A novel virus-derived adjuvant

描述（由申请人提供）：佐剂分子对树突状细胞（DC）的激活对于成功免疫至关重要。大多数传统和更新一代的佐剂来源于微生物来源，并通过结合和激活细胞表面上的Toll样受体（TLR）来发挥作用。我们已经发现了一种天然佐剂，其具有独特的激活人和小鼠DC的有效能力，并且可以在没有TLR的情况下发挥作用。这种独特的佐剂活性是由仙台病毒缺陷基因组提供的，仙台病毒缺陷基因组起源于仙台病毒复制的副产物。仙台病毒缺陷基因组的佐剂活性取决于它们通过病毒聚合酶的复制。刺激性DI基因组不产生病毒蛋白，因此，它们的活性不由编码蛋白提供。缺陷病毒基因组触发DC成熟的程度与我们测试的任何TLR配体或活病毒相同或上级。值得注意的是，与密切相关的病毒不同，有缺陷的病毒基因组可以诱导DC的完全激活，而不依赖于I型IFN信号传导的互补，并且在功能性病毒编码的免疫拮抗剂的存在下。我们假设独特的细胞事件是由特定的病毒RNA基序在缺陷基因组复制过程中暴露而触发的，从而允许其有效的佐剂活性。该基金的第一个目标将集中在研究参与缺陷病毒颗粒激活DC的细胞机制。我们提出，有缺陷的病毒基因组可以作为“金标准”，在病毒感染过程中有效激活DC成熟。参与缺陷病毒基因组应答的细胞途径的表征将有助于定义病毒感染期间实现最佳DC成熟的最低要求。我们将研究候选细胞信号通路的作用，缺陷病毒基因组的亚细胞定位，以及这些基因组与细胞蛋白的关联。我们预测，这些研究将揭示新的电路激活的树突状细胞，绕过病毒的拮抗作用和要求的I型IFN信号在感染标准病毒。第二个目标将致力于鉴定提供具有这种独特活性的缺陷基因组的病毒基序。基于我们的初步数据，我们将专注于研究缺陷基因组复制过程中产生或暴露的潜在RNA基序。我们期望从这一目标中收集的信息将对新佐剂分子的制备具有重要意义。最后，在第三个目标中，我们将研究在存在缺陷病毒颗粒的情况下在免疫过程中产生的免疫应答以及这些颗粒在小鼠中针对流感病毒免疫过程中的佐剂活性。这项研究将扩展我们的缺陷病毒基因组的佐剂活性在体内的表征，并在更详细的方法，其利用和交付的发展。公共卫生相关性：有缺陷的病毒基因组是免疫反应的有效天然刺激物，其功能机制与传统佐剂不同。在这个项目中，我们将研究参与缺陷病毒感染反应的细胞途径以及引发这种反应的病毒基序。我们的目标是获得新的免疫刺激方法的设计和新的佐剂分子的阐述的见解。此外，我们将在针对流感病毒感染的免疫过程中测试缺陷病毒颗粒作为佐剂。

DESCRIPTION (provided by applicant): The activation of dendritic cells (DCs) by adjuvant molecules is essential for successful immunization. Most of the traditional and newer generations of adjuvants are derived from microbial sources and function by binding and activating Toll like receptors (TLRs) on the cell surface. We have discovered a natural adjuvant that has a uniquely potent ability to activate human and mouse DCs and can function in the absence of TLRs. This distinctive adjuvant activity is provided by Sendai virus defective genomes that are originated as byproducts of Sendai virus replication. The adjuvant activity of Sendai virus defective genomes depends on their replication by the viral polymerase. Stimulatory DI genomes do not produce viral proteins and, therefore, their activity and is not provided by encoded proteins. Defective viral genomes trigger DC maturation to the same degree as or superior to that of any TLR ligand or live virus that we have tested. Notably, unlike closely related viruses, defective viral genomes can induce the complete activation of DCs independently of complementation by type I IFN signaling and in the presence of functional viral-encoded antagonists of immunity. We hypothesize that unique cellular events are triggered by a particular viral RNA motif exposed during the replication of the defective genomes allowing for their efficient adjuvant activity. The first aim of this grant will focus on the investigation of the cellular mechanisms involved in the activation of DCs in response to defective viral particles. We propose that defective viral genomes could be used as "gold standard" for efficient activation of DC maturation during virus infection. The characterization of the cellular pathways involved in the response to defective viral genomes will contribute to the definition of the minimal requirements to achieve optimal DC maturation during virus infection. We will study the role of candidate cellular signaling pathways, the subcellular localization of defective viral genomes, and the association of these genomes with cellular proteins. We predict that these studies will reveal novel circuits for the activation of DCs that bypass both viral antagonism and the requirement for type I IFN signaling present during infection with standard viruses. The second aim will address the identification of the viral motifs that provide defective genomes with this distinctive activity. Based on our preliminary data, we will focus on the study of potential RNA motifs generated or exposed during the replication of the defective genome. We expect that information gathered from this aim will be significant for the elaboration of new adjuvant molecules. Finally, in the third aim we will study the immune response developed during immunization in the presence of defective viral particles and the adjuvant activity of these particles during immunization against influenza virus in mice. This study will extend our characterization of the defective virus genome's adjuvant activity in vivo and precedes the development of more elaborated methods for their harnessing and delivery. Public Health Relevance: Defective viral genomes are potent natural stimulators of the immune response that function by mechanisms distinct from those of traditional adjuvants. In this project we will investigate the cellular pathways involved in the response to infection with defective viruses and the viral motifs that trigger this response. Our goal is to obtain insight for the design of new immunostimulatory approaches and the elaboration of novel adjuvant molecules. In addition, we will test defective viral particles as adjuvants during immunization against influenza virus infection.