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Abstract Background Pregnancy-associated malaria (PAM) is a serious consequence of Plasmodium falciparum-infected erythrocytes sequestration in the placenta through the adhesion to the placental receptor chondroitin sulfate A (CSA). Although women become resistant to PAM as they acquire transcending inhibitory immunity against CSA-binding parasites, hundreds of thousands of lives could be saved if a prophylactic vaccine targeting the surface proteins of placental parasites could be designed. Recent works point to the variant protein var2CSA as the key target for the development of a pregnancy-associated malaria vaccine. However, designing such a prophylactic vaccine has been hindered by the difficulty in identifying regions of var2CSA that could elicit broadly neutralizing and adhesion-blocking antibodies. Methods Var2CSA is a very large protein with an estimated molecular weight of 350 kDa, and can be divided into six cysteine rich Duffy binding-like domains (DBL). The human embryonic kidney 293 cell line (HEK293) was used to produce secreted soluble recombinant forms of var2CSA DBL domains. The Escherichia coli expression system was also assessed for the domains not expressed or expressed in low amount in the HEK293 system. To investigate whether var2CSA binding DBL domains can induce biologically active antibodies recognizing the native var2CSA and blocking the interaction, mice were immunized with the refolded DBL3-X or the HEK293 secreted DBL6-ε domains. Results Using the HEK293 expression system, DBL1-X, DBL4-ε and DBL6-ε were produced at relatively high levels in the culture supernatant, while DBL3-X and DBL5-ε were produced at much lower levels. DBL2-X and DBL3-X domains were obtained after refolding of the inclusion bodies produced in E. coli. Importantly, mice antisera raised against the recombinant DBL6-ε domain, specifically reacted against the surface of CSA-binding parasites and revealed adhesion blocking activity. Conclusion This is the first report showing inhibitory binding antibodies obtained through a var2CSA recombinant DBL domain immunization protocol. These results support the current strategies using var2CSA as immunogen in the aim of blocking placental sequestration of malaria parasites. This work is a step towards the development of a var2CSA based vaccine that will prevent pregnancy-associated malaria and improve pregnancy outcomes.

Maternal malaria is associated with the sequestration, in the placenta, of Plasmodium falciparum-infected erythrocytes onto chondroitin sulfate A (CSA), via the duffy binding-like (DBL)-γ3 domain of the P. falciparum erythrocyte membrane protein 1 (PfEMP1[SUPCSA]) (DBL-γ3[SUPCSA]). The production of antibodies against CSA-binding infected erythrocytes (Ies[SUPCSA]) is correlated with resistance to maternal malaria in multiparous women. We produced recombinant DBL-γ3[SUPCSA] (rDBL-γ3[SUPCSA]) in insect cells, corresponding to 2 variant DBL-γ3[SUPCSA] subtypes that mediate binding to CSA in laboratory lines and placental isolates. Both recombinant cysteine-rich DBL-γ3[SUPCSA] domains blocked Ies[SUPCSA] binding to CSA. Immunization of mice, with the rDBL-γ3[SUPCSA]-FCR3 and rDBL-γ3[SUPCSA]-3D7 domains, resulted in the generation of antibodies recognizing homologous and heterologous rDBL-γ3[SUPCSA], a finding indicating conserved epitopes inducing a pan-reactive immune response. Mouse monoclonal antibodies (MAbs) against both recombinant proteins were pan-reactive with various Ies[SUPCSA]. One MAb effi-ciently inhibited and reversed IE[SUPCSA] cytoadhesion to endothelial cells in vitro. Thus, DBL-γ3[SUPCSA] is the target of inhibitory and pan-reactive antibodies. Saimiri sciureus monkeys immunized with FCR3-rDBL-γ3[SUPCSA] developed pan-reactive and inhibitory antibodies, a finding suggesting that the development of a vaccine to prevent maternal malaria is feasible. [ABSTRACT FROM AUTHOR]

Although chondroitin-4-sulfate (CSA) is expressed throughout the microvasculature and CSA-binding infected erythrocytes (IE[sup CSA]) cytoadhere to lung and brain endothelial cells and sequester in male Saimiri sciureus, this phenotype seems to be dependent on the presence of a placenta to develop. This contradiction was investigated by modeling the interactions and cytoadhesion parameters in the microvasculature. Mixtures of IEs interacting with CSA, CD36, or intercellular adhesion molecule 1 were incubated with endothelial cells expressing the corresponding receptors, at physiological pH, under flow conditions. By use of suspensions composed of equal proportions of the phenotypes, cytoadhesion of ∼10 times as many IE[sup CSA] as of any other IE tested was observed. Adherent IE[sup CSA] resisted microvascular wall shear stresses 3-15 times more effectively than did the others. These results, which require confirmation with field isolates, demonstrate that the CSA phenotype is competitive and are consistent with this phenotype initiating microvessel occlusion and with CSA-mediated sequestration in microvessel conditions. [ABSTRACT FROM AUTHOR]

In natural Plasmodium falciparum infections, parasitized erythrocytes (PEs) circulate in the peripheral blood for a period corresponding roughly to the first part of the erythrocytic life cycle (ring stage). Later, in blood-stage development, parasite-encoded adhesion molecules are inserted into the erythrocyte membrane, preventing the circulation of the PEs. The principal molecule mediating PE adhesion is P. falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the polymorphic var gene family. The population of parasites is subject to clonal antigenic variation through changes in var expression, and a single PfEMP1 variant is expressed at the PE surface in a mutually exclusive manner. In addition to its role in immune evasion, switches in PfEMP1 expression may be associated with fundamental changes in parasite tissue tropism in malaria patients. A switch from CD36 binding to chondroitin sulphate A (CSA) binding may lead to extensive sequestration of PEs in placenta syncytiotrophoblasts. This is probably a key event in malaria pathogenesis during pregnancy. The CSA-binding phenotype of mature PEs is linked to another distinct adhesive phenotype: the recently described CSA-independent cytoadhesion of ring-stage PEs. Thus, a subpopulation of PEs that sequentially displays these two different phenotypes may bind to an individual endothelial cell or syncytiotrophoblast throughout the asexual blood-stage cycle. This suggests that non-circulating (cryptic) parasite subpopulations are present in malaria patients. [ABSTRACT FROM AUTHOR]

Maternal malaria is associated with the sequestration, in the placenta, of Plasmodium falciparum-infected erythrocytes onto chondroitin sulfate A (CSA), via the duffy binding-like (DBL)-gamma3 domain of the P. falciparum erythrocyte membrane protein 1 (PfEMP1(CSA)) (DBL-gamma3(CSA)). The production of antibodies against CSA-binding infected erythrocytes (IEs(CSA)) is correlated with resistance to maternal malaria in multiparous women. We produced recombinant DBL-gamma3(CSA) (rDBL-gamma3(CSA)) in insect cells, corresponding to 2 variant DBL-gamma3(CSA) subtypes that mediate binding to CSA in laboratory lines and placental isolates. Both recombinant cysteine-rich DBL-gamma3(CSA) domains blocked IEs(CSA) binding to CSA. Immunization of mice, with the rDBL-gamma3(CSA)-FCR3 and rDBL-gamma3(CSA)-3D7 domains, resulted in the generation of antibodies recognizing homologous and heterologous rDBL-gamma3(CSA), a finding indicating conserved epitopes inducing a pan-reactive immune response. Mouse monoclonal antibodies (MAbs) against both recombinant proteins were pan-reactive with various IEs(CSA). One MAb efficiently inhibited and reversed IE(CSA) cytoadhesion to endothelial cells in vitro. Thus, DBL-gamma3(CSA) is the target of inhibitory and pan-reactive antibodies. Saimiri sciureus monkeys immunized with FCR3-rDBL-gamma3(CSA) developed pan-reactive and inhibitory antibodies, a finding suggesting that the development of a vaccine to prevent maternal malaria is feasible.