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 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 (Bt) crystal protein 5B (Cry5B) has anthelmintic properties that could fill this void. Here we develop an Active Pharmaceutical Ingredient or API containing 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 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.

Purified cytosolic crystals from Bacillus thuringiensis as a novel active pharmaceutical ingredient (API)

“…For these and other reasons, using live Bt spore crystal lysates would be challenging from a regulatory and therapeutic vantage point as ingested anthelmintics ( e . g ., oral cures for intestinal nematodes) (14). The use of live spores also results in environmental contamination, which alters the natural bacterial composition and with potential implications for resistance (14).…”

“…g ., oral cures for intestinal nematodes) (14). The use of live spores also results in environmental contamination, which alters the natural bacterial composition and with potential implications for resistance (14). For all these considerations, we developed a novel form of Cry proteins delivered inside of dead bacteria.…”

“…For all these considerations, we developed a novel form of Cry proteins delivered inside of dead bacteria. This novel form is called IBaCC for I nactivated Ba cteria with C ytosolic C rystal(s) (14). In IBaCC, Bt crystals are formed during vegetative growth/stationary phase in sporulation-defective Bt.…”

“…In IBaCC, Bt crystals are formed during vegetative growth/stationary phase in sporulation-defective Bt. The Bt crystals are trapped in the cytosol of the non-sporulated Bacillus bacteria (14). Such Bacilli cells are easily killed by food grade essential oils, while at the same time nematode-active Bt crystals left inside their dead bacterial ghosts fully retain bioactivity (14).…”

Avaliação do potencial nematicida in vitro de formulações comerciais à base de Bacillus spp

“…Alternativas para uso de anti-helmínticos tem sido pesquisadas para reduzir a pressão de seleção sobre os fármacos sintéticos, como por exemplo, o controle biológico, fitoterápicos e sistemas de manejo integrado de pastagens (HOAI, 2020;AMORIM et al, 2021). Bactérias do gênero Bacillus e os metabólitos produzidos por elas se destacam há vários anos no controle parasitário, inclusive de helmintos (HU et al, 2018;LI et al, 2021;PINTO et al, 2017).…”