Harnessing ZBP1-driven cell death to improve influenza vaccine efficacy

项目摘要 接种疫苗是预防甲型流感病毒（IAV）感染的最有效手段。电流 IAV疫苗的方法主要靶向HA蛋白，但HA经历了显著的抗原漂移， 因此是持久体液免疫的不良靶点。开发通用IAV疫苗的替代方法 靶向病毒内部蛋白（如核蛋白），这些蛋白在大多数IAV之间也是高度保守的。 菌株然而，这种方法的一个主要障碍是缺乏触发正确免疫的佐剂。 免疫应答--"I型“（或Th 1/CD 8 + T细胞驱动的）应答--针对这些内部抗原。在这份提案中，我们概述了 一种寻求利用免疫原性细胞死亡作为激活针对IAV的I型免疫的手段的新策略 疫苗。我们已经确定了一个宿主信号通路，几乎所有的IAV激活的免疫原性 感染细胞死亡，包括原代气道上皮细胞、巨噬细胞和DC。这种细胞死亡途径 当宿主感受器蛋白ZBP 1检测到IAV基因组RNA并激活IAV的平行途径时， 程序性坏死（坏死性凋亡）和凋亡，以杀死感染的细胞，并引发CD 8 + T细胞介导的 适应性免疫反应增加ZBP 1启动的细胞死亡作为疫苗方法的吸引力的是 首先，我们发现IAV产生一种新形式的双链RNA --称为Z-RNA --来激活ZBP 1。 因此，Z-RNA是一种新的PAMP，并且可以容易地利用它来触发免疫原性细胞死亡和佐剂IAV。 疫苗。其次，ZBP 1从细胞核启动坏死性凋亡，导致核膜破裂和释放 高免疫原性的核DAMP和IAV抗原。核坏死性凋亡的免疫原性比 常规（细胞质引发的）坏死性凋亡。由于ZBP 1驱动的免疫原性细胞死亡（细胞凋亡和 核坏死性凋亡）对于有效的抗IAV细胞毒性T细胞应答是重要的，我们的发现使我们能够 提出Z-RNA是激活针对IAV的I型免疫应答的有效新佐剂，并且 经工程改造以产生Z-RNA的重组IAV代表新型I型免疫活化疫苗。在这 建议，三个实验室在IAV细胞死亡信号（Balachandran），重组IAV 技术（Langlois）和发病机理/疫苗策略（洛佩斯）将联合起来鉴定IAV产生的Z-RNA 激活ZBP 1（目的1）;确定哪些基质细胞和免疫细胞类型通过ZBP 1介导的机制死亡 启动和调节有效的适应性免疫应答（Aim 2）;并测试是否触发ZBP 1介导的细胞 合成和病毒产生的Z-RNA的死亡可以用来告知和提供下一代 能够对IAV提供有效交叉保护免疫的疫苗（目标3）。

Project Summary Vaccination represents the most effective means of preventing influenza A virus (IAV) infections. Current approaches to IAV vaccines primarily target the HA protein, but HA undergoes significant antigenic drift and is thus a poor target for lasting humoral immunity. An alternative approach to developing a universal IAV vaccine is to target internal viral proteins (such as the nucleoprotein), which are also highly conserved between most IAV strains. A major roadblock to this approach, however, is a lack of adjuvants that trigger the right kind of immune response - a ‘type I’ (or Th1/CD8+ T-cell driven) response - to these internal antigens. In this proposal, we outline a new strategy that seeks to harness immunogenic cell death as a means of activating type I immunity to IAV vaccines. We have identified a host signaling pathway which accounts for almost all IAV-activated immunogenic death in infected cells, including primary airway epithelial cells, macrophages and DCs. This cell death pathway is initiated when the host sensor protein ZBP1 detects IAV genomic RNA and activates parallel pathways of programmed necrosis (necroptosis) and apoptosis to kill the infected cell and instigate CD8+ T cell-mediated adaptive immune responses. Adding to the attractiveness of ZBP1-initiated cell death as a vaccine approach are our discoveries, first, that IAV produces a new form of double-stranded RNA - termed Z-RNA - to activate ZBP1. Z-RNA is thus a new PAMP, and can be readily harnessed to trigger immunogenic cell death and adjuvant IAV vaccines. Second, ZBP1 initiates necroptosis from the nucleus, resulting in nuclear envelope rupture and release of highly-immunogenic nuclear DAMPs and IAV antigens. Nuclear necroptosis is even more immunogenic than conventional (cytoplasm-initiated) necroptosis. As ZBP1-driven immunogenic cell death (both apoptosis and nuclear necroptosis) is important for effective anti-IAV cytotoxic T cell responses, our discoveries allow us to propose that Z-RNAs are potent new adjuvants for activating type I immune responses to IAV, and that recombinant IAVs engineered to produce Z-RNAs represent new type I immunity-activating vaccines. In this proposal, three labs with strong expertise in IAV cell death signaling (Balachandran), recombinant IAV technology (Langlois), and pathogenesis/vaccine strategies (Lopez) will unite to identify IAV-generated Z-RNAs that activate ZBP1 (Aim 1); determine which stromal and immune cell types die by ZBP1-mediated mechanisms to initiate and regulate effective adaptive immune responses (Aim 2); and test if triggering ZBP1-mediated cell death with synthetic and virus-generated Z-RNAs can be leveraged to inform and supply next-generation vaccines capable of providing effective cross-protective immunity to IAV (Aim 3).