Development of a Ferritin-Based Nanoparticle Vaccine against Classical Swine Fever

Abstract

The occurrence of classical swine fever (CSF) poses a significant threat to the global swine industry. Developing an effective and safe vaccine is crucial for preventing and controlling CSF. Here, we constructed self-assembled ferritin nanoparticles fused with the classical swine fever virus (CSFV) E2 protein and a derived B cell epitope (Fe-E2B) using a baculovirus expression system (BVES), demonstrating enhanced immunogenicity. Furthermore, we provide a detailed evaluation of the immunological efficacy of the FeE2B in rabbits. The results showed that robust and sustained antibody responses were detected in rabbits immunized with the Fe-E2B nanoparticle vaccine, comparable to those elicited by commercially available vaccines. Additionally, we demonstrated that the vaccine effectively activated crucial immune factors IFN-γ and IL-4 in vivo, increasing their levels by 1.41-fold and 1.39-fold, respectively. Immunization with Fe-E2B enabled rabbits to avoid viremia and stereotypic fever after CSFV challenge. In conclusion, this study highlights the potential of ferritin nanoparticles as antigen-presenting carriers to induce robust immune responses, proposing a candidate vaccine strategy for the prevention and control of CSF.

Keywords: B-cell antigenic epitope; E2 protein; classical swine fever virus; ferritin nanoparticles; immune responses.

Figure 1

Construction of recombinant transfer plasmids and identification of recombinant proteins. (A) Construction of the recombinant transfer vector. In immunofluorescence assay and Western blotting experiments, the recombinant proteins were validated using anti-His antibodies (B) and anti-E2 antibodies (C).

Figure 2

Recombinant protein expression and purification. (A–C) The protein expression trends of recombinant proteins E2B, Fe-E2, and Fe-E2B were observed under various multiplicities of infection (MOI = 1, 5, 10) of recombinant baculovirus and at different protein harvest times (48 h, 72 h, 96 h, 120 h). (A) Recombinant protein E2B (48 kDa). (B) Recombinant protein Fe-E2 (64 kDa). (C) Recombinant protein Fe-E2B (67 kDa). (D) The distribution of samples in the centrifuge tubes before and after ultracentrifugation. (E–G) Results of SDS-PAGE identification of the three protein bands after ultracentrifugation of each sample. (E) E2B samples. (F) Fe-E2 samples. (G) Fe-E2B samples. M: Marker. 1: Samples after ultrasonic fragmentation; 2: upper protein band after ultracentrifugation; 3: middle protein band after ultracentrifugation; 4: lower protein band after ultracentrifugation. The bands within the red boxes are the areas for grayscale value analysis.

Figure 3

Observation of the morphology of different ferritin nanoparticles under TEM. (A) Morphology of proteins (E2B) not containing ferritin under TEM. (B) Morphology of ferritin nanoparticle alone. (C) Morphology of ferritin-fused E2 protein nanoparticle. (D) Morphology of ferritin fused with E2 protein and B-cell antigenic epitopes nanoparticle.

Figure 4

Detection of antibody titers in rabbits. (A) Detection of specific antibody titers in rabbit serum using ELISA. (B) Detection of neutralizing antibody titers in rabbit serum using the virus neutralization test on PK-15 cells. ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 5

Assessment of lymphocyte proliferation and cytokine levels. (A) Lymphocyte proliferation assays were conducted using PBMC isolated from rabbits at 35 and 49 dpi. Sera of the experimental rabbits were collected at 35 and 49 dpi, and ELISA measured the levels of IFN-γ (B) and IL-4 (C). ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 6

Rectal temperature change curves in rabbits challenged with the C-strain over 120 continuous hours. The body temperature changes of five rabbits in each experimental group were plotted over the 120 h. (A) PBS group (No. 1–5). (B) Fe group (No. 6–10). (C) E2 group (No. 11–15). (D) Fe-E2 group (No. 15–20). (E) E2B group (No. 20–25). (F) Fe-E2B group (No. 25–30). (G) C group (No. 31–35).