Immunogenicity and Immunoprotection of PCV2 Virus-like Particles Incorporating Dominant T and B Cell Antigenic Epitopes Paired with CD154 Molecules in Piglets and Mice

Figure 1. Construction of shuttle plasmids and prediction of antigenic epitope structures. (A) Schematic diagram of the shuttle plasmid construction. (B) Secondary structure prediction of dominant antigenic epitope peptides.

Figure 2. Identification of recombinant protein expression, optimization of conditions and semi-quantitative analysis. (A) Normal sf9 cells and sf9 cells showed CPE after transfection. (B) Observation of recombinant protein expression by IFA. (C) Identification of recombinant protein expression by Western blot. (D) Optimization of expression conditions for recombinant proteins. (E) Semi-quantitative analysis of recombinant proteins. Lanes 1–6 are, in order, 1000 µg/mL, 750 µg/mL, 500 µg/mL, 250 µg/mL, 100 µg/mL, and 50 µg/mL concentrations of standard protein; lanes 7 and 9 are cell lysates infected with Ac-Cap and Ac-TBCap, respectively; lanes 8 and 10 are cell culture supernatants infected with Ac-Cap and Ac-TBCap, respectively.

Figure 3. Structural modeling and assembly of PCV2 VLP. (A) Three-dimensional reconstruction of the cryo-EM structure of the VLP assembled with the PCV2 cap as the basic unit. Cryo-EM density maps of full-length PCV2 VLPs are shown, with PCV2 VLP density indicated in grey. (B) Three-dimensional reconstruction of the cryo-EM structure of the VLP assembled with the TBCap as the basic unit. Again, showing typical icosahedral particle folds, antigenic epitopes on the surface of the PCV2 capsid are shown in different colors and an enlarged region of this capsid is shown. (C) The recombinant Cap and TBCap virus-like particles were identified by transmission electron microscopy. VLPs with diameters of approximately 17 nm and 20 nm, respectively, could be observed, consistent with the theoretical size.

Figure 4. The OD450 of each group at different days post-immunization (dpi) by PCV2 ELISA. Different superscripts (a, b, c, d, e) indicate significant differences among groups (p < 0.05).

Figure 5. Research on immunogenicity and attack protection tests in piglets. (A) Piglet immunization strategy. (B) Changes in piglet body temperature after challenge. (C) ELISA for PCV2-specific antibodies in piglets after immunization (S/P). (D) Lymph node virus load in piglets at autopsy. Different superscripts (a, b, c, e) indicate statistically significant differences in lymph node viral load between the test groups (p < 0.05).

Figure 6. Histopathological testing of piglets after challenge. (A) HE staining of piglet lung and inguinal lymph node tissue. (B) IHC of inguinal lymph node tissue in piglets, black arrows indicate tan-colored cells in the inguinal lymph node tissue.

Figure 7. Immunization strategies and immunogenicity in mice. (A) Mice immunization strategy. (B) ELISA for PCV2-specific antibodies in mice after immunization. (C) ADWG of mice after challenge. Different superscripts (a, b, c) indicate statistically significant differences in lymph node viral load between the test groups (p < 0.05).

Figure 8. Cytokine secretion and viral load. (A) The stimulation of splenic lymphocyte samples collected at 42 dpi. (B) Quantitative analysis of interleukin 4 (IL-4) in the supernatant of PCV2-stimulated splenic lymphocytes in immunized mice at 42 dpi. (C) Quantification of interferon-gamma (IFN-γ) in the supernatant of PCV2-stimulated splenic lymphocytes in immunized mice at 42 dpi. (D) PCV2 DNA loads (copies/mL) in blood collected at 7, 14 and 28 DPC. (E) PCV2 DNA loads (copies/g) in tissues collected on the day of necropsy. Different superscripts (a, b, c, d, e) indicate statistically significant differences in lymph node viral load between the test groups (p < 0.05).