Mitochondrial Hyperactivity and Reactive Oxygen Species Drive Innate Immunity to the Yellow Fever Virus-17D Live-Attenuated Vaccine

The yellow fever virus 17D (YFV-17D) live attenuated vaccine is considered one of the successful vaccines ever generated associated with high antiviral immunity, yet the signaling mechanisms that drive the response in infected cells are not understood. Here, we provide a molecular understanding of how metabolic stress and innate immune responses are linked to drive type I IFN expression in response to YFV-17D infection. Comparison of YFV-17D replication with its parental virus, YFV-Asibi, and a related dengue virus revealed that IFN expression requires RIG-I-like Receptor signaling through MAVS, as expected. However, YFV-17D uniquely induces mitochondrial respiration and major metabolic perturbations, including hyperactivation of electron transport to fuel ATP synthase. Mitochondrial hyperactivity generates reactive oxygen species (ROS) including peroxynitrite, blocking of which abrogated MAVS oligomerization and IFN expression in non-immune cells without reducing YFV-17D replication. Scavenging ROS in YFV-17D-infected human dendritic cells increased cell viability yet globally prevented expression of IFN signaling pathways. Thus, adaptation of YFV-17D for high growth imparts mitochondrial hyperactivity to meet energy demands, resulting in generation of ROS as the critical messengers that convert a blunted IFN response into maximal activation of innate immunity essential for vaccine effectiveness. Human monocyte-derived dendritic cells (DCs) were either mock-infected or infected with YFV-17D at an MOI of 0.1. We added fresh DC medium to the wells to the desired volume and MnTBAP (50 µM) or vehicle control was added to the appropriate wells at 0 and 24 h post-infection. At 48 hpi, we harvested hDCs by centrifugation, and lysed the cells with 1 mL of TRIzol reagent/sample (Thermo Fisher Scientific), and samples were stored at -80˚C before extraction and bulk RNA-seq. Differential expression was performed between infected and mock infected.

黄热病毒17D（yellow fever virus 17D, YFV-17D）减毒活疫苗被视为迄今开发最为成功的疫苗之一，可诱导强效抗病毒免疫应答，但其在感染细胞中触发免疫应答的信号传导机制尚未明确。本研究从分子层面阐明了代谢应激与先天免疫应答如何协同调控YFV-17D感染后I型干扰素（type I IFN）的表达。通过对比YFV-17D与其亲本病毒YFV-Asibi以及相关登革病毒的复制特征，研究发现干扰素的表达依赖于经MAVS介导的RIG-I样受体（RIG-I-like Receptor, RLR）信号通路，这与预期结果一致。但YFV-17D可特异性诱导线粒体呼吸作用及显著的代谢扰动，包括过度激活电子传递链以支撑ATP合酶的功能。线粒体过度激活会产生活性氧（reactive oxygen species, ROS），其中包括过氧亚硝酸盐；在非免疫细胞中阻断活性氧生成，可抑制MAVS的寡聚化及干扰素的表达，但不会降低YFV-17D的复制能力。在YFV-17D感染的人树突状细胞（human dendritic cells, DCs）中清除活性氧，可提升细胞存活率，但会全面阻断干扰素信号通路相关基因的表达。综上，YFV-17D为实现高效增殖而演化出适应性机制，诱导线粒体过度激活以满足能量需求，由此产生活性氧作为关键信使，将微弱的干扰素应答转化为足以支撑疫苗有效性的先天免疫最大化激活。本研究使用的人源单核细胞衍生树突状细胞（monocyte-derived dendritic cells, DCs）分为两组：模拟感染组与以感染复数（multiplicity of infection, MOI）为0.1的YFV-17D感染组。向各培养孔加入新鲜DC培养基至所需体积，并于感染后0小时和24小时，向对应培养孔中加入50 µM的MnTBAP或溶剂对照。于感染后48小时（hours post-infection, hpi），通过离心收集人源树突状细胞，使用1 mL TRIzol试剂（Thermo Fisher Scientific）裂解每一份细胞样品，将样品保存于-80℃直至RNA提取及批量RNA测序（bulk RNA-seq）。对感染组与模拟感染组的样品进行差异表达分析。