Production of Japanese Encephalitis Virus Antigens in Plants Using Bamboo Mosaic Virus-Based Vector

Abstract

Japanese encephalitis virus (JEV) is among the major threats to public health in Asia. For disease control and prevention, the efficient production of safe and effective vaccines against JEV is in urgent need. In this study, we produced a plant-made JEV vaccine candidate using a chimeric virus particle (CVP) strategy based on bamboo mosaic virus (BaMV) for epitope presentation. The chimeric virus, designated BJ2A, was constructed by fusing JEV envelope protein domain III (EDIII) at the N-terminus of BaMV coat protein, with an insertion of the foot-and-mouth disease virus 2A peptide to facilitate the production of both unfused and epitope-presenting for efficient assembly of the CVP vaccine candidate. The strategy allowed stable maintenance of the fusion construct over long-term serial passages in plants. Immuno-electron microscopy examination and immunization assays revealed that BJ2A is able to present the EDIII epitope on the surface of the CVPs, which stimulated effective neutralizing antibodies against JEV infection in mice. This study demonstrates the efficient production of an effective CVP vaccine candidate against JEV in plants by the BaMV-based epitope presentation system.

Keywords: Japanese encephalitis virus; bamboo mosaic virus-based vector; chimeric virus particles (CVPs); foot-and-mouth disease virus 2A; plant-made; vaccine.

FIGURE 1

Japanese encephalitis virus (JEV) EDIII is expressed in plants infected with chimeric BaMV. (A) Schematic representation of the recombinant constructs based on BaMV genome. (B) Infectivity and symptom of various recombinant BaMV construct on Chenopodium quinoa. Leaves inoculated with H₂O (mock) or recombinant plasmids pBS-d35CP, pB2A, pBJ, or pBJ2A were shown. The photos were taken at 10 days post-inoculation (dpi). (C) SDS-PAGE separation and immunoblot analysis of proteins extracted from inoculated or systemically infected leaves of N. benthamiana, as indicated on top of each panel. Leaves were H₂O-inoculated (mock) or inoculated with recombinant plasmids pBS-d35CP, pB2A, pBJ, or pBJ2A as indicated. Total proteins extracted from inoculated leaves (accounting for 1 mg fresh weight of leaf) from each treatment were separated in a 12% SDS-PAGE (Top panel), and stained with coomassie blue (CB). The proteins were transferred to PVDF membranes and reacted with antisera against BaMV CP (anti-CP, middle panel), or JEV EDIII (anti-EDIII, bottom panel), respectively. The relative molecular weights (in kDa) are given on the left of each panel, and positions of each target proteins on the right. IB, immuno-blot.

FIGURE 2

Analysis of the stability of the chimeric BJ2A over serial passages in C. quinoa plants by SDS-PAGE and immunoblot. Leaves were H₂O-inoculated (mock) or inoculated with recombinant plasmids pBS-d35CP, pB2A, pBJ, or pBJ2A, respectively. BJ2A P0 denotes the initial inoculation with the plasmid DNA as inoculum, whereas P1to P9 indicate the 1st to 9th passage using crude leaf sap from P0 as inocula, respectively. SDS-PAGE and immunoblot assays were performed as described in Figure 1C.

FIGURE 3

Immunoelectron microscopy for the identification of JEV EDIII on the surface of BJ2A virus particles. Purified BJ2A virions were incubated with pre-immune rabbit antiserum (A), or antisera specific for JEV EDIII (B) or BaMV CP (C), followed by gold-labeled goat anti-rabbit IgG secondary antibody, and subjected to examination by transmission electron microscopy. Scale bars, 100 nm.

FIGURE 4

Immune response of mice injected intraperitoneally with chimeric virus BJ2A as a vaccine candidate. (A) Determination of immune response by ELISA. Sera from mice immunized with saline, BJ2A, or rEDIII, were subjected to ELISA using rDEIII as the antigen. The titers of antisera were determined by blocking ELISA as described (Yang et al., 2007). The columns represent the mean O.D. (at 450 nm) values obtained with sera from individual mice with standard deviations shown as error bars. (B) Analysis of effective JEV EDIII-specific antibodies by indirect immunofluorescence assay. BHK-21 cells, infected with JEV or uninfected as indicated on the top, were fixed and stained with pooled sera prepared from mice immunized with saline, BJ2A, or rEDIII and an FITC-conjugated secondary antibody, followed by examination with an inverted fluorescent microscope (Leica) (panels denoted by “FITC”). Cell nuclei were stained by DAPI (panels denoted by “DAPI”).