Identification of a Potent Combination of Key Plasmodium falciparum Merozoite Antigens That Elicit Strain-Transcending Parasite-Neutralizing Antibodies

Blood-stage malaria vaccines that target single Plasmodium falciparum antigens involved in erythrocyte invasion have not induced optimal protection in field trials. Blood-stage malaria vaccine development has faced two major hurdles, antigenic polymorphisms and molecular redundancy, which have led to an inability to demonstrate potent, strain-transcending, invasion-inhibitory antibodies. Vaccines that target multiple invasion-related parasite proteins may inhibit erythrocyte invasion more efficiently. Our approach is to develop a receptor-blocking blood-stage vaccine against P. falciparum that targets the erythrocyte binding domains of multiple parasite adhesins, blocking their interaction with their receptors and thus inhibiting erythrocyte invasion. However, with numerous invasion ligands, the challenge is to identify combinations that elicit potent strain-transcending invasion inhibition. We evaluated the invasion-inhibitory activities of 20 different triple combinations of antibodies mixed in vitro against a diverse set of six key merozoite ligands, including the novel ligands P. falciparum apical asparagine-rich protein (PfAARP), EBA-175 (PfF2), P. falciparum reticulocyte binding-like homologous protein 1 (PfRH1), PfRH2, PfRH4, and Plasmodium thrombospondin apical merozoite protein (PTRAMP), which are localized in different apical organelles and are translocated to the merozoite surface at different time points during invasion. They bind erythrocytes with different specificities and are thus involved in distinct invasion pathways. The antibody combination of EBA-175 (PfF2), PfRH2, and PfAARP produced the most efficacious strain-transcending inhibition of erythrocyte invasion against diverse P. falciparum clones. This potent antigen combination was selected for coimmunization as a mixture that induced balanced antibody responses against each antigen and inhibited erythrocyte invasion efficiently. We have thus demonstrated a novel two-step screening approach to identify a potent antigen combination that elicits strong strain-transcending invasion inhibition, supporting its development as a receptor-blocking malaria vaccine.     

Proteolytic activity of Plasmodium falciparum subtilisin-like protease 3 on parasite profilin,a multifunctional protein

Subtilisin-like proteases of malaria parasite Plasmodium falciparum (PfSUB1, 2 and 3) are expressed at late asexual blood stages. PfSUB1 and 2 are considered important drug targets due to their essentiality for parasite blood stages and role in merozoite egress and invasion of erythrocytes. We have earlier shown the in vitro serine protease activity of PfSUB3 and its localization at asexual blood stages. In this study, we attempted to identify the biological substrate(s) of PfSUB3 and found parasite profilin (PfPRF) as a substrate of the protease. Eukaryotic profilins are multifunctional proteins with primary role in regulation of actin filament assembly. PfPRF possesses biochemical features of eukaryotic profilins and its rodent ortholog is essential in blood stages. Profilin from related apicomplexan parasite Toxoplasma gondii (TgPRF) is known to be involved in parasite motility, host cell invasion, active egress from host cell, immune evasion and virulence in mice. In this study, mature PfSUB3 proteolysed recombinant PfPRF in a dose-dependent manner in in vitro assays. Recombinant PfPRF was assessed for its proinflammatory activity and found to induce high level of TNF-α and low but significant level of IL-12 from mouse bone marrow-derived dendritic cells. Proteolysis of PfPRF by PfSUB3 is suggestive of the probable role of the protease in the processes of motility, virulence and immune evasion.     

The cysteine-rich regions of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> RON2 bind with host erythrocyte and AMA1 during merozoite invasion

Invasion of Plasmodium falciparum merozoites into host erythrocyte involves a series of highly specific and sequential interaction between merozoite and host erythrocyte surface protein. The key step in the invasion process is the formation of a tight protein–protein interaction between host and parasite called as moving junction. A number of parasite proteins secreted from two organelles, microneme and rhoptry, play a role in initial interaction and junction formation between merozoite with host red blood cells (RBCs) during the invasion process. In the present study, we investigated the role of different domains of a P. falciparum rhoptry neck protein PfRON2. Immunofluorescence assay revealed close association of PfAMA1 and PfRON2 in the merozoites during the invasion process. PfRON2 domains were expressed on COS-7 cell surface, and their interaction was analysed with host RBCs and PfAMA1 protein by rosetting assays. The rosetting assays suggest that the C-terminal cysteine-rich domain of PfRON2 plays a role in binding with host erythrocyte. The C-terminal as well as the central cysteine-rich domain of PfRON2 interact with PfAMA1; this binding can be inhibited by monoclonal antibody (mAb 4 G2) against PfAMA1, suggesting that the hydrophobic groove of PfAMA1 binds to PfRON2. These results suggest that PfRON2 plays a role in merozoite invasion and thus it can be an important vaccine candidate antigen.     

Time-course of synthesis,transport and incorporation of a protein identified in purified membranes of host erythrocytes infected with a knob-forming strain of Plasmodium falciparum

Membranes from host erythrocytes infected with a knob-positive strain of Plasmodium falciparum were purified by free-flow electrophoresis or gradient centrifugation. In these membranes the main parasite-derived protein was a 92 000 Da protein which was not present after infection of erythrocytes with the corresponding knob-negative strain. The protein is synthesized between 9–21 h after merozoite invasion and then synthesis ceases. At least 6 h elapses between the start of synthesis and the appearance of the protein in the erythrocyte membrane. No precursor proteins for the 92 000 Da protein were found. Since the purified erythrocyte membranes were free from contamination with whole parasites or parasite membranes, the 92 000 Da protein is clearly present in the host erythrocyte membrane.     

Surface proteins of schizont-infected erythrocytes and merozoites of Plasmodium falciparum

Schizont-infected erythrocytes and merozoites were isolated from in vitro cultures of the human parasite, Plasmodium falciparum labeled with various radioactive substrates. The isolated merozoites were viable since they were able to reinvade fresh erythrocytes. On the basis of sensitivity to specific enzymes, eleven proteins synthesised by the parasite, were localised on the surface of the schizont-infected erythrocyte. Eight of these were glycoproteins, six of which appeared to represent three doublets. Five merozoite surface proteins were identified on the basis of their sensitivity to trypsin and chymotrypsin, treatments which also rendered the merozoite incapable of erythrocyte invasion. Merozoites appeared not to contain any glycoproteins; all of the glycoproteins synthesised by the parasite were apparently transported to the surface of the schizont-infected erythrocyte.     

RALP1 Is a Rhoptry Neck Erythrocyte-Binding Protein of Plasmodium falciparum Merozoites and a Potential Blood-Stage Vaccine Candidate Antigen

Erythrocyte invasion by merozoites is an obligatory stage of Plasmodium infection and is essential to disease progression. Proteins in the apical organelles of merozoites mediate the invasion of erythrocytes and are potential malaria vaccine candidates. Rhoptry-associated, leucine zipper-like protein 1 (RALP1) of Plasmodium falciparum was previously found to be specifically expressed in schizont stages and localized to the rhoptries of merozoites by immunofluorescence assay (IFA). Also, RALP1 has been refractory to gene knockout attempts, suggesting that it is essential for blood-stage parasite survival. These characteristics suggest that RALP1 can be a potential blood-stage vaccine candidate antigen, and here we assessed its potential in this regard. Antibodies were raised against recombinant RALP1 proteins synthesized by using the wheat germ cell-free system. Immunoelectron microscopy demonstrated for the first time that RALP1 is a rhoptry neck protein of merozoites. Moreover, our IFA data showed that RALP1 translocates from the rhoptry neck to the moving junction during merozoite invasion. Growth and invasion inhibition assays revealed that anti-RALP1 antibodies inhibit the invasion of erythrocytes by merozoites. The findings that RALP1 possesses an erythrocyte-binding epitope in the C-terminal region and that anti-RALP1 antibodies disrupt tight-junction formation, are evidence that RALP1 plays an important role during merozoite invasion of erythrocytes. In addition, human sera collected from areas in Thailand and Mali where malaria is endemic recognized this protein. Overall, our findings indicate that RALP1 is a rhoptry neck erythrocyte-binding protein and that it qualifies as a potential blood-stage vaccine candidate.     

Plasmodium falciparum: a simplified technique for obtaining singly infected erythrocytes

We report the development of a simple technique involving 15 ml polypropylene tubes and a rotatory incubator for obtaining erythrocytes singly infected with Plasmodium falciparum. This technique will be useful for cloning of the parasite. Our finding that P. falciparum merozoite invasion is inhibited during rotation suggests that this method may also be useful for the study of parasite-erythrocyte interactions under dynamic circulatory conditions.     

Plasmodium falciparum rhoptry neck protein 4 has conserved regions mediating interactions with receptors on human erythrocytes and hepatocyte membrane

Plasmodium falciparum-related malaria represents a serious worldwide public health problem due to its high mortality rates. P. falciparum expresses rhoptry neck protein 4 (PfRON4) in merozoite and sporozoite rhoptries, it participates in tight junction-TJ formation via the AMA-1/RON complex and is refractory to complete genetic deletion. Despite this, which PfRON4 key regions interact with host cells remain unknown; such information would be useful for combating falciparum malaria. Thirty-two RON4 conserved region-derived peptides were chemically synthesised for determining and characterising PfRON4 regions having high host cell binding affinity (high activity binding peptides or HABPs). Receptor-ligand interaction/binding assays determined their specific binding capability, the nature of their receptors and their ability to inhibit in vitro parasite invasion. Peptides 42477, 42479, 42480, 42505 and 42513 had greater than 2% erythrocyte binding activity, whilst peptides 42477 and 42480 specifically bound to HepG2 membrane, both of them having micromolar and submicromolar range dissociation constants (Kd). Cell-peptide interaction was sensitive to treating erythrocytes with trypsin and/or chymotrypsin and HepG2 with heparinase I and chondroitinase ABC, suggesting protein-type (erythrocyte) and heparin and/or chondroitin sulphate proteoglycan receptors (HepG2) for PfRON4. Erythrocyte invasion inhibition assays confirmed HABPs’ importance during merozoite invasion. PfRON4 800–819 (42477) and 860–879 (42480) regions specifically interacted with host cells, thereby supporting their inclusion in a subunit-based, multi-antigen, multistage anti-malarial vaccine.     

Differential Recognition of Plasmodium falciparum Merozoite Surface Protein 2 Variants by Antibodies from Malaria Patients in Brazil

Four variants of merozoite surface protein 2 (MSP-2) of Plasmodium falciparum were used in serology to examine whether changes in repeat units affect its recognition by antibodies during infection with parasites of known MSP-2 types. The results indicate that variation in MSP-2 repeats may represent a mechanism for parasite immune evasion.     

Plasmodium falciparum Merozoite Surface Protein 1 (MSP-1)-MSP-3 Chimeric Protein: Immunogenicity Determined with Human-Compatible Adjuvants and Induction of Protective Immune Response

A chimeric gene, MSP-Fu₂₄, was constructed by genetically coupling immunodominant, conserved regions of the two leading malaria vaccine candidates, Plasmodium falciparum merozoite surface protein 1 (C-terminal 19-kDa region [PfMSP-1₁₉]) and merozoite surface protein 3 (11-kDa conserved region [PfMSP-3₁₁]). The recombinant MSP-Fu₂₄ protein was produced in Escherichia coli cells and purified to homogeneity by a two-step purification process with a yield of ∼30 mg/liter. Analyses of conformational properties of MSP-Fu₂₄ using PfMSP-1₁₉-specific monoclonal antibody showed that the conformational epitopes of PfMSP-1₁₉ that may be critical for the generation of the antiparasitic immune response remained intact in the fusion protein. Recombinant MSP-Fu₂₄ was highly immunogenic in mice and in rabbits when formulated with two different human-compatible adjuvants and induced an immune response against both PfMSP-1₁₉ and PfMSP-3₁₁. Purified anti-MSP-Fu₂₄ antibodies showed invasion inhibition of P. falciparum 3D7 and FCR parasites, and this effect was found to be dependent on antibodies specific for the PfMSP-1₁₉ component. The protective potential of MSP-Fu₂₄ was demonstrated by in vitro parasite growth inhibition using an antibody-dependent cell inhibition (ADCI) assay with anti-MSP-Fu₂₄ antibodies. Overall, the antiparasitic activity was mediated by a combination of growth-inhibitory antibodies generated by both the PfMSP-1₁₉ and PfMSP-3₁₁ components of the MSP-Fu₂₄ protein. The antiparasitic activities elicited by anti-MSP-Fu₂₄ antibodies were comparable to those elicited by antibodies generated with immunization with a physical mixture of two component antigens, PfMSP-1₁₉ and PfMSP-3₁₁. The fusion protein induces a protective immune response with human-compatible adjuvants and may form a part of a multicomponent malaria vaccine.Malaria is among the major parasitic diseases in tropical and subtropical countries. With as many as 300 to 500 million new cases each year, malaria accounts for the death of over 2 million people globally each year, and most are children (41). Among the four species of Plasmodium that infect humans, the most threatening is Plasmodium falciparum. The extensive spread of drug-resistant P. falciparum strains as well as the insecticide-resistant mosquito necessitates the development of a malaria vaccine on an urgent basis. Collectively, the major objective of the ongoing vaccine effort in this field is to develop a multistage, multivalent vaccine against P. falciparum (34).The blood-stage cycle of the parasite is responsible for malaria pathogenesis. Intervention at this stage of the parasite''s development through vaccination is likely to reduce malaria-related clinical symptoms. As a major interface between host and pathogen, the merozoite surface is an obvious target for the development of a malaria vaccine. A number of potential vaccine candidate antigens identified so far are located on or associated with the surface of the merozoite or in apical organelles. These include merozoite surface protein 1 (MSP-1), MSP-2, MSP-3, MSP-4, MSP-5, MSP-8, RAP1/2, AMA-1, and EBA-175, which are implicated in the process of merozoite invasion of the erythrocyte (23).MSP-1 is one of the most extensively studied proteins of P. falciparum (18). It is synthesized as a ∼200-kDa precursor and then processed in two steps: the primary processing step produces a complex of four fragments that are present on the merozoite surface, and the secondary processing step at invasion results in the shedding of the complex from the surface, except for the C-terminal 19-kDa domain (MSP-1₁₉), which remains anchored to the parasite surface by a glycosylphosphatidylinositol (GPI) moiety (2). The C-terminal 19-kDa fragment of MSP-1 is well conserved among P. falciparum isolates and contains two epidermal growth factor (EGF)-like domains that play a role in merozoite invasion. Substantial data from studies with P. falciparum MSP-1 and in vivo immunization studies of mice with Plasmodium yoelii and Plasmodium chabaudi indicate that the protective immune responses are directed against the C-terminal 19-kDa domain (10, 12, 15, 20, 27, 35). The inhibition of MSP-1 processing by conformation-specific antibodies (Abs) was previously proposed to be one of the possible mechanism for the inhibition of merozoite invasion (1).Another merozoite surface protein, MSP-3, was also shown to be the target of the protective immune responses in humans (29). The PfMSP-3 protein contains three blocks of four tandem heptad repeats based on the AXXAXX motif at the N terminus, a glutamic acid-rich domain, and a putative leucine zipper sequence at the C terminus (25). Although a clear surface localization of PfMSP-3 is known, it lacks any transmembrane domain or glycosylphosphatidylinositol (GPI) anchor site (24, 25) and is therefore considered to be loosely associated with the merozoite surface by interactions with other merozoite surface proteins. PfMSP-3 was identified as a candidate vaccine antigen by an antibody-dependent cellular inhibition (ADCI) assay using human immune sera (28). The potential of PfMSP-3 as a vaccine candidate was further illustrated by ADCI using mice antibodies and was further confirmed by the suppression of P. falciparum growth in an immunocompromised mouse after the passive transfer of human antibodies purified on MSP-3 peptides together with human monocytes (28, 40, 42). The immunization of Aotus and Saimiri monkeys with recombinant PfMSP-3 or its fragments provided protection against parasite challenge (6, 16). A 70-amino-acid-long conserved domain of PfMSP-3, referred to here as the PfMSP-3₁₁ region, was identified as the target of protective antibodies in human immune responses (40). The presence of high titers of cytophilic antibodies, IgG3, against this conserved region of MSP-3 has been correlated with protection against the parasite. In addition, immunization of humans with a synthetic peptide corresponding to this region was previously shown to induce antiparasitic antibodies that suppress parasite growth in an ADCI assay (11).It is generally believed that a combination vaccine for malaria is likely to be more effective than vaccines based on a single antigen, and attempts are being made to develop a malaria vaccine by using a mixture of more than one antigen or by combining immunologically relevant proteins of the target antigens as fusion proteins (31, 43, 45). In the present study, we have constructed a fusion chimera (MSP-Fu₂₄) consisting of PfMSP-1₁₉ and PfMSP-3₁₁ and produced the corresponding recombinant MSP-Fu₂₄ protein in Escherichia coli cells. The two individual components, PfMSP-1₁₉ and PfMSP-3₁₁, were also expressed and purified separately; the immunological properties of MSP-Fu₂₄ were compared with a physical mixture of the two individual components. MSP-Fu₂₄ retained the native conformation of the PfMSP-1₁₉ component and was highly immunogenic in small animals. The anti-MSP-Fu₂₄ antibodies inhibited parasite invasion into host red blood cells (RBCs) and also inhibited parasite growth in a monocyte-dependent manner, suggesting the potential of the fusion protein as a malaria vaccine candidate.     

Identification and characterization of the Plasmodium falciparum RhopH2 ortholog in Plasmodium vivax

Plasmodium vivax is one of the most important human malaria species that is geographically widely endemic and potentially affects a larger number of people than its more notorious cousin, Plasmodium falciparum. During invasion of red blood cells, the parasite requires the intervention of high molecular weight complex rhoptry proteins (RhopH) that are also essential for cytoadherence. PfRhopH2, a member of the RhopH multigene family, has been characterized as being crucial during P. falciparum infection. This study describes identifying and characterizing the pfrhoph2 orthologous gene in P. vivax (hereinafter named pvrhoph2). The PvRhopH2 is a 1,369-amino acid polypeptide encoded by PVX_099930 gene, for which orthologous genes have been identified in other Plasmodium species by bioinformatic approaches. Both P. falciparum and P. vivax genes contain nine introns, and there is a high degree of similarity between the deduced amino acid sequences of the two proteins. Moreover, PvRhopH2 contains a signal peptide at its N-terminus and 12 cysteines predominantly in its C-terminal half. PvRhopH2 is localized in one of the apical organelles of the merozoite, the rhoptry, and the localization pattern is similar to that of PfRhopH2 in P. falciparum. The recombinant PvRhopH2 protein is recognized by serum antibodies of patients naturally exposed to P. vivax, suggesting that PvRhopH2 is immunogenic in humans.     

Induction of Protective Immune Responses by Immunization with Linear Multiepitope Peptides Based on Conserved Sequences from Plasmodium falciparum Antigens

A cysteine-containing peptide motif, EWSPCSVTCG, is found highly conserved in the circumsporozoite protein (CSP) and the thrombospondin-related anonymous protein (TRAP) of all the Plasmodium species analyzed so far and has been shown to be crucially involved in the sporozoite invasion of hepatocytes. We have recently shown that peptide sequences containing this motif, and also the antibodies raised against the motif, inhibit the merozoite invasion of erythrocytes. However, during natural infection, and upon immunization with recombinant CSP, this motif represents a cryptic epitope. Here we present the results of immunization studies with two linear multiepitopic constructs, a 60-residue (P60) and a 32-residue (P32) peptide, containing the conserved motif sequence. Both the peptides per se generated high levels of specific antibodies in BALB/c mice. P32 was found to be genetically restricted to H-2^d and H-2^b haplotypes of mice, whereas P60 was found to be immunogenic in five different strains of mice. The antibody response was predominantly targeted to the otherwise cryptic, conserved motif sequence in P60. Anti-P60 antibodies specifically stained the asexual blood stages of Plasmodium falciparum and Plasmodium yoelii in an immunofluorescence assay, recognized a 60- to 65-kDa parasite protein in an immunoblot assay, and blocked P. falciparum merozoite invasion of erythrocytes in a dose-dependent manner. Immunization with P60 also induced significant levels of the cytokines interleukin-2 (IL-2), IL-4, and gamma interferon in BALB/c mice. Moreover, >60% of mice immunized with P60 survived a heterologous challenge infection with a lethal strain of P. yoelii. These results indicate that appropriate medium-sized synthetic peptides might prove useful in generating specific immune responses to an otherwise cryptic but critical and putatively protective epitope in an antigen and could form part of a multicomponent malaria vaccine.     

Immunogenicity and in vitro protective efficacy of recombinant Mycobacterium bovis bacille Calmette Guerin (rBCG) expressing the 19 kDa merozoite surface protein-1 (MSP-1(19)) 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-1₁₉) to evaluate its protective ability against merozoite invasion of red blood cells in vitro. A mutated version of MSP-1₁₉, 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-1₁₉ (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-1₁₉ 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-1₁₉ antigen as compared to those of rBCG026- and BCG-immunized animals. rBCG expressing the mutated version of MSP-1₁₉ 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.     

Identification of an Erythrocyte Binding Peptide from the Erythrocyte Binding Antigen, EBA-175, Which Blocks Parasite Multiplication and Induces Peptide-Blocking Antibodies

A biotinylated peptide covering a sequence of 21 amino acids (aa) from the erythrocyte binding antigen (EBA-175) of Plasmodium falciparum bound to human glycophorin A, an erythrocyte receptor for merozoites, as demonstrated by enzyme-linked immunosorbent assay (ELISA) and to erythrocytes as demonstrated by flow cytometry analysis. The peptide, EBA(aa1076–96), also bound to desialylated glycophorin A and glycophorin B when tested by ELISA. The peptide blocked parasite multiplication in vitro. The glycophorin A binding sequence was further delineated to a 12-aa sequence, EBA(aa1085–96), by testing the binding of a range of truncated peptides to immobilized glycophorin A. Our data indicate that EBA(aa1085–96) is part of a ligand on the merozoite for binding to erythrocyte receptors. This binding suggests that the EBA(aa1085–96) peptide is involved in a second binding step, independent of sialic acid. Antibody recognition of this peptide sequence may protect against merozoite invasion, but only a small proportion of sera from adults from different areas of malaria transmission showed antibody reactivities to the EBA(aa1076–96) peptide, indicating that this sequence is only weakly immunogenic during P. falciparum infections in humans. However, Tanzanian children with acute clinical malaria showed high immunoglobulin G reactivity to the EBA(aa1076–96) peptide compared to children with asymptomatic P. falciparum infections. The EBA(aa1076–96) peptide sequence from EBA-175 induced antibody formation in mice after conjugation of the peptide with purified protein derivative. These murine sera inhibited EBA(aa1076–96) peptide binding to glycophorin A.     

Typing of Plasmodium falciparum DNA from 2 years old Giemsa-stained dried blood spots using nested polymerase chain reaction assay

A panel of 129 Giemsa-stained thick blood spots (TBS) confirmed for Plasmodium falciparum infection having different levels of parasite density were collected from a malaria endemic area. DNA was extracted and nested polymerase chain reaction (PCR) assay was performed to amplify P. falciparum DNA. Nested PCR assay successfully amplified P. falciparum DNA at a very low parasitaemia of ~10 parasites/μl of blood. Current PCR assay is very simple and can be used retrospectively to monitor the invasion and prevalence of different Plasmodium species in endemic areas.     

Inside Scoop on Outside Proteins

Invasion into red blood cells is an essential step in the life cycle of parasites that cause human malaria. Antibodies targeting the key parasite proteins in this process are important for developing a protective immune response. In the current issue, Boyle and colleagues provide a detailed examination of Plasmodium falciparum invasion and specifically illuminate the fate of surface-exposed parasite proteins during and immediately after invasion.     

Sequential Processing of Merozoite Surface Proteins during and after Erythrocyte Invasion by Plasmodium falciparum

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.     

Effects of calcium signaling on Plasmodium falciparum erythrocyte invasion and post-translational modification of gliding-associated protein 45 (PfGAP45)

Plasmodium falciparum erythrocyte invasion is powered by an actin/myosin motor complex that is linked both to the tight junction and to the merozoite cytoskeleton through the Inner Membrane Complex (IMC). The IMC association of the myosin motor, PfMyoA, is maintained by its association with three proteins: PfMTIP, a myosin light chain, PfGAP45, an IMC peripheral membrane protein, and PfGAP50, an integral membrane protein of the IMC. This protein complex is referred to as the glideosome, and given its central role in erythrocyte invasion, this complex is likely the target of several specific regulatory effectors that ensure it is properly localized, assembled, and activated as the merozoite prepares to invade its target cell. However, little is known about how erythrocyte invasion as a whole is regulated, or about how or whether that regulation impacts the glideosome. Here we show that P. falciparum erythrocyte invasion is regulated by the release of intracellular calcium via the cyclic-ADP Ribose (cADPR) pathway, but that inhibition of cADPR-mediated calcium release does not affect PfGAP45 phosphorylation or glideosome association. By contrast, the serine/threonine kinase inhibitor, staurosporine, affects both PfGAP45 isoform distribution and the integrity of the glideosome complex. This data identifies specific regulatory elements involved in controlling P. falciparum erythrocyte invasion and reveals that the assembly status of the merozoite glideosome, which is central to erythrocyte invasion, is surprisingly dynamic.     

Rat monoclonal antibodies which inhibit the in vitro multiplication of plasmodium knowlesi

A total of 28 double cloned monoclonal antibodies specific for Plasmodium knowlesi were raised by fusion of Y3 rat myeloma cells with spleen cells of A0 rats immunized with W1 variant isolated merozoites. Four of these antibodies reacted positively in a solid phase radioimmunoassay against glutaraldehyde-fixed schizonts but gave no detectable reaction on indirect immunofluorescence against methanol-fixed schizonts or merozoites. The remaining 24 antibodies could be divided into 13 distinctive immunofluorescent categories on the basis of their patterns of binding to schizonts and merozoites and reactivity with Plasmodium falciparum. Eight antibodies were studied for their ability to inhibit the in vitro multiplication of W1 P. knowlesi as assessed by parasite incorporation of ³H-amino acids and parasite counts. Partially purified antibody preparations from ascitic fluids were all inhibitory for parasite growth; however, when fully purified antibodies were tested, six of the eight proved to be non-inhibitory. Two of the purified antibodies, both IgG2a isotype, inhibited the in vitro multiplication of P. knowlesi in a dose-dependent manner. Inhibition was not associated with detectable damage to intracellular parasites, suggesting that the inhibitory monoclonal antibodies act by blocking the reinfection of red cells by newly released merozoites. On immunofluorescent analysis both inhibitory antibodies bound to methanol-fixed schizonts, with the intensity increasing for progressively more mature parasites; both reacted diffusely with isolated merozoites, and neither cross-reacted with P. falciparum. Both bound specifically to a single metabolically labelled polypeptide which appears to be a minor parasite component and has an approximate molecular weight of 66,000 when analysed by SDS-PAGE fluorography. The putative protective antigen of P. knowlesi has potential interest as a vaccine against P. knowlesi malaria.