A Mouse Model to Define Immunovirologic Determinants of Polyomavirus CNS Disease

描述（由申请方提供）：进行性多灶性白质脑病（PML）是一种由人JC多瘤病毒引起的脱髓鞘脑病，是那他珠单抗治疗复发性多发性硬化症的危及生命的并发症。JCV血清阳性、既往免疫抑制和natalizumab治疗> 24个月是已知增加PML风险的唯一确定因素，但由于JCV感染的高患病率和患者在开始natalizumab治疗前接受的免疫抑制方案的异质性，其效用有限。无抗JCV药物可用。需要阐明PML的发病机制和控制JCV复制的免疫监视机制，以充分评估接受那他珠单抗治疗的MS个体的PML风险。多瘤病毒具有高度的物种特异性，人类是JCV的唯一宿主。迫切需要一种易于处理的动物模型来了解JCV诱导的PML的免疫学和病毒学决定因素，并提供一个临床前平台来评估新型抗JCV化合物的体内疗效。使用小鼠多瘤病毒（MPyV），我们已经开发了多瘤病毒诱导的CNS疾病的小鼠模型。我们提出应用该MPyV-CNS感染模型来解决基于以下假设的两个目的：（1）通过阻断T细胞外渗穿过血脑屏障进入CNS实质，那他珠单抗防止维持抗病毒免疫监视所需的脑驻留病毒特异性CD 8 T细胞;和（2）由于PML患者的JCV在其衣壳蛋白中具有新的突变，而衣壳蛋白是体液介导的免疫防御的主要靶点，病毒中和抗体选择获得向神经性的病毒变体。对于目标1，我们构建了MHC I类和II类四聚体以通过流式细胞术使MPyV特异性CD 8和CD 4 T细胞可视化，并开发了MPyV特异性TCR转基因小鼠以监测CNS中抗MPyV CD 8 T细胞的命运。对于目标2，我们将通过基于PCR的随机诱变创建衣壳突变体MPyV的文库，并通过将迭代病毒体内传代与下一代测序相结合，确定中和MPyV抗体是否选择赋予神经毒力的突变。我们还将尝试创建表达JCV的衣壳蛋白（以赋予JCV宿主细胞特异性）并具有MPyV的非编码控制元件、复制起点和非结构蛋白（以使其能够在小鼠中复制）的杂交病毒。这种新的杂交病毒将使我们能够确定JCV-PML变异病毒中衣壳突变的功能意义。

DESCRIPTION (provided by applicant): Progressive Multifocal Leukoencephalopathy (PML), a demyelinating encephalopathy caused by the human JC polyomavirus, is a life-threatening complication of natalizumab therapy for relapsing multiple sclerosis. Seropositivity for JCV, prior immunosuppression, and > 24 month of natalizumab treatment are the only identified factors known to increase risk for PML, but their utility is limited because of the high prevalence of JCV infection and the heterogeneity of immunosuppressive regimens patients receive before starting natalizumab. No anti-JCV agents are available. Elucidation of the pathogenesis of PML and immunosurveillance mechanisms that keep JCV replication in check are required to adequately assess risk for PML in MS individuals receiving natalizumab. Polyomaviruses are highly species specific, with humans being the only host reservoir for JCV. A tractable animal model is urgently needed to understand the immunologic and virologic determinants of JCV-induced PML, and to provide a preclinical platform to assess the in vivo efficacy of novel anti-JCV compounds. Using mouse polyomavirus (MPyV), we have developed a mouse model of polyomavirus-induced CNS disease. We propose applying this MPyV-CNS infection model to address two Aims based on the following hypotheses: (1) by blocking T cell extravasation across the blood-brain barrier into the CNS parenchyma, natalizumab prevents maintenance of brain-resident virus-specific CD8 T cells required for antiviral immunosurveillance; and (2) because JCV in PML patients harbor novel mutations in their capsid protein, the dominant target for humoral-mediated immune defense, virus-neutralizing antibodies select viral variants that acquire neurotropism. For Aim 1, we have constructed MHC class I and class II tetramers to visualize MPyV-specific CD8 and CD4 T cells by flow cytometry, and have developed an MPyV- specific TCR transgenic mouse to monitor the fate of anti-MPyV CD8 T cells in the CNS. For Aim 2, we will create a library of capsid mutant MPyVs by PCR-based random mutagenesis, and by combining iterative virus passaging in vivo with next-generation sequencing, determine whether neutralizing MPyV antibodies select mutations that confer neurovirulence. We will also attempt to create a hybrid virus expressing the capsid proteins of JCV (to confer JCV host cell specificity) and having the noncoding control elements, origin of replication, and nonstructural proteins of MPyV (to enable replication in the mouse). This novel hybrid virus will allow us to determine the functional significance of the capsid mutations in JCV-PML variant viruses.