Recombinant DNA and Protein Vaccines for Foot-and-mouth Disease Induce Humoral and Cellular Immune Responses in Mice

Abstract

Background: Foot-and-mouth disease virus (FMDV) is a small single-stranded RNA virus which belongs to the family Picornaviridae, genus Apthovirus. It is a principal cause of FMD which is highly contagious in livestock. In a wild type virus infection, infected animals usually elicit antibodies against structural and non-structural protein of FMDV. A structural protein, VP1, is involved in neutralization of virus particle, and has both B and T cell epitopes. A RNA-dependent RNA polymerase, 3D, is highly conserved among other serotypes and strongly immunogenic, therefore, we selected VP1 and 3D as vaccine targets.

Methods: VP1 and 3D genes were codon-optimized to enhance protein expression level and cloned into mammalian expression vector. To produce recombinant protein, VP1 and 3D genes were also cloned into pET vector. The VP1 and 3D DNA or proteins were co-immunized into 5 weeks old BALB/C mice.

Results: Antigen-specific serum antibody (Ab) responses were detected by Ab ELISA. Cellular immune response against VP1 and 3D was confirmed by ELISpot assay.

Conclusion: The results showed that all DNA- and protein-immunized groups induced cellular immune responses, suggesting that both DNA and recombinant protein vaccine administration efficiently induced Ag-specific humoral and cellular immune responses.

Keywords: B cell epitope peptide; DNA vaccine; FMDV; Recombinant protein vaccine.

Figure 1

FMDV type-O VP1, 3D cloning strategy and in vitro protein expression. FMDV VP1 (0.6 Kb) and 3D (1.4 Kb) PCR products were subcloned into pcDNA3.1V5/His mammalian expression vector and pET bacterial expression vector. (A) FMDV type-O VP1 and 3D cloning strategy (B) Protein expression of VP1 and 3D was determined in plasmids-transected RD cells, and its expression was visualized by FITC-labeling and DAPI staining for nucleus. (C) 293T cells were transiently transfected with pcDNA-VP1 (lane 1) and pcDNA-3D (lane 2) plasmids, and the expression was confirmed by Western blot analysis.

Figure 2

Purification of recombinant VP1 and 3D proteins, and biological activity of B cell epitope peptide polyclonal Ab. (A) pET-VP1 (lane 1) and -3D (lane 2) expressions in bacterial system were confirmed by Coomassie staining of SDS-PAGE gel. (B) The purified VP1 (lane 1) and 3D (lane 2) proteins were confirmed by B cell epitope peptide -specific polyclonal Abs.

Figure 3

Humoral immune responses in DNA vaccine-immunized mice. (A) In vivo DNA immunization scheme (B) The antigen-specific serum IgG responses in Balb/c mice after co-immunization with pcDNA-VP1 and pcDNA-3D at two different time points (□ 2 wks p.i., ■ 5 wks p.i.). (C) IgG isotyping analysis with sera harvested from the group at 9 wks from the first immunization and tested for antibodies at 1:50 dilution. (□ IgG1, ■ IgG2a). The result was obtained from averages of groups against each antigen. The data represent average±S.D.

Figure 4

Humoral immune responses in protein vaccine-immunized mice. (A) In vivo protein immunization scheme (B) The antigen-specific serum IgG responses in Balb/c mice after immunization against VP1 or 3D protein at two different time points. (□ 2 wks p.i., ■ 5 wks p.i.) (C) IgG isotyping analysis with sera from the group sampled at 7 wks from the first immunization and tested for antibodies at 1:50 dilution (□ IgG1, ■ IgG2a). The result was obtained from averages of four mice in each group. The data represent average±S.D.

Figure 5

Cellular immune response in DNA vaccine-immunized mice. Numbers of IFN-γ secreting cell spots from a pool of 4 mice splenocytes (4×10⁵ cells/well) were determined by recall Ag VP1 or 3D protein stimulation. The splenocytes were pooled from 4 mice harvested 3 weeks after the last vaccination with VP1 and 3D DNA. The ELISpot assay was performed after stimulating the cells with 10 µg/ml each of the recombinant proteins for 48 hrs.

Figure 6

Cellular immune responses in proteins vaccine-immunized mice. Numbers of IFN-γ secreting cell spots from a pool of 4 mice splenocytes (4×10⁵ cells/well) were determined by recall Ag VP1 or 3D protein stimulation. The splenocytes were pooled from 4 mice harvested 3 weeks after the last vaccination with VP1 and 3D protein. The ELISpot assay was performed after stimulating the cells with 10 µg/ml each of the recombinant proteins for 48 hrs.