1. Functional analysis of monoclonal antibodies against the Plasmodium falciparum PfEMP1-VarO adhesin
- Author
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Françoise Marchand, Farida Nato, Inès Vigan-Womas, Micheline Guillotte, Audrey Hessel, Odile Mercereau-Puijalon, Anita Lewit-Bentley, Graham A. Bentley, Alexandre Juillerat, Immunologie Moléculaire des Parasites, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Parasitologie moléculaire et Signalisation, Production de Protéines Recombinantes et d'Anticorps (Plate-Forme), Institut Pasteur [Paris] (IP), Biochimie Structurale et Cellulaire, Immunologie structurale, Immunologie moléculaire des parasites, Unité d'immunologie des maladies infectieuses [Antananarivo, Madagascar] (IPM), Institut Pasteur de Madagascar, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), This work was supported by the French National Research Agency (Agence Nationale de la Recherche, Programme Microbiologie, Immunologie et Maladies Emergentes, grant ANR-07-MIME-021-0). The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007–2013) under grant agreement No 242095., ANR-07-MIME-0021,ROSETTE,Analyses sérologiques, fonctionnelles et structurales des facteurs de virulence, PfEMP1, impliqués dans le rosetting et l'auto-agglutinantion(2007), European Project: 242095,EC:FP7:HEALTH,FP7-HEALTH-2009-single-stage,EVIMALAR(2009), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris], and Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]
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0301 basic medicine ,Antigenicity ,medicine.drug_class ,030231 tropical medicine ,Plasmodium falciparum ,Protozoan Proteins ,Enzyme-Linked Immunosorbent Assay ,Rosetting ,Monoclonal antibody ,Epitope ,03 medical and health sciences ,Mice ,Epitopes ,0302 clinical medicine ,parasitic diseases ,medicine ,Animals ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Binding site ,biology ,Research ,Antibodies, Monoclonal ,Rosette-disrupting antibodies ,biology.organism_classification ,Monoclonal antibodies (mAbs) ,Virology ,Molecular biology ,3. Good health ,Malaria ,Bacterial adhesin ,030104 developmental biology ,Infectious Diseases ,Biotinylation ,biology.protein ,Parasitology ,Antibody ,Cell Adhesion Molecules ,PfEMP1 adhesin ,Protein Binding - Abstract
Background Rosetting, 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. Methods Monoclonal 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. Results Domain-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. Conclusions Rosette-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 material The online version of this article (doi:10.1186/s12936-015-1016-5) contains supplementary material, which is available to authorized users.
- Published
- 2016