Immunostimulatory effects of multiple short-hairpin RNAs enhance foot-and-mouth disease vaccine-induced humoral immunity.

Small interfering RNAs are typically used to block gene expression via the sequence-specific degradation of mRNA. We previously developed a recombinant adenovirus expressing three short hairpin RNAs, targeting foot-and-mouth disease virus (FMDV) driven by the U6 promoter (Ad-3siRNA). These exhibited rapid antiviral effects against FMDV. Herein, we investigated the immunostimulatory effects of Ad-3siRNA in combination with an FMD inactivated vaccine. Ad-3siRNA induced various cytokines, including type I interferon, and upregulated interferon-stimulated genes in swine cells. Using the combination of Ad-3siRNA and a foot-and-mouth disease (FMD) inactivated vaccine, we observed enhanced protection efficacy until 1 week post-vaccination and enhanced neutralizing antibody levels from 1 week post-vaccination in mice. Furthermore, humoral immunity was significantly enhanced in pigs injected with a vaccine and a combination of Ad-3siRNA for 7 weeks post-vaccination. Ad-3siRNA induced various cytokines, such as IFN-α, IFN-γ, IL-12, IL-6, IL-15 and IL-18, in mice. In conclusion, we demonstrated that Ad-3siRNA, a novel immunostimulatory RNA delivered by a human adenoviral vector, could enhance protection and humoral immunity in combination with an FMD-inactivated vaccine. Exploring the applications of Ad-3siRNAs against other viral diseases and further detailed mechanistic studies are warranted.