VACCINATION AGAINST MYCOBACTERIUM TUBERCULOSIS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。结核病是一个巨大的国际公共卫生问题，仍然是世界范围内传染病造成死亡的头号原因。结核病主要是一种呼吸道疾病，引起结核病的细菌结核分枝杆菌（M.tb）通过吸入雾化飞沫传播。我们建议利用支气管镜下段性抗原激发技术引发肺对结核分枝杆菌蛋白抗原的免疫应答。该技术包括将支气管镜楔入特定的支气管，在局部注入稀释的抗原。在给予免疫反应足够的时间后，重复支气管镜检查，并对抗原挑战和控制肺段进行灌洗。我们将使用标准皮肤试验试剂结核菌素（也称为M.tb的纯化蛋白衍生物，或PPD）作为抗原。我们假设，对结核分枝杆菌抗原的保护性回忆反应可以在ppd阳性的个体中引起，因为以前的气溶胶暴露于结核分枝杆菌，并且这种反应在数量和质量上都不同于接种过卡介苗的未暴露个体。我们将通过以下具体目的来研究这一假设：1)确定结核分枝杆菌特异性淋巴细胞是否可以通过纯化结核分枝杆菌蛋白衍生物的肺段性攻击募集到肺部；2)从细胞类型、细胞因子产生、细胞毒能力和介导人单核吞噬细胞杀伤细胞内结核分枝杆菌的能力等方面描述PPD侵袭时细胞浸润肺泡节段的特征；3)比较呼吸暴露于结核分枝杆菌和未暴露于bcg疫苗的受试者对局部PPD攻击的肺部召回反应。我们的人体研究将包括ppd阳性受试者的肺段性挑战，这些受试者有明确的结核分枝杆菌或卡介苗接种史。抗原激发的设计改编自哮喘受试者的研究。受试者年龄在18-50岁之间，不吸烟，无哮喘或其他慢性呼吸道疾病史。有PPD皮肤试验反应大于30mm硬结史的个体，有PPD皮肤试验反应的局部溃疡史的个体，或有皮肤试验反应的全身性症状（如发热、寒战和肌痛）史的个体将被排除在研究之外。我们将进行初步支气管镜检查以注入PPD， 48小时后再进行一次重复检查。在初始支气管镜检查时，将经温无菌生理盐水稀释的PPD滴注于左肺舌支气管，并将等量生理盐水单独滴注于右中叶支气管。不进行支气管肺泡灌洗。第二次支气管镜检查，在PPD刺激后48小时进行，将包括刺激段和对照段的支气管肺泡灌洗。在一些研究中，还将获得血液样本，以便通过比较未分类的血液淋巴细胞的反应来确定肺部免疫反应引起的PPD的特异性。最初的研究将旨在确定诱导淋巴细胞流入受损支气管段所需的PPD的最小剂量。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Tuberculosis is an international public health problem of enormous magnitude and remains the number one cause of death from an infectious disease world-wide. Tuberculosis is primarily a respiratory disease, and Mycobacterium tuberculosis (M.tb), the bacteria which causes tuberculosis, is spread via the inhalation of aerosolized droplets. We propose to utilize the technique of bronchoscopic segmental antigen challenge to elicit pulmonary immune responses to protein antigens of M.tb. This technique involves wedging the bronchoscope into specific segmental bronchi to locally instill diluted antigens. After allowing sufficient time for an immune response to develop, repeat bronchoscopy is performed with lavage of antigen-challenged and control lung segments. We will utilize the standard skin test reagent tuberculin (also known as purified protein derivative of M.tb, or PPD) as the antigen. We hypothesize that a protective recall response to antigens of M.tb can be elicited in individuals who are PPD-positive because of previous aerosol exposure to M.tb, and that this response is both quantitatively and qualitatively different from that of non-exposed individuals who have been vaccinated with BCG. We will investigate this hypothesis using the following specific aims: 1) To determine whether M.tb-specific lymphocytes can be recruited to the lung by segmental pulmonary challenge with purified protein derivative of M.tb; 2) To characterize the cellular infiltrate into alveolar segments in response to PPD challenge in terms of cell type, cytokine production, cytotoxic capacity, and ability to mediate killing of intracellular M.tb with human mononuclear phagocytes; 3) To compare the local pulmonary recall response to segmental PPD challenge in individuals with respiratory exposure to M.tb and in unexposed BCG-vaccinated subjects. Our human studies will consist of segmental pulmonary challenge of PPD-positive subjects with a history of clear exposure to M.tb or of BCG vaccination. Design of antigen challenge is adapted from studies of asthmatic subjects. Subjects will be nonsmokers age 18-50 who have no history of asthma or other chronic respiratory disease. Individuals with a history of PPD skin test response greater than 30 mm of induration, with a history of local ulceration in response to PPD testing, or with a history of systemic symptoms (such as fever, chills, and myalgias) in response to skin testing will be excluded from the study. We will perform an initial bronchoscopy to instill PPD, and a repeat procedure 48 hours later. In the initial bronchoscopy, PPD diluted in warmed sterile saline will be instilled into the lingular bronchus of the left lung, and the same volume of saline alone will be instilled into the right middle lobe bronchus. No bronchoalveolar lavage will be performed. The second bronchoscopy, performed 48 hours after the PPD challenge, will consist of the performance of bronchoalveolar lavage of both the challenge and control segments. In some studies, blood samples will also be obtained to allow for determination of the specificity of the pulmonary immune response elicited PPD by comparison to responses of unsorted blood lymphocytes. Initial studies will be aimed at determining the minimal dose of PPD needed to induce influx of lymphocytes into the challenged bronchial segment.