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Plasmepsin X (PMX) is an aspartyl protease that processes proteins essential for Plasmodium parasites to invade and egress from host erythrocytes during the symptomatic asexual stage of malaria. PMX substrates possess a conserved cleavage region denoted by the consensus motif, SFhE (h=hydrophobic amino acid). Peptidomimetics reflecting the P

Plasmepsins IX (PMIX) and X (PMX) are essential aspartyl proteases for Plasmodium spp. egress, invasion, and development. WM4 and WM382 inhibit PMIX and PMX in Plasmodium falciparum and P. vivax. WM4 inhibits PMX, while WM382 is a dual inhibitor of PMIX and PMX. To understand their function, we identified protein substrates. Enzyme kinetic and structural analyses identified interactions responsible for drug specificity. PMIX and PMX have similar substrate specificity; however, there are distinct differences for peptide and protein substrates. Differences in WM4 and WM382 binding for PMIX and PMX map to variations in the S' region and engagement of the active site S3 pocket. Structures of PMX reveal interactions and mechanistic detail of drug binding important for development of clinical candidates against these targets.

Plasmepsin IX (PMIX) and X (PMX) are aspartyl proteases of Plasmodium spp. that play essential roles in parasite egress, invasion and development. Consequently, they are important drug targets for Plasmodium falciparum and P. vivax. WM4 and WM382 are potent inhibitors of PMIX and PMX that block invasion of liver and blood stages and transmission to mosquitoes. WM4 specifically inhibits PMX whilst WM382 is a dual inhibitor of PMIX and PMX. To understand the function of PMIX and PMX proteases we identified new protein substrates in P. falciparum and together with detailed kinetic analyses and structural analyses identified key molecular interactions in the active site responsible for the specificity of WM4 and WM382 inhibition. The crystal structures of PMX apo enzyme and the protease/drug complexes of PMX/WM382 and PMX/WM4 for P. falciparum and P. vivax have been solved. We show PMIX and PMX have similar substrate selectivity, however, there are distinct differences for both peptide and full-length protein substrates through differences in localised 3-dimensional structures for the enzyme substrate-binding cleft and substrate interface. The differences in affinities of WM4 and WM382 binding for PMIX and PMX map to variations in surface interactions with each protease in the S' region of the active sites. Crystal structures of PMX reveal interactions and mechanistic detail on the selectivity of drug binding which will be important for further development of clinical candidates against these important molecular targets.

PfSERA5, a significantly abundant protein present within the parasitophorous vacuole (PV) and essential for normal growth during the blood-stage life cycle of the malaria parasite Plasmodium falciparum, displays structural similarity to many other cysteine proteases. However, PfSERA5 does not exhibit any detectable protease activity and therefore the role of the PfSERA5 papain-like domain (PfSERA5E), thought to remain bound to its cognate prodomain, remains unknown. In this study, we present a revised structure of the central PfSERA5E domain at a resolution of 1.2 A, and the first structure of the "zymogen" of this papain-like domain including its cognate prodomain (PfSERA5PE) to 2.2 A resolution. PfSERA5PE is somewhat structurally similar to that of other known proenzymes, retaining the conserved overall folding and orientation of the prodomain through, and occluding, the archetypal papain-like catalytic triad "active-site" cleft, in the same reverse direction as conventional prodomains. Our findings are congruent with previously identified structures of PfSERA5E and of similar "zymogens" and provide a foundation for further investigation into the function of PfSERA5.

Plasmepsin V is an aspartyl protease that plays a critical role in the export of proteins bearing the Plasmodium export element (PEXEL) motif (RxLxQ/E/D) to the infected host erythrocyte, and thus the survival of the malaria parasite. Previously, development of transition state PEXEL mimetic inhibitors of plasmepsin V have primarily focused on demonstrating the importance of the P3 Arg and P1 Leu in binding affinity and selectivity. Here, we investigate the importance of the P2 position by incorporating both natural and non-natural amino acids into this position and show disubstituted beta-carbon amino acids convey the greatest potency. Consequently, we show analogues with either cyclohexylglycine or phenylglycine in the P2 position are the most potent inhibitors of plasmepsin V that impair processing of the PEXEL motif in exported proteins resulting in death of P. falciparum asexual stage parasites.

Plasmodium falciparum exports proteins into erythrocytes using the Plasmodium export element (PEXEL) motif, which is cleaved in the endoplasmic reticulum (ER) by plasmepsin V (PMV). A recent study reported that phosphatidylinositol-3-phosphate (PI(3)P) concentrated in the ER binds to PEXEL motifs and is required for export independent of PMV, and that PEXEL motifs are functionally interchangeable with RxLR motifs of oomycete effectors. Here we show that the PEXEL does not bind PI(3)P, and that this lipid is not concentrated in the ER. We find that RxLR motifs cannot mediate export in P. falciparum. Parasites expressing a mutated version of KAHRP, with the PEXEL motif repositioned near the signal sequence, prevented PMV cleavage. This mutant possessed the putative PI(3)P-binding residues but is not exported. Reinstatement of PEXEL to its original location restores processing by PMV and export. These results challenge the PI(3)P hypothesis and provide evidence that PEXEL position is conserved for co-translational processing and export., Export of Plasmodium falciparum proteins into infected erythrocytes relies upon the PEXEL motif in target proteins. Here Boddey et al. challenge the hypothesis that the PEXEL motif mediates export by binding PI(3)P and instead suggest it acts via cleavage by plasmepsin V.

Plasmepsin V is an aspartyl protease that plays a critical role in the export of proteins bearing the Plasmodium export element (PEXEL) motif (RxLxQ/E/D) to the infected host erythrocyte, and thus the survival of the malaria parasite. Previously, development of transition state PEXEL mimetic inhibitors of plasmepsin V have primarily focused on demonstrating the importance of the P

Plasmepsin V, an essential aspartyl protease of malaria parasites, has a key role in the export of effector proteins to parasite-infected erythrocytes. Consequently, it is an important drug target for the two most virulent malaria parasites of humans, Plasmodium falciparum and Plasmodium vivax. We developed a potent inhibitor of plasmepsin V, called WEHI-842, which directly mimics the Plasmodium export element (PEXEL). WEHI-842 inhibits recombinant plasmepsin V with a half-maximal inhibitory concentration of 0.2 nM, efficiently blocks protein export and inhibits parasite growth. We obtained the structure of P. vivax plasmepsin V in complex with WEHI-842 to 2.4-Å resolution, which provides an explanation for the strict requirements for substrate and inhibitor binding. The structure characterizes both a plant-like fold and a malaria-specific helix-turn-helix motif that are likely to be important in cleavage of effector substrates for export.

Increasing evidence suggests that antibodies against merozoite surface proteins (MSPs) play an important role in clinical immunity to malaria. Two unusual members of the MSP-3 family, merozoite surface protein duffy binding-like (MSPDBL)1 and MSPDBL2, have been shown to be extrinsically associated to MSP-1 on the parasite surface. In addition to a secreted polymorphic antigen associated with merozoite (SPAM) domain characteristic of MSP-3 family members, they also contain Duffy binding-like (DBL) domain and were found to bind to erythrocytes, suggesting that they play a role in parasite invasion. Antibody responses to these proteins were investigated in a treatment-reinfection study conducted in an endemic area of Papua New Guinea to determine their contribution to naturally acquired immunity. Antibodies to the SPAM domains of MSPDBL1 and MSPDBL2 as well as the DBL domain of MSPDBL1 were found to be associated with protection from Plasmodium falciparum clinical episodes. Moreover, affinity-purified anti-MSPDBL1 and MSPDBL2 were found to inhibit in vitro parasite growth and had strong merozoite opsonizing capacity, suggesting that protection targeting these antigens results from ≥2 distinct effector mechanisms. Together these results indicate that MSPDBL1 and MSPDBL2 are important targets of naturally acquired immunity and might constitute potential vaccine candidates.

Plasmodium falciparum causes the severe form of malaria that has high levels of mortality in humans. Blood-stage merozoites of P. falciparum invade erythrocytes, and this requires interactions between multiple ligands from the parasite and receptors in hosts. These interactions include the binding of the Rh5-CyRPA-Ripr complex with the erythrocyte receptor basigin1,2, which is an essential step for entry into human erythrocytes. Here we show that the Rh5-CyRPA-Ripr complex binds the erythrocyte cell line JK-1 significantly better than does Rh5 alone, and that this binding occurs through the insertion of Rh5 and Ripr into host membranes as a complex with high molecular weight. We report a cryo-electron microscopy structure of the Rh5-CyRPA-Ripr complex at subnanometre resolution, which reveals the organization of this essential invasion complex and the mode of interactions between members of the complex, and shows that CyRPA is a critical mediator of complex assembly. Our structure identifies blades 4-6 of the β-propeller of CyRPA as contact sites for Rh5 and Ripr. The limited contacts between Rh5-CyRPA and CyRPA-Ripr are consistent with the dissociation of Rh5 and Ripr from CyRPA for membrane insertion. A comparision of the crystal structure of Rh5-basigin with the cryo-electron microscopy structure of Rh5-CyRPA-Ripr suggests that Rh5 and Ripr are positioned parallel to the erythrocyte membrane before membrane insertion. This provides information on the function of this complex, and thereby provides insights into invasion by P. falciparum.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) mediates parasite sequestration to the cerebral microvasculature via binding of DBLβ domains to intercellular adhesion molecule 1 (ICAM1) and is associated with severe cerebral malaria. In a cohort of 187 young children from Papua New Guinea (PNG), we examined baseline levels of antibody to the ICAM1-binding PfEMP1 domain, DBLβ3PF11_0521, in comparison to four control antigens, including NTS-DBLα and CIDR1 domains from another group A variant and a group B/C variant. Antibody levels for the group A antigens were strongly associated with age and exposure. Antibody responses to DBLβ3PF11_0521 were associated with a 37% reduced risk of high-density clinical malaria in the follow-up period (adjusted incidence risk ratio [aIRR] = 0.63 [95% confidence interval {CI}, 0.45 to 0.88; P = 0.007]) and a 25% reduction in risk of low-density clinical malaria (aIRR = 0.75 [95% CI, 0.55 to 1.01; P = 0.06]), while there was no such association for other variants. Children who experienced severe malaria also had significantly lower levels of antibody to DBLβ3PF11_0521 and the other group A domains than those that experienced nonsevere malaria. Furthermore, a subset of PNG DBLβ sequences had ICAM1-binding motifs, formed a distinct phylogenetic cluster, and were similar to sequences from other areas of endemicity. PfEMP1 variants associated with these DBLβ domains were enriched for DC4 and DC13 head structures implicated in endothelial protein C receptor (EPCR) binding and severe malaria, suggesting conservation of dual binding specificities. These results provide further support for the development of specific classes of PfEMP1 as vaccine candidates and as biomarkers for protective immunity against clinical P. falciparum malaria.

Plasmodium falciparum is the causative agent of the most severe form of malaria in humans. The merozoite, an extracellular stage of the parasite lifecycle, invades erythrocytes in which they develop. The most abundant protein on the surface of merozoites is merozoite surface protein 1 (MSP1), which consists of four processed fragments. Studies indicate that MSP1 interacts with other peripheral merozoite surface proteins to form a large complex. Successful invasion of merozoites into host erythrocytes is dependent on this protein complex; however, the identity of all components and its function remain largely unknown. We have shown that the peripheral merozoite surface proteins MSPDBL1 and MSPDBL2 are part of the large MSP1 complex. Using surface plasmon resonance, we determined the binding affinities of MSPDBL1 and MSPDBL2 to MSP1 to be in the range of 2-4 × 10(-7) m. Both proteins bound to three of the four proteolytically cleaved fragments of MSP1 (p42, p38, and p83). In addition, MSPDBL1 and MSPDBL2, but not MSP1, bound directly to human erythrocytes. This demonstrates that the MSP1 complex acts as a platform for display of MSPDBL1 and MSPDBL2 on the merozoite surface for binding to receptors on the erythrocyte and invasion.

Plasmodium falciparum causes malaria disease during the asexual blood stages of infection when merozoites invade erythrocytes and replicate. Merozoite surface proteins (MSPs) are proposed to play a role in the initial binding of merozoites to erythrocytes, but precise roles remain undefined. Based on electron microscopy studies of invading Plasmodium merozoites, it is proposed that the majority of MSPs are cleaved and shed from the surface during invasion, perhaps to release receptor-ligand interactions. In this study, we demonstrate that there is not universal cleavage of MSPs during invasion. Instead, there is sequential and coordinated cleavage and shedding of proteins, indicating a diversity of roles for surface proteins during and after invasion. While MSP1 and peripheral surface proteins such as MSP3, MSP7, serine repeat antigen 4 (SERA4), and SERA5 are cleaved and shed at the tight junction between the invading merozoite and erythrocyte, the glycosylphosphatidylinositol (GPI)-anchored proteins MSP2 and MSP4 are carried into the erythrocyte without detectable processing. Following invasion, MSP2 rapidly degrades within 10 min, whereas MSP4 is maintained for hours. This suggests that while some proteins that are shed upon invasion may have roles in initial contact steps, others function during invasion and are then rapidly degraded, whereas others are internalized for roles during intraerythrocytic development. Interestingly, anti-MSP2 antibodies did not inhibit invasion and instead were carried into erythrocytes and maintained for approximately 20 h without inhibiting parasite development. These findings provide new insights into the mechanisms of invasion and knowledge to advance the development of new drugs and vaccines against malaria.

The development of effective malaria vaccines and immune biomarkers of malaria is a high priority for malaria control and elimination. Ags expressed by merozoites of Plasmodium falciparum are likely to be important targets of human immunity and are promising vaccine candidates, but very few Ags have been studied. We developed an approach to assess Ab responses to a comprehensive repertoire of merozoite proteins and investigate whether they are targets of protective Abs. We expressed 91 recombinant proteins, located on the merozoite surface or within invasion organelles, and screened them for quality and reactivity to human Abs. Subsequently, Abs to 46 proteins were studied in a longitudinal cohort of 206 Papua New Guinean children to define Ab acquisition and associations with protective immunity. Ab responses were higher among older children and those with active parasitemia. High-level Ab responses to rhoptry and microneme proteins that function in erythrocyte invasion were identified as being most strongly associated with protective immunity compared with other Ags. Additionally, Abs to new or understudied Ags were more strongly associated with protection than were Abs to current vaccine candidates that have progressed to phase 1 or 2 vaccine trials. Combinations of Ab responses were identified that were more strongly associated with protective immunity than responses to their single-Ag components. This study identifies Ags that are likely to be key targets of protective human immunity and facilitates the prioritization of Ags for further evaluation as vaccine candidates and/or for use as biomarkers of immunity in malaria surveillance and control.

Antibodies play a key role in acquired human immunity to Plasmodium falciparum (Pf) malaria and target merozoites to reduce or prevent blood-stage replication and the development of disease. Merozoites present a complex array of antigens to the immune system, and currently, there is only a partial understanding of the targets of protective antibodies and how responses to different antigens are acquired and boosted. We hypothesized that there would be differences in the rate of acquisition of antibodies to different antigens and how well they are boosted by infection, which impacts the acquisition of immunity. We examined responses to a range of merozoite antigens in 2 different cohorts of children and adults with different age structures and levels of malaria exposure. Overall, antibodies were associated with age, exposure, and active infection, and the repertoire of responses increased with age and active infection. However, rates of antibody acquisition varied between antigens and different regions within an antigen following exposure to malaria, supporting our hypothesis. Antigen-specific responses could be broadly classified into early response types in which antibodies were acquired early in childhood exposure and late response types that appear to require substantially more exposure for the development of substantial levels. We identified antigen-specific responses that were effectively boosted after recent infection, whereas other responses were not. These findings advance our understanding of the acquisition of human immunity to malaria and are relevant to the development of malaria vaccines targeting merozoite antigens and the selection of antigens for use in malaria surveillance.

Invasion of human red blood cells by Plasmodium falciparum involves interaction of the merozoite form through proteins on the surface coat. The erythrocyte binding-like protein family functions after initial merozoite interaction by binding via the Duffy binding-like (DBL) domain to receptors on the host red blood cell. The merozoite surface proteins DBL1 and -2 (PfMSPDBL1 and PfMSPDBL2) (PF10_0348 and PF10_0355) are extrinsically associated with the merozoite, and both have a DBL domain in each protein. We expressed and refolded recombinant DBL domains for PfMSPDBL1 and -2 and show they are functional. The red cell binding characteristics of these domains were shown to be similar to full-length forms of these proteins isolated from parasite cultures. Futhermore, metal cofactors were found to enhance the binding of both the DBL domains and the parasite-derived full-length proteins to erythrocytes, which has implications for receptor binding of other DBL-containing proteins in Plasmodium spp. We solved the structure of the erythrocyte-binding DBL domain of PfMSPDBL2 to 2.09 Å resolution and modeled that of PfMSPDBL1, revealing a canonical DBL fold consisting of a boomerang shaped α-helical core formed from three subdomains. PfMSPDBL2 is highly polymorphic, and mapping of these mutations shows they are on the surface, predominantly in the first two domains. For both PfMSPDBL proteins, polymorphic variation spares the cleft separating domains 1 and 2 from domain 3, and the groove between the two major helices of domain 3 extends beyond the cleft, indicating these regions are functionally important and are likely to be associated with the binding of a receptor on the red blood cell.

Plasmodium falciparum causes the virulent form of malaria and disease manifestations are linked to growth inside infected erythrocytes. In order to survive and evade host responses the parasite remodels the erythrocyte by exporting several hundred effector proteins beyond the surrounding parasitophorous vacuole membrane. A feature of exported proteins is a pentameric motif (RxLxE/Q/D) that is a substrate for an unknown protease. Here, we show the protein responsible for cleavage of this motif is Plasmepsin V, an aspartic acid protease located in the endoplasmic reticulum. Plasmepsin V cleavage reveals the export signal (xE/Q/D) at the N-terminus of cargo proteins. Expression of an identical mature protein with xQ at the N-terminus generated by signal peptidase was not exported demonstrating Plasmepsin V activity is essential and linked with other key export events. Identification of the protease responsible for export into erythrocytes provides a novel target for therapeutic intervention against this devastating disease.

Summary The Apicomplexan parasite responsible for the most virulent form of malaria, Plasmodium falciparum, invades human erythrocytes through multiple ligand–receptor interactions. The P. falciparum reticulocyte-binding protein homologue (PfRh or PfRBL) family have been implicated in the invasion process but their exact role is unknown. PfRh1 and PfRh4, members of this protein family, bind to red blood cells and function in merozoite invasion during which they undergo a series of proteolytic cleavage events before and during entry into the host cell. The ectodomain of PfRh1 and PfRh4 are processed to produce fragments consistent with cleavage in the transmembrane domain and released into the supernatant, at about the time of invasion, in a manner consistent with rhomboid protease cleavage. Processing of both PfRh1 and PfRh4, and by extrapolation all membrane-bound members of this protein family, is important for function and release of these proteins on the merozoite surface and they along with EBA-175 are important components of the tight junction, the transient structure that links the erythrocyte via receptor–ligand interactions to the actin–myosin motor in the invading merozoite.

The sera genes of the malaria-causing parasite Plasmodium encode a family of unique proteins that are maximally expressed at the time of egress of parasites from infected red blood cells. These multi-domain proteins are unique, containing a central papain-like cysteine-protease fragment enclosed between the disulfide-linked N- and C-terminal domains. However, the central fragment of several members of this family, including serine repeat antigen 5 (SERA5), contains a serine (S596) in place of the active-site cysteine. Here we report the crystal structure of the central protease-like domain of Plasmodium falciparum SERA5, revealing a number of anomalies in addition to the putative nucleophilic serine: (1) the structure of the putative active site is not conducive to binding substrate in the canonical cysteine-protease manner; (2) the side chain of D594 restricts access of substrate to the putative active site; and (3) the S(2) specificity pocket is occupied by the side chain of Y735, reducing this site to a small depression on the protein surface. Attempts to determine the structure in complex with known inhibitors were not successful. Thus, despite having revealed its structure, the function of the catalytic domain of SERA5 remains an enigma.

Virulence of Plasmodium falciparum, the most lethal parasitic disease in humans, results in part from adhesiveness and increased rigidity of infected erythrocytes. Pf332 is trafficked to the parasite-infected erythrocyte via Maurer's clefts, structures for protein sorting and export in the host erythrocyte. This protein has a domain similar to the Duffy-binding-like (DBL) domain, which functions by binding to receptors for adherence and invasion. To address structure of the Pf332 DBL domain, we expressed this region, and validated its fold on the basis of the disulphide bond pattern, which conformed to the generic pattern for DBL domains. The modelled structure for Pf332 DBL had differences compared with the erythrocyte-binding region of the alphaDBL domain of Plasmodium knowlesi Duffy-binding protein (Pk alpha-DBL). We addressed the function of Pf332 by constructing parasites that either lack expression of the protein or express an altered form. We found no evidence that Pf332 is involved in cytoadhesion or merozoite invasion. Truncation of Pf332 had a significant effect on deformability of the P. falciparum-infected erythrocyte, while loss of the full protein deletion did not. Our data suggest that Pf332 may contribute to the overall deformability of the P. falciparum-infected erythrocyte by anchoring and scaffolding.

The serine repeat antigen (SERA) proteins of the malaria parasitesPlasmodiumspp. contain a putative enzyme domain similar to that of papain family cysteine proteases. InPlasmodium falciparumparasites, more than half of the SERA family proteins, including the most abundantly expressed form, SERA5, have a cysteine-to-serine substitution within the putative catalytic triad of the active site. Although SERA5 is required for blood-stage parasite survival, the occurrence of a noncanonical catalytic triad casts doubt on the importance of the enzyme domain in this function. We used phage display to identify a small (14-residue) disulfide-bonded cyclic peptide (SBP1) that targets the enzyme domain of SERA5. Biochemical characterization of the interaction shows that it is dependent on the conformation of both the peptide and protein. Addition of this peptide to parasite cultures compromised development of late-stage parasites compared to that of control parasites or those incubated with equivalent amounts of the carboxymethylated peptide. This effect was similar in two different strains ofP. falciparumas well as in a transgenic strain where the gene encoding the related serine-type parasitophorous vacuole protein SERA4 was deleted. In compromised parasites, the SBP1 peptide crosses both the erythrocyte and parasitophorous vacuole membranes and accumulates within the parasitophorous vacuole. In addition, both SBP1 and SERA5 were identified in the parasite cytosol, indicating that the plasma membrane of the parasite was compromised as a result of SBP1 treatment. These data implicate an important role for SERA5 in the regulation of the intraerythrocytic development of late-stage parasites and as a target for drug development.

Glycosylphosphatidylinositol (GPI)-anchored proteins coat the surface of extracellular Plasmodium falciparum merozoites, of which several are highly validated candidates for inclusion in a blood-stage malaria vaccine. Here we determined the proteome of gradient-purified detergent-resistant membranes of mature blood-stage parasites and found that these membranes are greatly enriched in GPI-anchored proteins and their putative interacting partners. Also prominent in detergent-resistant membranes are apical organelle (rhoptry), multimembrane-spanning, and proteins destined for export into the host erythrocyte cytosol. Four new GPI-anchored proteins were identified, and a number of other novel proteins that are predicted to localize to the merozoite surface and/or apical organelles were detected. Three of the putative surface proteins possessed six-cysteine (Cys6) motifs, a distinct fold found in adhesive surface proteins expressed in other life stages. All three Cys6 proteins, termed Pf12, Pf38, and Pf41, were validated as merozoite surface antigens recognized strongly by antibodies present in naturally infected individuals. In addition to the merozoite surface, Pf38 was particularly prominent in the secretory apical organelles. A different cysteine-rich putative GPI-anchored protein, Pf92, was also localized to the merozoite surface. This insight into merozoite surfaces provides new opportunities for understanding both erythrocyte invasion and anti-parasite immunity.

Targeting antigen to dendritic cells (DC) in vivo might be an effective method of modulating immune responses. Given the functional specializations among DC subsets, we investigated how targeting different receptors on different DC subsets may influence antibody (Ab) production. We show here that targeting FIRE (F4/80-like receptor) or CIRE (C-type lectin receptor), two molecules expressed on the surface of immature CD8- DC in the mouse, increases Ab production 100-1000-fold over a non-targeted control. This response was equivalent to that achieved with CpG adjuvant. In contrast, targeting CD205, which is primarily expressed on CD8+ DC, did not elicit an Ab response unless an adjuvant was added. Strong Ab responses in FcRgamma-/- mice, and with the use of F(ab')2 fragments, confirmed that FIRE and CIRE targeting was due to specific rather than FcR or complement binding. Our findings may reflect differences in the ability of CD8+ and CD8- DC subsets to stimulate immune responses in vivo. Although the consensus view is that Ag presentation on DC in their steady state leads to tolerance, the Ab enhancement from FIRE and CIRE targeting in the apparent absence of any "danger" or inflammatory signal would suggest that targeting certain DC molecules can supplant the need for external adjuvants for eliciting immune responses.

Plasmodium falciparum apical membrane antigen 1 (AMA1) is a leading malaria vaccine candidate whose function has not been unequivocally defined. Partial complementation of function can be achieved by exchanging the AMA1 of P. falciparum (PfAMA1) with that of P. chabaudi (PcAMA1). In this study, parasites expressing chimeric AMA1 proteins were created to identify domains of PfAMA1 critical in erythrocyte invasion and which are important immune targets. We report that specific chimeric AMA1 proteins containing domains I to III from PfAMA1 and PcAMA1 were able to complement PfAMA1 function in erythrocyte invasion. We demonstrate that domain III does not contain dominant epitope targets of antibodies raised against Escherichia coli expressed and refolded PfAMA1 ectodomain. Furthermore, we generated a parasite line in which the N-terminal pro region of PfAMA1 does not undergo proteolytic cleavage and show that its removal is necessary for PfAMA1 function.

The relict plastid (apicoplast) of apicomplexan parasites synthesizes fatty acids and is a promising drug target. In plant plastids, a pyruvate dehydrogenase complex (PDH) converts pyruvate into acetyl-CoA, the major fatty acid precursor, whereas a second, distinct PDH fuels the tricarboxylic acid cycle in the mitochondria. In contrast, the presence of genes encoding PDH and related enzyme complexes in the genomes of five Plasmodium species and of Toxoplasma gondii indicate that these parasites contain only one single PDH. PDH complexes are comprised of four subunits (E1alpha, E1beta, E2, E3), and we confirmed four genes encoding a complete PDH in Plasmodium falciparum through sequencing of cDNA clones. In apicomplexan parasites, many nuclear-encoded proteins are targeted to the apicoplast courtesy of two-part N-terminal leader sequences, and the presence of such N-terminal sequences on all four PDH subunits as well as phylogenetic analyses strongly suggest that the P. falciparum PDH is located in the apicoplast. Fusion of the two-part leader sequences from the E1alpha and E2 genes to green fluorescent protein experimentally confirmed apicoplast targeting. Western blot analysis provided evidence for the expression of the E1alpha and E1beta PDH subunits in blood-stage malaria parasites. The recombinantly expressed catalytic domain of the PDH subunit E2 showed high enzymatic activity in vitro indicating that pyruvate is converted to acetyl-CoA in the apicoplast, possibly for use in fatty acid biosynthesis.

Plasmodium falciparum is the parasite responsible for the most acute form of malaria in humans. Recently, the serine repeat antigen (SERA) in P. falciparum has attracted attention as a potential vaccine and drug target, and it has been shown to be a member of a large gene family. To clarify the relationships among the numerous P. falciparum SERAs and to identify orthologs to SERA5 and SERA6 in Plasmodium species affecting rodents, gene trees were inferred from nucleotide and amino acid sequence data for 33 putative SERA homologs in seven different species. (A distance method for nucleotide sequences that is specifically designed to accommodate differing GC content yielded results that were largely compatible with the amino acid tree. Standard-distance and maximum-likelihood methods for nucleotide sequences, on the other hand, yielded gene trees that differed in important respects.) To infer the pattern of duplication, speciation, and gene loss events in the SERA gene family history, the resulting gene trees were then ‘‘reconciled’’ with two competing Plasmodium species tree topologies that have been identified by previous phylogenetic studies. Parsimony of reconciliation was used as a criterion for selecting a gene tree/species tree pair and provided (1) support for one of the two species trees and for the core topology of the amino acid‐derived gene tree, (2) a basis for critiquing fine detail in a poorly resolved region of the gene tree, (3) a set of predicted ‘‘missing genes’’ in some species, (4) clarification of the relationship among the P. falciparum SERA, and (5) some information about SERA5 and SERA6 orthologs in the rodent malaria parasites. Parsimony of reconciliation and a second criterion—implied mutational pattern at two key active sites in the SERA proteins—were also seen to be useful supplements to standard ‘‘bootstrap’’ analysis for inferred topologies.

Apical membrane antigen-1 (AMA-1) is a target of antibodies that inhibit invasion of Plasmodium falciparum into human erythrocytes and is a candidate for inclusion in a malaria vaccine. We have identified a line of P. falciparum (W2mef) less susceptible to anti-AMA1 antibodies raised to the protein from a heterologous parasite line (3D7). We have constructed transgenic P. falciparum expressing heterologous AMA-1 alleles. In vitro invasion assays show that these transgenic parasites differ from parental lines in susceptibility to inhibitory antibodies, providing direct evidence that sequence polymorphisms within AMA-1 are responsible for evasion of immune responses that inhibit parasite invasion. We also generated a parasite line that would express a chimeric AMA-1 protein, in which highly polymorphic residues within domain 1 were exchanged. Inhibition assays suggest that these residues are not sufficient for inhibition by invasion-blocking antibodies. This study is the first to use P. falciparum allelic exchange to examine the relationship between genetic diversity and susceptibility to protective antibodies. The findings have important implications for the development of an AMA-1-based malaria vaccine.

Serine repeat antigen 5 (SERA5) is an abundant antigen of the human malaria parasite Plasmodium falciparum and is the most strongly expressed member of the nine-gene SERA family. It appears to be essential for the maintenance of the erythrocytic cycle, unlike a number of other members of this family, and has been implicated in parasite egress and/or erythrocyte invasion. All SERA proteins possess a central domain that has homology to papain except in the case of SERA5 (and some other SERAs), where the active site cysteine has been replaced with a serine. To investigate if this domain retains catalytic activity, we expressed, purified, and refolded a recombinant form of the SERA5 enzyme domain. This protein possessed chymotrypsin-like proteolytic activity as it processed substrates downstream of aromatic residues, and its activity was reversed by the serine protease inhibitor 3,4-diisocoumarin. Although all Plasmodium SERA enzyme domain sequences share considerable homology, phylogenetic studies revealed two distinct clusters across the genus, separated according to whether they possess an active site serine or cysteine. All Plasmodia appear to have at least one member of each group. Consistent with separate biological roles for members of these two clusters, molecular modeling studies revealed that SERA5 and SERA6 enzyme domains have dramatically different surface properties, although both have a characteristic papain-like fold, catalytic cleft, and an appropriately positioned catalytic triad. This study provides impetus for the examination of SERA5 as a target for antimalarial drug design.

The Plasmodium falciparum serine repeat antigen (SERA) has shown considerable promise as a blood stage vaccine for the control of malaria. A related protein, SERPH, has also been described in P. falciparum. Whereas their biological role remains unknown, both proteins possess papain-like protease domains that may provide attractive targets for therapeutic intervention. Genomic sequencing has recently shown that SERA and SERPH are the fifth and sixth genes, respectively, in a cluster of eight SERA homologues present on chromosome 2. In this paper, the expression and functional relevance of these eight genes and of a ninth SERA homologue found on chromosome 9 were examined in blood stage parasites. Using reverse transcriptase-PCR and microarray approaches, we demonstrate that whereas mRNA to all nine SERA genes is synthesized late in the erythrocytic cycle, it is those genes in the central region of the chromosome 2 cluster that are substantially up-regulated at this time. Using antibodies specific to each SERA, it was apparent that SERA4 to -6, and possibly also SERA9, are synthesized in blood stage parasites. The reactivity of antibodies from malaria-immune individuals with the SERA recombinant proteins suggested that SERA2 and SERA3 are also expressed at least in some parasite populations. To examine whether SERA genes are essential to blood stage growth, each of the eight chromosome 2 SERA genes was targeted for disruption. Whereas genes at the periphery of the cluster were mostly dispensable (SERA2 and -3 and SERA7 and -8), those in the central region (SERA4 to -6) could not be disrupted. The inability to disrupt SERA4, -5, and -6 is consistent with their apparent dominant expression and implies an important role for these genes in maintenance of the erythrocytic cycle.

Background Polymorphism in antigens is a common mechanism for immune evasion used by many important pathogens, and presents major challenges in vaccine development. In malaria, many key immune targets and vaccine candidates show substantial polymorphism. However, knowledge on antigenic diversity of key antigens, the impact of polymorphism on potential vaccine escape, and how sequence polymorphism relates to antigenic differences is very limited, yet crucial for vaccine development. Plasmodium falciparum apical membrane antigen 1 (AMA1) is an important target of naturally-acquired antibodies in malaria immunity and a leading vaccine candidate. However, AMA1 has extensive allelic diversity with more than 60 polymorphic amino acid residues and more than 200 haplotypes in a single population. Therefore, AMA1 serves as an excellent model to assess antigenic diversity in malaria vaccine antigens and the feasibility of multi-allele vaccine approaches. While most previous research has focused on sequence diversity and antibody responses in laboratory animals, little has been done on the cross-reactivity of human antibodies. Methods We aimed to determine the extent of antigenic diversity of AMA1, defined by reactivity with human antibodies, and to aid the identification of specific alleles for potential inclusion in a multi-allele vaccine. We developed an approach using a multiple-antigen-competition enzyme-linked immunosorbent assay (ELISA) to examine cross-reactivity of naturally-acquired antibodies in Papua New Guinea and Kenya, and related this to differences in AMA1 sequence. Results We found that adults had greater cross-reactivity of antibodies than children, although the patterns of cross-reactivity to alleles were the same. Patterns of antibody cross-reactivity were very similar between populations (Papua New Guinea and Kenya), and over time. Further, our results show that antigenic diversity of AMA1 alleles is surprisingly restricted, despite extensive sequence polymorphism. Our findings suggest that a combination of three different alleles, if selected appropriately, may be sufficient to cover the majority of antigenic diversity in polymorphic AMA1 antigens. Antigenic properties were not strongly related to existing haplotype groupings based on sequence analysis. Conclusions Antigenic diversity of AMA1 is limited and a vaccine including a small number of alleles might be sufficient for coverage against naturally-circulating strains, supporting a multi-allele approach for developing polymorphic antigens as malaria vaccines. Electronic supplementary material The online version of this article (doi:10.1186/s12916-014-0183-5) contains supplementary material, which is available to authorized users.

We describe an approach for the rapid mapping of epitopes within a malaria antigen using a combination of phage display techniques. Phage display of antigen fragments identifies the location of the epitopes, then random peptide libraries displayed on phage are employed to identify accurately amino acids involved in the epitope. Finally, phage display of mutant fragments confirms the role of each residue in the epitope. This approach was applied to the apical membrane antigen-1 (AMA1), which is a leading candidate for inclusion in a vaccine directed against the asexual blood stages of Plasmodium falciparum. As part of the effort both to understand the function of AMA1 in the parasite life cycle and to define the specificity of protective immune responses, a panel of monoclonal antibodies (MAbs) was generated to obtain binding reagents to the various domains within the molecule. There is a pressing need to determine rapidly the regions recognized by these antibodies and the structural requirements required within AMA1 for high affinity binding of the MAbs. Using phage displaying random AMA1 fragments, it was shown that MAb5G8 recognizes a short linear epitope within the pro-domain of AMA1 whereas the epitope recognized by MAb 1F9 is reduction sensitive and resides within a disulphide-bonded 57 amino acid sub-domain of domain-1. Phage displaying random peptide libraries and mutant AMA1 fragments were employed for fine mapping of the MAb5G8 core epitope to a three-residue sequence in the AMA1 prodomain.

Plasmodium falciparum infections in malaria-naive individuals can lead to severe morbidity, which may be life threatening if untreated. Continued exposure to infection leads to a degree of immunity, and consequently, older children and adults living in areas of endemicity are protected from the severe clinical consequences of infection with P. falciparum. The effector mechanisms that mediate naturally acquired immunity to malaria are not completely understood, but antibodies to the asexual blood stage parasites play a role. This has been most clearly demonstrated by the reduction of parasitemias following the passive immunization of children with clinical malaria with immunoglobulin G (IgG) from malaria-immune adults (6, 7, 32). Antigens recognized by antibodies that are active in passive-immunization experiments are prime candidates for testing in a vaccine. Studies with an assay for antibody-dependent cellular inhibition have identified antibodies to MSP3 as an active component of passively transferred human IgG (28), but much evidence indicates that other merozoite antigens are capable of inducing antibodies that limit parasite development (13–15, 17, 26, 29, 33, 34). One of the prime candidate antigens for inclusion in a malaria vaccine is apical membrane antigen 1 (AMA1). AMA1 is an 83-kDa antigen that is synthesized in mature stages of the parasite and is initially localized in the necks of the rhoptry organelles (9, 30). At about the time of merozoite release, the full-length 83-kDa molecule is localized at the apical pole, and an N-terminally processed form of 66 kDa can be detected distributed around the merozoite surface (27, 30). Although the biological function of AMA1 is unknown, its location and stage specificity suggest that it may be involved in the process of erythrocyte invasion. AMA1 is one of only a few asexual blood stage antigens that have been identified in all Plasmodium species examined (42), and this has enabled the vaccine potential of AMA1 to be investigated using various animal models. Active immunization of monkeys or mice with either native (11) or recombinant (2, 8) forms of AMA1 has protected these animals against simian and rodent parasites, respectively. Much evidence indicates that anti-AMA1 antibodies mediate protection. Monoclonal antibodies raised against P. falciparum AMA1 and against PK66, the Plasmodium knowlesi homologue of AMA1, inhibit merozoite invasion in vitro (20, 35). Furthermore, passive immunization of AMA1-specific polyclonal antibodies into Plasmodium chabaudi-infected mice prevented lethal parasitemias (2). These protective antibodies react with conformational epitopes stabilized by disulfide bonds, as immunization with the reduced and alkylated AMA1 failed to protect mice against challenge with P. chabaudi (10). The sequence of AMA1 is relatively conserved among various Plasmodium spp., with the level of amino acid sequence identity exceeding 50% in pairwise comparisons among all known sequences (5, 12, 24, 25, 31, 42). AMA1 lacks the sequence repeats and marked polymorphisms found in other malaria antigens, such as the merozoite surface antigens MSP1 and MSP2 (3). However, some sequence variation, resulting from point mutations, is observed among alleles of AMA1 in P. falciparum (25, 30, 36), P. knowlesi (43), Plasmodium vivax (5), and P. chabaudi (10), and studies with the P. chabaudi-mouse model indicate that this variation is immunologically significant. Mice immunized with AMA1 or receiving passively transferred anti-AMA1 antibodies were not protected from a heterologous strain of P. chabaudi parasites, indicating that the protective antibodies recognized strain-specific epitopes. Early clinical trials with AMA1 have commenced, and it is important to determine the effect of sequence diversity on the efficacy of the recombinant AMA1 as a vaccine against P. falciparum. In this study, we demonstrate that immunization of rabbits with the refolded P. falciparum AMA1 ectodomain (the vaccine molecule) induces antibodies that inhibit merozoite invasion in vitro. The refolded antigen has also been used to affinity purify AMA1-specific antibodies from the plasma of individuals who have been exposed to chronic malaria infections. These naturally occurring human antibodies were also able to inhibit the invasion of erythrocytes by P. falciparum merozoites.

Apical membrane Ag 1 (AMA1) is a leading malaria vaccine candidate. Homologues of AMA1 can induce protection in mice and monkeys, but the mechanism of immunity is not understood. Mice immunized with a refolded, recombinant, Plasmodium chabaudi AMA1 fragment (AMA1B) can withstand subsequent challenge with P. chabaudi adami. Here we show that CD4+ T cell depletion, but not γδ T cell depletion, can cause a significant drop in antiparasite immunity in either immunized normal or immunized B cell KO mice. In normal mice, this loss of immunity is not accompanied by a decline in Ab levels. These observations indicate a role for AMA1-specific Ab-independent T cell-mediated immunity. However, the loss of immunity in normal CD4+ T cell-depleted mice is temporary. Furthermore, immunized B cell KO mice cannot survive infection, demonstrating the absolute importance of B cells, and presumably Ab, in AMA1-induced immunity. CD4+ T cells specific for a cryptic conserved epitope on AMA1 can adoptively transfer protection to athymic (nu/nu) mice, the level of which is enhanced by cotransfer of rabbit anti-AMA1-specific antisera. Recipients of rabbit antisera alone do not survive. Some protected recipients of T cells plus antisera do not develop their own AMA 1-specific Ab response, suggesting that AMA 1-specific CMI alone can protect mice. These data are the first to demonstrate the specificity of any protective CMI response in malaria and have important implications for developing a malaria vaccine.

Background Plasmodium falciparum serine repeat antigen 5 (PfSERA5) is an abundant blood stage protein that plays an essential role in merozoite egress and invasion. The native protein undergoes extensive proteolytic cleavage that appears to be tightly regulated. PfSERA5 N-terminal fragment is being developed as vaccine candidate antigen. Although PfSERA5 belongs to papain-like cysteine protease family, its catalytic domain has a serine in place of cysteine at the active site. Methods In the present study, we synthesized a number of peptides from the N- and C-terminal regions of PfSERA5 active domain and evaluated their inhibitory potential. Results The final proteolytic step of PfSERA5 involves removal of a C-terminal ~ 6 kDa fragment that results in the generation of a catalytically active ~ 50 kDa enzyme. In the present study, we demonstrate that two of the peptides derived from the C-terminal ~ 6 kDa region inhibit the parasite growth and also cause a delay in the parasite development. These peptides reduced the enzyme activity of the recombinant protein and co-localized with the PfSERA5 protein within the parasite, thereby indicating the specific inhibition of PfSERA5 activity. Molecular docking studies revealed that the inhibitory peptides interact with the active site of the protein. Interestingly, the peptides did not have an effect on the processing of PfSERA5. Conclusions Our observations indicate the temporal regulation of the final proteolytic cleavage step that occurs just prior to egress. General significance These results reinforce the role of PfSERA5 for the intra-erythrocytic development of malaria parasite and show the role of carboxy terminal ~ 6 kDa fragments in the regulation of PfSERA5 activity. The results also suggest that final cleavage step of PfSERA5 can be targeted for the development of new anti-malarials.

Background A highly effective vaccine against Plasmodium falciparum malaria should induce potent, strain transcending immunity that broadly protects against the diverse population of parasites circulating globally. We aimed to identify vaccine candidates that fulfill the criteria. Methods We have measured growth inhibitory activity of antibodies raised to a range of antigens to identify those that can efficiently block merozoite invasion for geographically diverse strains of P. falciparum. Results This has shown that the conserved Region III-V, of the P. falciparum erythrocyte-binding antigen (EBA)-175 was able to induce antibodies that potently inhibit merozoite invasion across diverse parasite strains, including those reliant on invasion pathways independent of EBA-175 function. Additionally, the conserved RIII-V domain of EBA-140 also induced antibodies with strong in vitro parasite growth inhibitory activity. Conclusion We identify an alternative, highly conserved region (RIV-V) of EBA-175, present in all EBA proteins, that is the target of potent, strain transcending neutralizing antibodies, that represents a strong candidate for development as a component in a malaria vaccine.

Apical membrane antigen-1 (AMA-1) of Plasmodium falciparum is one of the leading asexual blood stage antigens being considered for inclusion in a malaria vaccine. The ability of this molecule to induce a protective immune response has been shown to be dependent upon a conformation stabilized by disulfide bonds. In this study we have utilized the reversed-phase high performance liquid chromatography of dithiothreitol-reduced and nonreduced tryptic digests of Plasmodium chabaudi AMA-1 secreted from baculovirus-infected insect cells, in conjunction with N-terminal sequencing and electrospray-ionization mass spectrometry, to identify and assign disulfide-linked peptides. All 16 cysteine residues that are conserved in all known sequences of AMA-1 are incorporated into intramolecular disulfide bonds. Six of the eight bonds have been assigned unequivocally, whereas the two unassigned disulfide bonds connect two Cys-Xaa-Cys sequences separated by 14 residues. The eight disulfide bonds fall into three nonoverlapping groups that define three possible subdomains within the AMA-1 ectodomain. Although the pattern of disulfide bonds within subdomain III has not been fully elucidated, one of only two possible linkage patterns closely resembles the cystine knot motif found in growth factors. Sites of amino acid substitutions in AMA-1 that are well separated in the primary sequence are clustered by the disulfide bonds in subdomains II and III. These findings are consistent with the conclusion that these amino acid substitutions are defining conformational disulfide bond-dependent epitopes that are recognized by protective immune responses.

Plasmodium falciparum exports several hundred effector proteins that remodel the host erythrocyte and enable parasites to acquire nutrients, sequester in the circulation and evade immune responses. The majority of exported proteins contain the Plasmodium export element (PEXEL; RxLxE/Q/D) in their N-terminus, which is proteolytically cleaved in the parasite endoplasmic reticulum by Plasmepsin V, and is necessary for export. Several exported proteins lack a PEXEL or contain noncanonical motifs. Here, we assessed whether Plasmepsin V could process the N-termini of diverse protein families in P. falciparum. We show that Plasmepsin V cleaves N-terminal sequences from RIFIN, STEVOR and RESA multigene families, the latter of which contain a relaxed PEXEL (RxLxxE). However, Plasmepsin V does not cleave the N-terminal sequence of the major exported virulence factor erythrocyte membrane protein 1 (PfEMP1) or the PEXEL-negative exported proteins SBP-1 or REX-2. We probed the substrate specificity of Plasmepsin V and determined that lysine at the PEXEL P3 position, which is present in PfEMP1 and other putatively exported proteins, blocks Plasmepsin V activity. Furthermore, isoleucine at position P1 also blocked Plasmepsin V activity. The specificity of Plasmepsin V is therefore exquisitely confined and we have used this novel information to redefine the predicted P. falciparum PEXEL exportome.

Antipeptide antibodies have been produced which target regions either side of the cleavage activation sites of Newcastle disease virus (NDV) membrane glycoprotein precursors. Use of complementary pairs of antibodies in Western blot analysis of mercaptoethanol-reduced extracts of NDV-infected BHK-21 cells enabled analysis of the susceptibilities of NDV fusion protein precursors (Fo-proteins) to cleavage activation in these cells. In addition, it was possible to determine whether or not isolates produce haemagglutinin-neuraminidase (HN)-proteins in precursor forms (HNo-proteins). This assay system has been evaluated with a series of Australian isolates of NDV with well defined virulence properties in order to validate its use in pathotyping NDV isolates. Less well defined isolates also produced data consistent with their biological properties and an isolate was characterised which, hitherto, was not known to be present in Australian poultry. The applicability of this assay system in fundamental studies of the processes of cleavage activation of NDV Fo- and HNo-proteins and formatting of the antisera into ELISA systems are discussed.

Several merozoite surface proteins are being assessed as potential components of a vaccine against Plasmodium falciparum, the cause of the most serious form of human malaria. One of these proteins, merozoite surface protein 2 (MSP2), is unusually hydrophilic and contains tandem sequence repeats, characteristics of intrinsically unstructured proteins. A range of physicochemical studies has confirmed that recombinant forms of MSP2 are largely unstructured. Both dimorphic types of MSP2 (3D7 and FC27) are equivalently extended in solution and form amyloid-like fibrils although with different kinetics and structural characteristics. These fibrils have a regular underlying beta-sheet structure and both fibril types stain with Congo Red, but only the FC27 fibrils stain with Thioflavin T. 3D7 MSP2 fibrils seeded the growth of fibrils from 3D7 or FC27 MSP2 monomer indicating the involvement of a conserved region of MSP2 in fibril formation. Consistent with this, digestion of fibrils with proteinase K generated resistant peptides, which included the N-terminal conserved region of MSP2. A monoclonal antibody that reacted preferentially with monomeric recombinant MSP2 did not react with the antigen in situ on the merozoite surface. Glutaraldehyde cross-linking of infected erythrocytes generated MSP2 oligomers similar to those formed by polymeric recombinant MSP2. We conclude that MSP2 oligomers containing intermolecular beta-strand interactions similar to those in amyloid fibrils may be a component of the fibrillar surface coat on P. falciparum merozoites.

Serine repeat antigens (SERAs) are a family of secreted “cysteine-like” proteases of Plasmodium parasites. Several SERAs possess an atypical active-site serine residue in place of the canonical cysteine. The human malaria parasite Plasmodium falciparum possesses six “serine-type” ( SERA1 to SERA5 and SERA9 ) and three “cysteine-type” ( SERA6 to SERA8 ) SERAs. Here, we investigate the importance of the serine-type SERAs to blood-stage parasite development and examine the extent of functional redundancy among this group. We attempted to knock out the four P. falciparum serine-type SERA genes that have not been disrupted previously. SERA1 , SERA4 , and SERA9 knockout lines were generated, while only SERA5 , the most strongly expressed member of the SERA family, remained refractory to genetic deletion. Interestingly, we discovered that while SERA4 -null parasites completed the blood-stage cycle normally, they exhibited a twofold increase in the level of SERA5 mRNA. The inability to disrupt SERA5 and the apparent compensatory increase in SERA5 expression in response to the deletion of SERA4 provides evidence for an important blood-stage function for the serine-type SERAs and supports the notion of functional redundancy among this group. Such redundancy is consistent with our phylogenetic analysis, which reveals a monophyletic grouping of the serine-type SERAs across the genus Plasmodium and a predominance of postspeciation expansion. While SERA5 is to some extent further validated as a target for vaccine and drug development, our data suggest that the expression level of other serine-type SERAs is the only barrier to escape from anti-SERA5-specific interventions.

Merozoite surface proteins of the human malaria parasite Plasmodium falciparum are involved in initial contact with target erythrocytes, a process that begins a cascade of events required for successful invasion of these cells. In order to identify complexes that may play a role in invasion we purified detergent-resistant membranes (DRMs), known to be enriched in merozoite surface proteins, and used blue native-polyacrylamide gel electrophoresis (BN-PAGE) to isolate high molecular weight complexes for identification by mass spectrometry. Sixty-two proteins were detected and these mostly belonged to expected DRM proteins classes including GPI-anchored, multi-membrane spanning and rhoptry proteins. Proteins from seven known complexes were identified including MSP-1/7, the low (RAP1/2 and RAP1/3), and high (RhopH1/H2/H3) molecular weight rhoptry complexes, and the invasion motor complex (GAP45/GAP50/myosinA). Remarkably, a large proportion of identified spectra were derived from only 4 proteins: the GPI-anchored proteins MSP-1 and Pf92, the putative GPI-anchored protein Pf113 and RAP-1, the core component of the two RAP complexes. Each of these proteins predominated in high molecular weight species suggesting their aggregation in much larger complexes than anticipated. To demonstrate that the procedure had isolated novel complexes we focussed on MSP-1, which predominated as a distinct species at approximately 500 kDa by BN-PAGE, approximately twice its expected size. Chemical cross-linking supports the existence of a stable MSP-1 oligomer of approximately 500 kDa, probably comprising a highly stable homodimeric species. Our observations also suggests that oligomerization of MSP-1 is likely to occur outside the C-terminal epidermal growth factor (EGF)-like domains. Confirmation of MSP-1 oligomerization, together with the isolation of a number of known complexes by BN-PAGE, makes it highly likely that novel interactions occur amongst members of this proteome.

Phosphomannomutase (PMM) catalyses the conversion of mannose-6-phosphate to mannose-1-phosphate, an essential step in mannose activation and the biosynthesis of glycoconjugates in all eukaryotes. Deletion of PMM from Leishmania mexicana results in loss of virulence, suggesting that PMM is a promising drug target for the development of anti-leishmanial inhibitors. We report the crystallization and structure determination to 2.1 A of L. mexicana PMM alone and in complex with glucose-1,6-bisphosphate to 2.9 A. PMM is a member of the haloacid dehalogenase (HAD) family, but has a novel dimeric structure and a distinct cap domain of unique topology. Although the structure is novel within the HAD family, the leishmanial enzyme shows a high degree of similarity with its human isoforms. We have generated L. major PMM knockouts, which are avirulent. We expressed the human pmm2 gene in the Leishmania PMM knockout, but despite the similarity between Leishmania and human PMM, expression of the human gene did not restore virulence. Similarities in the structure of the parasite enzyme and its human isoforms suggest that the development of parasite-selective inhibitors will not be an easy task.

A dose escalating, placebo-controlled phase 1 trial was conducted to test the safety and immunogenicity of a vaccine containing recombinant Plasmodium falciparum apical membrane antigen 1 (AMA1) formulated in Montanide ISA720. Three groups of volunteers were vaccinated intramuscularly with 5 microg, 20 microg or 80 microg of AMA1, respectively, in 0.5 mL of formulation at 0, 3 and 6 months. Anti-AMA1 antibody levels and T cell stimulation indices were measured before and after each vaccination. No vaccine-related serious adverse events were recorded. Most subjects generated a mild to moderate, transient local reaction after the first vaccination. Three subjects developed a local reaction approximately 10 days following vaccination. Six of the 29 subjects seroconverted. Only one of these developed a high antibody titre. However, the interpretation of this trial was compromised by a loss of potency of the formulated vaccine during the course of the study.

Merozoite surface protein 1 (MSP1) is a highly polymorphic Plasmodium falciparum merozoite surface protein implicated in the invasion of human erythrocytes during the asexual cycle. It forms a complex with MSP6 and MSP7 on the merozoite surface, and this complex is released from the parasite around the time of erythrocyte invasion. MSP1 and many other merozoite surface proteins contain dimorphic elements in their protein structures, and here we show that MSP6 is also dimorphic. The sequences of eight MSP6 genes indicate that the alleles of each dimorphic form of MSP6 are highly conserved. The smaller 3D7-type MSP6 alleles are detected in parasites from all malarious regions of the world, whereas K1-type MSP6 alleles have only been detected in parasites from mainland Southeast Asia. Cleavage of MSP6, which produces the p36 fragment in 3D7-type MSP6 and associates with MSP1, also occurs in K1-type MSP6 but at a different site in the protein. Anti-3D7 MSP6 antibodies weakly inhibited erythrocyte invasion by homologous 3D7 merozoites but did not inhibit a parasite line expressing the K1-type MSP6 allele. Antibodies from hyperimmune individuals affinity purified on an MSP3 peptide cross-reacted with MSP6; therefore, MSP6 may also be a target of antibody-dependent cellular inhibition.

The potential of Plasmodium falciparum merozoite surface protein 3 as a component of an asexual-stage malaria vaccine is currently being assessed. The precursor form of MSP3 undergoes cleavage during schizogony to generate a mature processed form. It is unknown if this cleavage event is necessary for MSP3 function, but it may be an important consideration for assessing and developing MSP3 as an asexual-stage vaccine candidate. We have therefore determined the cleavage site in MSP3 by sequencing the N-terminus of the processed form of MSP3, which was isolated from parasite material. The position of the cleavage site indicates that the processed form of MSP3 retains the three blocks of alanine-rich heptad repeats, which are predicted to provide the structural framework for an intramolecular coiled-coil. The cleavage-site motif has many features in common with the published cleavage sites of MSP130, MSP636, and MSP722, which are all located on the merozoite surface and are implicated in the erythrocyte invasion process. The common cellular location and similar cleavage-site motifs suggest that these merozoite proteins may be cleaved by the same or related proteases.

Leishmania parasites synthesize a range of mannose-containing glycoconjugates thought to be essential for virulence in the mammalian host and sandfly vector. A prerequisite for the synthesis of these molecules is the availability of the activated mannose donor, GDP-Man, the product of the catalysis of mannose-1-phosphate and GTP by GDP-mannose pyrophosphorylase (GDP-MP). In contrast to the lethal phenotype in fungi, the deletion of the gene in Leishmania mexicana did not affect parasite viability but led to a total loss of virulence, making GDP-MP an ideal target for anti-Leishmania drug development. We show by immunofluorescence and subcellular fractionation that GDP-MP is a cytoplasmic protein, and we describe a colorimetric activity assay suitable for the high throughput screening of small molecule inhibitors. We expressed recombinant GDP-MP as a fusion with maltose-binding protein and separated the enzyme from maltose-binding protein by thrombin cleavage, ion-exchange, and size exclusion chromatography. Size exclusion chromatography and analytical ultracentrifugation studies demonstrate that GDP-MP self-associates to form an enzymatically active and stable hexamer. However, sedimentation studies show that the GDP-MP hexamer dissociates to trimers and monomers in a time-dependent manner, at low protein concentrations, at low ionic strength, and at alkaline pH. Circular dichroism spectroscopy reveals that GDP-MP is comprised of mixed alpha/beta structure, similar to its closest related homologue, N-acetyl-glucoseamine-1-phosphate uridyltransferase (Glmu) from Streptococcus pneumoniae. Our studies provide insight into the structure of a novel target for the development of anti-Leishmania drugs.

Apical membrane antigen-1 (AMA1) is a prime vaccine candidate for inclusion in a vaccine against malaria. It is known that the disulphide bond stabilised conformation of this antigen is important for eliciting a protective antibody response, however little is known about the epitopes within this molecule that are targeted by the immune response. We have used a peptide approach for the identification and characterisation of such regions. In this study, the in vitro refolded, recombinant ectodomain of AMA1 from the D strain of Plasmodium chabaudi adami, was digested with trypsin and individual peptide fragments examined for antigenic activity. We found that a tryptic fragment, which was derived from a loop-like structure within the putative domain I of the intact AMA1 molecule, was highly reactive with antibodies from the sera of hyperimmune mice. Two different synthetic peptide constructs incorporating this antigenically active fragment were assembled. The first consisted of two separate peptide chains which were linked through a disulphide bond formed using chemo-selective chemistry. A larger 45-mer loop peptide, generated by the oxidation of two cysteine residues close to the N- and C-termini of the 45-mer, represented the complete loop structure and incorporated the tryptic fragment. Each peptide construct was also able to elicit production of high titres of antibodies in mice and furthermore, the 45-residue loop peptide elicited antibodies capable of binding to AMA1 with titres comparable to those present in a mouse which had recovered from multiple exposures to P. chabaudi adami parasites. Passive immunisation with anti-loop antibodies did not suppress the development of parasitaemia in mice challenged with P. chabaudi adami suggesting that although highly immunogenic, the peptides represented inadequate or inappropriate epitopes for vaccination purposes.