AbstractMalaria is a major human parasitic disease. In P.falciparum-endemic areas, infections during gestation give rise to a form of malaria referred to as pregnancy associated malaria (PAM). During PAM, the sequestration of parasitized erythrocytes within the placenta leads to an inflammatory process with poor outcomes for the neonate. Implicated in this sequestration is the VAR2CSA protein, from the PfEMP1 family, which interacts with placental CSA. Observations have shown that multigravidae are less susceptible to PAM over successive pregnancies. Experimentally linked to a VAR2CSA targeted humoral immune response, this observation indicates promise for PAM vaccine development based on VAR2CSA. However, the VAR2CSA protein is structurally complex, presenting difficulites with recombinant large scale production. A strategy to circumvent this constraint is the derivation of fragments of the VAR2CSA protein which retain its ligand binding capacity (minimal binding regions) and which induce a cross-reactive adhesion inhibiting immune response.
This study focused on the development of a platform to interrogate the structural and functional properties of a fragment (DBL1-ID2a-His) derived from the recombinant VAR2CSA protein. The structural aspect of the study utilized circular dichroism spectroscopy to investigate secondary structure as well as stability of the DBL1-ID2a-His under different conditions. Results revealed no significant differences in secondary structure between reduced and non-reduced DBL1-ID2a-His. However, a dose and exposure-time dependent downward shift of melting temperature was observed. Molecular tools (Nanobodies®) were raised against the DBL1-ID2a-His which served to assess its functional properties under different conditions. These indicated a loss of epitopes as function of time of exposure to reducing conditions. Nanobodies® were further used to as probes to test the ability of the recombinant DBL1-ID2a-His to induce adhesion blocking antibodies against erythrocytes parasitized with the P. falciparum isolate FCR3. Results indicated a percentage drop in adhesion of parasitized erythrocytes to a CSA analog, chondroitin surface proteoglycan (CPSG) with 4 Nanobodies® (Nb2, Nb 6, Nb 27 and Nb 100) exhibiting the greatest inhibition.
The results of this study indicate the development of a platform for the characterization of the structural and functional properties of the minimal binding region of a VAR2CSA protein. It is envisaged that the tools obtained from this platform will be expanded in order to fully characterize the minimal binding region of the VAR2CSA.
|Date of Award||4 Jul 2014|
|Supervisor||Yann Sterckx (Advisor), Raluca Florea (Advisor) & Stefan Magez (Promotor)|
- Pregnancy associated malaria
- Structural Biology
- VAR2CSA protein
- minimal binding region