"As noted above, the immunogenicity of the polysaccharide antigen is determined by its molecular weight, the ability to form aggregate structures, so the highest immunogenicity is found out for exopolysaccharide fraction with molecular weight from 80 to 400 kD. Immunogenicity of the high molecular weight fraction of the exopolysaccharide exceeds more than 7 times the immunogenicity of the O-polysaccharide from bacterial cells LPS (Example 1C, FIG. 6), it is apparently determined by the presence in the molecule of a non-toxic lipid component--a non hydroxylated fatty acid contributing to supramolecular aggregate structures formation. Additionally, the exopolysaccharide is apyrogenic for rabbits when administered intravenously at a dose of no more than 0.050 mcg/kg in a rabbit pyrogenicity test (Example 1D). Exopolysaccharide vaccine formulation meets WHO Expert Committee requirements for polysaccharide vaccines pyrogenicity parameter (WHO TR--WHO Technical report No. 840, 1994).
"The claimed method for producing S. sonnei, phase I bacteria exopolysaccharide includes: (a) producing cultures of S. sonnei bacteria in liquid phase; (b) separating liquid phase from bacterial cells; © isolating polysaccharide from liquid phase. At the same time, the liquid phase, which maintains cell cultures viability, can be represented by a cultural medium of various composition and properties. Separating liquid phase from bacterial cells is preferably carried out while maintaining nativity of bacterial cells.
"Thus, the claimed method for producing a polysaccharide, which excludes the use of LPS as its source, does not contain the stage of LPS extraction from bacterial cell walls, resulting in the inevitable loss of bacterial cell nativity.
"Isolation of polysaccharide from liquid phase can be carried out by a method comprising: (i) removal of proteins and nucleic acids from liquid phase; (ii) ultrafiltration and (iii) dialysis of obtained solution.
"The claimed vaccine for prophylaxis and/or treatment of S. sonnei shigellosis contains prophylactically and/or therapeutically effective amounts of S. sonnei, phase I bacteria polysaccharides, consisting of 1-100 repeating disaccharide units of O-[4-amino-2-(N-acetyl)amino-2,4- dideoxy-.beta.-D-galactopyranosyl]-(1.fwdarw.4)-O-[2-(N-acetyl)amino-2-de- oxy-.alpha.-L-altrpyranuronic acid] connected by (1.fwdarw.3) bonds to form a polysaccharide chain, and obtained using S. sonnei bacteria, but without the use of lipopolysaccharides as its source.
"This polysaccharide is an exopolysaccharide, or capsular polysaccharide, secreted into the cultural medium by S. sonnei, phase I bacteria. The native exopolysaccharide includes a non-toxic lipid component, presented by non hydroxylated fatty acids from 16-18 carbon atoms in the molecule (FIG. 4). Its fatty acid content is less than 0.01% (w/w). Additionally, independently from the method of preparation with use S. sonnei bacteria, the polysaccharide does not include elements of the structure of LPS core domain (FIG. 4).
"Exopolysaccharide can be prepared by any method, including genetic engineering, using the genome of S. sonnei bacteria. Preferably the exopolysaccharide is produced using S. sonnei bacteria by a method comprising: (a) producing bacterial culture in liquid phase; (b) separating the liquid phase from bacterial cells; © isolating the polysaccharide from liquid phase. Meanwhile, in order to avoid destroying the cell walls and LPS entry into the liquid phase, separation it from the bacterial cells is advisable to carry out under conditions for maintain the nativity of bacterial cells. Isolating the polysaccharide from the liquid phase can be carried out by a method comprising: (i) removing proteins and nucleic acids from the liquid phase; (ii) ultrafiltration and (iii) dialysis of obtained solution.
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