To examine the effects of MPs in vivo, we assessed the levels of IgG1 and IgG2a PTd-specific antibody levels in sera at 14 and 28 days post immunization. At day 14, it was observed that animals immunized with Quadracel®, SOL and AQ formulations showed significantly higher amounts of IgG1 antibodies than the negative control ( Fig. 3A). At day 28, the IgG1 levels were similar in groups of mice immunized with Quadracel® SOL and AQ formulations ( Fig. 3A). The levels of IgG1 in the MP group were 10 times lower than other groups. At day 28, IgG2a levels
were significantly higher in SOL group than all the other groups, and were GSK2118436 supplier about 10 times less in both AQ and MP groups. Expectedly, Quadracel® induced only weak IgG2a responses ( Fig. 3B). In contrast, IgG2a responses were almost non-detectable in the Quadracel group, and about 100–1000 fold higher in mice immunized with microparticle-based or soluble adjuvant formulations. The ratio
of IgG2a and IgG1 was 1.58 in mice immunized with MP and was lower than 1 in mice immunized with Quadracel®, AQ and SOL formulations ( Fig. 3C). Interestingly the ratio of IgG2a and IgG1 titers was more than 1000-fold lower in mice immunized with Quadracel® indicating a Th2 skew in this group. To confirm if the antigen-specific antibody response was consistent with a cell-mediated response, the splenocytes of BI 6727 ic50 challenged mice were stimulated with PTd to assay the number of cells secreting IFN-γ and IL-4 by ELISPOT assay. The absolute number of IFN-γ spots in why the SOL
and MP formulations were significantly higher as compared to Quadracel® and AQ formulations (Fig. 4A). In contrast, the absolute number of IL-4 spots were higher in the Quadracel® group indicating that the presence of CpG and/or IDR in AQ, SOL, MP group shifted the immune response towards a Th1-type which was more clear when the ratio of IFN-γ and IL-4 spots were examined (Fig. 4C). While the ratio of 0.48 for Quadracel® reflected a predominantly Th2 response, the ratio was 0.8 and 1.0 for AQ and SOL groups, demonstrating that the presence of CpG ODN-IDR adjuvant complexes in the formulations induced more of a Th1 response. Importantly, the MP group had a ratio of 1.78, indicating a strong Th1 shift. We also looked at Th17 responses as it has been documented that IL-17 mediates the clearance of pathogens from airway epithelium  and . The number of IL-17 spots detected was statistically significantly higher in the MP group (Fig. 4D). Ultimately, whether an immune response is through induction of antibodies or cytokines, the best indicator for vaccine efficacy is its ability to clear the infectious agent, in this case B. pertussis. This was tested in an intranasal challenge with B. pertussis. Mice immunized with the microparticle-based adjuvant formulation displayed about 100 fold lower bacterial burden at day 7 post infections. Similar to mice immunized with Quadracel ( Fig. 5).