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 亏损(约10%/年)。BKPyV在儿童时期感染了大约80%-90%的人，并受到 宿主免疫系统。然而，在移植患者中，BKPyV重新激活会导致PVAN，原因是 抑制他们的免疫系统。目前的治疗方法是减少免疫抑制，这 增加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。病人通常都是 监测尿液和血浆中的BKPyV，为早期使用抗病毒药物治疗提供了机会， 在减少之前，需要免疫抑制。基于我们的机理研究，我们已经确定 原代肾细胞产生BKPyV所需的两条宿主途径，当被现有的 药物，降低病毒滴度。 我们发现，蛋白酶体抑制剂Bortezomib有效地将病毒滴度降低了3logs， 低剂量，不影响细胞健康。我们还将测试新一代蛋白酶体抑制剂 毒性较小和/或口服用药，卡菲佐米和咪唑米。 考虑到感染早期需要蛋白酶体，我们试图抑制第二个宿主 目标是减少感染后期的病毒产量，或者可能消灭受病毒感染的细胞。 BKPyV需要激活DNA损伤反应(DDR)才能使细胞周期停止，以延长S期 病毒复制和组装。抑制DDR激活的激酶(ATM和ATR)，迫使BKPyV 感染，而不是模拟感染，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标，蛋白酶体和DDR，它们都已经知道 抑制剂，一种已经得到FDA批准，另一种正在临床试验中(这两种药物都用于其他药物 适应症)。拟议的目标将决定这些抑制剂及其组合对 使用临床分离的病毒株感染原代肾近端的病毒滴度、细胞活力和细胞周期调节 肾小管上皮细胞，这是人类感染的自然细胞类型。我们会在之前对病毒进行排序 并在抑制研究后确定病毒基因组是否有变化。此概念验证数据 为了在临床试验中(超出本研究的范围)将抑制剂(S)作为抗病毒药物进行测试 可保护移植物免受BKPyV重新激活引起的移植物丢失。迫切需要一种能够 在免疫抑制之前或与免疫抑制联合预防或治疗BKPyV重新激活。

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.