title: Functional analysis of monoclonal antibodies against the Plasmodium falciparum PfEMP1-VarO adhesin.
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.
authors.affiliation: Institut Pasteur, Unité d'Immunologie Moléculaire des Parasites, 25-28 rue du Dr ROUX, 75015, Paris, France. micheline.guillotte-blisnick@pasteur.fr.|URA CNRS 2581, 25-28 rue du Dr ROUX, 75015, Paris, France. micheline.guillotte-blisnick@pasteur.fr.
authors.forename: Micheline
authors.initials: M
authors.lastname: Guillotte
authors.affiliation: Institut Pasteur, Plate-forme de Production de Protéines recombinantes et d'Anticorps (PF5), 25-28 rue du Dr ROUX, 75015, Paris, France. natofar@gmail.fr.
authors.forename: Farida
authors.initials: F
authors.lastname: Nato
authors.affiliation: Institut Pasteur, Unité d'Immunologie Structurale, 25-28 rue du Dr ROUX, 75015, Paris, France. alexandre.juillerat@cellectis.com.|CNRS URA 2185, 25-28 rue du Dr ROUX, 75015, Paris, France. alexandre.juillerat@cellectis.com.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Juillerat
authors.affiliation: Institut Pasteur, Unité d'Immunologie Moléculaire des Parasites, 25-28 rue du Dr ROUX, 75015, Paris, France. audrey.hessel@ibs.fr.|Institut Pasteur, Unité d'Immunologie Structurale, 25-28 rue du Dr ROUX, 75015, Paris, France. audrey.hessel@ibs.fr.
authors.forename: Audrey
authors.initials: A
authors.lastname: Hessel
authors.affiliation: Institut Pasteur, Plate-forme de Production de Protéines recombinantes et d'Anticorps (PF5), 25-28 rue du Dr ROUX, 75015, Paris, France. francoise.marchand@pasteur.fr.
authors.forename: Françoise
authors.initials: F
authors.lastname: Marchand
authors.affiliation: Institut Pasteur, Unité d'Immunologie Structurale, 25-28 rue du Dr ROUX, 75015, Paris, France. anita.bentley@orange.fr.|CNRS URA 2185, 25-28 rue du Dr ROUX, 75015, Paris, France. anita.bentley@orange.fr.
authors.forename: Anita
authors.initials: A
authors.lastname: Lewit-Bentley
authors.affiliation: Institut Pasteur, Unité d'Immunologie Structurale, 25-28 rue du Dr ROUX, 75015, Paris, France. graham.bentley@pasteur.fr.|CNRS URA 2185, 25-28 rue du Dr ROUX, 75015, Paris, France. graham.bentley@pasteur.fr.
authors.forename: Graham A
authors.initials: GA
authors.lastname: Bentley
authors.affiliation: Institut Pasteur, Unité d'Immunologie Moléculaire des Parasites, 25-28 rue du Dr ROUX, 75015, Paris, France. ines@pasteur.mg.|URA CNRS 2581, 25-28 rue du Dr ROUX, 75015, Paris, France. ines@pasteur.mg.|Institut Pasteur de Madagascar, Unité d'Immunologie des Maladies Infectieuses, BP 1274, Antananarivo 101, Madagascar. ines@pasteur.mg.
authors.forename: Inès
authors.initials: I
authors.lastname: Vigan-Womas
authors.affiliation: Institut Pasteur, Unité d'Immunologie Moléculaire des Parasites, 25-28 rue du Dr ROUX, 75015, Paris, France. odile.puijalon@pasteur.fr.|URA CNRS 2581, 25-28 rue du Dr ROUX, 75015, Paris, France. odile.puijalon@pasteur.fr.
authors.forename: Odile
authors.initials: O
authors.lastname: Mercereau-Puijalon
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism* / Q000494:pharmacology; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D051379:Mice; D010963:Plasmodium falciparum / Q000187:drug effects / Q000378:metabolism*; D011485:Protein Binding; D015800:Protozoan Proteins / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules; D015800:Protozoan Proteins