Vibrio cholerae strain CVD103Hg.sup.r, method of making same, and vaccines derived therefrom

A method of isolating deletion mutants of Vibrio cholerae Inaba comprising the steps of:

• (a) constructing a first plasmid comprising Vibrio cholerae flanking sequences of the coding sequence for the A.sub.1 subunit of Vibrio cholerae toxin and a gene for a first selectable marker of foreign origin ligated to said flanking sequences or coding sequence, wherein said flanking sequences are of sufficient length to promote detectable in vivo recombination;
• (b) mating virulent strain 569B of Vibrio cholerae with a first microorganism carrying the first plasmid;
• (c) selecting for Vibrio cholerae expressing the first selectable marker;
• (d) mating the selected product of step (c) with a second microorganism carrying a second plasmid with a second selectable marker, said second plasmid being incompatible with the first plasmid in said second microorganism;
• (e) selecting for Vibrio cholerae expressing both the first selectable marker and the second selectable marker to isolate an in vivo recombinant Vibrio cholerae organism;
• (f) mating said in vivo recombinant Vibrio cholerae with a third microorganism carrying a third plasmid, said third plasmid comprising flanking sequences of the coding sequence for the A.sub.1 subunit of Vibrio cholerae toxin, said coding sequence comprising a deleted fragment of sequence;
• (g) selecting for the loss of the first selectable marker;
• (h) mating the selected product of step (g) with a fourth microorganism carrying a fourth plasmid, said fourth plasmid encoding a gene conferring a resistance to mercury;
• and,
• (i) selecting for Vibrio cholerae expressing the gene conferring resistance to mercury.