Inhibit Merozoite Invasion Research Articles

PfRON3 is an erythrocyte-binding protein and a potential blood-stage vaccine candidate antigen

BackgroundErythrocyte invasion by merozoites is an essential step in Plasmodium falciparum infection and leads to subsequent disease pathology. Proteins both on the merozoite surface and secreted from the apical organelles (micronemes, rhoptries and dense granules) mediate the invasion of erythrocytes; some of the molecules have been regarded as targets in the development of an anti-malaria vaccine. Recently, a subgroup of rhoptry neck proteins (PfRON2, PfRON4 and PfRON5) associated with the microneme protein apical membrane antigen AMA1 has been described as components of the moving junction complex that assists merozoite invasion into erythrocytes. However, unlike PfRON2, PfRON4 and PfRON5, the latest study suggested that PfRON3 might be located in the rhoptry bulb and participates in a novel PfRON complex (PfRON2, 3 and 4), but does not form a complex with AMA1. Additionally, the full-length PfRON3 protein possesses three transmembrane regions at the N-terminus, which is highly conserved among RON3 orthologues in the genus Plasmodium, Toxoplasma gondii and Eimeria tenella. Overall, these findings suggest that PfRON3 may play an important role in merozoite invasion into erythrocytes.ResultsPfRON3 was primarily expressed during the late trophozoite stage, with a peak in transcription levels at 40 hours post-invasion. The subcellular localization of PfRON3 was confirmed that it is a merozoite rhoptry bulb protein. Additionally, the recombinant form of PfRON3 protein bound to the erythrocyte and was recognized by sera collected from malaria endemic areas in Africa, and anti-PfRON3 antibodies significantly inhibited merozoite invasion into erythrocytes.MethodsThe expression of PfRON3 was analysed via real-time quantitative PCR, and the recombinant PfRON3 proteins were generated with an Escherichia coli expression system. The subcellular localization of PfRON3 was assessed with immunoelectron microscopy and immunofluorescence assay (IFA). The recognition PfRON3 by malaria immune sera was analysed with an enzyme-linked immunosorbent assay (ELISA). Erythrocyte-binding assays were performed using recombinant PfRON3 proteins and invasion inhibition assays were carried out with PfRON3-specific antibodies.ConclusionThis study confirmed that PfRON3 is a rhoptry protein with an erythrocyte-binding property, which is likely associated red blood cell invasion. PfRON3 is a potential vaccine candidate.Electronic supplementary materialThe online version of this article (doi:10.1186/1475-2875-13-490) contains supplementary material, which is available to authorized users.

Plasmodium vivax Duffy binding protein: understanding targets of protective immunity

Plasmodium vivax merozoite invasion of reticulocytes is a rapid process that involves a series of specific, sequential interactions. These interactions are crucial for invasion and continued blood-stage infection, thus representing attractive targets for vaccine development. The Duffy binding protein (DBP) is a vital microneme protein associated with the decisive, irreversible step of junction formation during the later stages of the invasion process. The ligand domain of DBP, known as region II (DBPII), binds the Duffy Antigen Receptor for Chemokines (DARC) by dimerization of two DBP and two DARC molecules forming a heterotetrameric structure. Naturally acquired antibodies to DBPII are prevalent in residents of areas where vivax malaria is endemic. These antibodies can inhibit DBPII binding and merozoite invasion of human reticulocytes; however, they tend to be poorly inhibitory, strain-specific, and short-lived. Remarkably, strain-specific serologic activity can be altered by even single amino acid substitutions found in allelic variants of DBPII. While strain-transcending, broadly neutralizing antibodies are less prevalent, there is a significant correlation of their antibody reactivity to certain epitopes and inhibition of DBPII-receptor function. Our data indicate epitopes at or near the dimer interface of DBPII represent the primary targets of naturally occurring protective anti-DBPII antibodies that can inhibit merozoite invasion of host erythrocytes. However, these epitopes also contain most of the prevalent variable residues resulting from DBP allelic diversity. These data are consistent with the hypothesis that DBP variation is an immune evasion mechanism responsible for strain-specific immunity and stable, broadly neutralizing immunity is achieved when antibodies target functionally-conserved epitopes, thereby blocking DBP dimerization and inhibiting invasion. To overcome the natural tendency for eliciting a strain-specific response, synthetic DBPII alleles were evaluated for their ability to broaden efficacy of strain-transcending inhibition against diverse DBPII alleles. In an effort to identify potential conserved, protective epitopes we have prepared and evaluated a panel of anti-DBPII murine monoclonal antibodies. Inhibitory activities of these monoclonal antibodies were characterized with in vitro functional assays for DBPII and their respective minimal epitopes were mapped using phage display. These targets of putative strain-transcending inhibition identify epitopes that may be targeted for vaccine-induced anti-DBPII inhibitory antibodies.

Vaccination with Conserved Regions of Erythrocyte-Binding Antigens Induces Neutralizing Antibodies against Multiple Strains of Plasmodium falciparum

BackgroundA 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.MethodsWe 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.ResultsThis 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.ConclusionWe 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.

Comparative Immunogenicities of Full-Length Plasmodium falciparum Merozoite Surface Protein 3 and a 24-Kilodalton N-Terminal Fragment

Recombinant Plasmodium falciparum merozoite surface protein 3 (PfMSP3F) and a 24-kDa fragment from its N terminus (MSP3N) that includes the essential conserved domain, which elicits the maximum antibody (Ab)-dependent cellular inhibition (ADCI), were expressed as soluble proteins in Escherichia coli. Both proteins were found to be stable in both soluble and lyophilized forms. Immunization with MSP3F and MSP3N formulated separately with two human-compatible adjuvants, aluminum hydroxide (Alhydrogel) and Montanide ISA 720, produced significant antibody responses in mice and rabbits. Polyclonal Abs against both antigens recognized native MSP3 in the parasite lysate. These two Abs also recognized two synthetic peptides, previously characterized to possess B cell epitopes from the N-terminal region. Antibody depletion assay showed that most of the IgG response is directed toward the N-terminal region of the full protein. Anti-MSP3F and anti-MSP3N rabbit antibodies did not inhibit merozoite invasion or intraerythrocytic development but significantly reduced parasitemia in the presence of human monocytes. The ADCI demonstrated by anti-MSP3N antibodies was comparable to that exhibited by anti-MSP3F antibodies (both generated in rabbit). These results suggest that the N-terminal fragment of MSP3 can be considered a vaccine candidate that can form part of a multigenic vaccine against malaria.

Passive Immunoprotection of Plasmodium falciparum-Infected Mice Designates the CyRPA as Candidate Malaria Vaccine Antigen

An effective malaria vaccine could prove to be the most cost-effective and efficacious means of preventing severe disease and death from malaria. In an endeavor to identify novel vaccine targets, we tested predicted Plasmodium falciparum open reading frames for proteins that elicit parasite-inhibitory Abs. This has led to the identification of the cysteine-rich protective Ag (CyRPA). CyRPA is a cysteine-rich protein harboring a predicted signal sequence. The stage-specific expression of CyRPA in late schizonts resembles that of proteins known to be involved in merozoite invasion. Immunofluorescence staining localized CyRPA at the apex of merozoites. The entire protein is conserved as shown by sequencing of the CyRPA encoding gene from a diverse range of P. falciparum isolates. CyRPA-specific mAbs substantially inhibited parasite growth in vitro as well as in a P. falciparum animal model based on NOD-scid IL2Rγ(null) mice engrafted with human erythrocytes. In contrast to other P. falciparum mouse models, this system generated very consistent results and evinced a dose-response relationship and therefore represents an unprecedented in vivo model for quantitative comparison of the functional potencies of malaria-specific Abs. Our data suggest a role for CyRPA in erythrocyte invasion by the merozoite. Inhibition of merozoite invasion by CyRPA-specific mAbs in vitro and in vivo renders this protein a promising malaria asexual blood-stage vaccine candidate Ag.

Antibodies against a Plasmodium falciparum antigen PfMSPDBL1 inhibit merozoite invasion into human erythrocytes

One approach to develop a malaria blood-stage vaccine is to target proteins that play critical roles in the erythrocyte invasion of merozoites. The merozoite surface proteins (MSPs) and the erythrocyte-binding antigens (EBAs) are considered promising vaccine candidates, for they are known to play important roles in erythrocyte invasion and are exposed to host immune system. Here we focused on a Plasmodium falciparum antigen, PfMSPDBL1 (encoded by PF10_0348 gene) that is a member of the MSP3 family and has both Duffy binding-like (DBL) domain and secreted polymorphic antigen associated with merozoites (SPAM) domain. Therefore, we aimed to characterize PfMSPDBL1 as a vaccine candidate. Recombinant full-length protein (rFL) of PfMSPDBL1 was synthesized by a wheat germ cell-free system, and rabbit antiserum was raised against rFL. We show that rabbit anti-PfMSPDBL1 antibodies inhibited erythrocyte invasion of wild type parasites in vitro in a dose dependent manner, and the specificity of inhibitory activity was confirmed using PfMSPDBL1 knockout parasites. Pre-incubation of the anti-PfMSPDBL1 antibodies with the recombinant SPAM domain had no effect on the inhibitory activity suggesting that antibodies to this region were not involved. In addition, antibodies to rFL were elicited by P. falciparum infection in malaria endemic area, suggesting the PfMSLDBL1 is immunogenic to humans. Our results suggest that PfMSPDBL1 is a novel blood-stage malaria vaccine candidate.

A single amino acid change in the Plasmodium falciparum RH5 (PfRH5) human RBC binding sequence modifies its structure and determines species-specific binding activity

Identifying the ligands or regions derived from them which parasites use to invade their target cells has proved to be an excellent strategy for identifying targets for vaccine development. Members of the reticulocyte-binding homologue family (PfRH), including RH5, have been implicated in invasion as adhesins binding to specific receptors on erythrocyte surface. The regions mediating PfRH5-RBC specific interactions have been identified here by fine mapping the whole PfRH5 protein sequence. These regions, called high activity binding peptides (HABPs), bind to a receptor which is sensitive to trypsin treatment and inhibit merozoite invasion of RBCs by up to 80%, as has been found for HABP 36727. Our results show that a single amino acid change in the HABP 36727 sequence modifies a peptide's 3D structure, thereby resulting in a loss of specific binding to human RBCs and its inhibition ability, while binding to Aotus RBC remains unmodified. Such invasion differences and binding ability produced by replacing a single amino acid in an essential molecule, such as PfRH5, highlight the inherent difficulties associated with developing a fully effective vaccine against malaria.

Comparative Immunogenicities of Full-Length Plasmodium falciparum Merozoite Surface Protein 3 and a 24-Kilodalton N-Terminal Fragment

Recombinant Plasmodium falciparum merozoite surface protein 3 (PfMSP3F) and a 24-kDa fragment from its N terminus (MSP3N) that includes the essential conserved domain, which elicits the maximum antibody (Ab)-dependent cellular inhibition (ADCI), were expressed as soluble proteins in Escherichia coli. Both proteins were found to be stable in both soluble and lyophilized forms. Immunization with MSP3F and MSP3N formulated separately with two human-compatible adjuvants, aluminum hydroxide (Alhydrogel) and Montanide ISA 720, produced significant antibody responses in mice and rabbits. Polyclonal Abs against both antigens recognized native MSP3 in the parasite lysate. These two Abs also recognized two synthetic peptides, previously characterized to possess B cell epitopes from the N-terminal region. Antibody depletion assay showed that most of the IgG response is directed toward the N-terminal region of the full protein. Anti-MSP3F and anti-MSP3N rabbit antibodies did not inhibit merozoite invasion or intraerythrocytic development but significantly reduced parasitemia in the presence of human monocytes. The ADCI demonstrated by anti-MSP3N antibodies was comparable to that exhibited by anti-MSP3F antibodies (both generated in rabbit). These results suggest that the N-terminal fragment of MSP3 can be considered a vaccine candidate that can form part of a multigenic vaccine against malaria.

Reticulocyte and Erythrocyte Binding-Like Proteins Function Cooperatively in Invasion of Human Erythrocytes by Malaria Parasites

Plasmodium falciparum causes the most severe form of malaria in humans and invades erythrocytes using multiple ligand-receptor interactions. Two important protein families involved in erythrocyte binding are the erythrocyte binding-like (EBL) and the reticulocyte binding-like (RBL or P. falciparum Rh [PfRh]) proteins. We constructed P. falciparum lines lacking expression of EBL proteins by creating single and double knockouts of the corresponding genes for eba-175, eba-181, and eba-140 and show that the EBL and PfRh proteins function cooperatively, consistent with them playing a similar role in merozoite invasion. We provide evidence that PfRh and EBL proteins functionally interact, as loss of function of EBA-181 ablates the ability of PfRh2a/b protein antibodies to inhibit merozoite invasion. Additionally, loss of function of some ebl genes results in selection for increased transcription of the PfRh family. This provides a rational basis for considering PfRh and EBL proteins for use as a combination vaccine against P. falciparum. We immunized rabbits with combinations of PfRh and EBL proteins to test the ability of antibodies to block merozoite invasion in growth inhibition assays. A combination of EBA-175, PfRh2a/b, and PfRh4 recombinant proteins induced antibodies that potently blocked merozoite invasion. This validates the use of a combination of these ligands as a potential vaccine that would have broad activity against P. falciparum.

Immunogenicity and in vitro protective efficacy of recombinant Mycobacterium bovis bacille Calmette Guerin (rBCG) expressing the 19 kDa merozoite surface protein-1 (MSP-119) antigen of Plasmodium falciparum

Vaccine development against the blood-stage malaria parasite is aimed at reducing the pathology of the disease. We constructed a recombinant Mycobacterium bovis bacille Calmette Guerin (rBCG) expressing the 19 kDa C-terminus of Plasmodium falciparum merozoite surface protein-1 (MSP-1(19)) to evaluate its protective ability against merozoite invasion of red blood cells in vitro. A mutated version of MSP-1(19), previously shown to induce the production of inhibitory but not blocking antibodies, was cloned into a suitable shuttle plasmid and transformed into BCG Japan (designated rBCG016). A native version of the molecule was also cloned into BCG (rBCG026). Recombinant BCG expressing the mutated version of MSP-1(19) (rBCG016) elicited enhanced specific immune response against the epitope in BALB/c mice as compared to rBCG expressing the native version of the epitope (rBCG026). Sera from rBCG016-immunized mice contained significant levels of specific IgG, especially of the IgG2a subclass, against MSP-1(19) as determined by enzyme-linked immunosorbent assay. The sera was reactive with fixed P. falciparum merozoites as demonstrated by indirect immunofluorescence assay (IFA) and inhibited merozoite invasion of erythrocytes in vitro. Furthermore, lymphocytes from rBCG016-immunized mice demonstrated higher proliferative response against the MSP-1(19) antigen as compared to those of rBCG026- and BCG-immunized animals. rBCG expressing the mutated version of MSP-1(19) of P. falciparum induced enhanced humoral and cellular responses against the parasites paving the way for the rational use of rBCG as a blood-stage malaria vaccine candidate.

Antibody responses to 43 and 48 kDa antigens of blood-stage Plasmodium berghei in Balb/c mice

Progress towards a vaccine against malaria is advancing rapidly with several candidate antigens being tested for their safety and efficacy. In present investigation, two polypeptides (43 and 48kDa) of Plasmodium berghei (NK-65) were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immunogenicity and protective efficacy of both these polypeptides formulated in saponin has been compared in Balb/c mice against challenge infection with P. berghei. Antibody responses were evaluated by indirect fluorescent antibody test and enzyme-linked immunosorbent assay. Merozoite invasion inhibition assay and challenge infections revealed that 48kDa antigen is better immunogen as compared to 43kDa and provide better protection against rodent malaria infection.

Interaction between Plasmodium falciparum Apical Membrane Antigen 1 and the Rhoptry Neck Protein Complex Defines a Key Step in the Erythrocyte Invasion Process of Malaria Parasites

Invasion of host cells by apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii, is a multistep process. Central to invasion is the formation of a tight junction, an aperture in the host cell through which the parasite pulls itself before settling into a newly formed parasitophorous vacuole. Two protein groups, derived from different secretory organelles, the micronemal protein AMA1 and the rhoptry proteins RON2, RON4, and RON5, have been shown to form part of this structure, with antibodies targeting P. falciparum AMA1 known to inhibit invasion, probably via disruption of its association with the PfRON proteins. Inhibitory AMA1-binding peptides have also been described that block P. falciparum merozoite invasion of the erythrocyte. One of these, R1, blocks invasion some time after initial attachment to the erythrocyte and reorientation of the merozoite to its apical pole. Here we show that the R1 peptide binds the PfAMA1 hydrophobic trough and demonstrate that binding to this region prevents its interaction with the PfRON complex. We show that this defined association between PfAMA1 and the PfRON complex occurs after reorientation and engagement of the actomyosin motor and argue that it precedes rhoptry release. We propose that the formation of the AMA1-RON complex is essential for secretion of the rhoptry contents, which then allows the establishment of parasite infection within the parasitophorous vacuole.

Well-Defined Regions of the Plasmodium falciparum Reticulocyte Binding Protein Homologue 4 Mediate Interaction with Red Blood Cell Membrane

Two widely studied parasite protein families are considered attractive targets for developing a fully effective antimalarial vaccine: the erythrocyte binding antigen (EBA) family defining a sialic acid-dependent invasion pathway, and reticulocyte-binding homologue (RH) proteins associated with sialic acid-independent red blood cell (RBC) invasion. In this study, the micronemal invasive PfRH4 protein was finely mapped using 20-mer-long synthetic peptides spanning the entire protein length to identify protein regions that establish high affinity interactions with human RBCs. Twenty conserved, mainly alpha-helical high-activity binding peptides (HABPs) with nanomolar dissociation constants and recognizing 32, 25, 22, and 20 kDa RBC membrane molecules in a chymotrypsin and/or trypsin-sensitive manner were identified in this protein. Anti-PfRH4 rabbit sera and PfRH4 HABPs inhibited merozoite invasion in vitro, therefore suggesting the implication of these HABPs in Plasmodium falciparum invasion and supporting their inclusion in further structural and immunological studies to design potential components of a minimal subunit-based, multiantigenic, chemically synthesized antimalarial vaccine.

Identification of conserved erythrocyte binding regions in members of the Plasmodium falciparum Cys 6 lipid raft-associated protein family

Detergent-resistant lipid raft membrane-associated Pf12, Pf38 and Pf41 proteins belong to the Cys 6 family, whose members are implicated in Plasmodium falciparum invasion to erythrocytes. We have analyzed the interaction between 20-mer-long synthetic peptides spanning the entire Pf12, Pf38 and Pf41 sequences and erythrocytes. Eight high-activity binding peptides (HABPs) were identified in these proteins, which presented saturable bindings susceptible to erythrocytes’ enzymatic treatment, and β-turn, random coil and α-helical elements as principal structural features. Some of these HABPs inhibited merozoite invasion in vitro, suggesting a possible role of Pf12, Pf38 and Pf41 during erythrocyte invasion and supporting their inclusion in the design of a fully effective antimalarial vaccine.

Identification ofPlasmodium falciparumRhopH3 protein peptides that specifically bind to erythrocytes and inhibit merozoite invasion

The identification of sequences involved in binding to erythrocytes is an important step for understanding the molecular basis of merozoite-erythrocyte interactions that take place during invasion of the Plasmodium falciparum malaria parasite into host cells. Several molecules located in the apical organelles (micronemes, rhoptry, dense granules) of the invasive-stage parasite are essential for erythrocyte recognition, invasion, and establishment of the nascent parasitophorous vacuole. Particularly, it has been demonstrated that rhoptry proteins play an important role in binding to erythrocyte surface receptors, among which is the PfRhopH3 protein, which triggers important immune responses in patients from endemic regions. It has also been reported that anti-RhopH3 antibodies inhibit in vitro invasion of erythrocytes, further supporting its direct involvement in erythrocyte invasion processes. In this study, PfRhopH3 consecutive peptides were synthesized and tested in erythrocyte binding assays for identifying those regions mediating binding to erythrocytes. Fourteen PfRhopH3 peptides presenting high specific binding activity were found, whose bindings were saturable and presented nanomolar dissociation constants. These high-activity binding peptides (HABPs) were characterized by having alpha-helical structural elements, as determined by circular dichroism, and having receptors of a possible sialic acid-dependent and/or glycoprotein-dependent nature, as evidenced in enzyme-treated erythrocyte binding assays and further corroborated by cross-linking assay results. Furthermore, these HABPs inhibited merozoite in vitro invasion of normal erythrocytes at 200 microM by up to 60% and 90%, suggesting that some RhopH3 protein regions are involved in the P. falciparum erythrocyte invasion.

Characterization of Plasmodium falciparum integral membrane protein Pf25‐IMP and identification of its red blood cell binding sequences inhibiting merozoite invasion in vitro

The identification of proteins present on the surface of Plasmodium falciparum-infected red blood cells as well as of free merozoites has been widely considered as one of the main areas of research in the development of an antimalarial vaccine due to their involvement in the parasite's pathogenesis and invasion mechanisms. Major advances had been accomplished in this area thanks to the analysis of the reported genomic sequence of P. falciparum, allowing for the identification of genes encoding for putative integral membrane proteins. This study reports for the first time the transcription of the MAL8P1.3 gene, which codifies for a 25-kDa integral membrane protein of P. falciparum (FCB-2 strain), namely, Pf25-IMP. Western blot and immunofluorescence assays using goat polyclonal sera indicate that this protein is expressed in erythrocytic asexual blood stages. A highly robust, sensible, and specific receptor-ligand interaction assay allowed identification of two high activity binding peptides (HABPs) derived from Pf25-IMP: 30577 ((41)YKTANENVKLASSLSDRLSR(60)) and 30583 ((161)LNKKTVVRKIAEGLGYTIVF(180)). Both HABPs bound with high affinity to human red blood cells (RBCs), and such binding was susceptible to enzyme treatment with trypsin. A common RBC surface receptor of apparently 48 kDa was found for both HABPs, plus an additional 31-kDa receptor for HABP 30577. HABP 30577 inhibited merozoite invasion in vitro by 73%, while HABP 30583 showed a 59% inhibition at 200 microM concentration. The data suggest a possible role of Pf25-IMP in merozoite invasion to RBCs and support its inclusion in further immunological studies for evaluating its potential as vaccine candidates.

Plasmodium falciparum variant STEVOR antigens are expressed in merozoites and possibly associated with erythrocyte invasion

BackgroundPlasmodium falciparum STEVOR proteins, encoded by the multicopy stevor gene family have no known biological functions. Their expression and unique locations in different parasite life cycle stages evoke multiple functionalities. Their abundance and hypervariability support a role in antigenic variation.MethodsImmunoblotting of total parasite proteins with an anti-STEVOR antibody was used to identify variant antigens of this gene family and to follow changes in STEVOR expression in parasite populations panned on CSA or CD36 receptors. Immunofluorescence assays and immunoelectron microscopy were performed to study the subcellular localization of STEVOR proteins in different parasite stages. The capacity of the antibody to inhibit merozoite invasion of erythrocytes was assessed to determine whether STEVOR variants were involved in the invasion process.ResultsAntigenic variation of STEVORs at the protein level was observed in blood stage parasites. STEVOR variants were found to be present on the merozoite surface and in rhoptries. An insight into a participation in erythrocyte invasion was gained through an immunofluorescence analysis of a sequence of thin slides representing progressive steps in erythrocyte invasion. An interesting feature of the staining pattern was what appeared to be the release of STEVORs around the invading merozoites. Because the anti-STEVOR antibody did not inhibit invasion, the role of STEVORs in this process remains unknown.ConclusionThe localization of STEVOR proteins to the merozoite surface and the rhoptries together with its prevalence as a released component in the invading merozoite suggest a role of these antigens in adhesion and/or immune evasion in the erythrocyte invasion process. These observations would also justify STEVORs for undergoing antigenic variation. Even though a role in erythrocyte invasion remains speculative, an association of members of the STEVOR protein family with invasion-related events has been shown.

Cytokine-induced inhibition of Plasmodium falciparum erythrocytic growth in vitro.

The addition of recombinant cytokines to Plasmodium falciparum in vitro cultures retarded the growth of the parasite with the effect of recombinant IL-2 (rIL-2) > interferon-gamma (IFN-gamma) > tumour necrosis factor-beta (TNF-beta). The process was concentration dependent, being greatest at 30,000 U/ml and required a 72-h period of continuous exposure for maximum effect. Growth inhibition, as determined morphologically and radiometrically, was a consequence of defective schizont maturation rather than inhibition of merozoite invasion. It was cumulative and detectable within one erythrocytic (48 h) growth cycle.

Effect of GPI anchor moiety on the immunogenicity of DNA plasmids encoding the 19‐kDa C‐terminal portion of Plasmodium falciparum MSP‐1

The glycosylphosphatidylinositol (GPI)-anchored Plasmodium falciparum merozoite surface protein 1 (MSP-1) is a widely studied malaria vaccine candidate. The C-terminal 19-kDa portion of MSP-1 (MSP-1(19)) is of particular interest because this polypeptide moiety remains bound to the parasite even after erythrocyte invasion, while the remainder of MSP-1 is shed during invasion. Studies have shown that antibodies against MSP-1(19) inhibit merozoite invasion of erythrocytes efficiently, and that MSP-1(19) produces protective immunity in mice and monkeys. To investigate the efficacy of MSP-1(19 )DNA vaccine and role of GPI anchor moiety in the immunogenicity of MSP-1(19), we constructed expression vectors that produce MSP-1(19) as either secretory or GPI-anchored polypeptide. Both constructs efficiently expressed MSP-1(19) in transfected HEK-293 cells. While the recombinant plasmid lacking GPI anchor signal sequence expressed MSP-1(19) mainly as secreted polypeptide, that containing GPI anchor signal sequence produced GPI-anchored MSP-1(19 )on cell surface. In immunized mice, both constructs produced substantial levels of MSP-1(19)-specific IgG1, IgG2a, IgG2b, IgG3, IgA and IgM antibodies. In both cases, the IgG1 level was significantly higher than other isotypes. Interestingly, the plasmid containing GPI anchor signal sequence produced significantly higher levels of IgG2a and IgG2b than the plasmid that lacks GPI signal sequence.

Modelling Immune Response and Drug Therapy in Human Malaria Infection

A new intra‐host model of malaria that describes the dynamics of the blood stages of the parasite and its interaction with red blood cells and immune effectors is proposed. Local and global stability of the disease free equilibrium are investigated. Conditions for existence and uniqueness of the endemic equilibrium are derived. An intra‐host basic reproductive number is identified. We deduce that drugs based on inhibiting parasite production are more effective than those based on inhibiting merozoite invasion of erythrocytes. We extend the model to incorporate, in addition to immune response, drug therapy, following treatment with antimalarial drugs. Using stability analysis of the model, it is shown that infection can be eradicated within the host if the drug efficacy level exceeds a certain threshold value. It will persist if the efficacy is below this threshold. Numerical simulations are done to verify the analytic results and illustrate possible behaviour of the models.