Surface antigens in Plasmodium falciparum malaria : PfEMP1 and SURFIN4.2

Abstract: Plasmodium falciparum malaria is an infectious disease that on despite of the ongoing eradication efforts is still endemic in more than 100 countries, sometimes causing severe disease that leads to the death of around half a million people per year. Malaria pathology is tightly associated with the parasite cycle inside the human red blood cells (RBCs). Central to this cycle is the initial invasion by the merozoite and the extensive RBC modifications induced by the parasite, transporting proteins to the RBC cytoplasm and membrane. The P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) transported to the surface of the parasitized RBC (pRBC) and the surface-associated interspersed protein 4.2 (SURFIN4.2) present both at the pRBC surface as well as at the merozoite apex and surface, are the major focus of this thesis. PfEMP1 is the major surface antigen and mediates rosetting (binding of parasitized RBCs (pRBCs) to two or more RBCs), a parasite phenotype associated with the development of severe disease. The most N-terminal segment of this protein (the NTSDBL1α domain) has been identified as the ligand for rosetting and naturally acquired antibodies targeting this particular protein protect against severe disease development. In this study we wanted to address the specific regions in PfEMP1 and in other protein targets recognized by rosette-disrupting antibodies (generated upon immunization with recombinant PfEMP1 or naturally acquired during P. falciparum infection). We also wanted to explore other functional roles of these antibodies. A panel of antibodies (monoclonal and polyclonal) against rosette-mediating NTS-DBL1α domains was produced by animal immunization. The antibodies were analyzed with particular attention to their capacity to recognize the surface of the pRBC, disrupt the rosettes formed by homologous parasites and induce phagocytosis by monocytic cells. Additionally, the specific epitopes recognized by the majority of these antibodies were successfully mapped to a specific region of subdomain 3 (SD3) of the DBL1α domain, regardless of the parasite strain used. These results suggested this region as a major target of anti-rosetting antibodies. Most of these antibodies also induced opsonization for phagocytosis, a role that could be of great importance during pRBCs clearance in vivo. Interestingly, some of the antibodies with high opsonizing activity did not disrupt rosettes, indicating that other epitopes besides those involved in rosetting are exposed on the pRBC surface and are able to induce functional antibodies that could provide protection. The naturally acquired antibodies in sera from children living in a malaria endemic region were also investigated. The ability of these antibodies to recognize three parasite-derived surface proteins (PfEMP1, RIFIN-A and SURFIN4.2) was assessed. Different variables were also measured in the presence of these sera samples, including rosetting rate, surface reactivity and opsonization for phagocytosis on a rosetting model parasite grown in group O or group A RBCs. The data showed that the acquired immune response developed during natural infection could recognize the pRBC surface and more importantly could induce pRBC phagocytosis and in a few cases disrupt the rosettes formed by a heterologous parasite model. These activities however had limited access to the pRBCs inside a rosette formed with group A RBCs, where these cells act as a shield for the pRBCs, protecting it from antibodies’ recognition therefore impairing their effector function. This study also suggested that SURFIN4.2 previously identified at the pRBC surface could be involved in rosette formation, either as a direct ligand or as an accessory element for rosette strengthening. The suggestion of SURFIN4.2 as a possible mediator in rosetting prompted us to deepen the study of this protein, however, the initial results steered the approach to this protein from the rosetting phenomenon towards a more striking and understudied role of this protein during the invasion process. Using antibodies against the N-terminus, the protein was observed at the surface of the merozoite but more strikingly also in the neck of the rhoptries. The protein was shed into culture supernatant upon schizont rupture and was associated with GLURP (Glutamate Rich Protein) and RON-4 (Rhoptry Neck Protein 4) to form a complex we named SURGE (SURFIN4.2-RON-4-GLURP complEx). Importantly, SURFIN4.2 was detected at the apex of the merozoite during merozoite initial attachment and active invasion into the RBCs. The exact functional role of SURGE remains to be determined, but the presence of RON-4, a protein confined to the moving junction (MJ), strongly suggests a role in strengthening the stable contact between the merozoite apex and the RBC, possibly as and additional RBC adhesion molecule. Supporting the involvement of the protein complex during the invasion process, antibodies against the N-terminus of SURFIN4.2 partially inhibited invasion.