Role of CSL glycoprotein in infectivity and neutralization of Cryptosporidium parvum sporozoites

Abstract

Cryptosporidiosis, caused by the apicomplexan parasite Cryptosporidium parvum, has become a well recognized diarrheal disease of immunodeficient humans and other mammals throughout the world. Specific therapy and immunoprophylaxis are currently unavailable, but passive immunization with C. parvum-specific monoclonal antibodies (mAbs) has demonstrated efficacy in immunocompromised hosts. In particular, mAbs eliciting the circumsporozoite precipitate (CSP)-like reaction protected against C. parvum infection. The circumsporozoite-like antigen (CSL), an ∼1,300 kDa apical glycoprotein of sporozoites and merozoites, is the molecular species mechanistically bound by mAbs having the ability to elicit the CSP-like reaction. These findings indicated that CSL has a functional role in sporozoite infectivity. In the present study, a quantitative in vitro sporozoite infectivity assay was developed to evaluate neutralizing activity of mAbs. 3E2, a mAb which elicited the CSP-like reaction, demonstrated the greatest level of neutralizing activity against C. parvum infections. Here, I report that CSL has properties consistent with being a sporozoite ligand for epithelial cells. For these studies, CSL was isolated from sporozoites by isoelectric focusing (IEF). The 1,200-1,400 kDa Mᵣ region containing CSL in SDS-PAGE of sporozoites was comprised of 31 species when analyzed by two-dimensional electrophoresis. Eight species were present in IEF-isolated CSL. CSL specifically bound to Caco-2 cells in a dose-dependent, saturable, and self-displaceable manner with high affinity. CSL bound to Caco-2 cells inhibited the attachment and invasion of sporozoites in a dose-dependent manner. Characterization of the epitope recognized by 3E2 in a dot immunoblot indicated a β-glucose component. Sporozoites having undergone the CSP-like reaction after incubation with CSL-reactive 3E2, did not attach to or invade Caco-2 cells. These findings indicated that at least 1 of 8 CSL species isolated by IEF was a sporozoite ligand. The Caco-2 cell receptor for CSL was comprised of 16, 51, and 85 kDa molecules. Further, sporozoites incubated with isolated Caco-2 receptors failed to attach to and invade Caco-2 cells. Finally, CSL bound distinctly to receptors present on calf ileum. Based on these findings, I concluded that CSL ligand function is amenable to targeted disruption by CSL-reactive mAbs and that it is a rational target for immunization against cryptosporidiosis.