Theileria annulata sporozoite surface antigen (SPAG-1) contains neutralizing determinants in the C terminus

SPAG-1 is a surface antigen on Theileria annulata sporozoites that is a candidate both for inclusion in a subunit vaccine and as a ligand for host cell recognition. We have pinpointed major neutralizing epitopes to the C terminus. To facilitate this we expressed SPAG-1 as a series of defined fragments in the pGEX system. These constructs were validated by sequencing and by their spectrum of reactivity with monoclonal antibody (MoAb) BA4. This MoAb recognizes the elastin motif VGVAPG, that is predicted to occur three times in the N terminal half of SPAG-1. The recombinant proteins were then tested by Western blotting with a neutralizing MoAb (1A7) and two neutralizing bovine sera (10T and 34A). The results demonstrate that 1A7 and the bovine sera react with determinants unique to the C terminus. We mapped the neutralizing determinant recognized by MoAb 1A7 to a 16 residue sequence (residues 807-822) using synthetic peptides. Interestingly the bovine sera do not recognize the 1A7 epitope. The potential role of the C terminus as a ligand for host cell recognition and the implications for sub-unit vaccine production are discussed.