Development of optimal passive and active immunization strategies for cryptosporidiosis is dependent on the identification and characterization of functional parasite molecules which can be targeted. Each of a subset of five monoclonal antibodies (mAb) produced against immunoaffinity-isolated C. parvum antigens elicited distinct morphologic changes in sporozoites and merozoites, characterized by progressive formation and eventual release of membranous antigen-mAb precipitates from the sporozoite posterior m g g s MW, Stone AL, Arrowood MJ, Langer RC, Yount PA. Abstract # C46, Thud International Workshop on Pneumocystis, Toxoplarma, Cryptosporidium and Microsporidia (1994) Cleveland, OH; E g g s MW, Stone AL, Yount PA, Arrowood MJ, Langer RC, Bentley DL. Protective MQnQClOnd Antibodies Define a Ckcumspsrozoite-like Glycooproteh Exoantigen of Cryptosporidium parvum Sporozoites and Merozoites, submitted]. These changes were similar to, but more rapid in development and prominent than thoae elicited by therapeutic bovine colostral anti-C. parvum antibody we described previously [8], and resembled the malarial circumsporozoite precipitate (CSP) reaction [3]. Infectivity of sporozoites having undergone the CSP-like reaction was neutralized. Further, mAbs which elicited the CSP-like reaction provided marked passive protection against C. parvum oocyst challenge in neonatal BALB/c mice. The antigen mechanistically involved in the CSP-like reaction was released initially from the apical end of sporozoites. Cytoskeletal inactivation with cytochalasind did not inhibit apical release of the antigen, but did inhibit its posterior translocation. The CSP-like reaction was morphologically and antigenically distinct from P23 trail deposition [2] previously described for motile C. parvum sporozoites. The antigen recognized by mAbs which elicited the CSPlike reaction was localized to sporozoite apical complex organelles, including electrondense granules, by immunoelectron microscopy. In western blots of sporozoites or merozoites, the mAbs recognized multiple 46-230 kDa antigens and a high M, antigen, designated CSL, which variably migrated between 1200-1400 kDa in reducing SDS-

The apicomplexan protozoan parasite Cryptosporidium parvum causes a diarrheal disease in humans and other mammals for which specific therapy and immunoprophylaxis are unavailable. Passive immunization with Abs against whole C. parvum organisms has variable efficacy in immunocompromised or neonatal hosts. Because apical and surface-exposed zoite Ags of the Apicomplexa are critical to infectivity and targets of protective immunity, we examined the ability of mAbs generated against such Ags in C. parvum sporozoites to passively protect against infection and identify biologically relevant parasite molecules. A panel of mAbs was produced against affinity-purified native Ags using sporozoite apical- and surface-reactive mAb C4A1 as binding ligand. One resulting mAb, designated 3E2, elicited prominent morphologic changes in sporozoites and merozoites characterized by rapid and progressive formation, posterior movement, and release of membranous Ag-mAb precipitates. These changes had a striking resemblance to the malarial circumsporozoite precipitate (CSP) reaction. Sporozoite infectivity was completely neutralized after in vitro exposure to 3E2 and the CSP-like reaction. Furthermore, orally administered 3E2 completely prevented or markedly reduced infection in neonatal BALB/c mice. 3E2 bound to apical complex and surface molecules of zoites and was demonstrated in membranous precipitates by immunoelectron microscopy. In Western blots, 3E2 recognized multiple 46 to approximately 770 kDa sporozoite Ags and an approximately 1300-kDa Ag designated CSL, also expressed by merozoites. CSL was characterized as a soluble glycoprotein exoantigen released by infectious sporozoites. Further, CSL was determined to be the molecular species mechanistically involved in the CSP-like reaction by its identification in SDS-PAGE gels and Western blots of purified membranous precipitates. These findings indicate that CSL has a functional role in sporozoite infectivity and is a candidate molecular target for passive or active immunization against cryptosporidiosis.