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

描述(申请人提供)：近20亿人，即世界人口的三分之一，由于潜伏感染致病因子结核分枝杆菌(Mtb)而面临患活动性结核病(TB)的风险，每年发生近200万与结核病有关的死亡。艾滋病毒增加了结核病的风险，耐多药结核病(MDR-TB)，即同时对异烟肼(INH)和利福平耐药的结核病，在撒哈拉以南非洲的一些地区已成为一种公共卫生紧急情况，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，潜伏感染的治疗是全球消除结核病的必要方法，如果怀疑存在耐药性，就不能适当地针对潜伏感染，更重要的是，耐药的潜伏结核杆菌感染的范围尚不清楚。微生物学和传染病的一个核心原则认为，识别人类病原体中的抗生素耐药性需要直接接触到生物体。我们的总体假设挑战了这一范式，假设由于导致耐药性的基因突变(S)而特定发生的病原体蛋白质组的变化可以被宿主适应性免疫系统检测到。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mtb菌株中上调或特异性表达，这些mtb菌株具有与利福平耐药相关的rpoB单核苷酸多态(SNPs)，如果在临床疾病中存在，通常表明MDR-TB。此外，利福平耐药的rpoB突变体Mtb的特异性基因激活与包括枯草芽孢杆菌和变青链霉菌在内的模式生物的数据一致，因为rpoB突变可以显著激活野生型菌株不表达的休眠基因网络。在这项提案中，我们将分析博茨瓦纳艾滋病毒流行率近20%的艾滋病毒感染者和未感染者的细胞免疫反应，以应对之前确定的候选利福平耐药抗原。具体地说，我们将前瞻性地比较耐利福平肺结核病患者和易感利福平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创新的间接方法确定结核分枝杆菌药物敏感性的重要第一步，特别是在目前无法评估耐药性的潜伏感染期间。通过这样做，这项建议有可能改善结核病的临床护理，因为它首次允许研究耐药潜伏感染的流行病学，最重要的是，从根本上改变了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.