LAUSR

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.Genetic toxoids avoid MAL for reduced host toxicityCells expressing myelin and lymphocyte protein (MAL), the putative receptor for Clostridium perfringens’ epsilon toxin, can be sensitive to otherwise attenuated mutants of the toxin. Here, the team led by Richard Titball at United Kingdom’s University of Exeter found that a previous variant exhibits differential toxic effects when cells express sheep or human MAL. To circumvent this, Titball’s team applied site-directed mutagenesis of the receptor binding site to develop a new variant with enhanced reduction in toxicity towards MAL-expressing cells and able to induce high levels of neutralising antibodies upon immunisation of sheep. These findings suggests that testing genetic toxoids in cells expressing MAL from the target species might be relevant for enterotoxaemia vaccine development and warrant further studies into the role of MAL in epsilon toxin-mediated pathogenesis.