Abstract
BackgroundRosette-formation of Plasmodium falciparum parasitized erythrocytes is of importance in the development of severe malaria. The parasite-derived molecule PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1), central to rosetting, is suggested to be included in a multimeric vaccine targeting severe disease.MethodsThree recombinant NTS-DBL1α-domains of PfEMP1 were generated in Escherichia coli, purified and used for immunization of rats and goats. Antibody titres were determined in ELISA assays and responses were compared in-between different individual animals and species. Reactivity with the parasites was tested in live pRBC using FACS. B-cell epitopes prediction was carried out in silico and compared to the results obtained by peptide microarray. Screening for serological cross-reactivity with heterologous NTS-DBL1α variants was carried out by ELISA, peptide array and FACS on pRBC of different laboratory strains and patient isolates.ResultsAll three NTS-DBL1α-domains induced high titres of antibodies that were biologically active with no apparent difference between constructs covering slightly different parts of the DBL1α-sequence. The different animal species showed comparable titres of antibodies, while variations within individuals of the species could be observed.Mapping of the recognized epitopes revealed that most parts of the molecule were able to induce an antibody response with a tendency for the N and C terminal parts of the molecule for slightly higher recognition. Important differences to the epitopes predicted were found as some of the most conserved parts of the DBL1α-domain contained the main epitopes for antibody reactivity. ELISA assays and peptide microarray demonstrated substantial cross-reactivity to heterologous variants, while binding to native PfEMP1 was observed only in few combinations on the pRBC surface, underlining that mainly internal, conserved and not surface exposed parts of the DBL1α-domain are responsible for this observation.ConclusionBiologically active antibodies can be induced consistently, with high titres, in different animal species and the antibodies elicited by different constructs react with similar epitopes. Induced antibodies recognize epitopes localized in all subdomains of the DBL1α-sequence. Cross-reactivity between NTS-DBL1α-variants is common in ELISA, but rare with live pRBC emphasizing that also internal, conserved areas of PfEMP1 carry important highly immunogenic epitopes of the molecule.
Highlights
Rosette-formation of Plasmodium falciparum parasitized erythrocytes is of importance in the development of severe malaria
Rosetting has been found associated with severe malaria in many studies in Africa [1,2,3,4,5,6,7,8], has been described to lead to microvascular obstruction [9,10] and has been suggested as one of the most important factors bringing about severe disease [11,12]
The observations presented in this study suggest that B-cell epitope prediction is informative; there might be a tendency for skewing towards surface epitopes that are present in unstructured loops
Summary
Rosette-formation of Plasmodium falciparum parasitized erythrocytes is of importance in the development of severe malaria. The parasite-derived molecule PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1), central to rosetting, is suggested to be included in a multimeric vaccine targeting severe disease. The binding of parasitized to non-parasitized red blood cells (RBC), has been described as an important virulence factor of the Plasmodium falciparum parasite. The parasite ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1) binds serum proteins and receptors on the human RBC surface. Serum proteins, such as non-immune immunoglobulins, fibrinogen and albumin as well as blood group A and B antigen, heparan sulphate [13,14,15,16,17,18] and the complement receptor 1 (CR1) [19,20] have been identified to be involved in the rosetting phenomena. PfEMP1 vary in size between 200–400 kDa and are encoded by a repertoire of around 60 var genes per genome [26] responsible for the antigenic variation at the pRBC surface [27,28,29]
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