Nano-adjuvant based on lipo-imiquimod self-assembly for enhanced foot-and-mouth disease virus vaccine immune responses via intradermal immunization

Figures

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  Scheme 1. The Schematic representation of (a) the synthesis of IMQP and (b) proposed in vivo immune response through TLR7/MyD88-dependent NF-кB and MAPK signaling pathways.

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  Fig. 1. The characterization of IMQP. The ζ potential (a) and DLS (b) of IMQP, IMQP@FMDV and IMQP@OVA LNPs. TEM images of (c) IMQP NPs and (d) IMQP@OVA LNPs. (e) The adsorption rate of IMQP load OVA by BCA kit. (f) SDS-PAGE of the supernatant of OVA. All data were expressed as means ± SD, n = 3.

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  Fig. 2. Biosatety of IMQP. (a) The CCK-8 result with RAW 264.7 cells. (b) The hemolysis test by 1 % RBCs of SPF chicken. Inset: the optical photos of co-incubation of RBCs with IMQP (c) The H&E staining of IMQP with different concentrations (200 μg/mL, 1000 μg/mL). Scale bar: 20 μm. And (d) the complete Blood Count. The following parameters were measured: white blood cells, red blood cells, lymphocytes, neutrophils, monocytes, eosinophils and basophils. All blood analysis data fell well within the normal ranges. Results were expressed as means ± SD, n = 3.

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  Fig. 3. APCs activation in skin tissue from 1 to 7 days after immunization in mice. (a) H&E staining of the skin at injection site in 1, 3, 7 dpi. Scale Bar: 50 μm. (b) The activation percentage of DCs, LCs and GC-B cells after 1 dpi by FACS. (c) IHC staining of skin after 7 dpi. Scale Bar: 20 μm. Results were expressed as means ± SD, n = 3, (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ns = no significant differences between groups).

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  Fig. 4. Expression of specific antibodies and cytokines. The mice were immunized by intradermal on day 1 and strengthened-immune on day 14. The FMDV-specific antibody level of IgG (a–c), IgG1 (d), IgG2a (f), the ratio of IgG1/IgG2a, and cytokines (g–m) in serum were detected on day 14 or 28 after the first immunization. Results were expressed as means ± SD, n ≥ 3, (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ns = no significant differences between groups).

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  Fig. 5. IMQP promotes T cells activation in DLNs and spleen. (a–c) Proportion of differentiation into CD4+ and CD8+ from CD3+ T cells activation in DLNs, and (d) The ratio of CD4+/CD8+ T cells activation. (e) Proliferative responses of splenocytes responding to LPS and FMDV ex vivo. (f) The percentage of activated CD3+ T cells, and (g, h) proportion of differentiation into CD4+ and CD8+ from CD3+ T cells activation in splenocytes, and (I) The ratio of CD4+/CD8+ T cells activation. Results were presented as mean ± SD, n = 3, (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ns = no significant differences between groups).

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  Fig. 6. Effects of IMQP nano-adjuvant on BMDCs uptake and activation. (a) The CLSM images of IMQP@OVA LNPs to cellular uptake by BMDCs within 0.5–1.5 h. (b) Mean red fluorescence intensity corresponding to control, OVA-Rh B and IMQP@OVA-Rh B in different time. (c) The colocalization correlation image of the intensities of OVA-Rh B and Lyso-Tracker Green and (e–g) Expression of CD80+, CD86+ and MHC-II+ on BMDCs in 7 dpi by flow cytometry plots. Scale Bar: 5 μm. Results were presented as mean ± SD, n = 3, (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ns = no significant differences between groups).

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  Fig. 7. RNA-seq analysis of BMDCs treated with IMQP for 4 h. (a) Venn diagram of the differentially expressed genes in Mock and IMQP groups. (b) Volcano plot of fold changes of the genes identified at q < 0.05. (c) GO enrichment analysis showing the main 20 immune-related pathways in which the differentially expressed genes of IMQP versus Mock were enriched. (d) Heat map of immune-related gene expression in IMQP treated BMDCs. (e) Categorization of the differentially expressed genes of immune in IMQP treated BMDCs, as observed by KEGG enrichment analysis. (f) Enriched chord diagram of the differentially expressed genes based on KEGG enrichment analysis.