Attenuated Shigella flexneri 2a Delta guaBA strain CVD 1204 expressing enterotoxigenic Escherichia coli (ETEC) CS2 and CS3 fimbriae as a live mucosalvaccine against Shigella and ETEC infection

To construct a prototype hybrid vaccine against Shigella and enterotoxigeni c Escherichia coli (ETEC), the genes encoding the production of ETEC CS3 an d CS3 fimbriae were isolated and expressed in attenuated Shigella flexneri 2a guaBA strain CVD 1204. The CS2 cotA to -D gents, isolated from ETEC stra in C91F, and the CS3 cstA to -H genes, subcloned from plasmid pCS100, were cloned into similar to 15-copy-number-stabilized pGA1 behind the osmoticall y regulated ompC promoter, resulting in high expression of both fimbriae, U nder nonselective in vitro growth conditions, pGA1-CS2 and pGA1-CS3 were st able in CVD 1204, exhibiting a plasmid loss of only approximately 1% per du plication. Expression of CS2 and CS3 reduced the invasiveness of Shigella f or HeLa cells and slowed the intracellular growth rate, Guinea pigs immuniz ed intranasally with CVD 1204(pGA1-CS2) CVD 1204(pGA1-CS3), or with a mixtu re of these strains, developed secretory immunoglobulin A (IgA) in tears an d serum IgG antibodies against Shigella lipopolysaccharide CS2, and CS3 ant igens. Moreover, the animals mere protected against keratoconjunctivitis fo llowing conjunctival challenge with virulent S, flexneri 2a strain 2457T. A nimals immunized with Shigella expressing CS2 or CS3 developed serum antibo dies that agglutinated Shigella as well as an ETEC: strain bearing the homo logous fimbriae, whereas animals immunized with combined CVD 1204(pGA1-CS2) and CVD 1204(pGA1-CS3) developed antibodies that agglutinated all three te st strains, These observations support the feasibility of a multivalent vac cine against shigellosis and ETEC: diarrhea consisting of multiple Shigella live vectors expressing relevant ETEC antigens.