Protection against Shiga Toxin 1 Challenge by Immunization of Mice with Purified Mutant Shiga Toxin 1

Bacterial strains and culture conditions. E. coli O157:H7 strain 147, which produces only Stx1, was provided by K. Tamura (National Institute of Infectious Diseases, Tokyo, Japan). E. coli DH5α (Table 1) was used as a host strain for the wild-type and mutant Stx production. E. coli strain cultures were grown in antibiotic medium 3 (Difco Laboratories, Detroit, Mich.) at 37°C for 16 h with shaking.

Construction of mutant Stx1.A DNA region including the stx1 gene in E. coli O157:H7 strain 147 was amplified by PCR with the primers 5′-CTACGCATGCTGTTAAGGTTGCAGCTCTC-3′ and 5′-CACTGTCGACGCCCTGACCACATCGTAG-3′, ligated with the cloning vector pUC118 at SphI and SalI sites, and introduced into E. coli DH5α by transformation. Site-directed mutagenesis was carried out with four primers harboring HindIII sites (underlined), corresponding to the active center of the Stx1 A subunit: 5′-GTAAAGCTTGAGCTGTCACAGT-3′ for replacement of E by Q at position 167 [A₁₆₇(E→Q)], 5′-GTAAAGCTTTAGCTGTCACAGT-3′ for A₁₆₇(E→K), 5′-CTGAAGCTTTAGGTTTTCGCA-3′ for A₁₇₀(R→G), and 5′-CTGAAGCTTTACCTTTTCGCA-3′ for A₁₇₀(R→L). Amplified DNA fragments were digested with HindIII, ligated, and further ligated to pUC118 to construct plasmids to produce four kinds of mutant Stx1 (Table 1).

Purification of wild-type and mutant Stx1s.Cultures of E. coli strains harboring plasmids for producing the wild-type or mutant Stx1 were grown in 200 ml of medium and harvested by centrifugation. The bacterial cells were suspended in 0.1% polymyxin B in phosphate-buffered saline (PBS) (pH 7.4) and incubated at 37°C for 60 min. The bacterial cells were removed by centrifugation and filtration with a 0.45-μm-pore-size membrane, and the crude toxin preparation thus obtained was applied to a small column (1 or 2 ml) of Globotriose Fractogel (IsoSep AB, Tullinge, Sweden). After nonabsorbed proteins were washed out with 15 ml of PBS, Stx was eluted with 15 ml of 4 M MgCl₂ in PBS, dialyzed against PBS, and concentrated by ultrafiltration to a volume of approximately 500 μl. The purified Stx was divided into aliquots and stored at −80°C until use.

SDS-PAGE.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 15% acrylamide gel and the buffer system of Laemmli (17). The gel was stained by Coomassie brilliant blue to visualize protein bands.

Cytotoxicity.Vero cell cultures grown in minimal essential medium (MEM) (Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 5% fetal bovine serum (FBS) (Gibco BRL, Grand Island, N.Y.), 1.05 g of NaHCO₃/liter, 2.92 g of l-glutamic acid/liter, and 100 U of penicillin G (5% FBS-MEM)/ml were dispensed to each well of a 96-well culture plate (4 × 10⁴ cells/well) and incubated at 37°C for 16 h with 5% CO₂. After the culture medium was changed to the medium containing the wild-type or mutant Stx1s, the cell cultures were grown under the same conditions for 72 h. The cells were then washed twice with PBS and incubated with 5% FBS-MEM containing 10% Alamar blue (Biosource, Camarillo, Calif.) for 2 h, and the absorbance at 570 nm and 600 nm was measured to calculate the cell viability and 50% cytotoxic dose (CD₅₀) (13). The triplicate assays were repeated three times, and mean values were used for CD₅₀ calculation.

Mouse lethality.BALB/c mice were purchased from Charles River Japan, Inc. (Yokohama, Japan) and used after 1 week (at the age of 7 weeks). Mice were administered intraperitoneal doses of serial 10-fold dilutions (five mice for each dose) of purified wild-type or mutant Stx1s dissolved in 0.2 ml of PBS. Survival of the mice was observed for 4 days, and the 50% lethal dose (LD₅₀) was calculated by the method of Kärber (12). All animal experiments were done in accordance with the Kitasato University Guidelines for Animal Care and Experimentation.

Immunization of mice.Mice (Japan Charles River; 7 weeks old) were subcutaneously administered 10 μg of mutant Stx1 emulsified with an equal amount of Freund's complete adjuvant (Sigma Chemical Co., St. Louis, Mo.) and booster doses with the same dose of mutant Stx1 emulsified with an equal volume of Freund's incomplete adjuvant (Sigma Chemical Co.) by the same route on days 21 and 35. Sera were collected 2 weeks after the last immunization. In another experiment, the immunized mice were used for the Stx1 challenge experiments.

Challenge with Stx1.At 2 weeks after the last immunization, the immunized mice were challenged intraperitoneally with the wild-type Stx1 at serial 10-fold dilutions and observed for 4 days, and LD₅₀ values were calculated as described above.

ELISA.Enzyme-linked immunosorbent assays (ELISA) were used for the detection of immunoglobulin G (IgG) antibodies to Stx1. The purified wild-type Stx1 was dispensed to a 96-well plate (0.2 μg/well) and incubated at 4°C for 16 h for adsorption. Using peroxidase-conjugated goat anti-mouse IgG (Sigma Chemical Co.) as the secondary antibody, ELISA was performed as described in a previous paper (14). Triplicate assays were repeated three times, and standard deviations were calculated.

Western blotting.The ability of IgG antibodies induced by mutant Stx1s to recognize the wild-type Stx1 was examined by Western blotting assays. The purified wild-type Stx1 (5 μg) was applied to SDS-PAGE and electrotransferred to a nitrocellulose membrane filter after electrophoresis. Protein bands were detected by the sera prepared from mice immunized with mutant Stx1s and alkaline phosphatase-conjugated rabbit anti-mouse IgG (Jackson Immune Research Laboratories, West Grove, Pa.) as described elsewhere (27). The sera and the secondary antibody were diluted 200 and 1,000 times before use, respectively. A nonimmunized mouse serum was used as control.

Neutralization experiments.The wild-type Stx1 in 5% FBS-MEM was mixed with twofold serially diluted mouse serum and incubated at 37°C for 30 min. The final concentration of Stx1 was adjusted to 2 times the CD₅₀. The mixture of Stx and the sera was dispensed to Vero cells (4 × 10⁴ cells/well) in a 96-well plate, and the plate was incubated at 37°C for 72 h. The cell viability was measured by an Alamar blue assay as described above.

Yield and homogeneity of purified toxins.The purification took about 30 h after harvest of bacterial cells, and the yield of Stx1 was approximately 1 μg from 1 ml of culture broth. The purified wild-type and mutant Stx1s were analyzed by SDS-PAGE to check the homogeneity. As shown in Fig. 1, the wild-type Stx1 and the mutant Stx1s (STX111, STX112, STX 113, and STX114) were separated to two distinct protein bands with apparent molecular weights of 32,000 and 7,700, which correspond to the A and B subunits of Stx1, respectively. Compared with the A subunit of the wild-type Stx1, the A subunit bands of mutant Stx1s were thinner, suggesting that the association of A and B subunits is weaker in mutant toxins than in the wild-type toxin and that parts of the A subunits are lost during purification.

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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.

Toxicity of the mutant Stx1s.Vero-cell cytotoxicity of the mutant Stx1s was assayed by comparison with that of the wild-type Stx1. Neither decrease of viability nor change of cell morphology was observed for the cells mixed with 10 μg of the mutant Stx1s/ml, which showed that the CD₅₀ values for these mutant toxins were more than 10 μg/ml. On the other hand, the CD₅₀ of the wild-type Stx1 was determined to be 17 pg/ml.

The results of the mouse lethality assay are shown in Table 2. All mice administered 100 ng to 10 μg of the mutant Stx1s (STX111, STX112, STX113, and STX114) survived, indicating that the LD₅₀ values for these mutant toxins were more than 10 μg/mouse. In contrast, all mice administered 1 μg of the wild-type Stx1 died, and the LD₅₀ for the wild-type Stx1 was calculated as 200 ng/mouse (10 μg/kg of body weight). This value was higher than that for the wild-type Stx2 (approximately 1 ng/mouse) cloned, purified, and assayed by the same method used for Stx1.

Induction of anti-Stx1 antibody.An anti-Stx1 IgG antibody was induced in the sera by immunization with the mutant Stx1s. After the first booster injection on day 21, the ELISA titers were found to rise and were further enhanced by the second booster. As shown in Fig. 2, the antibody was detected on day 49 in the sera of all mice immunized with one of the four kinds of mutant Stx1s, although the titers were different among the mutant toxins. The higher titers were detected in the sera of the mice immunized with STX112 and STX114, indicating that these mutant toxins maintained the antigenicity of the intact Stx1 more rigorously than the other two mutant toxins, STX111 and STX113.

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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.

Western-blotting experiments revealed that the induced polyclonal antibody responded mainly to the A subunit of Stx1 (Fig. 3, lanes 2 to 5). However, faint but definite staining for B subunit bands was also observed, suggesting that the induced anti-Stx1 antibodies also responded to a monomeric form of the B subunit.

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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.

Neutralization of the cytotoxicity of Stx1.Vero cell cytotoxicity of the wild-type Stx1 was neutralized by incubation with the anti-Stx1 antiserum. At a 32-fold dilution, the sera from the mice immunized with STX112 and STX114 neutralized more than 80% of the cytotoxicity (Fig. 4). The neutralization ability of the sera from the mice immunized with STX111 and STX113 was lower than that of the other two sera, which correlated well with the antibody titers in these sera.

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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.

Protection against Stx1 challenge.Mice immunized with STX112 or STX114 were challenged by the wild-type Stx1. As shown in Table 3, all immunized mice survived after a challenge of Stx1 as high as 10 μg. These mice showed neither paralysis nor any sign of toxicity due to Stx1, and they survived for 6 months after the challenge. In contrast, all control mice injected with PBS alone died after the challenge of 1 μg of Stx1 (Table 3). Two out of five control mice survived at a dose of 100 ng, but they showed slight paralysis of the hind legs. These results clearly demonstrated that the mice were protected against the challenge of the wild-type Stx1 by vaccination with two of the mutant toxins, STX112 and STX114.