Combined monophosphoryl lipid A and alpha-Galactosyl ceramide adjuvants enhance the systemic immune response to intranasal inactivated influenza vaccination in a murine homologous challenge model

Inactivated influenza vaccines (IIVs) often fail to provide robust mucosal protection, necessitating the development of potent adjuvants for intranasal delivery. This study evaluated a novel adjuvant combination of Monophosphoryl lipid A (MPL), a TLR4 agonist, and alpha-Galactosyl Ceramide (α-GalCer), an NKT cell ligand, to enhance the efficacy of an intranasal inactivated A/PR8 (iPR8) influenza vaccine. In vitro results demonstrated that MPL+ α-GalCer increased the frequencies of CD4⁺ CD25⁺ , CD8⁺ CD25⁺ and CD4⁺ CD69⁺ T cells relative to both the vehicle control and either single adjuvant in splenocyte cultures, whereas in purified dendritic cell and macrophage cultures MPL+ α-GalCer was no more effective than MPL alone. Splenocyte supernatants showed higher elevated TNF- α, IL-2, IFN-γ, IL-4, and IL-13. This pre-activation drove robust allogeneic T-cell proliferation in a mixed lymphocyte reaction, indicating enhanced allostimulatory capacity rather than influenza-specific priming. In vivo , a single intranasal dose of iPR8+ MPL+ α-GalCer elicited high systemic IgG titers and was effective at inducing Th1-associated IgG2a alongside IgG1. Following an LD50-range PR8 challenge, the combined-adjuvant group showed 100% survival, which was not statistically significant and lower lung viral titers at 7 days post-infection. This group exhibited elevated post-challenge lung-associated antigen-specific IgA and a reduced viral burden, which was accompanied by lower inflammatory cell infiltration and cytokine levels. The protection of combination group was accompanied by increased frequencies of ex vivo antigen-responsive CD4 + and CD8 + T cells recovered post-challenge. Our findings suggest that the MPL+ α-GalCer combination enhances innate and adaptive immune responses, providing an effective adjuvant platform for homologous mucosal protection in a murine model.