Potency and stability of a trivalent, catalytically inactive vaccine against botulinum neurotoxin serotypes C, E and F (triCEF).

Botulism is an acute neuroparalytic affliction of the motor and autonomic neurons caused by the toxins produced from Clostridium botulinum and related bacterial strains. The botulinum neurotoxins, or BoNTs, consist of a phylogenetically diverse group of highly potent protein toxins. Current medical interventions for confirmed cases of botulism are limited to immediate administration of antitoxins and respiratory support. There is currently no licensed vaccine against botulism in the United States. The most widely distributed botulism vaccine was a pentavalent BoNT toxoid (PBT) against serotypes A-E administered until 2011 under an investigational new drug license. A binary vaccine composed of the recombinant, non-toxic, receptor binding domains (RBD) of serotypes/A1 and/B1 has completed a phase II clinical trial, but has yet to attain full licensure. We have previously published data demonstrating catalytically inactive, full length botulinum neurotoxin holoproteins (ciBoNT HPs) against serotypes/A1,/B1,/C1,/E1 and/F1 provide equivalent or superior potency against parental and dissimilar subtype toxins as compared the RBD vaccines. Here we describe the consistent potencies of the three independent lots each of ciBoNT/C1,/E1, and/F1 HPs against substantial monovalent challenges of the parental toxins. We also present data that a trivalent formulation of ciBoNT/C1,/E1 and/F1 (triCEF) maintains potency against both monovalent and polyvalent toxin challenges when stored as an adjuvanted vaccine at 4-8 °C for up to 2 years.

Recombinant Botulinum Neurotoxin Hc Subunit (BoNT Hc) and Catalytically Inactive Clostridium botulinum Holoproteins (ciBoNT HPs) as Vaccine Candidates for the Prevention of Botulism.

There are few available medical countermeasures against botulism and the discontinuation of the pentavalent botulinum toxoid vaccine by the Centers for Disease Control and Prevention in 2011 has resulted in the need for a safe and effective prophylactic alternative. Advances in genetic engineering have resulted in subsequent vaccine efforts being primarily focused on the production of highly purified recombinant protein antigens representing one or more domains of the botulinum neurotoxin. Recombinant subunit vaccines based on the carboxy one-third of the toxin (Hc) developed in our lab against serotypes A-F have been shown to be safe and effective. However, in response to the identification of an ever increasing number of BoNT subtypes with significant amino acid heterogeneity, we have developed catalytically inactive BoNT holoproteins (ciBoNT HPs) in an attempt to elicit greater protective immunity to address these toxin variants. Here we report the production of ciBoNT/B1 HP, ciBoNT/C1 HP, ciBoNT/E1 HP and ciBoNT/F1 HP and compare the immunological and protective abilities of ciBoNT HPs and BoNT/A Hc, BoNT/B Hc, BoNT/C Hc, BoNT/E Hc and BoNT/F Hc vaccines when challenged with homologous and heterologous toxins. Our results suggest the ciBoNT HP vaccines exhibit superior potency after single vaccinations but multiple vaccinations with BoNT/Hc antigens resulted in increased survival rates at the toxin challenge levels used.

Utilizing Ayurvedic literature for the identification of novel phytochemical inhibitors of botulinum neurotoxin A.

ETHNOPHARMACOLOGICAL RELEVANCE: Ayurveda, an ancient holistic system of health care practiced on the Indian subcontinent, utilizes a number of multi-plant formulations and is considered by many as a potential source for novel treatments, as well as the identification of new drugs. Our aim is to identify novel phytochemicals for the inhibition of bacterial exotoxin, botulinum neurotoxin A (BoNT/A) based on Ayurvedic literature. BoNT/A is released by Clostridium species, which when ingested, inhibits the release of acetylcholine by concentrating at the neuromuscular junction and causes flaccid paralysis, resulting in a condition termed as botulism, and may also lead to death due to respiratory arrest. METHODS: Fifteen plants were selected from the book 'Diagnosis and treatment of diseases in Ayurveda' by Vaidya Bhagwan Dash and Lalitesh Kashyap, based on their frequency of use in the formulations used for the treatment of six diseases with neuromuscular symptoms similar to botulism. Phytochemicals from these plants were screened using in silico, and in vitro methods. Structures of 570 reported phytochemicals from 14 plants were docked inside six reported BoNT/A light chain crystal structures using ensemble docking module in Maestro (Schrödinger, LLE). RESULTS: From the docking scores and structural diversity, nine compounds including acoric acid 1, three flavonoids, three coumarins derivatives, one kava lactone were selected and screened using an in vitro HPLC-based protease assay. The bioassay results showed that several compounds possess BoNT/A LC inhibition of 50-60% when compared to positive controls NSC 84094 and CB7967495 (80-95%). CONCLUSION: Further testing of the active compounds identified from Ayurvedic literature and structure-activity studies of acoric acid 1 using more sensitive bioassays is under way. The identification of acoric acid 1, a novel scaffold against BoNT/A, exemplifies the utility of Ayurvedic literature for the discovery of novel drug leads.