A Mouse Model to Define Immunovirologic Determinants of Polyomavirus CNS Disease

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性脑病，是纳塔利珠单抗治疗复发性多发性硬化症的一种危及生命的并发症。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感染的高流行率和患者在开始natalizumab之前接受的免疫抑制方案的异质性，它们的效用有限。没有抗jcv药物可用。阐明PML的发病机制和控制JCV复制的免疫监测机制是充分评估接受natalizumab治疗的MS患者PML风险的必要条件。多瘤病毒具有高度的物种特异性，人类是JCV的唯一宿主宿主。迫切需要一种易于处理的动物模型来了解jcv诱导PML的免疫学和病毒学决定因素，并为评估新型抗jcv化合物的体内疗效提供临床前平台。利用小鼠多瘤病毒（MPyV）建立了小鼠多瘤病毒诱导的中枢神经系统疾病模型。我们建议应用这种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细胞穿过血脑屏障外渗进入CNS实质，natalizumab阻止维持抗病毒免疫监视所需的脑驻留病毒特异性CD8 T细胞；(2)由于PML患者的JCV在其衣壳蛋白（体液介导的免疫防御的主要靶点）中含有新的突变，病毒中和抗体选择获得神经亲和性的病毒变体。在Aim 1中，我们构建了MHC I类和II类四聚体，通过流式细胞术可视化MPyV特异性CD8和CD4 T细胞，并开发了MPyV特异性TCR转基因小鼠来监测抗MPyV CD8 T细胞在中枢神经系统中的命运。对于Aim 2，我们将通过基于pcr的随机诱变建立一个衣壳突变MPyV文库，并通过将体内迭代病毒传代与下一代测序相结合，确定中和的MPyV抗体是否选择了赋予神经毒力的突变。我们还将尝试创建一种杂交病毒，表达JCV的衣壳蛋白（赋予JCV宿主细胞特异性），并具有MPyV的非编码控制元件、复制起源和非结构蛋白（使小鼠能够复制）。这种新型杂交病毒将使我们能够确定JCV-PML变异病毒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.