Deciphering Early Stages of Polyomavirus CNS Pathogenesis and Immunity

摘要 JC多瘤病毒(JCPyV)是一种普遍存在的人类病原体，在中国引起几种毁灭性的脑部疾病 免疫受损的个体。这些与JCPyV相关的中枢神经系统疾病中最值得注意的是 常常是致命的脱髓鞘脑病，进行性多灶性白质脑病(PML)。PML，一个 在CART之前的时代，艾滋病定义的病变已经成为威胁患者生命的并发症 自身免疫性和炎症性疾病的免疫调节剂和某些疾病的治疗 恶性血液病。在迅速扩大的与PML相关的生物制品清单中，Natalizumab (Tysabri®)发病率最高，对于以下多发性硬化症(MS)患者来说是不祥的后遗症 否则将受益于使用这种免疫调节剂大幅减少复发。戒毒， PML的唯一治疗选择往往是高死亡率的脑炎性反应。不是 有抗JCPyV的试剂可用。多瘤病毒具有物种特异性。缺乏可驯化的动物模型 多瘤病毒诱导的中枢神经系统疾病是阐明PML发病机制的公认瓶颈 控制JCPyV的免疫学机制，抑制多瘤病毒药物的体内评价 组织培养中的复制，并发现不可逆的JCPyV相关的早期事件 神经病理学。利用小鼠多瘤病毒(MuPyV)，我们建立了一种自然的病毒宿主模型 多瘤病毒相关的中枢神经系统疾病。在此R35应用程序中，我们计划利用我们最近三个关键字 发现：(1)将JCPyV-PML VP1衣壳蛋白突变映射到MuPyV的VP1使人能够逃脱病毒- 中和抗体(NAB)在保持中枢神经系统趋向性的同时；(2)高亲和力抗MuPyV产生的IL-21 大脑中的CD4T细胞是形成和维持MuPyV特异性大脑驻留记忆所必需的 CD8 T细胞(BTRM)；和(3)依赖STAT1的先天免疫限制室管膜的感染，a 脑实质感染的关键屏障。这些发现为以下三个关键问题奠定了基础 这里要说明的是：(1)室管膜是多瘤病毒侵袭大脑的舞台吗？ 实质？；(2)CD8 bTRM对持续感染反应的完整性取决于亚群吗？ 异质性？；以及(3)T细胞缺陷是否为NAB逃逸病毒变种的产生打开了大门？这个 拟议的研究将利用下一代测序的尖端进展来发现罕见的VP1 体内突变，定制冷冻EM图像重建方法来确定内源性VP1 NAB表位 和NAB逃逸机制，以及完整小鼠大脑的高分辨率3D成像以可视化病毒CNS 进入并扩散。这些研究的发现将回答关于先天和适应性的基本问题 多瘤病毒中枢神经系统感染的免疫控制和病毒传播的条件 在发展成不可逆转的神经病理之前，外周组织进入大脑。

ABSTRACT JC polyomavirus (JCPyV), a ubiquitous human pathogen, causes several devastating brain diseases in immune compromised individuals. The most notable of these JCPyV-associated CNS diseases is the frequently fatal demyelinating brain disease progressive multifocal leukoencephalopathy (PML). PML, an AIDS-defining lesion in the pre-cART epoch, has emerged as a life-threatening complication in patients receiving immunomodulatory agents for autoimmune and inflammatory disorders and treatment for certain hematological malignancies. Among the rapidly expanding list of PML-associated biologics, natalizumab (Tysabri®) has the highest incidence and is an ominous sequela for multiple sclerosis (MS) patients who otherwise benefit from dramatic reductions in relapses using this immunomodulatory agent. Drug withdrawal, the only therapeutic option for PML, is often complicated by a high-mortality cerebral inflammatory reaction. No anti-JCPyV agents are available. Polyomaviruses are species-specific. Lack of a tractable animal model of polyomavirus-induced CNS disease is an acknowledged bottleneck to elucidating PML pathogenesis, the immunological mechanisms that control JCPyV, in vivo evaluation of agents that inhibit polyomavirus replication in tissue culture, and uncovering early events that presage irreversible JCPyV-associated neuropathology. Using mouse polyomavirus (MuPyV), we developed a natural virus-host model of polyomavirus-associated CNS disease. In this R35 application, we plan to leverage our three recent key findings: (1) Mapping JCPyV-PML VP1 capsid protein mutations to MuPyV’s VP1 confers escape from virus- neutralizing antibodies (nAb) while preserving CNS tropism; (2) IL-21 produced by high-affinity anti-MuPyV CD4 T cells in the brain is required for formation and maintenance of MuPyV-specific brain resident-memory CD8 T cells (bTRM); and (3) STAT1-dependent innate immunity limits infection of the ventricular ependyma, a critical barrier to infection of the brain parenchyma. These findings lay the foundation for three key questions to be addressed here: (1) Is the ependyma the staging ground for polyomavirus invasion of the brain parenchyma?; (2) Does the integrity of the CD8 bTRM response to persistent infection depend on subset heterogeneity?; and (3) Does T cell deficiency open the door for outgrowth of nAb-escape virus variants? The proposed studies will make use of cutting edge advances in next-generation sequencing to uncover rare VP1 mutations in vivo, custom cryo EM image reconstruction approaches to define endogenous VP1 nAb epitopes and nAb escape mechanisms, and high-resolution 3D imaging of intact mouse brains to visualize virus CNS entry and spread. Findings from these studies will answer fundamental questions about innate and adaptive immune control of polyomavirus CNS infection and conditions underlying dissemination of virus from the periphery into the brain before development of irreversible neuropathology.