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Effective mucosal adjuvants enhance the magnitude and quality of the vaccine response. Cyclic di-GMP (CDG) is a promising mucosal vaccine adjuvant. However, its in vivo mechanisms are unclear. Here, we showed, in mice, that CDG elicits stronger Ab and T H  responses than the mammalian 2′3′-cyclic GMP-AMP (cGAMP), and generated better protection against  Streptococcus pneumoniae  infection than 2′3′-cGAMP adjuvanted vaccine. We identified two in vivo mechanisms of CDG. First, intranasally administered CDG greatly enhances Ag uptake, including pinocytosis and receptor-mediated endocytosis in vivo. The enhancement depends on MPYS (STING, MITA) expression in CD11C +  cells. Second, we found that CDG selectively activated pinocytosis-efficient-DCs, leading to T H  polarizing cytokines IL-12p70, IFNγ, IL-5, IL-13, IL-23, and IL-6 production in vivo. Notably, CDG induces IFNλ, but not IFNβ, in vivo. Our study revealed previously unrecognized in vivo functions of MPYS and advanced our understanding of CDG as a mucosal vaccine adjuvant.

The mucosal adjuvant cyclic di-GMP enhances antigen uptake and selectively activates pinocytosis-efficient cells in vivo