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Functional analysis of monoclonal antibodies against the Plasmodium falciparum PfEMP1-VarO adhesin
Micheline Guillotte1,2, Farida Nato3, Alexandre Juillerat4,5
1Institut Pasteur, Unité d'Immunologie Moléculaire des Parasites, 25-28 rue du Dr ROUX, 75015, Paris, France. micheline.guillotte-blisnick@pasteur.fr.
Insights
Monoclonal antibodies targeting Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) disrupt malaria rosettes by binding to conformation-dependent epitopes near the red blood cell binding site. This offers new strategies for malaria intervention.
Area of Science:
- Immunology
- Parasitology
- Molecular Biology
Background:
- Rosetting, where Plasmodium falciparum-infected red blood cells bind uninfected ones, is common in severe malaria.
- This binding involves PfEMP1 adhesins, encoded by var genes, interacting with serum components and red blood cell receptors.
- Rosette formation is a redundant phenotype with diverse underlying interactions and variant-specific antibodies.
Purpose of the Study:
- To investigate interactions involved in malaria rosetting.
- To map surface epitopes on PfEMP1.
- To characterize monoclonal antibodies for potential therapeutic strategies.
Main Methods:
- Isolated monoclonal antibodies from mice immunized with PfEMP1-VarO recombinant domains.
- Characterized antibody reactivity using ELISA and immunoblots.
- Assessed functionality via inhibition and disruption of rosette formation.
- Identified binding sites using competition ELISAs and mutant PfEMP1 domains.
Main Results:
- Obtained domain-specific, surface-reacting monoclonal antibodies for four PfEMP1 domains.
- Monoclonal antibodies targeting DBL1 potently inhibited and disrupted rosettes.
- Surface-reactive and rosette-interfering antibodies recognized disulfide bond-dependent PfEMP1 epitopes.
- Identified two distinct binding sites for rosette-disrupting antibodies near the red blood cell-binding site.
Conclusions:
- Rosette-inhibitory antibodies bind to conformation-dependent epitopes near the red blood cell-binding site.
- These findings offer novel insights for developing intervention strategies against malaria rosetting.
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.
