Dendritic Cell Targeting Enhances Flavivirus Vaccine Efficacy

描述（由申请人提供）：黄病毒家族中的病毒填充NIAID的A、B和C类病毒列表中的所有三种病毒。多种黄病毒疾病（尤其是登革热）需要疫苗，而黄病毒如何与先天免疫系统相互作用的详细知识对于疫苗开发至关重要。为了满足这一需求，我们开发了一种生产黄病毒样病毒颗粒（VLPs）的系统。VLPs是包装的，基因缺失的黄病毒，能够在培养细胞中启动复制周期。VLPs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、RNA和非结构蛋白合成。然而，vlp感染的细胞不会产生能够传播到其他细胞的功能性病毒。除了VLPs，我们还生产了第二种名为RepliVAX的黄病毒颗粒。RepliVAX显示出VLPs的非传播特性，但除了编码由VLPs产生的病毒产物外，RepliVAX还产生一种亚病毒颗粒（SVP），具有诱导人抗病毒免疫的能力。RepliVAX在小鼠身上具有显著的效力，目前正在开发一种疫苗。在足垫中接种了VLPs的小鼠产生了高水平的IFNa，而接种了紫外线灭活的VLPs的小鼠则不产生任何IFNa。VLP接种小鼠的引流淋巴结（LN）含有西尼罗河病毒抗原和基因组，以及高水平的IFNa mRNA。这些体内结果表明，VLPs的目标是引流LN，在那里它们诱导IFNa合成。体外，来自非免疫系统细胞的细胞系的VLP（和WNV）感染诱导产生不同的I型IFN亚型（IFN¿），并且诱导依赖于病毒复制。WNV感染人单核细胞衍生树突状细胞（mDCs）诱导的IFN也依赖于病毒复制，但mDCs产生的IFN属于IFNa亚型。另一方面，人浆细胞样dc （pDCs）在暴露于活的或灭活的西尼罗河病毒时诱导高水平的IFNa。有趣的是，西尼罗河病毒不会有效地感染pDCs。我们假设西尼罗河病毒感染周围的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pDCs并招募T和B细胞。此外，我们假设dc中心先天信号通路的有效参与有助于我们的RepliVAX疫苗的效力。为了解决这些假设，我们将确定LN中RepliVAX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的产生，并检查这些细胞的激活对给予RepliVAX的免疫反应的影响。这些研究将为先天性免疫和适应性免疫之间的联系提供重要的见解，从而导致对黄病毒感染和疫苗接种产生保护性免疫。

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.