Innate Immune Recognition Enhances Flavivirus Vaccine Efficacy

描述（由申请方提供）：黄病毒属属于NIAID的A、B和C类病毒列表。目前迫切需要针对多种黄病毒疾病，特别是登革热和西尼罗河脑炎的疫苗。为了开发更有效的疫苗，需要全面了解黄病毒如何引发免疫反应。为了满足这一需求，我们开发了单循环黄病毒和包装细胞系统，以高滴度生产它们。这些单循环病毒包括西尼罗河病毒（WNV）衍生的病毒复制子颗粒（VRP）和名为davax的WN脑炎候选疫苗。这两种颗粒编码自我复制的WNV RNA基因组（复制子），基因组包装所需的一个或多个基因缺陷。VRP和davax模拟WNV感染的许多方面（受体结合、摄取、RNA释放和基因组复制）。然而，VRP和davax不能在正常细胞中产生子代病毒体，因此不是致病性的。尽管如此，davax编码保护性免疫原，亚病毒颗粒（SVP），并已被证明是一个非常有效的疫苗候选者。VRP可以被修饰以表达标记基因（以允许体内追踪），进一步增强其效用。我们的单循环颗粒靶向小鼠的引流淋巴结，在那里它们感染巨噬细胞并诱导高水平的I型干扰素（IFN），这表明IFN对于指导对我们疫苗的适应性免疫应答至关重要。然而，缺乏I型IFN受体的小鼠产生的抗体应答与WT动物无法区分，表明IFN不连接先天免疫和适应性免疫。在了解西尼罗河病毒如何诱导IFN的过程中，我们研究了小鼠骨髓来源的树突状细胞的体外培养物对西尼罗河病毒VRP感染的反应。这些研究表明，蛋白激酶R（PKR）似乎是作为一种模式识别受体（PRR），触发IFN的合成。此外，VRP接种的PKR缺陷小鼠产生的IFN水平和WNV特异性抗体水平低于WT小鼠，表明PRR途径（PRRP）控制先天性和适应性反应。基于这些研究，我们假设：通过PRR识别抗疟药会导致多个PRRP的参与，这些PRRP可能独立或协同作用，以诱导先天性和适应性免疫。将在3个特定目标中评估该假设，其中我们将阐明巨噬细胞耗竭和特异性PRRP缺陷对接种这些独特单循环黄病毒颗粒的小鼠中的1）先天性、2）体液和3）细胞免疫应答的影响。

DESCRIPTION (provided by applicant): Flaviviruses populate the NIAID's Category A, B, and C lists of viruses. Vaccines are urgently needed for multiple flavivirus diseases, notably dengue and West Nile encephalitis. To develop more effective vaccines a comprehensive understanding of how flaviviruses trigger immune responses is needed. To address this need, we developed single-cycle flaviviruses and packaging cell systems that produce them at high titers. These single-cycle viruses include West Nile virus (WNV) -derived viral replicon particles (VRPs) and a WN encephalitis vaccine candidate named Replivax. Both particles encode self-replicating WNV RNA genomes (replicons) deficient in one or more genes required for genome packaging. VRPs and Replivax mimic many aspects of WNV infection (receptor binding, uptake, RNA release, and genome replication). However, VRPs and Replivax cannot produce progeny virions in normal cells, and hence are not pathogenic. Nevertheless, Replivax encodes a protective immunogen, the sub-viral particle (SVP), and has proven to be a remarkably potent vaccine candidate. VRPs can be modified to express marker genes (to allow tracking in vivo), further enhancing their utility. Our single-cycle particles target the draining lymph nodes of mice, where they infect macrophages and induce high levels of type I interferon (IFN), suggesting that IFN is critical for directing the adaptive immune response to our vaccine. However, mice deficient in type I IFN receptors produced antibody responses to Replivax indistinguishable from WT animals, suggesting that IFN was not linking innate and adaptive immunity. In the process of learning how WNV induces IFN, we examined how ex vivo cultures of murine bone marrow-derived dendritic cells responded to WNV VRP infection. These studies showed that protein kinase R (PKR) appeared to be acting as a pattern-recognition receptor (PRR) that triggered IFN synthesis. Further, VRP-inoculated PKR-deficient mice produced lower levels of IFN and lower levels of WNV-specific antibodies than WT mice, indicating that PRR pathways (PRRPs) control both innate and adaptive responses. Based on these studies we hypothesize that: Recognition of Replivax via PRRs results in the engagement of multiple PRRPs, which may act independently, or in concert, to induce innate and adaptive immunity. This hypothesis will be evaluated in 3 specific aims in which we will elucidate the effect of macrophage depletion and deficiencies in specific PRRPs on the 1) innate, 2) humoral, and 3) cellular immune response in mice inoculated with these unique single-cycle flavivirus particles.