CVI Accepts, published online ahead of print on 28 January 2009

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Clin. Vaccine Immunol. doi:10.1128/CVI.00328-08
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

DNA vaccine encoding the enterohemorragic Escherichia coli (EHEC) Shiga-like toxin 2 (Stx2) A₂ and B subunits confers protective immunity to Stx challenge in the murine model

Leticia V. Bentancor, Marcos Bilen, Romina J. Fernández Brando, María Victoria Ramos, Luis C.S. Ferreira, Pablo D. Ghiringhelli, and Marina S. Palermo^*

División Inmunología, Instituto de Leucemia Experimental (CONICET), Instituto de Investigationes Hematológicas, Academia Nacional de Medicina, Buenos Aires, Argentina; Laboratorio de Ingeniería Genética y Biología Celular y Molecular, Universidad Nacional de Quilmes, Pcia. de Buenos Aires, Argentina; Departamento de Microbiologia, Universidade de São Paulo, São Paulo, Brasil

^* To whom correspondence should be addressed. Email: mspalermo{at}hematologia.anm.edu.ar.

Production of verocytotoxin or shiga-like toxin (Stx), particularly Stx2, is the basis of the hemolytic uremic syndrome (HUS), a frequently lethal outcome of subjects infected with Stx2-producing enterohemorrhagic Escherichia coli (EHEC) strains, The toxin is formed by a single A subunit that promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide (Gb3) at the surface of host cells. Host enzymes cleave the A subunit into the A₁ peptide, endowed with N-glycosidase activity to the 28S rRNA, and the A₂ peptide that confers stability to the B pentamer. We report the construction of a DNA vaccine (pStx2AB) that expresses a non-toxic Stx2 mutated form consisting of the last 32 aminoacids of A₂ sequence and the complete B subunit, as two non-fused polypeptides. Immunization trials carried out with the DNA vaccine in BALB/c mice, alone or in combination with another DNA vaccine encoding GM-CSF, resulted in systemic Stx-specific antibody responses targeting both A and B subunits of the native Stx2. Moreover, anti-Stx2 antibodies raised in mice immunized with pStx2AB showed toxin neutralization activity in vitro and, more importantly, confered partial protection to Stx2 challenge. The present vector represents the second DNA vaccine so far reported to induce protective immunity to Stx2 and may contribute, either alone or in combination with other procedures, to the development of prophylatic or therapeutic interventions aiming amelioration of the EHEC infection-associated sequels.