In vitro tests for the measurement of clostridial toxins, toxoids and antisera

Knight, Pamela; Queminet, J.; Blanchard, J.H.; Tilleray, J.H.

doi:10.1016/1045-1056(90)90028-x

Cited by 26 publications

(3 citation statements)

References 4 publications

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“…Relatively few cell lines are susceptible to the toxin and most in vitro studies on Etx have been carried out using the highly susceptible Madin-Darby Canine Kidney (MDCK) cell line 24, 25. Other toxin-sensitive cell lines include the mouse kidney cell line mpkCCD cl4 23 and the recently identified human renal adenocarcinoma cell line ACHN 26.…”

Section: Introductionmentioning

confidence: 99%

Clostridium perfringens epsilon toxin H149A mutant as a platform for receptor binding studies

et al. 2013

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Clostridium perfringens epsilon toxin (Etx) is a pore-forming toxin responsible for a severe and rapidly fatal enterotoxemia of ruminants. The toxin is classified as a category B bioterrorism agent by the U.S. Government Centres for Disease Control and Prevention (CDC), making work with recombinant toxin difficult. To reduce the hazard posed by work with recombinant Etx, we have used a variant of Etx that contains a H149A mutation (Etx-H149A), previously reported to have reduced, but not abolished, toxicity. The three-dimensional structure of H149A prototoxin shows that the H149A mutation in domain III does not affect organisation of the putative receptor binding loops in domain I of the toxin. Surface exposed tyrosine residues in domain I of Etx-H149A (Y16, Y20, Y29, Y30, Y36 and Y196) were mutated to alanine and mutants Y30A and Y196A showed significantly reduced binding to MDCK.2 cells relative to Etx-H149A that correlated with their reduced cytotoxic activity. Thus, our study confirms the role of surface exposed tyrosine residues in domain I of Etx in binding to MDCK cells and the suitability of Etx-H149A for further receptor binding studies. In contrast, binding of all of the tyrosine mutants to ACHN cells was similar to that of Etx-H149A, suggesting that Etx can recognise different cell surface receptors. In support of this, the crystal structure of Etx-H149A identified a glycan (β-octyl-glucoside) binding site in domain III of Etx-H149A, which may be a second receptor binding site. These findings have important implications for developing strategies designed to neutralise toxin activity.

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Section: Introductionmentioning

confidence: 99%

Clostridium perfringens epsilon toxin H149A mutant as a platform for receptor binding studies

et al. 2013

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“…Most cell lines tested are resistant to ε-toxin [ 25 ]. Some are highly sensitive to the toxin, for example Madin Darby Canine Kidney (MDCK) cells [ 26 ], with a CT 50 dose of 1–10 nM [ 27 , 28 ] and MOLT-4 human T cells with a CT 50 dose of 11 nM [ 28 ]. Aerolysin is active towards a broad range of cell lines, and when tested using a sensitive cell line, aerolysin is a more potent cytolytic agent than ε-toxin with EL4 T-cells killed by concentrations as low as 10 pM [ 24 ].…”

Section: The Receptor-binding Domainmentioning

confidence: 99%

The Molecular Architecture and Mode of Action of Clostridium perfringens ε-Toxin

Titball

2024

Toxins

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Clostridium perfringens ε-toxin has long been associated with a severe enterotoxaemia of livestock animals, and more recently, was proposed to play a role in the etiology of multiple sclerosis in humans. The remarkable potency of the toxin has intrigued researchers for many decades, who suggested that this indicated an enzymatic mode of action. Recently, there have been major breakthroughs by finding that it is a pore-forming toxin which shows exquisite specificity for cells bearing the myelin and lymphocyte protein (MAL) receptor. This review details the molecular structures of the toxin, the evidence which identifies MAL as the receptor and the possible roles of other cell membrane components in toxin binding. The information on structure and mode of action has allowed the functions of individual amino acids to be investigated and has led to the creation of mutants with reduced toxicity that could serve as vaccines. In spite of this progress, there are still a number of key questions around the mode of action of the toxin which need to be further investigated.

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“…The potency of the final product is often quantified by the induction of antibodies in rabbits, with the neutralizing capacities of the induced antibodies assessed in naïve mice. All these tests all have lethal endpoints, for which the btoxin is responsible (17), except for the rabbits, and inflict severe suffering.…”

Section: Introductionmentioning

confidence: 99%

An in vitro assay for toxicity testing of Clostridium perfringens type C β-toxin

Hoonakker,

Zariri,

de Brouwer

et al. 2024

Front. Immunol.

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IntroductionVeterinary vaccines against Clostridium perfringens type C need to be tested for absence of toxicity, as mandated by pharmacopoeias worldwide. This toxicity testing is required at multiple manufacturing steps and relies on outdated mouse tests that involve severe animal suffering. Clostridium perfringens type C produces several toxins of which the β-toxin is the primary component responsible for causing disease. Here, we describe the successful development of a new cell-based in vitro assay that can address the specific toxicity of the β-toxin.MethodsDevelopment of the cell-based assay followed the principle of in vitro testing developed for Cl. septicum vaccines, which is based on Vero cells. We screened four cell lines and selected the THP-1 cell line, which was shown to be the most specific and sensitive for β-toxin activity, in combination with a commercially available method to determine cell viability (MTS assay) as a readout. ResultsThe current animal test is estimated to detect 100 – 1000-fold dilutions of the Cl. perfringens type C non-inactivated antigen. When tested with an active Cl. perfringens type C antigen preparation, derived from a commercial vaccine manufacturing process, our THP-1 cell-based assay was able to detect toxin activity from undiluted to over 10000-fold dilution, showing a linear range between approximately 1000- and 10000-fold dilutions. Assay specificity for the β-toxin was confirmed with neutralizing antibodies and lack of reaction to Cl. perfringens culture medium. In addition, assay parameters demonstrated good repeatability.ConclusionsHere, we have shown proof of concept for a THP-1 cell-based assay for toxicity testing of veterinary Cl. perfringens type C vaccines that is suitable for all vaccine production steps. This result represents a significant step towards the replacement of animal-based toxicity testing of this veterinary clostridial antigen. As a next step, assessment of the assay’s sensitivity and repeatability and validation of the method will have to be performed in a commercial manufacturing context in order to formally implement the assay in vaccine quality control.

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In vitro tests for the measurement of clostridial toxins, toxoids and antisera

Cited by 26 publications

References 4 publications

Clostridium perfringens epsilon toxin H149A mutant as a platform for receptor binding studies

Clostridium perfringens epsilon toxin H149A mutant as a platform for receptor binding studies

The Molecular Architecture and Mode of Action of Clostridium perfringens ε-Toxin

An in vitro assay for toxicity testing of Clostridium perfringens type C β-toxin

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