Antiviral treatment of BK polyomavirus reactivation

项目摘要 BK多瘤病毒（BKPyV）相关性肾病（PVAN）是肾移植的主要原因 损失（~10%/年）。BKPyV在儿童时期感染大约80 - 90%的人，并通过以下方式进行控制： 宿主的免疫系统然而，在移植患者中，BKPyV再激活引起PVAN， 抑制他们的免疫系统。目前的治疗方法是减少免疫抑制， 增加了移植物排斥反应的风险，但未经治疗的PVAN将导致同种异体移植物丢失。患者通常 监测尿液和血浆中的BKPyV，提供早期使用抗病毒药物治疗的机会， 在需要减少免疫抑制之前。根据我们的机制研究，我们已经确定 BKPyV在原代肾细胞中产生所需的两种宿主途径，当被现有的 降低病毒滴度 我们发现，硼替佐米，一种蛋白酶体抑制剂，有效地降低病毒滴度3个对数， 低剂量且不影响细胞健康。我们还将测试新一代蛋白酶体抑制剂， 毒性较低和/或可口服，卡非佐米和ixazomib。 考虑到蛋白酶体在感染早期是必需的，我们试图抑制第二个宿主 靶点可以在感染后期减少病毒产生，或者可以潜在地消除病毒感染的细胞。 BKPyV需要激活DNA损伤反应（DDR）以使细胞周期停滞，从而延长S期， 病毒复制和装配。抑制DDR激活激酶（ATM和ATR），迫使BKPyV 感染但非模拟感染的细胞退出S期，病毒滴度降低。 我们的研究揭示了两个有效的靶点，蛋白酶体和DDR，这两个靶点都是已知的。 抑制剂，一种已经被FDA批准，另一种正在临床试验中（两者都被用于其他疾病）。 适应症）。拟议的目标将确定这些抑制剂的影响，以及它们的组合， 病毒滴度、细胞活力和细胞周期调节，使用临床病毒分离株感染原发性肾近端 肾小管上皮细胞，是人类感染的天然细胞类型。我们会先给病毒排序 并在抑制研究后确定病毒基因组是否有变化。这个概念验证数据 为了在临床试验（超出本研究范围）中将抑制剂作为抗病毒药物进行检测， 可以防止由BKPyV再激活引起的移植物损失。我们迫切需要抗病毒药物， 在免疫抑制之前或与免疫抑制一起预防或治疗BKPyV再活化。

Project Summary BK polyomavirus (BKPyV) associated nephropathy (PVAN) is the leading cause of kidney transplant loss (~10%/year). BKPyV infects approximately 80-90% of people during childhood and is kept in check by the host immune system. However, in transplant patients BKPyV reactivation causes PVAN due to suppression of their immune system. The current treatment is reduction of immunosuppression, which increases the risk of graft rejection, but untreated PVAN will result in allograft loss. Patients are routinely monitored for BKPyV in the urine and the plasma, providing an opportunity to treat early with antivirals, before decreasing immunosuppression is required. Based on our mechanistic studies, we have identified two host pathways required for BKPyV production in primary kidney cells that, when inhibited by existing drugs, reduce viral titers. We discovered that bortezomib, a proteasome inhibitor, potently decreases viral titers by 3 logs, at low doses and without affecting cell fitness. We will also test newer generation proteasome inhibitors that are less toxic and/or orally available, carfilzomib and ixazomib. Given that the proteasome is required early during infection, we sought to inhibit a second host target that would reduce viral production late in infection, or could potentially eliminate virally infected cells. BKPyV requires activation of the DNA damage response (DDR) for cell cycle arrest, to prolong S phase for viral replication and assembly. Inhibiting the DDR activating kinases (ATM and ATR), forces BKPyV infected, but not mock infected, cells exit S phase, and viral titers are reduced. Our studies revealed two potent targets, the proteasome and the DDR, which both have known inhibitors, one is already FDA approved, and the other is in clinical trials (both are being used for other indications). The proposed aims will determine the effects of these inhibitors, and combinations of them, on viral titers, cell viability and cell cycle regulation, using clinical viral isolates to infect primary renal proximal tubular epithelial cells, which are the natural cell type infected in humans. We will sequence the virus before and after inhibition studies to determine if there are changes to the viral genome. This proof-of-concept data is required in order to test the inhibitor(s) in clinical trials (beyond the scope of this study) as antivirals that can protect against graft loss caused by BKPyV reactivation. There is an urgent need for antivirals that can either prevent or treat BKPyV reactivation prior to or in conjunction with immunosuppression.