Passive Oral Immunization by Egg Yolk Immunoglobulin (IgY) to Vibrio cholerae Effectively Prevents Cholera

In an attempt to prepare egg yolk immunoglobulin (IgY) to treat and prevent cholera, hens were immunized by a mixture of heat- or formalin-killed Vibrio cholerae O1 and O139 organisms, or by the recombinant cholera toxin B subunit (CTB). The IgYs were partially purified from egg yolk and orally administered to suckling mice before or after challenge with live O1 or O139 cells. The anti-O1 and O139 IgYs and the mixture of either IgY with anti-CTB IgY significantly protected the occurrence of cholera caused by both O1 and O139 infection. Since large amounts of IgY can be prepared very easily and at low cost, this seems to be a useful procedure for preventing and treating cholera

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

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

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

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