Immunization against the colonization factor antigen I of enterotoxigenic Escherichia coli by administration of a bivalent Salmonella typhimurium aroA strain

Abstract

An expression plasmid (pCFA-1) carrying the cfaB gene that codes for the enterotoxigenic Escherichia coli (ETEC) fimbrial adhesin colonization factor antigen I (CFA/I) subunit was constructed and used to transform a derivative of the attenuated Salmonella typhimurium aroA vaccine strain SL3261 carrying an F'lacI q . Treatment of the transformed strain with isopropyl-ß-D-thiogalactopyranoside (IPTG) resulted in elevated in vitro expression of the CFA/I subunit. Although flagellar function and lipopolysaccharide (LPS) synthesis were similar in both the parental and the recombinant strains, spleen colonization was reduced in the recombinant strain. All BALB/c mice parenterally inoculated with the recombinant strain developed significant anti-CFA/I and anti-LPS serum antibody titers (P<0.05). Moreover, 2 of 5 mice orally inoculated with the engineered Salmonella strain developed anti-CFA/I intestinal IgA (P>0.05) while 4/5 of the same mice developed anti-LPS IgA (P<0.05). The results indicate that the vaccine strain elicited an antibody response against the bacterial host both after oral and intravenous immunization while the response against the CFA/I antigen was significant only after inoculation by the intravenous route.

New vaccine strategies against enterotoxigenic Escherichia coli: I: DNA vaccines against the CFA/I fimbrial adhesin

“…Mice were bled by retro-orbital puncture before and 2 weeks after the second immunization. Secreted IgA responses were evaluated in fecal extracts and small intestinal homogenates, as previously described (16). Enzyme-linked immunosorbent assays (ELISA) with heat-denatured CFA/I subunits as solid-phase bound antigen were used to analyze sera, fecal extracts or intestinal homogenates of immunized mice.…”

Adjuvant Activity of a Nontoxic Mutant ofEscherichia coliHeat-Labile Enterotoxin on Systemic and Mucosal Immune Responses Elicited against a Heterologous Antigen Carried by a LiveSalmonella entericaSerovar Typhimurium Vaccine Strain

“…In this study we propose a new orally delivered vaccine formulation consisting of the combined use of a live bivalent Salmonella enterica serovar Typhimurium aroA strain expressing the major subunit of the colonization factor antigen I (CFA/I), a fimbrial antigen involved with the colonization of the human gut epithelium by ETEC (1,10), and LT (R192G) . The hybrid ETEC-Salmonella vaccine strain (HG3) has previously been shown to elicit weak systemic and mucosal antibody responses against the heterologous antigen after repeated oral dosing to mice (15).…”

“…The cloned cfaB gene contained the complete sequence of the mature fimbrial subunit (herein referred as the CFA/I subunit), which is composed of 143 amino acids (M r 15,057) and a signal peptide of 23 amino acids (11,19). To avoid overexpression of the CFA/I subunit under noninducing conditions, an FЈ plasmid carrying the lacI q gene was also introduced into the vaccine strain (15). The HG3 strain could only express the CFA/I subunit in the presence of inducer (isopropyl-␤-D-thiogalactopyranoside [IPTG]) during in vitro growth, a feature probably associated with the inherently low immunogenicity of this construction following oral delivery to BALB/c mice (15).…”

“…To avoid overexpression of the CFA/I subunit under noninducing conditions, an FЈ plasmid carrying the lacI q gene was also introduced into the vaccine strain (15). The HG3 strain could only express the CFA/I subunit in the presence of inducer (isopropyl-␤-D-thiogalactopyranoside [IPTG]) during in vitro growth, a feature probably associated with the inherently low immunogenicity of this construction following oral delivery to BALB/c mice (15). ETEC strains 258909-3 (CFA/I, O128:H?, and ST/LT) and 258909-3M, a CFA/I-negative derivative (13), were used in inhibition of hemagglutination assays (IHA).…”

“…Serum samples collected from the retroorbital plexus before immunization and by cardiac puncture 1 week after the last inoculation were kept at Ϫ20°C. Intestine homogenates for mucosal immunoglobulin A (IgA) assays were obtained as previously described (2,15) and stored at Ϫ70°C until assayed. Serum IgG and intestinal IgA specific for CFA/I, lipopolysaccharide (LPS), and LT were detected in MaxiSorp plates (Nalge Nunc International, Naperville, Ill.) previously coated with either 0.1 g of heat-dissociated CFA/I subunits, 0.2 g of serovar Typhimurium LPS (Sigma), or 0.2 g of LT (R192G) per well, respectively.…”