Subunit vaccine of PCV3 capsid protein produced by sf9 cells with double knockout of Caspase-1 and Dronc induces strong immune response in mice.

Porcine circovirus type 3 (PCV3) associated with multisystemic clinicopathological diseases in swine herds has caused economic losses and there is no available commercial vaccine. Production of PCV3 capsid protein (Cap) by Spodoptera frugiperda 9 (sf9) cells using baculovirus expression vector system (BEVS) is a valid strategy to develop vaccines. Here, we report that subunit vaccine of PCV3 produced by sf9 cells with double knockout of Caspase-1 and Dronc genes induces strong immune response in mice. Three kinds of knockout sf9 cells aimed at Caspase-1 gene, Dronc gene and both genes were successfully generated by clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) system, and sequence analysis confirmed this. The anti-apoptosis ability of three kinds of knockout sf9 cells was assessed, and double knockout sf9 cells are the best. The expression of PCV3 Cap was enhanced in double knockout sf9 cells compared to wild type sf9 cells, and subunit vaccines were produced by PCV3 Cap expressed from double knockout sf9 cells and wild type cells, respectively. Results of immunological experiment in mice showed subunit vaccine of PCV3 Cap from double knockout sf9 cells induces higher level of serum antibody, stimulates lymphocyte proliferation and enhances expression of IL-2, IFN-γ, IL-4 and IL-10 compared to wild type cells. These results present knockout sf9 cells to enhance the expression of protein in BEVS, and provide a technical platform for vaccine development of PCV3.