Plasmodium Falciparum Surface Research Articles

Surveillance for Malaria Elimination in Swaziland: A National Cross-Sectional Study Using Pooled PCR and Serology

BackgroundTo guide malaria elimination efforts in Swaziland and other countries, accurate assessments of transmission are critical. Pooled-PCR has potential to efficiently improve sensitivity to detect infections; serology may clarify temporal and spatial trends in exposure.Methodology/Principal FindingsUsing a stratified two-stage cluster, cross-sectional design, subjects were recruited from the malaria endemic region of Swaziland. Blood was collected for rapid diagnostic testing (RDT), pooled PCR, and ELISA detecting antibodies to Plasmodium falciparum surface antigens. Of 4330 participants tested, three were RDT-positive yet false positives by PCR. Pooled PCR led to the identification of one P. falciparum and one P. malariae infection among RDT-negative participants. The P. falciparum-infected participant reported recent travel to Mozambique. Compared to performing individual testing on thousands of samples, PCR pooling reduced labor and consumable costs by 95.5%. Seropositivity was associated with age ≥20 years (11·7% vs 1·9%, P<0.001), recent travel to Mozambique (OR 4.4 [95% CI 1.0–19.0]) and residence in southeast Swaziland (RR 3.78, P<0.001).ConclusionsThe prevalence of malaria infection and recent exposure in Swaziland are extremely low, suggesting elimination is feasible. Future efforts should address imported malaria and target remaining foci of transmission. Pooled PCR and ELISA are valuable surveillance tools for guiding elimination efforts.

Immunization with different Pf AMA1 alleles in sequence induces clonal imprint humoral responses that are similar to responses induced by the same alleles as a vaccine cocktail in rabbits

BackgroundAntibodies to key Plasmodium falciparum surface antigens have been shown to be important effectors that mediate clinical immunity to malaria. The cross-strain fraction of anti-malarial antibodies may however be required to achievestrain-transcending immunity. Such antibody responses against Plasmodium falciparum apical membrane antigen 1 (PfAMA1), a vaccine target molecule that is expressed in both liver and blood stages of the parasite, can be elicited through immunization with a mixture of allelic variants of the parasite molecule. Cross-strain antibodies are most likely elicited against epitopes that are shared by the allelic antigens in the vaccine cocktail.MethodsA standard competition ELISA was used to address whether the antibody response can be further focused on shared epitopes by exclusively boosting these common determinants through immunization of rabbits with different PfAMA1 alleles in sequence. The in vitro parasite growth inhibition assay was used to further evaluate the functional effects of the broadened antibody response that is characteristic of multi-allele vaccine strategies.ResultsA mixed antigen immunization protocol elicited humoral responses that were functionally similar to those elicited by a sequential immunization protocol (p > 0.05). Sequential exposure to the different PfAMA1 allelic variants induced immunological recall of responses to previous alleles and yielded functional cross-strain antibodies that would be capable of optimal growth inhibition of variant parasites at high enough concentrations.ConclusionsThese findings may have implications for the current understanding of the natural acquisition of clinical immunity to malaria as well as for rational vaccine design.

The IC 50 of anti-Pfs25 antibody in membrane-feeding assay varies among species

Plasmodium falciparum surface protein 25 (Pfs25) is a candidate for transmission-blocking vaccines (TBVs). Anti-Pfs25 antibodies block the development of oocysts in membrane-feeding assays and we have shown the activity correlates with antibody titer. In this study, we purified Pfs25-specific IgGs to convert antibody titer to μg/mL and determined the amount of antibody required to inhibit 50% of oocyst development (IC 50). The IC 50 were, 15.9, 4.2, 41.2, and 85.6 μg/mL for mouse, rabbit, monkey and human, respectively, and the differences among species were significant. Anti-Pfs25 sera from rabbit, monkey and human showed different patterns of competition against 6 mouse monoclonal antibodies, and the avidity of antibodies among four species were also different. These data suggests that information obtained from animal studies which assess efficacy of TBV candidates may be difficult to translate to human immunization.

Rapid and specific biotin labelling of the erythrocyte surface antigens of both cultured and ex-vivo Plasmodium parasites.

BackgroundSensitive detection of parasite surface antigens expressed on erythrocyte membranes is necessary to further analyse the molecular pathology of malaria. This study describes a modified biotin labelling/osmotic lysis method which rapidly produces membrane extracts enriched for labelled surface antigens and also improves the efficiency of antigen recovery compared with traditional detergent extraction and surface radio-iodination. The method can also be used with ex-vivo parasites.MethodsAfter surface labelling with biotin in the presence of the inhibitor furosemide, detergent extraction and osmotic lysis methods of enriching for the membrane fractions were compared to determine the efficiency of purification and recovery. Biotin-labelled proteins were identified on silver-stained SDS-polyacrylamide gels.ResultsDetergent extraction and osmotic lysis were compared for their capacity to purify biotin-labelled Plasmodium falciparum and Plasmodium chabaudi erythrocyte surface antigens. The pellet fraction formed after osmotic lysis of P. falciparum-infected erythrocytes is notably enriched in suface antigens, including PfEMP1, when compared to detergent extraction. There is also reduced co-extraction of host proteins such as spectrin and Band 3.ConclusionBiotinylation and osmotic lysis provides an improved method to label and purify parasitised erythrocyte surface antigen extracts from both in vitro and ex vivo Plasmodium parasite preparations.

Reduced Production of RNA Transcripts from Individual DNA Plasmids Given in a Multivalent DNA Vaccine Formula

We conducted transient transfection studies using two DNA vaccines constructs encodingtwo Plasmodium falciparum surface proteins, PfCSP and PfSSP2, and UM449 melanoma cells todetermine transcription and translation efficiencies. Plasmids were transfected individually or incombination with an empty control plasmid with and without a functional CMV IE promoter.Western blot analysis using NSF1, a monoclonal antibody specific for PfCSP, and UM449 celllysate revealed an abrogation in expression of PfCSP when a plasmid carrying the Pfcsp genewas co-transfected with an empty control plasmid with a functional CMV IE promoter. When acontrol plasmid without a functional CMV IE promoter was substituted in the expression study,normal levels of PfCSP were detected by Western blot. Total RNA was isolated followingtransfection and reverse transcriptase quantitative (RTQ)-PCR was performed. Levels of Pfcspand Pfssp2 transcripts decreased significantly when co-transfected with a control plasmidcontaining a functional CMV IE promoter while transcript levels of Pfcsp and Pfssp2 weresignificantly higher in cells co-transfected with a control plasmid without a functional CMV IEpromoter. The presence of multiple copies of a functional CMV IE promoter leads to a decreasein expression of malaria antigens present in a multivalent vaccine mixture when transfected invitro.

SERUM ANTIBODY LEVELS TO GLYCOSYLPHOSPHATIDYLINOSITOLS IN SPECIMENS DERIVED FROM MATCHED MALIAN CHILDREN WITH SEVERE OR UNCOMPLICATED PLASMODIUM FALCIPARUM MALARIA AND HEALTHY CONTROLS

Neutralizing antibodies to glycosylphosphatidylinositols (GPIs), which are Plasmodium falciparum surface protein anchor molecules implicated in malaria pathogenesis, are thought to protect against symptomatic malaria. Index cases of severe malaria in Malian children 3 months to 14 years of age were matched by age and residence to uncomplicated malaria and healthy controls. Serum antibodies to GPI (IgM and IgG) were measured at the time of severe malaria and after the malaria transmission season. The mean optical density values for IgM and IgG antibodies were higher in children with severe or uncomplicated malaria compared with healthy controls. Similarly, higher percentages of children with IgM and IgG antibodies to GPI were observed in the severe malaria group compared with matched healthy controls. IgG antibody levels to GPI were highest among children with cerebral malaria and children who died. The IgG antibody levels to GPI peaked during periods of malaria transmission and decreased after malaria transmission ended. A direct correlation between age and parasitemia and IgG antibodies to GPI was observed. In summary, higher levels of IgM and IgG antibodies to GPI in young children were associated with disease severity and were short-lived.

Effect of the malaria vaccine Combination B on merozoite surface antigen 2 diversity

Extensive genetic polymorphism is generally found in Plasmodium falciparum surface antigens. This poses a considerable obstacle to the development of a malaria vaccine. In order to assess possible effects of a polymorphic vaccine, we have analyzed the genetic diversity of parasites collected in the course of a phase 2b field trial of the blood stage vaccine Combination B in Papua New Guinea. The full-length 3D7 allele of the merozoite surface protein 2 (MSP2) was included in Combination B as one of three subunits. Vaccinees had a lower prevalence of parasites carrying a 3D7-type allele (corresponding to that in the vaccine) and selection appeared to favour the alternative FC27-type alleles resulting in a higher incidence of morbid episodes associated with FC27-type parasites. We sequenced MSP2 alleles detected in study participants after vaccination to identify breakthrough genotypes. Extensive genetic diversity of MSP2 was observed in both the repetitive and family-specific domains, but alleles occurring in vaccine recipients were no different from those found in placebo recipients. A phylogenetic analysis showed no clustering of 3D7-type breakthrough infections from vaccine recipients. The repeat unit present in the vaccine molecule occurred in a number of alleles from the trial area and was also observed in vaccinated individuals. Thus the anti-repeat immune response did not lead to elimination of parasites carrying the same repeat unit. We conclude that the conserved epitopes in the family-specific domain were the most important determinants of the vaccine effect against new 3D7-type infections and that the hypervariable domains were not subject to selective effects of the vaccine.

Dissection of Merozoite Surface Protein 3, a Representative of a Family of Plasmodium falciparum Surface Proteins, Reveals an Oligomeric and Highly Elongated Molecule

Vaccination with the merozoite surface protein 3 (MSP3) of Plasmodium falciparum protects against infection in primates and is under development as a vaccine against malaria in humans. MSP3 is secreted and associates with the parasite membrane but lacks a predicted transmembrane domain or a glycosylphosphatidylinositol anchor. Its role in the invasion of red blood cells is unclear. To study MSP3, we produced recombinant full-length protein and found by size exclusion chromatography that the apparent size of MSP3 was much larger than predicted from its sequence. To investigate this, we used several biophysical techniques to characterize the full-length molecule and four smaller polypeptides. The MSP3 polypeptides contain a large amount of alpha-helix and random coil secondary structure as measured by circular dichroism spectroscopy. The full-length MSP3 forms highly elongated dimers and tetramers as revealed by chemical cross-linking and analytical ultracentrifugation. The dimer is formed through a leucine zipper-like domain located between residues 306 and 362 at the C terminus. Two dimers interact through their C termini to form a tetramer with an apparent association constant of 3 mum. Sedimentation velocity experiments determined that the MSP3 molecules are highly extended in solution (some with f/f(0) > 2). These data, in light of the recent discoveries of three other Plasmodium proteins containing very similar C-terminal sequences, suggest that the members of this newly identified family may adopt highly extended and oligomeric novel structures capable of interacting with a red blood cell at relatively long distances.

Differential antibody responses to Plasmodium falciparum glycosylphosphatidylinositol anchors in patients with cerebral and mild malaria

Glycosylphosphatidylinositol (GPI) membrane anchors of Plasmodium falciparum surface proteins are thought to be important factors contributing to malaria pathogenesis, and anti-GPI antibodies have been suggested to provide protection by neutralizing the toxic activity of GPIs. In this study, IgG responses against P. falciparum GPIs and a baculovirus recombinant MSP1p19 antigen were evaluated in two distinct groups of 70 patients each, who were hospitalized with malaria. Anti-GPI IgGs were significantly lower in patients hospitalized with confirmed cerebral malaria compared to those with mild malaria ( P < 0.01) but did not discriminate for fatal outcome. In contrast, a specific marker of the anti-parasite immunity, as monitored by the anti-MSP1p19 IgG response, was similar in both cerebral and mild malaria individuals, although it was significantly lower in a subgroup with fatal outcomes. These results are consistent with a potential anti-toxin role for anti-GPI antibodies associated with protection against cerebral malaria.

Synthesis of a malaria candidate glycosylphosphatidylinositol (GPI) structure: a strategy for fully inositol acylated and phosphorylated GPIs.

A congener of the glycosylphosphatidylinositol (GPI) membrane anchor present on the cell surface of the malaria pathogen Plasmodium falciparum has been synthesized. This GPI is an example of a small number of such membrane anchors that carry a fatty acyl group at O-2 of the inositol. Although the acyl group plays crucial roles in GPI biosynthesis, it rarely persits in mature molecules. Other notable examples are the mammalian GPIs CD52 and AchE. The presence of bulky functionalities at three contiguous positions of the inositol moiety creates a very crowded environment that poses difficulties for carrying out selective chemical manipulations. Thus installations of the axial long-chain acyl group and neighboring phosphoglyceryl complex were fraught with obstacles. The key solution to these obstacles in the successful synthesis of the malarial candidate and prototype structures involved stereoelectronically controlled opening of a cyclic ortho ester. The reaction proceeds in very good yields, the desired axial diastereomer being formed predominantly, even more so in the case of long-chain acyl derivatives. The myoinositol precursor was prepared from methyl alpha-d-glucopyranoside by the biomimetic procedure of Bender and Budhu. For the glycan array, advantage was taken of the fact that (a). n-pentenyl ortho ester donors are rapidly and chemospecifically activated upon treatment with ytterbium triflate and N-iodosuccinimide and (b). coupling to an acceptor affords alpha-coupled product exclusively. A strategy for obtaining the GPI's alpha-glucosaminide component from the corresponding alpha-mannoside employed Deshong's novel azide displacement procedure. Thus all units of the glycan array were obtained from a beta-d-manno-n-pentenyl ortho ester, this being readily prepared from d-mannose in three easy, high-yielding steps. The "crowded environment" at positions 1 and 2, noted above, could conceivably be relieved by migration of the acyl group to the neighboring cis-O-3-hydroxyl in the natural product. However, study of our synthetic intermediates and prototypes indicate that the O-2 acyl group is quite stable, and that such migration does not occur readily.

Proteolysis of Plasmodium falciparum surface antigen, Pfs230, during gametogenesis

During Plasmodium falciparum gametogenesis, proteolysis of Pfs230, a 360 kDa gametocyte surface protein, generates two large polypeptides, 307 and 300 kDa, that remain associated with the surface of the newly formed gamete. Using peptide specific antibodies, the amino termini of the 307 and 300 kDa forms have been mapped to between aa 477–487 and aa 523–555, respectively, which is the region between the glutamate rich repeats and the cysteine motif domains. Concomitantly, two peptides, 47 and 35 kDa, corresponding to the region upstream from the cleavage site are released into the medium. The membrane permeant cysteine protease inhibitor, E64d, blocks production of the 300 and 35 kDa forms of Pfs230, but does not alter the formation of the 307 or 47 kDa forms. In contrast, E64, which has been shown to inhibit the development of P. falciparum trophozoites, does not block proteolytic processing of Pfs230. Production of both the 307 and 300 kDa forms was reduced by a metallo-protease inhibitor, 1,10-phenanthroline, whereas the rest of the protease inhibitors tested had no effect on Pfs230 processing. This is the first study of proteolysis during gametogenesis and it demonstrates that the two large forms of Pfs230 produced are generated by proteases with different specificities. The data also suggest that Pfs230 undergoes proteolytic processing prior to emergence from the red blood cell.

Processing and localisation of a GPI-anchored Plasmodium falciparum surface protein expressed by the baculovirus system

We describe the expression, in insect cells using the baculovirus system, of two protein fragments derived from the C-terminus of merozoite surface protein 1(MSP-1) of the human malaria parasite Plasmodium falciparum, and their glycosylation and intracellular location. The transport and intracellular localisation of the intact C-terminal MSP-1 fragment, modified by addition of a signal sequence for secretion, was compared with that of a similar control protein in which translation of the GPI-cleavage/attachment site was abolished by insertion of a stop codon into the DNA sequence. Both proteins could only be detected intracellularly, most likely in the endoplasmic reticulum. This lack of transport to the cell surface or beyond, was confirmed for both proteins by immunofluorescence with a specific antibody and characterisation of their N-glycans. The N-glycans had not been processed by enzymes localised in post-endoplasmic reticulum compartments. In contrast to MSP-1, the surface antigen SAG-1 of Toxoplasma gondii was efficiently transported out of the endoplasmic reticulum of insect cells and was located, at least in part, on the cell surface. No GPI-anchor could be detected for either of the MSP-1 constructs or SAG-1, showing that the difference in transport is a property of the individual proteins and cannot be attributed to the lack of a GPI-anchor. The different intracellular location and post-translational modification of recombinant proteins expressed in insect cells, as compared to the native proteins expressed in parasites, and the possible implications for vaccine development are discussed.

Flow cytometric analysis of IgG reactive to parasitized red blood cell membrane antigens in Plasmodium falciparum-immune individuals

Antigens exposed at the surface of Plasmodium falciparum parasitized red blood cells (pRBCs) represent potential targets for protective antibodies involved in opsonization and immune phagocytosis of pRBCs. We measured the recognition of parasitized red blood cell membrane associated antigens by IgG in the plasma of clinically immune individuals by flow cytometry and ELISA. The plasmas were selected on the basis of preexisting IgG antibodies to pRBC membrane associated recombinant proteins. In every plasma sample IgG could bind the surface of live pRBCs in flow cytometry. In addition, there was a significant correlation between the level of IgG recognition of live pRBCs and of pRBC membrane ghost proteins or major identified antigens by ELISA. Flow cytometry thus represents a technique suitable to test for the accessibility and potential functionality of IgG antibodies directed to antigens expressed by the surface of pRBCs.

Fertilization and wounding of the style induce the expression of a highly conserved plant gene homologous to a Plasmodium falciparum surface antigen in the wild potato Solanum chacoense Bitt.

Pistil tissues are actively involved in pollen tube growth and respond to the presence of the growing pollen tubes by modulating the expression of specific genes. Once fertilization has occurred, complex developmental programs lead to embryogenesis, ovary maturation, and seed set. In order to understand the early events that follow pollination and fertilization we have used a subtractive hybridization approach to characterize genes which are related to pollination and fertilization events. One cDNA clone isolated and named SPP30 (Solanum pollinated pistil) was found to share significant sequence identities with a Plasmodium falciparum (malaria parasite) surface antigen and a yeast gene of unknown function. Searches in recent EST databases also revealed that SPP30 homologues are found in both monocot and dicot species. The presence of this conserved gene in evolutionarily distant organisms such as yeast, Plasmodium, and plants suggests that it codes for an essential cellular function. This is also strengthened by its extremely high sequence conservation in both monocots and dicots where virtually all substitutions tolerated are conservative.

Characterization and localization of Plasmodium falciparum surface antigens on infected erythrocytes from west African patients

The malaria-induced surface antigens on Plasmodium falciparum-infected erythrocytes from West African patients were characterized by agglutination of infected cells by human sera, surface immunofluorescence of live infected cells, inhibition of cytoadherence to C32 melanoma cells by human sera, immunoelectron microscopy (immunoEM), and immunoprecipitation. In a nonimmune individual, serum antibody reactivity to surface antigens of infected cells was acquired during convalescence, as tested by all five methods, and was generally parasite isolate-specific. By contrast, adult hyperimmune West African sera reacted with many isolates, including isolates from geographically distinct regions. A quantitative correlation was established between agglutination and surface immunofluorescence assay titers, and between surface immunofluorescence assay and immunoEM reactivity, suggesting that a single antigen or a set of coexpressed antigens is being detected. Surface iodination of infected cells identified trypsin-sensitive high M, antigens in the sodium dodecyl sulfate extract. All sera tested that agglutinated infected cells also immunoprecipitated these antigens. The same surface antigens were immunoprecipitated by the homologous convalescent serum as by adult sera. By immunoEM these antigens were localized exclusively at the knob-like protrusions of infected cells, where they may participate in adherence to vascular endothelium.

Genes for Plasmodium falciparum surface antigens cloned by expression in COS cells.

Two genes encoding membrane antigens of Plasmodium falciparum were isolated by transient expression in mammalian cells and selection with human immune sera from African adults exposed to P. falciparum malaria. COS-7 cells were transfected with a plasmid expression library constructed from P. falciparum genomic DNA, and cells expressing reactive malaria antigens on their surface were enriched by adherence to antibody-coated dishes. One of the genes isolated is distinctive in that it does not contain repeat sequences typical of many malarial genes cloned by immunoscreening of bacterial expression libraries. The second gene apparently encodes a polymorphic version of the P. falciparum merozoite surface antigen Ag513, since the two sequences are identical in the 5' and 3' coding regions but diverge completely in the center. The COS-7 expression system provides an alternate means for cloning genes encoding malarial membrane antigens by using those antibodies in complex immune sera that bind membrane-associated, nondenatured molecules.

Structural diversity in the 45-kilodalton merozoite surface antigen of Plasmodium falciparum

An integral membrane protein associated with the merozoite surface of Plasmodium falciparum termed merozoite surface antigen 2 (the 45-kDa merozoite surface antigen), occurs in antigenically diverse forms. Here we report the sequences of the MSA 2 gene from two other isolates of P. falciparum. The 43 N-terminal residues and the 74 C-terminal residues of all three MSA 2 sequences are highly conserved, but between these conserved regions there are dramatic differences among the alleles. Instead of the two copies of a 32-amino-acid repeat present in the MSA 2 of isolate FC27, MSA 2 from clone 3D7 and isolate Indochina 1 contain 5 and 12 copies respectively of the four amino acid sequence Gly Gly Ser Ala. The sequences flanking the repeats also differ among the three antigens. The repeats in MSA 2 appear to be immunodominant during natural infection, and antibodies to the repeat regions of different alleles react with a restricted number of parasite isolates.

Pox virus DNA sequences and recombinant vectors e.g. vaccinia virus carrying Plasmodium falciparum surface antigen: gene expression and use in vaccination against malaria etc.

Pox virus DNA sequences and recombinant vectors e.g. vaccinia virus carrying Plasmodium falciparum surface antigen: gene expression and use in vaccination against malaria etc.

Immunization with synthetic peptides of a Plasmodium falciparum surface antigen induces antimerozoite antibodies.

Polypeptides expressed on the surface of merozoites, the invasive stage of the asexual blood cycle, are good candidates for the development of malaria vaccines. Five synthetic peptides with predetermined specificity deduced from a genomic DNA clone coding for the NH2-terminal portion of the main merozoite surface polypeptide of Plasmodium falciparum were evaluated for their capability to raise antibodies that react with the P. falciparum merozoites. Antibodies induced by two of the peptides (3 and 5) reacted with the membrane surfaces of seven of seven isolates of P. falciparum from different geographic areas. Antibodies against peptide 4, which contains a repeated amino acid sequence (Gly-Gly-Ser and Val-Ala-Ser), reacted with six of seven isolates. Structural analysis of the deduced polypeptides suggests that peptide 3 is exposed at the surface of merozoites. When it was used to immunize monkeys, three of the four animals were partially protected from a challenge infection that induced a fulminant infection in control animals.