Genetic detoxification and adjuvant-activity retention of Escherichia coli enterotoxin LT

Intensive effort has been invested in the search for new effective vaccines that can be conveniently administered, with minimal handling of birds. Heat-labile enterotoxin (LT) of Escherichia coli H10407 is known as a powerful adjuvant for injection and oral administration. However, to use the toxin as an immunostimulator in animals, its toxicity must be neutralized. To this aim, we modified the LT gene by changing two amino acids (threonine 50 and valine 53) of the A subunit to proline. The modified non-toxic LT gene (nLT) was cloned and expressed in E. coli as a soluble protein. The protein was efficiently purified, reaching levels of about 20 mg active hexameric nLT per litre of induced culture. The mutated protein, nLT, and the wild-type protein (wtLT) were administered orally to chickens. Those treated with the wtLT exhibited diarrhoea, whereas chickens treated with the nLT showed no signs of disease compared with untreated birds. The new non-toxic, purified nLT stimulated antibody production in birds treated by injection or by oral administration. A field trial with layers that included a series of injections of Bovine Serum Albumin mixed with nLT showed this modified LT's ability to act as an adjuvant for the antigen mixed with it. This study demonstrates the efficient expression and purification of LT, in which toxicity was neutralized by genetic modification. Such an approach will enable the use of a non-toxic LT molecule with a modified A subunit by the poultry industry, to enhance immune responses against antigens co-vaccinated with it.