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

Abstract

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 (BZ) anthelmintics. However, BZ 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 (Bt) crystal protein 5B (Cry5B) has anthelmintic properties that could fill this void. Here we develop an API (Active Pharmaceutical Ingredient) form of Bt Cry5B compatible with MDA. We expressed Cry5B in asporogenous Bt during vegetative phase, forming cytosolic crystals. These Bacteria with Cytosolic Crystals (BaCC) were rendered inviable (inactivated BaCC or 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 Cry5B IBaCC (the curative single dose is 40 mg/kg) was non-toxic 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

“…We recently developed a new Active Pharmaceutical Ingredient (API) containing Cry5B called IBaCC for inactivated bacterium with cytosolic crystal ( Fig. 3 A ( Li et al, 2020 )). In this form, Cry5B is expressed in a sporulation defective Bt strain under control of a non-sporulating promoter, giving rise to production of a Cry5B crystal inside a vegetative Bt cell.…”

“…Our data show that Cry5B crystals from IBaCC will survive the rumen and that H. contortus adults can ingest Cry5B since they are intoxicated in vitro . How Cry5B is ingested by H. contortus in the abomasum is an area for investigation but there is precedent for a blood feeder accessing Cry5B in vivo with hookworms ( Kalkofen, 1970 ; Cappello et al, 2006 ; Hu et al, 2012 , 2013b , 2018a , 2018b ; Wu et al, 2015 ; Li et al, 2020 ).…”

“…Feces from sheep experimentally mono-infected with H. contortus were shipped overnight from Virginia Tech to the Aroian Lab for these studies. Cry5B IBaCC was prepared as described ( Li et al, 2020 ).…”

“…Cry5B crystals were isolated from Cry5B IBaCC ( Li et al, 2020 ) and incubated in duplicate in cow rumen fluid for 12, 24, or 48 h as described ( Goeser, 2008 ; Goeser et al, 2009 ; Goeser and Combs, 2009 ). Incubations were carried out courtesy of Zinpro Corporation.…”

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

“…Continuing human MDA campaigns and veterinary parasite control strategies would therefore benefit substantially with the addition of new anthelmintics against ascarid parasites with the following requirements: highly and broadly potent against this family of helminths, and safe, inexpensive, and massively scalable for MDA. We have recently described an active pharmaceutical ingredient (API) called IBaCC (see below) containing the Bacillus thuringiensis (Bt) Crystal (Cry) protein Cry5B that is effective against human hookworm infections in rodents and against Haemonchus contortus infections in sheep and that meets all the requirements for MDA [ 31 , 32 ]. Cry5B itself was shown to be active against A. suum in pigs when delivered as a live spore-crystal lysate (SCL) [ 33 ].…”