Immune responses to epitopes inserted in Salmonella flagellin

Abstract

Plasmid pLS408 includes gene fliC(d) specifying Salmonella flagellin of antigenic type d with an in vitro deletion of a 48 base-pair EcoRV fragment in its central hypervariable antigenically-determinant region IV. Oligonucleotides specifying peptide epitopes of antigens of unrelated pathogens inserted, in correct orientation, at the unique EcoRV site of pLS408 specify chimeric flagellins and, in many instances, cause production of functional flagella when the plasmid is placed in a flagellin-deficient delta aroA live-vaccine strain of Salmonella dublin. The foreign epitope is then exposed at the surface of the flagellar filaments, as shown by the immobilizing effect of anti-epitope antibody and by immunogold electron-microscopy. The live-vaccine strain with a foreign epitope at the surface of its flagella when administered to mice by injection nearly always causes production of antibody with affinity for the foreign epitope and, sometimes, also for the source protein. Repeated injection of the live vaccine with an epitope of Streptococcus pyogenes type 5 M protein as insert caused production of opsonizing antibody and conferred partial protection against Streptococcus challenge. Injection of semi-purified chimeric flagella or flagellin, alone or with adjuvant, likewise causes antibody production, in one instance sufficient to give partial protection against influenza A virus challenge. Plasmid pLS408 with some inserts does not confer motility, either because the filaments produced are non-functional or because flagellin is made but not assembled or because little or no flagellin is produced. The features of a sequence which as insert determine production or non-production of functional flagella are not known. The effect of insertion of known T-cell epitopes and cellular immune responses to epitope inserts in flagellin are as yet little explored.