1 Expressed in Escherichia coli

ABSTRACT The apical membrane antigen 1 (AMA1) has emerged as a promising vaccine candidate against malaria. Advanced evaluation of its protective efficacy in humans requires the production of highly purified and correctly folded protein. We describe here a process for the expression, fermentation, refolding, and purification of the recombinant ectodomain of AMA1 (amino acids 83Gly to 531Glu) of Plasmodium falciparum (3D7) produced in Escherichia coli. A synthetic gene containing an E. coli codon bias was cloned into a modified pET32 plasmid, and the recombinant protein was produced by using a redox-modified E. coli strain, Origami (DE3). A purification process was developed that included Sarkosyl extraction followed by affinity purification on a Ni-nitrilotriacetic acid column. The recombinant AMA1 was refolded in the presence of reduced and oxidized glutathione and further purified by using two ion-exchange chromatographic steps. The final product, designated AMA1/E, was homogeneous, monomeric, and >99% pure and had low endotoxin content and low host cell contamination. Analysis of AMA1/E showed that it had the predicted primary sequence, and tertiary structure analysis confirmed its compact disulfide-bonded nature. Rabbit antibodies made to the protein recognized the native parasite AMA1 and inhibited the growth of the P. falciparum homologous 3D7 clone in an in vitro assay. Reduction-sensitive epitopes on AMA1/E were shown to be necessary for the production of inhibitory anti-AMA1 antibodies. AMA1/E was recognized by a conformation-dependent, growth-inhibitory monoclonal antibody, 4G2dc1. The process described here was successfully scaled up to produce AMA1/E protein under GMP conditions, and the product was found to induce highly inhibitory antibodies in rabbits.

Invasion-inhibitory antibodies inhibit proteolytic processing of apical membrane antigen 1 of Plasmodium falciparum merozoites

Apical membrane antigen 1 (AMA-1) is a promising vaccine candidate for Plasmodium falciparum malaria. Antibodies against AMA-1 of P. falciparum (PfAMA-1) interrupt merozoite invasion into RBCs. Initially localized within the apical complex, PfAMA-1 is proteolytically processed and redistributed circumferentially on merozoites at about the time of their release and invasion into RBCs. An 83-kDa precursor form of PfAMA-1 is processed to 66-kDa and then to 48- and 44-kDa products. We show that, even at low concentrations, IgG antibodies against correctly folded recombinant PfAMA-1 cross-linked and trapped the 52-, 48-, and 44-kDa proteolytic products on merozoites. These products are normally shed into the culture medium. At higher concentrations antibodies inhibited invasion into RBCs and caused a reduction in the amount of 44- and 48-kDa products, both on merozoites and in the culture medium. A corresponding increase also occurred in the amount of the 66- and 52-kDa forms detected on the merozoites. These antibodies also prevented circumferential redistribution of AMA-1. In contrast, monovalent invasion-inhibitory Fab fragments caused accumulation of 66- and 52-kDa forms, with no cross-linking, trapping, or prevention of redistribution. Antibodies at low concentrations can be used as trapping agents for intermediate and soluble forms of AMA-1 and are useful for studying proteolytic processing of AMA-1. With this technique, it was confirmed that protease inhibitor chymostatin and Ca2+ chelators can inhibit the breakdown of the 66-kDa form. We propose that antibodies to AMA-1 capable of inhibiting erythrocyte invasion act by disrupting proteolytic processing of AMA-1.

Molecular factors and biochemical pathways induced by febrile temperature in intraerythrocytic Plasmodium falciparum parasites.

ABSTRACT 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 deoxynucleotidyltransferase-mediated 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.

Selective stage-specific changes in the permeability to small hydrophilic solutes of human erythrocytes infected with Plasmodium falciparum

The permeability characteristics of Plasmodium falciparum-infected human erythrocytes to various 3H-labelled solutes were measured during the maturation of the parasites in sorbitol-synchronised cultures. Using [14C]inulin as the extracellular marker, estimates were made of the influx kinetics of [3H]amino acids into trichloroacetic acid (TCA)-soluble pools within the erythrocyte and concomitant incorporation into TCA-precipitable material. These measurements provided values of the rates of protein synthesis by the parasite and the initial influx rates for the transport of precursor amino acids into the erythrocyte. For about 12-15 h after parasitisation, the influx of L-[3H]glutamine remained at a low level comparable to that in the uninfected cell (2-9 nmol g-1 cells min-1). As pigment appeared in the trophozoite, the initial rate of influx of L-glutamine increased to a value up to 100-fold higher than in the uninfected erythrocyte. The increase in permeability affected only the parasitised cells in a culture of partially infected erythrocytes, and was selective with respect to substrate since the influx kinetics for both [3H]isoleucine and [3H]arginine were not affected by parasitisation. The permeability changes occurred mainly over a 4-8 h period in the development of the young trophozoite, during which time [3H]glycine influx was enhanced by a factor of 3-10, with a comparable increase in the uptake of myo-[3H]inositol. L-[3H]glutamate, which did not penetrate significantly into uninfected erythrocytes, entered red cells infected with mature trophozoites at a rate which was much less than 1% of the parasite-induced-L-glutamine influx. At the stages when the permeability to L-glutamine was markedly enhanced, parasitised cells remained impermeable to [3H]sucrose. An analysis of the relative 3H activities in glutathione and free amino acid pools indicated that, if L-glutamine permeation did not increase during parasite maturation beyond the ring stage, or was blocked by a potential antimalarial compound, an insufficient supply of L-glutamine would be available for the increased rates of parasite protein synthesis and glutathione turnover within the red cell.

Characterization of the 175-kilodalton erythrocyte binding antigen of Plasmodium falciparum

EBA-175 is a soluble 175-kDa Plasmodium falciparum antigen that is released into culture supernatants during rupture of schizont-infected erythrocytes. EBA-175 binds to erythrocytes and binding is sialic acid-dependent. A clone expressing the gene encoding EBA-175 was obtained previously by screening a genomic DNA expression library with antibodies that had been affinity-purified from EBA-175. Antibodies were raised against a 43-amino-acid peptide (EBA-peptide 4) synthesized according to the deduced amino acid sequence. Antibodies to peptide 4 and affinity-purified antibodies specific for EBA-175 were used to characterize further EBA-175 giving the following results: (1) EBA-175 differs biochemically and immunologically from other reported malarial antigens; (2) the EBA-175s from six geographical isolates of P. falciparum are antigenically conserved; (3) EBA-175 is expressed during schizogony as a 190-kDa protein which is larger than the culture supernatant form of the antigen. The 190-kDa form of the protein is recovered from the cell pellet in schizont-infected erythrocytes and partitions to the soluble fraction when extracted with detergent; (4) release of soluble EBA-175 into the culture supernatant coincides with schizont rupture; (5) there was no observable change in pI (pI = 6.86) by isoelectric focusing between the cellular and supernatant species of the protein; and (6) release of EBA-175 into the culture supernatant is inhibited by the addition of chymostatin and leupeptin. The continued research into the role of EBA-175 during erythrocyte invasion may aid in vaccine development for malaria.

High antibody titer against apical membrane antigen-1 is required to protect against malaria in the Aotus model.

A Plasmodium falciparum 3D7 strain Apical Membrane Antigen-1 (AMA1) vaccine, formulated with AS02A adjuvant, slowed parasite growth in a recent Phase 1/2a trial, however sterile protection was not observed. We tested this AS02A, and a Montanide ISA720 (ISA) formulation of 3D7 AMA1 in Aotus monkeys. The 3D7 parasite does not invade Aotus erythrocytes, hence two heterologous strains, FCH/4 and FVO, were used for challenge, FCH/4 AMA1 being more homologous to 3D7 than FVO AMA1. Following three vaccinations, the monkeys were challenged with 50,000 FCH/4 or 10,000 FVO parasites. Three of the six animals in the AMA+ISA group were protected against FCH/4 challenge. One monkey did not become parasitemic, another showed only a short period of low level parasitemia that self-cured, and a third animal showed a delay before exhibiting its parasitemic phase. This is the first protection shown in primates with a recombinant P. falciparum AMA1 without formulation in Freunds complete adjuvant. No animals in the AMA+AS02A group were protected, but this group exhibited a trend towards reduced growth rate. A second group of monkeys vaccinated with AMA+ISA vaccine was not protected against FVO challenge, suggesting strain-specificity of AMA1-based protection. Protection against FCH/4 strain correlated with the quantity of induced antibodies, as the protected animals were the only ones to have in vitro parasite growth inhibitory activity of >70% at 1∶10 serum dilution; immuno-fluorescence titers >8,000; ELISA titers against full-length AMA1 >300,000 and ELISA titer against AMA1 domains1+2 >100,000. A negative correlation between log ELISA titer and day 11 cumulative parasitemia (Spearman rank r = −0.780, p value = 0.0001), further confirmed the relationship between antibody titer and protection. High titers of cross-strain inhibitory antibodies against AMA1 are therefore critical to confer solid protection, and the Aotus model can be used to down-select future AMA1 formulations, prior to advanced human trials.