Construction of an auxotrophic Shigella flexneri strain for use as a live vaccine

Shigella flexneri 2a Strain CVD 1207, with Specific Deletions in virG , sen , set , and guaBA , Is Highly Attenuated in Humans

“…of genes encoding virulence attributes met with only limited success; at doses producing immune responses (10 8 or more CFU), shigellosis-like adverse reactions (diarrhea, fever, and dysentery) were observed in many vaccinees (18,22). The findings in this paper are noteworthy because they demonstrate that the introduction of multiple rational mutations has attenuated strain 2457T to a degree not previously achieved.…”

“…Lindberg and colleagues constructed SFL 1070 from S. flexneri 2a strain 2457T using a chromosomal mutation in aroD, which encodes an enzyme in the aromatic amino acid biosynthesis pathway that is necessary to sustain bacterial replication within human cells (22). In volunteer trials, SFL 1070 was measurably attenuated compared to the wild type but still induced residual dose-related reactogenicity (gastrointestinal symptoms and fever) in 10 to 44% of the subjects who received three doses of 10 5 to 10 9 CFU (13).…”

Expression of Shigella dysenteriae serotype 1 O-antigenic polysaccharide by Shigella flexneri aroD vaccine candidates and different S. flexneri serotypes

“…The live oral vaccines S. flexneri serotype Y strain SFL124 and S. flexneri serotype 2a strain SFL1070, carrying deletions encompassing all or nearly all of the coding sequence of the aroD gene, have been described previously (14,16,21,28). Strains of the different S. flexneri serotypes isolated from patients, 1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, and X, were from the strain collection of the Division of Clinical Bacteriology, Huddinge Hospital, Karolinska Institute, Stockholm, Sweden.…”

“…The presence of only low-molecular-weight LPS fractions was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Selected colonies were subsequently tested for their abilities to bind Congo red dye, an indication of their invasiveness, according to the method of Payne and Finkelstein (24), and for their abilities to invade HeLa cells (21). Finally, the aroD mutation was assessed by determining growth dependence upon aromatic amino acids with minimal agar with or without an Aro-mix, containing tyrosine, phenylalanine, tryptophan, and 2,3-dihydroxybenzoic acid.…”

“…One vaccine candidate, SFL124, was developed by attenuation of the virulent S. flexneri serotype Y strain SFL. The attenuation involved transduction of a Tn10-inactivated aroD gene from Escherichia coli K-12 NK5131 (21,22) into SFL1 to produce the tetracycline-resistant aro::Tn10 transductant strain SFL114. Subsequent growth on fusaric acid medium supplemented with 2,3-dihydroxybenzoic acid permitted selection of the tetracycline-sensitive mutant SFL124, which is deleted for most of the aroD gene (16).…”