Functional analysis of monoclonal antibodies against the Plasmodium falciparum PfEMP1-VarO adhesin

Abstract

BackgroundRosetting, namely the capacity of the Plasmodium falciparum-infected red blood cells to bind uninfected RBCs, is commonly observed in African children with severe malaria. Rosetting results from specific interactions between a subset of variant P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesins encoded by var genes, serum components and RBC receptors. Rosette formation is a redundant phenotype, as there exists more than one var gene encoding a rosette-mediating PfEMP1 in each genome and hence a diverse array of underlying interactions. Moreover, field diversity creates a large panel of rosetting-associated serotypes and studies with human immune sera indicate that surface-reacting antibodies are essentially variant-specific. To gain better insight into the interactions involved in rosetting and map surface epitopes, a panel of monoclonal antibodies (mAbs) was investigated.MethodsMonoclonal antibodies were isolated from mice immunized with PfEMP1-VarO recombinant domains. They were characterized using ELISA and reactivity with the native PfEMP1-VarO adhesin on immunoblots of reduced and unreduced extracts, as well as SDS-extracts of Palo Alto 89F5 VarO schizonts. Functionality was assessed using inhibition of Palo Alto 89F5 VarO rosette formation and disruption of Palo Alto 89F5 VarO rosettes. Competition ELISAs were performed with biotinylated antibodies against DBL1 to identify reactivity groups. Specificity of mAbs reacting with the DBL1 adhesion domain was explored using recombinant proteins carrying mutations abolishing RBC binding or binding to heparin, a potent inhibitor of rosette formation.ResultsDomain-specific, surface-reacting mAbs were obtained for four individual domains (DBL1, CIDR1, DBL2, DBL4). Monoclonal antibodies reacting with DBL1 potently inhibited the formation of rosettes and disrupted Palo Alto 89F5 VarO rosettes. Most surface-reactive mAbs and all mAbs interfering with rosetting reacted on parasite immunoblots with disulfide bond-dependent PfEMP1 epitopes. Based on competition ELISA and binding to mutant DBL1 domains, two distinct binding sites for rosette-disrupting mAbs were identified in close proximity to the RBC-binding site.ConclusionsRosette-inhibitory antibodies bind to conformation-dependent epitopes located close to the RBC-binding site and distant from the heparin-binding site. These results provide novel clues for a rational intervention strategy that targets rosetting.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-1016-5) contains supplementary material, which is available to authorized users.

Host-parasite interactions during Plasmodium infection: Implications for immunotherapies

“…The binding of Pf EMP1 to CSA receptors allows the sequestration of iRBCs in the placenta and VAR2CSA antibodies function mainly by inhibiting parasite adhesion to RBCs and sequestration along with other effector mechanisms ( 235 ). In addition, monoclonal antibodies against Pf EMP1 inhibited the formation of rosettes ( 236 ). Interestingly, a new class of receptor-based monoclonal antibodies generated by the insertion of a host receptor (collagen-binding inhibitory receptor, LAIR1) into an antibody gene have been shown to agglutinate iRBCs and opsonize them for phagocytosis by monocytes, thereby aiding parasite clearance ( 237 ).…”