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Infection and Immunity, January 2000, p. 221-226, Vol. 68, No. 1
0019-9567/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Development of a Delta glnA Balanced Lethal Plasmid System for Expression of Heterologous Antigens by Attenuated Vaccine Vector Strains of Vibrio cholerae

Edward T. Ryan,1 Thomas I. Crean,1 Sims K. Kochi,2 Manohar John,1 Angel A. Luciano,1 Kevin P. Killeen,2 Karl E. Klose,3 and Stephen B. Calderwood1,4,*

Division of Infectious Diseases, Massachusetts General Hospital, Boston, Massachusetts 021141; AVANT Immunotherapeutics Inc., Needham, Massachusetts 024942; Department of Microbiology, University of Texas Health Science Center, San Antonio, Texas 782843; and Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 021154

Received 27 July 1999/Returned for modification 22 September 1999/Accepted 20 October 1999

We have previously shown that more prominent immune responses are induced to antigens expressed from multicopy plasmids in live attenuated vaccine vector strains of Vibrio cholerae than to antigens expressed from single-copy genes on the V. cholerae chromosome. Here, we report the construction of a Delta glnA derivative of V. cholerae vaccine strain Peru2. This mutant strain, Peru2Delta glnA, is unable to grow on medium that does not contain glutamine; this growth deficiency is complemented by pKEK71-NotI, a plasmid containing a complete copy of the Salmonella typhimurium glnA gene, or by pTIC5, a derivative of pKEK71-NotI containing a 1.8-kbp fragment that directs expression of CtxB with a 12-amino-acid epitope of the serine-rich Entamoeba histolytica protein fused to the amino terminus. Strain Peru2Delta glnA(pTIC5) produced 10-fold more SREHP-12-CtxB in supernatants than did ETR3, a Peru2-derivative strain containing the same fragment inserted on the chromosome. To assess immune responses to antigens expressed by this balanced lethal system in vivo, we inoculated germfree mice on days 0, 14, 28, and 42 with Peru2Delta glnA, Peru2Delta glnA(pKEK71-NotI), Peru2(pTIC5), Peru2Delta glnA(pTIC5), or ETR3. All V. cholerae strains were recoverable from stool for 8 to 12 days after primary inoculation, including Peru2Delta glnA; strains containing plasmids continued to harbor pKEK71-NotI or pTIC5 for 8 to 10 days after primary inoculation. Animals were sacrificed on day 56, and serum, stool and biliary samples were analyzed for immune responses. Vibriocidal antibody responses, reflective of in vivo colonization, were equivalent in all groups of animals. However, specific anti-CtxB immune responses in serum (P <=  0.05) and bile (P <=  0.001) were significantly higher in animals that received Peru2Delta glnA(pTIC5) than in those that received ETR3, confirming the advantage of higher-level antigen expression in vivo. The development of this balanced lethal system thus permits construction and maintenance of vaccine and vector strains of V. cholerae that express high levels of immunogenic antigens from plasmid vectors without the need for antibiotic selection pressure.

* Corresponding author. Mailing address: Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA 02114. Phone: (617) 726-3811. Fax: (617) 726-7416. E-mail: scalderwood{at}partners.org.

Infection and Immunity, January 2000, p. 221-226, Vol. 68, No. 1
0019-9567/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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