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

Abstract

The coccidian parasite Cryptosporidium parvum causes diarrhea in humans, calves, and other mammals. Neither immunization nor parasite-specific pharmaceuticals that are consistently effective against this organism are available. While polyclonal antibodies against whole C. parvum reduce infection, their efficacy and predictability are suboptimal. We hypothesized that passive immunization against cryptosporidiosis could be improved by using neutralizing monoclonal antibodies (MAbs) targeting functionally defined antigens on the infective stages. We previously reported that the apical complex and surface-exposed zoite antigens CSL, GP25-200, and P23 are critical in the infection process and are therefore rational targets. In the present study, a panel of 126 MAbs generated against affinity-purified CSL, GP25-200, and P23 was characterized to identify the most efficacious neutralizing MAb formulation targeting each antigen. To identify neutralizing MAbs, sporozoite infectivity following exposure to individual MAbs was assessed by enzyme-linked immunosorbent assay. Of 126 MAbs evaluated, 47 had neutralizing activity. These were then evaluated individually in oocystchallenged neonatal mice, and 14 MAbs having highly significant efficacy were identified for further testing in formulations. Epitope specificity assays were performed to determine if candidate MAbs recognized the same or different epitopes.

Characterization of an Intestinal Epithelial Cell Receptor Recognized by theCryptosporidium parvumSporozoite Ligand CSL

“…Studies to investigate this possibility resulted in the recent identification of the CSL ligand (47,77). Our continuing studies have been directed towards an improved understanding of the biology of parasitehost interactions in cryptosporidiosis, as such information is essential to targeted drug discovery, immunization, and other specific modalities for life cycle disruption (72,75,80). Thus, in the present study the host cell receptor for CSL, designated CSL-R, was characterized.…”

“…Clearly, disruption of zoite attachment and invasion events essential to the life cycle of C. parvum would prevent initiation of primary infection or allow termination of existing infection. To this end, our studies have focused on CSL, an ϳ1,300-kDa conserved apical complex glycoprotein expressed by the infective sporozoite and merozoite stages of C. parvum (47,73,77,80). CSL was originally identified by a monoclonal antibody (MAb), designated 3E2, which prevents sporozoite attachment and invasion in vitro and passively protects against C. parvum infection in vivo (77,80).…”

“…To this end, our studies have focused on CSL, an ϳ1,300-kDa conserved apical complex glycoprotein expressed by the infective sporozoite and merozoite stages of C. parvum (47,73,77,80). CSL was originally identified by a monoclonal antibody (MAb), designated 3E2, which prevents sporozoite attachment and invasion in vitro and passively protects against C. parvum infection in vivo (77,80). Consistent with these observations, we subsequently determined that CSL contains a zoite ligand which is involved in attachment to intestinal epithelial cells during the infection process (47).…”

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).…”

Systemic Antibody Responses to the Immunodominant p23 Antigen and p23 Polymorphisms in Children with Cryptosporidiosis in Bangladesh

“…[11][12][13][14] Monoclonal and bovine colostral antibodies to p23 protect against Cryptosporidium parvum challenge in mice and calves, respectively. 11,12,[15][16][17][18] The p23 antigen induces serum, mucosal, humoral, and cell-mediated immune responses in experimentally infected or immunized animals, [19][20][21][22][23][24][25][26] and active immunization with DNA or peptide vaccines targeting p23 has been shown to confer varying degrees of protection in animal models. [27][28][29] The p23 antigen is one of the most immunodominant Cryptosporidium antigens and is consistently recognized by serum from actively infected or previously exposed humans.…”