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Infection and Immunity, March 2003, p. 1352-1360, Vol. 71, No. 3
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.3.1352-1360.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Genetic Characterization and Immunogenicity of Coli Surface Antigen 4 from Enterotoxigenic Escherichia coli when It Is Expressed in a Shigella Live-Vector Strain

Zeev Altboum,{dagger} Myron M. Levine, James E. Galen, and Eileen M. Barry*

Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland 21201

Received 16 October 2002/ Returned for modification 14 November 2002/ Accepted 27 November 2002

The genes that encode the enterotoxigenic Escherichia coli (ETEC) CS4 fimbriae, csaA, -B, -C, -E, and -D', were isolated from strain E11881A. The csa operon encodes a 17-kDa major fimbrial subunit (CsaB), a 40-kDa tip-associated protein (CsaE), a 27-kDa chaperone-like protein (CsaA), a 97-kDa usher-like protein (CsaC), and a deleted regulatory protein (CsaD'). The predicted amino acid sequences of the CS4 proteins are highly homologous to structural and assembly proteins of other ETEC fimbriae, including CS1 and CS2, and to CFA/I in particular. The csaA, -B, -C, -E operon was cloned on a stabilized plasmid downstream from an osomotically regulated ompC promoter. pGA2-CS4 directs production of CS4 fimbriae in both E. coli DH5{alpha} and Shigella flexneri 2a vaccine strain CVD 1204, as detected by Western blot analysis and bacterial agglutination with anti-CS4 immune sera. Electron-microscopic examination of Shigella expressing CS4 confirmed the presence of fimbriae on the bacterial surface. Guinea pigs immunized with CVD 1204(pGA2-CS4) showed serum and mucosal antibody responses to both the Shigella vector and the ETEC fimbria CS4. Among the seven most prevalent fimbrial antigens of human ETEC, CS4 is the last to be cloned and sequenced. These findings pave the way for CS4 to be included in multivalent ETEC vaccines, including an attenuated Shigella live-vector-based ETEC vaccine.

* Corresponding author. Mailing address: Center for Vaccine Development, University of Maryland School of Medicine, 685 West Baltimore St., Baltimore, MD 21201. Phone: (410) 706-5328. Fax: (410) 706-6205. E-mail: ebarry{at}medicine.umaryland.edu.

Editor: D. L. Burns

{dagger} Current address: Israel Institute for Biological Research, Ness-Ziona, Israel 70450.

Infection and Immunity, March 2003, p. 1352-1360, Vol. 71, No. 3
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.3.1352-1360.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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