A novel CpG ODN compound adjuvant enhances immune response to spike subunit vaccines of porcine epidemic diarrhea virus

Figures

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  Figure 1. Stimulation of porcine peripheral blood mononuclear cells (PBMCs) proliferation by CpG oligodeoxynucleotides (ODNs). (A) PBMCs proliferation was stimulated with 5 μg/mL CpG ODNs. (B) PBMCs proliferation was stimulated with 10 μg/mL CpG ODNs. (C) Dose-dependent study of CpG5. The data was expressed as the means ± SEM (n = 5; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001).

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  Figure 2. CpG oligodeoxynucleotides (ODNs) stimulate the secretion of interferon-gamma (IFN-γ) from porcine peripheral blood mononuclear cells (PBMCs). (A) Stimulation with 5 μg/mL of CpG ODNs. (B) Stimulation with 10 μg/mL of CpG ODNs. The data was expressed as the means ± SEM (n = 5; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001). The symbols above the bar chart represent statistical significance compared to the GC group.

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  Figure 3. Preparation and characterization of porcine epidemic diarrhea virus (PEDV) antigens. (A) Schematics illustrating the structure of PEDV spike protein. (B) Analysis of PEDV spike protein by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blotting. (C) Transmission electron microscope image of PEDV spike protein.

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  Figure 4. Humoral immune response of porcine epidemic diarrhea virus (PEDV) subunit vaccines in BALB/c mice. (A) Schematic of the immunization procedure for BALB/c mice. (B) The endpoint titer of PEDV IgG was detected at each time point in BALB/c immunized mice. (C) The endpoint titer of PEDV IgG1 was detected at each time point in BALB/c immunized mice. (D) The endpoint titer of PEDV IgG2a was detected at each time point in BALB/c immunized mice. (E) Measurement of PEDV neutralizing antibody titer in immunized BALB/c mice. The data was expressed as the means ± SEM (n = 6; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

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  Figure 5. Evaluation of the cellular immune response of a porcine epidemic diarrhea virus (PEDV) subunit vaccine in mice. (A) Concentration of interleukin (IL)-2 was determined using enzyme-linked immunosorbent assay (ELISA). (B) Concentration of IL-4 was determined using ELISA. (C) Concentration of IL-10 was determined using ELISA. (D) Concentration of interferon-gamma (IFN-γ) was determined using ELISA. Splenic lymphocytes from immunized mice were isolated at day 21 post-immunization. The data was expressed as the means ± SEM (n = 3; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001).

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  Figure 6. Antigen-specific lymphocyte proliferation. Lymphocyte proliferation was stimulated by spike protein of porcine epidemic diarrhea virus. Splenic lymphocyte from immunized mice were isolated at day 21 post-immunization. The data was expressed as the means ± SEM (n = 3; ns, not significant, ***P < 0.001).

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  Figure 7. Humoral immune response of porcine epidemic diarrhea virus (PEDV) subunit vaccine in piglets. (A) Schematic of the piglet immunization procedure. (B) The endpoint titer of PEDV IgG was detected at each time point in immunized piglets. (C) Detection of PEDV-neutralizing antibody titers in immunized piglets. The data was expressed as the means ± SEM (n = 4; ns, not significant, *P < 0.05, ***P < 0.001).

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  Figure 8. T cell responses in immunized piglets. (A) Representative gating strategy for evaluating T cells. (B) Proportion of CD3+/CD8+ T cells in PBMCs of different groups. (C) Number of CD3+/CD8+ T cells in PBMCs of different groups. (D) Proportion of CD3+/CD4+ T cells in PBMCs of different groups. (E) Number of CD3+/CD4+ T cells in PBMCs of different groups. The data was expressed as the means ± SEM (n = 4; ns, not significant, *P < 0.05).