Botulinum neurotoxins (BoNTs) are highly poisonous substances that are also effective medicines. Accidental BoNT poisoning often occurs through ingestion of Clostridium botulinum-contaminated food. Here, we present the crystal structure of a BoNT in complex with a clostridial non-toxic non-hemagglutinin (NTNHA) protein at 2.7 angstrom. Biochemical and functional studies show that NTNHA provides large and multivalent binding interfaces to protect BoNT from gastrointestinal degradation. Moreover, the structure highlights key residues in BoNT that regulate complex assembly in a pH-dependent manner. Collectively, our findings define the molecular mechanisms by which NTNHA shields BoNT in the hostile gastrointestinal environment and releases it upon entry into the circulation. These results will assist in the design of small molecules for inhibiting oral BoNT intoxication, and of delivery vehicles for oral administration of biologics.

Our results indicate that although NAb formation does play a role in secondary treatment failure with BoNT-A, this is not the cause in all patients, and the influence of other factors needs to be investigated. Gaining a better understanding of the underlying mechanisms for secondary treatment failure may help in the prediction, diagnosis, management, and prevention of this problem.

ObjectiveTo investigate the prevalence of neutralizing antibodies (NAbs) against botulinum neurotoxin type A (BoNT/A) during long-term BoNT/A treatment in different neurologic indications.MethodsIn this monocentric, observational cross-sectional study, 596 outpatients treated with BoNT/A for different indications were tested for BoNT/A binding antibodies by ELISA. Positive samples were investigated for NAbs with the mouse hemidiaphragm test. The prevalence of NAbs was analyzed for different indications: facial hemispasm, blepharospasm, cervical dystonia, other dystonia, and spasticity. Besides the rate of NAb-positive patients overall and per patient subgroup, a Kaplan-Meier analysis of the probability of remaining NAb negative with duration of treatment is provided, and a stepwise binary logistic regression analysis is performed to identify factors significantly contributing to the induction of NAbs.ResultsOverall, 83 of 596 patients (13.9%) had measurable NAbs. The probability of developing NAbs increased with the single and cumulative dose of treatment and was influenced by the BoNT/A formulation, while all other factors analyzed, including disease entity and treatment duration, had no additional influence.ConclusionsWe present the largest study to date of the prevalence of BoNT/A NAbs in a large unbiased cohort of patients including the relevant neurologic indications. Repeated injections of BoNT/A inevitably bear the risk of developing NAbs. However, in addition to avoiding booster injections and providing short intervals between injections, reducing the individual injected doses may diminish the risk of NAb induction independently of the indication for which BoNT/A is used.

Different types of botulinum neurotoxin (BoNT) block different proteins of the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein complex within cholinergic nerve terminals, producing blockade of cholinergic neuromuscular and autonomic synapses. Animal studies indicate the longest duration of action for BoNT type A (BoNTA) followed by types B, F, and E. Diffusion to adjacent and remote muscles may be related to protein composition, dilutions, volume, target muscle selection, and injection technique. A review of head-to-head, randomized, controlled trials of BoNTA preparations (Botox and Dysport) suggests that Dysport tends to have higher efficacy, longer duration, and higher frequency of adverse effects. Conversion factors between the preparations varied, however, and remain controversial. In clinical settings, a Botox:Dysport conversion ratio of 1:3 may be appropriate. Animal studies suggest a conversion ratio of 1:2.5-3. When therapeutic effects between these preparations are attempting to be equalized, Dysport seems to produce more adverse effects. In mice, Botox appears to have a better safety margin than Dysport and BoNTB. In rats, diffusion margins are similar for Botox and Dysport. Jitter derived from stimulation single-fiber EMG of injected and remote muscles show no differences between Botox and Dysport. Atrophy of extrafusal muscle fibers of injected and remote muscles do not differ between the BoNTA preparations.