Production and immunogenicity of a deoxyribonucleic acid Alphavirus vaccine expressing classical swine fever virus E2-Erns protein and porcine Circovirus Cap-Rep protein

Figures

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  FIGURE 1. Diagram representation of the recombinant plasmid pSCA1-E2-Erns-Cap-Rep. CMV is the eukaryotic promoter. Stop codons are the termination signal. SV40 poly (A) is the strong transcription termination signal, which could normally terminate transcription and further enhance its stability. The ampicillin resistance is for selection and maintenance in Escherichia coli. NsP1–4 are nsP1, nsP2, nsP3, and nsP4, which were four non-structural proteins and play important role in RNA synthesis. CMV, cytomegalovirus; Amp, ampicillin; Ori, origin site; nsP1–4, non-structural proteins 1–4; SV40 poly (A), Simian virus 40 polyadenylic acid A.

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  FIGURE 2. mRNA expression levels of E2, Erns, Cap, and Rep in vitro after the transfection of pSCA1-E2-Erns-Cap-Rep. (A–D) The mRNA level of E2, Erns, Cap, and Rep was normalized based on β-actin expression in PK15 cells, respectively. (E–H) The mRNA level of E2, Erns, Cap, and Rep was normalized based on β-actin expression in 3D4/21 macrophage cells, respectively. Three independent qPCR experiments always obtained consistent conclusion, and one of the results was shown. * denotes the significant difference between different groups (*P < 0.05, **P < 0.01, and ***P < 0.001).

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  FIGURE 3. Evaluation of pSCA1-E2-Erns-Cap-Rep plasmid vaccine immunogenicity. (A,B) CSFV and PCV2 antibody assay of pSCA1-E2-Erns-Cap-Rep plasmid in serum of mice among 50, 100, and 200 μg groups, respectively, were determined by ELISA assay after immunization at different times. (C,D) The production of CSFV and PCV2 antibodies by the pSCA1-E2-Erns-Cap-Rep plasmid vaccine after immunization was compared with different doses. * denotes the significant difference between the pSCA1-E2-Erns-Cap-Rep plasmid vaccine group (50, 100, and 200 μg groups) and PBS group (*P < 0.05, **P < 0.01, and ***P < 0.001). All data are represented as mean ± SD (n = 4).

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  FIGURE 4. Adjuvants enhanced the CSFV and PCV2 antibody levels after pSCA1-E2-Erns-Cap-Rep plasmid vaccine immunization in vivo. (A–C) Compared with the 50, 100, and 200 μg pSCA1-E2-Erns-Cap-Rep plasmid vaccine group, the 50, 100, and 200 μg + adjuvant groups, respectively, stimulated the increase of CSFV antibody levels in mice; (D–F) Compared with the 50, 100, and 200 μg pSCA1-E2-Erns-Cap-Rep plasmid vaccine group, the 50, 100, and 200 μg + adjuvant groups, respectively, stimulated the increase of PCV2 antibody levels in mice. *, **, and *** denote the significant difference between different groups (*P < 0.05, **P < 0.01, and ***P < 0.001). All data are represented as mean ± SD (n = 4).

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  FIGURE 5. Determination of the neutralized CSFV effect of the pSCA1-E2-Erns-Cap-Rep plasmid vaccine. Representative immunofluorescence images of PK15 cells infected the remaining CSFV neutralized by neutralizing antibody stained with nucleus (DAPI) and CSFV (green).

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  FIGURE 6. Viral neutralizing responses determination post-vaccination. (A) Neutralization assay using indirect immunofluorescence for CSFV. (B) Neutralization assay using ELISA for PCV2. Significance is presented as *p < 0.05, **p < 0.01, and ***p < 0.001. Error bars represent SD.

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  FIGURE 7. The expression level of IFN-γ and TNF-α after the pSCA1-E2-Erns-Cap-Rep plasmid vaccine immunization in spleens. (A) The IFN-γ was consistently increased with the increasing dose of plasmid vaccine with adjuvant and no adjuvant groups. (B) The TNF-α was consistently increased with the increasing dose of plasmid vaccine with adjuvant and no adjuvant groups. Three independent qPCR experiments always obtained consistent conclusions and one of the results was shown. * denotes the significant difference between different groups (**P < 0.01 and ***P < 0.001).