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
