Abstract
Intranasal administration of vaccine adjuvants directly deliver therapeutic agents to the lungs to induce potent lung mucosal immune responses. Cyclic di-GMP (CDG) is a promising mucosal vaccine adjuvant candidate capable of inducing protective immunity. This protocol describes an in vivo approach to induce and detect mucosal (lung) and systemic (blood and spleen) vaccine adjuvant responses of CDG. This protocol also includes the methods to detect both humoral and cellular immune responses of CDG adjuvant. Last, this protocol can be used to study other cyclic dinucleotides as mucosal vaccine adjuvants.
Keywords: Cyclic dinucleotides, Mucosal vaccine adjuvant, Intranasal immunization, Humoral and cellular vaccine responses, Ex vivo recall assay
Background
Cyclic diguanylate monophosphate (CDG) is part of a new class of vaccine adjuvants called cyclic dinucleotides currently being used in clinical trials for the treatment of cancer and infectious diseases. In vitro and in vivo administration of CDG can induce strong immune responses by engaging the STING pathway to induce production of type I interferon and TNFα (McWhirter et al., 2009; Blaauboer et al., 2014). As a potential mucosal vaccine adjuvant, CDG is well known to induce protective immunity against bacterial pathogens by producing strong antibody and Th1/Th2/Th17 responses in the lung (Ebensen et al., 2007; Yan et al., 2009; Madhun et al., 2011). Mechanistically, this mucosal adjuvant acts through the lung resident dendritic cells (Blaauboer et al., 2014; Mansouri et al., 2019). To date, the cellular and molecular mechanisms of CDG have been mainly characterized by In vitro experiments with murine and human cultured cells. In vitro studies often use transfection reagents to administer CDG into cells. As CDG has two phosphate groups preventing it from directly passing through the cell membrane, the method of transfection may be artificial. Interestingly, CDG was found to be taken up by cells via pinocytosis in vivo (Blaauboer et al., 2015). This protocol describes a more physiological relevant approach to studying the adjuvant effect of CDG in vivo through intranasal immunization.
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
This protocol was adapted from previously published work (Blaauboer et al., 2014 and 2015; Mansouri et al., 2019). The work was supported by R01AI110606 and R21AI125999 to LJ.
Competing interests
The authors declare no conflict of interests.
Ethics
Mice were housed and bred in the Animal Research Facility at the University of Florida. All experiments with mice were performed by the regulations and approval of the Institutional Animal Care and Use Committee from the University of Florida (Protocol number 201609362, 05/09/2016~05/08/2019).
References
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