An efficient method for viable cryopreservation and recovery of hookworms and other gastrointestinal nematodes in the laboratory.

Hookworms (genera Ancylostoma and Necator) are amongst the most prevalent and important parasites of humans globally. These intestinal parasites ingest blood, resulting in anemia, growth stunting, malnutrition, and adverse pregnancy outcomes. They are also critical parasites of dogs and other animals. In addition, hookworms and hookworm products are being explored for their use in treatment of autoimmune and inflammatory diseases. There is thus a significant and growing interest in these mammalian host-obligate parasites. Laboratory research is hampered by the lack of good means of cryopreservation and recovery of parasites. Here, we describe a robust method for long-term (≥3 year) cryopreservation and recovery of both Ancylostoma and Necator hookworms that is also applicable to two other intestinal parasites that passage through the infective L3 stage, Strongyloides ratti and Heligmosomoides polygyrus bakeri. The key is a revised recovery method, in which cryopreserved L1s are thawed and raised to the infective L3 stage using activated charcoal mixed with uninfected feces from a permissive host. This technique will greatly facilitate research on and availability of gastrointestinal parasitic nematodes with great importance to global health, companion animal health, and autoimmune/inflammatory disease therapies.

An efficient method for viable cryopreservation of hookworms and other gastrointestinal nematodes in the laboratory.

Hookworms (genera Ancylostoma and Necator ) are amongst of the most prevalent and important parasites of humans globally. These intestinal parasites ingest blood, resulting in anemia, growth stunting, malnutrition, and adverse pregnancy outcomes. They are also critical parasites of dogs and other animals. In addition, hookworms and hookworm products are being explored for their use in treatment of autoimmune and inflammatory diseases. There is thus a significant and growing interest in these mammalian host-obligate parasites. Laboratory research is hampered by the lack of good means of cryopreservation. Here, we describe a robust method for long-term (≥3 year) cryoprotection and recovery of both Ancylostoma and Necator hookworms that is also applicable to two other intestinal parasites that passages through the infective third larval stage, Strongyloides ratti and H eligmosomoides polygyrus bakeri . The key is the use cryo-preserved first-staged larvae raised to the infective third larval stage using activated charcoal mixed with uninfected feces from a permissive host. This technique will greatly facilitate research on and availability of gastrointestinal parasitic nematodes with great importance to global health, companion animal health, and autoimmune/inflammatory disease therapies.

Efficient and Scalable Process to Produce Novel and Highly Bioactive Purified Cytosolic Crystals from Bacillus thuringiensis.

Bacillus thuringiensis (Bt) is a Gram-positive soil bacterium that is widely and safely applied in the environment as an insecticide for combatting insect pests that damage crops or are disease vectors. Dominant active ingredients made by Bt are insect-killing crystal (Cry) proteins released as crystalline inclusions upon bacterial sporulation. Some Bt Cry proteins, e.g., Cry5B (formally Cry5Ba1), target nematodes (roundworms) and show exceptional promise as anthelmintics (cures for parasitic nematode diseases). We have recently described inactivated bacteria with cytosolic crystal(s) (IBaCC) in which bioactive Bt Cry crystals (containing Cry5B) are fully contained within the cytosol of dead bacterial ghosts. Here, we demonstrate that these IBaCC-trapped Cry5B crystals can be liberated and purified away from cellular constituents, yielding purified cytosolic crystals (PCC). Cry5B PCC contains ~95% Cry5B protein out of the total protein content. Cry5B PCC is highly bioactive against parasitic nematode larvae and adults in vitro. Cry5B PCC is also highly active in vivo against experimental human hookworm and Ascaris infections in rodents. The process was scaled up to the 100-liter scale to produce PCC for a pilot study to treat two foals infected with the ascarid Parascaris spp. Single-dose Cry5B PCC brought the fecal egg counts of both foals to zero. These studies describe the process for the scalable production of purified Bt crystals and define a new and attractive pharmaceutical ingredient form of Bt Cry proteins. IMPORTANCE Bacillus thuringiensis crystal proteins are widely and safely used as insecticides. Recent studies have shown they also can cure gastrointestinal parasitic worm (nematode) infections when ingested. However, reproducible, scalable, and practical techniques for purifying these proteins have been lacking. Here, we address this severe limitation and present scalable and practical methods for large-scale purification of potently bioactive B. thuringiensis crystals and crystal proteins. The resultant product, called purified cytosolic crystals (PCC), is highly compatible with ingestible drug delivery and formulation. Furthermore, there are growing applications in agriculture and insect control where access to large quantities of purified crystal proteins is desirable and where these methods will find great utility.

Yeast Particle Encapsulation of Scaffolded Terpene Compounds for Controlled Terpene Release.

Terpenes are naturally occurring compounds produced by plants that are of great commercial interest in the food, agricultural, cosmetic, and pharmaceutical industries due to their broad spectra of antibacterial, antifungal, anthelmintic, membrane permeation enhancement, and antioxidant biological activities. Applications of terpenes are often limited by their volatility and the need for surfactants or alcohols to produce stable, soluble (non-precipitated) products. Yeast particles (YPs) are hollow, porous microspheres that have been used for the encapsulation of terpenes (YP terpenes) by passive diffusion of terpenes through the porous YP cell walls. We here report the development of a second generation YP encapsulated terpene technology that incorporates the stimuli-responsive control of terpene release using biodegradable pro-terpene compounds (YP pro-terpenes). YP terpenes and YP pro-terpenes were both produced, in which high levels of carvacrol, eugenol, thymol and geraniol were encapsulated. The YP pro-terpenes show higher encapsulation stability than YP terpenes due to pro-terpenes being non-volatile solids at room temperature and stable in suspensions at neutral pH. YP pro-terpenes and YP terpenes were evaluated for biological activity in antibacterial, antifungal and anthelmintic assays. The YP pro-terpenes retained the full biological activity of the parent terpene compound.

An inactivated bacterium (paraprobiotic) expressing Bacillus thuringiensis Cry5B as a therapeutic for Ascaris and Parascaris spp. infections in large animals.

Ascaris and Parascaris are important parasites in the family Ascarididae, large, ubiquitous intestinal-dwelling nematodes infecting all classes of vertebrates. Parasitic nematode drug resistance in veterinary medicine and drug recalcitrance in human medicine are increasing worldwide, with few if any new therapeutic classes on the horizon. Some of these parasites are zoonotic, e.g., Ascaris is passed from humans to pigs and vice versa. The development of new therapies against this family of parasites would have major implications for both human and livestock health. Here we tested the therapeutic ability of a paraprobiotic or dead probiotic that expresses the Bacillus thuringiensis Cry5B protein with known anthelmintic properties, against zoonotic Ascaris suum and Parascaris spp. This paraprobiotic, known as IBaCC, intoxicated A. suum larvae in vitro and was highly effective in vivo against intestinal A. suum infections in a new mouse model for this parasite. Fermentation was scaled up to 350 l to treat pigs and horses. Single dose Cry5B IBaCC nearly completely cleared A. suum infections in pigs. Furthermore, single dose Cry5B IBaCC drove fecal egg counts in Parascaris-infected foals to zero, showing at least parity with, and potential superiority to, current efficacy of anthelmintics used against this parasite. Cry5B IBaCC therefore represents a new, paraprobiotic One Health approach towards targeting Ascarididae that is safe, effective, massively scalable, stable, and useful in human and veterinary medicine in both the developed and developing regions of the world.

Recombinant Paraprobiotics as a New Paradigm for Treating Gastrointestinal Nematode Parasites of Humans.

Gastrointestinal nematodes (GINs) of humans, e.g., hookworms, negatively impact childhood growth, cognition, nutrition, educational attainment, income, productivity, and pregnancy. Hundreds of millions of people are targeted with mass drug administration (MDA) of donated benzimidazole anthelmintics. However, benzimidazole efficacy against GINs is suboptimal, and reduced/low efficacy has been seen. Developing an anthelmintic for human MDA is daunting: it must be safe, effective, inexpensive, stable without a cold chain, and massively scalable. Bacillus thuringiensis crystal protein 5B (Cry5B) has anthelmintic properties that could fill this void. Here, we developed an active pharmaceutical ingredient (API) containing B. thuringiensis Cry5B compatible with MDA. We expressed Cry5B in asporogenous B. thuringiensis during vegetative phase, forming cytosolic crystals. These bacteria with cytosolic crystals (BaCC) were rendered inviable (inactivated BaCC [IBaCC]) with food-grade essential oils. IBaCC potency was validated in vitro against nematodes. IBaCC was also potent in vivo against human hookworm infections in hamsters. IBaCC production was successfully scaled to 350 liters at a contract manufacturing facility. A simple fit-for-purpose formulation to protect against stomach digestion and powdered IBaCC were successfully made and used against GINs in hamsters and mice. A pilot histopathology study and blood chemistry workup showed that five daily consecutive doses of 200 mg/kg body weight Cry5B IBaCC (the curative single dose is 40 mg/kg) was nontoxic to hamsters and completely safe. IBaCC is a safe, inexpensive, highly effective, easy-to-manufacture, and scalable anthelmintic that is practical for MDA and represents a new paradigm for treating human GINs.

A new paraprobiotic-based treatment for control of Haemonchus contortus in sheep.

Haemonchus contortus is a critical parasite of goats and sheep. Infection by this blood-feeding gastrointestinal nematode (GIN) parasite has significant health consequences, especially in lambs and kids. The parasite has developed resistance to virtually all known classes of small molecule anthelmintics used to treat it, giving rise in some areas to multidrug resistant parasites that are very difficult to control. Thus, new anthelmintics are urgently needed. Bacillus thuringiensis (Bt) crystal protein 5B (Cry5B), a naturally occurring protein made by a bacterium widely and safely used around the world as a bioinsecticide, represents a new non-small molecule modality for treating GINs. Cry5B has demonstrated anthelmintic activities against parasites of monogastric animals, including some related to those that infect humans, but has not yet been studied in a ruminant. Here we show that H. contortus adults are susceptible to Cry5B protein in vitro. Cry5B produced in its natural form as a spore-crystal lysate against H. contortus infections in goats had no significant efficacy. However, a new Active Pharmaceutical Ingredient (API) paraprobiotic form of Cry5B called IBaCC (Inactivated Bacterium with Cytosolic Crystals), in which Cry5B crystals are encapsulated in dead Bt cell wall ghosts, showed excellent efficacy in vitro against larval stages of H. contortus and relative protein stability in bovine rumen fluid. When given to sheep experimentally infected with H. contortus as three 60 mg/kg doses, Cry5B IBaCC resulted in significant reductions in fecal egg counts (90%) and parasite burdens (72%), with a very high impact on female parasites (96% reduction). These data indicate that Cry5B IBaCC is a potent new treatment tool for small ruminants in the battle against H. contortus.