Mechanistic Exploration of cGAS-STING-Mediated Vaccine Enhancement

项目总结 本R01提案的目标包括疫苗佐剂的分子和免疫学特征。 与干扰素基因蛋白刺激物(STING)介导的过程相关的活性。两个至关重要的因素 目前在佐剂人用疫苗方面的不足是显而易见的。首先，很少有佐剂是 美国批准临床使用第二，佐剂相关免疫的确切机制基础 人们对增强作用知之甚少。有效佐剂可触发快速、局部的先天免疫反应 跟随他们的管理。从根本上说，先天信号是通过模式的参与而启动的 识别受体(PRRs)的配体指示或模仿微生物感染。这反过来又会导致 最终指导适应性免疫反应的免疫调节因子和促炎因子的表达 能够清除受感染的组织。代表识别环二核苷酸(CDN)的PRR，一个 细胞酶cGMP-AMP合成酶(CGAS)检测胞质dsDNA后的产物 来源于微生物、线粒体或细胞核。刺介导的表型最终由 由被激活的蛋白质转录诱导的基因。这与转录因子密切相关。 干扰素调节因子3(IRF3)和核因子κB(NF-κB)，它们还合成了不同本体的mRNAs 它们的功能作用大多还未被发掘。此外，刺似乎控制着生理过程， 免疫系统的细胞类型之间存在显著差异，包括基质细胞、髓系细胞和T细胞。有趣的是， 在小鼠模型中通过药物诱导刺痛依赖活性大大增强了疫苗的效力，因为 以对不同病原体产生的保护性免疫为标志。不幸的是，精确的分子和 获得性免疫增强与刺痛活性之间的内在关联仍未得到很大程度的检验。 此外，刺痛佐剂是否在灵长类动物中引起同样有效的免疫原性结果尚未得到证实。 检查过了。我们计划将这些与我们强大的CRISPR和转录切割技术结合起来，以获得 对刺痛介导的免疫结果的基本基础的深入洞察。我们假设 与基于刺激剂的佐剂相关的抗原导向获得性免疫增强在功能上是 与使用这些模型可以识别的分子和细胞过程相关联。我们还确定了一个 一流的小分子，激活跨物种的cGAS刺痛并增强对寨卡病毒的免疫原性 病毒抗原。在体外、小鼠和非人类灵长类动物(NHP)模型中将其与CDN一起使用将允许 美国将：1)验证和表征cGAS作为一种新的免疫治疗靶点；2)展示刺痛佐剂 在高度临床相关的模式物种中的疗效；以及3)确定特定物种的异同 关于叮咬介导的免疫反应。

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.