CRISPR/Cas9-mediated soc knockout enables T4 phage display of the PCV2d capsid protein and enhances the immunogenicity of chimeric T4 phage nanoparticles

Porcine circovirus type 2 (PCV2) is a major pathogen that seriously threatens the global swine industry. Currently available commercial vaccines provide limited cross-protection against circulating strains and induce insufficient cellular immune responses. In this study, we constructed PCV2d capsid protein-displaying T4 phage nanoparticles (PCV2-T4 NPs) and evaluated their immunogenicity. Specifically, CRISPR/Cas9 was used to knock out the soc gene in T4 phage, generating the Soc-deficient mutant T4ΔSoc. A Soc-Cap fusion gene was then constructed, expressed in E. coli , and purified by affinity chromatography. The purified fusion protein was assembled in vitro with T4ΔSoc to form PCV2-T4 NPs. BALB/c mice were immunized, and PCV2-specific antibody responses, cytokine secretion, and neutralizing titers were assessed. PCV2-T4 NPs induced high-titer PCV2-specific IgG antibodies reaching a titer of 1:12,800, and significantly increased IFN-γ and IL-4 secretion, indicating a balanced Th1/Th2 immune response. In the in vitro neutralization assay used in this study, sera from mice immunized with PCV2-T4 NPs exhibited significantly higher neutralizing antibody titres than those from the protein–adjuvant and commercial vaccine groups ( P < 0.01). These results suggest that PCV2-T4 NPs can induce both humoral and cellular immune responses in mice, supporting their further evaluation as a candidate PCV2 vaccine.