Immune-based detection of rifampicin-resistance in HIV/TB

描述（由申请人提供）：近20亿人（占世界人口的三分之一）面临活动性结核病（TB）的风险，因为他们潜伏感染了病原体结核分枝杆菌（Mtb），每年发生近200万与结核病相关的死亡。艾滋病毒增加了结核病的风险，耐多药结核病（MDR-TB）被定义为对异烟肼（INH）和利福平都具有耐药性的结核病，在撒哈拉以南非洲的一些地区已成为突发公共卫生事件，那里的艾滋病毒流行率和结核病发病率是世界上最高的。结核病控制的一个主要障碍是，检测结核分枝杆菌耐药性需要收集微生物样本，而在许多艾滋病毒活动性疾病病例和所有潜伏感染病例中都无法获得微生物样本。事实上，如果怀疑存在耐药性，那么治疗潜伏性感染这一全球消除结核病的必要方法就无法得到适当的针对性，而且重要的是，耐药潜伏性结核杆菌感染的范围是未知的。微生物学和传染病的一个核心原则认为，鉴定人类病原体的抗生素耐药性需要直接接触生物体。我们的总体假设挑战了这一范式，假设病原体蛋白质组的变化是由于基因突变导致耐药性的结果，可以被宿主适应性免疫系统免疫检测到。这一假说有两个部分：1)某些基因被耐药菌株特异性上调，2)这些基因的产物可以被宿主适应性免疫系统识别。我们最近发表的蛋白质组学实验已经确定了许多特定的蛋白质，这些蛋白质在rpoB中具有单核苷酸多态性（snp）的结核分枝杆菌菌株中上调或特异性表达，从而对利福平产生耐药性，如果在临床疾病中存在，通常表明是耐多药结核病。此外，耐利福平的rpoB突变型结核分枝杆菌的特异性基因激活与模式生物（包括枯草芽孢杆菌和lividans链霉菌）的数据一致，即rpoB突变可以显著激活野生型菌株不表达的休眠基因网络。在本研究中，我们将分析博茨瓦纳HIV感染者和未感染者对先前确定的候选“利福平耐药抗原”的细胞免疫反应。具体来说，我们将前瞻性地比较利福平耐药和利福平敏感肺结核患者对一组新抗原的记忆免疫反应，因为感染结核分枝杆菌分离株的潜在药物敏感性可以在活动性疾病中确定。这项概念验证研究是评估一种确定结核分枝杆菌药物敏感性的创新间接方法的重要第一步，特别是在潜伏感染期间，目前无法评估耐药性。通过这样做，这一建议有可能通过首次允许对耐药潜伏感染的流行病学进行研究来改善结核病临床护理，最重要的是，从根本上改变全球结核病控制的公共卫生方法。

DESCRIPTION (provided by applicant): Nearly 2 billion people, one third of the World's population, are at risk of active tuberculosis (TB) because they are latently infected with the causative agent Mycobacterium tuberculosis (Mtb), and almost 2 million TB- related deaths occur annually. HIV increases the risk of TB, and multi-drug resistant TB (MDR-TB), defined as TB resistant to both isoniazid (INH) and rifampicin, has become a public health emergency in some parts of sub-Saharan Africa, where the HIV prevalence and TB incidence rates are the highest in the world. A major barrier to TB control is that detection of Mtb drug resistance requires collection of microbiological samples, which are not available in many cases of active disease in HIV and in all cases of latent infection. Indeed, treatment of latent infection, a necessary approach to global TB elimination, cannot be properly targeted if drug resistance is suspected and, importantly, the scope of drug-resistant latent Mtb infection is unknown. A central tenet of microbiology and infectious diseases holds that identification of antibiotic resistance in human pathogens requires direct access to the organism. Our overarching hypothesis challenges this paradigm by hypothesizing that the changes in the proteome of pathogens that specifically occur as a result of genetic mutation(s) conferring drug resistance can be immunologically detected by the host adaptive immune system. This hypothesis has two parts - 1) that certain genes are specifically upregulated by drug resistant strains, and 2) that te products of these genes can be recognized by the host adaptive immune system. Our recently published proteomics experiments have identified numerous specific proteins that are either upregulated in or specifically expressed by Mtb strains that have single nucleotide polymorphisms (SNPs) in rpoB conferring resistance to rifampicin, which, if present in clinical disease, usually indicates MDR-TB. Furthermore, specific gene activation by rifampicin-resistant, rpoB-mutant Mtb is consistent with data from model organisms, including Bacillus subtilis and Streptomyces lividans, in that rpoB mutation can dramatically activate dormant gene networks not expressed by wild-type strains. In this proposal, we will analyze cellular immune responses to previously identified candidate "rifampicin-resistance antigens" in HIV- infected and uninfected adults in Botswana, where the prevalence of HIV is nearly 20%. Specifically, we will prospectively compare memory immune responses to a panel of novel antigens in individuals with rifampicin- resistant versus rifampicin-susceptible pulmonary TB, as the underlying drug-susceptibility of the infecting Mtb isolates can be determined in active disease. This proof-of-concept study represents an important first step to evaluating an innovative, indirect method to determine drug susceptibility in Mtb, particularly during latent infection, wher drug resistance currently cannot be assessed. By so doing, this proposal has the potential to improve TB clinical care by allowing, for the first time, study of the epidemiology of drug-resistant latent infection and, most importantly, radically transforming public health approaches to global TB control.