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. 2018 Aug;8(2):287-294.
doi: 10.1016/j.ijpddr.2018.05.001. Epub 2018 May 4.

Bacillus Thuringiensis Cry5B Protein as a New Pan-Hookworm Cure

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Free PMC article

Bacillus Thuringiensis Cry5B Protein as a New Pan-Hookworm Cure

Yan Hu et al. Int J Parasitol Drugs Drug Resist. .
Free PMC article

Abstract

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 ∼80% cure rate that is lower (<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.

Keywords: Ancylostoma; Anthelmintic; Bacillus thuringiensis; Cry5B; Hookworm; Necator.

Figures

Image 1
Fig. 1
Fig. 1
Efficacy of Cry5B SCL against A. caninum infections in dogs. Two dogs were pre-treated with famotidine and dexmedetomidine, treated with Cry5B, and post-treated with atipamezole. The dose of Cry5B was 40 mg/kg on day 42 and day 44 p. i. Shown are (A) average hookworm burdens based on capsule endoscopy and (B) average fecal egg counts (eggs per gram of feces or EPG) five days after the second treatment.
Fig. 2
Fig. 2
Efficacy of Cry5B against N. americanus and A. ceylanicum. (A) Intoxication of N. americanus adults in vitro over time at a dose of 250 μg/mL Cry5B purified protein (n = 6/dose/repeat, average of 2 repeats). (B) N. americanus hookworm burdens in placebo (water control) versus Cry5B treatment. Here and in similar figures, each dot represents hookworm burden in an individual hamster; long horizontal bar represents the average and short horizontal bars represent standard error. (C) N. americanus fecal egg counts (EPG) before treatment and after treatment with placebo (water control) or Cry5B. Here and in similar figures, each bar represents the average of all hamsters; error bar represents standard error. (D) A. ceylanicum hookworm burdens in placebo (water control) versus Cry5B treatment. (E) A. ceylanicum fecal egg counts (EPG) before treatment and after treatment with placebo (water control) or Cry5B. The dose in vivo for B-E is 40 mg/kg Cry5B, delivered as SCL.
Fig. 3
Fig. 3
Inhibition of pepsin via cimetidine improves Cry5B efficacy. (A) A. ceylanicum hookworm burdens in hamsters treated with spore lysate (SL), cimetidine alone (Cim only), or Cry5B SCL at the indicated dose without or with (+Cim) a dose of cimetidine that inhibits pepsin activity 15 min prior to SCL treatment. (B) Corresponding fecal egg counts (EPG) for experiment in A before treatment and after treatment. (C, D) Same experiment as in (A, B) except using purified Cry5B.
Fig. 4
Fig. 4
Cry5B is efficacious in the absence of a Th2 immune response. (A) H. polygyrus bakeri worm burdens in placebo (SL control) versus Cry5B SCL treatment in STAT6−/− mice. (B) H. polygyrus bakeri fecal egg counts in placebo (SL control) versus Cry5B SCL treatment in STAT6−/− mice. The Cry5B dose is 2 × 40 mg/kg. For comparison, a single 100 mg/kg dose reduced H. polygyrus bakeri burdens 67% in immunocompetent mice (Hu et al., 2010a).
Fig. 5
Fig. 5
Repeated dosing with Cry5B does not abrogate efficacy. (A) Schematic of repeated dosing experiment. (B) Hookworm burdens from hamsters infected with A. ceylanicum hookworms and treated with Cry5B SCL, SL (no Cry5B), or water control. “Pre-dose” refers to hamsters treated with SCL or SL two times prior to infection and subsequent “treatment dose.” All hamsters were given a treatment dose of water, Cry5B, or SL (no Cry5B) 18 days post-infection. (C) Same experiment as in B except purified Cry5B protein was used for “pre-doses” and “treatment doses.” The dose of Cry5B used in all “pre-doses” and “treatment doses” was 10 mg/kg.

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