Novel Immunoassays for Mycobacteria Detection in Non-human Primates

描述（由申请人提供）： 非人灵长类动物感染结核分枝杆菌（M。结核病）和M.牛会发展疾病，因此不适合用于育种或研究项目。慢性感染的携带者很难诊断，并且对于维持用于生物医学研究的健康、高质量动物群体存在重大问题。目前，筛查的主要方法是结核菌素皮肤试验和血细胞刺激试验。这些常规诊断系统由于特异性和灵敏度的问题而具有局限性。此外，潜伏感染（或低水平持续感染）的动物难以通过这些方法识别。因此，该R24资助提案的目的是开发新的免疫测定法，该免疫测定法灵敏、特异、快速且经济，用于检测非人灵长类动物的分枝杆菌感染。假设是基于常规和新型免疫测定方法的多种测试的组合将准确且可靠地确定M.tb./ M.非人类灵长类动物的牛传染病。该项目建立在（1）分枝杆菌基因组学，蛋白质组学和免疫学的知识和（2）传染病的新型诊断仪器的最新进展。该项目采取双管齐下的方法，利用实验接种的猕猴来表征抗原特异性免疫反应和自然感染的猕猴来评估免疫测定性能。以下具体目标将开发和实施这些检测：（1）鉴定M。TB.这些蛋白质是实验性接种M.结核病，(2)开发和优化用于检测靶向特定目的1中鉴定的抗原的体液和细胞介导的免疫应答的新型多重免疫测定，（3）在感染的急性和潜伏期在实验接种的非人灵长类动物中测试这些新型多重和基于细胞的免疫测定，和（4）在自然感染的动物中评价这些免疫测定（即，隔离期间和圈养动物中的疫情）。本提案中开发的新型免疫测定方法对于建立和维持非人灵长类动物的无特异性病原体（SPF）菌落至关重要，以便最佳地用于生物医学研究，包括使用猕猴进行艾滋病研究。

DESCRIPTION (provided by applicant): Non-human primates infected with Mycobaterium tuberculosis (M. tb.) and M. bovis develop disease and are thus not suitable for use in breeding or research projects. Chronically infected carriers are difficult to diagnose and present significant problems for maintaining colonies of healthy, high-quality animals for biomedical research. Currently, the main methods for screening are the tuberculin skin test and a blood cell stimulation assay. These conventional diagnostic systems have limitations due to problems with specificity and sensitivity. In addition, animals with latent infection (or low-level persistent infection) are difficult to identify by these methods. Accordingly, the objective of this R24 grant proposal is to develop novel immunoassays that are sensitive, specific, rapid, and economical for detecting infection of non-human primates with mycobacteria. The hypothesis is that a combination of multiple tests, based on conventional and novel immunoassay methods, will accurately and reliably determine M.tb./M. bovis infection of nonhuman primates. This project builds on recent advances in (1) knowledge of mycobacteria genomics, proteomics, and immunology and (2) novel diagnostic instrumentation for infectious disease. This project takes a two-pronged approach that utilizes experimentally inoculated macaques to characterize antigenspecific immune responses and naturally infected macaques to assess immunoassay performance. The following Specific Aims will develop and implement these assays: (1) to identify M. tb. proteins that are targets of humoral and cell-mediated immune responses in non-human primates experimentally inoculated with M. tb., (2) to develop and optimize novel multiplex immunoassays for detecting both humoral and cell-mediated immune responses targeted against the antigens identified in Specific Aim 1, (3) to test these novel multiplex and cell-based immunoassays in experimentally inoculated non-human primates at acute and latent stages of infection, and (4) to evaluate these immunoassays in naturally infected animals (i.e., during quarantine and in outbreaks in captive animals). The novel immunoassay methods developed in this proposal will be essential for establishing and maintaining specific-pathogen-free (SPF) colonies of nonhuman primates for optimal use in biomedical research, including the use of macaques for AIDS research.