Identification of a 15-kilodalton surface glycoprotein on sporozoites of Cryptosporidium parvum

Abstract

An immunoglobulin A monoclonal antibody (MAb5C3) was developed against a 15-kDa surface glycoprotein (GP15) of Cryptosporidium parvum sporozoites. Indirect immunofluorescence and colloidal gold immunoelectron microscopy revealed that the antibody reacted with both the sporozoite and merozoite surface plasma membranes. On Western immunoblots, MAb5C3 binding was found to be strongly inhibited when 200 mM N-acetylglucosamine was used as a competing sugar. N-Acetylgalactosamine inhibited binding of the antibody only slightly, whereas glucose, mannose, and galactose failed to inhibit binding. MAb5C3 was found to recognize a similar 15-kDa epitope associated with a Cryptosporidium sp. isolated from guinea pigs. However, MAb5C3 failed to react with any proteins or glycoproteins associated with C. baileyi from chickens, Cryptosporidium sp. (= bovine C. muris) from cattle, C. serpentis from a rat snake, bradyzoites of Besnoitia darlingi from an opossum, sporozoite/oocyst extracts of Caryospora bigenetica from an eastern diamondback rattlesnake, sporozoites of Eimeria nieschulzi and E. papillata from rats and mice, or tachyzoites of Toxoplasma gondii (RH strain). When hybridoma supernatants containing MAb5C3 were administered orally to suckling mice experimentally infected with C. parvum, a 75% reduction in developmental stages was seen histologically at 72 h postinfection and a 67.5% reduction in mean oocyst output was found at 6 days postinfection.

Speculation on whether a vaccine against cryptosporidiosis is a reality or fantasy

“…Six C. parvum sporozoite proteins have been cloned so far. Five were localised to either the sporozoite surface, CP15/60 [62,63], CP15 [64] and P23 [65,66], or to the micronemes, the thrombospondin-related adhesive protein of Cryptosporidium-1 (TRAP-C1) [67] and GP900 [68], while the precise location of the sporozoite cysteine-rich protein (SCRP) (Spano et al, unpublished) is presently under study. CP15/60, CP15 and P23 are low mol.…”

“…polypeptides of 148, 119 and 111 aa, respectively, whose genes were cloned by screening C. parvum expression libraries with mAb or polyclonal antibodies raised against oocyst/sporozoite lysates [63,64,66,69]. Despite the lack of a putative leader peptide for extracellular targeting and of transmembrane or GPI-anchoring signals in the aa sequence of CP15/60, CP15 and P23, immuno¯uorescence analyses suggested that these three proteins are located on the membrane of sporozoites, and in the case of CP15/60 and P23 on the membrane of merozoites [62,65]. Subcellular fractionation shows that P23 can be found in the micronemes of C. parvum sporozoites (Petry et al, unpublished).…”

“…A 15 kDa antigen was recognised by serum/ faecal antibodies from Cryptosporidium-infected humans, BALB/c mice, rabbits, lambs, calves, piglets and goat kids [93±95]. This antigen comigrated with the sporozoite surface antigen designated CP15/60 (see Section 5.1), de®ned by the mAb 5C3 [62]. Oral administration of this anti-CP15/60 IgA mAb to suckling mice provided protection against infection [62].…”

“…This antigen comigrated with the sporozoite surface antigen designated CP15/60 (see Section 5.1), de®ned by the mAb 5C3 [62]. Oral administration of this anti-CP15/60 IgA mAb to suckling mice provided protection against infection [62]. A recent study compared the antigenicity of two dierent recombinant forms of CP15/60, one produced in a prokaryotic expression system and the other in a eukaryotic one.…”

Both CP15 and CP25 are left as trails behind gliding sporozoites of Cryptosporidium parvum (Apicomplexa)

“…Previous studies have identified a 15 kDa surface glycoprotein on sporozoites of C. paruum, which was thought to be sloughed from the surface of sporozoites during gliding [4]. mAbs were recently made to this and other parasite molecules in our laboratory [5], some of which were employed to determine whether CP15 may be a second antigen involved in sporozoite substrate attachment.…”