Development of Shigella sonnei live oral vaccines based on defined rfb(Inaba) deletion mutants of Vibro cholerae expressing the Shigella serotype D O polysaccharide

Previous experimentation has highlighted a number of difficulties in the development of carrier-based bivalent vaccines (J.-F. Viret and D. Favre, Biologicals 22:361-372, 1994). In an attempt to obviate these problems, we decided to express the Shigella sonnei serotype D O polysaccharide (O-PS) in rough Vibrio cholerae carrier strains. Toward this aim, a series of defined rfb(Inaba) deletion (Delta rfb(Inaba)) mutants of the cholera vaccine strain V. cholerae CVD103-HgR (O1 Inaba serotype) and derivatives bearing the chromosomally integrated locus encoding the S. sonnei O-PS were constructed and characterized, The various mutations disrupt genes thought to be involved in either the synthesis of perosamine, the synthesis of 3-deoxy-L-glycero tetronic acid, or the O-PS transport functions together with synthesis of the perosamine synthetase, Some deletions were obtained only in strains expressing the heterologous lipopolysaccharide (LPS). Viable Delta rfb(Inaba) deletions in CVD103-HgR profoundly altered some of its phenotypic properties, The same deletions present in CVD103-HgR derivatives expressing the heterologous LPS affected their phenotypes only to a lesser extent, Only in strains in which perosamine synthesis was specifically abolished could high amounts of core-bound S. sonnei O-PS be synthesized, Two such strains (CH21, which expresses both the R1 core and the S. sonnei O-PS, and CH22, which expresses only the latter antigenic determinant) were further analyzed and were found to be indistinguishable from CVD103-HgR with regard to lack of enterotoxin activity, choleragenoid production, mercury resistance, pilin production, and, for CH22, motility, Mice immunized with CH22 produced high titers of S. sonnei O-PS-specific antibodies.