Analysis of antibody induction upon immunization with distinct NTS-DBL1α-domains of PfEMP1 from rosetting Plasmodium falciparum parasites.
BACKGROUND
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.


METHODS
Three 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.


RESULTS
All 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.


CONCLUSIONS
Biologically 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.
Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, SE-17177, Sweden.
Davide
D
Angeletti
Letusa
L
Albrecht
Mats
M
Wahlgren
Kirsten
K
Moll
D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood*; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D018985:Epitopes, B-Lymphocyte / Q000276:immunology; D004912:Erythrocytes / Q000469:parasitology; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D006041:Goats; D006801:Humans; D017780:Malaria Vaccines / Q000008:administration & dosage / Q000276:immunology*; D010963:Plasmodium falciparum / Q000235:genetics / Q000276:immunology; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000276:immunology
D000913:Antibodies, Protozoan; D018985:Epitopes, B-Lymphocyte; D017780:Malaria Vaccines; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; C085731:erythrocyte membrane protein 1, Plasmodium falciparum