Protective Monoclonal Antibodies Define a Distinct, Conserved Epitope on an Apical Complex Exoantigen of Cryptosporidium parvum Sporozoites

Abstract

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-

Novel Bumped Kinase Inhibitors Are Safe and Effective Therapeutics in the Calf Clinical Model for Cryptosporidiosis

“…The propagation of C. parvum (Iowa isolate) in calves and oocyst purification [34][35][36][37], synthesis of BKIs [22,25,27,38], in vitro CpCDPK1 enzyme assay [22,25,38], in vitro determination of C. parvum BKI sensitivity [22,25,38,39], neonatal mouse [36,40] and calf models of C. parvum infection [37,[41][42][43], and pharmacologic measurement of BKI plasma and stool levels and plasma protein binding have all been previously described [27,44]. BKI-1294 and BKI-1553 were synthesized on the pyrazolo [2,3-d] pyrimidine scaffold, while BKI-1517 was synthesized on a 5-aminopyrazole-4-carboxamide scaffold [25] (Figure 1).…”

Characterization and Formulation of Multiple Epitope-Specific Neutralizing Monoclonal Antibodies for Passive Immunization against Cryptosporidiosis

“…Therefore, a panel of second-generation MAbs was produced against immunoaffinity-purified GP25-200 (39) and P23 (42) to determine whether the antigens contain additional neutralizationsensitive epitopes that could be targeted to enhance efficacy. We recently reported that one of the resulting MAbs produced from GP25-200-immunized mice, designated 3E2, elicited the circumsporozoite precipitate (CSP)-like reaction (36,39). This reaction, named after the malarial CSP reaction (7), is characterized by the progressive posterior formation and release of membranous antigen-MAb complexes, after which zoites are rendered noninfective (36,39).…”

“…Therefore, effective neutralization of the infective stages could prevent initiation of the life cycle or interrupt and terminate the cycle in an existing infection. The C. parvum antigens GP25-200 (1,39), CSL (39,40), and P23 (1,23) were selected as targets for the present study. Each antigen is involved in the pathogenesis of infection, expressed on the surface of both infective zoite stages, and conserved on diverse C. parvum isolates (1,23,31,39,40).…”

“…The C. parvum antigens GP25-200 (1,39), CSL (39,40), and P23 (1,23) were selected as targets for the present study. Each antigen is involved in the pathogenesis of infection, expressed on the surface of both infective zoite stages, and conserved on diverse C. parvum isolates (1,23,31,39,40). GP25-200 was originally defined by MAb C4A1 as a sporozoite apical and surface pellicle glycoprotein complex comprised of multiple 25-to 200-kDa species identified in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (1).…”

Stable expression of Cryptosporidium parvum glycoprotein gp40/15 in Toxoplasma gondii

“…These proteins, as well as proteins on the surface of the zoite, facilitate attachment to the cell surface, gliding motility, and subsequent invasion and intracellular development of the parasite. However, unlike those of other apicomplexans, most of the Cryptosporidium surface and apical complex antigens that have been characterized are glycoproteins [9][10][11][12][13][14]. Two of these, gp900 [10] and gp40/15 [11,13,14], are mucin-like glycoproteins, proteins with high percentages of serine and threonine residues that are sites for O-linked glycosylation.…”