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

描述（由申请人提供）：结核（TB）疾病是由结核分枝杆菌（Mtb）感染引起的，是全世界5岁以下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岁以下儿童占患者总数的30-40%，而这些受感染的儿童往往患有更严重、往往致命的结核病。造成5岁以下儿童结核病致死率的一个重要因素是标准结核病诊断在这一年龄组的表现不佳，特别是与成人相比。诊断不准确导致诊断延误和漏诊，进而导致儿童发病率和死亡率增加。目前，需要的是一种简单、可靠的免疫诊断血液检测，将儿童结核性肺炎与非结核性肺炎区分开来。我们假设CD8+ T细胞对结核分枝杆菌肽的检测可用于区分幼儿结核性肺炎与非结核性肺炎。在这方面，CD8+ T细胞优先识别严重Mtb感染的细胞，作为OHSU的研究人员，我们在乌干达5岁以下的儿童中观察到Mtb反应性CD8+ T细胞在结核病儿童中检测到，而在无症状的Mtb感染/暴露儿童中未检测到。综上所述，这些数据表明CD8+ T细胞与细菌负荷相关。同时，通过我们的大规模抗原发现项目，我们已经定义了45种免疫优势、临床验证的CD8 TB抗原，这些抗原是OHSU独家授权给ViTi， Inc.的。为了改进儿童诊断，在我们的I期研究中，我们使用生物信息学方法确定了45种免疫优势CD8抗原中与HLA高亲和力结合的富集区域。我们选择了166条代表这些可能包含免疫原性表位簇的区域的肽，并使用IFN-？ELISPOT法检测了从有和没有潜伏性结核感染（LTBI）的个体中分离的CD8+ T细胞对这些肽的识别。从这些数据中，我们选择了包括两个肽库的肽集；一种针对结核分枝杆菌感染的敏感性进行优化，另一种针对结核分枝杆菌感染的特异性进行优化，用于原型免疫诊断血液检测，我们随后将其分别称为ViTi ONESENS和ViTi ONESPEC。在这个诊断准确性的第二阶段建议中，我们将首先在乌干达5岁以下肺炎住院儿童队列中确定这些原型免疫诊断血液测试的敏感性和特异性。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公司的其他新型免疫显性CD8抗原，以创建用于ViTi TWOSENS和ViTi TWOSPEC的改进肽库。最后，我们将确定ViTi TWOSENS和ViTi TWOSPEC在乌干达5岁以下肺炎住院儿童的同一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.