A Four-Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotypes C and D

Human botulism can be caused by botulinum neurotoxin (BoNT) serotypes A to G. Here, we present an antibody-based antitoxin composed of four human monoclonal antibodies (mAbs) against BoNT/C, BoNT/D, and their mosaic toxins. This work built on our success in generating protective mAbs to BoNT /A, B and E serotypes. We generated mAbs from human immune single-chain Fv (scFv) yeast-display libraries and isolated scFvs with high affinity for BoNT/C, BoNT/CD, BoNT/DC and BoNT/D serotypes. We identified four mAbs that bound non-overlapping epitopes on multiple serotypes and mosaic BoNTs. Three of the mAbs underwent molecular evolution to increase affinity. A four-mAb combination provided high-affinity binding and BoNT neutralization of both serotypes and their mosaic toxins. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing and neutralizing BoNT/C and BoNT/D serotypes and their mosaic toxins. A derivative of the four-antibody combination (NTM-1634) completed a Phase 1 clinical trial (Snow et al., Antimicrobial Agents and Chemotherapy, 2019) with no drug-related serious adverse events.     

Novel application of human neurons derived from induced pluripotent stem cells for highly sensitive botulinum neurotoxin detection

Human induced pluripotent stem cells (hiPSC) hold great promise for providing various differentiated cell models for in vitro toxigenicity testing. For Clostridium botulinum neurotoxin (BoNT) detection and mechanistic studies, several cell models currently exist, but none examine toxin function with species-specific relevance while exhibiting high sensitivity. The most sensitive cell models to date are mouse or rat primary cells and neurons derived from mouse embryonic stem cells, both of which require significant technical expertise for culture preparation. This study describes for the first time the use of hiPSC-derived neurons for BoNT detection. The neurons used in this study were differentiated and cryopreserved by Cellular Dynamics International (Madison, WI) and consist of an almost pure pan-neuronal population of predominantly gamma aminoisobutyric acidergic and glutamatergic neurons. Western blot and quantitative PCR data show that these neurons express all the necessary receptors and substrates for BoNT intoxication. BoNT/A intoxication studies demonstrate that the hiPSC-derived neurons reproducibly and quantitatively detect biologically active BoNT/A with high sensitivity (EC(50) ～0.3 U). Additionally, the quantitative detection of BoNT serotypes B, C, E, and BoNT/A complex was demonstrated, and BoNT/A specificity was confirmed through antibody protection studies. A direct comparison of BoNT detection using primary rat spinal cord cells and hiPSC-derived neurons showed equal or increased sensitivity, a steeper dose-response curve and a more complete SNARE protein target cleavage for hiPSC-derived neurons. In summary, these data suggest that neurons derived from hiPSCs provide an ideal and highly sensitive platform for BoNT potency determination, neutralizing antibody detection and for mechanistic studies.     

In vitro potency determination of botulinum neurotoxin B based on its receptor-binding and proteolytic characteristics

Botulinum neurotoxins (BoNTs) are the most potent toxins known. However, the paralytic effect caused by BoNT serotypes A and B is taken advantage of to treat different forms of dystonia and in cosmetic procedures. Due to the increasing areas of application, the demand for BoNTs A and B is rising steadily. Because of the high toxicity, it is mandatory to precisely determine the potency of every produced BoNT batch, which is usually accomplished by performing toxicity testing (LD₅₀ test) in mice. Here we describe an alternative in vitro assay for the potency determination of the BoNT serotype B. In this assay, the toxin is first bound to its specific receptor molecules. After the proteolytic subunit of the toxin has been released and activated by chemical reduction, it is exposed to synaptobrevin, its substrate protein. Finally the proteolytic cleavage is quantified by an antibody-mediated detection of the neoepitope, reaching a detection limit below 0.1 mouse LD₅₀/ml. Thus, the assay, named BoNT/B binding and cleavage assay (BoNT/B BINACLE), takes into account the binding as well as the protease function of the toxin, thereby measuring its biological activity.     

Practical Application of Novel Test Methods to Evaluate the Potency of Botulinum Toxin: A Comparison Analysis among Widely Used Products in Korea

The safe and effective dosing of botulinum neurotoxins (BoNTs) requires accurate and reliable methods to measure their potency. Several novel methods have been introduced over the past decade; however, only few studies have compared the potency of BoNT products with that of the LD50 and other alternative assays. Therefore, the objective of this study was to comparatively evaluate widely used BoNT products using various test methods. Four types of BoNTs (prabotulinumtoxin A, onabotulinumtoxin A, neubotulinumtoxin A, and letibotulinumtoxin A) were used in this study. The estimated potency was assessed using the LD50 assay, and the total BoNT type A protein levels were measured using the enzyme-linked immunosorbent assay (ELISA). The in vitro efficacy of the BoNTs was determined using fluorescence resonance energy transfer (FRET) and surface plasmon resonance (SPR) assays. The results showed differences in the total amount of BoNT protein and the cleavage activity of SNAP-25 within all types of BoNTs. The SPR study seemed to be useful for evaluating the potency by specifically measuring intact 19S neurotoxin, and these results provide new insights for assessing different BoNT products.     

New Modified Recombinant Botulinum Neurotoxin Type F with Enhanced Potency

Botulinum neurotoxins (BoNTs) are notorious toxins and powerful agents and can be lethal, causing botulism, but they are also widely used as therapeutics, particularly to treat neuromuscular disorders. As of today, the commercial BoNT treatments available are from native A or B serotypes. Serotype F has shown efficacy in a clinical trial but has scarcely been used, most likely due to its medium duration of effect. Previously, the uniqueness of the light chain of the F7 subtype was identified and reported, showing an extended interaction with its substrates, VAMPs 1, 2 and 3, and a superior catalytic activity compared to other BoNT/F subtypes. In order to more extensively study the properties of this neurotoxin, we engineered a modified F7 chimera, mrBoNT/F7-1, in which all the regions of the neurotoxin were identical to BoNT/F7 except the activation loop, which was the activation loop from BoNT/F1. Use of the activation loop from BoNT/F1 allowed easier post-translational proteolytic activation of the recombinant protein without otherwise affecting its properties. mrBoNT/F7-1 was expressed, purified and then tested in a suite of in vitro and in vivo assays. mrBoNT/F7-1 was active and showed enhanced potency in comparison to both native and recombinant BoNT/F1. Additionally, the safety profile remained comparable to BoNT/F1 despite the increased potency. This new modified recombinant toxin F7 could be further exploited to develop unique therapeutics to address unmet medical needs.     

Use of botulinum toxins in cancer therapy

A recent study has demonstrated for the first time that botulinum neurotoxin (BoNT) briefly opens tumour vessels, allowing more effective destruction of cancer cells by radiotherapy and chemotherapy. This review discusses the implications of BoNTs in cancer treatment. After briefly reviewing the different BoNT serotypes, their pharmacological activities and their general use in medicine, the authors focus on their possible application in cancer and describe how BoNTs have been used so far to treat spasm related to tumour or to therapies. By dissecting the mechanisms of action leading to a potentiation of anticancer therapy, it can be seen that BoNTs act by an effect on the tumour microenvironment rather than by a direct cytotoxic effect on tumour cells.     

Comparison of oral toxicological properties of botulinum neurotoxin serotypes A and B

Botulinum neurotoxins (BoNTs) are among the most potent biological toxins for humans. Of the seven known serotypes (A-G) of BoNT, serotypes A, B and E cause most of the foodborne intoxications in humans. BoNTs in nature are associated with non-toxic accessory proteins known as neurotoxin-associated proteins (NAPs), forming large complexes that have been shown to play important roles in oral toxicity. Using mouse intraperitoneal and oral models of botulism, we determined the dose response to both BoNT/B holotoxin and complex toxins, and compared the toxicities of BoNT/B and BoNT/A complexes. Although serotype A and B complexes have similar NAP composition, BoNT/B formed larger-sized complexes, and was approximately 90 times more lethal in mouse oral intoxications than BoNT/A complexes. When normalized by mean lethal dose, mice orally treated with high doses of BoNT/B complex showed a delayed time-to-death when compared with mice treated with BoNT/A complex. Furthermore, we determined the effect of various food matrices on oral toxicity of BoNT/A and BoNT/B complexes. BoNT/B complexes showed lower oral bioavailability in liquid egg matrices when compared to BoNT/A complexes. In summary, our studies revealed several factors that can either enhance or reduce the toxicity and oral bioavailability of BoNTs. Dissecting the complexities of the different BoNT serotypes and their roles in foodborne botulism will lead to a better understanding of toxin biology and aid future food risk assessments.     

Association of botulinum neurotoxins with synaptic vesicle protein complexes

Botulinum neurotoxins (BoNTs) elicit flaccid paralysis by cleaving SNARE proteins within peripheral neurons. BoNTs are classified into seven serotypes, termed A-G, based on antibody cross-neutralization. Clostridia produce BoNTs as single-chain toxins that are cleaved into a di-chain protein that comprises an N-terminal zinc metalloprotease domain that is linked by a disulfide bond to the C-terminal translocation/receptor-binding domain. BoNT/A and BoNT/B utilize synaptic vesicle protein 2 (SV2) and synaptotagmin, respectively, as receptors for entry into neurons. Using affinity chromatography, BoNT/A and BoNT/B were found to bind a synaptic vesicle protein complex in CHAPS extracts of synaptic vesicles. Mass spectroscopy identified synaptic vesicle protein 2, synaptotagmin I, synaptophysin, vesicle-associated membrane protein 2, and the vacuolar ATPase-proton pump as components of the BoNT-synaptic vesicle protein complex. BoNT/A and BoNT/B possessed unique density-gradient profiles when bound to synaptic vesicle protein complexes. The identification of BoNT/A and BoNT/B bound to synaptic vesicle protein complexes provides insight into the interactions of BoNT and neuronal receptors.     

Botulinum Toxin: An Update on Pharmacology and Newer Products in Development

Since its introduction as a treatment for strabismus, botulinum toxin (BoNT) has had a phenomenal journey and is now recommended as first-line treatment for focal dystonia, despite short-term clinical benefits and the risks of adverse effects. To cater for the high demand across various medical specialties, at least six US Food and Drug Administration (FDA)-approved formulations of BoNT are currently available for diverse labelled indications. The toxo-pharmacological properties of these formulations are not uniform and thus should not be used interchangeably. Synthetic BoNTs and BoNTs from non-clostridial sources are not far from clinical use. Moreover, the study of mutations in naturally occurring toxins has led to modulation in the toxo-pharmacokinetic properties of BoNTs, including the duration and potency. We present an overview of the toxo-pharmacology of conventional and novel BoNT preparations, including those awaiting imminent translation from the laboratory to the clinic.     

Structural Features of Clostridium botulinum Neurotoxin Subtype A2 Cell Binding Domain

Botulinum neurotoxins (BoNT) are a group of clostridial toxins that cause the potentially fatal neuroparalytic disease botulism. Although highly toxic, BoNTs are utilized as therapeutics to treat a range of neuromuscular conditions. Several serotypes (BoNT/A-/G, /X) have been identified with vastly differing toxicological profiles. Each serotype can be further sub-categorised into subtypes due to subtle variations in their protein sequence. These minor changes have been attributed to differences in both the duration of action and potency for BoNT/A subtypes. BoNTs are composed of three domains—a cell-binding domain, a translocation domain, and a catalytic domain. In this paper, we present the crystal structures of the botulinum neurotoxin A2 cell binding domain, both alone and in complex with its receptor ganglioside GD1a at 1.63 and 2.10 Å, respectively. The analysis of these structures reveals a potential redox-dependent Lys-O-Cys bridge close to the ganglioside binding site and a hinge motion between the H_CN and H_CC subdomains. Furthermore, we make a detailed comparison with the previously reported H_C/A2:SV2C structure for a comprehensive structural analysis of H_C/A2 receptor binding.     

Recommended Immunological Strategies to Screen for Botulinum Neurotoxin-Containing Samples

Botulinum neurotoxins (BoNTs) cause the life-threatening neurological illness botulism in humans and animals and are divided into seven serotypes (BoNT/A–G), of which serotypes A, B, E, and F cause the disease in humans. BoNTs are classified as “category A” bioterrorism threat agents and are relevant in the context of the Biological Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection, quantification and discrimination capabilities of 23 expert laboratories from the health, food and security areas. Here we describe three immunological strategies that proved to be successful for the detection and quantification of BoNT/A, B, and E considering the restricted sample volume (1 mL) distributed. To analyze the samples qualitatively and quantitatively, the first strategy was based on sensitive immunoenzymatic and immunochromatographic assays for fast qualitative and quantitative analyses. In the second approach, a bead-based suspension array was used for screening followed by conventional ELISA for quantification. In the third approach, an ELISA plate format assay was used for serotype specific immunodetection of BoNT-cleaved substrates, detecting the activity of the light chain, rather than the toxin protein. The results provide guidance for further steps in quality assurance and highlight problems to address in the future.     

Development of sensitive colorimetric capture elisas for Clostridium botulinum neurotoxin serotypes A and B.

Sensitive and specific enzyme-linked immunosorbent assays were developed to detect Clostridium botulinum neurotoxin serotypes A (BoNT A) and B (BoNT B) in assay buffer and human serum. The assay is based upon affinity-purified horse polyclonal antibodies directed against the approximately 50 kDa C-fragments of each toxin. Standard curves were linear over the range of 0.1-10 ng mL. Detection was possible at 0.2 ng mL (20 pg/well) and accurate quantitation at 0.5 ng/mL (50 pg well) in assay buffer and 10% human serum. Variations between triplicates was typically 5-10%. Less than 1% cross reactivity occurred between other serotypes when each assay was performed against serotypes A, B and E. When tested against toxins complexed to their associated nontoxic proteins, interference was absent (BoNT B) or < 25% (BoNT A). These assays demonstrate sensitivity close to that of the mouse bioassay without the use of animals and in a much simpler format than other reported assays of similar sensitivity.     

Central injection of botulinum neurotoxins: behavioural effects in mice

Strains of Clostridium botulinum produce seven antigenically distinct botulinum neurotoxins (BoNTs) designated as serotypes A-G. All serotypes interfere with neural transmission by blocking the release of acetylcholine in cholinergic neurons. They cleave specific sites on proteins of the SNARE [soluble n-ethylmaleimide-sensitive factor (NSF) attachment protein receptor] complex, which play a key role in neuroexocytosis. This study assessed the behavioural effects due to central administration of BoNTs in mice. CD1 mice were injected intracerebroventricularly (icv) with sub-lethal doses of BoNT/A or /B and their behavioural responses in conditioning of active avoidance, object recognition test and pharmacologically induced locomotor activity were tested. Compared to control mice, BoNT-treated mice showed: (1) a reduced capacity to discriminate a novel object within a familiar environment; (2) an enhanced stimulant effect by scopolamine and a depressant effect by oxotremorine on locomotor activity. In contrast, central injection of BoNTs did not alter active avoidance acquisition. These results suggest an in vivo functional alteration due to the action of BoNTs directly administered into the central nervous system. The present data demonstrate that BoNTs may represent an analytical tool for studying the functional role of cholinergic neurons.     

Development of sensitive colorimetric capture ELISAs for Clostridium botulinum neurotoxin serotypes E and F.

Sensitive and specific enzyme-linked immunosorbent assays (ELISAs) were developed to detect Clostridium botulinum neurotoxin serotypes E (BoNT E) and F (BoNT F) in assay buffer and human serum. The assay is based upon affinity-purified horse polyclonal antibodies directed against the approximately 50 kD C-fragments of each toxin. Standard curves were linear over 0.5-10 ng/ml (BoNT E) or 2-20 ng/ml (BoNT F). Accurate measurements were achieved at 0.5 ng/ml (BoNT E) or 2 ng/ml (BoNT F) in assay buffer and 10% human serum. Variation between triplicates was typically 5-10%. Less than 1% cross-reactivity occurred between other serotypes A, B, E or F). When tested against toxins complexed to their neurotoxin-associated proteins, interference was absent for BoNT F. However, pure BoNT E and that complexed to associated proteins demonstrated significant quantitative differences. We believe these differences arise from trypsin activation of the toxin. These assays demonstrated sensitivities close to that of the mouse bioassay, without the use of animals, in a much simpler format than other reported assays of similar sensitivity.     

Camelid single domain antibodies (VHHs) as neuronal cell intrabody binding agents and inhibitors of Clostridium botulinum neurotoxin (BoNT) proteases

Botulinum neurotoxins (BoNTs) function by delivering a protease to neuronal cells that cleave SNARE proteins and inactivate neurotransmitter exocytosis. Small (14 kDa) binding domains specific for the protease of BoNT serotypes A or B were selected from libraries of heavy chain only antibody domains (VHHs or nanobodies) cloned from immunized alpacas. Several VHHs bind the BoNT proteases with high affinity (K_D near 1 nM) and include potent inhibitors of BoNT/A protease activity (K_i near 1 nM). The VHHs retain their binding specificity and inhibitory functions when expressed within mammalian neuronal cells as intrabodies. A VHH inhibitor of BoNT/A protease was able to protect neuronal cell SNAP25 protein from cleavage following intoxication with BoNT/A holotoxin. These results demonstrate that VHH domains have potential as components of therapeutic agents for reversal of botulism intoxication.     

Recommended Mass Spectrometry-Based Strategies to Identify Botulinum Neurotoxin-Containing Samples

Botulinum neurotoxins (BoNTs) cause the disease called botulism, which can be lethal. BoNTs are proteins secreted by some species of clostridia and are known to cause paralysis by interfering with nerve impulse transmission. Although the human lethal dose of BoNT is not accurately known, it is estimated to be between 0.1 μg to 70 μg, so it is important to enable detection of small amounts of these toxins. Our laboratory previously reported on the development of Endopep-MS, a mass-spectrometric‑based endopeptidase method to detect, differentiate, and quantify BoNT immunoaffinity purified from complex matrices. In this work, we describe the application of Endopep-MS for the analysis of thirteen blinded samples supplied as part of the EQuATox proficiency test. This method successfully identified the presence or absence of BoNT in all thirteen samples and was able to successfully differentiate the serotype of BoNT present in the samples, which included matrices such as buffer, milk, meat extract, and serum. Furthermore, the method yielded quantitative results which had z-scores in the range of −3 to +3 for quantification of BoNT/A containing samples. These results indicate that Endopep-MS is an excellent technique for detection, differentiation, and quantification of BoNT in complex matrices.     

Challenges in searching for therapeutics against Botulinum Neurotoxins

Introduction: Botulinum neurotoxins (BoNTs) are the most potent toxins known. BoNTs are responsible for botulism, a deadly neuroparalytic syndrome caused by the inactivation of neurotransmitter release at peripheral nerve terminals. Thanks to their specificity and potency, BoNTs are both considered potential bio-weapons and therapeutics of choice for a variety of medical syndromes. Several variants of BoNTs have been identified with individual biological properties and little antigenic relation. This expands greatly the potential of BoNTs as therapeutics but poses a major safety problem, increasing the need for finding appropriate antidotes.

Areas covered: The authors describe the multi-step molecular mechanism through which BoNTs enter nerve terminals and discuss the many levels at which the toxins can be inhibited. They review the outcomes of the different strategies adopted to limit neurotoxicity and counter intoxication. Potential new targets arising from the last discoveries of the mechanism of action and the approaches to promote neuromuscular junction recovery are also discussed.

Expert opinion: Current drug discovery efforts have mainly focused on BoNT type A and addressed primarily light chain proteolytic activity. Development of pan-BoNT inhibitors acting independently of BoNT immunological properties and targeting a common step of the intoxication process should be encouraged.     

A protein chip membrane-capture assay for botulinum neurotoxin activity

Botulinum neurotoxins A and B (BoNT/A and B) are neuromuscular blocking agents which inhibit neurotransmission by cleaving the intra-cellular presynaptic SNARE proteins SNAP-25 and VAMP2, localized respectively in plasma membrane and synaptic vesicles. These neurotoxins are both dangerous pathogens and powerful therapeutic agents with numerous clinical and cosmetic applications. Consequently there is a need for in vitro assays of their biological activity to screen for potential inhibitors and to replace the widely used in vivo mouse assay. Surface plasmon resonance (SPR) was used to measure membrane vesicle capture by antibodies against SNAP-25 and VAMP2. Substrate cleavage by BoNTs modified capture providing a method to assay toxin activity. Firstly using synaptic vesicles as a substrate, a comparison of the EC₅₀s for BoNT/B obtained by SPR, ELISA or flow cytometry indicated similar sensitivity although SPR assays were more rapid. Sonication of brain or neuronal cultures generated plasma membrane fragments with accessible intra-cellular epitopes adapted to measurement of BoNT/A activity. SPR responses were proportional to antigen concentration permitting detection of as little as 4 pM SNAP-25 in crude lysates. BoNT/A activity was assayed using monoclonal antibodies that specifically recognize a SNAP-25 epitope generated by the proteolytic action of the toxin. Incubation of intact primary cultured neurons with BoNT/A yielded an EC₅₀ of 0.5 pM. The SPR biosensor method was sensitive enough to monitor BoNT/A and B activity in cells cultured in a 96-well format providing an alternative to experimental animals for toxicological assays.     

Generation and Characterization of Six Recombinant Botulinum Neurotoxins as Reference Material to Serve in an International Proficiency Test

The detection and identification of botulinum neurotoxins (BoNT) is complex due to the existence of seven serotypes, derived mosaic toxins and more than 40 subtypes. Expert laboratories currently use different technical approaches to detect, identify and quantify BoNT, but due to the lack of (certified) reference materials, analytical results can hardly be compared. In this study, the six BoNT/A1–F1 prototypes were successfully produced by recombinant techniques, facilitating handling, as well as improving purity, yield, reproducibility and biosafety. All six BoNTs were quantitatively nicked into active di-chain toxins linked by a disulfide bridge. The materials were thoroughly characterized with respect to purity, identity, protein concentration, catalytic and biological activities. For BoNT/A₁, B₁ and E₁, serotypes pathogenic to humans, the catalytic activity and the precise protein concentration were determined by Endopep-mass spectrometry and validated amino acid analysis, respectively. In addition, BoNT/A₁, B₁, E₁ and F₁ were successfully detected by immunological assays, unambiguously identified by mass spectrometric-based methods, and their specific activities were assigned by the mouse LD₅₀ bioassay. The potencies of all six BoNT/A1–F1 were quantified by the ex vivo mouse phrenic nerve hemidiaphragm assay, allowing a direct comparison. In conclusion, highly pure recombinant BoNT reference materials were produced, thoroughly characterized and employed as spiking material in a worldwide BoNT proficiency test organized by the EQuATox consortium.     

Clinical Uses of Botulinum Neurotoxins: Current Indications,Limitations and Future Developments

Botulinum neurotoxins (BoNTs) cause flaccid paralysis by interfering with vesicle fusion and neurotransmitter release in the neuronal cells. BoNTs are the most widely used therapeutic proteins. BoNT/A was approved by the U.S. FDA to treat strabismus, blepharospam, and hemificial spasm as early as 1989 and then for treatment of cervical dystonia, glabellar facial lines, axillary hyperhidrosis, chronic migraine and for cosmetic use. Due to its high efficacy, longevity of action and satisfactory safety profile, it has been used empirically in a variety of ophthalmological, gastrointestinal, urological, orthopedic, dermatological, secretory, and painful disorders. Currently available BoNT therapies are limited to neuronal indications with the requirement of periodic injections resulting in immune-resistance for some indications. Recent understanding of the structure-function relationship of BoNTs prompted the engineering of novel BoNTs to extend therapeutic interventions in non-neuronal systems and to overcome the immune-resistance issue. Much research still needs to be done to improve and extend the medical uses of BoNTs.