Presence of a circumsporozoite-like protein in micronemes of blood-stage merozoites of malaria parasites.

Presence of a circumsporozoite-like protein in micronemes of blood-stage merozoites of malaria parasites A.H. Cochrane,1 S. Uni,2 M. Maracic,' L. di Giovanni,1 M. Aikawa,2 & R.S. Nussenzweig1 We demonstrate for the first time the presence of a circumsporozoite (CS)-like protein in invasive blood stages of malaria parasites. Immunogold electron microscopy using antisporozoite monoclonal antibodies localized these antigens in the micronemes of merozoites. Western immunoblot and two-dimensional gel electrophoresis of mature blood-stage extracts of Plasmodium falciparum, P. berghei, P. cynomolgi, and P. brasilianum identified polypeptides having the same apparent molecular mass and isoelectric points as the corresponding sporozoite (CS) proteins. The CS-like protein of merozoites is present in relatively minor amounts, compared to the CS protein of sporozoites. Mice with long-term P. berghei blood-induced infections develop antibodies which react with sporozoites. Introduction Sporozoites are the invasive stage of plasmodia in- oculated into the vertebrate host by the bite of An- opheles mosquitos. Their surface membrane is covered by the circumsporozoite (CS) protein which arises from higher M, (relative molecular mass) precursors. All CS proteins contain an immunodominant region consisting of a single epitope which is tandemly re- peated and species specific. Monoclonal antibodies (mAbs) directed against this repeat region inhibit sporozoite invasion of target cells in vitro and their passive administration protects mice against sporo- zoite challenge (reviewed in 1). Synthetic peptides and recombinant CS proteins based on the immunodom- inant epitope of Plasmodium falciparum have been used in human immunization trials and have induced some degree of protection (2, 3). Merozoites are the invasive stage of plasmodia released by rupture of infected red blood cells (IRBC) containing mature parasites. Ultrastructural data sug- gest that merozoite entry into the RBC is mediated by contents of rhoptries which are tear-shaped organelles located anteriorly in both merozoites and sporozoites (4). Microneme and rhoptry contents appear to be antigenically distinct (reviewed in 5). Microneme anti- gens are poorly characterized but have been shown to be released into the medium during in vitro growth of IRBC (6). Recently, the CS protein and/or its pre- 1 Department of Medical and Molecular Parasitology, New York University School of Medicine, New York, NY 10016, USA. 2 Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA. cursors have been demonstrated in the micronemes of sporozoites (7). Methods and results CS proteins, like the protective response they induce, have been considered to be stage specific (reviewed in 1). In the present study, using antisporozoite mAbs we have demonstrated, for the first time, the presence of CS-like proteins in the micronemes of blood-stage merozoites of P. falciparum (NF 54 strain), P. berghei (NK 65 strain), P. cynomolgi (Berok strain), and P. brasilianum (Colombian strain). This finding can- not be explained by "carry over" of the antigen from sporozoites or exoerythrocytic stages since all para- sites that were used were derived after several passages of blood-induced infections. Immunogold electron microscopy (8) was per- formed using several P. brasilianum antisporozoite mAbs which react with the immunodominant repeat sequence of the CS protein of P. brasilianum and P. malariae sporozoites. Gold particles were clustered over micronemes of budding merozoites within the segmenters but not over their rhoptries. There was no labelling of young parasites or of the cytoplasm or membrane of the IRBC. Using the same mAbs, we also detected microneme as well as surface membrane localization of the CS antigen in sporozoites. Extracts of sporozoites and RBC containing schizonts/segmenters of P. falciparum, P. berghei, P. cynomolgi, and P. brasilianum were analysed by Western immunoblot (8). For each species, the major merozoite antigen detected by the homologous anti- sporozoite mAb had the same M, as did the corres- ponding CS protein precursor. A weaker band having Bulletin of the World Health Orgunizatlon, 66 (Suppl.): 181-183 (190) 181 A.H. Cochrane et al. the same Mr as the mature CS protein was also detected in blood-stage extracts of P. berghei and P. brasilianum. The isoelectric points (pI) of the merozoite CS- like antigens were compared with the PI of the corresponding CS proteins of sporozoites using two- dimensional gel electrophoresis (9) and Western immunoblot. For P. brasilianum, a single antigen was detected, the pl of which corresponded exactly to the pl of the CS precursor molecule. For P. berghei, the PIs of the two merozoite-associated CS-like antigens were exactly the same as those of the CS protein and its precursor. Our preliminary data, obtained using extract of schizonts/segmenters of P. cynomolgi metabolically labelled with "5S-methionine, indicate that the CS- like protein is actively synthesized during develop- ment of blood stages. Two proteins, having the same electrophoretic mobilities as the P. cynomolgi CS protein and its precursor, were precipitated from the radiolabelled blood-stage extract by the homologous antisporozoite mAb. Characteristic of the immunodominant epitope of the CS protein of sporozoites, the CS-like mole- cules found in merozoites of P. falciparum, P. cyno- molgi, P. brasilianum, and P. berghei contain an epitope which is represented more than once as determined by a two-site one-antibody immunoradio- metric assay (10). The precise amount of CS-like protein in mero- zoites is difficult to quantitate because it appears only in some, and not all, mature schizonts. Furthermore, the amount of CS-like protein appears to vary in different plasmodial species, with P. brasilianum having the greatest amount. For this species we have estimated, using an IRMA and extracts of mostly mature parasites, that the amount of CS-like protein in each segmenter is approximately 100- to 1000-fold less than the amount ofCS protein in each sporozoite (AHC, unpublished data). Studies are now in progress to assess the presence ofmRNA transcripts for the CS protein in blood stages and to characterize the mech- anisms which regulate the stage-related level of expression of this protein. To determine if the CS-like protein is immuno- genic and can induce the formation of antibodies which react with sporozoites, we blood-induced P. berghei infections in mice and allowed the mice to develop a long-term infection of high parasitaemia. Immune sera of these mice were reactive in the CSP assay, by immunofluorescence using glutaraldehyde- fixed sporozoites as antigen, and by an IRMA using as antigen the P. berghei recombinant CS protein (11). In addition, by Western blot, immune sera of several of these mice detected the CS protein (Pb44) and its precursor. The fact that mice with long-term blood-induced P. berghei infections produced some antibodies which react with the corresponding sporozoites raises the possibility that the merozoite CS-like antigen contrib- utes to the antisporozoite antibody response of indi- viduals living in malaria endemic areas. This does not appear to be a major contributing factor since sero- logical studies in endemic areas have shown low antisporozoite antibody responses in young children with high levels of parasitaemia (12), and no signific- ant differences in the levels of antisporozoite anti- bodies in the presence or absence of chemoprophyl- axis (13). Acknowledgements We acknowledge the support of the Agency for Interna- tional Development (DPE-0453-A-405012-00 and DPE- 0453-A-00-4027-00) and the U.S. Public Health Service (grant Al-10645 from the National Institutes of Health). We also thank the UNDP/World Bank/WHO Special Pro- gramme for Research and Training in Tropical Diseases for their support. 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