Induction of a protective immune response to rabies virus in sheep after oral immunization with transgenic maize, expressing the rabies virus glycoprotein.

The introduction of exogenous genes into plants permits the development of a new generation of biological products, i.e., edible vaccines. Cereals, especially maize, have been the systems of choice for the expression of antigenic proteins because the proteins can be expressed at high levels in the kernel and stored for prolonged periods without excessive deterioration. The utilization of plant-derived antigens for oral delivery provides an alternative strategy for the control of pathogens in animals compared to the current vaccine administration methods, such as injection. However, there is some doubt about the efficacy of these types of vaccines in polygastric animals due to the features of their digestive system. Here, we report the efficacy of an edible vaccine against rabies evaluated in sheep. Kernels containing different doses of G protein (0.5, 1, 1.5 and 2mg) were given in a single dose by the oral route. Cumulative survival was better in groups that received 2mg of G protein and for the positive control (inactivated rabies vaccine); this observation was supported by the presence of neutralizing antibodies. Animals in the control group died after challenge. The degree of protection achieved for 2mg of G protein was comparable to that conferred by a commercial vaccine. In conclusion, this is the first study in which an orally administered edible vaccine showed efficacy in a polygastric model.

Expression of rabies virus G protein in carrots (Daucus carota).

Antigens derived from various pathogens can readily be synthesized at high levels in plants in their authentic forms. Such antigens administered orally can induce an immune response and, in some cases, result in protection against a subsequent challenge. We here report the expression of rabies virus G protein into carrots. The G gene was subcloned into the pUCpSSrabG vector and then used to transform carrot embryogenic cells by particle bombardment. The carrot cells were selected in liquid medium, a method previously unreported. The presence of the transgene was verified by PCR, and by RT-PCR. By western blot, G protein transgene was identified in 93.3% of adult carrot roots. The G protein was quantified by densitometric analysis (range 0.4-1.2%). The expressed protein was antigenic in mice. This confirms that the carrot is an adequate system for antigen expression.

Fruit-specific expression of the human immunodeficiency virus type 1 tat gene in tomato plants and its immunogenic potential in mice.

The human immunodeficiency virus type 1 (HIV-1) Tat protein is considered a potential candidate vaccine antigen. In an effort to design a strategy for noninvasive vaccination against HIV-1, we developed transgenic tomatoes expressing the Tat protein. Two independent plants testing positive in transgene detection analysis were selected and grown to maturity. Monoclonal antibodies against Tat recognized a protein of the expected size. Interestingly, expression of Tat seemed to be toxic to the plant, as in all cases the fruit exhibited underdeveloped reproductive structures and no seeds. Nine groups of 10 pathogen-free BALB/c male mice were primed either orally, intraperitoneally, or intramuscularly with 10 mg of tomato fruit extract derived from transgenic or wild-type plants and with 10 microg of Tat86 recombinant protein. Mice were immunized at days 0, 14, and 28, and given boosters after 15 weeks; sera were drawn 7 days after each booster, and the antibody titer was determined by enzyme-linked immunosorbent assay. All three immunization approaches induced the development of a strong anti-Tat immunological response, which increased over time. Isotype subclass determination showed the presence of mucosal (immunoglobulin A) immunity soon after the beginning of the oral immunization protocol, and the data were confirmed by the presence of anti-Tat antibodies in fecal pellets and in vaginal washes. We also demonstrated that sera from immunized mice inhibited with high efficiency recombinant Tat-dependent transactivation of the HIV-1 long terminal repeat promoter. This neutralization activity might be relevant for the suppression of extracellular Tat activities, which play an important role in HIV disease development.

Expression of Escherichia coli heat-labile enterotoxin b subunit (LTB) in carrot (Daucus carota L.).

We expressed the B subunit of enterotoxigenic Escherichia coli heat-labile enterotoxin (LTB) encoded by a synthetic codon-optimized gene in carrot. An Agrobacterium-mediated transformation method was used. Thirty independent transgenic lines were regenerated via somatic embryogenesis after 6 months in culture and were transferred to a greenhouse. GM1-ELISA assay was used to assess LTB protein content in mature taproots. Some transgenic lines expressed LTB up to 0.3% of the total soluble protein, which is tenfold higher than the expression levels reported earlier using the native bacterial gene in plants. Immunological assay confirmed proper assembly of the pentameric complex and in vitro activity of the recombinant LTB protein, suggesting that it can be functional in prevention of diarrhea.