Immunization with a Chimera Consisting of the B Subunit of Shiga Toxin Type 2 and Brucella Lumazine Synthase Confers Total Protection against Shiga Toxins in Mice

Abstract

The striking feature of enterohemorrhagic Escherichia coli (EHEC) infections is the production of Shiga toxins (Stx) implicated in the development of the life-threatening hemolytic uremic syndrome. Despite the magnitude of the social impact of EHEC infections, no licensed vaccine or effective therapy is available for human use. One of the biggest challenges is to develop an effective and safe immunogen to ensure nontoxicity, as well as a strong input to the immune system to induce long-lasting, high-affinity Abs with anti-Stx–neutralizing capacity. The enzyme lumazine synthase from Brucella spp. (BLS) is a highly stable dimer of pentamers and a scaffold with enormous plasticity on which to display foreign Ags. Taking into account the advantages of BLS and the potential capacity of the B subunit of Stx2 to induce Abs that prevent Stx2 toxicity by blocking its entrance into the host cells, we engineered a new immunogen by inserting the B subunit of Stx2 at the amino termini of BLS. The resulting chimera demonstrated a strong capacity to induce a long-lasting humoral immune response in mice. The chimera induced Abs with high neutralizing capacity for Stx2 and its variants. Moreover, immunized mice were completely protected against i.v. Stx2 challenge, and weaned mice receiving an oral challenge with EHEC were completely protected by the transference of immune sera. We conclude that this novel immunogen represents a promising candidate for vaccine or Ab development with preventive or therapeutic ends, for use in hemolytic uremic syndrome–endemic areas or during future outbreaks caused by pathogenic strains of Stx-producing E. coli.

Retinoid Levels Influence Enterohemorrhagic Escherichia coli Infection and Shiga Toxin 2 Susceptibility in Mice

“…We have previously shown that EHEC infection of weaned BALB/c mice can be lethal, as a consequence of systemic damage related to Stx2 toxicity (39). In addition, we have also demonstrated that renal dysfunction secondary to EHEC infection is blocked when anti-Stx2-polyclonal serum is simultaneously inoculated, confirming that Stx is responsible for renal damage and an increased plasma urea concentration (43).…”

“…All control or VA-D mice that died after infection displayed signs of renal damage, compatible with Stx2-related toxicity, as we have previously shown (43), confirming that Stx2-related complications were the cause of death in the experiments performed, even in VA-D mice. It is well known that retinoids play an important role in gut homeostasis and intestinal epithelial integrity (21).…”

Protection of Mice against Shiga Toxin 2 (Stx2)-Associated Damage by Maternal Immunization with a Brucella Lumazine Synthase-Stx2 B Subunit Chimera

“…(i) BLS-Stx2B. BLS-Stx2B was expressed and purified as previously described (20). Briefly, inclusion bodies containing BLS-Stx2B were solubilized by overnight incubation in 8 M urea-50 mM Tris-HCl-5 mM EDTA (pH 8) buffer and dialyzed against 1 M urea-50 mM Tris-HCl-5 mM EDTA (pH 8.5) buffer.…”

“…It assembles as a remarkably stable dimer of pentamers, with 10 N-terminal sites of linkage that allow the insertion of small protein domains without disturbing its conformation (19). We have recently developed a fusion protein between BLS and Stx2B (BLS-Stx2B) that provides high titers of neutralizing antibodies and protective capacity against intravenous challenge with Stx2 in adult immunized mice (20).…”

Comparative Genomics and Immunoinformatics Approach for the Identification of Vaccine Candidates for Enterohemorrhagic Escherichia coli O157:H7

“…Fusion proteins comprising of Stx-derived peptides and T3SS-related proteins are promising vaccine candidates. St2B-Tir-Stx1B-Zot, Stx2B-Stx1B-Int281, EspAStx2A1, EspA-IntiminC300-Stx2B, and Stx2B-BLS fusions have been demonstrated to reduce EHEC colonization in animal models, such as mice and goats (20)(21)(22)(23)(24)(25)(26)(27). Overall, cumulative information indicates that mucosal delivery routes seem to be an effective way to induce immune responses to block the adhesion of EHEC in the intestine, mainly through expression of secretory immunoglobulin A (sIgA) (4).…”