Enhanced Immunogenicity of Foot-and-Mouth Disease Virus-like Particles Using a Water-in-Oil-in-Water Adjuvant

Figures

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  Figure 1. Schematic diagram of the experimental procedure, including preparation, lymphocyte proliferation in vitro, and a series of immune responses in vivo.

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  Figure 2. Physicochemical properties of VLPs+WT. Appearance (A), viscosity (C), particle size and PDI (D) of VLPs+WT before and after centrifugation for 15 min at 3000 r/min. (B) The morphology of VLPs+WT under CLSM. Scale bar = 1 µm. ns = no significant differences.

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  Figure 3. The biosafety of VLPs+WT in vivo. H&E-stained tissues of the vital organs (heart, liver, spleen, lung, kidney, skin) in BALB/c mice from different groups were examined at 14 days after immunization. Scale bar = 100 μm. Data are presented as the mean ± SEM, n = 3.

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  Figure 4. Experimental design, detection of specific antibodies, antibody subtypes, and cytokines. (A) Schematic illustration of the experimental process. s.c.: subcutaneous. (B) Specific IgG levels in serum were assessed using the FMD O-type antibody liquid-phase blocking ELISA on days 14, 21, 28, 56, 70, and 84 after vaccination. Data shown are endpoint titers at a dilution of 1:64. The concentration of IgG1 (C), IgG2a (D), IL-4 (E), and IFN-γ (F) in serum on day 21 after vaccination. Data were represented as mean ± SEM, n = 5. (ns = no significant differences; *** p < 0.001; **** p < 0.0001).

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  Figure 5. The proliferation and differentiation of spleen T lymphocytes. (A) Lymphocyte proliferation under LPS and VLPs stimulation were measured by CCK-8 assay. (B) Flow cytometry analysis of CD3+CD4+ and CD3+CD8+ T cells in the splenocytes after being immunized with PBS, VLPs, VLPs+WT, and VLPs+201. Data are shown as mean ± SEM, n = 3. (** p < 0.01, *** p < 0.001, **** p < 0.0001).

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  Figure 6. Western blot, the number of DEGs, enrichment analysis, and RT-qPCR results. (A) MyD88, PI3K, AKT, p65, p-p65 and β-actin protein bands were observed in the VLP group and VLPs+WT group. Mice (n = 3/group) were subcutaneously immunized with VLPs or VLPs+WT. Splenocytes were isolated from the spleens harvested 2 weeks after immunization and used for transcriptome sequencing. (B) The volcano plot shows the numbers of up-regulated and down-regulated genes. (C) GO functional enrichment analysis. GO terms with fold change ≥ 2 and FDR < 0.01 were considered significantly enriched by DEGs. (D) Enrichment of KEGG pathways analysis. The x-axis means the value of the gene ratio and the y-axis means the enrichment term of the pathway. The size of the dot on behalf of the number of DEGs and the color represents q values. (E) Heatmap of some DEGs in spleen cells. The color key indicates reads per kilobase per million reads normalized log2 transformed counts. (F) Validation of DEGs with RT-qPCR. Data were normalized to the expression of GAPDH, n = 3.