Specific Egg Yolk Immunoglobulin as a New Preventive Approach for Shiga-Toxin-Mediated Diseases

Shiga toxins (Stxs) are involved in the development of severe systemic complications associated with enterohemorrhagic Escherichia coli (EHEC) infection. Various neutralizing agents against Stxs are under investigation for management of EHEC infection. In this study, we immunized chickens with formalin-inactivated Stx-1 or Stx-2, and obtained immunoglobulin Y (IgY) from the egg yolk. Anti-Stx-1 IgY and anti-Stx-2 IgY recognized the corresponding Stx A subunit and polymeric but not monomeric B subunit. Anti-Stx-1 IgY and anti-Stx-2 IgY suppressed the cytotoxicity of Stx-1 and Stx-2 to HeLa 229 cells, without cross-suppressive activity. The suppressive activity of these IgY was abrogated by pre-incubation with the corresponding recombinant B subunit, which suggests that the antibodies directed to the polymeric B subunits were predominantly involved in the suppression. In vivo, the intraperitoneal or intravenous administration of these IgY rescued mice from death caused by intraperitoneal injection of the corresponding toxin at a lethal dose. Moreover, oral administration of anti-Stx-2 IgY reduced the mortality of mice infected intestinally with EHEC O157:H7. Our results therefore suggest that anti-Stx IgY antibodies may be considered as preventive agents for Stx-mediated diseases in EHEC infection

Western blot analysis and dot blot analysis.

<p>(A–C) Western blot analysis of anti-Stx-1 IgY and anti-Stx-2 IgY. hpStx-1 and hpStx-2 were subjected to SDS-PAGE at 2.5 µg/lane. (A) CBB staining of SDS-PAGE gel. (B) Anti-Stx-1 IgY and (C) anti-Stx-2 IgY were applied to the membrane at 20 µg/ml. The marks in the figures indicate A subunit, A1 fragment and monomeric B subunit. (D, E) Dot blot analysis of anti-Stx-1 IgY and anti-Stx-2 IgY. hpStx-1 and hpStx-2 were blotted at 4 µg/dot, and Stx-1BH and Stx-2BH at 2 µg/dot. Anti-Stx-1 IgY and anti-Stx-2 IgY were applied to the membrane at 2 µg/ml and 4 µg/ml, respectively.</p

Time course of IgY concentration in feces of mice given anti-Stx-2 IgY orally.

<p>ddY mice were given 200 mg/kg anti-Stx-2 IgY orally. IgY concentration was measured by ELISA. Each point represents the mean ± SEM of five mice.</p

Anti-Stx-1 IgY and anti-Stx-2 IgY preparations.

a<p>assayed using DC™ Protein assay (BioRad, Hercules, CA, USA).</p>b<p>assayed using Chicken IgG ELISA Quantitation Kit™ (Bethyl Laboratories, Inc., Montgomery, TX, USA).</p

Mortality of mice administered anti-Stx-1 IgY and anti-Stx-2 IgY after challenge with ppStx-1 and ppStx-2.

<p>ddY mice were challenged i.p. with 2.5 times LD₅₀ ppStx-1 and 2 times LD₅₀ ppStx-2. Anti-Stx-1 IgY and anti-Stx-2 IgY were given to mice i.v. at a dose of 100 mg/kg at various timing on the challenge.</p><p>***There is a statistical significant difference at <i>p</i><0.001 vs. the control (log rank test).</p

Neutralizing activity of anti-Stx IgY.

<p>(A) hpStx-1, (B) hpStx-2 and (C) hpStx-2A1B were mixed with serial dilutions of anti-Stx-1 IgY and anti-Stx-2 IgY, and incubated for 1 h at 37°C. The mixture was subjected to the cytotoxicity assay. Final concentration of hpStx-1, hpStx-2 and hpStx-2A1B was five times CD₅₀. N: cells not treated with Stx. C: cells treated with Stx alone (control). Each column represents the mean ± SEM of triplicate wells. There was a significant difference at *<i>p</i><0.05, **<i>p</i><0.01 and ***<i>p</i><0.001 vs. the control (Dunnet's comparison test).</p

Effect of anti-Stx-1 IgY and anti-Stx-2 IgY on mortality of mice challenged with ppStx-1 and ppStx-2.

<p>ddY mice were administered i.p. with anti-Stx-1 IgY or anti-Stx-2 IgY, followed 10 min later by an i.p. challenge with ppStx-1 (2.5 times LD₅₀) or ppStx-2 (2 times LD₅₀). Death of mice was observed for 10 days after the challenges.</p><p>***There is a statistical significant difference at <i>p</i><0.001 vs. the control (log rank test).</p

Neutralizing activity of anti-Stx IgY on Stx bound to target cells.

<p>(A) hpStx-1 and (C) hpStx-2 were added to HeLa 229 cells at a concentration of 20 times CD₅₀, and kept on ice for 30 min. Cells were extensively washed with cold DMEM to remove unbound Stx. (A) Anti-Stx-1 IgY and (C) anti-Stx-2 IgY were added to the Stx-pulsed cells and the cytotoxicity assay was performed. (B) Anti-Stx-1 IgY and (D) anti-Stx-2 IgY were added to HeLa 229 cells just before addition of (B) hpStx-1 and (D) hpStx-2 at a concentration of five times CD₅₀, and the cytotoxicity assay was performed. N: cells not treated with Stx. C: cells treated with Stx alone (control). Each column represents the mean ± SEM of quadruplicate wells. There was a significant difference at *<i>p</i><0.05, **<i>p</i><0.01 and ***<i>p</i><0.001 vs. the control (Dunnet's comparison test).</p

EHEC or <i>E. coli</i> strains used in this study.

a<p>see “bacterial strains” in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026526#s4" target="_blank">Materials and Methods</a> for detail.</p>b<p>ppStx-1 and</p>c<p>ppStx-2 were used for <i>in vivo</i> experiments that required a large amount of Stxs.</p

Effect of orally administered anti-Stx IgY on mortality of mice infected with EHEC O157:H7.

<p>(A) GPU993-S and (B) GPU96MM-S were inoculated intragastrically to Balb/c mice. GPU993-S and GPU96MM-S were resistant to streptomycin. Mice inoculated with these bacteria were given drinking water supplemented with streptomycin throughout the experimental period. MMC was injected i.p. at 18, 21 and 24 h, and anti-Stx-1 IgY or anti-Stx-2 IgY was given at 16, 19, 22 and 40 h after inoculation. Control mice were given PBS instead of anti-Stx IgY. Each group included 6 mice. There was a significant difference at *<i>p</i><0.05 vs. the control (log rank test).</p