Receptor Affinity-Based Purification of PfEMP1 Proteins.

The virulence of Plasmodium falciparum is linked to the ability of infected erythrocytes (IEs) to bind a range of human receptors. This binding is mediated by a family of highly polymorphic proteins known as P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 proteins are expressed on the surface of IEs and are composed of extracellular domains (NTS, CIDR, DBL), a transmembrane region and an acidic C-terminal segment. Subdomains of the extracellular N-terminal part of PfEMP1 molecules have been shown to bind specific receptors.In this chapter, we describe how to purify PfEMP1 proteins by a receptor affinity-based method. This includes how to prepare affinity columns and how to subsequently test the functionality of the purified PfEMP1 protein in an ELISA-based assay.

Affinity Purification of PfEMP1-Specific Antibodies from Human Blood.

Acquired immunity against Plasmodium falciparum infections relies heavily on IgG antibodies specific for PfEMP1 proteins expressed on the surface of infected erythrocytes. Purified human antibodies can be used, for example, to study the interactions between specific PfEMP1 proteins and receptors expressed by human endothelial cells, and to identify which IgG antibodies play a functional role in natural acquired immunity.This chapter describe how to affinity purify PfEMP1-specific human antibodies on an affinity column coupled with PfEMP1 protein. We include ELISA-based methods for identification of human plasma samples reactive against PfEMP1, and for testing of affinity purified IgG antibodies prior to their use in more advanced procedures.

Production of anti-PfEMP1 Polyclonal Antisera in Rats and Mice.

Plasmodium falciparum-infected erythrocytes (IEs) bind various host receptors via members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family expressed on the surface of the IEs. Antibody reagents are needed to investigate interactions between specific PfEMP1 proteins and receptors expressed by human endothelial cells. This protocol describes the production of rat and mouse polyclonal anti-PfEMP1 antibodies. Polyclonal antibodies are relatively easy to produce and have advantages compared to monoclonal antibodies (see Chapters 28 - 30 ) for some applications. An ELISA-based method to test the polyclonal antibodies before their use in more advanced procedures is also presented.

Analysis of Antibody Inhibition of PfEMP1 Binding by Competition ELISA.

Plasmodium falciparum express variant antigens on the surface of infected erythrocytes (IEs). These include proteins of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. PfEMP1 proteins mediate binding of IEs to various human endothelial receptors and induce antibodies response during natural infections. In this chapter, we describe a competition ELISA that is an assay for antibodies neutralizing binding of PfEMP1 proteins to their cognate receptor.

ICAM-1-binding Plasmodium falciparum erythrocyte membrane protein 1 variants elicits opsonic-phagocytosis IgG responses in Beninese children.

Members of the highly polymorphic Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family expressed on the surface of infected erythrocytes (IEs) are important virulence factors, which mediate vascular adhesion of IEs via endothelial host receptors and are targets of naturally acquired immunity. The PfEMP1 family can be divided into clinically relevant subgroups, of which some bind intercellular adhesion molecule 1 (ICAM-1). While the acquisition of IgG specific for ICAM-1-binding DBLß domains is known to differ between PfEMP1 groups, its ability to induce antibody-dependent cellular phagocytosis (ADCP) is unclear. We therefore measured plasma levels of DBLß-specific IgG, the ability of such IgG to inhibit PfEMP1-binding to ICAM-1, and its ability to opsonize IEs for ADCP, using plasma from Beninese children with severe (SM) or uncomplicated malaria (UM). IgG specific for DBLß from group A and B ICAM-1-binding PfEMP1 were dominated by IgG1 and IgG3, and were similar in SM and UM. However, levels of plasma IgG inhibiting ICAM-1-binding of group A DBLß of PFD1235w was significantly higher in children with UM than SM, and acute UM plasma induced a higher ADCP response than acute SM plasma.

Progress and new horizons toward a VAR2CSA-based placental malaria vaccine.

Introduction: Several malaria vaccines are under various phases of development with some promising results. In placental malaria (PM) a deliberately anti-disease approach is considered as many studies have underlined the key role of VAR2CSA protein, which therefore represents the leading vaccine candidate. However, evidence indicates that VAR2CSA antigenic polymorphism remains an obstacle to overcome.Areas covered: This review analyzes the progress made thus far in developing a VAR2CSA-based vaccine, and addresses the current issues and challenges that must be overcome to develop an effective PM vaccine.Expert opinion: Phase I trials of PAMVAC and PRIMVAC VAR2CSA vaccines have shown more or less satisfactory results with regards to safety and immunogenicity. The second generation of VAR2CSA-based vaccines could benefit from optimization approaches to broaden the activity spectrum against various placenta-binding isolates through continued advances in the structural understanding of the interaction with CSA.

Antibacterial and antifungal activities and phytochemical profile of leaf extract from different extractants of Ricinus communis against selected pathogens.

OBJECTIVES: Ricinus communis leaves are used in herbal preparations for treating candidiasis, skin and wound infections in Ghana. This study aimed at comparing the phytochemical profile of aqueous, methanol, petroleum ether, ethyl acetate and ethanolic extracts of the leaves of Ricinus communis and determine the growth inhibitory activities, bactericidal, bacteriostatic and fungicidal effects of the respective extracts on Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumonaie and Candida albicans. RESULTS: The aqueous, methanol and ethanol extracts were shown to contain most of the phytochemicals analyzed. All solvents extracts exhibited inhibitory activity against the growth of all microorganisms under study. The methanol extract showed highest zones of inhibition and was found to be statistically significant (P < 0.05) compared to other solvents extracts. All solvents extracts exhibited both bacteriostatic and bactericidal effects on the test organisms at varying concentration, with MIC values ranging from 3.13 to 25.0 mg/ml and MBCs were from 200 to 400 mg/ml. MFCs of Candida albicans was between 200 and 400 mg/l. Our data confirm the anti-bacterial and anti-fungal properties of R. communis and showed that the biologically relevant phytochemicals from the leaves of this plant can be extracted with the solvents aqueous, methanol and ethanol.