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Infection and Immunity, November 2000, p. 6391-6397, Vol. 68, No. 11
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Construction of a Vibrio cholerae Vaccine Candidate Using Transposon Delivery and FLP Recombinase-Mediated Excision

Su L. Chiang and John J. Mekalanos^*

Department of Microbiology and Molecular Genetics and Shipley Institute of Medicine, Harvard Medical School, Boston, Massachusetts

Received 25 April 2000/Returned for modification 13 July 2000/Accepted 11 August 2000

Recent efforts to develop a vaccine against the diarrheal disease cholera have focused on the use of live attenuated strains of the causative organism, Vibrio cholerae. The Ogawa lipopolysaccharide phenotype is expressed by many epidemic strains, and motility defects reduce the risk of reactive diarrhea in vaccine recipients. We therefore converted a motile Inaba⁺ vaccine candidate, Peru-2, to a nonmotile Ogawa⁺ phenotype using a mariner-based transposon carrying rfbT, the gene required for expression of the Ogawa phenotype. Analysis of 22 nonmotile Peru-2 mutants showed that two were Ogawa⁺, and both of these strains had insertions in the flgE gene. It was possible to convert these strains to antibiotic sensitivity by introducing a recombinase that acts on sites flanking the antibiotic marker on the transposon. The resulting strains are competent for colonization in infant mice and may therefore be suitable as vaccine candidates for use either independently or in a combination with strains of different biotypes and serotypes.

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^* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics and Shipley Institute of Medicine, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 432-1935. Fax: (617) 738-7664. E-mail: john_mekalanos{at}hms.harvard.edu.

Present address: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115.

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Infection and Immunity, November 2000, p. 6391-6397, Vol. 68, No. 11
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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