Mtb and HIV/SIV antigen peptide signatures as blood biomarkers to detect early infection to active disease in young children and NHP

摘要 每年有100万儿童患结核病;但目前的结核病诊断在儿童中表现不佳， 每年死于结核病相关原因的205，000名儿童中的绝大多数（96%）没有得到 治疗这些儿童通常表现出非特异性症状和少杆菌结核病，特别是那些 合并感染艾滋病毒，但未被诊断，然后可能进展为播散性或肺外结核 在没有适当治疗的情况下，病情会迅速恶化。在幼儿中， 大多数一线结核病诊断使用的痰液样本降低了准确诊断结核病的能力， 监测其对治疗的反应。因此，迫切需要非痰基诊断来解决这一问题 这是一个问题，但目前版本的这种测定要么表现出对活动性结核病的敏感性差，要么不能 区分活动性疾病和潜伏性结核感染或准确监测治疗反应。 本文报道了血清中结核分枝杆菌（Mtb）毒力因子的检测， 可诊断儿童所有形式的结核病（肺结核和肺外结核），包括少杆菌和艾滋病毒- 结核病相关病例。我们最近表明，多重检测血清中的HIV和Mtb衍生蛋白， 可以灵敏地诊断幼儿的艾滋病毒和结核病，同时监测他们对艾滋病毒的反应， 肺结核治疗。这对于那些死亡风险最高的人来说是一个关键问题; 在出生后的头两年，在哺乳期间暴露和/或感染这两种病原体。基于我们 在鉴定病原体特异性肽生物标志物和开发相应的诊断测定方面的成功， 我们建议开发一种多重HIV和TB检测试剂盒，用于改进HIV病毒载量的检测和监测 以及从早期感染到活动性结核病的所有阶段， 或艾滋病/结核病合并感染。我们建议通过追求三个相互关联的具体目标来实现这一目标 我们将：1）建立一种用于多重定量TB衍生因子相关血清水平的测定法， 随着结核病发展的每个阶段，从早期感染到婴儿非人灵长类结核病的活动性结核病， 该模型密切概括了艾滋病毒和结核病感染和合并感染的病理学; 2）开发预测性 该模型基于结核病阶段标志物、免疫反应和结核病病理之间的相关性 模型;以及3）对该多重测定进行多中心验证，以诊断HIV感染和潜伏性TB 感染和早期和活动性结核病在艾滋病毒暴露的儿科队列仔细注释的临床， 放射学和细菌学数据。采用HIV/TB共感染的非人灵长类动物模型， 定量蛋白质组学将使我们能够识别与症状发展相对应的结核病相关变化 以及与疾病进展相关的免疫变化，这些变化不能通过使用人类免疫系统检测到。 同伙在多个特征良好的儿科队列中对我们的检测方法进行综合评价， 这些生物标志物的快速转化为实践和我们的便携式设备为基础的测定的发展。

ABSTRACT One million children develop TB annually; but current TB diagnostics exhibit poor performance in children, and the vast majority (96%) of the 205,000 children who die of TB-related causes each year do not receive treatment. Such children often present with non-specific symptoms and paucibacillary TB – particularly those co-infected with HIV – and are not diagnosed, and may then progress to disseminated or extrapulmonary TB cases that can rapidly progress in the absence of appropriate treatment. In young children, difficulty obtaining sputum samples used by most front-line TB diagnostics reduces the ability to accurately diagnose TB and monitor its response to treatment. Thus, non-sputum-based diagnostics are urgently needed to address this problem, but current versions of such assays either demonstrate poor sensitivity for active TB or cannot differentiate active disease from latent TB infection or accurately monitor treatment responses. We have reported that detection of virulence factors secreted by Mycobacterial tuberculosis (Mtb) in serum can diagnose all forms of TB in children (pulmonary and extrapulmonary TB), including paucibacillary and HIV- associated TB cases. We have recently shown that multiplex detection of HIV- and Mtb-derived protiens in serum can sensitively diagnose HIV and TB in young children, and simultaneously monitor their response to HIV and TB treatment. This is a critical issue for those at highest risk for mortality; very young children who may be exposed and/or infected with both pathogens while breastfeeding, during the first two years of life. Based on our success in identifying pathogen-specific peptide biomarkers and developing corresponding diagnostic assays, we propose to develop a multiplex HIV and TB assay for improved detection and monitoring of HIV viral load and all stages of TB, from early infection to active TB disease, in young children suspected HIV and TB infections or HIV/TB co-infections. We propose to achieve this goal through the pursuit of three interlinked specific aims where we will: 1) establish an assay for multiplex quantification of serum levels of TB-derived factors associated with each stage of TB development, from early infection to active TB disease in an infant non-human primate TB model that closely recapitulates the pathology of HIV and TB infection and co-infection; 2) develop a predictive model based on correlations between TB stage markers, immune responses and TB pathology in this disease model; and 3) conduct a multi-center validation of this multiplex assay to diagnose HIV infection and latent TB infections and early and active TB disease in HIV-exposed pediatric cohorts with carefully annotated clinical, radiological and bacteriological data. Employing a non-human primate model of HIV/TB co-infection and quantitative proteomics will allow us to identify TB-associated changes corresponding to symptom development and immune changes associated with disease progression that could not feasibly be detected using human cohorts. The comprehensive evaluation of our assay in multiple well-characterized pediatric cohorts will facillitate the rapid translation of these biomarkers into practice and the development of our portable device-based assay.