Fc-Mediated E2-Dimer Subunit Vaccines of Atypical Porcine Pestivirus Induce Efficient Humoral and Cellular Immune Responses in Piglets

Figures

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  Figure 1. Identification of the recombinant Drosophila cell lines. The recombinant plasmids pMT-Bip-aE2 (A), pMT-Bip-aE2Fc (B), and pMT-Bip-aE2ΔFc (C) were digested with EcoR I and Xho I and analyzed via 1% agarose gel electrophoresis. M, DL 5000 DNA Marker. (D) Western blotting analysis of APPV E2, E2Fc, and E2ΔFc proteins expressed in S2 cells. The pMT-Bip-V5-HisA vector was used as a negative control. The S2 cell lines, which stably express APPV E2, E2Fc, and E2ΔFc proteins, were induced by CuSO4 and cultured at 27 °C for 4 days. M, protein marker.

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  Figure 2. Purification of APPV E2, E2Fc, and E2ΔFc fusion proteins from S2 cells. (A) Western blotting analysis of APPV E2, E2Fc, and E2ΔFc proteins before purification. Elution peak curve of APPV E2 (B), E2Fc (C), and E2ΔFc (D) fusion proteins. The SDS-PAGE profiles were shown in the upper right corner of the elution curve. The S2 cell lines, which stably express APPV E2, E2Fc, and E2ΔFc proteins, were induced by CuSO4 and cultured at 27 °C for 4 days. M, protein marker.

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  Figure 3. Characterization of APPV E2, E2Fc, and E2ΔFc fusion proteins. (A) Schematic design of APPV E2, E2Fc, and E2ΔFc proteins. APPV E2, E2Fc, and E2ΔFc recombinant proteins were analyzed via SDS-PAGE (B) and western blotting with a monoclonal antibody against APPV E2 (C) in the presence (+) or absence (−) of the reducing agent β-mercaptoethanol. M, Marker; β-ME, β-mercaptoethanol. The black arrow indicates the bands of the Fc or ΔFc stabilized dimer. Structures of APPV E2Fc (D) and E2ΔFc (E) proteins. The models were built using the SWISS-MODEL server (https://swissmodel.expasy.org/interactive, accessed on 2 June 2021), with SMTL ID: 2yq2.1.A (aE2), 5w38.1.A (IgG3Fc), and 5hsf.1.A (IgG3ΔFc) used as the templates.

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  Figure 4. Functionality and antigenicity of APPV E2, E2Fc, or E2ΔFc fusion proteins. (A) Co-localization of FcγRI on macrophages and APPV E2Fc or E2ΔFc fusion protein. Immunofluorescent microscopic photomicrographs displayed localization of FcγRI on macrophages and the pAb FcγRI (CD64)-immunoreactive green fluorescence was detected by Alexa Fluor 488 goat anti-rabbit antibody. While the localization of E2 in FcγRI was analyzed through the mAb E2-immunoreactive red fluorescence using Alexa Fluor 555 goat anti-mouse antibody. The nuclei (blue) were labeled with DAPI. Binding affinity of APPV E2, E2Fc, and E2ΔFc fusion proteins to APPV-specific (aE2) mAb (B) and FcγRI (C). ELISA was performed to detect the binding affinity of APPV E2, E2Fc, and E2ΔFc fusion proteins to APPV-specific (aE2) mAb and FcγRI. Binding affinity was characterized as OD450 value. CSFV-specific (cE2) mAb was assessed to investigate the cross-reactivity. PBS was used as a negative control. Different letters (a, b) indicate a statistically significant difference between different experimental groups (p < 0.05). Experiments were conducted independently in triplicates. Data are represented as the mean ± SD (n = 3).

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  Figure 5. Detection of APPV E2-specific antibodies. (A) Schematic outline of the experimental design. (B) APPV E2-specific IgG detected via indirect ELISA at indicated dpi. Different letters (a, b, c) indicate a statistically significant difference between different experimental groups (p < 0.05). Experiments were conducted independently in triplicates. Data are represented as the mean ± SD (n = 5).

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  Figure 6. Lymphocyte proliferation assay and detection of cytokines. (A) Detection of lymphocyte proliferative responses. (B–E) Detection of the levels of cytokines in the supernatants of stimulated lymphocytes from piglets immunized with the APPV E2 subunit vaccines. Different letters (a, b, c) indicate a statistically significant difference between different experimental groups (p < 0.05). Experiments were conducted independently in triplicates. Data are represented as the mean ± SD (n = 3).