Antigenic variation and virulence in Plasmodium falciparum malaria: Studies on the surface protein PfEMP1

Approximately 40% of the world’s population is at risk of contracting malaria, a disease caused by the intracellular protozoan Plasmodium. The species Plasmodium falciparum is responsible for the majority of severe morbidity and mortality. A major virulence factor of the falciparum parasite is its ability to cause accumulation of parasitized red blood cells in the microvasculature of different organs, through binding to endothelial cells (cytoadhesion) and to unparasitized red blood cells (rosetting). The binding is mediated by members of the adhesive surface protein, PfEMP1 (P. falciparum erythrocyte membrane protein 1), which is encoded by the variable var gene family. One var gene is activated at a time and the var gene expression can be switched in order to avoid antibody response, a mechanism called antigenic variation. This makes PfEMP1 pivotal for the virulence of the P. falciparum parasite. The studies presented in this thesis aim at enhancing the understanding of PfEMP1, both at a phenotypic and a genotypic level, with special focus on clinical isolates. We developed a precise method to study var gene transcription and applied it to elucidate var gene transcription dynamics in clinical isolates from Uganda as well as in laboratory strains. The results show that the var gene transcription profile is unique for each parasite isolate and strain, and that clinical isolates have more complex transcriptional profiles than in vitro strains. Clinical isolates were found to switch away from the var genes associated with severe disease upon in vitro adaptation. We therefore conclude that it is crucial to study var genes directly after parasite collection so that it reflects the expression in the patient. A model parasite clone for severe malaria was used in order to confirm that the method correctly identified the var gene that is transcribed, translated into PfEMP1 and transported to the parasitized red blood cell surface. Heparan sulfate has been found to be a PfEMP1 receptor that is frequently recognized in clinical isolates. To explore this finding, we generated a low anti-coagulant heparin (LAH) to study its ability to disrupt rosettes in fresh clinical isolates. We found that LAH is able to disrupt rosettes in clinical isolates from children infected with malaria. The rosette disruption effect was more pronounced in isolates from children with complicated malaria than in isolates from children with mild malaria indicating that this compound in the future might have a place in the treatment of severe malaria. Further, we identified a surface-exposed sequence in PfEMP1, which is associated with severe malaria. The sequence includes a motif that is able to induce a cross-reactive antibody response, in which the generated antibodies recognize parasitized red blood cells in a subset of clinical isolates and laboratory strains. In addition, the antibodies reacted selectively with the sequence motif in a peptide-array of different PfEMP1 domains. Residues within the sequence motif were found to be important for antibody binding, and one third of degenerate peptide-sequences of Ugandan patient isolates were shown to react with the antibody. We conclude that the sequence motif, which is associated with severe malaria, generates strain-transcending antibodies that recognize the parasitized red blood cell surface. In conclusion, this thesis provides insights into var gene transcription dynamics in clinical isolates, it enhances the understanding of low anticoagulant heparin as a treatment for severe malaria, and it describes a surface-exposed epitope in PfEMP1 associated with severe malaria generating strain-transcending antibodies

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

High expression of cyclin D1 is associated to high proliferation rate and increased risk of mortality in women with ER-positive but not in ER-negative breast cancers

Cyclin D1 has a central role in cell cycle control and is an important component of estrogen regulation of cell cycle progression. We have previously shown that high cyclin D expression is related to aggressive features of ER-positive but not ER-negative breast cancer. The aims of the present study were to validate this differential ER-related effect and furthermore explore the relationship between cyclin D overexpression and CCND1 gene amplification status in a node-negative breast cancer case-control study. Immunohistochemical nuclear expression of cyclin D1 (n = 364) and amplification of the gene CCND1 by fluorescent in situ hybridization (n = 255) was performed on tissue microarray sections from patients with T1-2N0M0 breast cancer. Patients given adjuvant chemotherapy were excluded. The primary event was defined as breast cancer death. Breast cancer-specific survival was analyzed in univariate and multivariable models using conditional logistic regression. Expression of cyclin D1 above the median (61.7%) in ER breast cancer was associated with an increased risk for breast cancer death (OR 3.2 95% CI 1.5-6.8) also when adjusted for tumor size and grade (OR 3.1). No significant prognostic impact of cyclin D1 expression was found among ER-negative cases. Cyclin D1 overexpression was significantly associated to high expression of the proliferation markers cyclins A (rho 0.19, p = 0.006) and B (rho 0.18, p = 0.003) in ER-positive tumors, but not in ER-negative cases. There was a significant association between CCND1 amplification and cyclin D1 expression (p = 0.003), but CCND1 amplification was not statistically significantly prognostic (HR 1.4, 95% CI 0.4-4.4). We confirmed our previous observation that high cyclin D1 expression is associated to high proliferation and a threefold higher risk of death from breast cancer in ER-positive breast cancer.Peer reviewe

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

Montana Kaimin, January 30, 2008

Student newspaper of the University of Montana, Missoula.https://scholarworks.umt.edu/studentnewspaper/6138/thumbnail.jp

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