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.TB)和牛分枝杆菌会发生疾病，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，为生物医学研究维持健康、高质量的动物群体带来了重大问题。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。由于特异性和敏感性的问题，这些传统的诊断系统具有局限性。此外，潜伏感染(或低水平持续感染)的动物很难通过这些方法识别。因此，这项R24赠款计划的目标是开发灵敏、特异、快速和经济的新的免疫分析方法来检测非人类灵长类动物感染分枝杆菌。这个假设是，基于传统和新型免疫分析方法的多种检测方法的组合，将准确和可靠地确定非人类灵长类动物的牛分枝杆菌/牛分枝杆菌感染。该项目建立在(1)分枝杆菌基因组学、蛋白质组学和免疫学知识以及(2)新的传染病诊断工具方面的最新进展。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，并利用自然感染的猕猴来评估免疫分析性能。制定和实施这些检测方法的具体目标如下：(1)鉴定结核分枝杆菌。(2)开发和优化新的多重免疫分析方法，以检测针对特定目标1中确定的抗原的体液和细胞介导的免疫反应，(3)在实验接种的非人类灵长类动物中，在感染的急性和潜伏期测试这些新的多重和基于细胞的免疫分析方法，以及(4)在自然感染的动物中(即，在检疫期间和在圈养动物中暴发)评估这些免疫分析方法。在这项建议中开发的新的免疫分析方法对于建立和维护非人类灵长类动物的无特定病原体(SPF)群体以在生物医学研究中的最佳使用至关重要，包括将猕猴用于艾滋病研究。

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.