Regulation of Clostridium tetani Neurotoxin Expression by Culture Conditions

Background: Ensuring consistency of tetanus neurotoxin (TeNT) production by Clostridium tetani could help to ensure consistent product quality in tetanus vaccine manufacturing, ultimately contributing to reduced animal testing. The aim of this study was to identify RNA signatures related to consistent TeNT production using standard and non-standard culture conditions. Methods: We applied RNA sequencing (RNA-Seq) to study C. tetani gene expression in small-scale batches under several culture conditions. Results: We identified 1381 time-dependent differentially expressed genes (DEGs) reflecting, among others, changes in growth rate and metabolism. Comparing non-standard versus standard culture conditions identified 82 condition-dependent DEGs, most of which were specific for one condition. The tetanus neurotoxin gene (tetX) was highly expressed but showed expression changes over time and between culture conditions. The tetX gene showed significant down-regulation at higher pH levels (pH 7.8), which was confirmed by the quantification data obtained with the recently validated targeted LC-MS/MS approach. Conclusions: Non-standard culture conditions lead to different gene expression responses. The tetX gene appears to be the best transcriptional biomarker for monitoring TeNT production as part of batch-to-batch consistency testing during tetanus vaccine manufacturing.

Disruption of synaptic transmission in the Bed Nucleus of the Stria Terminalis reduces seizure-induced death in DBA/1 mice and alters brainstem E/I balance

Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death in refractory epilepsy patients. Accumulating evidence from recent human studies and animal models suggests that seizure-related respiratory arrest may be important for initiating cardiorespiratory arrest and death. Prior evidence suggests that apnea onset can coincide with seizure spread to the amygdala and that stimulation of the amygdala can reliably induce apneas in epilepsy patients, potentially implicating amygdalar regions in seizure-related respiratory arrest and subsequent postictal hypoventilation and cardiorespiratory death. This study aimed to determine if an extended amygdalar structure, the dorsal bed nucleus of the stria terminalis (dBNST), is involved in seizure-induced respiratory arrest (S-IRA) and death using DBA/1 mice, a mouse strain which has audiogenic seizures and a high incidence of postictal respiratory arrest and death. The presence of S-IRA significantly increased c-Fos expression in the dBNST of DBA/1 mice. Furthermore, disruption of synaptic output from the dBNST via viral-induced tetanus neurotoxin significantly improved survival following S-IRA in DBA/1 mice without affecting baseline breathing or hypercapnic and hypoxic ventilatory response. This disruption in the dBNST resulted in changes to the balance of excitatory/inhibitory synaptic events in the downstream brainstem regions of the lateral parabrachial nucleus (PBN) and the periaqueductal gray (PAG). These findings suggest that the dBNST is a potential subcortical forebrain site necessary for the mediation of seizure-induced respiratory arrest, potentially through its outputs to brainstem respiratory regions.

Novel Small Molecule Inhibitors That Prevent the Neuroparalysis of Tetanus Neurotoxin

Tetanus neurotoxin (TeNT) is a protein exotoxin produced by Clostridium tetani that causes the deadly spastic neuroparalysis of tetanus. It consists of a metalloprotease light chain and of a heavy chain linked via a disulphide bond. TeNT binds to the neuromuscular junction (NMJ) and it is retro-axonally transported into vesicular compartments to the spinal cord, where it is released and taken up by inhibitory interneuron. Therein, the catalytic subunit is translocated into the cytoplasm where it cleaves its target protein VAMP-1/2 with consequent blockage of the release of inhibitory neurotransmitters. Vaccination with formaldehyde inactivated TeNT prevents the disease, but tetanus is still present in countries where vaccination coverage is partial. Here, we show that small molecule inhibitors interfering with TeNT trafficking or with the reduction of the interchain disulphide bond block the activity of the toxin in neuronal cultures and attenuate tetanus symptoms in vivo. These findings are relevant for the development of therapeutics against tetanus based on the inhibition of toxin molecules that are being retro-transported to or are already within the spinal cord and are, thus, not accessible to anti-TeNT immunoglobulins.

Interaction between a Novel Oligopeptide Fragment of the Human Neurotrophin Receptor TrkB Ectodomain D5 and the C-Terminal Fragment of Tetanus Neurotoxin

This article presents experimental evidence and computed molecular models of a potential interaction between receptor domain D5 of TrkB with the carboxyl-terminal domain of tetanus neurotoxin (Hc-TeNT). Computational simulations of a novel small cyclic oligopeptide are designed, synthesized, and tested for possible tetanus neurotoxin-D5 interaction. A hot spot of this protein-protein interaction is identified in analogy to the hitherto known crystal structures of the complex between neurotrophin and D5. Hc-TeNT activates the neurotrophin receptors, as well as its downstream signaling pathways, inducing neuroprotection in different stress cellular models. Based on these premises, we propose the Trk receptor family as potential proteic affinity receptors for TeNT. In vitro, Hc-TeNT binds to a synthetic TrkB-derived peptide and acts similar to an agonist ligand for TrkB, resulting in phosphorylation of the receptor. These properties are weakened by the mutagenesis of three residues of the predicted interaction region in Hc-TeNT. It also competes with Brain-derived neurotrophic factor, a native binder to human TrkB, for the binding to neural membranes, and for uptake in TrkB-positive vesicles. In addition, both molecules are located together In Vivo at neuromuscular junctions and in motor neurons.

Novel neutralizing human monoclonal antibodies against tetanus neurotoxin

Tetanus is a fatal disease caused by tetanus neurotoxin (TeNT). TeNT is composed of a light chain (Lc) and a heavy chain, the latter of which is classified into two domains, N-terminus Hn and C-terminus Hc. Several TeNT-neutralizing antibodies have been reported, but it remains unclear which TeNT domains are involved in neutralization. To further understand the mechanism of these antibodies, we isolated TeNT-reactive human antibody clones from peripheral blood mononuclear cells. We then analyzed the reactivity of the isolated antibody clones to each protein domain and their inhibition of Hc-ganglioside GT1b binding, which is critical for TeNT toxicity. We also investigated the TeNT-neutralizing ability of isolated antibody clones and showed that an Hn-reactive clone protected strongly against TeNT toxicity in mice. Furthermore, combination treatment of Hn-reactive antibody clones with both Hc-reactive and TeNT mix (the mixture of Hc, Hn, and Lc proteins)–reactive antibody clones enhanced the neutralizing effect. These results indicated that antibody clones targeting Hn effectively neutralized TeNT. In addition, the use of a cocktail composed of Hc-, Hn-, and TeNT mix–reactive antibodies provided enhanced protection compared to the use of each antibody alone.

Extremely potent human monoclonal antibodies for the prophylaxis and therapy of Tetanus

Human monoclonal antibodies were used here to study the mechanism of neuron intoxication by tetanus neurotoxin protein toxins and as a safe preventive and therapeutic substitute of hyperimmune sera. By screening memory B cells of immune donors, we selected two monoclonal antibodies specific for tetanus neurotoxin with exceptionally high neutralizing activities, which have been extensively characterized both structurally and functionally. We found that these antibodies interfere with the binding and translocation of the neurotoxin into neurons by interacting with two epitopes, whose definition pinpoints crucial events in the cellular pathogenesis of tetanus. Some mechanistic aspects of tetanus neurotoxin intoxication were revealed, explaining at the same time, the unprecedented neutralization ability of these antibodies. Importantly, these antibodies are exceptionally efficient in preventing experimental tetanus when injected in mice long before the neurotoxin. Moreover, their Fab derivatives neutralize tetanus neurotoxin in post-exposure experiments, suggesting their potential therapeutic use upon intrathecal injection. As such, these human monoclonal antibodies, as well as their Fab derivatives, meet all requirements for being considered for prophylaxis and therapy of human tetanus and are ready for clinical trials.

Novel neutralizing human monoclonal antibodies against tetanus neurotoxin

Tetanus is a fatal disease caused by tetanus neurotoxin (TeNT). TeNT is composed of a light chain (Lc) and a heavy chain, the latter of which is classified into two domains, N-terminus Hn and C-terminus Hc. Several TeNT-neutralizing antibodies have been reported, but it remains unclear which TeNT domains are involved in neutralization. To further understand the mechanism of these antibodies, we isolated TeNT-reactive human antibody clones from peripheral blood mononuclear cells. We then analyzed the reactivity of the isolated antibody clones to each protein domain and their inhibition of Hc-ganglioside GT1b binding, which is critical for TeNT toxicity. We also investigated the TeNT-neutralizing ability of isolated antibody clones and showed that Hn-reactive clones protected strongly against TeNT toxicity in mice. Furthermore, combination treatment of Hn-reactive antibody clones with both Hc-reactive and TeNT mix–reactive antibody clones enhanced the neutralizing effect. These results indicated that antibody clones targeting Hn effectively neutralized TeNT. In addition, the use of a cocktail composed of Hc-, Hn-, and TeNT mix–reactive antibodies provided enhanced protection compared to the use of each antibody alone.