title: Expression of an Escherichia coli antigenic fusion protein comprising the heat labile toxin B subunit and the heat stable toxin, and its assembly as a functional oligomer in transplastomic tobacco plants.
abstract: Enterotoxigenic Escherichia coli (ETEC) strains are important pathogens in developing countries. Some vaccine formulations containing the heat labile toxin B subunit (LTB) have been used in clinical trials; however, the induction of neutralizing antibodies against the heat-stable toxin (ST), a poor immunogenic peptide, is necessary, as most ETEC strains can produce both toxins. In this study, a plant optimized synthetic gene encoding for the LTB-ST fusion protein has been introduced into plastids of tobacco leaf tissues, using biolistic microprojectile bombardment, in an effort to develop a single plant-based candidate vaccine against both toxins. Transplastomic tobacco plants carrying the LTB-ST transgene have been recovered. Transgene insertion into the plastid was confirmed by both PCR and Southern blot analysis. GM1-ELISA revealed that the LTB-ST fusion protein retained its oligomeric structure, and displayed antigenic determinants for both LTB and ST. Western blot analysis, using LTB antisera, confirmed the presence of a 17-KDa protein in transplastomic lines, with the correct antigenicity of the fusion protein. Expression levels of this fusion protein in different lines reached up to 2.3% total soluble protein. Oral immunization of mice with freeze-dried transplastomic tobacco leaves led to the induction of both serum and mucosal LTB-ST specific antibodies. Following cholera toxin challenge, a decrease of intestinal fluid accumulation was observed in mice immunized with LTB-ST-containing tobacco. These findings suggest that tobacco plants expressing LTB-ST could serve as a plant-based candidate vaccine model providing broad-spectrum protection against ETEC-induced diarrhoeal disease.
authors.affiliation: Department of Natural Resources and Environmental Sciences, University of Illinois, Urbana, IL 61801, USA.
authors.forename: Sergio
authors.initials: S
authors.lastname: Rosales-Mendoza
authors.forename: Angel Gabriel
authors.initials: AG
authors.lastname: Alpuche-Solís
authors.forename: Ruth Elena
authors.initials: RE
authors.lastname: Soria-Guerra
authors.forename: Leticia
authors.initials: L
authors.lastname: Moreno-Fierros
authors.forename: Luzmila
authors.initials: L
authors.lastname: Martínez-González
authors.forename: Areli
authors.initials: A
authors.lastname: Herrera-Díaz
authors.forename: Schuyler S
authors.initials: SS
authors.lastname: Korban