Deborah A. Schaefer

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 oocyst-challenged 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. Formulations of two or three neutralizing MAbs, each recognizing distinct epitopes, were then evaluated. A formulation of MAbs 3E2 (anti-CSL [αCSL]), 3H2 (αGP25-200), and 1E10 (αP23) provided highly significant additive efficacy over that of either individual MAbs or combinations of two MAbs and reduced intestinal infection by 86 to 93%. These findings indicate that polyvalent neutralizing MAb formulations targeting epitopes on defined antigens may provide optimal passive immunization against cryptosporidiosis.

Efficacy of Monoclonal Antibodies against Defined Antigens for Passive Immunotherapy of Chronic Gastrointestinal Cryptosporidiosis

ABSTRACT Cryptosporidium parvum is an important cause of diarrhea in humans and calves and can persistently infect immunocompromised hosts. Presently, there are no consistently effective parasite-specific drugs for cryptosporidiosis. We hypothesized that neutralizing monoclonal antibodies (MAbs) targeting the apical complex and surface antigens CSL, GP25-200, and P23 could passively immunize against cryptosporidiosis. We recently reported that a formulation of MAbs 3E2 (anti-CSL), 3H2 (anti-GP25-200), and 1E10 (anti-P23) provided significant additive prophylactic efficacy over that of the individual MAbs in neonatal ICR mice. In the present study, these MAbs were evaluated for therapeutic efficacy against persistent infection in adult gamma interferon-depleted SCID mice. 3E2 demonstrated the most significant and consistent therapeutic effect, reducing intestinal infection in two experiments. In one experiment, 3E2 plus 3H2 and 3E2 plus 3H2 plus 1E10 also significantly reduced infection; however, no significant increase in efficacy over 3E2 alone was apparent. The results indicate that anti-CSL MAb 3E2 has highly significant efficacy in reducing, but not eliminating, persistent C. parvum infection.

Characterization of an Intestinal Epithelial Cell Receptor Recognized by the Cryptosporidium parvum Sporozoite Ligand CSL

ABSTRACT The protozoan parasite Cryptosporidium parvum is a leading cause of diarrhea in humans and neonatal calves. The absence of approved parasite-specific drugs, vaccines, and immunotherapies for cryptosporidiosis relates in part to limited knowledge on the pathogenesis of zoite attachment and invasion. We recently reported that the C. parvum apical complex glycoprotein CSL contains a zoite ligand for intestinal epithelial cells which is defined by monoclonal antibody (MAb) 3E2. In the present study, the host cell receptor for CSL was characterized. For these studies, a panel of epithelial and mesenchymal cell lines was examined for permissiveness to C. parvum and the ability to bind CSL. Cells of epithelial origin were significantly more permissive and bound significantly greater quantities of CSL than cells of mesenchymal origin. Caco-2 intestinal cells were selected from the epithelial panel for further characterization of the CSL receptor. Immunoelectron microscopy demonstrated that CSL bound initially to the surface of Caco-2 cells and was rapidly internalized. The molecule bound by CSL was identified as an 85-kDa Caco-2 cell surface protein by radioimmunoprecipitation and CSL affinity chromatography. Sporozoite incubation with the isolated 85-kDa protein reduced binding of MAb 3E2. Further, attachment and invasion were significantly inhibited when sporozoites were incubated with the 85-kDa protein prior to inoculation onto Caco-2 cells. These observations indicate that the 85-kDa protein functions as a Caco-2 cell receptor for CSL. CSL also bound specifically to intestinal epithelium from calves, indicating receptor expression in a second important host species. Molecular characterization of the CSL receptor may lead to novel avenues for disrupting ligand-receptor interactions in the pathogenesis of C. parvum infection.

Cathelicidin-like Helminth Defence Molecules (HDMs) Absence of Cytotoxic, Anti-microbial and Anti-protozoan Activities Imply a Specific Adaptation to Immune Modulation

Host defence peptides (HDPs) are expressed throughout the animal and plant kingdoms. They have multifunctional roles in the defence against infectious agents of mammals, possessing both bactericidal and immune-modulatory activities. We have identified a novel family of molecules secreted by helminth parasites (helminth defence molecules; HDMs) that exhibit similar structural and biochemical characteristics to the HDPs. Here, we have analyzed the functional activities of four HDMs derived from Schistosoma mansoni and Fasciola hepatica and compared them to human, mouse, bovine and sheep HDPs. Unlike the mammalian HDPs the helminth-derived HDMs show no antimicrobial activity and are non-cytotoxic to mammalian cells (macrophages and red blood cells). However, both the mammalian- and helminth-derived peptides suppress the activation of macrophages by microbial stimuli and alter the response of B cells to cytokine stimulation. Therefore, we hypothesise that HDMs represent a novel family of HDPs that evolved to regulate the immune responses of their mammalian hosts by retaining potent immune modulatory properties without causing deleterious cytotoxic effects.

Phospholipases and Cationic Peptides Inhibit Cryptosporidium parvum Sporozoite Infectivity by Parasiticidal and Non-Parasiticidal Mechanisms

abstract:  The apicomplexan parasite Cryptosporidium parvum is an important cause of diarrhea in humans and cattle, and it can persistently infect immunocompromised hosts. No consistently effective parasite-specific pharmaceuticals or immunotherapies for control of cryptosporidiosis are presently available. The innate immune system represents the first line of host defense against a range of infectious agents, including parasitic protozoa. Several types of antimicrobial peptides and proteins, collectively referred to herein as biocides, constitute a major effector component of this system. In the present study, we evaluated lactoferrin, lactoferrin hydrolysate, 5 cationic peptides (lactoferricin B, cathelicidin LL37, indolicidin, &bgr;-defensin 1, &bgr;-defensin 2), lysozyme, and 2 phospholipases (phospholipase A2, and phosphatidylinositol-specific phospholipase C) for anti-cryptosporidial activity. The biocides were evaluated either alone or in combination with 3E2, a monoclonal antibody (MAb) against C. parvum that inhibits sporozoite attachment and invasion. Sporozoite viability and infectivity were used as indices of anti-cryptosporidial activity in vitro. All biocides except lactoferrin had a significant effect on sporozoite viability and infectivity. Lactoferrin hydrolysate and each of the 5 cationic peptides were highly parasiticidal and strongly reduced sporozoite infectivity. While each phospholipase also had parasiticidal activity, it was significantly less than that of lactoferrin hydrolysate and each of the cationic peptides. However, each phospholipase reduced sporozoite infectivity comparably to that observed with lactoferrin hydrolysate and the cationic peptides. Moreover, when 3 of the cationic peptides (cathelicidin LL37, &bgr;-defensin 1, and &bgr;-defensin 2) were individually combined with MAb 3E2, a significantly greater reduction of sporozoite infectivity was observed over that by 3E2 alone. In contrast, reduction of sporozoite infectivity by a combination of either phospholipase with MAb 3E2 was no greater than that by 3E2 alone. These collective observations suggest that cationic peptides and phospholipases neutralize C. parvum by mechanisms that are predominantly either parasiticidal or non-parasiticidal, respectively.

Antibodies fused to innate immune molecules reduce initiation of Cryptosporidium parvum infection in mice.

ABSTRACT At present no completely effective treatments are available for Cryptosporidium parvum infections in humans and livestock. Based on previous data showing the neutralizing potential of a panel of monoclonal antibodies developed against C. parvum, and based on the fact that innate immune peptides and enzymes have anticryptosporidial activity, we engineered several of these antibodies into antibody-biocide fusion proteins. We hypothesized that the combination of high-affinity antibody targeting with innate immune molecule-mediated killing would result in a highly effective new antiprotozoal agent. To test this hypothesis, we expressed antibody-biocide fusion proteins in a mammalian cell culture system and used the resulting products for in vitro and in vivo efficacy experiments. Antibody-biocide fusion proteins efficiently bound to, and destroyed, C. parvum sporozoites in vitro through a membrane-disruptive mechanism. When antibody-biocide fusion proteins were administered orally to neonatal mice in a prophylactic model of cryptosporidiosis, the induction of infection was reduced by as much as 81% in the mucosal epithelium of the gut, as determined on the basis of histopathological scoring of infectious stages. Several versions of antibody fusion proteins that differed in antigen specificity and in the biocide used had strong inhibitory effects on the initiation of infection. The results lay the groundwork for the development of a new class of antimicrobials effective against Cryptosporidium.

ASSOCIATION OF IL-10 EXPRESSION BY MUCOSAL LYMPHOCYTES WITH INCREASED EXPRESSION OF CRYPTOSPORIDIUM PARVUM EPITOPES IN INFECTED EPITHELIUM

The objective of this study was to determine whether changes in the ileal intraepithelial lymphocyte (IEL) phenotype and function occurred prior to development of diarrhea in Cryptosporidium parvum–infected calves. Calves were orally inoculated with 108 oocysts and maintained in enteric pathogen-free conditions until their use in experiments. Age-matched uninfected calves were used for comparisons. Ileal IELs were isolated and phenotyped to determine whether changes in lymphocyte population dynamics had occurred by 3 days postinoculation (PI). Ex vivo reverse transcriptase–polymerase chain reaction of messenger ribonucleic acid (mRNA) from IELs from infected calves was compared with controls to determine whether changes in cytokine expression had occurred by 3 days PI. No significant changes in lymphocyte population dynamics were documented; however, IELs isolated from 4 out of 8 infected calves, but not from 8 out of 8 control calves, expressed mRNA for interleukin-10 (IL-10). IL-10 expression by IELs was associated with the expression of a significantly larger (P < 0.001) proportion (0.75) of monoclonal antibody–defined C. parvum epitopes within infected ileal epithelium, as compared with a much smaller proportion (0.30) of epitopes with IL-10− lymphocytes. The results suggest that a temporal association exists between the expression of IL-10 by ileal IELs and the expression of C. parvum antigens in infected calf epithelium prior to development of cryptosporidiosis.

Bumped-Kinase Inhibitors for Cryptosporidiosis Therapy

Bumped kinase inhibitors (BKIs) of Cryptosporidium parvum calcium-dependent protein kinase 1 (CpCDPK1) are leading candidates for treatment of cryptosporidiosis-associated diarrhea. Potential cardiotoxicity related to anti-human ether-à-go-go potassium channel (hERG) activity of the first-generation anti-Cryptosporidium BKIs triggered further testing for efficacy. A luminescence assay adapted for high-throughput screening was used to measure inhibitory activities of BKIs against C. parvum in vitro. Furthermore, neonatal and interferon γ knockout mouse models of C. parvum infection identified BKIs with in vivo activity. Additional iterative experiments for optimum dosing and selecting BKIs with minimum levels of hERG activity and frequencies of other safety liabilities included those that investigated mammalian cell cytotoxicity, C. parvum proliferation inhibition in vitro, anti-human Src inhibition, hERG activity, in vivo pharmacokinetic data, and efficacy in other mouse models. Findings of this study suggest that fecal concentrations greater than parasite inhibitory concentrations correlate best with effective therapy in the mouse model of cryptosporidiosis, but a more refined model for efficacy is needed.

ANTIBODY FUSIONS REDUCE ONSET OF EXPERIMENTAL CRYPTOSPORIDIUM PARVUM INFECTION IN CALVES

Abstract Cryptosporidium parvum is one of the main causes of diarrhea in neonatal calves resulting in significant morbidity and economic losses for producers worldwide. We have previously demonstrated efficacy of a new class of antimicrobial antibody fusions in a neonatal mouse model for C. parvum infection. Here, we extend efficacy testing of these products to experimental infection in calves, the principal target species. Neonatal calves were challenged with C. parvum oocysts and concomitantly treated with antibody–biocide fusion 4H9-G1-LL37 over the course of four days. This resulted in reduced severity of the disease when compared to control animals. Overall clinical health parameters showed significant improvement in treated animals. Oocyst shedding was reduced in treated when compared to control animals. Control of oocyst shedding is a prerequisite for breaking the cycle of re-infection on dairy farms. Antibody–biocide fusion products thus have the potential to reduce the impact of the infection in both individual animals and in the herd.

Antigenic differences within the Cryptosporidium hominis and Cryptosporidium parvum surface proteins P23 and GP900 defined by monoclonal antibody reactivity

The biological basis for the specificity of host infectivity patterns of Cryptosporidium spp., in particular C. hominis and C. parvum, has yet to be fully elucidated. Comparison of the C. parvum and C. hominis P23 and GP900 predicted amino acid sequences revealed 3 differences in P23 and 4 and 17 differences in GP900 domains 1 and 5, respectively. Using monoclonal antibodies developed against the surface (glyco)proteins P23 and GP900 of the C. parvum Iowa isolate, solubilized glycoprotein from three C. hominis isolates was screened for reactivity using Western immunoblots. One of ten P23 MAbs and three of 21 GP900 MAbs were not reactive with any of the three C. hominis isolates. The non-reactive P23 MAb binds to a peptide epitope, while the non-reactive GP900 MAbs bind to either carbohydrate/carbohydrate-dependent or peptide epitopes of C. parvum. These results demonstrate phenotypic differences between C. hominis and C. parvum within two (glyco)proteins that are involved in parasite gliding motility and attachment/invasion.