A 140-megadalton plasmid (pWR110), which has previously been associated with virulence in Shigella flexneri, was transferred to Escherichia coli K-12. Segments of S. flexneri chromosomal material were then transferred to the plasmid-bearing K-12 strains. The virulence of these transconjugant hybrids was assessed in the HeLa cell model, in ligated rabbit ileal loops, or in the Sereny test. A K-12 strain which harbored only pWR110 invaded HeLa cells, produced minimal lesions in the rabbit ileal mucosa, and was negative in the Sereny test. Plasmid-containing K-12 hybrids which had incorporated various shigella chromosomal regions gave differential reactions in the rabbit ileal loops and in the Sereny test. Analysis of these transconjugants indicated that three regions were linked with virulent phenotypes. These included the his region (when the genes responsible for 0-antigen synthesis were cotransferred) and the kcp locus (linked to the lac-gal region). Either of these chromosomal regions was sufficient to allow invasion of the rabbit ileal mucosa. In addition to both of these regions, another shigella chromosomal segment linked to the arg and mtl loci was necessary for fluid production in the rabbit ileal loop and for a positive Sereny reaction. Thus, derivatives of an E. coli K-12 strain, constructed by the stepwise conjugal transfer of a large plasmid and three chromosomal segments from S. flexneri, appeared to contain the necessary determinants for full pathogenicity in a variety of laboratory models.

A living oral vaccine, designed to protect against Shigella flexneri 2a infections, was constructed by using Escherichia coli K-12 as a carrier strain. The hybrid strain, designated EC104, contained both chromosomal and plasmid genes from S. flexneri donor strains, In addition to expressing the S. flexneri 2a somatic antigen, it had inherited the property of epithelial-celi invasion. After the oral administration to rhesus monkeys, EC104 was isolated from the feces for up to 3 days, but by day 4 all stool cultures were negative. The serum antibody response against S. flexneri 2a somatic antigen was variable, but the vaccine conferred significant protection against an oral challenge with virulent S. flexneri 2a.

The form-1 antigen of Shigella sonnei was transferred to the avirulent Salmonella typhi strain 21a and the resulting 5076-1C transconjugate strain was tested for safety and immunogenicity as a candidate oral vaccine. The transconjugant strain was shown to be well tolerated and safe in 19 human volunteers who were fed from 1 X 10(7) to 1 X 10(10) organisms. Only two of 10 volunteers tested had developed a rise in antibody titer to the lipopolysaccharide of the hybrid 5076-1C strain.

The requirement for both plasmid and chromosomal genes in the biosynthesis of Shigella dysenteriae 1 lipopolysaccharide O antigen was demonstrated in Escherichia coli-Shigella hybrids. A 6-megadalton S. dysenteriae 1 plasmid, designated pWR23, was phenotypically tagged with the Tn3 ampicillin-resistance transposon. The tagged plasmid, designated pWR24, was transferred by transformation or conjugal mobilization to a rough E. coli K-12 recipient. Although the resultant hybrids were agglutinated in S. dysenteriae 1 antiserum, they did not remove all of the anti-Shiga agglutinins in absorption experiments. Modified lipid A core structure was detected in these hybrids, but Shiga O antigen was not expressed. When the his+ locus of the S. dysenteriae 1 chromosome was transferred by transduction to E. coli K-12 containing pWR24, complete Shiga O antigen was expressed. Lipopolysaccharide extracted from these hybrids was indistinguishable chemically, electrophoretically, and serologically from native S. dysenteriae 1 lipopolysaccharide.

No abstract