Multiplexed quantification of circulating peptidomic signatures for EBOLA early diagnosis

用于埃博拉早期诊断的循环肽组特征的多重定量

• 批准号: 9387209
• 负责人: Tony Y. Hu
• 金额: $ 17.41万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-07 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9387209
• 关键词: Address; Africa; Amplifiers; Animals; Antibodies; Area; Biological Assay; Biological Markers; Calibration; Caring; Categories; Centers for Disease Control and Prevention (U.S.); Characteristics; Cleaved cell; Cohort Studies; Communicable Diseases; Containment; Custom; Data; Democratic Republic of the Congo; Detection; Diagnosis; Diagnostic; Diagnostic tests; Digestion; Disease; Disease Outbreaks; Early Diagnosis; Ebola Hemorrhagic Fever; Ebola virus; Emergency Situation; Endemic Diseases; Epidemic; Equipment; Event; Exhibits; Filoviridae; Filovirus; Formic Acids; Frankfurt-Marburg Syndrome Virus; Future; Hour; Human; Human Resources; Hydrolysis; Immune response; Individual; Infection; Infectious Agent; International; Laboratories; Lassa Fever; MALDI-TOF Mass Spectrometry; Malaria; Mass Spectrum Analysis; Mediating; Medical Research; Meningitis; Methodology; Methods; Modeling; Molecular; Morbidity - disease rate; Nanostructures; Nanotechnology; Peptides; Physicians; Pilot Projects; Plasma; Pre-Clinical Model; Preparation; Primates; Procedures; Proteins; Protocols documentation; RNA; Recombinant Proteins; Recombinants; Recovery; Reproducibility; Research Institute; Resolution; Reston; Safety; Sampling; Sensitivity and Specificity; Serum; Serum Proteins; Signal Transduction; Silicon; Site; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sudan; Surface; Symptoms; System; Testing; Time; Typhoid Fever; United States Food and Drug Administration; United States National Institutes of Health; Variant; Viral; Viral Antigens; Viral Hemorrhagic Fevers; Viral load measurement; Virion; Virus Diseases; antibody conjugate; base; clinical application; diagnostic assay; disease diagnosis; effective therapy; forest; hospital laboratories; improved; instrument; ionization; microwave electromagnetic radiation; miniaturize; mortality; multiplex detection; nanodisk; nanostructured; new technology; nonhuman primate; novel; pathogen; point of care; pre-clinical; protein complex; rapid diagnosis; stable isotope; viral RNA; viral detection

ABSTRACT Ebola virus (EBOV) is classified as a filamentous, enveloped, non-segmented negative-sense RNA carrying Category A Priority Pathogen by the NIH and CDC. Infections with EBOV cause severe hemorrhagic fever and high mortality in humans and non-human primates (NHPs). During the Ebola outbreak of late 2014 in West Africa, the lack of laboratory tests for early Ebola-virus-disease (EVD) detection (preferably before the onset of symptoms) and lack of effective treatments limited the containment and management of the eventual epidemic. To address this critical need for early EVD diagnosis, we have recently customized a platform we developed to enhance biomarker detection to allow sensitive and specific EBOV biomarker quantification in serum samples. Our method uses antibody-modified porous silicon nanodisks (pSiNDs) and high-throughput, high-resolution mass spectrometry (MS) – together referred to as pSiND-MS – to greatly enhance the specificity and sensitivity of target peptide detection from complex protein samples. In our customized EVD assay, these pSiNDs are functionalized with custom antibodies to specific peptides of the EBOV matrix protein VP40 in order to efficiently capture these peptides from non-enzymatically-digested serum samples of EBOV-infected hosts. Notably, the starting non-enzymatic digestion step of this assay is used to both inactivate live EBOV particles and to site- specifically digest all serum proteins for subsequent pSiND-MS analysis. We have observed that VP40 peptides are increased in the plasma of infected animals even before EBOV RNA is detectable with currently available diagnostic assays. We have identified EBOV- and EBOV-species-specific VP40 peptides, and hypothesize that multiplex detection of our two selected VP40 peptides will robustly indicate EVD, EBOV species and EBOV load in potentially-infected individuals. We will thus apply pSiND-MS to quantify low-abundance VP40 peptides for early diagnosis of EBOV using a rapid, single-test assay, where we propose to: 1) optimize the pSiND-MS platform for detection of these biomarkers and 2) apply pSiND-MS to longitudinally quantify VP40 levels in sera from EBOV-infected non-human primates at the U.S. Army Medical Research Institute of Infectious Diseases, to produce an informative preclinical EBOV-detection profile. Major advantages of our approach include the simplicity and safety of sample preparation; the use of matrix- assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS equipment found in most hospitals and laboratories (and future use of simplified, miniaturized MS systems); the potential to read the assay on MS/MS instruments for marker sequence confirmation; the specificity and sensitivity of biomarker detection; the use of rapidly-customizable, modular multiplex assay; and rapid diagnosis (2hrs from sample). These characteristics are ideal for a point-of-need application. Successful completion of this proposal will provide a novel technology that can overcome significant hurdles of current diagnostic methodologies for Ebola and other infectious agents, potentially transforming the protocols for care, treatment, and containment of future epidemics.

摘要 埃博拉病毒(Ebola Virus，EBOV)是一种丝状、囊膜、非节段性负义RNA载体 美国国立卫生研究院和美国疾病控制与预防中心的A类优先病原体。感染EBOV会导致严重的出血热和 人类和非人类灵长类动物(NHP)的高死亡率。在2014年底西非爆发埃博拉疫情期间， 缺乏早期埃博拉病毒病(EVD)检测的实验室检测(最好是在 病症)和缺乏有效的治疗方法限制了对最终疫情的控制和管理。 为了满足对早期EVD诊断的这一迫切需求，我们最近定制了一个我们开发的平台 增强生物标记物检测，以便在血清样本中进行敏感和特异的EBOV生物标记物定量。 我们的方法使用抗体修饰的多孔硅纳米盘(PSiND)和高通量、高分辨率 质谱学(MS)-统称为pSiND-MS-极大地提高了特异性和灵敏度 从复杂蛋白质样品中检测目标多肽的方法。在我们定制的EVD检测中，这些pSiND是 使用针对EBOV基质蛋白VP40的特定多肽的定制抗体来功能化，以便有效地 从EBOV感染宿主的非酶消化血清样本中捕获这些多肽。值得注意的是， 本试验的非酶消化步骤用于灭活活的EBOV颗粒和定位EBOV颗粒。 为后续的pSiND-MS分析专门消化所有血清蛋白。我们观察到VP40多肽 甚至在目前可用的EBOV RNA检测到之前，感染动物的血浆中的EBOV就增加了 诊断化验。我们已经确定了EBOV和EBOV物种特异性的VP40多肽，并假设 我们选择的两个VP40多肽的多重检测将有力地指示EVD、EBOV种类和EBOV载量 在可能感染的个体中。因此，我们将应用pSiND-MS来量化低丰度的VP40多肽 使用快速、单次检测方法进行EBOV早期诊断，我们建议：1)优化pSiND-MS 这些生物标志物的检测平台和2)应用pSiND-MS纵向定量血清中的VP40水平 从美国陆军传染病医学研究所感染EBOV的非人类灵长类动物，到 制作一份信息丰富的临床前EBOV检测档案。 我们方法的主要优点包括样品制备的简单性和安全性；使用基质- 辅助激光解吸/电离飞行时间(MALDI-TOF)MS设备在大多数医院和 实验室(以及简化、小型化MS系统的未来使用)；在MS/MS上读取化验结果的可能性 标记序列确认的仪器；生物标记检测的特异性和敏感性； 可快速定制的模块化多重分析；以及快速诊断(从样本开始2小时)。这些特点 是按需应用程序的理想选择。这项提案的成功完成将提供一项新的技术 这可以克服目前埃博拉和其他感染性病原体诊断方法的重大障碍， 有可能改变护理、治疗和遏制未来流行病的方案。