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BACKGROUND: The ability of Plasmodium falciparum to undergo antigenic variation, by switching expression among protein variants encoded by multigene families, such as var, rif and stevor, is key to the survival of this parasite in the human host. The RIFIN protein family can be divided into A and B types based on the presence or absence of a 25 amino acid motif in the semi-conserved domain. A particular type B RIFIN, PF13_0006, has previously been shown to be strongly transcribed in the asexual and sexual stages of P. falciparum in vitro. METHODS: Antibodies to recombinant PF13_0006 RIFIN were used in immunofluorescence and confocal imaging of 3D7 parasites throughout the asexual reproduction and sexual development to examine the expression of PF13_0006. Furthermore, reactivity to recombinant PF13_0006 was measured in plasma samples collected from individuals from both East and West African endemic areas. RESULTS: The PF13_0006 RIFIN variant appeared expressed by both released merozoites and gametes after emergence. 7.4% and 12.1% of individuals from East and West African endemic areas, respectively, carry plasma antibodies that recognize recombinant PF13_0006, where the antibody responses were more common among older children. CONCLUSIONS: The stage specificity of PF13_0006 suggests that the diversity of RIFIN variants has evolved to provide multiple specialized functions in different stages of the parasite life cycle. These data also suggest that RIFIN variants antigenically similar to PF13_0006 occur in African parasite populations.

Since 2000, under the Fifth and subsequent Framework Programmes, the European Commission has funded research to spur the development of a malaria vaccine. This funding has contributed to the promotion of an integrated infrastructure consisting of European basic, applied and clinical scientists in academia and small and medium enterprises, together with partners in Africa. Research has added basic understanding of what is required of a malaria vaccine, allowing selected candidates to be prioritized and some to be moved forward into clinical trials. To end the health burden of malaria, and its economic and social impact on development, the international community has now essentially committed itself to the eventual eradication of malaria. Given the current tentative advances towards elimination or eradication of malaria in many endemic areas, malaria vaccines constitute an additional and almost certainly essential component of any strategic plan to interrupt transmission of malaria. However, funding for malaria vaccines has been substantially reduced in the Seventh Framework Programme compared with earlier Framework Programmes, and without further support the gains made by earlier European investment will be lost.

Placental malaria infections are caused by Plasmodium falciparum-infected red blood cells sequestering in the placenta by binding to chondroitin sulfate A, mediated by VAR2CSA, a variant of the PfEMP1 family of adhesion antigens. Recent studies have shown that many P. falciparum genomes have multiple genes coding for different VAR2CSA proteins, and parasites with >1 var2csa gene appear to be more common in pregnant women with placental malaria than in nonpregnant individuals. We present evidence that, in pregnant women, parasites containing multiple var2csa-type genes possess a selective advantage over parasites with a single var2csa gene. Accumulation of parasites with multiple copies of the var2csa gene during the course of pregnancy was also correlated with the development of antibodies involved in blocking VAR2CSA adhesion. The data suggest that multiplicity of var2csa-type genes enables P. falciparum parasites to persist for a longer period of time during placental infections, probably because of their greater capacity for antigenic variation and evasion of variant-specific immune responses.

The cell division cycle and mitosis of intra-erythrocytic (IE) Plasmodium falciparum are poorly understood aspects of parasite development which affect malaria molecular pathogenesis. Specifically, the timing of the multiple gap (G), DNA synthesis (S) and chromosome separation (M) phases of parasite mitosis are not well defined, nor whether genome divisions are immediately followed by cleavage of the nuclear envelope. Curiously, daughter merozoite numbers do not follow the geometric expansion expected from equal numbers of binary divisions, an outcome difficult to explain using the standard model of cell cycle regulation. Using controlled synchronisation techniques, confocal microscopy to visualise key organelles and fluorescence in situ hybridization (FISH) to follow the movements and replication of genes and telomeres, we have re-analysed the timing and progression of mitotic events. The asynchronous duplications of the P. falciparum centrosome equivalents, the centriolar plaques, are established and these are correlated with chromosome and nuclear divisions in a new model of P. falciparum schizogony. Our results improve the resolution of the cell cycle and its phases during P. falciparum IE development, showing that asynchronous, independent nuclear division occurs during schizogony, with the centriolar plaques playing a major role in regulating mitotic progression.

Malaria during pregnancy is a major health problem for African women. The disease is caused by Plasmodium falciparum malaria parasites, which accumulate in the placenta by adhering to chondroitin sulfate A (CSA). The interaction between infected erythrocytes and the placental receptor is mediated by a parasite expressed protein named VAR2CSA. A vaccine protecting pregnant women against placental malaria should induce antibodies inhibiting the interaction between VAR2CSA and CSA. Much effort has been put into defining the part of the 350 kDa VAR2CSA protein that is responsible for binding. It has been shown that full-length recombinant VAR2CSA binds specifically to CSA with high affinity, however to date no sub-fragment of VAR2CSA has been shown to interact with CSA with similar affinity or specificity. In this study, we used a biosensor technology to examine the binding properties of a panel of truncated VAR2CSA proteins. The experiments indicate that the core of the CSA-binding site is situated in three domains, DBL2X-CIDR(PAM) and a flanking domain, located in the N-terminal part of VAR2CSA. Furthermore, recombinant VAR2CSA subfragments containing this region elicit antibodies with high parasite adhesion blocking activity in animal immunization experiments.

The PFD1235w Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigen is associated with severe malaria in children and can be expressed on the surface of infected erythrocytes (IE) adhering to ICAM1. However, the exact three-dimensional structure of this PfEMP1 and its surface-exposed epitopes are unknown. An insect cell and Escherichia coli based system was used to express single and double domains encoded by the pfd1235w var gene. The resulting recombinant proteins have been evaluated for yield and purity and their ability to induce rat antibodies, which react with the native PFD1235w PfEMP1 antigen expressed on 3D7PFD1235w-IE. Their recognition by human anti-malaria antibodies from previously infected Tanzanian donors was also analysed.

Udgivelsesdato: 2010-null, The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigens play a major role in cytoadhesion of infected erythrocytes (IE), antigenic variation, and immunity to malaria. The current consensus on control of variant surface antigen expression is that only one PfEMP1 encoded by one var gene is expressed per cell at a time. We measured var mRNA transcript levels by real-time Q-PCR, analysed var gene transcripts by single-cell FISH and directly compared these with PfEMP1 antigen surface expression and cytoadhesion in three different antibody-selected P. falciparum 3D7 sub-lines using live confocal microscopy, flow cytometry and in vitro adhesion assays. We found that one selected parasite sub-line simultaneously expressed two different var genes as surface antigens, on single IE. Importantly, and of physiological relevance to adhesion and malaria pathogenesis, this parasite sub-line was found to bind both CD31/PECAM1 and CD54/ICAM1 and to adhere twice as efficiently to human endothelial cells, compared to infected cells having only one PfEMP1 variant on the surface. These new results on PfEMP1 antigen expression indicate that a re-evaluation of the molecular mechanisms involved in P. falciparum adhesion and of the accepted paradigm of absolutely mutually exclusive var gene transcription is required.

Udgivelsesdato: 2010-Apr-19, BACKGROUND: Infected humans make protective antibody responses to the PfEMP1 adhesion antigens exported by Plasmodium falciparum parasites to the erythrocyte membrane, but little is known about the kinetics of this antibody-receptor binding reaction or how the topology of PfEMP1 on the parasitized erythrocyte membrane influences antibody association with, and dissociation from, its antigenic target. METHODS: A Quartz Crystal Microbalance biosensor was used to measure the association and dissociation kinetics of VAR2CSA PfEMP1 binding to human monoclonal antibodies. Immuno-fluorescence microscopy was used to visualize antibody-mediated adhesion between the surfaces of live infected erythrocytes and atomic force microscopy was used to obtain higher resolution images of the membrane knobs on the infected erythrocyte to estimate knob surface areas and model VAR2CSA packing density on the knob. RESULTS: Kinetic analysis indicates that antibody dissociation from the VAR2CSA PfEMP1 antigen is extremely slow when there is a high avidity interaction. High avidity binding to PfEMP1 antigens on the surface of P. falciparum-infected erythrocytes in turn requires bivalent cross-linking of epitopes positioned within the distance that can be bridged by antibody. Calculations of the surface area of the knobs and the possible densities of PfEMP1 packing on the knobs indicate that high-avidity cross-linking antibody reactions are constrained by the architecture of the knobs and the large size of PfEMP1 molecules. CONCLUSIONS: High avidity is required to achieve the strongest binding to VAR2CSA PfEMP1, but the structures that display PfEMP1 also tend to inhibit cross-linking between PfEMP1 antigens, by holding many binding epitopes at distances beyond the 15-18nm sweep radius of an antibody. The large size of PfEMP1 will also constrain intra-knob cross-linking interactions. This analysis indicates that effective vaccines targeting the parasite's vulnerable adhesion receptors

Udgivelsesdato: 2009-null, BACKGROUND: The var2csa gene encodes a Plasmodium falciparum adhesion receptor which binds chondroitin sulfate A (CSA). This var gene is more conserved than other PfEMP1/var genes and is found in all P. falciparum isolates. In isolates 3D7, FCR3/It4 and HB3, var2csa is transcribed from a sub-telomeric position on the left arm of chromosome 12, but it is not known if this location is conserved in all parasites. Genome sequencing indicates that the var2csa gene is duplicated in HB3, but whether this is true in natural populations is uncertain. METHODOLOGY/PRINCIPAL FINDINGS: To assess global variation in the VAR2CSA protein, sequence variation in the DBL2X region of var2csa genes in 54 P.falciparum samples was analyzed. Chromosome mapping of var2csa loci was carried out and a quantitative PCR assay was developed to estimate the number of var2csa genes in P.falciparum isolates from the placenta of pregnant women and from the peripheral circulation of other malaria patients. Sequence analysis, gene mapping and copy number quantitation in P.falciparum isolates indicate that there are at least two loci and that both var2csa-like genes can be transcribed. All VAR2CSA DBL2X domains fall into one of two distinct phylogenetic groups possessing one or the other variant of a large (approximately 26 amino acid) dimorphic motif, but whether either motif variant is linked to a specific locus is not known. CONCLUSIONS/SIGNIFICANCE: Two or more related but distinct var2csa-type PfEMP1/var genes exist in many P. falciparum isolates. One gene is on chromosome 12 but additional var2csa-type genes are on different chromosomes in different isolates. Multiplicity of var2csa genes appears more common in infected placentae than in samples from non-pregnant donors indicating a possible advantage of this genotype in pregnancy associated malaria.

Udgivelsesdato: 2009-Dec, The var genes encode a family of adhesion receptor proteins, Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), which profoundly influence malaria pathogenesis. Only a single var gene is transcribed and one PfEMP1 expressed per P.falciparum parasite. Here we present the in vivo transcript distribution of var genes in a P. falciparum-infected non-immune individual and show that the initial expression of PfEMP1 is based on a strategy that allows all or most variants of PfEMP1s to be expressed by the parasite population at the onset of the blood stage infection.

Udgivelsesdato: 2008-null, There is a need for improved methods for in situ localization of surface proteins on Plasmodium falciparum-infected erythrocytes to help understand how these antigens are trafficked to, and positioned within, the host cell membrane. This protocol for confocal immunofluorescence microscopy combines surface antigen labeling on live cells with subsequent fixation and permeabilization, which enables antibodies to penetrate the cell and label internal antigens. The key steps of the protocol are as follows: indirect labeling of the surface antigen using a fluorescently tagged secondary antibody; fixation and permeabilization; indirect labeling of the internal antigen using a secondary antibody tagged with a spectrally distinct fluorescent dye; and detection of the differentially labeled antigens using a laser scanning confocal microscope. The protocol can be completed in approximately 7 h. Although the protocol is discussed here in the context of malaria parasite-infected cells, it can also be modified to visualize the membrane and intracellular distribution of surface and internal proteins in other eukaryotic cells.

Udgivelsesdato: 2008-Sep, Immunogenicity testing of Plasmodium falciparum antigens being considered as malaria vaccine candidates was undertaken in rabbits. The antigens compared were recombinant baculovirus MSP-1(19) and five Pichia pastoris candidates, including two versions of MSP-1(19), AMA-1 (domains I and II), AMA-1+MSP-1(19), and fused AMA-1/MSP-1(19)). Animals were immunized with equimolar amounts of each antigen, formulated in Montanide ISA720. The specificities and titers of antibodies were compared using immunofluorescence assays and enzyme-linked immunosorbent assay (ELISA). The antiparasite activity of immunoglobulin G (IgG) in in vitro cultures was determined by growth inhibition assay, flow cytometry, lactate dehydrogenase assay, and microscopy. Baculovirus MSP-1(19) immunizations produced the highest parasite-specific antibody titers in immunofluorescence assays. In ELISAs, baculovirus-produced MSP-1(19) induced more antibodies than any other single MSP-1(19) immunogen and three times more MSP-1(19) specific antibodies than the AMA-1/MSP-1(19) fusion. Antibodies induced by baculovirus MSP-1(19) gave the highest levels of growth inhibition in HB3 and 3D7 parasite cultures, followed by AMA-1+MSP-1(19) and the AMA-1/MSP-1(19) fusion. With the FCR3 isolate (homologous to the AMA-1 construct), antibodies to the three AMA-1-containing candidates gave the highest levels of growth inhibition at high IgG concentrations, but antibodies to baculovirus MSP-1(19) inhibited as well or better at lower IgG concentrations. The two P. pastoris-produced MSP-1(19)-induced IgGs conferred the lowest growth inhibition. Comparative analysis of immunogenicity of vaccine antigens can be used to prioritize candidates before moving to expensive GMP production and clinical testing. The assays used have given discriminating readouts but it is not known whether any of them accurately reflect clinical protection.

Udgivelsesdato: 2008-null, BACKGROUND: The insertion of parasite antigens into the host erythrocyte membrane and the structure and distribution of Plasmodium falciparum adhesion receptors on that membrane are poorly understood. Laser scanning confocal microscopy (LSCM) and a novel labelling and fixation method have been used to obtain high resolution immuno-fluorescent images of erythrocyte surface PfEMP1 and internal antigens which allow analysis of the accumulation of PfEMP1 on the erythrocyte membrane during asexual development. METHODS: A novel staining technique has been developed which permits distinction between erythrocyte surface PfEMP1 and intracellular PfEMP1, in parasites whose nuclear material is exceptionally well resolved. Primary antibody detection by fluorescence is carried out on the live parasitized erythrocyte. The surface labelled cells are then fixed using paraformaldehyde and permeabilized with a non-ionic detergent to permit access of antibodies to internal parasite antigens. Differentiation between surface and internal antigens is achieved using antibodies labelled with different fluorochromes and confocal microscopy RESULTS: Surface exposed PfEMP1 is first detectable by antibodies at the trophozoite stage of intracellular parasite development although the improved detection method indicates that there are differences between different laboratory isolates in the kinetics of accumulation of surface-exposed PfEMP1. CONCLUSION: A sensitive method for labelling surface and internal PfEMP1 with up to three different fluorochromes has been developed for laser scanning confocal optical microscopy and the analysis of the developmental expression of malaria adhesion antigens.

Udgivelsesdato: 2007-null, BACKGROUND: The var multigene family encodes PfEMP1, which are expressed on the surface of infected erythrocytes and bind to various host endothelial receptors. Antigenic variation of PfEMP1 plays a key role in malaria pathogenesis, a process partially controlled at the level of var gene transcription. Transcriptional levels, throughout the intra-erythrocytic cycle, of 59 var genes of the NF54 clone were measured simultaneously by quantitative real-time PCR. The timing of var transcript abundance, the number of genes transcribed and whether transcripts were correctly spliced for protein expression were determined. Two parasite populations were studied; an unselected population of NF54 and a selected population, NF54VAR2CSA, to compare both the transcription of var2csa and the expression pattern of the corresponding protein. METHODS: Synchronized parasites were harvested at different time points along the 48 hours intra-erythrocytic cycle for extraction of RNA and for analysis of expression of variant surface antigens by flow cytometry. Total RNA from each parasite sample was extracted and cDNA synthesized. Quantitative real-time PCR was performed using gene-specific primers for all var genes. Samples for flow cytometry were labelled with rabbit IgG targeting DBL5epsilon of VAR2CSA and serum IgG from malaria-exposed men and pregnant women. RESULTS: var transcripts were detected at all time points of the intra-erythrocytic cycle by quantitative real-time PCR, although transcription peaked in ring-stage parasites. There was no difference in the timing of appearance of group A, B or C transcripts, and dominant and subdominant var transcripts appeared to be correctly spliced at all time points. VAR2CSA appeared on the surface of infected erythrocytes 16 hours after invasion, consistent with previous studies of other PfEMP1. Transcription of the pseudogene var1csa could not be detected in NF54VAR2CSA cells. CONCLUSION: The optimal sampling point for analysis of var transc

Udgivelsesdato: 2004-Sep-6, BACKGROUND: The ability of Plasmodium falciparum-infected erythrocytes to adhere to the microvasculature endothelium is thought to play a causal role in malaria pathogenesis. Cytoadhesion to endothelial receptors is generally found to be highly sensitive to trypsinization of the infected erythrocyte surface. However, several studies have found that parasite adhesion to placental receptors can be markedly less sensitive to trypsin. This study investigates whether chondroitin sulphate A (CSA) binding parasites express trypsin-resistant variant surface antigens (VSA) that bind female-specific antibodies induced as a result of pregnancy associated malaria (PAM). METHODS: Fluorescence activated cell sorting (FACS) was used to measure the levels of adult Scottish and Ghanaian male, and Ghanaian pregnant female plasma immunoglobulin G (IgG) that bind to the surface of infected erythrocytes. P. falciparum clone FCR3 cultures were used to assay surface IgG binding before and after selection of the parasite for adhesion to CSA. The effect of proteolytic digestion of parasite erythrocyte surface antigens on surface IgG binding and adhesion to CSA and hyaluronic acid (HA) was also studied. RESULTS: P. falciparum infected erythrocytes selected for adhesion to CSA were found to express trypsin-resistant VSA that are the target of naturally acquired antibodies from pregnant women living in a malaria endemic region of Ghana. However in vitro adhesion to CSA and HA was relatively trypsin sensitive. An improved labelling technique for the detection of VSA expressed by CSA binding isolates has also been described. CONCLUSION: The VSA expressed by CSA binding P. falciparum isolates are currently considered potential targets for a vaccine against PAM. This study identifies discordance between the trypsin sensitivity of CSA binding and surface recognition of CSA selected parasites by serum IgG from malaria exposed pregnant women. Thus, the complete molecular definition of an antigenic P.

Udgivelsesdato: 2003-Aug, Binding of immunoglobulin M (IgM) antibodies from normal human serum to the surface of Plasmodium falciparum-infected red blood cells (iRBC) has previously been demonstrated only in parasites that form rosettes with uninfected red cells. We show that natural, nonspecific IgM but not IgG, IgA, IgD, or IgE also binds to the surface of iRBC selected for adhesion to chondroitin sulfate A (CSA), a placental receptor for parasites associated with malaria in pregnancy. The protease sensitivity of IgM-binding appears to match that of CSA binding, suggesting that the two phenotypes may be mediated by the same parasite molecule. We also show that a wide range of mouse monoclonal antibodies of the IgM class bind nonspecifically to CSA-selected iRBC, an important consideration in the interpretation of immunological assays performed on these parasite lines.