2007

Joensuu, J. J.; Van Molle, I.; Verdonck, F.; Kotiaho, M.; Buts, L.; Ehrström, A.; Peltola, M.; Siljander-Rasi, H.; Nuutila, Anna-Maria; Oksman-Caldentey, Kirsi-Marja; Teeri, T. H.; Bouckaert, J.; Wyns, L.; De Greve, H.; Panjikar, S.; Cox, E.; Goddeeris, B. M.; Niklander-Teeri, V.

F4 fimbriae are the major colonization factors associated with porcine neonatal and postweaning diarrhea caused by enterotoxigenic Escherichia coli (ETEC). Via the chaperone/usher pathway, the F4 are assembled as long polymers of the major subunit FaeG, which also possesses the adhesive properties of the fimbriae. Being highly stable and mucosally immunogenic F4/FaeG offers a unique model system to study oral vaccination against ETEC-induced postweaning diarrhea (PWD). PWD is a major problem in piggeries worldwide and results in significant economic losses. No vaccine is currently available to protect piglets against PWD. Transgenic plants provide an economically feasible platform for large-scale production of vaccine antigens for animal health. Here, the capacity of transgenic plants to produce FaeG was evaluated. Using the model plant tobacco, FaeG was directed to different subcellular compartments by specific targeting signals. Targeting of FaeG into chloroplasts offered a superior accumulation level of 1% of total soluble proteins over the endoplasmic reticulum and the apoplast. Intrinsically, the incomplete fold of fimbrial subunits renders them unstable and susceptible to aggregation and/or proteolytical degradation in the absence of a specific periplasmic chaperone. The chloroplast-targeted FaeG was purified from tobacco and crystallized. The crystal structure shows that chloroplasts circumvent the absence of the fimbrial assembly machinery by assembling FaeG into strand-swapped dimers. Moreover, the FaeG-dimers retained the key properties of an oral vaccine, i.e. stability in gastrointestinal conditions, binding to porcine intestinal F4 receptors, and inhibition of the F4+ ETEC attachment to F4R. To investigate the oral immunogenicity, the FaeG protein was expressed in the crop plants alfalfa and barley. Desiccated alfalfa plants and barley grains stored FaeG in a stable form for years. When the transgenic alfalfa plants and cholera toxin were orally co-administered to weaned piglets, F4-specific systemic and mucosal immune responses were induced and the duration and number of F4+ E. coli excretion following F4+ ETEC challenge were reduced. In conclusion, these results suggest that transgenic plants producing the FaeG subunit protein could be used for production and delivery of oral vaccines against porcine PWD.