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18 pages, 2211 KiB

Open AccessArticle

Optimization of SNAP-25 and VAMP-2 Cleavage by Botulinum Neurotoxin Serotypes A–F Employing Taguchi Design-of-Experiments

by Laura von Berg, Daniel Stern, Jasmin Weisemann, Andreas Rummel, Martin Bernhard Dorner and Brigitte Gertrud Dorner

Toxins 2019, 11(10), 588; https://doi.org/10.3390/toxins11100588 - 11 Oct 2019

Cited by 3 | Viewed by 3983

Abstract

The detection of catalytically active botulinum neurotoxins (BoNTs) can be achieved by monitoring the enzymatic cleavage of soluble NSF (N-ethylmaleimide-sensitive-factor) attachment protein receptor (SNARE) proteins by the toxins’ light chains (LC) in cleavage-based assays. Thus, for sensitive BoNT detection, optimal cleavage conditions for [...] Read more.

The detection of catalytically active botulinum neurotoxins (BoNTs) can be achieved by monitoring the enzymatic cleavage of soluble NSF (N-ethylmaleimide-sensitive-factor) attachment protein receptor (SNARE) proteins by the toxins’ light chains (LC) in cleavage-based assays. Thus, for sensitive BoNT detection, optimal cleavage conditions for the clinically relevant A–F serotypes are required. Until now, a systematic evaluation of cleavage conditions for the different BoNT serotypes is still lacking. To address this issue, we optimized cleavage conditions for BoNT/A–F using the Taguchi design-of-experiments (DoE) method. To this aim, we analyzed the influence of buffer composition (pH, Zn²⁺, DTT (dithiothreitol), NaCl) as well as frequently used additives (BSA (bovine serum albumin), Tween 20, trimethylamine N-oxide (TMAO)) on BoNT substrate cleavage. We identified major critical factors (DTT, Zn²⁺, TMAO) and were able to increase the catalytic efficiency of BoNT/B, C, E, and F when compared to previously described buffers. Moreover, we designed a single consensus buffer for the optimal cleavage of all tested serotypes. Our optimized buffers are instrumental to increase the sensitivity of cleavage-based assays for BoNT detection. Furthermore, the application of the Taguchi DoE approach shows how the method helps to rationally improve enzymatic assays. Full article

(This article belongs to the Special Issue Characterization and Quantitative Analysis of Botulinum Neurotoxin)

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14 pages, 1622 KiB

Open AccessArticle

Development and Characterization of Monoclonal Antibodies to Botulinum Neurotoxin Type E

by Candace S. Bever, Miles Scotcher, Luisa W. Cheng, Robert M. Hnasko and Larry H. Stanker

Toxins 2019, 11(7), 407; https://doi.org/10.3390/toxins11070407 - 13 Jul 2019

Cited by 3 | Viewed by 5880

Abstract

Botulism is a devastating disease caused by botulinum neurotoxins (BoNTs) secreted primarily by Clostridium botulinum. Mouse bioassays without co-inoculation with antibodies are the standard method for the detection of BoNTs, but are not capable of distinguishing between the different serotypes (A–G). Most [...] Read more.

Botulism is a devastating disease caused by botulinum neurotoxins (BoNTs) secreted primarily by Clostridium botulinum. Mouse bioassays without co-inoculation with antibodies are the standard method for the detection of BoNTs, but are not capable of distinguishing between the different serotypes (A–G). Most foodborne intoxications are caused by serotypes BoNT/A and BoNT/B. BoNT/E outbreaks are most often observed in northern coastal regions and are associated with eating contaminated marine animals and other fishery products. Sandwich enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of BoNT/E3. Monoclonal antibodies (mAbs) were generated against BoNT/E3 by immunizing with recombinant peptide fragments of the light and heavy chains of BoNT/E3. In all, 12 mAbs where characterized for binding to both the recombinant peptides and holotoxin, as well as their performance in Western blots and sandwich ELISAs. The most sensitive sandwich assay, using different mAbs for capture and detection, exhibited a limit of detection of 0.2 ng/ml in standard buffer matrix and 10 ng/mL in fish product matrices. By employing two different mAbs for capture and detection, a more standardized sandwich assay was constructed. Development of sensitive and selective mAbs to BoNT/E would help in the initial screening of potential food contamination, speeding diagnosis and reducing use of laboratory animals. Full article

(This article belongs to the Special Issue Characterization and Quantitative Analysis of Botulinum Neurotoxin)

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12 pages, 756 KiB

Open AccessArticle

Content/Potency Assessment of Botulinum Neurotoxin Type-A by Validated Liquid Chromatography Methods and Bioassays

by Bruna Xavier, Rafaela Ferreira Perobelli, Maurício Elesbão Walter, Francielle Santos da Silva and Sérgio Luiz Dalmora

Toxins 2019, 11(1), 35; https://doi.org/10.3390/toxins11010035 - 12 Jan 2019

Cited by 4 | Viewed by 5147

Abstract

Botulinum neurotoxin type-A (BoNTA) is one of the seven different serotypes (A to G) produced by Clostridium botulinum. A stability-indicating size-exclusion chromatography (SEC) method was developed and validated, and the specificity was confirmed by forced degradation study, interference of the excipients, and peaks [...] Read more.

Botulinum neurotoxin type-A (BoNTA) is one of the seven different serotypes (A to G) produced by Clostridium botulinum. A stability-indicating size-exclusion chromatography (SEC) method was developed and validated, and the specificity was confirmed by forced degradation study, interference of the excipients, and peaks purity. The method was applied to assess the content and high-molecular-weight (HMW) forms of BoNTA in biopharmaceutical products, and the results were compared with those of the LD₅₀ mouse bioassay, the T−47D cell culture assay, and the reversed-phase chromatography (RPC) method, giving mean values of 0.71% higher, 0.36% lower, and 0.87% higher, respectively. Aggregated forms showed significant effects on cytotoxicity, as well as a decrease in the bioactivity (p < 0.05). The employment of the proposed method in conjunction with the optimized analytical technologies for the analysis of the intact and altered forms of the biotechnology-derived medicines, in the correlation studies, enabled the demonstration of the capability of each one of the methods and allowed for great improvements, thereby assuring their safe and effective use. Full article

(This article belongs to the Special Issue Characterization and Quantitative Analysis of Botulinum Neurotoxin)

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Review

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23 pages, 738 KiB

Open AccessReview

Critical Analysis of Neuronal Cell and the Mouse Bioassay for Detection of Botulinum Neurotoxins

by Sabine Pellett, William H. Tepp and Eric A. Johnson

Toxins 2019, 11(12), 713; https://doi.org/10.3390/toxins11120713 - 7 Dec 2019

Cited by 17 | Viewed by 5945

Abstract

Botulinum Neurotoxins (BoNTs) are a large protein family that includes the most potent neurotoxins known to humankind. BoNTs delivered locally in humans at low doses are widely used pharmaceuticals. Reliable and quantitative detection of BoNTs is of paramount importance for the clinical diagnosis [...] Read more.

Botulinum Neurotoxins (BoNTs) are a large protein family that includes the most potent neurotoxins known to humankind. BoNTs delivered locally in humans at low doses are widely used pharmaceuticals. Reliable and quantitative detection of BoNTs is of paramount importance for the clinical diagnosis of botulism, basic research, drug development, potency determination, and detection in clinical, environmental, and food samples. Ideally, a definitive assay for BoNT should reflect the activity of each of the four steps in nerve intoxication. The in vivo mouse bioassay (MBA) is the ‘gold standard’ for the detection of BoNTs. The MBA is sensitive, robust, semi-quantitative, and reliable within its sensitivity limits. Potential drawbacks with the MBA include assay-to-assay potency variations, especially between laboratories, and false positives or negatives. These limitations can be largely avoided by careful planning and performance. Another detection method that has gained importance in recent years for research and potency determination of pharmaceutical BoNTs is cell-based assays, as these assays can be highly sensitive, quantitative, human-specific, and detect fully functional holotoxins at physiologically relevant concentrations. A myriad of other in vitro BoNT detection methods exist. This review focuses on critical factors and assay limitations of the mouse bioassay and cell-based assays for BoNT detection. Full article

(This article belongs to the Special Issue Characterization and Quantitative Analysis of Botulinum Neurotoxin)

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12 pages, 311 KiB

Open AccessEditor’s ChoiceReview

Tables of Toxicity of Botulinum and Tetanus Neurotoxins

by Ornella Rossetto and Cesare Montecucco

Toxins 2019, 11(12), 686; https://doi.org/10.3390/toxins11120686 - 22 Nov 2019

Cited by 77 | Viewed by 11449

Abstract

Tetanus and botulinum neurotoxins are the most poisonous substances known, so much so as to be considered for a possible terrorist use. At the same time, botulinum neurotoxin type A1 is successfully used to treat a variety of human syndromes characterized by hyperactive [...] Read more.

Tetanus and botulinum neurotoxins are the most poisonous substances known, so much so as to be considered for a possible terrorist use. At the same time, botulinum neurotoxin type A1 is successfully used to treat a variety of human syndromes characterized by hyperactive cholinergic nerve terminals. The extreme toxicity of these neurotoxins is due to their neurospecificity and to their metalloprotease activity, which results in the deadly paralysis of tetanus and botulism. Recently, many novel botulinum neurotoxins and some botulinum-like toxins have been discovered. This large number of toxins differs in terms of toxicity and biological activity, providing a potential goldmine for novel therapeutics and for new molecular tools to dissect vesicular trafficking, fusion, and exocytosis. The scattered data on toxicity present in the literature require a systematic organization to be usable by scientists and clinicians. We have assembled here the data available in the literature on the toxicity of these toxins in different animal species. The internal comparison of these data provides insights on the biological activity of these toxins. Full article

(This article belongs to the Special Issue Characterization and Quantitative Analysis of Botulinum Neurotoxin)

23 pages, 951 KiB

Open AccessEditor’s ChoiceReview

Antibodies and Vaccines against Botulinum Toxins: Available Measures and Novel Approaches

by Christine Rasetti-Escargueil and Michel R. Popoff

Toxins 2019, 11(9), 528; https://doi.org/10.3390/toxins11090528 - 12 Sep 2019

Cited by 38 | Viewed by 11135

Abstract

Botulinum neurotoxin (BoNT) is produced by the anaerobic, Gram-positive bacterium Clostridium botulinum. As one of the most poisonous toxins known and a potential bioterrosism agent, BoNT is characterized by a complex mode of action comprising: internalization, translocation and proteolytic cleavage of a [...] Read more.

Botulinum neurotoxin (BoNT) is produced by the anaerobic, Gram-positive bacterium Clostridium botulinum. As one of the most poisonous toxins known and a potential bioterrosism agent, BoNT is characterized by a complex mode of action comprising: internalization, translocation and proteolytic cleavage of a substrate, which inhibits synaptic exocytotic transmitter release at neuro-muscular nerve endings leading to peripheral neuroparalysis of the skeletal and autonomic nervous systems. There are seven major serologically distinct toxinotypes (A–G) of BoNT which act on different substrates. Human botulism is generally caused by BoNT/A, B and E. Due to its extreme lethality and potential use as biological weapon, botulism remains a global public health concern. Vaccination against BoNT, although an effective strategy, remains undesirable due to the growing expectation around therapeutic use of BoNTs in various pathological conditions. This review focuses on the current approaches for botulism control by immunotherapy, highlighting the future challenges while the molecular underpinnings among subtypes variants and BoNT sequences found in non-clostridial species remain to be elucidated. Full article

(This article belongs to the Special Issue Characterization and Quantitative Analysis of Botulinum Neurotoxin)

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29 pages, 2681 KiB

Open AccessReview

Vaccine Production to Protect Animals Against Pathogenic Clostridia

by Nicolas E. Zaragoza, Camila A. Orellana, Glenn A. Moonen, George Moutafis and Esteban Marcellin

Toxins 2019, 11(9), 525; https://doi.org/10.3390/toxins11090525 - 11 Sep 2019

Cited by 45 | Viewed by 15447

Abstract

Clostridium is a broad genus of anaerobic, spore-forming, rod-shaped, Gram-positive bacteria that can be found in different environments all around the world. The genus includes human and animal pathogens that produce potent exotoxins that cause rapid and potentially fatal diseases responsible for countless [...] Read more.

Clostridium is a broad genus of anaerobic, spore-forming, rod-shaped, Gram-positive bacteria that can be found in different environments all around the world. The genus includes human and animal pathogens that produce potent exotoxins that cause rapid and potentially fatal diseases responsible for countless human casualties and billion-dollar annual loss to the agricultural sector. Diseases include botulism, tetanus, enterotoxemia, gas gangrene, necrotic enteritis, pseudomembranous colitis, blackleg, and black disease, which are caused by pathogenic Clostridium. Due to their ability to sporulate, they cannot be eradicated from the environment. As such, immunization with toxoid or bacterin-toxoid vaccines is the only protective method against infection. Toxins recovered from Clostridium cultures are inactivated to form toxoids, which are then formulated into multivalent vaccines. This review discusses the toxins, diseases, and toxoid production processes of the most common pathogenic Clostridium species, including Clostridium botulinum, Clostridium tetani, Clostridium perfringens, Clostridium chauvoei, Clostridium septicum, Clostridium novyi and Clostridium hemolyticum. Full article

(This article belongs to the Special Issue Characterization and Quantitative Analysis of Botulinum Neurotoxin)

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22 pages, 2742 KiB

Open AccessEditor’s ChoiceReview

Immunogenicity Associated with Botulinum Toxin Treatment

by Steven Bellows and Joseph Jankovic

Toxins 2019, 11(9), 491; https://doi.org/10.3390/toxins11090491 - 26 Aug 2019

Cited by 114 | Viewed by 13063

Abstract

Botulinum toxin (BoNT) has been used for the treatment of a variety of neurologic, medical and cosmetic conditions. Two serotypes, type A (BoNT-A) and type B (BoNT-B), are currently in clinical use. While considered safe and effective, their use has been rarely complicated [...] Read more.

Botulinum toxin (BoNT) has been used for the treatment of a variety of neurologic, medical and cosmetic conditions. Two serotypes, type A (BoNT-A) and type B (BoNT-B), are currently in clinical use. While considered safe and effective, their use has been rarely complicated by the development of antibodies that reduce or negate their therapeutic effect. The presence of antibodies has been attributed to shorter dosing intervals (and booster injections), higher doses per injection cycle, and higher amounts of antigenic protein. Other factors contributing to the immunogenicity of BoNT include properties of each serotype, such as formulation, manufacturing, and storage of the toxin. Some newer formulations with purified core neurotoxin devoid of accessory proteins may have lower overall immunogenicity. Several assays are available for the detection of antibodies, including both structural assays such as ELISA and mouse-based bioassays, but there is no consistent correlation between these antibodies and clinical response. Prevention and treatment of antibody-associated non-responsiveness is challenging and primarily involves the use of less immunogenic formulations of BoNT, waiting for the spontaneous disappearance of the neutralizing antibody, and switching to an immunologically alternate type of BoNT. Full article

(This article belongs to the Special Issue Characterization and Quantitative Analysis of Botulinum Neurotoxin)

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