The roles of the glycosylphosphatidylinositol anchor on the production andimmunogenicity of recombinant ookinete surface antigen Pbs21 of Plasmodiumberghei when prepared in a baculovirus expression system

Malarial ookinetes express an immunodominant surface protein (P28) that is a priority candidate for the development of transmission-blocking vaccines. The full length P28 gene from Plasmodium berghei [Pbs21(1-213)] and a dele tion construct [Pbs21(1-188)] encoding a protein that lacks the 25 C-termin al amino acids, including the glycosylphosphatidylinositol (GPI) anchor sig nal, were expressed in insect cells using baculovirus vectors. Pbs21(1-213) protein is strongly hydrophobic, found in the cytoplasm and on the surface of Spodoptera Sf21 cells, and in the culture medium. Pbs21(1-188) protein was largely found in the aqueous phase of the medium and in the cytoplasm o f Sf21 cells, but was not detected on the cell surface. The presence of 25 C-terminal amino acids is therefore critical to the attachment of recombina nt Pbs21 to the parasite plasma membrane. Mice were immunized subcutaneousl y or intramuscularly with affinity purified recombinant Pbs21(1-213) Pbs21( 1-188) or native Pbs21 proteins. Following two immunizations native Pbs21 i nduces higher titres when administered by either route, than the recombinan t protein bearing an insect GPI anchor, which in turn is markedly more immu nogenic than the recombinant polypeptide lacking a GPI anchor. When specifi c anti Pbs21 antibody titres exceeded 1 mg/ml all three antigens were capab le of inducing transmission blockade greater than or equal to 90%, below 1 mg/ml blockade did not correlate with antibody concentration.