High Prevalence of Natural Antibodies against Plasmodium Falciparum 83-kilodalton Apical Membrane Antigen (pf83/ama-1) as Detected by Capture-enzyme-linked Immunosorbent Assay Using Ftjll-length B Aculovirus Recombinant Pf83/ama- 1

The 83-kilodalton (kD) apical membrane antigen of Plasinodiuin falciparum (PF83/AMA1) is a potential asexual blood stage vaccine component. This antigen has been expressed as a full-length, nonfusion, recombinant baculovirus protein (PF83-7G81) using the authentic predicted signal peptide for appropriate postsynthetic routing. When purified by a novel high-performance, ion exchange chromatography (HPIEC) method, PF83-7G8-1 induced polyclonal antibodies in rats that immunoprecipitated both 83and 66-kD forms of PF83/AMA-1 from 35S-methionine metabolically labeled parasite extracts. Using HPEC-purified PF83-7G8-1 in combination with a rat monoclonal antibody against the highly conserved carboxy-terminal (CT) region of PF83/AMA-1, we developed a CTcapture-enzyme-linked immunosorbent assay to measure naturally acquired responses against the entire PF83/AMA-l molecule. Analysis of populations from villages in GuineaBissau and in an area of high malarial transmission in Senegal demonstrated a very high prevalence (94100%) of naturally acquired serum IgG responses to PF83/AMA-I. Analysis of these natural responses showed that PF83/AMA-l may be a well-recognized asexual parasite antigen. A statistically significant age-related change in antibody levels to PF83l AMA-1 was observed in Guinea-Bissau. No such correlation was observed in the Senegalese population, although an age-related antibody response was seen for total parasite antigen. No significant correlation was observed between PF83/AMAI responses and the parameters of parasite load and malaria-related fever. Current efforts to develop a vaccine against Plasniodiuriz falciparuiiz target all vertebrate life cycle stages of the parasite for immunointervention. Within the erythrocytic cycle, which is responsible for clinical disease, several asexual blood stage proteins have been characterized and are,putative components for inclusion in a malaria vaccine.'-' The P. falciparuin merozoite 83kilodalton (kD) apical membrane antigen (PF83/ AMA-1) (Thomas AW, unpublished data)'V2 is one of these molecules. A strong basis for the vaccine candidacy of PF83lAMA-1 is the result of work by Deans and others,s who identified a 66-kD P. kizowlesi late-stage apical protein (subsequently called PK66), which was the target of inhibitory monoclonal antibodies (MAbs) in vitro. These antibodies retained their activity as Fab fragments and appeared to inhibit parasite development in a manner independent of schizont maturation: suggesting a functional role for PK66 in erythrocyte invasion. Strong protective responses were induced in rhesus monkeys by immunization with PK66 in combination with exposure to P. krzo~les i . '~ The PF83/AMA-I antigen is an 83-kD analog of PK66 (Thomas AW, unpublished data).', Molecular analysis of PF83/AMA-1 and PK66/AMA-1 has shown that the two species are biologically conserved with regard to late-stage synthesis in mature schizonts and differential subcellular localization within schizonts and free merozoites.Il In addition, initial work suggests that polymorphism in PF83/ AMA1 is limited.I2 Serum antibodies acquired during the course of naturally occumng infec_ _ 730