During the first two days after challenge little effect of the virus infection was seen. By day three animals started to loose

weight. This weight loss was higher in the mice which had been immunized with adjuvanted vaccines than in non-immunized mice and BLU9931 mw in mice which received unadjuvanted vaccines. Weight loss correlated with the strength of the induced immune responses but not with GPI-0100 dose. Three days after virus challenge, the animals were sacrificed and virus titers were determined in lung homogenates to evaluate protection elicited by the vaccines. The HNE buffer group showed an average lung virus titer of 6.45 10log (Fig. 4). The average titer in lungs of mice receiving a low dose of unadjuvated HA (0.04 and 0.2 μg) was not statistically different from that of the buffer group. Only mice receiving 1 μg unadjuvanted HA showed a statistically significant reduction in lung virus titers (p < 0.05). Immunization with GPI-0100-adjuvanted vaccine resulted in significantly decreased lung virus titer at all tested antigen doses (p values between buffer and the adjuvanted vaccines were ≤0.01 for all antigen doses tested, p values between unadjuvanted and adjuvanted vaccines were ≤0.05 or ≤0.01, at HA doses of 1 μg or 0.04 and 0.2 μg, SNS 032 respectively). The result shows that GPI-0100 improves vaccine-elicited protection against influenza virus infection even at an extremely low antigen dose of 0.04 μg HA. GPI-0100 is a stable semi-synthetic

saponin derivative, which has been demonstrated to stimulate both the humoral and the cellular arm of the immune system [10], [11], [12], [17] and [22]. In the present study we evaluated the immunogenicity and protective efficacy of GPI-0100-adjuvanted A/PR8 influenza subunit vaccine in mice. The results show that GPI-0100 boosts influenza-specific antibody

responses of the IgG1 and especially Phosphatidylinositol diacylglycerol-lyase the IgG2a subtype in a dose-dependent manner. There was also a trend towards higher numbers of influenza-specific cytokine-producing T cells in mice immunized with GPI-0100 adjuvanted vaccine though differences were not significant for all antigen doses studied. Furthermore, GPI-0100-enhanced immune responses provided better protection against influenza virus infection as demonstrated by reduced lung virus titers after challenge. Remarkably, an adjuvanted 0.04 μg HA dose presented a better formulation than an unadjuvanted 1 μg HA dose for all immune parameters studied. In line with earlier studies using OVA, HagB antigen of P. gingivalis and gD antigen of HSV-1, here we confirm that GPI-0100 boosts antigen-specific antibody responses with a Th1 IgG isotype profile in a dose-dependent manner [11], [12], [14] and [16]. High levels of antigen-specific IgG2a titers were induced in addition to IgG1 titers, resulting in a more balanced Th1/Th2 antibody response. In addition, we observed that GPI-0100 stimulates antigen-specific IFN-γ responses, which has also been reported previously in OVA studies [11].