Neutralizing Monoclonal Antibody Protects Against Cryptosporidium parvum Infection by Inhibiting Sporozoite Attachment and Invasion

Monoclonal antibody (mAb) 3E2, previously reported to elicit the circumsporozoite precipitate (CSP)-like reaction and protect neonatal BALBlc mice against C. parvwn oocyst challenge, recognizes multiple sporozoite antigens ranging from 46-230 kDa, and a variably migrating 1,200-1,400 kDa antigen in western blots [8,9]. The 1,200-1,400 kDa antigen species recognized by mAb 3E2, designated CSL, was determined to be mechanistically involved in the neutralization-associated CSP-like reaction [9]. To further define the mechanism of mAb 3E2-mediated neutralization and the role of CSL, sporozoite attachment and invasion assays were performed in vitro. allowing examination of sporozoite-host cell interactions not readily studied in vivo. MATERIALS AND METHODS. C. parvum oocysts (Iowa isolate) were purified and excysted (45 min, 37 C) in WRC 935 medium containing 0.75% (wlv) taurocholic acid. Sporozoites were purified by filtration through a polycarbonate filter (2 pm pore size). Sporozoites ( I .2 X 103 were incubated (15 min, 37 C, 10% CO,) with 1.0 pg of either mAb 3E2 or an isotype matched control mAb of irrelevant specificity, both derived from hybridoma culture supernatant, in a total volume of 200 pl minimum essential medium (MEM). Sporozoite-mAb preparations were then inoculated onto confluent Caco-2 cell monolayers, in triplicate (1.2 X I(Y sporozoiteslreplicate) and incubated (2 h, 37 C, 10% CO,). Caco-2 cell monolayers had been grown on glass cover slips (12 mm diam) in MEM containing 1% nonessential amino acids and 10% FBS. Cultures were then fed 1 ml medium and at 24 h post-inoculation were washed with PBS, methanol fixed (30 s) and blocked (30 min, 37 C) with PBS containing 3.2% (wlv) fish gelatin and 2% (w/v) BSA (blocking buffer). To detect intracellular stages, cover slip cultures were then incubated (30 min, 37 C) with C. parvum-specific mAb 4B10, which recognizes intracellular stages through 72 h development in culture, washed, incubated with affinity-purified fluoresceinated goat anti-mouse IgGMA containing 0.001% (v/v) Evan’s blue, washed and examined by epifluorescence microscopy. Infection was systematically quantitated by counting intracellular stages present in each cover slip culture, using a grid format to ensure complete coverage of the entire cover slip. The mean numbers of parasites for each treatment group were examined for statistically significant differences by Student’s one-tailed f test. Because CSL is mechanistically involved in the CSP-like reaction, it was purified for further study of its potential role in the infection process. Excysted oocysts were solubilized in lysis buffer (50 mM Tris, 5.0 mM EDTA, 0.1 mM TLCK, 5.0 mM iodoacetamide, 1 .O mM PMSF, 0.01 mM leupeptin, 0.01 mM pepstatin A and 1.0% (wlv) octyl-glucoside) by sonication (4 C) and freeze-thaw, then centrifuged (15,000 X g, 10 min) to remove insoluble particulates. C. parvum molecules in the soluble fraction were then separated by preparative isoelectric focusing (IEF) according to the manufacturer’s protocol (Rotofor, Biorad). Purity of isolated CSL was determined by silver staining the preparation after resolving in 10-2076 and 2-12% gradient SDS-PAGE reducing gels. Immunoreactivity and identity of isolated CSL were determined by western blotting of the preparation resolved in 10-20% and 2-12% SDSPAGE reducing gels [8,9]. Lanes were probed with mAb 3E2 or isotype control mAb (8 pg/ml), followed by affinity-purified phosphataseconjugated goat anti-mouse IgM and phosphatase substrate. To determine if CSL could specifically bind to Caco-2 cells and potentially function as a sporozoite ligand, an in vitro binding assay was performed. Four pg of solubilized 1) IEF-puritied CSL, 2) Percoll density gradient-purified C. parvurn oocyst shells, 3) C. parvum sporozoites, or 4) Heligniosomoides polygyrrts nematodes, were incubated (I5 min, 22 C) with Caco-2 cell monolayers cultured as above. All preparations were ultracentrifuged to remove insoluble particulates (50,000 X g, 30 min) and the soluble fractions extensively dialyzed against PBS prior to incubation with Caco-2 cells. After incubation, monolayers were washed with PBS, methanol fixed, blocked, and incubated (30 min, 37 C) with the following: I ) mAb 3E2 for cultures incubated with CSL or solubilized sporozoites, 2) oocyst shell glycoprotein-specific mAb 2B2 for cultures incubated with solubilized oocyst shells, 3) H. polygyrus-specific mAb Hp8 for cultures incubated with solubilized H. polygyncs, or 4) isotype control mAbs for each culture preparation. Monolayers were then washed, processed and examined by epifluorescence microscopy as above. To determine if CSL binding to Caco-2 cells would affect sporozoite attachment and invasion, the binding assay described above was repeated. After incubation with the preparations, monolayers were inoculated, in triplicate, with purified sporozoites ( I .2 X 10’lcover slip),, incubated (2 h, 37 C, 10% COJ and fed 1 ml medium. At 24 h post-inoculation, monolayers were washed with PBS and methanol fixed. Infection was quantitated by epifluorescence microscopy as above using C. parvumspecific mAb 7B6, which is non-reactive with CSL and recognizes intracellular stages through 72 h of development. RESULTS AND DISCUSSION. Caco-2 cell monolayers inoculated with mAb 3E2-treated sporozoites having undergone the CSP-like reaction contained significantly fewer intracellular stages than monolayers inoculated with isotype control mAb-treated sporozoites (Table 1). These data suggested that mAb 3E2 neutralized infectivity by inhibiting sporozoite attachment and invasion rather than by entering host cells bound to sporozoites and subsequently arresting intracellular development. Both mechanisms of antibody-mediated neutralization have been described for Plasmodium sp. [5,6].