Mechanistic Exploration of cGAS-STING-Mediated Vaccine Enhancement

项目摘要 该R 01提案的目标包括疫苗佐剂的分子和免疫学表征 与干扰素基因刺激因子（STING）蛋白介导的过程相关的活性。两个重要 目前关于含佐剂的人疫苗的不确定性是明显的。首先，很少有佐剂 第二，促排剂相关免疫的确切机制基础， 增强的理解很少。有效的佐剂触发快速、局部的先天免疫应答 在他们的管理之后。从根本上说，先天信号是通过参与模式启动的， 通过指示或模拟微生物感染的配体识别受体（PRR）。这反过来又导致了 最终指导适应性免疫应答的免疫调节和促炎因子的表达 能够清除受感染的组织。STING代表感测环状二核苷酸（CDN）的PRR， 细胞酶环GMP-AMP合酶（cGAS）在检测到细胞质dsDNA后的产物 来源于微生物、线粒体或细胞核。STING介导的表型最终由 由活化的蛋白质转录诱导的基因。这关键地涉及转录因子 IFN调节因子3（IRF 3）和核因子κB（NF-κB），它们合成不同本体的mRNA，但它们的表达水平不同。 其功能性作用大多未被探索。此外，STING似乎控制生理过程， 在免疫系统的细胞类型之间存在显著差异，包括基质细胞、骨髓细胞和T细胞。有趣的是， 在鼠模型中STING依赖性活性的药理学诱导大大增强了疫苗功效， 由针对不同病原体引起的保护性免疫指示。不幸的是，精确的分子和 与STING活性相关的适应性免疫增强的先天相关性在很大程度上仍未得到检验。 此外，STING佐剂是否在灵长类动物中引发类似有效的免疫原性结果还没有被证实。 考察我们计划将这些与我们强大的CRISPR和转录组技术结合起来， 深入了解STING介导的免疫结果的基本基础。我们假设 与基于STING的佐剂相关的抗原导向的适应性免疫的增强在功能上是 与分子和细胞的过程联系起来，这些过程可以用这些模型来识别。我们还发现了一个 第一个在物种间激活cGAS-STING并增强Zika免疫原性的小分子 病毒抗原在体外、鼠和非人灵长类动物（NHP）模型中将其与CDN一起包括在内， 我们要：1）鉴定和表征cGAS作为新的免疫靶点; 2）证明STING佐剂 在高度临床相关的模型物种中的功效;以及3）识别物种特异性的相似性和差异 关于针刺介导的免疫反应。

PROJECT SUMMARY The goals of this R01 proposal include the molecular and immunological characterization of vaccine adjuvant activity associated with processes mediated by the protein Stimulator of Interferon Genes (STING). Two vital insufficiencies are currently evident regarding adjuvanted human vaccines. First, very few adjuvants are approved for clinical use in the U.S. Second, the precise mechanistic bases of adjuvant-associated immune augmentation are poorly understood. Effective adjuvants trigger rapid, localized innate immune responses following their administration. Fundamentally, the innate signaling is initiated through engagement of pattern recognition receptors (PRRs) by ligands indicative or imitative of microbial infection. This, in turn, leads to expression of immunomodulatory and proinflammatory factors that ultimately direct adaptive immune responses capable of eliminating infected tissues. STING represents the PRR that senses cyclic dinucleotides (CDN), a product of the cellular enzyme cyclic GMP-AMP synthase (cGAS) following its detection of cytoplasmic dsDNA derived from microbes, mitochondria, or the nucleus. STING-mediated phenotypes are ultimately conferred by genes that are transcriptionally induced by the activated protein. This crucially involves the transcription factors IFN regulatory factor 3 (IRF3) and nuclear factor κB (NF-κB), which synthesize mRNAs of distinct ontologies yet whose functional roles are mostly unexplored. Moreover, STING appears to control physiological processes that differ dramatically between cell types of the immune system including stromal, myeloid, and T cells. Intriguingly, pharmacologic induction of STING-dependent activity in murine models greatly enhances vaccine efficacy as indicated by protective immunity elicited against diverse pathogens. Unfortunately, the precise molecular and innate correlates of adaptive immune potentiation associated with STING activity remain largely unexamined. Furthermore, whether STING adjuvants elicit similarly effective immunogenic outcomes in primates has not been examined. We plan to couple these with our powerful CRISPR and transcriptomic technologies to obtain penetrative insight into the fundamental bases of STING-mediated immune outcomes. We hypothesize that the enhancement of antigen-directed adaptive immunity associated with STING-based adjuvants is functionally linked to molecular and cellular processes that are discernable using these models. We have also identified a first-in-class small molecule that activates cGAS-STING across species and enhances immunogenicity to Zika virus antigen. Including this alongside CDN in in vitro, murine, and nonhuman primate (NHP) models will allow us to: 1) Validate and characterize cGAS as a new immunotherapeutic target; 2) Demonstrate STING adjuvant efficacy in a highly clinically relevant model species; and 3) Identify species-specific similarities and differences with respect to STING-mediated immune responses.