Induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes

Abstract

Background: Plant viruses can be employed as versatile vectors for the production of vaccines by expressing immunogenic epitopes on the surface of chimeric viral particles. Although several viruses, including tobacco mosaic virus, potato virus X and cowpea mosaic virus, have been developed as vectors, we aimed to develop a new viral vaccine delivery system, a bamboo mosaic virus (BaMV), that would carry larger transgene loads, and generate better immunity in the target animals with fewer adverse environmental effects.

Methods: We engineered the BaMV as a vaccine vector expressing the antigenic epitope(s) of the capsid protein VP1 of foot-and-mouth disease virus (FMDV). The recombinant BaMV plasmid (pBVP1) was constructed by replacing DNA encoding the 35 N-terminal amino acid residues of the BaMV coat protein with that encoding 37 amino acid residues (T128-N164) of FMDV VP1.

Results: The pBVP1 was able to infect host plants and to generate a chimeric virion BVP1 expressing VP1 epitopes in its coat protein. Inoculation of swine with BVP1 virions resulted in the production of anti-FMDV neutralizing antibodies. Real-time PCR analysis of peripheral blood mononuclear cells from the BVP1-immunized swine revealed that they produced VP1-specific IFN-gamma. Furthermore, all BVP1-immunized swine were protected against FMDV challenge.

Conclusion: Chimeric BaMV virions that express partial sequence of FMDV VP1 can effectively induce not only humoral and cell-mediated immune responses but also full protection against FMDV in target animals. This BaMV-based vector technology may be applied to other vaccines that require correct expression of antigens on chimeric viral particles.

Figure 1

Schematic representation of plasmids of wild-type BaMV-S as well as recombinant pBS-d35CP and pBVP1. (a) pBaMV-S is an infectious BaMV cDNA plasmid under the control of cauliflower mosaic virus 35S promoter. The BaMV genome comprises 5 open reading frames (ORFs). The ORF1 encodes for viral replicase, whereas ORF2, ORF3 and ORF4 are called a "Triple-gene-block" that encodes viral movement proteins. ORF5 (CP) encodes a structural protein for virion formation. (b) pBS-d35CP is a mutated BaMV plasmid whose DNA coding for the N-terminal 35 amino acids of CP has been truncated, and multiple cloning sites have been engineered. (c) pBVP1 is a recombinant plasmid derived from pBS-d35CP by inserting the DNA coding for the 37 amino acid VP1 peptide to replace that coding for the N-terminal 35 amino acids of CP.

Figure 2

Detection of VP1 antigenic epitopes in Chenopodium quinoa (C. quinoa) leaves inoculated with chimeric virus BVP1. Leaves of C. quinoa were mock-inoculated (lane 1) or inoculated with wild-type pBaMV-S (lane 2), pBS-d35CP (lane 3) or pBVP1 (lane 4). Total proteins were prepared as described in Methods and electrophoretically separated on a 12% SDS-polyacrylamide gel, stained with Coomassie blue (panel A), or transferred to PVDF membrane, and detected with anti-BaMV-S CP serum (panel B), rabbit anti-FMDV VP1 serum (panel C), or serum from FMDV-infected swine (panel D).

Figure 3

Analysis of purified BVP1 virions on SDS- PAGE. Various amounts of BVP1 virions purified from infected leaves by CsCl₂ density gradient centrifugation were analyzed by electrophoresis through a 12.5% polyacrylamide and stained with PlusOne Silver Staining Kit (Amersham Bioscience, Sweden). Lane M, molecular weight standards. The relative molecular weights were as indicated on the left.

Figure 4

Immunoelectron microscopy for identification of BaMV CP and FMDV VP1 on the surface of virus particles. Leaf dips from C. quinoa infected with pBVP1 (A, B) or pBaMV-S (C) were obtained 10 days post-inoculation. Grids were first incubated with leaf extract and coated with diluted anti-BaMV CP serum (A) or anti-FMDV VP1 serum (B, C) followed by gold-labeled goat anti-rabbit IgG complexes. Grids were inspected in a Philips CM100 electron microscope. All bars represent 250 nm.

Figure 5

Serum titers of swine immunized with chimeric virus BVP1. Groups of swine were immunized with 0.5 mg (▲), 5 mg (◆), 10 mg (■) of chimeric virus BVP1 or 5 mg of wild-type virus BaMV-S (○) or PBS (□). Swine sera were collected at the indicated time after immunization. Anti-VP1 titers were determined by ELISA. The animals inoculated with 0.5 mg BVP1 were boosted with the same amount of BVP1 four weeks after priming while those inoculated with 5 mg and 10 mg of BVP1 were boosted six weeks after priming.