Attenuated Shigella flexneri 2a vaccine strain CVD 1204 expressing colonization factor antigen I and mutant heat-labile enterotoxin of enterotoxigenic Escherichia coli

A multivalent live oral vaccine against both Shigella spp. and enterotoxige nic Escherichia coli (ETEC) is being developed based on the hypothesis that protection can be achieved if attenuated shigellae express ETEC fimbrial c olonization factors and genetically detoxified heat-labile toxin from a hum an 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 c loned separately on expression plasmids downstream from the CFA/I operon. F ollowing electroporation into S. flexneri 2a vaccine strain CVD 1204, coexp ression of CFA/I and LThK63 or LThR72 was demonstrated by Western blot anal ysis, GM(1) binding assays, and agglutination with anti-CFA/I antiserum. He magglutination and electron microscopy confirmed surface expression of CFA/ I. Guinea pigs immunized intranasally on days 0 and 15 with CVD 1204 expres sing CFA/I and LThK63 or LThR72 exhibited high titers of both serum immunog lobulin G (IgG) and mucosal secretory IgA anti-CFA/I; 40% of the animals pr oduced antibodies directed against LTh. All immunized guinea pigs also prod uced 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 demonstrate s the feasibility of expressing multiple ETEC antigens on a single plasmid in an attenuated Shigella vaccine strain and engendering immune responses a gainst both the heterologous antigens and vector strain.