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* Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717;
Laboratory of Zoonotic Pathogens, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840;
Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80521; and
Physics Department, Montana State University, Bozeman, MT 59717
The gut provides a large area for immunization enabling the development of mucosal and systemic Ab responses. To test whether the protective Ags to Yersinia pestis can be orally delivered, the Y. pestis caf1 operon, encoding the F1-Ag and virulence Ag (V-Ag) were cloned into attenuated Salmonella vaccine vectors. F1-Ag expression was controlled under a promoter from the caf1 operon; two different promoters (P), PtetA in pV3, PphoP in pV4, as well as a chimera of the two in pV55 were tested. F1-Ag was amply expressed; the chimera in the pV55 showed the best V-Ag expression. Oral immunization with Salmonella-F1 elicited elevated secretory (S)-IgA and serum IgG titers, and Salmonella-V-Ag(pV55) elicited much greater S-IgA and serum IgG Ab titers than Salmonella-V-Ag(pV3) or Salmonella-V-Ag(pV4). Hence, a new Salmonella vaccine, Salmonella-(F1+V)Ags, made with a single plasmid containing the caf1 operon and the chimeric promoter for V-Ag allowed the simultaneous expression of F1 capsule and V-Ag. Salmonella-(F1+V)Ags elicited elevated Ab titers similar to their monotypic derivatives. For bubonic plague, mice dosed with Salmonella-(F1+V)Ags and Salmonella-F1-Ag showed similar efficacy (>83% survival) against 
1000 LD50 Y. pestis. For pneumonic plague, immunized mice required immunity to both F1- and V-Ags because the mice vaccinated with Salmonella-(F1+V)Ags protected against 100 LD50 Y. pestis. These results show that a single Salmonella vaccine can deliver both F1- and V-Ags to effect both systemic and mucosal immune protection against Y. pestis.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Public Health Service Grant AI-56286 and was supported in part by Montana Agricultural Station and U.S. Department of Agriculture Formula Funds and also supported in part by National Aeronautics and Space Administration-Experimental Program to Stimulate Competitive Research under Grant NCC5-579. The Veterinary Molecular Biology BSL-3 facility was in part supported by National Institutes of Health/NCRR COBRE P20 RR-020185, and the challenge studies were in part supported by Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, and the Rocky Mountain Research Center of Excellence, National Institutes of Health Grant U54 AI-06537.
2 Address correspondence and reprint requests to Dr. David W. Pascual, Veterinary Molecular Biology, Montana State University, P.O. Box 173610, Bozeman, MT 59717-3610. E-mail address: dpascual{at}montana.edu
3 Abbreviations used in this paper: V-Ag, virulence Ag; BHI, brain-heart infusion; CFC, cytokine-forming cell; LB, Luria-Bertani; P, promoter; sPBS, sterile PBS.
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