Clostridium perfringens epsilon toxin vaccine candidate lacking toxicity to cells expressing myelin and lymphocyte protein

Helen Morcrette,Nick Lewis,Monika Bokori-Brown,Leo Bennett,Brendan W Wren,Richard W Titball,Stephanie Ong

doi:10.1038/s41541-019-0128-2

Helen Morcrette, Nick Lewis + Show 5 more

Open Access

https://doi.org/10.1038/s41541-019-0128-2

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Abstract

A variant form of Clostridium perfringens epsilon toxin (Y30A-Y196A) with mutations, which shows reduced binding to Madin–Darby canine kidney (MDCK) cells and reduced toxicity in mice, has been proposed as the next-generation enterotoxaemia vaccine. Here we show that, unexpectedly, the Y30A-Y196A variant does not show a reduction in toxicity towards Chinese hamster ovary (CHO) cells engineered to express the putative receptor for the toxin (myelin and lymphocyte protein; MAL). The further addition of mutations to residues in a second putative receptor binding site of the Y30A-Y196A variant further reduces toxicity, and we selected Y30A-Y196A-A168F for further study. Compared to Y30A-Y196A, Y30A-Y196A-A168F showed more than a 3-fold reduction in toxicity towards MDCK cells, more than a 4-fold reduction in toxicity towards mice and at least 200-fold reduction in toxicity towards CHO cells expressing sheep MAL. The immunisation of rabbits or sheep with Y30A-Y196A-A168F induced high levels of neutralising antibodies against epsilon toxin, which persisted for at least 1 year. Y30A-Y196A-A168F is a candidate for development as a next-generation enterotoxaemia vaccine.

Highlights

Epsilon toxin is one of the major toxins produced by Clostridium perfringens.[1]
Using a plasmid template, which encoded the Y30A-Y196A variant form of epsilon prototoxin,[20] residues V72, F92, H149, V166 or A168 were mutated to alanine (F92, H149, V166) or phenylalanine (V72, A168) and the His-tagged proteins encoded by the mutated genes were expressed in E. coli and purified (Fig. 1b)
We showed that a combination of Y30A and Y196A mutations markedly reduced the ability of the toxin to bind to and kill Madin–Darby canine kidney (MDCK) cells

Summary

Introduction

Epsilon toxin is one of the major toxins produced by Clostridium perfringens.[1] The toxin is produced by the bacterium as a prototoxin, which is activated by proteolysis, with the consequent release of carboxy-terminal and amino-terminal peptides from the protein.[2,3,4] This toxin is important in the field of veterinary medicine because it plays a major role in the development of enterotoxaemia of sheep and goats, and occasionally of other livestock animals. The disease is often called pulpy kidney, lesions in the kidneys of affected animals are not observed. Evidence has been presented that epsilon toxin may play a role in the development of multiple sclerosis in humans.[8,9]

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