Dendritic Cell Targeting Enhances Flavivirus Vaccine Efficacy

描述（由申请方提供）：黄病毒家族中的病毒属于NIAID的A、B和C类病毒列表中的所有三种。多种黄病毒疾病需要疫苗，最明显的是登革热，详细了解黄病毒如何与先天免疫系统相互作用对疫苗开发至关重要。为了满足这一需求，我们开发了一种生产黄病毒病毒样颗粒（VLP）的系统。VLP是包装的、基因缺失的黄病毒，能够在培养的细胞中启动复制周期。VLP在感染的许多方面与正常黄病毒相同，即：受体结合、摄取、RNA释放以及RNA和非结构蛋白合成。然而，VLP感染的细胞不产生能够扩散到其他细胞的功能性病毒。除了VLP之外，我们还产生了第二种类型的黄病毒颗粒，命名为DEVAX。<$VAX显示出VLP的非扩散特性，但除了编码由VLP产生的病毒产物外，<$VAX还产生亚病毒颗粒（SVP），该亚病毒颗粒（SVP）具有在人体中诱导抗病毒免疫的能力。<$VAX在小鼠中非常有效，并且正在开发作为疫苗。在足垫中用VLP接种的小鼠产生高水平的IFNa，与用UV灭活的VLP接种的小鼠相反，其不产生任何IFNa。从VLP接种的小鼠收获的引流淋巴结（LN）含有WNV抗原和基因组以及高水平的IFNa mRNA。这些体内结果表明，VLP靶向引流LN，在那里它们诱导IFNa合成。在体外，来自非免疫系统细胞的细胞系的VLP（和WNV）感染诱导不同的I型IFN亚型（IFN？）的产生，并且诱导依赖于病毒复制。通过WNV感染人单核细胞衍生的树突状细胞（mDC）的IFN诱导也依赖于病毒复制，但由mDC产生的IFN是IFNa亚型。另一方面，人浆细胞样DC（pDC）响应于暴露于活的或灭活的WNV而诱导高水平的IFNa。有趣的是，WNV不会有效地感染pDC。我们假设WNV感染外周的DC（可能是朗格汉斯细胞），导致它们迁移到LN，在那里它们激活pDC并募集T和B细胞。此外，我们假设以DC为中心的先天信号传导途径的有效参与有助于我们的dVAX疫苗的效力。为了解决这些假设，我们将鉴定LN中dVAX感染细胞的确切性质，确定这些细胞（或邻近细胞）是否负责IFN产生，并检查这些细胞的活化对dVAX施用的免疫应答的影响。这些研究将为先天免疫和适应性免疫之间的联系提供重要的见解，这些免疫导致产生对黄病毒感染和疫苗接种的保护性免疫。

Description (provided by applicant): Viruses in the flavivirus family populate all three of NIAID's Category A, B, and C lists of viruses. Vaccines are needed for multiple flavivirus diseases, most notably dengue, and detailed knowledge of how flaviviruses interact with the innate immune system is critical for vaccine development. To address this need, we have developed a system to produce flavivirus virus-like particles (VLPs). VLPs are packaged, gene-deleted flaviviruses capable of initiating a replication cycle in cells in culture. VLPs are identical to normal flaviviruses in many aspects of infection, namely: receptor binding, uptake, RNA release, and RNA and nonstructural protein synthesis. However, VLP-infected cells do not produce functional virus capable of spreading to other cells. In addition to VLPs, we have produced a second type of flavivirus particle named RepliVAX. RepliVAX displays the non-spreading properties of VLPs, but in addition to encoding the viral products made by VLPs, RepliVAX also produces a sub-viral particle (SVP) with demonstrated capacity to induce antiviral immunity in man. RepliVAX is remarkably potent in mice and is being developed as a vaccine. Mice inoculated in the footpad with VLPs produce high levels of IFNa, in contrast to mice inoculated with UV-inactivated VLPs, which do not produce any IFNa. Draining lymph nodes (LN) harvested from VLP- inoculated mice contain WNV antigen and genome, and high levels of IFNa mRNA. These in vivo results suggest that VLPs are targeted to the draining LN where they induce IFNa synthesis. In vitro, VLP (and WNV) infection of cell lines derived from non-immune system cells induces production of a different type I IFN subtype (IFN¿), and induction is dependent on viral replication. IFN induction by WNV infection of human monocyte-derived dendritic cells (mDCs) is also dependent on viral replication, but the IFN produced by mDCs is of the IFNa subtype. On the other hand, human plasmacytoid DCs (pDCs), induce high levels of IFNa in response to exposure to live or inactivated WNV. Intriguingly, WNV does not productively infect pDCs. We hypothesize that WNV infects DCs (likely Langerhans cells) in the periphery, leading to their migration to the LN where they activate pDCs and recruit T and B cells. Further, we hypothesize that effective engagement of DC-centered innate signaling pathways contributes to the potency of our RepliVAX vaccine. To address these hypotheses, we will identify the precise nature of RepliVAX-infected cells in LN, determine if these cells (or neighboring cells) are responsible for the IFN production, and examine the effect of activation of these cells on the immune response to RepliVAX administration. These studies will provide important insights into links between innate and adaptive immunity that lead to the production of protective immunity to flavivirus infection and vaccination.