Application of Imaging to Development of Tuberculosis Interventions

描述（由申请人提供）：结核病目前困扰着世界上近三分之一的人口，但由于结核分枝杆菌（病原体）生长速度缓慢以及需要通过菌落形成单位（cfu）评估生存能力，干预措施的开发进展受到阻碍。拟议的研究旨在通过开发和验证可用于评估结核病新预防和治疗策略的成像技术来克服这一主要障碍。我们以前的研究已经导致了结核病的荧光和生物发光成像策略的发展，但其中最敏感的已被证明是报告酶荧光（REF），其在肺部感染期间的检测阈值约为104 cfu。我们还表明，REF成像允许在体外和动物中治疗后24-48小时内进行治疗评价。这项工作为拟议的研究奠定了基础，这些研究旨在将REF成像应用于确定治疗效果，跟踪动物中的Mtb感染和分析结核病的疫苗接种策略。我们已经证明了成像定量和跟踪肺部和皮下感染的能力，为跟踪结核病的疫苗接种和挑战剂量提供了独特的机会，这在以前是不可行的，特别是在实时使用活动物。在当前的研究中，我们将通过测试REF成像评估各种治疗方法的功效的能力，改进当前REF成像检测阈值并证明疫苗功效的成像研究的实用性，来建立我们早期的研究。具体而言，我们建议：1）改善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该技术达到每个器官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，成像可用于测量治疗结果，并有可能改善现有的REF基板。在这个目标中，我们将比较成像与cfu的治疗效果评估，分析和改进检测阈值的底物，并仔细评估我们准确跟踪Mtb感染的能力。 动物模型2)利用影像学分析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的结合将使我们能够比传统的基于CFU的方法更快速，更详细地评估结核疫苗的疗效。我们的初步研究表明，我们可以利用显微内窥镜成像来跟踪用疫苗的报告菌株的皮下接种和Mtb肺部感染的REF成像。为此，我们将构建稳定的报告基因疫苗株，并将其用于在小鼠和豚鼠的整个疫苗接种过程中跟踪疫苗接种和攻毒剂量，以更好地了解疫苗接种期间的活疫苗和攻毒剂量活力动力学。

DESCRIPTION (provided by applicant): 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 wil 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.