A plant-based recombinant plpE vaccine for fowl cholera

Abstract

Fowl cholera, caused by infection with Pasteurella multocida is an economically significant infectious disease of domestic birds and a major cause of economic loss in poultry industries worldwide. Commercial vaccines consist of either whole cell bacterins or live attenuated bacteria, which have disadvantages of lack of cross-protection against different strains or reversion to virulence, respectively. Moreover, those vaccines are delivered systemically. Since P. multocida infects via the respiratory route, a subunit mucosal vaccine eliciting systemic and mucosal immunity as well as cross-protection might offer a better solution. PlpE is an outer-membrane protein of P. multocida, which protects mice and chickens against fowl cholera, both in soluble and insoluble forms when produced in E. coli and administered systemically. Subunit vaccine antigens produced in transgenic plants can elicit protective immunity in model and target animals when administered either systemically or orally. In this thesis, the author reports the production of plant-based recombinant PlpE vaccines in Nicotiana benthamiana leaves and N. tabacum cell lines and the efficacy of those vaccines in mice and chickens against fowl cholera. Crude plant extracts were prepared from pooled vaccine batches prepared from the homogenized freeze-dried leaf powder of N. benthamiana and cell suspension powder of N. tabacum cell line containing transient and stable transgenic plant-PlpE, respectively. An 8-10 µg dose of transient plant-PlpE vaccine adjuvanted with Alhydrogel® protected 64% of the mice against a lethal challenge infection with P. multocida X-73. During the second mouse vaccination trial, 100 µg transient plant-PlpE adjuvanted with Alhydrogel® protected 70% of the animals against lethal challenge with P. multocida X-73. Mice immunized subcutaneously with 100 µg of stable transgenic plant-PlpE with adjuvant were not protected, whereas only 10% of the mice immunized with stable transgenic plant-PlpE without adjuvant were protected. In a chicken trial, transient plant-PlpE was tested along with soluble and insoluble E. coli PlpE. Chickens were immunized with 100 µg of the plant extract in Alhydrogel® and challenged with P. multocida strain VP161. Sera from the birds in all 3 vaccinated groups reacted with recombinant E. coli PlpE in Western blot analysis. Eighty and fifty percent of birds were protected in groups of chickens immunized with of soluble and insoluble E. coli PlpE, respectively. No birds in the plant-PlpE immunized group were protected; however, the mean time to death was significantly delayed in birds immunized with transient plant-PlpE. Lastly, a feeding trial was performed in mice to test the efficacy of the transient plant-PlpE as an oral vaccine. Mice fed 100 µg of transient plant-PlpE powder adjuvanted with saponin produced circulating PlpE-specific antibodies. Though no mice survived the challenge with P. multocida X-73, the mean time to death was significantly delayed in mice fed on transient plant-PlpE vaccine. This is the first report of expression of any P. multocida protein in plants, and the first report of a plant-based recombinant fowl cholera vaccine, which protected mice against infection. Additionally, this is the first report of any bacterial antigen produced in plants that elicited a strong antibody response in chickens.