Self-Assembling E2-Based Nanoparticles Improve Vaccine Thermostability and Protective Immunity against CSFV

Figures

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  Figure 1. Expression and self-assembly analysis of CSFV E2-mi3 NPs. Diagram of plasmid E2 (A), mi3 NPs (C), and E2-mi3 NPs (E). SDS-PAGE (left) and Western blot (right) analysis of the purified E2 protein (B), mi3 NPs protein (D), and E2-mi3 NPs protein (F). The size distribution of the mi3 NPs (G) and E2-mi3 NPs (H) was analyzed by DLS. TEM analysis of self-assembly capacity of mi3 NPs (I) and E2-mi3 NPs (J), scale bar = 200 nm.

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  Figure 2. Cartoon of the tertiary structure modeling of the CSFV E2 protein and E2-mi3 protein. (A) 3D model of the CSFV E2 protein. (B) 3D model of the CSFV E2-mi3 protein (mi3 skeleton protein tertiary structures are shown in blue). (C) The 3D model of the CSFV E2 protein and E2-mi3 protein were analyzed by Pymol.

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  Figure 3. Thermostability and long-term storage stability evaluation of the CSFV E2-mi3 NPs. (A) E2-mi3 NP protein remained soluble after high temperatures. (B) Size distribution of the E2-mi3 NP protein at different temperatures. (C) E2-mi3 NP integrity following long-term storage. ImageJ2x (1.4.3.67) software was used to count the number of E2-mi3 NPs. (D) Effective antigens analysis of the E2 protein and E2-mi3 NPs protein by Dot blotting.

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  Figure 4. Cellular uptake efficiency evaluation of CSFV E2-mi3 NPs. (A) The amount of E2 protein and E2-mi3 NP protein in RAW264.7 cells was measured by Western blot. (B) Gray value analysis of RAW264.7 cells. Note: * p < 0.05.

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  Figure 5. Specific antibody responses induced by E2-mi3 NPs in rabbits and pigs. Rabbit serum samples for the detection of CSFV-specific antibodies (A) through the CSFV antibody test kit and mi3 protein specific antibodies (C) with indirect-ELISA. Pig serum samples for the detection of CSFV-specific antibodies (B) via the CSFV antibody test kit and mi3 protein specific antibodies (D) through indirect-ELISA. Note: * p < 0.05.

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  Figure 6. Protective efficacy of E2-mi3 NPs in rabbits. Note: Rectal temperatures of the rabbits following CSFV C-strain challenge.

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  Figure 7. Detection of viral RNA in the blood samples from the immunized rabbits after being challenged with the C-strain by RT-qPCR (viral RNA values were obtained by Log10 processing). Note: Samples were considered negative when CSFV RNA loads/500 μL were less than 103.

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  Figure 8. Immunohistochemical analysis of viral replication in rabbits after challenge. (A) Representative immunohistochemical examination of immunized rabbits challenged with the CSFV C-strain (scale bar = 100 μm). (B) The proportions of positive cell area in these tissues (heart, liver, spleen, lung, and kidney). Note: * p < 0.05.

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  Figure 9. Humoral and cellular immune responses in pigs induced by E2-mi3 NPs. (A) Lymphocyte proliferation assay was performed to assess the cellular immune responses. (B) The percentages of CD4+ and CD8+ T-lymphocytes in peripheral blood lymphocytes were analyzed by flow cytometry. (C) ELISpot assay of IFN-γ secreted by porcine peripheral blood lymphocytes. (D) Cytokine levels (IFN-γ, IL-2, IL-4, and IL-12p70) of sera from pigs immunized were measured by ELISA. Note: * p < 0.05.