A novel virus-derived adjuvant

描述(由申请人提供)：佐剂分子激活树突状细胞(DC)是成功免疫的关键。大多数传统和新一代佐剂来自微生物来源，其功能是通过结合和激活细胞表面的Toll样受体(TLRs)来发挥作用。我们已经发现了一种天然佐剂，它具有激活人类和小鼠DC的独特强大能力，并且可以在没有TLRs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，这些基因组起源于仙台病毒复制的副产品。仙台病毒缺陷基因组的佐剂活性取决于病毒聚合酶的复制。刺激性DI基因组不产生病毒蛋白，因此它们的活性也不是由编码蛋白提供的。有缺陷的病毒基因组触发DC成熟的程度与我们测试的任何TLR配体或活病毒相同或更高。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型干扰素信号的互补，也不依赖于病毒编码的免疫拮抗剂的存在。我们假设，独特的细胞事件是由缺陷基因组复制过程中暴露的特定病毒RNA基序触发的，从而允许它们有效的佐剂活性。这笔赠款的第一个目的将集中在研究DC对缺陷病毒颗粒的反应中所涉及的细胞机制。我们认为有缺陷的病毒基因组可以作为病毒感染过程中有效激活DC成熟的“金标准”。对参与缺陷病毒基因组反应的细胞通路的表征将有助于定义在病毒感染期间实现最佳DC成熟所需的最低要求。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，这些研究将揭示树突状细胞激活的新电路，绕过病毒拮抗和感染标准病毒期间存在的I型干扰素信号要求。第二个目标将致力于识别为有缺陷的基因组提供这种独特活动的病毒基序。基于我们的初步数据，我们将重点研究在缺陷基因组复制过程中产生或暴露的潜在RNA基序。我们希望从这一目标收集的信息将对新佐剂分子的阐述具有重要意义。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免疫过程中产生的免疫反应，以及这些颗粒在小鼠对流感病毒免疫期间的佐剂活性。这项研究将扩展我们对有缺陷的病毒基因组在体内的佐剂活性的表征，并领先于开发更详细的方法来控制和传递它们。与公共卫生相关：有缺陷的病毒基因组是免疫反应的强大自然刺激因子，其作用机制与传统佐剂不同。在这个项目中，我们将研究参与感染缺陷病毒反应的细胞通路以及触发这种反应的病毒基序。我们的目标是为设计新的免疫刺激方法和阐述新的佐剂分子获得洞察力。此外，我们将在流感病毒感染的免疫接种中测试有缺陷的病毒颗粒作为佐剂。

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.