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

Crystal protein Cry5B as a novel and powerful anthelmintic

Soil-transmitted helminths (STHs), most notably, hookworms, whipworms, and Ascaris, are nematodes that infect more than 1.5 billion of the poorest people and are amongst the leading causes of morbidity worldwide. Only two classes of de-worming drugs (anthelmintics) are available for treatment, and only one is commonly used in mass drug administrations. New anthelmintics are urgently needed to overcome emerging resistance and to produce higher cure rates. Crystal (Cry) proteins, in particular Cry5B, made by Bacillus thuringiensis (Bt) are promising new candidates. Cry5B has excellent anthelmintic properties against many free-living and parasitic nematodes, including in vivo efficacy against multiple STH infections in rodents (Heligomasmidoes polygyrus and Ancylostoma ceylanicum) and in pigs (Ascaris suum). An enormous challenge for STHs, very different from most diseases worked on in the developing world, is the requirement that therapies be very cheap (the people infected are very poor and current drugs costs pennies a dose), massively scalable (over 4 billion people are at risk from infection), and have a long shelf life in harsh environments, that have high temperature and humidity and no cold chain. We will update our progress in several key areas. We will present new data on the in vivo activity of Cry5B against a major human parasite in humans. We will also present data on the whether or not the immune system is required for Cry5B action in vivo. We will also present on our development efforts to produce a deployable version of Cry5B that is cheap, safe, scalable, and stable. These efforts are currently focused on bacterial engineering, expression, and formulation

Terpenes as ‘resistance-busting” anthelmintic drug

There is an urgent need for new therapies for parasitic helminthic diseases affecting 1.5-2 billion people worldwide due to the threat of wide-spread resistance development to existing treatments and due to problems of incomplete efficacies. Terpenes are plant secondary metabolites and major essential oil constituents. Historically, the terpene thymol was successfully used to cure hookworm infections in the 1900’s. Although effective, large doses were needed and thymol treatment had significant side effects. Because free terpenes are absorbed in the stomach, less than 10% of oral terpenes entered the site where the parasites live. To overcome these problems we have developed microparticle encapsulated terpenes and enteric coated terpene capsules. We screened 20 terpenes for anthelmintic activity in vitro against adult stages of the hookworm and whipworm parasitic nematodes Ancylostoma ceylanicum and Trichuris muris. Here we will present results of this work, which shows the promising potential for some terpenes as pan-nematode anthelmintics. This work has allowed us to classify terpenes into at least two groups based on their in vitro killing kinetics. We have also shown that some terpenes are effective against an albendazole-resistant Caenorhabditis elegans strain suggesting that terpenes may play an important role in overcoming helminthic drug resistance. We will also present our work on optimizing lead terpene formulations in vitro and in vivo in animal models of parasitic nematode infection in order to overcome the challenges and realize the potential of “resistance-busting” terpene-based anthelmintic therapies

Collaborating to Cure the Most Common Parasites on the Planet

Soil-transmitted helminths (STHs), most notably, hookworms, whipworms, and Ascaris, are nematodes that infect more than 1.5 billion of the poorest people and are leading causes of morbidity worldwide. Only one class of de-worming drugs (anthelmintic) is commonly used in mass drug administrations. New anthelmintics are urgently needed to overcome emerging resistance and to produce higher cure rates. Crystal (Cry) proteins, in particular Cry5B, made by Bacillus thuringiensis (Bt) are promising new candidates. Cry5B has excellent anthelmintic properties against many free-living and parasitic nematodes, including in vivo efficacy against multiple STH infections in rodents (Heligomasmidoes polygyrus and Ancylostoma ceylanicum) and in pigs (Ascaris suum). An enormous challenge for STHs, very different from most diseases worked on in the developing world, is the requirement that therapies be very cheap (the people infected are very poor and current drugs costs pennies a dose), massively scalable (over 4 billion people are at risk from infection), and have a long shelf life in harsh environments, that have high temperature and humidity and no cold chain. Working together, we have made excellent progress in our development efforts to produce a deployable version of Cry5B that is cheap, safe, scalable, and stable. These efforts are focused on microbiology, bacterial engineering, expression, and formulation. In the process of this work, we have discovered a novel bacterial expression system that meets these key requirements. In addition, we will provide latest information about the broad spectrum of activity of Cry5B against key parasites that make this therapeutic a very attractive alternative from current treatments

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

An Inactivated Bacterium (Paraprobiotic) Expressing \u3ci\u3eBacillus thuringiensis\u3c/i\u3e Cry5B as a Therapeutic for \u3ci\u3eAscaris\u3c/i\u3e and \u3ci\u3eParascaris\u3c/i\u3e 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

Bacillus thuringiensis Cry5B protein as a new pan-hookworm cure

Hookworms are intestinal nematode parasites that infect nearly half a billion people and are globally one of the most important contributors to iron-deficiency anemia. These parasites have significant impacts in developing children, pregnant women and working adults. Of all the soil-transmitted helminths or nematodes (STNs), hookworms are by far the most important, with disease burdens conservatively estimated at four million DALYs (Disability-Adjusted Life Years) and with productivity losses of up to US$139 billion annually. To date, mainly one drug, albendazole is used for hookworm therapy in mass drug administration, which has on average approximately 80% cure rate that is lower ( \u3c 40%) in some places. Given the massive numbers of people needing treatment, the threat of parasite resistance, and the inadequacy of current treatments, new and better cures against hookworms are urgently needed. Cry5B, a pore-forming protein produced by the soil bacterium Bacillus thuringiensis (Bt) has demonstrated good efficacy against Ancylostoma ceylanicum hookworm infections in hamsters. Here we broaden studies of Cry5B to include tests against infections of Ancylostoma caninum hookworms in dogs and against infections of the dominant human hookworm, Necator americanus, in hamsters. We show that Cry5B is highly effective against all hookworm parasites tested in all models. Neutralization of stomach acid improves Cry5B efficacy, which will aid in practical application of Cry5B significantly. Importantly, we also demonstrate that the anti-nematode therapeutic efficacy of Cry5B is independent of the host immune system and is not itself negated by repeated dosing. This study indicates that Bt Cry5B is a pan-hookworm anthelmintic with excellent properties for use in humans and other animals

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

Small Molecule Inhibitors of Metabolic Enzymes Repurposed as a New Class of Anthelmintics

We thank Qi Wang for her technical assistance related to clustering compounds and identifying representatives for screening. This work was supported by National Institute of Allergy and Infectious Diseases (NIAID) grant AI081803 to M.M. The study was also partly supported by NIAID grant AI056189 to R.V.A.Peer reviewedPostprin

Hypoxia and the Hypoxic Response Pathway Protect against Pore-Forming Toxins in C. elegans

Pore-forming toxins (PFTs) are by far the most abundant bacterial protein toxins and are important for the virulence of many important pathogens. As such, cellular responses to PFTs critically modulate host-pathogen interactions. Although many cellular responses to PFTs have been recorded, little is understood about their relevance to pathological or defensive outcomes. To shed light on this important question, we have turned to the only genetic system for studying PFT-host interactions—Caenorhabditis elegans intoxication by Crystal (Cry) protein PFTs. We mutagenized and screened for C. elegans mutants resistant to a Cry PFT and recovered one mutant. Complementation, sequencing, transgenic rescue, and RNA interference data demonstrate that this mutant eliminates a gene normally involved in repression of the hypoxia (low oxygen response) pathway. We find that up-regulation of the C. elegans hypoxia pathway via the inactivation of three different genes that normally repress the pathway results in animals resistant to Cry PFTs. Conversely, mutation in the central activator of the hypoxia response, HIF-1, suppresses this resistance and can result in animals defective in PFT defenses. These results extend to a PFT that attacks mammals since up-regulation of the hypoxia pathway confers resistance to Vibrio cholerae cytolysin (VCC), whereas down-regulation confers hypersusceptibility. The hypoxia PFT defense pathway acts cell autonomously to protect the cells directly under attack and is different from other hypoxia pathway stress responses. Two of the downstream effectors of this pathway include the nuclear receptor nhr-57 and the unfolded protein response. In addition, the hypoxia pathway itself is induced by PFT, and low oxygen is protective against PFT intoxication. These results demonstrate that hypoxia and induction of the hypoxia response protect cells against PFTs, and that the cellular environment can be modulated via the hypoxia pathway to protect against the most prevalent class of weapons used by pathogenic bacteria