Falciparum-infected Erythrocytes Research Articles

Functional Characterization of the Plasmodium falciparum Chloroquine-Resistance Transporter (PfCRT) in Transformed Dictyostelium discoideum Vesicles

BackgroundChloroquine (CQ)-resistant Plasmodium falciparum malaria has been a global health catastrophe, yet much about the CQ resistance (CQR) mechanism remains unclear. Hallmarks of the CQR phenotype include reduced accumulation of protonated CQ as a weak base in the digestive vacuole of the erythrocyte-stage parasite, and chemosensitization of CQ-resistant (but not CQ-sensitive) P. falciparum by agents such as verapamil. Mutations in the P. falciparum CQR transporter (PfCRT) confer CQR; particularly important among these mutations is the charge-loss substitution K→T at position 76. Dictyostelium discoideum transformed with mutant PfCRT expresses key features of CQR including reduced drug accumulation and verapamil chemosensitization.Methodology and FindingsWe describe the isolation and characterization of PfCRT-transformed, hematin-free vesicles from D. discoideum cells. These vesicles permit assessments of drug accumulation, pH, and membrane potential that are difficult or impossible with hematin-containing digestive vacuoles from P. falciparum-infected erythrocytes. Mutant PfCRT-transformed D. discoideum vesicles show features of the CQR phenotype, and manipulations of vesicle membrane potential by agents including ionophores produce large changes of CQ accumulation that are dissociated from vesicular pH. PfCRT in its native or mutant form blunts the ability of valinomycin to reduce CQ accumulation in transformed vesicles and decreases the ability of K+ to reverse membrane potential hyperpolarization caused by valinomycin treatment.ConclusionIsolated vesicles from mutant-PfCRT-transformed D. discoideum exhibit features of the CQR phenotype, consistent with evidence that the drug resistance mechanism operates at the P. falciparum digestive vacuole membrane in malaria. Membrane potential apart from pH has a major effect on the PfCRT-mediated CQR phenotype of D. discoideum vesicles. These results support a model of PfCRT as an electrochemical potential-driven transporter in the drug/metabolite superfamily that (appropriately mutated) acts as a saturable simple carrier for the facilitated diffusion of protonated CQ.

A Simplified, Sensitive Phagocytic Assay for Malaria Cultures Facilitated by Flow Cytometry of Differentially-Stained Cell Populations

BackgroundPhagocytosis of infected and uninfected erythrocytes is an important feature of malaria infections. Flow cytometry is a useful tool for studying phagocytic uptake of malaria-infected erythrocytes in vitro. However, current approaches are limited by the inability to discriminate between infected and uninfected erythrocytes and a failure to stain the early developmental ring stages of infected erythrocytes. The majority of infected erythrocytes in circulation are of the ring stage and these are therefore important targets to study.Methodology/Principal Findings In vitro P. falciparum cultures comprising infected and uninfected erythrocytes were labeled and exposed to cells derived from the human monocytic THP-1 cell line. Phagocytosis was assayed by flow cytometry. Dual labeling of Plasmodium DNA and erythrocyte cytoplasm with dihydroethidium and CellTrace™ Violet respectively allowed, for the first time, the detection and enumeration of phagocytes with ingested erythrocytes from both early ring- and late schizont-stage P, falciparum cultures. The sensitivity of the method was tested using varying conditions including phagocyte type (monocytes versus macrophages), parasite stage (rings versus schizonts), and negative (incubation with cytochalasin D) and positive (incubation with immune sera) effectors of phagocytosis. The current assay clearly demonstrated uptake of infected and uninfected erythrocytes exposed to phagocytes; the extent of which was dependent on the conditions mentioned.ConclusionsWe describe a simple, sensitive and rapid method for quantifying phagocytosis of P. falciparum-infected erythrocytes, by flow cytometry. This approach can be applied for studying parasite-phagocyte interactions under a variety of conditions. The investigation of phagocytosis of P. falciparum-infected erythrocytes can extend from looking solely at late-staged infected erythrocytes to include early-staged ones as well. It does away with the need to purify infected cells, allowing the study of effects on neighboring uninfected cells. This method may also be translated for use with different types of phagocytes.

Interleukin-10 (IL-10) Polymorphisms Are Associated with IL-10 Production and Clinical Malaria in Young Children

The role of interleukin-10 (IL-10) in malaria remains poorly characterized. The aims of this study were to investigate (i) whether genetic variants of the IL-10 gene influence IL-10 production and (ii) whether IL-10 production as well as the genotypes and haplotypes of the IL-10 gene in young children and their mothers are associated with the incidence of clinical malaria in young children. We genotyped three IL-10 single nucleotide polymorphisms in 240 children and their mothers from a longitudinal prospective cohort and assessed the IL-10 production by maternal peripheral blood mononuclear cells (PBMCs) and cord blood mononuclear cells (CBMCs). Clinical episodes of Plasmodium falciparum malaria in the children were documented until the second year of life. The polymorphism IL-10 A-1082G (GCC haplotype of three SNPs in IL-10) in children was associated with IL-10 production levels by CBMC cultured with P. falciparum-infected erythrocytes (P = 0.043), with the G allele linked to low IL-10 production capacity. The G allele in children was also significantly associated with a decreased risk for clinical malaria infection in their second year of life (P = 0.016). Furthermore, IL-10 levels measured in maternal PBMCs cultured with infected erythrocytes were associated with increased risk of malaria infection in young children (P < 0.001). In conclusion, IL-10 polymorphisms and IL-10 production capacity were associated with clinical malaria infections in young children. High IL-10 production capacity inherited from parents may diminish immunological protection against P. falciparum infection, thereby being a risk for increased malaria morbidity.

Metabolomic Analysis of Patient Plasma Yields Evidence of Plant-Like α-Linolenic Acid Metabolism in Plasmodium falciparum

Metabolomics offers a powerful means to investigate human malaria parasite biology and host-parasite interactions at the biochemical level, and to discover novel therapeutic targets and biomarkers of infection. Here, we used an approach based on liquid chromatography and mass spectrometry to perform an untargeted metabolomic analysis of metabolite extracts from Plasmodium falciparum-infected and uninfected patient plasma samples, and from an enriched population of in vitro cultured P. falciparum-infected and uninfected erythrocytes. Statistical modeling robustly segregated infected and uninfected samples based on metabolite species with significantly different abundances. Metabolites of the α-linolenic acid (ALA) pathway, known to exist in plants but not known to exist in P. falciparum until now, were enriched in infected plasma and erythrocyte samples. In vitro labeling with (13)C-ALA showed evidence of plant-like ALA pathway intermediates in P. falciparum. Ortholog searches using ALA pathway enzyme sequences from 8 available plant genomes identified several genes in the P. falciparum genome that were predicted to potentially encode the corresponding enzymes in the hitherto unannotated P. falciparum pathway. These data suggest that our approach can be used to discover novel facets of host/malaria parasite biology in a high-throughput manner.

A 95 kDa protein of Plasmodium vivax and P. cynomolgi visualized by three‐dimensional tomography in the caveola–vesicle complexes (Schüffner's dots) of infected erythrocytes is a member of the PHIST family

Plasmodium vivax and P. cynomolgi produce numerous caveola-vesicle complex (CVC) structures within the surface of the infected erythrocyte membrane. These contrast with the electron-dense knob protrusions expressed at the surface of Plasmodium falciparum-infected erythrocytes. Here we investigate the three-dimensional (3-D) structure of the CVCs and the identity of a predominantly expressed 95 kDa CVC protein. Liquid chromatography - tandem mass spectrometry analysis of immunoprecipitates by monoclonal antibodies from P. cynomolgi extracts identified this protein as a member of the Plasmodium helical interspersed subtelomeric (PHIST) superfamily with a calculated mass of 81 kDa. We named the orthologous proteins PvPHIST/CVC-81(95) and PcyPHIST/CVC-81(95) , analysed their structural features, including a PEXEL motif, repeated sequences and a C-terminal PHIST domain, and show that PHIST/CVC-81(95) is most highly expressed in trophozoites. We generated images of CVCs in 3-D using electron tomography (ET), and used immuno-ET to show PHIST/CVC-81(95) localizes to the cytoplasmic side of the CVC tubular extensions. Targeted gene disruptions were attempted in vivo. The pcyphist/cvc-81(95) gene was not disrupted, but parasites containing episomes with the tgdhfr selection cassette were retrieved by selection with pyrimethamine. This suggests that PHIST/CVC-81(95) is essential for survival of these malaria parasites.

Deciphering the Resistance-Counteracting Functions of Ferroquine in Plasmodium falciparum-Infected Erythrocytes.

The aminoquinoline chloroquine (CQ) has been widely used for treating malaria since World War II. Resistance to CQ began to spread around 1957 and is now found in all malarious areas of the world. CQ resistance is caused by multiple mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT). These mutations result in an increased efflux of CQ from the acidic digestive vacuole (DV) to the cytosol of the parasite. This year, we proposed a strategy to locate and quantify the aminoquinolines in situ within infected red blood cells (iRBCs) using synchrotron based X-ray nanoprobe fluorescence. Direct measurements of unlabeled CQ and ferroquine (FQ) (a ferrocene-CQ conjugate, extremely active against CQ-resistant strains) enabled us to evidence fundamentally different transport mechanisms from the cytosol to the DV between CQ and FQ in the CQ-susceptible strain HB3. These results inspired the present study of the localization of CQ and FQ in the CQ-resistant strain W2. The introduction of the ferrocene core in the lateral side chain of CQ has an important consequence: the transporter is unable to efflux FQ from the DV. We also found that resistant parasites treated by FQ accumulate a sulfur-containing compound, credibly glutathion, in their DV.

Understanding How Plasmodium falciparum Binds to the Placenta and Produces Pathology Provides a Rationale for Pregnancy-Associated Malaria Vaccine Development

By adulthood repeated exposure to Plasmodium falciparum, the causative agent of the most severe form of malaria in humans, can induce semi-immunity to clinical infection. During pregnancy, however, there is a striking recurrence of severe disease, a syndrome described as pregnancy-associated malaria (PAM). This is caused by P. falciparum-infected erythrocytes binding to receptors on the placental endothelium that are expressed uniquely during pregnancy. This subset of parasites binds by virtue of expressing a cell surface antigen that is the ligand for the corresponding endothelial receptor, thereby providing a selective pressure for its clonal expansion. The antigen also triggers a protective antibody-mediated immune response. With subsequent pregnancies and further exposure to these variant parasites, antibodies develop earlier and women may experience milder infections. Women with PAM exhibit more severe malaria infections than do non-gravid women, with 2,500-10,000 deaths each year due to haemolytic anaemia. PAM also has a detrimental effect on the foetus, increasing the risk of low birth weight, which causes 60,000- 200,000 infant deaths annually in sub-Saharan Africa. Current research aims to produce a vaccine to stimulate development of antibodies which would be protective against infection with wild-type parasites. This is hampered by significant diversity in variant surface antigens and available vaccine candidates may only protect women living in malaria-endemic areas. While placental-parasite interactions are not sufficiently understood, incremental progress is being made towards producing an effective vaccine. This review focuses on the molecular structures involved in binding to consider the prospects for a vaccine mimicking naturally-acquired immunity to PAM.

Plasmodium falciparum-Infected Erythrocytes Induce Granzyme B by NK Cells through Expression of Host-Hsp70

In the early immune response to Plasmodium falciparum-infected erythrocytes (iRBC), Natural Killer (NK) cells are activated, which suggests an important role in innate anti-parasitic immunity. However, it is not well understood whether NK cells directly recognize iRBC or whether stimulation of NK cells depends mainly on activating signals from accessory cells through cell-to-cell contact or soluble factors. In the present study, we investigated the influence of membrane-bound host Heat shock protein (Hsp) 70 in triggering cytotoxicity of NK cells from malaria-naïve donors or the cell line NK92 against iRBC. Hsp70 and HLA-E membrane expression on iRBC and potential activatory NK cell receptors (NKG2C, CD94) were assessed by flow cytometry and immunoblot. Upon contact with iRBC, Granzyme B (GzmB) production and release was initiated by unstimulated and Hsp70-peptide (TKD) pre-stimulated NK cells, as determined by Western blot, RT-PCR and ELISPOT analysis. Eryptosis of iRBC was determined by Annexin V-staining. Our results suggest that presence of Hsp70 and absence of HLA-E on the membrane of iRBC prompt the infected host cells to become targets for NK cell-mediated cytotoxicity, as evidenced by impaired parasite development. Contact of iRBC with NK cells induced release of GzmB. We propose that following GzmB uptake, iRBC undergo eryptosis via a perforin-independent, GzmB-mediated mechanism. Since NK activity toward iRBC could be specifically enhanced by TKD peptide and abrogated to baseline levels by blocking Hsp70 exposure, we propose TKD as an innovative immunostimulatory agent to be tested as an adjunct to anti-parasitic treatments in vivo.

HIV-1 Inhibits Phagocytosis and Inflammatory Cytokine Responses of Human Monocyte-Derived Macrophages to P. falciparum Infected Erythrocytes

HIV-1 infection increases the risk and severity of malaria by poorly defined mechanisms. We investigated the effect of HIV-1Ba-L infection of monocyte-derived macrophages (MDM) on phagocytosis of opsonised P. falciparum infected erythrocytes (IE) and subsequent proinflammatory cytokine secretion. Compared to mock-infected MDM, HIV-1 infection significantly inhibited phagocytosis of IE (median (IQR) (10 (0–28) versus (34 (27–108); IE internalised/100 MDM; p = 0.001) and decreased secretion of IL-6 (1,116 (352–3,387) versus 1,552 (889–6,331); pg/mL; p = 0.0078) and IL-1β (16 (7–21) versus 33 (27–65); pg/mL; p = 0.0078). Thus inadequate phagocytosis and cytokine production may contribute to impaired control of malaria in HIV-1 infected individuals.

High Levels of Antibodies to Multiple Domains and Strains of VAR2CSA Correlate with the Absence of Placental Malaria in Cameroonian Women Living in an Area of High Plasmodium falciparum Transmission

Placental malaria, caused by sequestration of Plasmodium falciparum-infected erythrocytes in the placenta, is associated with increased risk of maternal morbidity and poor birth outcomes. The parasite antigen VAR2CSA (variant surface antigen 2-chondroitin sulfate A) is expressed on infected erythrocytes and mediates binding to chondroitin sulfate A, initiating inflammation and disrupting homeostasis at the maternal-fetal interface. Although antibodies can prevent sequestration, it is unclear whether parasite clearance is due to antibodies to a single Duffy binding-like (DBL) domain or to an extensive repertoire of antibodies to multiple DBL domains and allelic variants. Accordingly, plasma samples collected longitudinally from pregnant women were screened for naturally acquired antibodies against an extensive panel of VAR2CSA proteins, including 2 to 3 allelic variants for each of 5 different DBL domains. Analyses were performed on plasma samples collected from 3 to 9 months of pregnancy from women living in areas in Cameroon with high and low malaria transmission. The results demonstrate that high antibody levels to multiple VAR2CSA domains, rather than a single domain, were associated with the absence of placental malaria when antibodies were present from early in the second trimester until term. Absence of placental malaria was associated with increasing antibody breadth to different DBL domains and allelic variants in multigravid women. Furthermore, the antibody responses of women in the lower-transmission site had both lower magnitude and lesser breadth than those in the high-transmission site. These data suggest that immunity to placental malaria results from high antibody levels to multiple VAR2CSA domains and allelic variants and that antibody breadth is influenced by malaria transmission intensity.

A Plasma Survey Using 38 PfEMP1 Domains Reveals Frequent Recognition of the Plasmodium falciparum Antigen VAR2CSA among Young Tanzanian Children

PfEMP1 proteins comprise a family of variant antigens that appear on the surface of P. falciparum-infected erythrocytes and bind to multiple host receptors. Using a mammalian expression system and BioPlex technology, we developed an array of 24 protein constructs representing 38 PfEMP1 domains for high throughput analyses of receptor binding as well as total and functional antibody responses. We analyzed the reactivity of 561 plasma samples from 378 young Tanzanian children followed up to maximum 192 weeks of life in a longitudinal birth cohort. Surprisingly, reactivity to the DBL5 domain of VAR2CSA, a pregnancy malaria vaccine candidate, was most common, and the prevalence of reactivity was stable throughout early childhood. Reactivity to all other PfEMP1 constructs increased with age. Antibodies to the DBL2βC2PF11_0521 domain, measured as plasma reactivity or plasma inhibition of ICAM1 binding, predicted reduced risk of hospitalization for severe or moderately severe malaria. These data suggest a role for VAR2CSA in childhood malaria and implicate DBL2βC2PF11_0521 in protective immunity.

In situ nanochemical imaging of label-free drugs: a case study of antimalarials in Plasmodium falciparum-infected erythrocytes

We report here for the first time the in vitro localization of unlabeled antimalarial drugs with high spatial resolution. This strategy further enhances our understanding of the action mechanisms of antimalarial drugs. Our approach may be applied to a wide range of domains where quantitative chemical imaging of drugs at the sub-cellular level appears critical.

Opening up the advantages of the ruthenocenic bioprobes of ferroquine: distribution and localization in Plasmodium falciparum-infected erythrocytes

A ferrocene-quinoline conjugate named ferroquine (FQ or SSR97193) is active against both chloroquine-susceptible and chloroquine-resistant Plasmodium falciparum and P. vivax strains and/or isolates. FQ was shown to be efficient for the treatment of uncomplicated malaria in humans (phase IIb of clinical trials). However, the molecular basis of FQ's mechanism of action is still unknown because few approaches (such as radioactive labelling or immunofluorescence) are available for that purpose. Previous reports from our laboratory suggest that the intramolecular hydrogen bond in the lateral side chain plays a crucial role in the antimalarial activity of the drug. We used two ruthenocenic bioprobes of FQ (with and without an intramolecular hydrogen bond) to study their localization and quantification in Plasmodium falciparum-infected erythrocytes. We first used Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) analysis to trace ruthenoquine (RQ, with an intramolecular hydrogen bond) and methylruthenoquine (Me-RQ, without an intramolecular hydrogen bond) in the infected red blood cells (iRBCs). We showed that RQ accumulates faster in the digestive vacuole of the iRBCs than Me-RQ. We next examined the ruthenium distribution at the ultrastructural level by transmission electron microscopy (TEM). We showed that RQ accumulates faster in the parasitic digestive vacuole (DV) close to its membranes than Me-RQ.

Three Dimensional Imaging of Maurer's Clefts in “Unroofed”Plasmodium Falciparum-Infected Erythrocytes by Transmission Electron Microscopy

Plasmodium falciparum human malaria parasites establish unique membrane systems in the cytoplasm of their host erythrocytes. These systems include Maurer's Clefts (MCs), which are evident as small membranous structures in host erythrocyte periphery and are thought to support the trafficking of P. falciparum proteins to the host cell surface. Three-dimensional structures of MCs and their tethering to the host erythrocyte membrane have been reported from reconstructions of transmission electron microscopy (TEM) and fluorescence images (Hanssen et al.

Synthesis and evaluation of novel 7-trifluoromethyl-4-(4-substituted anilino)quinolines as antiparasitic and antineoplastic agents.

Several novel derivatives bearing the 7-trifluoromethyl-4-(4-substituted anilino)-quinoline skeleton were synthesized and evaluated for their in vitro activity against the blood streaming form of the parasites Trypanosoma brucei rhodesiense, the trypomastigotes of Trypanosoma cruzi cultured in rat skeletal myoblasts, the amastigotes form of Leishmania donovani, Plasmodium falciparum (K1 strain) infected erythrocyte suspension, as well as their toxicity towards rat skeletal L-6 cells. In addition, three of the synthesized compounds were tested for their in vitro antitumor activity towards 60 human tumor cell lines by the National Cancer Institute (NCI). Compound 1-(4-[[7-(trifluoromethyl)quinolin-4-yl]amino]phenyl)ethan-1-one thiosemicarbazone 23 exhibited potential activity against T. b. rhodesiense, T. cruzi and P. falciparum with IC50's of 0.278, 0.85 and 0.417 microgram/ml, respectively. These values are even more valuable since this compound was clearly non-toxic (cytotoxicity IC50 > 90 micrograms/ml), leading to in vitro therapeutic indices of > 323, > 106 and > 216, respectively. Meanwhile, compound N-[4-[5-(4-chlorophenyl)-4,5-dihydroisoxazol-3-yl]phenyl]-7-(trifluoromethyl) quinolin-4-amine 21 showed broad spectrum antitumor activity with full panel median growth inhibition GI50 (MG-MID) of 1.95 mumol and total growth inhibition TGI (MG-MID) of 6.87 mumol, respectively. Compound 23 represents a very good prospective as a lead compound able to combat three tropical diseases at a time. However, the pattern for the antitumor activity did not parallel any of the antiparasitic ones, indicating that non-common mechanisms of action may exist among these activities.

Clag9 Is Not Essential for PfEMP1 Surface Expression in Non-Cytoadherent Plasmodium falciparum Parasites with a Chromosome 9 Deletion

BackgroundThe expression of the clonally variant virulence factor PfEMP1 mediates the sequestration of Plasmodium falciparum infected erythrocytes in the host vasculature and contributes to chronic infection. Non-cytoadherent parasites with a chromosome 9 deletion lack clag9, a gene linked to cytoadhesion in previous studies. Here we present new clag9 data that challenge this view and show that surface the non-cytoadherence phenotype is linked to the expression of a non-functional PfEMP1.Methodology/Principal FindingsLoss of adhesion in P. falciparum D10, a parasite line with a large chromosome 9 deletion, was investigated. Surface iodination analysis of non-cytoadherent D10 parasites and COS-7 surface expression of the CD36-binding PfEMP1 CIDR1α domain were performed and showed that these parasites express an unusual trypsin-resistant, non-functional PfEMP1 at the erythrocyte surface. However, the CIDR1α domain of this var gene expressed in COS-7 cells showed strong binding to CD36. Atomic Force Microscopy showed a slightly modified D10 knob morphology compared to adherent parasites. Trafficking of PfEMP1 and KAHRP remained functional in D10. We link the non-cytoadherence phenotype to a chromosome 9 breakage and healing event resulting in the loss of 25 subtelomeric genes including clag9. In contrast to previous studies, knockout of the clag9 gene from 3D7 did not interfere with parasite adhesion to CD36.Conclusions/SignificanceOur data show the surface expression of non-functional PfEMP1 in D10 strongly indicating that genes other than clag9 deleted from chromosome 9 are involved in this virulence process possibly via post-translational modifications.

Broad inhibition of plasmodium falciparum cytoadherence by (+)-epigallocatechin gallate

BackgroundThe surface antigen PfEMP-1 is a key virulence factor of the human malaria parasite implicated in the cytoadherence of Plasmodium falciparum infected erythrocytes to a range of receptors on host endothelium. Among these host receptors, binding to ICAM-1 is related to cerebral malaria. The majority of the mortality in children with cerebral malaria is seen within 24 h of hospital admission despite the use of effective anti-parasite drugs, therefore, the development of adjunctive therapies is urgently needed.The polyphenolic compound (+)-epigallocatechin gallate ((+)-EGCG) has been previously evaluated for anti-adhesive properties using a small number of laboratory parasite isolates. Here, this property is further explored using a new panel of ICAM-1-binding patient isolates of P. falciparum to ascertain if (+)-EGCG might be effective as a broad spectrum inhibitor of ICAM-1-based cytoadherence.MethodsPlasmodium falciparum lines, including A4 and ItG as positive controls and nine new ICAM-1 binding patient isolates, were allowed to bind with ICAM-1-Fc protein under static assay conditions in the presence and absence of 50 μM (+)-EGCG. Adhesion levels of all the parasite strains were quantified by microscopy as the mean number of infected erythrocyte (IE) bound per mm2 of surface area and statistical comparisons were made to demonstrate the effect of (+)-EGCG on the binding of various parasite variants to human ICAM-1.ResultsThis study revealed that binding of patient isolates to ICAM-1 was reduced significantly with inhibition levels of 37% in patient isolate BC-12 up to a maximum of 80% in patient isolate 8146 at 50 μM (+)-EGCG.ConclusionEvaluation of the anti-adhesive property of (+)-EGCG against a new panel of ICAM-1-binding patient isolates of P. falciparum showed that this inhibitor, identified as potential mimic of the L43 loop of human ICAM-1, was effective at blocking cytoadherence.

Limited response of NK92 cells to Plasmodium falciparum-infected erythrocytes

BackgroundMechanisms by which anti-malarial immune responses occur are still not fully clear. Natural killer (NK) cells are thought to play a pivotal role in innate responses against Plasmodium falciparum. In this study, the suitability of NK92 cells as models for the NK mechanisms involved in the immune response against malaria was investigated.MethodsNK92 cells were assessed for several signs of activation and cytotoxicity due to contact to parasites and were as well examined by oligonucleotide microarrays for an insight on the impact P. falciparum-infected erythrocytes have on their transcriptome. To address the parasite side of such interaction, growth inhibition assays were performed including non-NK cells as controls.ResultsBy performing microarrays with NK92 cells, the impact of parasites on a transcriptional level was observed. The findings show that, although not evidently activated by iRBCs, NK92 cells show transcriptional signs of priming and proliferation. In addition, decreased parasitaemia was observed due to co-incubation with NK92 cells. However, such effect might not be NK-specific since irrelevant cells also affected parasite growth in vitro.ConclusionsAlthough NK92 cells are here shown to behave as poor models for the NK immune response against parasites, the results obtained in this study may be of use for future investigations regarding host-parasites interactions in malaria.

The case for PfEMP1-based vaccines to protect pregnant women against Plasmodium falciparum malaria

Vaccines are very cost-effective tools in combating infectious disease mortality and morbidity. Unfortunately, vaccines efficiently protecting against infection with malaria parasites are not available and are not likely to appear in the near future. An alternative strategy would be vaccines protecting against the disease and its consequences rather than against infection per se, by accelerating the development of the protective immunity that is normally acquired after years of exposure to malaria parasites in areas of stable transmission. This latter strategy is being energetically pursued to develop a vaccine protecting pregnant women and their offspring against mortality and morbidity caused by the accumulation of Plasmodium falciparum-infected erythrocytes in the placenta. It is based on a detailed understanding of the parasite antigen and the host receptor involved in this accumulation, as well as knowledge regarding the protective immune response that is acquired in response to placental P. falciparum infection. Nevertheless, it remains controversial in some quarters whether such a vaccine would have the desired impact, or indeed whether the strategy is meaningful. This article critically examines the relevance of several perceived obstacles to development of a vaccine against placental malaria.

The NTS-DBL2X Region of VAR2CSA Induces Cross-Reactive Antibodies That Inhibit Adhesion of Several Plasmodium falciparum Isolates to Chondroitin Sulfate A

Binding to chondroitin sulfate A by VAR2CSA, a parasite protein expressed on infected erythrocytes, allows placental sequestration of Plasmodium falciparum-infected erythrocytes. This leads to severe consequences such as maternal anemia, stillbirths, and intrauterine growth retardation. The latter has been clearly associated to increased morbidity and mortality of the infants. Acquired anti-VAR2CSA antibodies have been associated with improved pregnancy outcomes, suggesting a vaccine could prevent the syndrome. However, identifying functionally important regions in the large VAR2CSA protein is difficult. Using genetic immunization, we raised polyclonal antisera against overlapping segments of VAR2CSA in mice and rabbits. The adhesion-inhibition capacities of induced antisera and of specific antibodies purified from plasma of malaria-exposed pregnant women were assessed on laboratory-adapted parasite lines and field isolates expressing VAR2CSA. Competition enzyme-linked immunosorbent assay (ELISA) was employed to analyze functional resemblance between antibodies induced in animals and those naturally acquired by immune multigravidae. Antibodies targeting the N-terminal sequence (NTS) up to DBL2X (NTS-DBL2X) efficiently blocked parasite adhesion to chondroitin sulfate A in a manner similar to that of antibodies raised against the entire VAR2CSA extracellular domain. Interestingly, naturally acquired antibodies and those induced by vaccination against NTS-DBL2X target overlapping strain-transcendent anti-adhesion epitopes. This study highlights an important step achieved toward development of a protective vaccine against placental malaria.