The structures of the polysaccharide part of lipopolysaccharides isolated from eight Escherichia coli K12lShigella dysenteriae type 1 hybrids have been determined using sugar and methylation analysis plus 'H-and I3C-nuclear magnetic resonance spectroscopy. The hybrids express parts of the S. dysenteriae type 1 0-antigen tetrasaccharide repeating unit because of the presence of pSS3, a plasmid expressing an a-galactosyl : lipopolysaccharide transferase and pSS9, a pBR322 plasmid expressing S. dysenteriae type 1 rj+h genes. The various classes of hybrids are the result of transposon Tn 1000 insertions in pSS9 inactivating different rj+h genes. The following structural elements were found.E. coli K12 (pSS3) and E. coli K12 (pSS3, pSS9-6; a class I hybrid); a-D-Galp(l+3)P-~-GlcpNAc(l+. Class IV hybrids: E. coli K12 (pSS3, pSS9-36); (pSS3, pSS9-107) and (pSS3, pSS9-114); a-~-Rhap(l+2)a-~-Galp(l+3)/?-~-GlcpNAc(l+. Class V hybrids: E. coli K12 (pSS3, pSS9-78) and (pSS3, pSS9-111); a-~-Rhap(l+3)a-~-Rhap(l+2 )a-~-Galp(l+3)~-~-GlcpNAc(l+.
Vaccine candidates against Shigella dysenteriae type 1, which is associated with the most severe cases of bacillary dysentery, were constructed. The rfp and rfb gene clusters, which code for S. dysenteriae 1 O antigen biosynthesis, were randomly integrated into either the chromosome or the virulence plasmid of the rough attenuated Shigella flexneri aroD strain SFL124-27 with a minitransposon carrying an arsenite resistance selection marker. The recombinant clones efficiently expressed the recombinant O antigen, exhibited a normal growth pattern, were able to invade and survive within eukaryotic cells to the same extent as the parental strain, and expressed the recombinant antigen within invaded cells. A clone was selected as the vaccine candidate, which was demonstrated to be immunogenic and safe in animal models, leading to 47% full protection and 53% partial protection against challenge with the wild-type strain.
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