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100万儿童患上结核病；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20.5万名儿童中的绝大多数(96%)没有收到 治疗。这类儿童通常表现出非特异性症状和少杆菌结核病--尤其是那些 合并感染艾滋病毒--未被诊断，然后可能发展为播散性或肺外结核病 在缺乏适当治疗的情况下可以迅速进展的病例。在幼儿中，很难获得 大多数一线结核病诊断使用的痰样本降低了准确诊断结核病和 监测其对治疗的反应。因此，迫切需要非基于痰的诊断来解决这一问题 有问题，但当前版本的此类检测要么对活动性结核病的敏感度很低，要么不能 区分活动性疾病和潜伏性结核病感染或准确监测治疗反应。 我们已经报道了检测血清中结核分枝杆菌(Mtb)分泌的毒力因子 可以诊断所有形式的儿童结核病(肺结核病和肺外结核病)，包括少杆菌和艾滋病毒- 相关的结核病病例。我们最近发现，血清中HIV和结核分枝杆菌衍生蛋白的多重检测 可以敏感地诊断幼儿中的艾滋病毒和结核病，同时监测他们对艾滋病毒和结核病的反应 结核病治疗。对于那些死亡风险最高的人来说，这是一个关键问题；非常年幼的儿童可能 在生命的头两年，在母乳喂养期间接触和/或感染这两种病原体。基于我们的 成功识别病原体特异性多肽生物标记物并开发相应的诊断分析方法， 我们建议开发一种艾滋病毒和结核病的多重检测方法，以改进对艾滋病毒病毒载量的检测和监测 以及所有阶段的结核病，从早期感染到活动性结核病，在疑似感染艾滋病毒和结核病的幼儿中 或艾滋病毒/结核病混合感染。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)建立一种多重量化血清结核病相关因子水平的检测方法 随着结核病发展的每个阶段，从早期感染到婴儿非人类灵长类结核病的活动性结核病 紧密概括艾滋病毒和结核病感染及混合感染的病理的模型；2)开发预测 基于结核病分期标记物、免疫反应和结核病病理相关性的模型 模型；以及3)对这种多重检测方法进行多中心验证，以诊断HIV感染和潜伏结核病 艾滋病毒暴露的儿童队列中的感染和早期活动性结核病与仔细注释的临床 放射学和细菌学数据。采用HIV/TB混合感染的非人类灵长类动物模型 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，这些变化无法用人类检测到 一群人。在多个特征良好的儿科队列中对我们的分析进行综合评估将是可行的 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分析的发展。

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.