Protection against Shiga toxin 1 challenge by immunization of mice with purified mutant Shiga toxin 1

Abstract

Shiga toxin 1 (Stx1) of enterohemorrhagic Escherichia coli O157:H7 was cloned, and four mutant Stx1s were constructed by site-directed mutagenesis with PCR. The wild-type and mutant Stx1s with amino acid replacements at positions 167 and 170 of the A subunit were purified by one-step affinity chromatography with commercially available Globotriose Fractogel, and the mutant Stxs were used for the immunization of mice. The mutant toxins were nontoxic to Vero cells in vitro and to mice in vivo and induced the immunoglobulin G antibody against the wild-type Stx1, which neutralized the cytotoxicity of Stx1. The induced antibody titers depended on the mutation at position 170 of the A subunit. The mice immunized with the mutant Stx1s were protected against a challenge of approximately 100 times the 50% lethal dose of the wild-type Stx1, suggesting that the mutant toxins are good candidates for toxoid vaccines for infection by Stx1-producing E. coli.

FIG. 1.

SDS-PAGE of the purified wild-type and mutant Stxs. The wild-type and mutant toxins (10 μg) were applied to a 15% polyacrylamide gel. MW, molecular weight standards; lane 1, wild-type Stx1; lane 2, STX111; lane 3, STX112; lane 4, STX113; lane 5, STX114.

FIG. 2.

Anti-Stx1 antibody titers induced by immunization with the mutant Stx1s. Groups of mice (n = 5) were immunized subcutaneously with 10 μg of the mutant Stx1s as described in Materials and Methods. Levels of Stx1 IgG antibodies were determined by ELISA. The serum dilution at which the difference of absorbance between the sample and the control became less than 0.2 was defined as the antibody titer. The data are expressed as means ± standard deviation.

FIG. 3.

Response to the subunits of Stx1 by the sera from the mutant Stx1-immunized mice. The wild-type Stx1 (5 μg) was subjected to SDS-PAGE and stained by Coomassie brilliant blue (lane 1). The wild-type Stx1 (5 μg) was subjected to a Western blotting assay. The sera from the mice immunized with STX111 (lane 2), STX112 (lane 3), STX113 (lane 4), or STX114 (lane 5) or with nonimmunized serum (lane 6) were used as primary antibodies, and alkaline phosphatase-conjugated rabbit anti-mouse IgG was used as a secondary antibody.

FIG. 4.

Neutralization of the Stx1 cytotoxicity by the mouse antisera. The wild-type Stx1 serum and the sera from the mice immunized with the mutant Stx1s were incubated at 37°C for 30 min before the cytotoxicity assay. ⧫, STX111; ▪, STX112; Δ, STX113; ○, STX114; ▿, nonimmunized.