Proteome-wide screen for M. tuberculosis antigens

描述(由申请人提供)：目前诊断结核病(TB)的方法并不理想。结核菌素皮肤试验的敏感性和特异性较低。较新的干扰素-伽马释放分析更具特异性，但在某些情况下缺乏敏感性，价格昂贵，需要相对复杂的实验室。由于几个原因，活动性结核病的微生物学诊断在许多患者中是困难的。在一些人，例如儿童和艾滋病毒-结核病混合感染者中，较低的生物体负担往往限制了痰涂片显微镜用于诊断的用途。培养和基于核酸的诊断技术也受到这种低生物负担以及样本处理和运送到中央实验室的困难的限制，特别是在资源有限的情况下。具有重叠表现的疾病使结核病的临床诊断不可靠。血清学试验用于诊断许多传染病，具有灵敏度高、特异度高、方法学成熟、适应护理点试验形式等优点。然而，使用一种或几种抗原的基于抗体的结核病检测受到灵敏度不足的限制。新的血清学检测纳入了在感染过程中特异表达的抗原，可能会提高敏感性和特异性。体内表达的抗原也有可能识别疾病阶段特异性抗体反应。除了它们开发新诊断的潜力外，识别疾病阶段特定的蛋白表达可能有助于深入了解结核病感染和疾病发病机制。这项研究的总体目标是在蛋白质组范围内寻找由人类体液免疫反应识别的结核分枝杆菌(Mtb)蛋白抗原。在这个项目的第一个目标中，我们将为大约3,000个Mtb蛋白质合成核酸可编程蛋白质阵列(NAPPA)。在第二个目标中，这些阵列将用从结核杆菌感染兔的精心定义的结核病感染阶段获得的血清进行探测，结核杆菌感染兔是人类结核病的优秀模型，包括潜伏性结核病和免疫抑制后的重新激活。为实现这一目标，还将对有充分记录的结核病患者的库人血清进行筛查。这些研究将确定要在后续研究中分析的候选抗原，以筛选结核病患者广泛识别的抗原，以及在结核病感染的特定阶段引发抗体反应的抗原。这些数据可能导致改进对活动性和潜伏性结核病的诊断测试，并为潜伏性和活动性结核病的发病机制提供新的见解。公共卫生相关性：结核病仍然是疾病和死亡的主要原因，特别是在生活在资源有限的环境中的人和艾滋病毒感染者中。这项拟议的研究旨在为诊断结核病的新方法提供基础。更早、更简单、更敏感和更具体的结核病诊断可能会对美国和世界各地结核病患者的识别和治疗产生重大影响。

DESCRIPTION (provided by applicant): Current approaches to the diagnosis of tuberculosis (TB) are not optimal. The tuberculin skin test has low sensitivity and specificity. Newer interferon-gamma release assays are more specific but lack sensitivity in some settings, are expensive, and require a relatively sophisticated laboratory. The microbiologic diagnosis of active TB disease is difficult in many patients for several reasons. In some, e.g. children and persons with HIV-TB co-infection, the low organism burden often limits the utility of sputum smear microscopy for diagnosis. Culture and nucleic acid-based diagnostic techniques are also limited by this low organism burden, as well as by the difficulties of sample handling and transport to central laboratories, especially in resource-limited settings. Illnesses with overlapping manifestations make clinical diagnosis of TB unreliable. Serologic tests are used to diagnose many infectious diseases, and have the advantages of high sensitivity and specificity, established methodologies, and adaptability to point of care test formats. Antibody-based tests for TB, however, using one or a few antigens, have been limited by inadequate sensitivity. New serologic tests that incorporate antigens that are specifically expressed during infection may have enhanced sensitivity and specificity. In vivo-expressed antigens also have the potential to identify disease stage-specific antibody responses. In addition to their potential for the development of new diagnostics, identification of disease stage-specific protein expression may provide insight into TB infection and disease pathogenesis. The overall objective of this research is to undertake a proteome-wide search for protein antigens of Mycobacterium tuberculosis (Mtb) that are recognized by the human humoral immune response. In the first aim of this project, we will synthesize nucleic acid programmable protein arrays (NAPPA) for approximately 3,000 Mtb proteins. In the second aim, these arrays will be probed with serum obtained at carefully defined stages of TB infection from Mtb-infected rabbits, an excellent model of human TB, including latent TB and reactivation after immune suppression. Screening of banked human serum from patients with well-documented TB will also be undertaken in this aim. These studies will identify candidate antigens to be analyzed in subsequent studies to screen for antigens that are broadly recognized by humans with TB, and antigens that provoke an antibody response at specific stages of TB infection. These data may lead to improved diagnostic tests for active and latent TB, and provide new insights into the pathogenesis of latent and active TB. PUBLIC HEALTH RELEVANCE: TB remains a major cause of illness and death, particularly in people living in resource-limited settings and persons who are HIV-infected. The proposed research is designed to provide the basis for new methods to diagnose TB. Earlier, simpler, and more sensitive and specific TB diagnosis could have a major impact on the identification and treatment of people with TB in the U.S. and worldwide.