Biochemical and immunological characterization of a Plasmodium chitinase, a malaria transmission-blocking vaccine candidate

Malaria parasites penetrate the mosquito's chitin-contaimng peritrophic matrix that surrounds the blood meal in the midgut, using a parasite-secreted chitinase activated by mosquito-derived midgut proteases. Thus chitinase is a candidate for a transmission-blocking vaccine because inhibition of chitinase blocks mosquito invasion by the Plasmodium ookinete. We have further characterized the chitinase produced by ookinetes of the avian malaria parasite, P. gallinaceum, a model system for human malaria transmission. Supernatants of ookinete cultures were subjected to anion exchange and size exclusion chromatography; chitinase activity was identified by the use of a 4-methylumbelliferone chitotrioside substrate. Silver staining of a chitinasecontaimng fraction on SDS-PAGE revealed a single band of ∼50-kDa. Rabbits vaccinated with partially purified chitinase elicited antibodies that recognized by immunoblot a ∼50-kDa band that was found to be present in extracts of ookinetes but not zygotes. Antiserum made to a synthetic peptide derived from the active site of the Entamoeba histolytica chitinase also recognized a -50-kDa band in ookinetes but not in zygotes. Soluble extracts of ookinetes were passed over a Protein G-sepharose column to which the anti-peptide antiserum was bound. Chitinase activity eluted from the column with 0.2M glycine pH 2.0. These data indicate that the chitinase of Plasmodium gallinaceum is approximately 50 kD. Sera derived from vaccination of animals with purified protein fractions containing chitinase activity and a synthetic peptide derived from another protozoal chitinase recognize an ookinete stage-specific ∼50 kD protein. Such sera should be useful for immunoscreening expression libraries to isolate the Plasmodium gallinaceum chitinase gene.