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

Abstract

To construct a prototype hybrid vaccine against Shigella and enterotoxigenic Escherichia coli (ETEC), the genes encoding the production of ETEC CS2 and CS3 fimbriae were isolated and expressed in attenuated Shigella flexneri 2a guaBA strain CVD 1204. The CS2 cotA to -D genes, isolated from ETEC strain C91F, and the CS3 cstA to -H genes, subcloned from plasmid pCS100, were cloned into ~15-copy-number-stabilized pGA1 behind the osmotically regulated ompC promoter, resulting in high expression of both fimbriae. Under nonselective in vitro growth conditions, pGA1-CS2 and pGA1-CS3 were stable in CVD 1204, exhibiting a plasmid loss of only approximately 1% per duplication. Expression of CS2 and CS3 reduced the invasiveness of Shigella for HeLa cells and slowed the intracellular growth rate. Guinea pigs immunized intranasally with CVD 1204(pGA1-CS2) or CVD 1204(pGA1-CS3), or with a mixture of these strains, developed secretory immunoglobulin A (IgA) in tears and serum IgG antibodies against Shigella lipopolysaccharide, CS2, and CS3 antigens. Moreover, the animals were protected against keratoconjunctivitis following conjunctival challenge with virulent S. flexneri 2a strain 2457T. Animals immunized with Shigella expressing CS2 or CS3 developed serum antibodies that agglutinated Shigella as well as an ETEC strain bearing the homologous fimbriae, whereas animals immunized with combined CVD 1204(pGA1-CS2) and CVD 1204(pGA1-CS3) developed antibodies that agglutinated all three test strains. These observations support the feasibility of a multivalent vaccine against shigellosis and ETEC diarrhea consisting of multiple Shigella live vectors expressing relevant ETEC antigens.

FIG. 1

Maps of CS2 and CS3 fimbria-expressing plasmids. (A) Map of the cloning vector pGA1, derived from pGEN91 by replacing gfp with a synthetic linker containing multiple cloning sites. (B) Map of pGA1-CS2, constructed by cloning 5,696 bp of the CS2 operon into pGA1. (C) Map of pGA1-CS3, constructed by cloning 4,746 bp of the CS3 operon into pGA1.

FIG. 2

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and Western blots of CS2- and CS3-expressing CVD 1204. (A) Fast-Page BluPrint satin; (B) Western blot probed with anti-CS3 polyclonal antibody; (C) Western blot probed with anti-CS2 polyclonal antibody. Lane 1, CVD 1204(pGA1-CS3); lane 2, CVD 1204(pGA1); lane 3, purified CS3 pili; lane 4, CVD 1204(pGA1-CS2); lane 5, CVD 1204(pGA1); lane 6, purified CS2 pili.

FIG. 3

Induction of fimbrial expression by salt concentration. (A) Western blot probed with anti-CS2 polyclonal antibody. Lane 1, purified CS2 pili; lanes 2 to 5, CVD 1204(pGA1-CS2) grown in the indicated NaCl concentrations (millimolar). (B) Western blot probed with anti-CS3 polyclonal antibody. Lane 1, purified CS3 pili; lanes 2 to 5, CVD 1204(pGA1-CS3) grown in the indicated NaCl concentrations.

FIG. 4

Production of sIgA and serum IgG antigen-specific antibodies in guinea pigs immunized with Shigella strains expressing CS2 and CS3 fimbriae. The immunized groups were as follows: group 1, CVD 1204; group 2, CVD 1204(pGA1-CS3); group 3, CVD 1204(pGA1-CS2); and group 4, a mixture of CVD 1204(pGA1-CS3) and CVD 1204(pGA1-CS2). Antibody titers were determined in preimmune sera, following a single immunization (day 14) and following two immunizations (day 28). Antibodies elicited against Shigella LPS, CS2, and CS3 antigens were quantitated. The titers of tear sIgA are presented in panels A (LPS), B (CS3), and C (CS2); the titers of serum IgG are presented in panels D (LPS), E (CS3), and F (CS2).