Results are

expressed as means ± standard deviation (SD) and were compared using an unpaired Student’s t test. To determine the effectiveness of the sublingual immunization, mice were immunized with 25k-hagA, 25k-hagA-MBP, or PBS. Sublingual immunization with 25k-hagA-MBP induced significant serum IgG and IgA 7 days after the final immunization (Fig. 1a). In contrast, 25k-hagA-immunized and nonimmunized mice induced low or no detectable titers, respectively, after sublingual immunization. In addition, the serum IgG and IgA Ab responses MAPK Inhibitor Library molecular weight induced by 25k-hagA-MBP persisted for almost 1 year (Fig. 1b). When the subclasses of antigen-specific IgG antibodies induced by sublingual 25k-hagA or 25k-hagA-MBP

Roxadustat challenge were determined, all IgG subclasses were significantly enhanced in 25k-hagA-MBP group. On the other hand, 25k-hagA-immunized group showed a low level of IgG1 (and sparse IgG2b) (Fig. 1c). Sublingual immunization of 25k-hagA-MBP induced high levels of 25k-hagA-MBP-specific IgA Ab responses in saliva (Fig. 2a). In contrast, essentially no IgA was detected in the saliva of mice sublingually treated with 25k-hagA or PBS. The most 25k-hagA-MBP-specific IgA AFCs were detected in the salivary glands suspensions (Fig. 2b). As sublingual immunization with 25k-hagA-MBP elicited 25k-hagA-MBP-specific Ab responses in both mucosal and systemic compartments, establishing the nature of the T cell help supporting the responses was important. When mononuclear cells from the SMLs of immunized mice were restimulated with 25k-hagA-MBP in vitro, significant levels of proliferative responses were induced (Fig. 3a). In contrast, no significant proliferation or cytokine production was observed in hagA-immunized mice (data not shown). Furthermore, mononuclear cells isolated from SMLs immunized with 25k-hagA-MBP showed higher production

of IL-4, IFN-γ, and TGF-β (Fig. 3b). These data Mirabegron indicate that sublingually immunized 25k-hagA-MBP-specific Th1-type and Th2-type responses are induced in SMLs. Given that sublingual immunization with 25k-hagA-MBP elicited long-term antigen-specific Ab responses in sera, we sought to determine whether these antibodies were capable of suppressing the alveolar bone absorption caused by P. gingivalis infection. Thus, mice given 25k-hagA, 25k-hagA-MBP, and PBS were infected orally with P. gingivalis 7 days after the last immunization. Mice immunized with 25k-hagA-MBP showed a significant protection and reduced bone loss caused by P. gingivalis infection (Fig. 4). In contrast, mice immunized with 25k-hagA alone did not show the reduced level of bone loss by P. gingivalis infection. These findings indicate that sublingual immunization with 25k-hagA-MBP is protective against oral infection by P. gingivalis.