A Novel Proteomic Approach to TB Biomarker Discovery using Human Exosomes

描述（由申请人提供）：活动性结核病（TB）的标准治疗方案已有40年的历史，但效果不理想，因为它需要每天坚持服药50年。6个月，在症状消失很久之后。现在，几十年来第一次，结核病药物管道中充满了新的、高效的和可能缩短治疗时间的药物，接近为2/3期临床试验做好准备。不幸的是，评估这些药物面临重大挑战，因为样本量大（约2500例患者）和随访时间长（约30个月），需要证明它们在预防治疗失败和复发方面与标准方案相比具有非劣效性。虽然目前使用两个月分枝杆菌培养转化作为替代标记，但它有许多缺点，例如需要从不再咳嗽的患者收集痰，将其运送到专门的参考实验室，并等待数周才能得到统计能力差的二分类结果。本提案的目的是利用针对人类外泌体的蛋白质组学方法开发一种定量的、基于血液的结核病生物标志物测定方法。外泌体是宿主细胞在活动性感染期间释放的囊状纳米颗粒。外泌体含有高浓度的结核分枝杆菌（Mtb）蛋白，这与它们在免疫信号传导中的假设作用一致，似乎随着感染小鼠体内结核分枝杆菌的清除而减少。因此，我们假设外泌体蛋白可以作为合适的生物标志物，用于预测临床试验中随机分配到不同药物方案的人群以及标准方案的个体对结核病治疗的微生物反应。一种非痰液依赖、非生长为基础的生物标志物检测方法可以应用于上述两种情况中的一种或两种情况的早期治疗，从而改变结核病药物开发和全球结核病控制的速度和范围。该检测也可用于监测儿童结核病、肺外结核和HIV-TB合并感染中常见的少杆菌病的治疗。为了实现这一目标，我们组建了一支由结核病研究人员组成的多学科团队，他们具有外泌体生物学、蛋白质组学、诊断、新型预测方法和临床试验方面的专业知识。我们将以我的K23导师的nih资助的肺炎队列研究的现有基础设施为基础，收集患者的连续血清标本，随访到培养证实的肺结核治疗的起始阶段。我们将利用这些数据和样本，1)优化处理血清外泌体用于结核分枝杆菌生物标志物检测的方法；2)评估血清学、基于外泌体的mtb生物标志物组对治疗反应的动力学，并确定评估该反应的最佳采样框架。在完成这些原理证明研究后，我们期望有初步的数据来证明更大规模的临床研究，以确定准确性

DESCRIPTION (provided by applicant): The standard treatment regimen for active tuberculosis (TB) is >40 years old and has suboptimal effectiveness, because it requires daily drug adherence for ?6 months, long after symptoms have resolved. Now, for the first time in decades, the TB drug pipeline is full of new, highly potent and possibly treatment- shortening agents nearing readiness for phase 2/3 clinical trials. Unfortunately, evaluating these drugs poses major challenges, because of the large sample sizes (~2500 patients) and long follow-up periods (~30 months) required to show their non-inferiority to the standard regimen in preventing treatment failure and relapse. Although two-month mycobacterial culture conversion is currently used as a surrogate marker, it has numerous disadvantages, such as the need to collect sputum from patients who are no longer coughing, transport it to specialized reference laboratories, and wait weeks for a dichotomous result which has poor statistical power. The objective of this proposal is to develop a quantitative, blood-based, TB biomarker assay using a proteomic approach that targets human exosomes. Exosomes are vesicular nanoparticles released by host cells during active infection. Consistent with their postulated role in immune signaling, exosomes contain high concentrations of M. tuberculosis (Mtb) proteins, which seem to decrease with clearance of Mtb from infected mice. Thus, we hypothesize that exosome proteins can serve as suitable biomarkers for predicting microbiologic response to TB treatment among groups randomized to different drug regimens in clinical trials, as well as among individuals on standard regimens. A non-sputum-dependent, non-growth-based biomarker assay that could be applied early in treatment in either or both of these scenarios could transform the pace and scope of TB drug development and global TB control. This assay would also be useful for monitoring treatment of paucibacillary disease as is common in childhood TB, extrapulmonary TB, and HIV-TB co-infection. To achieve this goal, we have assembled a multidisciplinary team of TB researchers with expertise in exosome biology, proteomics, diagnostics, novel prediction methods, and clinical trials. We will build on the existing infrastructure of my K23 mentor's NIH-funded cohort study of pneumonia by collecting serial serum specimens from patients followed through the initiation phase of treatment for culture-confirmed pulmonary TB. We will use these data and samples to 1) optimize methods for processing serum exosomes for Mtb- biomarker assays; 2) evaluate the kinetics of a serologic, exosome-based Mtb-biomarker panel in response to treatment, and determine the optimal sampling frame for assessing that response. Upon completing these proof-of-principle studies, we expect to have the preliminary data to justify larger clinical studies to determine the accuracy of the marker in predicting treatment response in test and validation cohorts; to further develop and validate a clinical testing platform for these markers; and ultimately to evaluate the resultin refined assay as a surrogate marker in Phase 3 clinical trial banks currently under development.