Mice orally immunized with a transgenic plant expressing the glycoprotein of Crimean-Congo hemorrhagic fever virus

Abstract

While Crimean-Congo hemorrhagic fever (CCHF) has a high mortality rate in humans, the associated virus (CCHFV) does not induce clinical symptoms in animals, but animals play an important role in disease transmission to humans. Our aim in this study was to examine the immunogenicity of the CCHFV glycoprotein when expressed in the root and leaf of transgenic plants via hairy roots and stable transformation of tobacco plants, respectively. After confirmatory analyses of transgenic plant lines and quantification of the expressed glycoprotein, mice were either fed with the transgenic leaves or roots, fed the transgenic plant material and injected subcutaneously with the plant-made CCHFV glycoprotein (fed/boosted), vaccinated with an attenuated CCHF vaccine (positive control), or received no treatment (negative control). All immunized groups had a consistent rise in anti-glycoprotein IgG and IgA antibodies in their serum and feces, respectively. The mice in the fed/boosted group showed a significant rise in specific IgG antibodies after a single boost. Our results imply that oral immunization of animals with edible materials from transgenic plants is feasible, and further assessments are under way. In addition, while the study of CCHF is challenging, our protocol should be further used to study CCHFV infection in the knockout mouse model and virus neutralization assays in biosafety level 4 laboratories.

Fig. 1.

Schematic of the T-DNA region of binary vectors used for the expression of the G1/G2 glycoprotein gene. The synthetic G1/G2 glycoprotein gene consisting of the coding sequences for the G1 and G2 portions of the CCHFV glycoprotein was expressed under the control of the CaMV 35S promoter. RB, right border; LB, left border.

Fig. 2.

PCR and Southern blot analyses of genomic DNA extracts from transgenic plants. (a) Gene-specific primers were used to amplify a 602-bp fragment. The G1/G2 gene was present in seven lines of tobacco plants (lanes 1 to 7, right) and four lines of hairy root (lanes 1 to 4, left). (b) The corresponding genomic DNA was examined by Southern blotting using a 602-bp gene-specific probe on tobacco plant lines (lanes 1 to 7, right) and hairy root lines (lanes 1 to 4, left). PC, positive control; WT, wild-type genome as a negative control; Lad, 100-bp DNA ladder.

Fig. 3.

Analysis of expression of the G1/G2 glycoprotein in transgenic plants by Western blotting and ELISA using anti-G1/G2 mouse serum. (a) Western blot of three transgenic tobacco lines (lanes 1 to 3) and one transgenic tobacco hairy root (HR) line. All samples were standardized to contain 40 μg of TSP. (b) Quantification of the G1/G2 glycoprotein in 1 g (fresh weight) of the transgenic lines using a standard curve of the purified G1/G2 glycoprotein. WT, wild-type plant; Lad, protein molecular mass marker.

Fig. 4.

Anti-G1/G2 antibody quantification by ELISA in serum and fecal samples from mice immunized with transgenic plant material. The curves show anti-G1/G2 IgG antibodies in serum (a) and anti-G1/G2 IgA antibodies in serum and feces (b). The six groups of mice included those fed leaf or root, those fed leaf or root and given a booster injection of the purified G1/G2, those subcutaneously vaccinated by the CCHF vaccine, and a nonimmunized group (as a negative control [NC]). In the IgA assay, the same groups were pooled as fed, fed/boosted, and vaccinated group.

Fig. 5.

Specificity and cross-reactivity of anti-G1/G2 murine serum with both plant-made G1/G2 glycoprotein and CCHF vaccine containing inactivated CCHF virus as antigen determined by ELISA and Western blotting. (a) The ELISA shows that anti-G1/G2 IgG antibodies in serum reacted with both antigens. (b) Illustration of cross-reaction of the antiserum with both antigens by Western blot analysis. Lane 1, CCHF vaccine containing inactivated CCHF virus; lane 2, plant-made G1/G2 glycoprotein. Ag, antigen.