Search Results (92)

Botulinum neurotoxins (BoNTs) are known to inhibit synaptic transmission by targeting SNARE proteins, but their selectivity toward central excitatory and inhibitory pathways is not yet fully understood. In this study, the interaction of serotypes A (BoNT/A) and B (BoNT/B) with the glutamatergic and GABAergic systems has been investigated using a pharmacological approach in an animal model of inflammatory pain, i.e., the formalin test in mice. BoNTs were administered intracerebroventricularly, three days before testing, followed 15 min before testing by systemic administration of sub-analgesic doses of MK801, an NMDA receptor antagonist, or muscimol, a GABA_A receptor agonist. BoNT/A reduced the second phase of the formalin test without affecting both the first phase and the interphase, suggesting a selective action on excitatory glutamatergic circuits while sparing GABAergic inhibition. Co-administration of MK801 with BoNT/A did not enhance analgesia, and muscimol did not further reduce interphase, confirming preserved GABAergic transmission. In contrast, BoNT/B abolished the interphase, consistent with impaired GABA release. Co-administration of MK801 or muscimol with BoNT/B restored the interphase, indicating compensatory rebalancing of excitatory-inhibitory networks. These results demonstrate that BoNT/A and BoNT/B exert distinct effects on central neurotransmission and support the hypothesis that BoNT/A preferentially targets excitatory synapses, while BoNT/B targets inhibitory synapses. This work contributes to a deeper understanding of anti-inflammatory mechanisms of BoNTs and their selective interaction with central pain pathways. Full article

Recombinant monoclonal antibody (mAb) botulinum neurotoxin (BoNT) antitoxins, consisting of three mAbs that bind non-overlapping epitopes, are highly potent. However, the three-mAb mixtures pose unique development and manufacturing challenges. Combining even more mAbs to create multivalent antitoxin drugs multiplies those challenges. We previously reported that a single tri-epitopic IgG1-based mAb (TeAb) containing the variable domains of the three parental BoNT/A mAbs and an Fc was as potent as the combination of three IgGs in the mouse neutralization assay (MNA). Here, we extended the tri-epitopic strategy to three other BoNT serotypes. Each TeAb (TeAb-B for BoNT/B, TeAb-E for BoNT/E, and TeAb-F for BoNT/F) binding was measured using fluorescence-activated cell sorting and flow fluorimetry, and the potency was tested in the MNA. The three TeAbs displayed binding affinities that were the same within error of the parental IgGs for each epitope, and all had higher avidity to each serotype of BoNT than that of the parental mAbs. The potency of the BoNT/B, BoNT/E, and BoNT/F TeAbs was similar to the combinations of the three parental IgGs binding BoNT/B, BoNT/E, and BoNT/F in the MNA. We now have four examples of a single TeAb recapitulating the affinity and in vivo potency of a three-mAb antitoxin. The tri-epitopic strategy could be applied to streamline the production and bioanalytics of antibody drugs where three-mAb binding is required for activity. Full article

Botulinum neurotoxin serotype A (BoNT/A), the most toxic of the seven serotypes produced by Clostridium botulinum, poses significant public health risks because of its involvement in foodborne outbreaks and potential use in bioterrorism. In this study, we developed high-affinity monoclonal antibodies for BoNT/A detection using single-cell fluorescence-activated cell sorting and nested PCR. The optimized antibody pair demonstrated exceptional sensitivity, detecting recombinant BoNT/A at concentrations as low as 0.02 ng/mL with a linear range of 0.02–10 ng/mL, while maintaining high specificity against BoNT/B, E, and F. Biolayer interferometry confirmed superior binding kinetics, and a time-resolved fluoroimmunoassay (TRFIA) demonstrated consistent performance in complex food matrices, including ham sausage and soybean paste. These rabbit-derived monoclonal antibodies enable ultrasensitive detection of BoNT/A across diverse food matrices, offering a powerful tool for food safety monitoring and biosecurity. Full article

TrenibotulinumtoxinE (trenibotE), a botulinum neurotoxin serotype E (BoNT/E), is being developed for clinical use, and can fill a unique treatment gap for patients who are seeking neurotoxin treatment with a rapid onset and short duration of effect. This preclinical study characterized the pharmacological activity of trenibotE using the mouse Digit Abduction Score (DAS) assay. A comparative analysis was also performed between trenibotE and an equi-efficacious dose of the botulinum neurotoxin serotype A (BoNT/A) onabotulinumtoxinA (onabotA). TrenibotE showed a dose-dependent increase in peak DAS and duration of effect. A comparison of onabotA and trenibotE in this assay at approximate equi-efficacious doses showed trenibotE to have a faster onset of effect (trenibotE yielded a significantly greater effect as early as 6 h post-injection), shorter time to peak effect (24–27 h vs. 2 days), and an overall shorter duration of response (3 days vs. 14 days). The unique temporal characteristics of trenibotE and pharmacological differentiation from onabotA observed in this preclinical assay support the clinical development of this molecule. Full article

Background: Botulinum toxin (BoNT), produced by Clostridium botulinum, has transitioned from being a lethal neurotoxin to a versatile therapeutic agent. Its ability to inhibit neurotransmitter release by targeting Soluble N-ethylmaleimide-sensitive factor Attachment Protein Receptor (SNARE) proteins underpins its applications in treating conditions such as spasticity, dystonia, chronic pain, and overactive bladder. The clinical and pharmacological properties of BoNT have been extensively studied, with significant advancements in its therapeutic use, safety profile, and understanding of associated adverse effects. Objective: This comprehensive review aims to consolidate historical developments, molecular mechanisms, clinical applications, and challenges associated with BoNT, with a focus on expanding its therapeutic scope while ensuring safety and efficacy. Method: A narrative approach was used to analyze and synthesize insights from 155 references spanning experimental studies, clinical trials, and reviews. Key topics included BoNT’s historical milestones, mechanisms of action, therapeutic applications, and adverse events. Findings: BoNT demonstrates remarkable efficacy in a wide range of medical and cosmetic applications. In movement disorders such as dystonia and spasticity, it reduces muscle overactivity and improves functional outcomes. In chronic pain management, including migraines and neuropathic pain, BoNT significantly alleviates symptoms by modulating neurotransmitter activity. Cosmetic use for conditions like glabellar lines and hyperhidrosis highlights its precision and safety when administered appropriately. For conditions like strabismus and blepharospasm, BoNT effectively restores muscle control, reducing involuntary contractions. In urological applications, BoNT has proven to be an effective therapy for overactive bladder, offering significant symptom relief in refractory cases. However, concerns about long-distance effects, where the toxin may spread beyond the injection site to affect distant muscles or systems, have been reported in certain high-dose or sensitive populations. These findings emphasize the importance of dose optimization and patient-specific approaches. Adverse effects such as localized pain, hematoma, dysphagia, and systemic effects, particularly in high-risk groups, underscore the need for careful monitoring. The development of immunogenicity, leading to neutralizing antibodies, remains a challenge that impacts long-term therapeutic efficacy. Emerging research on novel serotypes, including BoNT/X, and innovations in delivery mechanisms, offer promising avenues to address current limitations. Advances in optimizing dosing regimens and refining injection techniques have also contributed to minimizing complications and improving outcomes across diverse patient populations. Conclusions: BoNT remains a cornerstone in neurology and cosmetic medicine, with its therapeutic potential still expanding. The balance between efficacy and safety, driven by innovations in formulation and application, underscores the importance of continued research. Future directions should focus on minimizing adverse effects, reducing immunogenicity, and exploring novel indications to further enhance its clinical utility. Full article

Botulinum neurotoxins (BoNTs) are the most potent toxins on Earth and are classified as Category A biological agents. BoNTs lead to paralysis in humans and cause botulism. Antibody therapeutics can effectively treat toxin-mediated infectious diseases. In this study, we generated a pharmaceutical humanized monoclonal antibody (HZ45 mAb) to prevent or treat botulism. HZ45 binds to the heavy chain receptor (HCR) domain of the toxin, preventing the toxin from entering the cell. The mAb was produced using hybridoma technology and phage display. We evaluated HZ45 mAb for the neutralization of BoNT serotype A (BoNT/A) in mice and rabbits. The survival results showed that pretreatment with HZ45 mAb provided 100% protection at a dose of 0.1 mg per mouse against a maximum of 100 LD₅₀ of BoNT/A. To assess the therapeutic efficacy of HZ45 mAb in New Zealand white rabbits (NZWs), a 5 mg dose was administered 4 or 8 h after challenge with 10 LD₅₀. The results indicated that 5 mg of HZ45 could treat the NZWs within 8 h after exposure to 10 LD₅₀ botulinum. Consequently, in an in vivo context, including mice and rabbits, HZ45 mAb could protect against botulinum type A intoxication. Full article

Background/Objectives. Bovine botulism, although relatively rare, presents significant economic losses due to high mortality rates and restrictions on livestock product trade. Vaccination remains the most effective strategy for preventing botulism-related mortality. This study evaluated the efficacy of a bivalent recombinant vaccine targeting the C-terminal portion of the heavy chain (Hc) of botulinum neurotoxin serotype C (BoNT/C) (Hc BoNT/C) and botulinum neurotoxin serotype D (BoNT/D) (Hc BoNT/D) in inducing neutralizing antibodies against these toxins and their mosaic variants BoNT/CD and BoNT/DC in cattle. This comparison aims to improve the design of an optimal recombinant vaccine for preventing bovine botulism caused by the most common serotypes. Methods. Twenty, four-month-old Holstein Friesian calves were randomly assigned to two groups of ten animals: vaccinated group and control group. Sera were collected at various time points to assess antibody titers using ELISA and neutralizing antibody titers using a mouse protection assay. Neutralizing antibody titers were compared to those obtained with a commercially available toxoid vaccine. Results. The recombinant vaccine elicited significant increases in anti-HcBoNT/C and anti-HcBoNT/D IgG antibody levels in vaccinated animals compared to controls animals with no adverse effects. Specifically, post-vaccination, the calves showed no local reactions (swelling, warmth) or behavioral changes suggestive of systemic illness. Neutralizing antibody titers against BoNT/C and BoNT/D were significantly higher in the recombinant vaccine group compared to the toxoid vaccine group. However, the recombinant vaccine showed lower neutralizing activity against BoNT/DC compared to the toxoid vaccine. Conclusions. The bivalent recombinant vaccine demonstrated promising immunogenicity in cattle, inducing high neutralizing antibody titers against BoNT/C and BoNT/D. While effective against these toxins, the lower efficacy against BoNT/DC highlights the need for further research to optimize the vaccine formulation, potentially by incorporating a BoNT/DC Hc component, to provide broader protection against bovine botulism. Full article

Botulinum neurotoxins (BoNTs), ricin, and many other biological toxins are called AB toxins possessing heterogeneous A and B subunits. We propose herein a quick and safe sensing approach to AB toxins based on their unique quaternary structures. The proposed approach utilizes IgG antibodies against their A-subunits in combination with those human cell-membrane glycolipids that act as the natural ligands of B-subunits. In practice, an IgG antibody against the A-subunit of a target toxin is selected from commercially available sources and immobilized on the surface of Au nanoparticles to constitute a multivalent IgG/Au nanoconjugate. The derived IgG/Au conjugate is used in the pretreatment process of test samples for deactivating biological toxins in the form of a ternary toxin/antibody/Au complex. This process is implemented in advance to reduce the risk of handling biological toxins in laboratory work. On the other hand, the human glycolipid is immobilized on a tiny glass plate and used as a biosensor chip. The biosensor chip is set in the chamber of a flow sensing system using localized surface plasmon resonance (LSPR) spectrometry available in portable size at relatively low cost. In principle, the LSPR sensing system enables us to perform a rapid and selective detection for different kinds of biological toxins if the human glycolipid is correctly selected and installed in the sensing system. In the present LSPR sensing approach, a target AB toxin may have been deactivated during the pretreatment process. The test sample containing the deactivated AB toxin becomes a real target to be analyzed by the sensing system. In the present, we describe the concept of employing the commercially available IgG antibody in the pretreatment process followed by a typical procedure for converting it into the multivalent antibody/Au nanoconjugate and its preliminary applications in the LSPR detection of a ricin homologue (RCA₁₂₀) and BoNTs in different serotypes. The tested LSPR sensing approach has worked very well for the ricin homologue and certain serotypes of botulinum neurotoxins like BoNT/A, indicating that the prior deactivation process at their A-domains causes no significant damage to the function of their B-domains with respect to determining the host cell-membrane glycolipid. The experimental results also indicated that LSPR responses from these pretreated AB toxins are significantly amplified. That is obviously thanks to the presence of Au nanoparticles in the multivalent IgG/Au nanoconjugate. We suggest in conclusion that the proposed LSPR sensing approach will provide us with a safe and useful tool for the study of biological AB toxins based on their unique quaternary protein structures. Full article

Botulinum neurotoxins (BoNTs) are highly toxic proteins that require high-affinity immunocapture reagents for use in endopeptidase-based assays. Here, 30 novel and 2 earlier published llama single-domain antibodies (VHHs) against the veterinary-relevant BoNT serotypes C and D were yeast-produced. These VHHs recognized 10 independent antigenic sites, and many cross-reacted with the BoNT/DC and CD mosaic variants. As VHHs are highly suitable for genetically linking to increase antigen-binding affinity, 52 VHH multimers were produced and their affinity for BoNT/C, D, DC, and CD was determined. A selection of 15 multimers with high affinity (K_D < 0.1 nM) was further shown to be resilient to a high salt wash that is used for samples from complex matrices and bound native BoNTs from culture supernatants as shown by Endopep-MS. High-affinity multimers suitable for further development of a highly sensitive Endopep-MS assay include four multimers that bind both BoNT/D and CD with K_D of 14–99 pM, one multimer for BoNT/DC (65 pM) that also binds BoNT/C (75 pM), and seven multimers for BoNT/C (<1–19 pM), six of which also bind BoNT/DC with lower affinity (93–508 pM). In addition to application in diagnostic tests, these VHHs could be used for the development of novel therapeutics for animals or humans. Full article

Clostridium botulinum serotypes C and D cause botulism in livestock, a neuroparalytic disease that results in substantial economic losses. Vaccination with aluminium-based toxoid vaccines is widely used to control the spread of botulism. Aluminium-based adjuvants are preferred owing to their apparent stimulation of the immune responses to toxoid vaccines when compared to other adjuvants. The aim of our study was to evaluate aluminium hydroxide nanoparticles as a potential substitute for alhydrogel in the botulism bivalent vaccine. Botulism vaccines were formulated with either alhydrogel or nanoalum and comparative efficacy between the two formulations was conducted by evaluating the immune response in vaccinated guinea pigs. A significant increase in immunological parameters was observed, with the antibody titres higher in the serum of guinea pigs (20 IU/mL of anti-BoNT C/D) injected with nanoalum-containing vaccine than guinea pigs inoculated with the standard alhydrogel-containing vaccine (8.7 IU/mL and 10 IU/mL of anti-BoNT C and anti-BoNT D, respectively). Additionally, the nanoalum-containing vaccine demonstrated potency in a multivalent vaccine (20 IU/mL of anti-BoNT C/D), while the standard alhydrogel-containing vaccine showed a decline in anti-BoNT C (5 IU/mL) antibody titres. Full article

Botulinum neurotoxins (BoNTs) are zinc endopeptidases produced by the Clostridium genus of anerobic bacteria, largely known for their ability to cleave synaptic proteins, leading to neuromuscular paralysis. In the central nervous system, BoNTs are known to block the release of glutamate neurotransmitter, and for this reason, researchers explored the possible therapeutic action in disorders characterized by neuronal hyperactivity, such as epilepsy. Thus, using multidisciplinary approaches and models of experimental epilepsy, we investigated the pharmacological potential of BoNT/E serotype. In this review, written in memory of Prof. Matteo Caleo, a pioneer in these studies, we go back over the hypotheses and experimental approaches that led us to the conclusion that intrahippocampal administration of BoNT/E (i) displays anticonvulsant effects if prophylactically delivered in a model of acute generalized seizures; (ii) does not have any antiepileptogenic action after the induction of status epilepticus; (iii) reduces frequency of spontaneous seizures in a model of recurrent seizures if delivered during the chronic phase but in a transient manner. Indeed, the control on spontaneous seizures stops when BoNT/E effects are off (few days), thus limiting its pharmacological potential in humans. Full article

Botulinum neurotoxins are a varied group of protein toxins that share similar structures and modes of activity. They include at least seven serotypes and over forty subtypes that are produced by seven different clostridial species. These bacterial species are not limited strictly to BoNT-producers as neuro-toxigenic and non-neuro-toxigenic members have been identified within each species. The nomenclature surrounding these toxins and associated bacteria has been evolving as new isolations and discoveries have arisen, resulting in challenges in diagnostic reporting, epidemiology and food safety studies, and in the application of therapeutic products. An understanding of the intricacies regarding the nomenclature of BoNTs and BoNT-producing clostridia is crucial for communication that allows for accurate reporting of information that is pertinent to each situation. Full article

The objective of this study is to clarify the effect of restoring the lowered masticatory muscle functional pressure and correcting bilateral differences in masticatory muscle functional pressure on jawbone growth during growth and development with a quantitative evaluation of the changes in the micro/nanostructural characteristics of entheses. Male Wistar rats aged 4 weeks were divided into an experimental group injected with a botulinum toxin serotype A (BoNT/A) formulation to reduce muscle function (BTX group) and a control group (CTRL group). They were euthanised after 6, 8, 10, 12, and 16 weeks after measuring the difference between the midline of the upper and lower incisors. The mandibles were harvested for histological examination, second harmonic generation imaging, and the quantitative evaluation of biological apatite (BAp) crystal alignment. The midline difference decreased with age in weeks. In rats from 6 weeks after BoNT/A administration to 12 weeks after administration, the collagen fibre bundle diameter was significantly smaller in the BTX group; the difference between the two groups decreased with increasing age. BAp crystal alignment was significantly different on the x-axis and the y-axis on the BTX group from 6 weeks after BoNT/A administration to 10 weeks after administration. Asymmetry of mandibular bone formation caused by load imbalance during growth could be corrected by the adjustment of the function of the masseter muscle on either side. Full article

Clostridium botulinum neurotoxins (BoNTs) are the most potent toxins known, causing the deadly disease botulism. They function through Zn²⁺-dependent endopeptidase cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, preventing vesicular fusion and subsequent neurotransmitter release from motor neurons. Several serotypes of BoNTs produced by Clostridium botulinum (BoNT/A-/G and/X) have been well-characterised over the years. However, a BoNT-like gene (homologue of BoNT) was recently identified in the non-clostridial species, Enterococcus faecium, which is the leading cause of hospital-acquired multi-drug resistant infections. Here, we report the crystal structure of the catalytic domain of a BoNT homologue from Enterococcus faecium (LC/En) at 2.0 Å resolution. Detailed structural analysis in comparison with the full-length BoNT/En AlphaFold2-predicted structure, LC/A (from BoNT/A), and LC/F (from BoNT/F) revealed putative subsites and exosites (including loops 1–5) involved in recognition of LC/En substrates. LC/En also appears to possess a conserved autoproteolytic cleavage site whose function is yet to be established. Full article

Equine-derived antitoxin (BAT^®) is the only treatment for botulism from botulinum neurotoxin serotype G (BoNT/G). BAT^® is a foreign protein with potentially severe adverse effects and is not renewable. To develop a safe, more potent, and renewable antitoxin, humanized monoclonal antibodies (mAbs) were generated. Yeast displayed single chain Fv (scFv) libraries were prepared from mice immunized with BoNT/G and BoNT/G domains and screened with BoNT/G using fluorescence-activated cell sorting (FACS). Fourteen scFv-binding BoNT/G were isolated with K_D values ranging from 3.86 nM to 103 nM (median K_D 20.9 nM). Five mAb-binding non-overlapping epitopes were humanized and affinity matured to create antibodies hu6G6.2, hu6G7.2, hu6G9.1, hu6G10, and hu6G11.2, with IgG K_D values ranging from 51 pM to 8 pM. Three IgG combinations completely protected mice challenged with 10,000 LD₅₀s of BoNT/G at a total mAb dose of 6.25 μg per mouse. The mAb combinations have the potential for use in the diagnosis and treatment of botulism due to serotype G and, along with antibody combinations to BoNT/A, B, C, D, E, and F, provide the basis for a fully recombinant heptavalent botulinum antitoxin to replace the legacy equine product. Full article