Epitope Mapping and Topographic Analysis of VAR2CSA DBL3X Involved in P. falciparum Placental Sequestration

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Abstract
Pregnancy-associated malaria is a major health problem, which mainly affects primigravidae living in malaria endemic areas. The syndrome is precipitated by accumulation of infected erythrocytes in placental tissue through an interaction between chondroitin sulphate A on syncytiotrophoblasts and a parasite-encoded protein on the surface of infected erythrocytes, believed to be VAR2CSA. VAR2CSA is a polymorphic protein of approximately 3,000 amino acids forming six Duffy-binding-like (DBL) domains. For vaccine development it is important to define the antigenic targets for protective antibodies and to characterize the consequences of sequence variation. In this study, we used a combination of in silico tools, peptide arrays, and structural modeling to show that sequence variation mainly occurs in regions under strong diversifying selection, predicted to form flexible loops. These regions are the main targets of naturally acquired immunoglobulin gamma and accessible for antibodies reacting with native VAR2CSA on infected erythrocytes. Interestingly, surface reactive anti-VAR2CSA antibodies also target a conserved DBL3X region predicted to form an α-helix. Finally, we could identify DBL3X sequence motifs that were more likely to occur in parasites isolated from primi- and multigravidae, respectively. These findings strengthen the vaccine candidacy of VAR2CSA and will be important for choosing epitopes and variants of DBL3X to be included in a vaccine protecting women against pregnancy-associated malaria. Pregnancy-associated malaria caused by Plasmodium falciparum is characterized by the accumulation of parasite-infected red blood cells in the placenta and is a major health problem in Africa. VAR2CSA is a parasite protein expressed on the surface of malaria-infected red blood cells and mediates the binding to the placental receptor, chondroitin sulphate A. It is believed that a vaccine based on VAR2CSA will protect pregnant women against the adverse effects of pregnancy-associated malaria. However, due to the size and polymorphism of VAR2CSA it is required to define smaller regions that can be included in a vaccine and to analyze the degree and consequences of sequence variation to ensure a broadly protective immune response. The authors have characterized the chondroitin sulphate A-binding DBL3X domain of VAR2CSA with regard to epitopes targeted by naturally acquired antibodies and the influence of sequence variation by bioinformatics and experimental data based on a VAR2CSA peptide array. They identify both variable and conserved surface-exposed epitopes that are targets of naturally acquired immunoglobulin gamma in pregnant women with placental malaria. These findings will be imperative for choosing epitopes and variants of DBL3X to be included in a vaccine protecting pregnant women against malaria.