Application of Imaging to Development of Tuberculosis Interventions

项目摘要 结核病目前困扰着世界上近三分之一的人口，但在发展结核病方面取得的进展， 由于结核分枝杆菌的生长速度缓慢， 需要通过菌落形成单位（cfu）来评估生存力。这项研究旨在克服 这一主要障碍，通过开发和验证成像技术，可以应用于 评价结核病新的预防和治疗策略。我们之前的研究表明 在结核病的荧光和生物发光成像策略的发展，但最 其中敏感的已被证明是报告酶荧光（REF），其检测阈值为 肺部感染时约为104 cfu。我们还表明，REF成像允许 体外和动物治疗后24-48小时内的治疗评价。这项工作已经确定了 拟议研究的基础，旨在将REF成像应用于治疗效果 确定、追踪动物中的结核杆菌感染以及分析结核病疫苗接种策略。我们 已经证明成像能够量化和跟踪肺部和皮下感染， 提供了跟踪结核病疫苗接种和挑战剂量的独特机会， 以前是可行的，特别是在实时使用活动物。在目前的研究中，我们将建立在 我们早期的研究通过测试REF成像的能力来评估各种治疗方法的疗效， 改善当前REF成像检测阈值，并证明成像研究的实用性 疫苗功效。具体而言，我们建议：1）改善REF成像，以促进结核病 research.我们的工作假设是，REF成像的催化性质将使这项技术能够 达到每个器官10 cfu的阈值，足以分析结核病毒力和治疗方法。 我们的初步研究表明，REF成像是非常敏感的，成像可以用来测量 治疗结果，并有可能改善现有的REF底物。为此，我们将 比较成像与cfu的疗效评价，分析并改进阈值的底物 检测和仔细评估我们在动物模型中准确跟踪Mtb感染的能力。2)分析 利用成像技术研究疫苗对结核病的有效性。我们的工作假设是， 显微内窥镜和REF将使我们能够更快地评估结核疫苗的疗效， 比传统的基于CFU的方法更详细。我们的初步研究表明，我们可以利用微- 皮下接种疫苗和REF报告菌株后的内窥镜成像 肺结核感染的影像学检查为此，我们将构建稳定的报告疫苗株， 在小鼠和豚鼠的整个疫苗接种过程中，使用它们来跟踪疫苗接种和攻毒剂量 更好地了解活疫苗和疫苗接种期间攻毒剂量的活力动态。

PROJECT SUMMARY Tuberculosis currently afflicts nearly one-third of the world's population, but progress in development of interventions is hamstrung by the slow growth rate of Mycobacterium tuberculosis, the causative agent, and the need to evaluate viability by colony forming units (cfu). The proposed study is designed to overcome this major roadblock through the development and validation of imaging technologies that can be applied to evaluation of novel prevention and treatment strategies for tuberculosis. Our previous studies have resulted in development of both fluorescent and bioluminescent imaging strategies for tuberculosis, but the most sensitive of these has proven to be reporter enzyme fluorescence (REF), which has a detection threshold of approximately 104 cfu during pulmonary infection. We have also shown that REF imaging allows therapeutic evaluation within 24-48 h post-treatment both in vitro and in animals. This work has set the foundation for the proposed studies designed to allow application of REF imaging to therapeutic efficacy determination, tracking Mtb infections in animals and analysis of vaccination strategies for tuberculosis. We have demonstrated the ability of imaging to quantify and track both pulmonary and subcutaneous infections, offering the unique opportunity to track both vaccination and challenge doses for tuberculosis, which has not been previously feasible, particularly in real-time using live animals. In the current study, we will build upon our earlier studies by testing the ability of REF imaging to evaluate efficacy of a diverse set of therapeutics, improvement of the current REF imaging threshold of detection and demonstrate the utility of imaging study of vaccine efficacy. Specifically, we propose to: 1) Improve REF imaging to facilitate tuberculosis research. Our working hypothesis is that the catalytic nature of REF imaging will allow this technology to achieve thresholds of 10 cfu per organ, sufficient for analysis of tuberculosis virulence and therapeutics. Our preliminary studies demonstrate that REF imaging is very sensitive, imaging can be used to measure therapeutic outcome and that there is potential to improve existing REF substrates. In this aim we will compare therapeutic efficacy evaluation for imaging with cfu, analyze and improve substrates for thresholds of detection and carefully evaluate our ability to accurately track Mtb infection in animal models. 2) Analyze vaccine efficacy against tuberculosis using imaging. Our working hypothesis is that the combination of micro-endoscopy and REF will allow us to evaluate tuberculosis vaccine efficacy more rapidly and in more detail than conventional cfu-based methods. Our preliminary studies demonstrate that we can utilize micro- endoscopic imaging to follow sub-cutaneous inoculation with reporter strains of the vaccine and REF imaging for pulmonary infection with Mtb. In this aim we will construct stable reporter vaccine strains and use them to follow both the vaccination and challenge dose throughout vaccination in mice and guinea pigs to better understand the live vaccine and challenge dose viability dynamics during vaccination.