Mechanisms of STING-Mediated Mucosal Vaccine Adjuvant Activity of Cyclic di-GMP

描述（由申请人提供）：本提案的目的是揭示STING（干扰素基因刺激因子）介导环二gmp （CDG）粘膜疫苗佐剂活性的体内机制。粘膜免疫最能有效地诱导粘膜保护性免疫反应。然而，目前批准的大多数人类疫苗都是全身注射，通常不能引起有效的粘膜免疫。粘膜减毒活疫苗存在安全性和可接受性问题，而经粘膜途径接种的纯化抗原通常免疫原性较差。CDG具有很强的粘膜免疫原性，因此被认为是一种很有前途的粘膜疫苗佐剂。CDG发挥其粘膜佐剂作用的机制尚不清楚，这阻碍了CDG作为有效粘膜佐剂的进一步发展。STING，也被称为MPYS/MITA，是通过细胞质感知DNA产生I型IFN （IFN-I）所必需的。最近，我们发现STING通过CDG介导IFN-I的产生。随后的结构研究发现，STING的细胞质尾部与CDG结合。有人提出，STING是CDG的直接传感器，导致IFN-I的产生。然而，STING在cdg诱导的疫苗佐剂活性中的体内作用尚不清楚。在本研究中，我们发现STING-/-小鼠在经鼻免疫抗原和CDG后不能产生抗原特异性抗体反应。此外，CDG免疫的STING-/-小鼠中不产生Th1/Th2/Th17细胞因子、促炎细胞因子和IFN-I。令人惊讶的是，我们发现IFN-I信号并不是CDG粘膜佐剂活性所必需的，因为IFNAR1-/-小鼠与野生型小鼠具有相同的抗原特异性抗体反应。这与sting介导的DNA疫苗佐剂活性不同，后者需要IFN-I信号传导。我们提出了两个特定的目标来解决sting介导的CDG粘膜佐剂活性的体内细胞和体外分子机制。目的1：树突状细胞（DC）在体内表达STING对CDG的粘膜疫苗佐剂活性至关重要。我们将使用我们生成的条件STING-/-和条件STING-/ wt小鼠在体内确定STING介导CDG佐剂活性的细胞机制；目的2：确定CDG介导sting介导的DC激活的IFN-I刺激独立机制。我们发现CDG促进sting依赖而IFN-I信号独立的DC成熟。我们将使用生化和分子生物学方法来鉴定介导这种IFN-I刺激无关功能的STING的近端信号转导器。我们将利用KO小鼠进一步验证其在CDG体内佐剂活性中的重要性。目前，还没有一种含有粘膜佐剂的疫苗制剂被批准用于人类。我们的研究所产生的知识可以帮助推进CDG作为人类使用的有效粘膜疫苗佐剂的发展。

DESCRIPTION (provided by applicant): The objective of this proposal is to uncover the in vivo mechanisms by which STING (Stimulator of interferon genes) mediates the mucosal vaccine adjuvant activity of cyclic di-GMP (CDG). Protective mucosal immune responses are most effectively induced by mucosal immunization. However, most of the currently approved human vaccines are administered systemically and generally fail to elicit effective mucosal immunity. Live attenuated mucosal vaccines present safety and acceptability issues while purified antigens are generally poor immunogenics when administered by the mucosal route. CDG exhibits potent mucosal immunogenicity thus, has been explored as a promising mucosal vaccine adjuvant. The mechanism by which CDG executes its mucosal adjuvant activity is unknown, which hinders the further development of CDG as an efficacious mucosal adjuvant. STING, also known as MPYS/MITA, is essential for type I IFN (IFN-I) production by cytosolic sensing of DNA. Recently, we showed that STING mediates IFN-I production by CDG. Subsequent structure studies found that the cytoplasmic tail of STING binds to CDG. It is proposed that STING is a direct sensor for CDG, leading to IFN-I production. However, the in vivo role of STING in CDG-induced vaccine adjuvant activity is not known. In this proposal, we found that STING-/- mice fail to generate antigen-specific antibody response after intranasal immunization of an antigen and CDG. Furthermore, the production of Th1/Th2/Th17 cytokines, proinflammatory cytokines and IFN-I are absent in CDG immunized STING-/- mice. Surprisingly, we found that IFN-I signaling is NOT required for the mucosal adjuvant activity of CDG because IFNAR1-/- mice have the same antigen-specific antibody response as the wild-type mice. This is distinct from STING-mediated DNA vaccine adjuvant activity, which requires IFN-I signaling. We propose two specific Aims to address the in vivo cellular and in vitro molecular mechanisms underlying STING-mediated CDG mucosal adjuvant activity. Aim 1: Dendritic Cells (DC) Expression of STING is essential for the mucosal vaccine adjuvant activity of CDG in vivo. We will use conditional STING-/- and conditional STINGwt/wt mice generated by us to determine the cellular mechanism of STING-mediated CDG adjuvant activity in vivo; Aim 2: Determine the IFN-I stimulation independent mechanism underlying STING-mediated DC activation by CDG. We found that CDG promotes STING-dependent but IFN-I signaling-independent DC maturation. We will use biochemical and molecular biological approaches to identify the proximal signaling transducer of STING that mediates this IFN-I stimulation-independent function. We will further verify its importance in the adjuvant activity of CDG in vivo using KO mice. Currently, there is no vaccine formulation containing a mucosal adjuvant approved for human use. The knowledge generated by our studies can help advance the development of CDG as an efficacious mucosal vaccine adjuvant for human use.