Studies on Plasmodium falciparum antigens obtained by eukaryotic expression cloning in COS cells

by Taylor, Brian John

Plasmodirtm faiciparum is the causative organism of the most severe form of malaria, which kills millions of people each year. The existence of drug-resistant P. falcipantm strains and the failure of existing malaria vaccines emphasizes the need for new vaccines incorporating novel parasite antigens. Parasite proteins associated with cellular membranes are particularly promising as vaccine candidates. An efficient way to isolate genes encoding these antigens is by using eukaryotic expression cloning in COS cells and selection with hyper-immune hurnan serum. In 1989, Elliott and colleagues used this method to clone a completely new cysteine-rich P. fnlcipar~tmantigen called Pf12. The major goal of this thesis was to determine the sub-cellular location and time of expression of Pf12. In preliminary panning experiments, three genes encoding parasite antigens were identified in addition io Pf 12. As these three antigens had been previousiy characterized, Pf12 was chosen as the sole candidate for further study. Recombinant subfragments of Pf12 were expressed using E. coli, baculovirus, and DNA immunization expression systems. Two of these proteins, Pfl2ec A2 and Pfl2ec B2, were produced in E. coli and antiserum specific for each purified subfragment was used to charactenze wild-type Pf 12. The anti-Pfl2ec 82 antiserum. which had the strongest specificity for Pf12, was used to identify Pf12 as a 37 kDa protein expressed on the surface of mature schizonts within the infected RBC (iRBC), the parasitophorous vacuolar membrane, the merozoite surface, and perhaps the inner leaflet of the iRBC membrane. The anti-Pfl2ec A2 and B2 antisera also had significant specificity for human serum albumin (HSA). The time of expression and localization of Pf12 suggest that it may be involved in release of merozoites from the iRBC, stabilization the iRBC, re-invasion of =Cs, or perhaps evasion of merozoites from the host immune response by mimicking host proteins such as HSA. These studies suggest that Pf12 would make an excellent asexual stage vaccine candidate.