A CD8+ T cell diagnostic to identify children with pulmonary tuberculosis

描述(由申请人提供)：结核病(TB)是由结核分枝杆菌(Mtb)感染引起的疾病，是全球5岁儿童感染性发病率和死亡率的主要原因。在结核病流行地区，世界上每年900万成人结核病病例中的绝大多数居住在这些地区，5岁以下的儿童占患者总数的30%-40%，这些被感染的儿童往往患有更严重的、往往是致命的结核病。5岁儿童结核病致死率的一个重要因素是这个年龄段的标准结核病诊断表现不佳，特别是与成年人相比。糟糕的诊断会导致延迟和漏诊，进而导致儿童发病率和死亡率的增加。目前，需要的是一种简单、强大的免疫诊断血液测试，将儿童结核病肺炎与非结核病肺炎区分开来。我们推测，针对结核分枝杆菌多肽的CD8+T细胞的检测可以用于区分患有肺结核肺炎的幼儿和非结核肺炎患儿。在这方面，CD8+T细胞优先识别严重感染结核分枝杆菌的细胞，作为OHSU调查人员，我们在乌干达5岁儿童中观察到，在患有结核病的儿童中检测到具有结核分枝杆菌反应性的CD8+T细胞，而在感染/接触结核杆菌的无症状儿童中未检测到。综上所述，这些数据表明CD8+T细胞与细菌负荷有关。同时，我们通过我们的大规模抗原发现计划定义了45种免疫优势、经临床验证的CD8结核抗原，这些抗原从OHSU独家授权给Viti，Inc.。为了开发一种改进的儿童诊断方法，在我们的第一阶段研究中，我们使用生物信息学方法来定义这45个免疫优势CD8抗原中与HLA高亲和力结合的富集区。我们选择了166个多肽，代表这些可能包含免疫原性表位簇的区域，并使用干扰素？？ELISPOT分析，检测从有和没有潜伏性结核病感染(LTBI)的个体分离的CD8+T细胞对这些肽的识别能力。从这些数据中，我们选择了一组包含两个肽库的多肽；一个针对结核分枝杆菌感染的敏感性进行了优化，另一个针对结核分枝杆菌感染的特异性进行了优化，用于原型免疫诊断血液测试，我们随后将分别称为VITI ONESENS和VITI ONESPEC。在这项诊断准确性的第二阶段提案中，我们将首先确定这些原型免疫诊断血液测试在乌干达5岁肺炎住院儿童队列中的敏感性和特异性。然后，为了进一步改进这些原型测试，我们将把我们在第一阶段研究中使用的相同策略应用于OHSU授权给VITI公司的其他新的免疫优势CD8抗原，以创建用于VITI TWOSENS和VITI TWOSPEC的改进的肽库。最后，我们将确定Viti TWOSENS和Viti TWOSPEC在同样的乌干达肺炎住院儿童队列中的敏感性和特异性。

DESCRIPTION (provided by applicant): Tuberculosis (TB) disease, which results from infection with Mycobacterium tuberculosis (Mtb), is a leading cause of infectious morbidity and mortality in children < 5 years old worldwide. In TB endemic regions, in which the vast majority of the world's annual 9 million adult cases of TB disease reside, children < 5 years old account for 30-40% of the total patients and those children who are infected tend to have more severe, often fatal forms of TB. A significant contributor to the deadliness of TB in children < 5 years ol is the poor performance of standard TB diagnostics in this age group, especially as compared to adults. Poor diagnostics result in delayed and missed diagnoses, which in turn lead to increased morbidity and mortality in children. Currently, what is needed is a simple, robust immunodiagnostic blood test that will differentiate childhood TB pneumonia from pneumonia not due to TB. We hypothesize that the detection of CD8+ T cells directed toward Mtb peptides can be utilized to distinguish young children with TB pneumonia from those with pneumonia not due to TB. In this regard, CD8+ T cells preferentially recognize heavily Mtb-infected cells and as OHSU investigators, we have observed in Ugandan children < 5 years old, that Mtb-reactive CD8+ T cells are detected in children with TB and not detected in asymptomatic children with Mtb infection/exposure. Taken together, these data suggest that CD8+ T cells correlate with bacterial burden. In parallel, we have defined 45 immunodominant, clinically-validated CD8 TB antigens through our large scale antigen discovery program that are exclusively licensed to ViTi, Inc. from OHSU. To develop an improved diagnostic for children, in our Phase I study, we used a bioinformatic approach to define regions enriched for high affinity binding to HLA within these 45 immunodominant CD8 antigens. We selected 166 peptides representing these regions likely to contain clusters of immunogenic epitopes, and using IFN-? ELISPOT assay, tested the recognition of these peptides by CD8+ T cells isolated from individuals with and without latent TB infection (LTBI). From these data, we selected sets of peptides comprising two peptide pools; one optimized for sensitivity and one for specificity for Mtb infection to be used in prototype immunodiagnostic blood tests, which we will subsequently refer to as ViTi ONESENS and ViTi ONESPEC, respectively. In this Phase II proposal of diagnostic accuracy, we will first determine the sensitivity and specificity of these prototype immunodiagnostic blood tests, in cohorts of Ugandan children < 5 years old hospitalized with pneumonia. Then to further improve upon on these prototype tests, we will apply the same strategy we used in the Phase 1 study to additional novel immunodominant CD8 antigens licensed by OHSU to ViTi Inc. to create improved peptide pools to be used in ViTi TWOSENS and ViTi TWOSPEC. Finally, we will determine the sensitivity and specificity of ViTi TWOSENS and ViTi TWOSPEC in the same cohorts of Ugandan children < 5 years old hospitalized with pneumonia.