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

描述(申请人提供)：这项建议的目的是揭示体内机制，通过STING(干扰素基因刺激物)介导的粘膜疫苗佐剂活性的环状二-GMP(CDG)。黏膜免疫最有效地诱导保护性黏膜免疫反应。然而，目前批准的大多数人类疫苗都是系统接种的，通常无法诱导有效的粘膜免疫。减毒活粘膜疫苗存在安全性和可接受性问题，而纯化的抗原通过粘膜途径接种时，免疫原性一般较差。CDG具有很强的粘膜免疫原性，被认为是一种很有前途的黏膜疫苗佐剂。CDG发挥其粘膜佐剂活性的机制尚不清楚，这阻碍了CDG作为一种有效的黏膜佐剂的进一步发展。SING，也被称为MPYS/MITA，是通过胞浆感应DNA产生I型干扰素(干扰素-I)所必需的。最近，我们发现STING介导CDG产生干扰素-I。随后的结构研究发现，STING的细胞质尾巴与CDG结合。有人认为，STING是CDG的直接传感器，导致干扰素-I的产生。然而，在体内，刺痛在CDG诱导的疫苗佐剂活性中的作用尚不清楚。在这个方案中，我们发现，经鼻腔免疫抗原和CDG后，刺痛/-小鼠不能产生抗原特异性抗体反应。此外，CDG免疫的STING-/-小鼠缺乏Th1/Th2/Th17细胞因子、促炎细胞因子和干扰素-I的产生。令人惊讶的是，我们发现CDG的粘膜佐剂活性并不需要干扰素-I信号，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，后者需要干扰素-I信号。我们提出了两个特定的目标，以解决体内细胞和体外分子机制潜在的刺痛介导的CDG粘膜佐剂活性。目的1：树突状细胞(DC)的表达是CDG黏膜疫苗佐剂活性所必需的。我们将利用我们建立的条件性STING-/-和条件性STINGwt/wt小鼠，在体内确定STING介导的CDG佐剂活性的细胞机制；目的2：确定CDG通过STING介导的DC激活背后的干扰素-I刺激独立机制。我们发现CDG促进刺痛依赖但不依赖于干扰素-I信号的DC成熟。我们将使用生化和分子生物学的方法来确定STIN的近端信号转导通路，它介导了这种干扰素-I刺激非依赖的功能。我们将通过KO小鼠进一步验证其在CDG体内佐剂活性中的重要性。目前，还没有包含被批准用于人类的粘膜佐剂的疫苗配方。我们的研究所产生的知识有助于推动CDG作为一种有效的人类使用的黏膜疫苗佐剂的发展。

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.