ORIGINAL PAPER Immunogenicity of nuclear-encoded LTB:ST fusion protein from Escherichia coli expressed in tobacco plants Sergio Rosales-Mendoza • Ruth E. Soria-Guerra • Leticia Moreno-Fierros • Dania O. Govea-Alonso • Areli Herrera-Dı ´az • Schuyler S. Korban • A ´ ngel G. Alpuche-Solı ´s Received: 3 January 2011 / Revised: 21 January 2011 / Accepted: 21 January 2011 / Published online: 12 February 2011 Ó Springer-Verlag 2011 Abstract Enterotoxigenic Escherichia coli (ETEC) is one of the main causative agents of diarrhea in infants and for travelers. Inclusion of a heat-stable (ST) toxin into vaccine formulations is mandatory as most ETEC strains can produce both heat-labile (LT) and ST enterotoxins. In this study, a genetic fusion gene encoding for an LTB:ST protein has been constructed and transferred into tobacco via Agrobacterium tumefaciens-mediated transformation. Transgenic tobacco plants carrying the LTB:ST gene are then subjected to GM1-ELISA revealing that the LTB:ST has assembled into pentamers and displays antigenic determinants from both LTB and ST. Protein accumulation of up to 0.05% total soluble protein is detected. Subse- quently, mucosal and systemic humoral responses are elicited in mice orally dosed with transgenic tobacco leaves. This has suggested that the plant-derived LTB:ST is immunogenic via the oral route. These findings are critical for the development of a plant-based vaccine capable of eliciting broader protection against ETEC and targeting both LTB and ST. Features of this platform in comparison to transplastomic approaches are discussed. Keywords Oral immunization Á Chimeric protein Á Broader protective vaccine Introduction The annual burden of diarrhea among infants living in the developing world is estimated to be around 1.5 billion episodes, and accounting for 3 million deaths. Entero- toxigenic Escherichia coli (ETEC) is the most frequently isolated enteropathogen, resulting in approximately 210 million diarrheal episodes, and approximately 380,000 deaths per year (WHO 2010). ETEC is capable of producing two forms of toxins, the heat-labile (LT) and the heat-stable (ST) toxins. LT is an A-5B toxin wherein the A subunit (LTA) has a toxic ADP ribosyl transferase activity, while the B subunit is associ- ated as a pentamer, but lacks toxicity (Field 1979). ST is a 19 aa peptide that contributes to secretion of fluids and electrolytes by activating the transmembrane guanylate cyclase C receptor (Sato and Shimonishi 2004). It has been demonstrated that the anti-LT immune response provides at least short term protection (Walker et al. 2007). However, neutralizing antibodies against ST must be elicited to protect against strains producing both toxins (Taxt et al. 2010). Due to lack of ST immunoge- nicity, genetic fusions between ST and carrier proteins have been made in efforts to successfully immunize against Communicated by P. Lakshmanan. S. Rosales-Mendoza Á R. E. Soria-Guerra Á D. O. Govea-Alonso Laboratorio de Biofarmace ´uticos recombinantes, Facultad de Ciencias Quı ´micas, Universidad Auto ´noma de San Luis Potosı ´, Av. Dr. Manuel Nava 6, 78210 San Luis Potosı ´, SLP, Mexico L. Moreno-Fierros Inmunidad en Mucosas, UBIMED, FES-Iztacala, Universidad Nacional Auto ´noma de Me ´xico, Avenida de los Barrios 1, 54090 Tlalnepantla, Mexico A. Herrera-Dı ´az Á A ´ . G. Alpuche-Solı ´s (&) Laboratorio de Biologı ´a Molecular de Plantas, Divisio ´n de Biologı ´a Molecular, Instituto Potosino de Investigacio ´n Cientı ´fica y Tecnolo ´gica, Camino a la Presa San Jose ´ 2055, 78216 San Luis Potosı ´, SLP, Mexico e-mail: alpuche@ipicyt.edu.mx S. S. Korban Department of Natural Resources and Environmental Sciences, University of Illinois, Urbana, IL 61801, USA 123 Plant Cell Rep (2011) 30:1145–1152 DOI 10.1007/s00299-011-1023-0