Salmonella enterica serovar Typhimurium vaccine strains expressing a nontoxic Shiga-like toxin 2 derivative induce partial protective immunity to the toxin expressed by enterohemorrhagic Escherichia coli

Abstract

Shiga-like toxin 2 (Stx2)-producing enterohemorrhagic Escherichia coli (referred to as EHEC or STEC) strains are the primary etiologic agents of hemolytic-uremic syndrome (HUS), which leads to renal failure and high mortality rates. Expression of Stx2 is the most relevant virulence-associated factor of EHEC strains, and toxin neutralization by antigen-specific serum antibodies represents the main target for both preventive and therapeutic anti-HUS approaches. In the present report, we describe two Salmonella enterica serovar Typhimurium aroA vaccine strains expressing a nontoxic plasmid-encoded derivative of Stx2 (Stx2DeltaAB) containing the complete nontoxic A2 subunit and the receptor binding B subunit. The two S. Typhimurium strains differ in the expression of flagellin, the structural subunit of the flagellar shaft, which exerts strong adjuvant effects. The vaccine strains expressed Stx2DeltaAB, either cell bound or secreted into the extracellular environment, and showed enhanced mouse gut colonization and high plasmid stability under both in vitro and in vivo conditions. Oral immunization of mice with three doses of the S. Typhimurium vaccine strains elicited serum anti-Stx2B (IgG) antibodies that neutralized the toxic effects of the native toxin under in vitro conditions (Vero cells) and conferred partial protection under in vivo conditions. No significant differences with respect to gut colonization or the induction of antigen-specific antibody responses were detected in mice vaccinated with flagellated versus nonflagellated bacterial strains. The present results indicate that expression of Stx2DeltaAB by attenuated S. Typhimurium strains is an alternative vaccine approach for HUS control, but additional improvements in the immunogenicity of Stx2 toxoids are still required.

FIG. 1.

Expression of FliC flagellin and Stx2ΔAB by the S. Typhimurium vaccine strains. (A) Detection of FliC expression in the S. Typhimurium vaccine strains. FliC was detected in Western blots developed with polyclonal anti-FliC antibodies and whole-cell extracts of the bacterial strains. Lanes: 1, LDV326; 2, LDV327; 3, LDV328; 4, LDV329. (B) Detection of Stx2ΔAB, encoded by the S. Typhimurium vaccine strains, in culture supernatants (lanes 2, 4, 6, and 8) and whole-cell extracts (lanes 3, 5, 7, and 9) of the strains. Lanes: 1, purified recombinant StxB; 2 and 3, LDV326; 4 and 5, LDV327; 6 and 7, LDV328; 8 and 9, LDV329. (C) In vitro motilities of recombinant S. Typhimurium vaccine strains LDV326 (▴), LDV327 (▾), LDV328 (▪), and LDV329 (⧫) at 37°C measured by the diameter of the bacterial growth halos.

FIG. 2.

Colonization of mouse guts by the S. Typhimurium vaccine strains. Five Peyer's patches (PP) obtained from each mouse orally dosed with 10¹⁰ bacterial cells were homogenized and serially diluted in PBS before being plated onto ampicillin-LB plates. The graph represents the number of CFU obtained 72 h after the initial inoculation of the S. Typhimurium strain. Asterisks indicate statistically significant differences from results for mice immunized with S. Typhimurium strain LDV326 or LDV328 (P < 0.05).

FIG. 3.

Serum IgG and fecal IgA responses to Stx2B elicited in mice orally immunized with the recombinant S. Typhimurium vaccine strains. The anti-Stx2B antibody titers were determined by ELISA using the purified recombinant Stx2 B subunit. (A) Kinetics of anti-Stx2B IgG responses detected in pools of sera from mice immunized with one of the three S. Typhimurium strains. Mice immunized with one (open bars), two (shaded bars), or three (filled bars) doses of the vaccine strain are distinguished. (B) Individual serum anti-Stx2B IgG titers elicited in mice immunized with three doses of one of the tested S. Typhimurium vaccine strains. (C) Fecal anti-Stx2B IgA titers detected in mice immunized with three doses of one of the tested S. Typhimurium vaccine strains. Fecal IgA levels were measured in sample pools collected from animals of the same immunization group. Asterisks indicate statistically significant differences from results for mice immunized with S. Typhimurium strain LDV326 or LDV328 (P < 0.05).