Application of Imaging to Development of Tuberculosis Interventions

项目总结 结核病目前困扰着世界近三分之一的人口，但在发展结核病方面取得的进展 干预措施受到结核分枝杆菌缓慢生长速度的阻碍，结核杆菌是病原体，以及 需要用集落形成单位(CFU)来评价细胞的活性。拟议的研究旨在克服 这一主要障碍是通过开发和验证可应用于 结核病防治新策略的评价。我们之前的研究结果是 在开发针对结核病的荧光和生物发光成像策略方面，但大多数 其中敏感的是报告酶荧光(REF)，其检测阈值为 肺部感染期间约有104个CFU。我们还展示了REF成像允许 在体外和动物体内进行治疗后24-48小时内的疗效评价。这项工作奠定了 旨在将REF成像应用于治疗效果的拟议研究的基础 确定、跟踪动物中的结核分枝杆菌感染情况，并分析结核病疫苗接种策略。我们 已经证明了成像能够量化和跟踪肺部和皮下感染， 为跟踪结核病的疫苗接种和挑战剂量提供了独特的机会，而结核病尚未 以前是可行的，特别是在使用活动物的实时检测中。在目前的研究中，我们将在 我们早期的研究通过测试REF成像评估各种治疗方法的疗效， 提高目前REF成像的检测阈值，论证影像研究的实用性 疫苗的效力。具体地说，我们建议：1)改进REF成像，以促进结核病 研究。我们的工作假设是，REF成像的催化性质将允许这项技术 达到每个器官10个CFU的阈值，足以分析结核病的毒力和治疗。 我们的初步研究表明，REF成像非常敏感，成像可以用来测量 治疗结果，并有潜力改善现有的REF底物。为了实现这一目标，我们将 比较影像与CFU的疗效评价，分析和改进底物的阈值 并仔细评估我们在动物模型中准确跟踪结核分枝杆菌感染的能力。2)分析 应用影像学方法研究结核病疫苗的疗效。我们的工作假设是 显微内窥镜和REF将使我们能够更快和更多地评估结核病疫苗的效果 比传统的基于CFU的方法更详细。我们的初步研究表明，我们可以利用微型 内窥镜成像跟随皮下接种报告菌株的疫苗和REF 结核杆菌肺部感染的影像诊断。为此，我们将构建稳定的报告疫苗株并 在小鼠和豚鼠的整个接种过程中，使用它们跟踪接种和挑战剂量 为了更好地了解活疫苗，并在疫苗接种过程中挑战剂量存活动态。

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.