Development of an o-pthalaldehyde (OPA) assay to measure protein content in Ricin Vaccine E. coli (RVEc™).

A recombinant ricin vaccine from E. coli (RVEc™), was developed at the US Army Medical Research Institute of Infectious Diseases (USAMRIID) and assessed in an FDA sponsored Phase 1a clinical trial. At the maximum dosage, two of the study participants developed physiological responses that were elevated to the level of severe adverse reactions.

Camelid VHH Antibodies that Neutralize Botulinum Neurotoxin Serotype E Intoxication or Protease Function.

Botulinum neurotoxin (BoNT) serotype E is one of three serotypes that cause the preponderance of human botulism cases and is a Tier 1 Select Agent. BoNT/E is unusual among BoNT serotypes for its rapid onset and short duration of intoxication. Here we report two large panels of unique, unrelated camelid single-domain antibodies (VHHs) that were selected for their ability to bind to BoNT/E holotoxin and/or to the BoNT/E light chain protease domain (LC/E).

Engineering of Botulinum Neurotoxins for Biomedical Applications.

Botulinum neurotoxins (BoNTs) have been used as therapeutic agents in the clinical treatment of a wide array of neuromuscular and autonomic neuronal transmission disorders. These toxins contain three functional domains that mediate highly specific neuronal cell binding, internalization and cytosolic delivery of proteolytic enzymes that cleave proteins integral to the exocytosis of neurotransmitters. The exceptional cellular specificity, potency and persistence within the neuron that make BoNTs such effective toxins, also make them attractive models for derivatives that have modified properties that could potentially expand their therapeutic repertoire.

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.

A matrix-focused structure-activity and binding site flexibility study of quinolinol inhibitors of botulinum neurotoxin serotype A.

Our initial discovery of 8-hydroxyquinoline inhibitors of BoNT/A and separation/testing of enantiomers of one of the more active leads indicated considerable flexibility in the binding site. We designed a limited study to investigate this flexibility and probe structure-activity relationships; utilizing the Betti reaction, a 36 compound matrix of quinolinol BoNT/A LC inhibitors was developed using three 8-hydroxyquinolines, three heteroaromatic amines, and four substituted benzaldehydes. This study has revealed some of the most effective quinolinol-based BoNT/A inhibitors to date, with 7 compounds displaying IC values ⩽1μM and 11 effective at ⩽2μM in an ex vivo assay.

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.

What next for botulism vaccine development?

Botulism is a severe neuroparalytic disease caused by the toxins produced from several Clostridium species. Botulinum neurotoxins (BoNTs) cause flaccid paralysis by inducing a blockade at voluntary motor and autonomic cholinergic junctions that, if not treated, can be fatal. Vaccination to elicit protective circulating antibodies that bind, neutralize and clear toxins before they can be internalized and affect cholinergic neurons remains the most effective form of protection against BoNT.

A novel strategy for development of recombinant antitoxin therapeutics tested in a mouse botulism model.

Antitoxins are needed that can be produced economically with improved safety and shelf life compared to conventional antisera-based therapeutics. Here we report a practical strategy for development of simple antitoxin therapeutics with substantial advantages over currently available treatments. The therapeutic strategy employs a single recombinant 'targeting agent' that binds a toxin at two unique sites and a 'clearing Ab' that binds two epitopes present on each targeting agent.

Discovery of a novel enzymatic cleavage site for botulinum neurotoxin F5.

Botulinum neurotoxins (BoNTs) cause botulism by cleaving proteins necessary for nerve transmission. There are seven serotypes of BoNT, A-G, characterized by their response to antisera. Many serotypes are further distinguished into differing subtypes based on amino acid sequence, some of which result in functional differences.

Production of catalytically inactive BoNT/A1 holoprotein and comparison with BoNT/A1 subunit vaccines against toxin subtypes A1, A2, and A3.

A recombinant, catalytically inactive Clostridium botulinum neurotoxin A1 holoprotein (ciBoNT/A1 HP) was constructed by introducing amino acid substitutions H223A, E224A, and H227A in the active site to ablate proteolytic activity. ciBoNT/A1 HP was produced in the yeast Pichia pastoris and the purified product was evaluated as a vaccine candidate by comparison against recombinant BoNT/A1 LC, LC-belt, LC-H(n), and H(c) antigens and a LC-H(n)+H(c) combination in mouse potency and efficacy bioassays when challenged with BoNT/A subtypes /A1, /A2, and /A3. A single dose of ciBoNT/A1 HP provided equivalent or greater protective immunity, not only against the homologous toxin, but also against two distinct toxin subtypes with significant amino acid divergence.

Protection with recombinant Clostridium botulinum C1 and D binding domain subunit (Hc) vaccines against C and D neurotoxins.

Recombinant botulinum Hc (rBoNT Hc) vaccines for serotypes C1 and D were produced in the yeast Pichia pastoris and used to determine protection against four distinct BoNT C and D toxin subtypes. Mice were vaccinated with rBoNT/C1 Hc, rBoNT/D Hc, or with a combination of both vaccines and challenged with BoNT C1, D, C/D, or D/C toxin. Mice receiving monovalent vaccinations were partially or completely protected against homologous toxin and not protected against heterologous toxin.