Recombinant Shiga toxin B-subunit-keyhole limpet hemocyanin conjugate vaccine protects mice from Shigatoxemia

Abstract

Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis in humans and, in a subgroup of infected subjects, a more serious condition called hemolytic-uremic syndrome (HUS). These conditions arise because EHEC produces two antigenically distinct forms of Shiga toxin (Stx), called Stx1 and Stx2. Despite this, the production of Stx2 by virtually all EHEC serotypes and the documented role this toxin plays in HUS make it an attractive vaccine candidate. Previously, we assessed the potential of a purified recombinant Stx2 B-subunit preparation to prevent Shigatoxemia in rabbits. This study revealed that effective immunization could be achieved only if endotoxin was included with the vaccine antigen. Since the presence of endotoxin would be unacceptable in a human vaccine, the object of the studies described herein was to investigate ways to safely augment, in mice, the immunogenicity of the recombinant Stx2 B subunit containing <1 endotoxin unit per ml. The study revealed that sera from mice immunized with such a preparation, conjugated to keyhole limpet hemocyanin and administered with the Ribi adjuvant system, displayed the highest Shiga toxin 2 B-subunit-specific immunoglobulin G1 (IgG1) and IgG2a enzyme-linked immunosorbent assay titers and cytotoxicity-neutralizing activities in Ramos B cells. As well, 100% of the mice vaccinated with this preparation were subsequently protected from a lethal dose of Stx2 holotoxin. These results support further evaluation of a Stx2 B-subunit-based human EHEC vaccine.

FIG. 1.

Survival plots for mice immunized with the Stx2 B subunit in admixture with various adjuvants or saline control containing no Stx2 B subunit. Saline (- - -), Quil A (  ), Quil A plus LPS (- - - - ), Alhydrogel (...), Alhydrogel plus MPL (—), RAS-TDM (), and RAS-TDM plus MPL (). (A) Groups of five mice were used per immunization protocol; (B) groups of 15 mice were used per immunization protocol.

FIG. 2.

Analysis of the recombinant Stx2 B-subunit-KLH conjugate by SDS-PAGE. Lane A, low-molecular-mass prestained standard proteins used to calibrate the gel; lane B, KLH; lane C, Stx2 B subunit; lane D, Stx2 B-subunit-KLH conjugate vaccine. The gel was stained with Coomassie blue.

FIG. 3.

ELISA analysis for antigen-specific IgG1 (A and B) and IgG2a (C and D) titers of sera from individual mice after two injections of KLH only (▴) or the Stx2 B-subunit-KLH conjugate (⋄) administered with Alhydrogel (A and C) or RAS-TDM (B and D). The data represent the average of triplicate determinations for each point.

FIG. 4.

Ramos B-cell apoptosis neutralizing activity of sera obtained from individual mice immunized with KLH only or the Stx2 B-subunit-KLH conjugate admixed with RAS-TDM or Alhydrogel. The error bars represent the standard errors of the mean values from triplicate determinations. The unshaded bars represent serum samples that significantly (P ≤ 0.05, Student's t test) reduced Stx2 toxicity relative to the activity of control sera. K106 is a positive control serum sample obtained from a rabbit immunized with the recombinant Stx2 B subunit in the presence of endotoxin, as described in our previous article (30).

FIG. 5.

Survival plots for mice immunized with KLH alone or the Stx2 B-subunit-KLH conjugate admixed with RAS-TDM or Alhydrogel (n = 10 mice per group). KLH mixed with Alhydrogel (), Stx2 B-subunit-KLH mixed with Alhydrogel (—), KLH mixed with RAS-TDM (- - -), and Stx2 B-subunit-KLH mixed with RAS-TDM (). The differences in the survival rates of mice immunized with the Stx2 B-subunit-KLH conjugate preparations and those of the mice immunized with KLH alone were highly significant (P = 0.025 and 0.011, Stx2 B-subunit-KLH administered with Alhydrogel or RAS-TDM, respectively, Fisher's exact test).