It has been shown previously that intranasal administration of c-di-GMP as an adjuvant for influenza vaccines can induce multifunctional influenza-specific

CD4+ Th1 cells in the spleen of immunized mice [8] and [9]. Furthermore, multifunctional Th1 cells have also been shown to be present in the blood of vaccinated human volunteers and in the non-inflamed normal 17-AAG manufacturer human lung tissue, as determined by their potential to produce IL-2, IFN-γ and/or TNF-α upon re-activation [31] and [32]. Consistent with the cytokine profile of influenza-specific multifunctional Th1 cells, our study showed increased IL-2 and IFN-γ levels in antigen re-stimulated PCLS of mice vaccinated with HAC1/c-di-GMP. The induction of Th1 cytokines in re-stimulated PCLS indicates that the antigen was recognized by HAC1-specific memory T-cells. These results are in line with the hypothesis by Jul-Larsen and colleagues Panobinostat who discussed that addition of an adjuvant improves the efficacy of HAC1 toward the induction of a robust T-cell response [32]. Additionally, our results aligned with previous studies on intranasally administered c-di-GMP showing an induction of a

Th1-biased cytokine profile in re-stimulated splenocytes against target antigen [8], [9] and [33]. Yet, our study also showed a mild induction of the Th2 cytokine IL-5 and the anti-inflammatory cytokine IL-10 in re-stimulated PCLS of intratracheally c-di-GMP-vaccinated mice. The fold induction of the Th1 cytokines for the double-adjuvanted vaccinated mice, however, far exceeded the level of Th2 cytokines that were induced (IFN-γ:IL-5, about 119-fold; IFN-γ:IL-10, about 39-fold). Nevertheless, the double-adjuvanted vaccine, as well as the c-di-GMP admixed antigen, induced IL-10 secretion in PCLS upon antigenic re-stimulation which exceeded the non-stimulated IL-10 baseline level. Among other cytokines, IL-10 can be released already by influenza-specific

CD4+ memory T-cells and has been described as having a putatively crucial role in regulating inflammation during acute influenza infection [34]. The fact that the double-adjuvanted vaccine induced IL-10-competent cells might also contribute to a reduced level of inflammation in the lungs with repeated exposure to the virus post vaccination. Overall, the data presented in the current study demonstrate that the double-adjuvanted HAC1 vaccine is immunogenic in the mouse model when administered intratracheally. Even though the protective efficacy of the double-adjuvanted HAC1 vaccine needs to be evaluated in a relevant animal model, the present study demonstrates that the double-adjuvanted HAC1 induces systemic functional antibody response as well as local humoral and cellular immune responses when administered via the respiratory tract, indicating potential for future needle-free vaccine applications. The authors would like to thank Olaf Macke, Sabine Schild, Sarah Dunker and Olga Danov for their technical assistance. The authors would like to thank Dr.