Vibrio cholerae O139 conjugate vaccines

A method for preparing a conjugate molecule, comprising:

• (a) contacting a recombinant mutated diphtheria toxin comprising CRMH21G with adipic acid dihydrazide in the presence of a carboxyl activating reagent;
• and
• (b) contacting the product of (a) with capsular polysaccharide of Vibrio cholerae O139, in the presence of a carboxyl activating reagent, thereby preparing the conjugate molecule comprising capsular polysaccharide of Vibrio cholerae O139, covalently bound to the recombinant mutated diphtheria toxin comprising CRMH21G which functions as a carrier protein, in which the toxin is covalently bound to the polysaccharide by coupling with a dicarboxylic acid dihydrazide linker and wherein the dicarboxylic acid dihydrazide linker comprises adipic acid dihydrazide, whereby, the conjugate elicits serum antibodies vibriocidal to at least Vibrio cholerae O139.

A method for preparing a conjugate molecule, comprising:

• (a) contacting the capsular polysaccharide of Vibrio cholerae O139 with adipic acid dihydrazide in the presence of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate;
• and
• (b) contacting the product of (a) with a recombinant mutated diphtheria toxin comprising CRMH21G in the presence of a carboxyl activating reagent, thereby preparing the conjugate molecule comprising capsular polysaccharide of Vibrio cholerae O139, covalently bound to the recombinant mutated diphtheria toxin comprising CRMH21G which functions as a carrier protein, in which the toxin is covalently bound to the polysaccharide by coupling with a dicarboxylic acid dihydrazide linker and wherein the dicarboxylic acid dihydrazide linker comprises adipic acid dihydrazide, whereby, the conjugate elicits serum antibodies vibriocidal to at least Vibrio cholerae O139.