Expression of the rabies virus nucleoprotein in plants at high-levels and evaluation of immune responses in mice

Abstract

Transgenic plants have been employed successfully as a low-cost system for the production of therapeutically valuable proteins including antibodies, antigens and hormones. Here, we report expression of a full-length nucleoprotein gene of rabies virus in transgenic tomato plants. The nucleoprotein was also transiently expressed in Nicotiana benthamiana plants by agroinfiltration. In both cases, the nucleoprotein was expressed at high levels, 1-5% of total soluble protein in tomato and 45% in N. benthamiana. Previously, only epitopes of the nucleoprotein had been expressed in plants. The presence and expression of the transgene was verified by PCR, Southern, northern and western blots. Mice were immunized both intraperitoneally (i.p.) and orally with tomato protein extracts containing the N protein induced the production of antibodies. The antibody titer of mice immunized i.p., was at least four times higher than that of mice immunized orally. These results were reflected in the challenge experiments where i.p.-immunized mice were partially protected against a peripheral virus challenge whereas orally immunized mice were not. This protection was comparable to that obtained in previous experiments employing different expression systems. Work is in progress to express both G and N proteins in transgenic plants and evaluate protection in mice.

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

“…This effect has already been observed in another study in which the adjuvant and the antigen had to be co-expressed in order to enhance the immune response (Jin-Tao et al 2006). In other experiments, mice immunized through intraperitoneal injection with plant-derived N protein were partially protected; whereas mice immunized orally were not (Perea-Arango et al 2008).…”

Comparison of the protective efficacy between single and combination of recombinant adenoviruses expressing complete and truncated glycoprotein, and nucleoprotein of the pathogenic street rabies virus in mice

“…Inoculation with N protein expressed in insect cells increased the production of Nab prior to a booster vaccination, elicited T- and B-cell responses, and protected against a lethal RABV challenge in mice [7]. Fifty percent of mice immunized intraperitoneally with tomato extract containing N protein survived a peripheral virus challenge [8]. IM immunization with N protein expressed in silkworm larvae contributed to 90% protection in mice challenged with an intracerebral inoculation of the CVS strain [9].…”

Current Developments and Future Prospects for Plant-Made Biopharmaceuticals Against Rabies

“…extracts from tomato fruits containing 100 lg of the N protein. Mice were boosted with N-protein-enriched chromatographic fraction containing 100 lg of the N protein [49]. In a challenge experiment consisting of an intraperitoneal inoculation of a lethal Rabies virus dose, mice i.p.…”