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Infection and Immunity, September 2000, p. 4884-4892, Vol. 68, No. 9
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Attenuated Shigella flexneri 2a Vaccine Strain CVD 1204 Expressing Colonization Factor Antigen I and Mutant Heat-Labile Enterotoxin of Enterotoxigenic Escherichia coli

Hilary Koprowski II,1 Myron M. Levine,1 Richard J. Anderson,1,dagger Genevieve Losonsky,1 Mariagrazia Pizza,2 and Eileen M. Barry1,*

Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland 21201,1 and IRIS, Chiron Spa, Siena, Italy2

Received 15 February 2000/Returned for modification 6 April 2000/Accepted 12 May 2000

A multivalent live oral vaccine against both Shigella spp. and enterotoxigenic Escherichia coli (ETEC) is being developed based on the hypothesis that protection can be achieved if attenuated shigellae express ETEC fimbrial colonization factors and genetically detoxified heat-labile toxin from a human ETEC isolate (LTh). Two detoxified derivatives of LTh, LThK63 and LThR72, were engineered by substitution---serine to lysine at residue 63, or lysine to arginine at residue 72. The genes encoding these two derivatives were cloned separately on expression plasmids downstream from the CFA/I operon. Following electroporation into S. flexneri 2a vaccine strain CVD 1204, coexpression of CFA/I and LThK63 or LThR72 was demonstrated by Western blot analysis, GM1 binding assays, and agglutination with anti-CFA/I antiserum. Hemagglutination and electron microscopy confirmed surface expression of CFA/I. Guinea pigs immunized intranasally on days 0 and 15 with CVD 1204 expressing CFA/I and LThK63 or LThR72 exhibited high titers of both serum immunoglobulin G (IgG) and mucosal secretory IgA anti-CFA/I; 40% of the animals produced antibodies directed against LTh. All immunized guinea pigs also produced mucosal IgA (in tears) and serum IgG anti-S. flexneri 2a O antibodies. Furthermore, all immunized animals were protected from challenge with wild-type S. flexneri 2a. This prototype Shigella-ETEC hybrid vaccine demonstrates the feasibility of expressing multiple ETEC antigens on a single plasmid in an attenuated Shigella vaccine strain and engendering immune responses against both the heterologous antigens and vector strain.

* 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.

dagger Present address: Wellcome Centre for Human Genetics, Oxford OX3 7BN, United Kingdom.

Infection and Immunity, September 2000, p. 4884-4892, Vol. 68, No. 9
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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