Var transcription profiling of Plasmodium falciparum 3D7: assignment of cytoadherent phenotypes to dominant transcripts

Background\ud Cytoadherence of Plasmodium falciparum -infected red blood cells is mediated by var gene-encoded P. falciparum erythrocyte membrane protein-1 and host receptor preference depends in most cases on which of the 50–60 var genes per genome is expressed. Enrichment of phenotypically homogenous parasites by panning on receptor expressing cells is fundamental for the identification of the corresponding var transcript.\ud \ud \ud Methods\ud P. falciparum 3D7 parasites were panned on several transfected CHO-cell lines and their var transcripts analysed by i) reverse transcription/PCR/cloning/sequencing using a universal DBLα specific oligonucleotide pair and ii) by reverse transcription followed by quantitative PCR using 57 different oligonucleotide pairs.\ud \ud \ud Results\ud Each cytoadherence selected parasite line also adhered to untransfected CHO-745 cells and upregulation of the var gene PFD995/PFD1000c was consistently associated with cytoadherence to all but one CHO cell line. In addition, parasites panned on different CHO cell lines revealed candidate var genes which reproducibly associated to the respective cytoadherent phenotype. The transcription profile obtained by RT-PCR/cloning/sequencing differed significantly from that of RT-quantitative PCR.\ud \ud \ud Conclusion\ud Transfected CHO cell lines are of limited use for the creation of monophenotypic cytoadherent parasite lines. Nevertheless, 3D7 parasites can be reproducibly selected for the transcription of different determined var genes without genetic manipulation. Most importantly, var transcription analysis by RT-PCR/cloning/sequencing may lead to erroneous interpretation of var transcription profiles.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) [06/51873-0]CNP

var gene transcription and PfEMP1 expression in the rosetting and cytoadhesive Plasmodium falciparum clone FCR3S1.2

<p>Abstract</p> <p>Background</p> <p>The pathogenicity of <it>Plasmodium falciparum </it>is in part due to the ability of the parasitized red blood cell (pRBC) to adhere to intra-vascular host cell receptors and serum-proteins. Binding of the pRBC is mediated by <it>Plasmodium falciparum </it>erythrocyte membrane protein 1 (PfEMP1), a large multi-variant molecule encoded by a family of ≈60 <it>var </it>genes.</p> <p>Methods</p> <p>The study of <it>var </it>gene transcription in the parasite clone FCR3S1.2 was performed by semi-quantitative PCR and quantitative PCR (qPCR). The expression of the major PfEMP1 in FCR3S1.2 pRBC was analysed with polyclonal sera in rosette disruption assays and immunofluorecence.</p> <p>Results</p> <p>Transcripts from <it>var</it>1 (FCR3S1.2_<it>var</it>₁; IT4<it>var</it>21) and other <it>var </it>genes were detected by semi-quantitative PCR but results from qPCR showed that one <it>var </it>gene transcript dominated over the others (FCR3S1.2_<it>var</it>₂; IT4<it>var</it>60). Antibodies raised in rats to the recombinant NTS-DBL1α of <it>var</it>2 produced in <it>E. coli </it>completely and dose-dependently disrupted rosettes (≈95% at a dilution of 1/5). The sera reacted with the Maurer's clefts in trophozoite stages (IFA) and to the infected erythrocyte surface (FACS) indicating that FCR3S1.2_{<it>var2 </it>}encodes the dominant PfEMP1 expressed in this parasite.</p> <p>Conclusion</p> <p>The major transcript in the rosetting model parasite FCR3S1.2 is FCR3S1.2_<it>var</it>₂(IT4<it>var</it>60). The results suggest that this gene encodes the PfEMP1-species responsible for the rosetting phenotype of this parasite. The activity of previously raised antibodies to the NTS-DBL1α of FCR3S1.2_<it>var</it>₁is likely due to cross-reactivity with NTS-DBL1α of the <it>var</it>2 encoded PfEMP1.</p

B-Cell Epitopes in NTS-DBL1 alpha of PfEMP1 Recognized by Human Antibodies in Rosetting Plasmodium falciparum

Plasmodium falciparum is the most lethal of the human malaria parasites. the virulence is associated with the capacity of the infected red blood cell (iRBC) to sequester inside the deep microvasculature where it may cause obstruction of the blood-flow when binding is excessive. Rosetting, the adherence of the iRBC to uninfected erythrocytes, has been found associated with severe malaria and found to be mediated by the NTS-DBL1 alpha-domain of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1). Here we show that the reactivity of plasma of Cameroonian children with the surface of the FCR3S1.2-iRBC correlated with the capacity to disrupt rosettes and with the antibody reactivity with a recombinant PfEMP1 (NTS-DBL1 alpha of IT4(var60)) expressed by parasite FCR3S1.2. the plasma-reactivity in a microarray, consisting of 96 overlapping 15-mer long peptides covering the NTS-DBL1 alpha domain from IT4var60 sequence, was compared with their capacity to disrupt rosettes and we identified five peptides where the reactivity were correlated. Three of the peptides were localized in subdomain-1 and 2. the other two peptide-sequences were localized in the NTS-domain and in subdomain-3. Further, principal component analysis and orthogonal partial least square analysis generated a model that supported these findings. in conclusion, human antibody reactivity with short linear-peptides of NTS-DBL1 alpha of PfEMP1 suggests subdomains 1 and 2 to hold anti-rosetting epitopes recognized by anti-rosetting antibodies. the data suggest rosetting to be mediated by the variable areas of PfEMP1 but also to involve structurally relatively conserved areas of the molecule that may induce biologically active antibodies.Swedish Research Council (VR)Swedish Academy of Sciences (KVA, Soderberg Foundation)Karolinska Institutet-DPAEU Network of Excellence EviMalarKarolinska Inst, Dept Microbiol Tumor & Cell Biol MTC, Stockholm, SwedenKarolinska Inst, Dept Lab Med, Therapeut Immunol TIM, Stockholm, SwedenKarolinska Univ Hosp, CAST, Huddinge, SwedenUniv Estadual Campinas, Dept Biochem, Campinas, SP, BrazilWeb of Scienc

A Sequence in Subdomain 2 of DBL1a of Plasmodium falciparum Erythrocyte Membrane Protein 1 Induces Strain Transcending Antibodies

Immunity to severe malaria is the first level of immunity acquired to Plasmodium falciparum. Antibodies to the variant antigen PfEMP1 (P. falciparum erythrocyte membrane protein 1) present at the surface of the parasitized red blood cell (pRBC) confer protection by blocking microvascular sequestration. Here we have generated antibodies to peptide sequences of subdomain 2 of PfEMP1-DBL1a previously identified to be associated with severe or mild malaria. A set of sera generated to the amino acid sequence KLQTLTLHQVREYWWALNRKEVWKA, containing the motif ALNRKE, stained the live pRBC. 50% of parasites tested (7/14) were positive both in flow cytometry and immunofluorescence assays with live pRBCs including both laboratory strains and in vitro adapted clinical isolates. Antibodies that reacted selectively with the sequence REYWWALNRKEVWKA in a 15-mer peptide array of DBL1a-domains were also found to react with the pRBC surface. By utilizing a peptide array to map the binding properties of the elicited anti-DBL1a antibodies, the amino acids WxxNRx were found essential for antibody binding. Complementary experiments using 135 degenerate RDSM peptide sequences obtained from 93 Ugandan patient-isolates showed that antibody binding occurred when the amino acids WxLNRKE/D were present in the peptide. The data suggests that the ALNRKE sequence motif, associated with severe malaria, induces strain-transcending antibodies that react with the pRBC surfac

Plasmodium vivax vaccine: What is the best way to go?

Malaria is one of the most devastating human infectious diseases caused by Plasmodium spp. parasites. A search for an effective and safe vaccine is the main challenge for its eradication. Plasmodium vivax is the second most prevalent Plasmodium species and the most geographically distributed parasite and has been neglected for decades. This has a massive gap in knowledge and consequently in the development of vaccines. The most significant difficulties in obtaining a vaccine against P. vivax are the high genetic diversity and the extremely complex life cycle. Due to its complexity, studies have evaluated P. vivax antigens from different stages as potential targets for an effective vaccine. Therefore, the main vaccine candidates are grouped into preerythrocytic stage vaccines, blood-stage vaccines, and transmission-blocking vaccines. This review aims to support future investigations by presenting the main findings of vivax malaria vaccines to date. There are only a few P. vivax vaccines in clinical trials, and thus far, the best protective efficacy was a vaccine formulated with synthetic peptide from a circumsporozoite protein and Montanide ISA-51 as an adjuvant with 54.5% efficacy in a phase IIa study. In addition, the majority of P. vivax antigen candidates are polymorphic, induce strain-specific and heterogeneous immunity and provide only partial protection. Nevertheless, immunization with recombinant proteins and multiantigen vaccines have shown promising results and have emerged as excellent strategies. However, more studies are necessary to assess the ideal vaccine combination and test it in clinical trials. Developing a safe and effective vaccine against vivax malaria is essential for controlling and eliminating the disease. Therefore, it is necessary to determine what is already known to propose and identify new candidates

Immunization with the MAEBL M2 domain protects against lethal Plasmodium yoelii infection

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOMalaria remains a world-threatening disease largely because of the lack of a long-lasting and fully effective vaccine. MAEBL is a type 1 transmembrane molecule with a chimeric cysteine-rich ectodomain homologous to regions of the Duffy binding-like erythrocyte binding protein and apical membrane antigen 1 (AMA1) antigens. Although MAEBL does not appear to be essential for the survival of blood-stage forms, ectodomains M1 and M2, homologous to AMA1, seem to be involved in parasite attachment to erythrocytes, especially M2. MAEBL is necessary for sporozoite infection of mosquito salivary glands and is expressed in liver stages. Here, the Plasmodium yoelii MAEBL-M2 domain was expressed in a prokaryotic vector. C57BL/6J mice were immunized with doses of P. yoelii recombinant protein rPyM2-MAEBL. High levels of antibodies, with balanced IgG1 and IgG2c subclasses, were achieved. rPyM2-MAEBL antisera were capable of recognizing the native antigen. Anti-MAEBL antibodies recognized different MAEBL fragments expressed in CHO cells, showing stronger IgM and IgG responses to the M2 domain and repeat region, respectively. After a challenge with P. yoelii YM (lethal strain)-infected erythrocytes (IE), up to 90% of the immunized animals survived and a reduction of parasitemia was observed. Moreover, splenocytes harvested from immunized animals proliferated in a dose-dependent manner in the presence of rPyM2-MAEBL. Protection was highly dependent on CD4(+), but not CD8(+), T cells toward Th1. rPyM2-MAEBL antisera were also able to significantly inhibit parasite development, as observed in ex vivo P. yoelii erythrocyte invasion assays. Collectively, these findings support the use of MAEBL as a vaccine candidate and open perspectives to understand the mechanisms involved in protection.Malaria remains a world-threatening disease largely because of the lack of a long-lasting and fully effective vaccine. MAEBL is a type 1 transmembrane molecule with a chimeric cysteine-rich ectodomain homologous to regions of the Duffy binding-like erythr831037813792FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOsem informaçãosem informaçã

Plasmodium falciparum Rosetting Epitopes Converge in the SD3-Loop of PfEMP1-DBL1α

The ability of Plasmodium falciparum parasitized RBC (pRBC) to form rosettes with normal RBC is linked to the virulence of the parasite and RBC polymorphisms that weaken rosetting confer protection against severe malaria. The adhesin PfEMP1 mediates the binding and specific antibodies prevent sequestration in the micro-vasculature, as seen in animal models. Here we demonstrate that epitopes targeted by rosette disrupting antibodies converge in the loop of subdomain 3 (SD3) which connects the h6 and h7 α-helices of PfEMP1-DBL1α. Both monoclonal antibodies and polyclonal IgG, that bound to epitopes in the SD3-loop, stained the surface of pRBC, disrupted rosettes and blocked direct binding of recombinant NTS-DBL1α to RBC. Depletion of polyclonal IgG raised to NTS-DBL1α on a SD3 loop-peptide removed the anti-rosetting activity. Immunizations with recombinant subdomain 1 (SD1), subdomain 2 (SD2) or SD3 all generated antibodies reacting with the pRBC-surface but only the sera of animals immunized with SD3 disrupted rosettes. SD3-sequences were found to segregate phylogenetically into two groups (A/B). Group A included rosetting sequences that were associated with two cysteine-residues present in the SD2-domain while group B included those with three or more cysteines. Our results suggest that the SD3 loop of PfEMP1-DBL1α is an important target of anti-rosetting activity, clarifying the molecular basis of the development of variant-specific rosette disrupting antibodies

Asymptomatic infection with Plasmodium falciparum and Plasmodium vivax in the Brazilian Amazon Basin: to treat or not to treat?

In this study, we determined whether the treatment of asymptomatic parasites carriers (APCs), which are frequently found in the riverside localities of the Brazilian Amazon that are highly endemic for malaria, would decrease the local malaria incidence by decreasing the overall pool of parasites available to infect mosquitoes. In one village, the treatment of the 19 Plasmodium falciparum-infected APCs identified among the 270 residents led to a clear reduction (Z = -2.39, p = 0.017) in the incidence of clinical cases, suggesting that treatment of APCs is useful for controlling falciparum malaria. For vivax malaria, 120 APCs were identified among the 716 residents living in five villages. Comparing the monthly incidence of vivax malaria in two villages where the APCs were treated with the incidence in two villages where APCs were not treated yielded contradictory results and no clear differences in the incidence were observed (Z = -0.09, p = 0.933). Interestingly, a follow-up study showed that the frequency of clinical relapse in both the treated and untreated APCs was similar to the frequency seen in patients treated for primary clinical infections, thus indicating that vivax clinical immunity in the population is not species specific but only strain specific

Fucosylated Chondroitin Sulfate Inhibits Plasmodium Falciparum Cytoadhesion And Merozoite Invasion.

Sequestration of Plasmodium falciparum-infected erythrocytes (Pf-iEs) in the microvasculature of vital organs plays a key role in the pathogenesis of life-threatening malaria complications, such as cerebral malaria and malaria in pregnancy. This phenomenon is marked by the cytoadhesion of Pf-iEs to host receptors on the surfaces of endothelial cells, on noninfected erythrocytes, and in the placental trophoblast; therefore, these sites are potential targets for antiadhesion therapies. In this context, glycosaminoglycans (GAGs), including heparin, have shown the ability to inhibit Pf-iE cytoadherence and growth. Nevertheless, the use of heparin was discontinued due to serious side effects, such as bleeding. Other GAG-based therapies were hampered due to the potential risk of contamination with prions and viruses, as some GAGs are isolated from mammals. In this context, we investigated the effects and mechanism of action of fucosylated chondroitin sulfate (FucCS), a unique and highly sulfated GAG isolated from the sea cucumber, with respect to P. falciparum cytoadhesion and development. FucCS was effective in inhibiting the cytoadherence of Pf-iEs to human lung endothelial cells and placenta cryosections under static and flow conditions. Removal of the sulfated fucose branches of the FucCS structure virtually abolished the inhibitory effects of FucCS. Importantly, FucCS rapidly disrupted rosettes at high levels, and it was also able to block parasite development by interfering with merozoite invasion. Collectively, these findings highlight the potential of FucCS as a candidate for adjunct therapy against severe malaria.581862-7