4C). However, the c-di-GMP-adjuvanted HAC1 antigen induced cells to secret slightly elevated levels of IL-5 upon HAC1 re-stimulation

(2.2 ± 0.1 and 2.4 ± 0.1 for single- and double-adjuvanted, respectively) compared to non-stimulated PCLS. The release of the anti-inflammatory cytokine IL-10 was at baseline levels in PCLS from the non-adjuvanted and positive control groups (fold induction ≤ 2; Fig. 4D) as well as HAC1/SiO2 immunized mice. In contrast, IL-10 levels were enhanced in PCLS samples from HAC1/c-di-GMP as well as HAC1/SiO2/c-di-GMP vaccinated mice, when re-stimulated with HAC1 (12 ± 4 and 7 ± 2, respectively). The present study evaluated the systemic and local immunogenicity

of a double-adjuvanted CDK inhibitor influenza vaccine (HAC1/SiO2/c-di-GMP) delivered via the respiratory tract. The vaccine is intended Compound Library to be used as an inhalable needle-free vaccine targeting the upper and lower respiratory tract. However, for the work described here, we administered the vaccine intratracheally as a practical alternative to evaluate effects of the vaccine in the deeper lung before conducting an inhalation study prior to the challenge experiments. Minne and colleagues described the impact of vaccine delivery site on the immune responses and concluded that targeting the lower lungs for an inhaled influenza vaccination can induce systemic and local immune responses most efficiently [23]. Recent results with the NP-admixed antigen in a human lung Org 27569 tissue model showed that HAC1/SiO2 was able to re-activate formerly primed T-cells [12]. Even though HAC1/SiO2 had a re-activating potential in human PCLS, vaccination of mice intratracheally

was barely able to induce seroprotection (HAI titer >1:40). Moreover, it did not induce any local immune response, such as antigen-specific Ig secretion or T-cell induction upon re-stimulation, when administered at a lower antigen dose (5 μg HAC1). However, addition of the mucosal adjuvant c-di-GMP to HAC1/SiO2 induced HAI and IgG antibodies and T-cells that are considered potential markers for systemic and local protective immune responses against influenza infection. Importantly, no adverse side effects or clinical signs of decreased well-being of the study animals were observed after intratracheal administration of the double-adjuvanted vaccine. These increased antigen-specific immune responses demonstrated the synergistic effect of the combination of nontoxic concentrations of SiO2 and c-di-GMP and were in line with the work of Svindland et al. [9]. Although mucosal IgG and IgA were induced by the single-adjuvanted vaccine HAC1/c-di-GMP, a higher antigen dose was required.