Construction and immunogenicity evaluation of a bivalent nanoparticle based on mi3 displaying porcine circovirus type 2 and type 3 capsid proteins.

Porcine circovirus type 2 (PCV2) and type 3 (PCV3) pose severe threats to swine health worldwide. PCV2 field strains continuously mutate with the co-circulation of multiple genotypes, resulting in potential immune escape from existing vaccines. PCV3, an emerging pathogen, currently has no commercially available vaccines. Clinically, PCV2-PCV3 co-infection is prevalent, thus highlighting an urgent need for a bivalent vaccine. This study aimed to construct a broad-spectrum, high-efficiency bivalent nanoparticle antigen (PCV2-PCV3 Nano) based on mi3 nanocages and to evaluate its immunological efficacy. First, amino acid sequences of PCV2 and PCV3 capsid (Cap) proteins were optimized through analysis of currently prevalent strains. SpyCatcher (SC) or SpyTag (ST) domains were genetically fused to mi3, PCV2 Cap, or PCV3 Cap, respectively. The fusion sequences were codon-optimized and heterologously expressed in Escherichia coli. After purification, PCV2 Cap and PCV3 Cap were covalently anchored onto mi3 nanocages via SC-ST bioconjugation, successfully constructing PCV2-PCV3 Nano. Immunization of BALB/c mice demonstrated that PCV2-PCV3 Nano significantly induced higher antibody levels and conferred immune protection against PCV2b, PCV2d, and PCV3 infections compared with conventional antigens and commercial PCV2 vaccines. This study establishes a critical foundation for the industrial application of PCV2-PCV3 Nano, and it provides technical support for the development of mi3-based multivalent vaccines.