Development of a nano-emulsion and evaluation of its intradermal adjuvant function of Swine Influenza H3N2 Vaccine

BACKGROUND: Intradermal (ID) immunization is a potent vaccination strategy, but its potential is limited by the lack of adjuvants that can effectively enhance immune responses while minimizing local skin reactogenicity. OBJECTIVES: To develop and evaluate a novel composite nano-emulsion adjuvant, designated VP, for enhancing the efficacy of intradermal vaccination against swine influenza virus (SIV). METHODS: A novel adjuvant VP, formulated with squalene, sea buckthorn oil, and the TLR3 agonist Poly(I: C), was designed. Its physicochemical properties (particle size, zeta potential, viscosity) were characterized. The immunogenicity and protective efficacy of the VP-adjuvanted inactivated H3N2 vaccine were systematically evaluated in mice, comparing intradermal (ID) and intramuscular (IM) routes. Assessments included humoral immunity (serum and bronchoalveolar lavage fluid (BALF) IgG/HAI titers, duration), cellular immunity (splenic T cell subsets, cytokine profiles), and protection against homologous viral challenge (viral load, lung pathology, weight loss). Local skin reactogenicity and inflammatory responses were also investigated. RESULTS: The VP adjuvant exhibited a potent immune-stimulatory effect, comparable to commercial adjuvants. The VP/H3N2 formulation formed a stable nano-emulsion with an average particle size of ~ 137 nm, a near-neutral surface charge, and low viscosity (~ 1.3cP), facilitating antigen transport to skin-draining lymph nodes. Immunization induced a robust, Th1-biased immune response characterized by high IgG2a/IgG1 ratios and elevated IFN-γ secretion. Notably, the VP-adjuvanted vaccine elicited the highest proportions of IFN-γ⁺ CD4⁺, IFN-γ⁺ CD8⁺and memory-type CD44⁺ CD4⁺ T cells among all experimental groups. ID administration was superior to IM, yielding significantly higher and more durable serum/BALF antibody titers and longer-lasting immunity. VP-adjuvanted vaccination provided protection against challenge, with minimal lung pathology and markedly reduced viral loads. Crucially, ID immunization with VP elicited only a moderate, transient local inflammatory response with rapid absorption and no significant adverse skin reactions. Mechanistic studies revealed that VP functioned by upregulating key chemokines (e.g., CX3CL1, CCL19) at the injection site, significantly enhancing antigen uptake by dendritic cells in vitro, and promoting targeted antigen delivery to draining lymph nodes in vivo. CONCLUSIONS: This study successfully developed a novel composite nano-emulsion adjuvant, VP, which demonstrated high efficacy in intradermal SIV vaccination by potently enhancing both humoral and cellular immunity, providing durable protection, and maintaining an acceptable safety profile. The adjuvant mechanism involves enhanced local immune activation, promotion of Dendritic cells (DCs) phagocytosis, and improved lymphoid trafficking. These findings provide a theoretical and practical foundation for developing next-generation influenza vaccine adjuvants.