Simvastatin Combined with CpG Enhances the Immunogenicity of the H9N2 Inactivated Vaccine

The H9N2 virus has severely harmed the livestock and bird farming industry. Currently, it is mainly prevented through vaccination immunization. However, conventional vaccines often fail to induce durable immune responses and long-lasting immunoprotection. In this research, we used Simvastatin (Sim) and CpG as adjuvants for the H9N2 inactivated vaccine to evaluate the vaccine's immunogenicity in chickens. We evaluate vaccine immunogenicity through antibody testing, T lymphocyte phenotyping, and RNA-sequencing analysis. The results indicated that the Sim + CpG/H9N2 formulation significantly enhanced specific IgY and hemagglutination inhibition (HI) antibody titers. It also increased the proportions of CD4⁺ T cells and CD8⁺ T cells, promoted immune organ development, and stimulated the formation of germinal centers. RNA-sequencing analysis revealed that Sim + CpG/H9N2 vaccination significantly upregulated immune-related genes, which were enriched in pathways associated with stress response activation, immune cell recruitment, and inflammatory signaling. Overall, these findings demonstrate that Sim + CpG/H9N2 markedly enhances the immunogenicity of the inactivated H9N2 vaccine and provides new insights into the application of vaccine adjuvants for improved immune protection.