The first steps of this project are to produce the recombinant protein, investigate refolding conditions and develop a process for purifying the refolded recombinant protein.? A gene encoding the Plasmodium falciparum Circumsporozoite (CS) protein has been designed, synthesized, and cloned into an E. coli expression vector. A clone expressing the recombinant CS protein (rCSP) was selected. The rCSP, expressed as inclusion bodies, has an intact N-terminus and is being characterized for disulfide linkages. The current effort is to develop a process for scaled production of the rCSP. Efforts are also being made to produce the CSP in the Pichia pastoris expression system.? We have generated several monoclonal antibodies (mAbs) using the C-terminal domain of a yeast produced CS protein as immunizing antigen. Six mAbs recognized the sporozoites and the native CS protein. Three of them are conformation-dependent mAbs, recognizing at least 2 independent epitopes. The other 3 mAbs recognized the CS repeats and are thus conformation independent. Two of these antibodies have the ability to inhibit sporozoite invasion of HepG2 cells in an in vitro assay.? A mouse model using a transgenic berghei parasite expressing falciparum CSP on its sporozoite surface is being developed and tested as an assay to evaluate protection imparted by a CSP-based vaccine. We have also created an expression construct to generate a transgenic knowelsi parasite, expressing falciparum CSP on its sporozoite surface. This transgenic parasite, once obtained, will be used to establish a rhesus model to evaluate protection imparted by a CSP-based vaccine in monkeys.
