Cryptosporidium spp is a ubiquitous parasite that has long been recognized as a frequent cause of protozoal diarrhea in humans. While infections in immunocompetent hosts are usually self-limiting, immunocompromised individuals can develop severe, chronic, and life-threatening illness. Vaccine development or immunotherapy that prevents disease or reduces the severity of infection is a relevant option since efficacious drug treatments are lacking. In particular, children in developing countries might benefit the most from a vaccine since cryptosporidiosis in early childhood has been reported to be associated with subsequent impairment in growth, physical fitness, and intellectual capacity. In this review, immunotherapies that have been used clinically are described as well as experimental vaccines and their evaluation in vivo.

Glycoproteins expressed by Cryptosporidium parvum are immunogenic in infected individuals but the nature of the epitopes recognised in C. parvum glycoproteins is poorly understood. Since a known immunodominant antigen of Cryptosporidium, the 17. kDa glycoprotein, has previously been shown to bind to lectins that recognise the Tn antigen (GalNAcα1-Ser/Thr-R), a large number of glycopeptides with different Tn valency and presentation were prepared. In addition, glycopeptides were synthesised based on a 40. kDa cryptosporidial antigen, a polymorphic surface glycoprotein with varying numbers of serine residues, to determine the reactivity with sera from C. parvum-infected humans. These glycopeptides and non-glycosylated peptides were used to generate a glycopeptide microarray to allow screening of sera from C. parvum-infected individuals for the presence of IgM and IgG antibodies. IgG but not IgM in sera from C. parvum-infected individuals bound to multivalent Tn antigen epitopes presented on glycopeptides, suggesting that glycoproteins from C. parvum that contain the Tn antigen induce immune responses upon infection. In addition, molecular differences in glycosylated peptides (e.g. substituting Ser for Thr) as well as the site of glycosylation had a pronounced effect on reactivity. Lastly, pooled sera from individuals infected with either Toxoplasma or Plasmodium were also tested against the modified Cryptosporidium peptides and some sera showed specific binding to glycopeptide epitopes. These studies reveal that specific anti-glycopeptide antibodies that recognise the Tn antigen may be useful diagnostically and in defining the roles of parasite glycoconjugates in infections.

Cryptosporidium parasites are a major cause of diarrhea and malnutrition in the developing world, a frequent cause of waterborne disease in the developed world, and a potential bioterrorism agent. Currently, available treatment is limited, and Cryptosporidium drug discovery remains largely unsuccessful. As a result, the pharmacokinetic properties required for in vivo efficacy have not been established. We have been engaged in a Cryptosporidium drug discovery program targeting IMP dehydrogenase (CpIMPDH). Here, we report the activity of eight potent and selective inhibitors of CpIMPDH in the interleukin-12 (IL-12) knockout mouse model, which mimics acute human cryptosporidiosis. Two compounds displayed significant antiparasitic activity, validating CpIMPDH as a drug target. The best compound, P131 (250 mg/kg of body weight/day), performed equivalently to paromomycin (2,000 mg/kg/day) when administered in a single dose and better than paromomycin when administered in three daily doses. One compound, A110, appeared to promote Cryptosporidium infection. The pharmacokinetic, uptake, and permeability properties of the eight compounds were measured. P131 had the lowest systemic distribution but accumulated to high concentrations within intestinal cells. A110 had the highest systemic distribution. These observations suggest that systemic distribution is not required, and may be a liability, for in vivo antiparasitic activity. Intriguingly, A110 caused specific alterations in fecal microbiota that were not observed with P131 or vehicle alone. Such changes may explain how A110 promotes parasitemia. Collectively, these observations suggest a blueprint for the development of anticryptosporidial therapy.

BMS906024, a γ-secretase inhibitor that blocks Notch signaling, was previously shown to inhibit Cryptosporidium parvum growth in vitro. A structure–activity relationship (SAR) analysis of BMS906024 reported herein demonstrates the importance of the stereochemistry of the C-3 benzodiazepine and the succinyl β-substituent. However, concomitant removal of the succinyl α-substituent and switching the primary amide with secondary amides was tolerated. For example, 32 (SH287) inhibited C. parvum growth in HCT-8 host cells with an EC50 = 6.4 nM and an EC90 = 16 nM; however, blocking C. parvum growth with BMS906024 derivatives was correlative with inhibition of Notch signaling, highlighting that additional SAR analysis will be needed to separate these two activities.

Cryptosporidium spp. are opportunistic protozoan parasites that infect epithelial cells of the small intestine and cause diarrheal illness in both immunocompetent and immunodeficient individuals. These infections may be more severe in immunocompromised individuals and young children, especially in children under 2 in developing countries. The parasite has a global distribution and is an important cause of childhood diarrhea where it may result in cognitive impairment and growth deficits. Current therapies are limited with nitazoxanide being the only FDA-approved drug. However, it is not efficacious in immunocompromised patients. Additionally, there are no vaccines for cryptosporidiosis available. While acquired immunity is needed to clear Cryptosporidium parasites completely, innate immunity and early responses to infection are important in keeping the infection in check so that adaptive responses have time to develop. Infection is localized to the epithelial cells of the gut. Therefore, host cell defenses are important in the early response to infection and may be triggered through toll receptors or inflammasomes which induce a number of signal pathways, interferons, cytokines, and other immune mediators. Chemokines and chemokine receptors are upregulated which recruit immune cells such neutrophils, NK cells, and macrophages to the infection site to help in host cell defense as well as dendritic cells that are an important bridge between innate and adaptive responses. This review will focus on the host cell responses and the immune responses that are important in the early stages of infection.

The Cryptosporidium parvum acidic ribosomal protein P2 (CpP2) is an important immunodominant marker in C. parvum infection. In this study, the CpP2 antigen was evaluated as a vaccine candidate using a DNA vaccine model in adult C57BL/6 IL-12 knockout (KO) mice, which are susceptible to C. parvum infection. Our data show that subcutaneous immunization in the ear with DNA encoding CpP2 (CpP2-DNA) cloned into the pUMVC4b vector induced a significant anti-CpP2 IgG antibody response that was predominantly of the IgG1 isotype. Compared to control KO mice immunized with plasmid alone, CpP2-immunized mice demonstrated specific in vitro spleen cell proliferation as well as enhanced IFN-γ production to recombinant CpP2. Further, parasite loads in CpP2 DNA-immunized mice were compared to control mice challenged with C. parvum oocysts. Although a trend in reduction of infection was observed in the CpP2 DNA-immunized mice, differences between groups were not statistically significant. These results suggest that a DNA vaccine encoding the C. parvum P2 antigen is able to provide an effective means of eliciting humoral and cellular responses and has the potential to generate protective immunity against C. parvum infection but may require using alternative vectors or adjuvant to generate a more potent and balanced response.

Cryptosporidium parvum (Cp) is a potential biowarfare agent and major cause of diarrhea and malnutrition. This protozoan parasite relies on inosine 5′-monophosphate dehydrogenase (IMPDH) for the production of guanine nucleotides. A CpIMPDH-selective N-aryl-3,4-dihydro-3-methyl-4-oxo-1- phthalazineacetamide inhibitor was previously identified in a high throughput screening campaign. Herein we report a structure-activity relationship study for the phthalazinone-based series that resulted in the discovery of benzofuranamide analogs that exhibit low nanomolar inhibition of CpIMPDH. In addition, the antiparasitic activity of select analogs in a Toxoplasma gondii model of C. parvum infection is also presented.

In a previous study, we observed an increase in the severity of cryptosporidial infection corresponding to decreased levels of short-chain fatty acids (SCFAs). Therefore, we decided to examine the effect of SCFAs on Cryptosporidium growth in human ileocecal adenocarcinoma (HTC-8) cells. HTC-8 cells were infected with 1 × 105 C. parvum oocysts. After 48 h of incubation with selected SCFAs, cells were fixed and labeled with monoclonal antibody directed to all intracellular stages, and the number of parasites was quantitated using a fluorescent microscope. Acetate, butyrate, propionate and valproate significantly inhibited growth, with an EC50 between 4 and 10 mM. Additionally, when combined, butyrate, acetate and propionate showed increased efficacy. Butyrate also inhibited growth when incubated with sporozoites prior to infection of host cell monolayers. In addition, we looked at possible mechanisms of action of inhibition. A combination of C. parvum infection and butyrate treatment led to increases in apoptosis and certain inflammatory cytokines. We conclude that acetate, propionate and butyrate have direct inhibitory activities in host cells against C. parvum, and butyrate can also affect sporozoite infectivity directly. While not preventing infection, SCFAs may help in keeping the infection low or in check.

Cryptosporidium spp. are opportunistic protozoan parasites that infect epithelial cells of the small intestine, causing diarrheal illness in humans. Differences in severity may be due to the immunological status of the host, malnutrition or prior exposure but may also be due to differences in the host gut flora. We examined changes in bacterial flora following antibiotic treatment to determine how cryptosporidial infections and gut integrity were affected by alterations in the microbiome. DNA was extracted from fecal and intestinal samples during peak infection. V4 region amplicons were generated and sequenced using 16sRNA on an Illumina MiSeq. Species evenness and richness were estimated using the Shannon diversity index. There was a significant decrease in anaerobes and overgrowth of Enterobacteriaceae in mice treated with cloxacillin. We also examined levels of short-chain fatty acids in fecal samples. There was a significant decrease in acetate, propionate, and butyrate in these same mice. Concurrent with the shift in bacterial infection was a significant increase in severity of cryptosporidial infection and increase in gut permeability. Treatment with other antibiotics significantly altered the microbiome but did not change the infection, suggesting that specific alterations in the host microbiome allow for more favorable growth of the parasite.