Identifying and characterising the Plasmodium falciparum RhopH3 Plasmodium vivax homologue

Four Plasmodium species cause malaria in humans, Plasmodium falciparum being the most widely studied to date. All Plasmodium species have paired club-shaped organelles towards their apical extreme named rhoptries that contain many lipids and proteins which are released during target cell invasion. P. falciparum RhopH3 is a rhoptry protein triggering important immune responses in patients from endemic regions. It has also been shown that anti-RhopH3 antibodies inhibit in vitro invasion of erythrocytes. Recent immunisation studies in mice with the Plasmodium yoelii and Plasmodium berghei RhopH3 P. falciparum homologue proteins found that they are able to induce protection in murine models. This study described identifying and characterising RhopH3 protein in Plasmodium vivax; it is encoded by a seven exon gene and expressed during the parasite's asexual stage. PvRhopH3 has similar processing to its homologue in P. falciparum and presents a cellular immunolocalisation pattern characteristic of rhoptry proteins.     

Identification and characterisation of the Plasmodium vivax rhoptry-associated protein 2

Plasmodium vivax is currently the most widespread of the four parasite species causing malaria in humans around the world. It causes more than 75 million clinical episodes per year, mainly on the Asian and American continents. Identifying new antigens to be further tested as anti-P. vivax vaccine candidates has been greatly hampered by the difficulty of maintaining this parasite cultured in vitro. Taking into account that one of the most promising vaccine candidates against Plasmodium falciparum is the rhoptry-associated protein 2, we have identified the P. falciparum rhoptry-associated protein 2 homologue in P. vivax in the present study. This protein has 400 residues, having an N-terminal 21 amino-acid stretch compatible with a signal peptide and, as occurs with its falciparum homologue, it lacks repeat sequences. The protein is expressed in asexual stage P. vivax parasites and polyclonal sera raised against this protein recognised a 46 kDa band in parasite lysate in a Western blot assay.     

Plasmodium falciparum infection and exoerythrocytic development in mice with chimeric human livers

The exoerythrocytic stage of Plasmodium falciparum has remained a difficult phase of the parasite life-cycle to study. The host and tissue specificity of the parasite requires the experimental infection of humans or non-human primates and subsequent surgical recovery of parasite-infected liver tissue to analyze this stage of the parasites development. This type of study is impossible in humans due to obvious ethical considerations and the cost and complexity in working with primate models has precluded their use for extensive studies of the exoerythrocytic stage. In this study we assessed, for the first time, the use of transgenic, chimeric mice containing functioning human hepatocytes as an alternative for modeling the in vivo interaction of P. falciparum parasites and human hepatocytes. Infection of these mice with P. falciparum sporozoites produced morphologically and antigenically mature liver stage schizonts containing merozoites capable of invading human red blood cells. Additionally, using microdissection, highly enriched P. falciparum liver stage parasites essentially free of hepatocyte contamination, were recovered for molecular studies. Our results establish a stable murine model for P. falciparum that will have a wide utility for assessing the biology of the parasite, potential anti-malarial chemotherapeutic agents and vaccine design.     

RAP1 controls rhoptry targeting of RAP2 in the malaria parasite Plasmodium falciparum

Rhoptry associated protein 1 (RAP1) and 2 (RAP2), together with a poorly described third protein RAP3, form the low molecular weight complex within the rhoptries of Plasmodium falciparum. These proteins are thought to play a role in erythrocyte invasion by the extracellular merozoite and are important vaccine candidates. We used gene-targeting technology in P.falciparum blood-stage parasites to disrupt the RAP1 gene, producing parasites that express severely truncated forms of RAP1. Immunoprecipitation experiments suggest that truncated RAP1 species did not complex with RAP2 and RAP3. Consistent with this were the distinct subcellular localizations of RAP1 and 2 in disrupted RAP1 parasites, where RAP2 does not traffic to the rhoptries but is instead located in a compartment that appears related to the lumen of the endoplasmic reticulum. These results suggest that RAP1 is required to localize RAP2 to the rhoptries, supporting the hypothesis that rhoptry biogenesis is dependent in part on the secretory pathway in the parasite. The observation that apparently host-protective merozoite antigens are not essential for efficient erythrocyte invasion has important implications for vaccine design.     

Pv12, a 6-Cys antigen of Plasmodium vivax, is localized to the merozoite rhoptry

Pf12 in Plasmodium falciparum has been characterized as a merozoite surface protein and the Pf12 gene is actively transcribed during schizont stage. An orthologous gene, Pv12, has been identified in genome of P. vivax, but the protein product has not been characterized. The Pv12 is a 362 amino acid long polypeptide encoded by a single exon gene PVX_113775, for which orthologous genes have been identified in other Plasmodium species by bioinformatic approaches. Pv12 contains two predicted six-cysteine (6-Cys) domains, which may be constrained by predicted disulfide bonds, and a transmembrane domain and a predicted GPI anchor attachment site in C-terminal region. The recombinant Pv12 protein is recognized by serum antibodies of patients naturally exposed to P. vivax and the native Pv12 protein from parasite extract is also recognized by immune mouse serum. The Pv12 is localized in rhoptry; an apical organelle of the merozoite, and the localization pattern of Pv12 is distinct from that of Pf12 in P. falciparum. The present study suggests that Pv12 is immunogenic in humans during parasite infection and it could play an important role in erythrocyte invasion.     

The crystal structure of C-terminal merozoite surface protein 1 at 1.8 A resolution, a highly protective malaria vaccine candidate

The C-terminal proteolytic processing product of merozoite surface protein 1 (MSP1) appears essential for successful erythrocyte invasion by the malarial parasite, Plasmodium. We have determined the crystal structure at 1.8 A resolution of a soluble baculovirus-recombinant form of the protein from P. cynomolgi, which confers excellent protective efficacy in primate vaccination trials. The structure comprises two EGF-like domains, and sequence comparisons strongly suggest that the same conformation is present in all species of Plasmodium, including P. falciparum and P. vivax, which are pathogenic in man. In particular, conserved interdomain contacts between the two EGF modules should preserve the compact form of the molecule in all species. Implications of the crystal structure for anti-malarial vaccine development are discussed.     

Cloning, expression, and characterisation of a Plasmodium vivax MSP7 family merozoite surface protein

Plasmodium vivax remains the most widespread Plasmodium parasite species around the world, producing about 75 million malaria cases, mainly in South America and Asia. A vaccine against this disease is of urgent need, making the identification of new antigens involved in target cell invasion, and thus potential vaccine candidates, a priority. A protein belonging to the P. vivax merozoite surface protein 7 (PvMSP7) family was identified in this study. This protein (named PvMSP7(1)) has 311 amino acids displaying an N-terminal region sharing high identity with P. falciparum MSP7, as well as a similar proteolytical cleavage pattern. This protein's expression in P. vivax asexual blood stages was revealed by immuno-histochemical and molecular techniques.     

Plasmodium vivax thrombospondin related adhesion protein: immunogenicity and protective efficacy in rodents and Aotus monkeys

The thrombospondin related adhesion protein (TRAP) is a malaria pre-erythrocytic antigen currently pursued as malaria vaccine candidate to Plasmodium falciparum. In this study, a long synthetic peptide (LSP) representing a P. vivax TRAP fragment involved in hepatocyte invasion was formulated in both Freund and Montanide ISA 720 adjutants and administered by IM and subcutaneous routes to BALB/c mice and Aotus monkeys. We measured specific humoral immune responses in both animal species and performed a sporozoite challenge in Aotus monkeys to assess the protective efficacy of the vaccine. After immunization both mice and Aotus seroconverted as shown by ELISA, and the specific anti-peptide antibodies cross reacted with the parasite in IFAT assays. Only two out of six immunized animals became infected after P. vivax sporozoite challenge as compared with four out of six animals from the control group. These results suggest that this TRAP fragment has protective potential against P. vivax malaria and deserves further studies as vaccine candidate.     

Allelic polymorphisms in apical membrane antigen-1 are responsible for evasion of antibody-mediated inhibition in Plasmodium falciparum

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.     

Aotus monkeys: their great value for anti-malaria vaccines and drug testing

Non-human primates represent a valuable resource for testing potential vaccines candidates and drugs for human use. Malaria remains one of the greatest burdens for the humanity represented by approximately 500 million new clinical cases per year worldwide and at least two million deaths caused annually. Additional control measures such as vaccines and new anti-malarial compounds are therefore urgently needed. Safety and protective efficacy studies in animal models are critical steps for vaccines and drugs development and primate models are probably the most appropriate for this purpose. Although Aotus genus provides several species susceptible to both Plasmodium falciparum and Plasmodium vivax, having different susceptibility to malaria, Aotus lemurinus griseimembra represents the best current malaria primate model because of its high susceptibility to infection by blood forms and sporozoites of both species of Plasmodium. Although the ultimate validation of this model depends upon human trials, over the past two decades these monkeys have proved very useful to test multiple malaria vaccine candidates prior to trials in humans. A good correlation between the B- and T-cell epitopes recognised by humans and by immunised monkeys has been documented, and cross reactivity between reagents for human and Aotus cytokines and lymphocyte markers have been identified and are facilitating the selection of vaccine candidates for clinical trials. Aotus also represents a good model for the screening of anti-malarial drugs and the understanding of malaria pathogenesis as well. In view of the decreasing availability of these primates, breeding programs and biomedical research facilities must be improved in countries of primate origin.     

Cross-reactivity studies of an anti-Plasmodium vivax apical membrane antigen 1 monoclonal antibody: binding and structural characterisation

Apical membrane antigen 1 (AMA1) has an important, but as yet uncharacterised, role in host cell invasion by the malaria parasite, Plasmodium. The protein, which is quite conserved between Plasmodium species, comprises an ectoplasmic region, a single transmembrane segment and a small cytoplasmic domain. The ectoplasmic region, which can induce protective immunity in animal models of human malaria, is a leading vaccine candidate that has entered clinical trials. The monoclonal antibody F8.12.19, raised against the recombinant ectoplasmic region of AMA1 from Plasmodium vivax, cross-reacts with homologues from Plasmodium knowlesi, Plasmodium cynomolgi, Plasmodium berghei and Plasmodium falciparum, as shown by immunofluorescence assays on mature schizonts. The binding of F8.12.19 to recombinant AMA1 from both P. vivax and P. falciparum was measured by surface plasmon resonance, revealing an apparent affinity constant that is about 100-fold weaker for the cross-reacting antigen when compared to the cognate antigen. Crystal structure analysis of Fab F8.12.19 complexed to AMA1 from P. vivax and P. falciparum shows that the monoclonal antibody recognises a discontinuous epitope located on domain III of the ectoplasmic region, the major component being a loop containing a cystine knot. The structures provide a basis for understanding the cross-reactivity. Antibody contacts are made mainly to main-chain and invariant side-chain atoms of AMA1; contact antigen residues that differ in sequence are located at the periphery of the antigen-binding site and can be accommodated at the interface between the two components of the complex. The implications for AMA1 vaccine development are discussed.     

Getting down to malarial nuts and bolts: the interaction between Plasmodium vivax merozoites and their host erythrocytes

Of the four Plasmodium species that routinely cause malaria in humans, Plasmodium falciparum is responsible for the majority of malaria mortality and consequently gets most of the headlines. Outside Africa, however, more malaria cases are caused by its distant cousin Plasmodium vivax, resulting in a daunting morbidity and economic burden for countries across Asia and the Americas. Plasmodium life cycles are complex, but the symptoms and pathology of malaria occur during the blood phase, when merozoites recognize and invade erythrocytes, initiating a developmental programme that culminates in lysis of the erythrocyte and release of multiple daughter merozoites. P. vivax merozoites are dependent on a single host cell receptor for erythrocyte invasion, the Duffy antigen receptor for chemokines, and humans that do not express this receptor on the surface of their erythrocytes are immune to P. vivax infection. This essential receptor-ligand interaction is addressed from both the host and parasite side in two papers in this issue of Molecular Microbiology, with important implications for plans to develop a P. vivax vaccine.     

Biochemical, biophysical, and functional characterization of bacterially expressed and refolded receptor binding domain of Plasmodium vivax duffy-binding protein

Invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions. Plasmodium vivax is completely dependent on interaction with the Duffy blood group antigen to invade human erythrocytes. The P. vivax Duffy-binding protein, which binds the Duffy antigen during invasion, belongs to a family of erythrocyte-binding proteins that also includes Plasmodium falciparum sialic acid binding protein and Plasmodium knowlesi Duffy binding protein. The receptor binding domains of these proteins lie in a conserved, N-terminal, cysteine-rich region, region II, found in each of these proteins. Here, we have expressed P. vivax region II (PvRII), the P. vivax Duffy binding domain, in Escherichia coli. Recombinant PvRII is incorrectly folded and accumulates in inclusion bodies. We have developed methods to refold and purify recombinant PvRII in its functional conformation. Biochemical, biophysical, and functional characterization confirms that recombinant PvRII is pure, homogeneous, and functionally active in that it binds Duffy-positive human erythrocytes with specificity. Refolded PvRII is highly immunogenic and elicits high titer antibodies that can inhibit binding of P. vivax Duffy-binding protein to erythrocytes, providing support for its development as a vaccine candidate for P. vivax malaria. Development of methods to produce functionally active recombinant PvRII is an important step for structural studies as well as vaccine development.     

Plasmodium vivax: cloning and expression of a major blood-stage surface antigen

Plasmodium vivax is a highly prevalent malaria pathogen of man; the following report is the first to describe the cloning and expression of a major asexual erythrocytic stage antigen of this species. The screening of a genomic DNA expression library with a monoclonal antibody directed against a 200-kDa surface component (Pv200) of the more mature schizonts of P. vivax led to the selection of a recombinant bacterial clone which produced a fusion protein. Mouse and rabbit immune sera raised against the purified fusion protein recognized the 200-kDa parasite antigen on Western blots and reacted with the surface of segmenters by immunofluorescence. Sequencing of the 1.9-kb P. vivax DNA insert coding for this fusion protein revealed a 45-47% homology at the nucleotide level with the P. falciparum gene of a parasite surface antigen, Pf195, which has been shown to be a promising candidate for a malaria vaccine in primates and in man.     

Cross-reactive anti-PfCLAG9 antibodies in the sera of asymptomatic parasite carriers of Plasmodium vivax

The PfCLAG9 has been extensively studied because their immunogenicity. Thereby, the gene product is important for therapeutics interventions and a potential vaccine candidate. Antibodies against synthetic peptides corresponding to selected sequences of the Plasmodium falciparum antigen PfCLAG9 were found in sera of falciparum malaria patients from Rondônia, in the Brazilian Amazon. Much higher antibody titres were found in semi-immune and immune asymptomatic parasite carriers than in subjects suffering clinical infections, corroborating original findings in Papua Guinea. However, sera of Plasmodium vivax patients from the same Amazon area, in particular from asymptomatic vivax parasite carriers, reacted strongly with the same peptides. Bioinformatic analyses revealed regions of similarity between P. falciparum Pfclag9 and the P. vivax ortholog Pvclag7. Indirect fluorescent microscopy analysis showed that antibodies against PfCLAG9 peptides elicited in BALB/c mice react with human red blood cells (RBCs) infected with both P. falciparum and P. vivax parasites. The patterns of reactivity on the surface of the parasitised RBCs are very similar. The present observations support previous findings that PfCLAG9 may be a target of protective immune responses and raises the possibility that the cross reactive antibodies to PvCLAG7 in mixed infections play a role in regulate the fate of Plasmodium mixed infections.     

Sequence analysis of the Rhop-3 gene of Plasmodium yoelii

The 110 kDa/Rhop-3 rhoptry protein of Plasmodium falciparum is non-covalently associated with two other proteins, the 140 kDa Rhop-1 and the 130 kDa Rhop-2. cDNAs encoding Rhop-3 from Plasmodium yoelii were isolated using rhoptry-specific antisera from Plasmodium falciparum, P. yoelii, and Plasmodium chabaudi. The cDNAs encoded peptides with partial homology to the C-terminal region (residues 541-861) of P. falciparum Rhop-3. Core regions of homology to the P. falciparum gene will be useful in determining the biological role of Rhop-3 and its potential as a vaccine candidate for malaria.     

Recombinant Mycobacterium bovis BCG producing the circumsporozoite protein of Plasmodium falciparum FCC-1/HN strain induces strong immune responses in BALB/c mice

The current vaccine against tuberculosis, Mycobacterium bovis strain bacillus Calmette-Guerin (BCG), offers potential advantages as a live, innately immunogenic vaccine vehicle for expression and delivery of protective recombinant antigens. Malaria is one of the severest parasitic diseases in humans especially in the developing world. No efficacious vaccine is currently available. However, circumsporozoite protein (CSP) is a malaria vaccine candidate currently undergoing clinical trials. We analyzed the immune response to recombinant BCG (rBCG) vaccine expressing Plasmodium falciparum CSP (BCG-CSP) under the control of heat shock protein 70 promoter in BALB/c mice. The lymphocytes proliferative response to P. falciparum soluble antigen was significantly higher than those in the groups of BCG and normal saline, and the production of cytokines (IFN-gamma and IL-2) in response to malaria antigen was significantly higher in rBCG and BCG groups than control group of normal saline. A specific IgG antibody response against P. falciparum antigen of CSP was also characterized. The booster injection could enhance the production of cytokine, proliferation responses of spleen lymphocytes and the antibodies titer of BCG-CSP. The results in the study demonstrated that rBCG vaccine producing CSP is an appropriate vaccine for further evaluation in non-human primates.     

Plasmodium vivax: in vitro susceptibility of blood stages to synthetic trioxolane compounds and the diamidine DB75

Plasmodium vivax is an important human pathogen causing malaria in more temperate climates of the world. Similar to Plasmodium falciparum, the causative agent for malaria tropica, drug resistance is beginning to emerge for this parasite species and this hampers adequate treatment of infection. We have used a short-term ex vivo drug assay to monitor activity of OZ277 (RBx-11160), a fully synthetic anti-malarial peroxide, and the diamidine DB75 against P. vivax. For both compounds as well as the anti-malarial reference compounds artesunate, artemether, and chloroquine, the in vitro IC(50) values were determined in one-cycle hypoxanthine incorporation assays. Results from such assays were found to be very similar compared to IC(50) values obtained from one-cycle P. falciparum hypoxanthine assays. We demonstrate the anti-parasite activity of OZ277 and the reference compounds to be faster than that of DB75. These data warrant clinical testing of OZ277 against P. vivax malaria and support recent data on clinical activity against P. vivax for DB75.