Characterization of novel surface proteins of "Plasmodium falciparum" and their assessment as molecular targets for a malaria subunit vaccine

Each year there are more than 250 million cases of malaria, claiming nearly one million deaths of which most are among children below the age of five from sub-Saharan Africa. An effective malaria vaccine could prove to be the most cost-effective and efficacious means of preventing severe disease and death from malaria. To date, no approved malaria vaccine is available and only a few candidate vaccines were able to induce some protective efficacy. Limited success in the development of a malaria vaccine may partly be due to the reliance on a hand full of antigens discovered more than 20 years ago. Since the fully annotated Plasmodium falciparum genome has become available in 2002, reverse vaccinology presents a new opportunity to identify novel malaria vaccine candidate antigens.
In this thesis, we anticipated the rational selection of novel malaria subunit vaccine candidates. Proteins on the surface of extracellular stages of the malaria parasite are accessible to immune surveillance by antibodies. Thus, our strategy is based on the selection of hypothetical surface proteins of extracellular parasite stages and subsequent functional characterization using specific monoclonal antibodies.
Functional in vitro and in vivo assays require antibodies capable of recognizing the endogenous antigen in its native context. We developed an entirely cell-based approach that bypasses the problematic step of protein purification. By presenting the antigen on the surface of mammalian cells in its native conformation for immunisation and hybridoma selection, this procedure promotes the generation of monoclonal antibodies capable of binding to the native endogenous target proteins. This was exemplified by three hypothetical surface proteins of P. falciparum, whereof two contain complex folds comprising numerous disulfide bonds. The developed new strategy for the generation of monoclonal antibodies may be applied for a wide range of cell-surface proteins.
The generated monoclonal antibodies were used to study the selected hypothetical P. falciparum surface proteins. PF14_0325 was found to be expressed in late asexual blood stages and PFF0620c, a member of the 6-cysteine protein family implicated in cell-cell interactions, in gametocytes and sporozoites. Both proteins await further investigation in assay systems assessing sporozoite and sexual blood stage inhibition. A third candidate, designated cysteine-rich protective antigen (CyRPA), is expressed in merozoites and localizes to its apical pole. CyRPA-specific antibodies inhibited parasite growth in vitro as well as in vivo. The inhibitory mechanism was found to be independent of immune cells or complement, although complement augmented the effect in vivo. Antibodies blocked invasion of merozoites into erythrocytes, indicating that CyRPA has a function in merozoite invasion. The protein was found to be highly conserved. Together, this renders CyRPA a candidate component for a malaria blood stage vaccine.
The in vivo growth inhibitory effect of antibodies specific for malaria P. falciparum blood stage antigens was assessed by passive immunisation experiments in P. falciparum infected NOD/scid-IL2Rγnull mice engrafted with human erythrocytes. In contrast to previously described passive immunisation studies in other SCID mouse lines, this model evinced a dose-response relationship. Thus we propose this model for comparison of the relative in vivo inhibitory potency of malaria specific antibodies.
Applying the principle of reverse vaccinology, we identified and characterized surface proteins of extracellular malaria stages. Thereby we identified CyRPA as target of merozoite invasion-inhibitory antibodies. We expect that characterization of further hypothetical parasite proteins with this strategy will identify additional vaccine candidate antigens from the extracellular stages of P. falciparum. This will increase the choice of vaccine antigens for incorporation into an effective multivalent, multi-stage malaria subunit vaccine.