Fig. 1. Schematic representation of the manufacturing process of probiotic E. coli MVs expressing pneumococcal CPS14.

A large quantity of MVs with reduced endotoxin content was isolated from the supernatant of a flagella-deficient probiotic E. coli clone (EcN ΔflhD) cultured in LB broth supplemented with 1% glycine^16,17. A pneumococcal CPS14 expression vector containing the entire CPS biosynthesis-related gene locus¹⁰ was introduced into the EcN ΔflhD strain. The resultant MVs expressed a chimeric LPS in which an E. coli O6 antigen repeating unit was replaced by a pneumococcal CPS composed of the serotype 14-specific tetra-saccharide repeating unit galactose-1-phophate-(β1-4)-N-acetylglucosamine-1-phosphate-(β1-3)-galactose-1-phophate-(β1-4)-glucose-¹⁸.