Enhanced POC assay for TB in HIV-infected children based on the ultrasensitive detection of the urinary form of the lipoarabinomannan antigen

摘要 结核病仍然是流行地区幼儿死亡的主要原因，特别是在 艾滋病感染率很高。这些儿童很难用现有的检测方法诊断，据信， 在2017年死于结核病的超过23万名儿童中，许多人本可以通过更好的诊断来挽救生命。 一个非常有前途的诊断结核病的生物标志物是脂阿拉伯甘露聚糖（LAM），一个主要的分枝杆菌表面 糖脂在绝大多数活动性感染的TB患者中以不同浓度积累。我们 实验室最近对了解抗原特性的多样性做出了重大贡献， LAM和针对该抗原的体液免疫应答的复杂性。一个惊人的发现 我们的研究结果是，尿型结核LAM（uLAM）在抗原性上不同于细菌型， 相关形式（ManLAM），我们已经确定了新的单克隆抗体组合，允许 灵敏和特异的检测uLAM。最近，这导致了一种新的侧向流测定法的产生。 (the Fujifilm SILVAMP TB LAM测定），其对uLAM的敏感性显著高于Fujifilm SILVAMP TB LAM测定。 商业测定。然而，尽管有这种改进，该测定法的灵敏度和稳健性仍然不是最好的。 足以满足WHO对最佳测定的要求。这项建议将以我们以前的工作为基础， 进一步提高我们现有试剂的亲和力和特异性，并确定新的试剂， uLAM的灵敏度和准确性。为了更好地了解uLAM的性质并提供标准化样本 对于未来的测定，我们将从具有一定范围uLAM水平的阳性患者中分离大量（~ 2L）的尿液， 并将这些样本的多个等分试样冷冻起来以备将来使用。一组选定的尿液将用于 从多个患者中纯化高浓度的uLAM，以及uLAM的结构和免疫学性质。 将比较这些抗原。我们设计了现有的捕获和检测试剂， 对ManLAM的亲和力/亲合力提高，并且将这些试剂的灵敏度与那些 现有试剂和侧流分析对几个队列的儿科尿液样本，符合 该RFA的要求（<5岁，>20% HIV+）。我们将筛选记忆B细胞群， 鉴定对uLAM具有高亲和力和/或特异性的新单克隆抗体，并选择酵母展示文库 通过对我们的先导检测试剂的关键CDR区域进行随机化生成，用于具有更高 亲和力和特异性。这些新mAb的结合特性将是 将表征它们，并测试它们在检测儿科样品中的uLAM中的功效。抗体 与现有试剂相比，具有明显更好灵敏度的组合将转移到我们的商业 富士胶片和其他公司的合作者在其平台上进行整合和测试。总目标 本研究的目的是开发一种改进的LAM检测方法， (TPP)世卫组织提出的用于结核病检测的高优先级生物标志物、非痰液POC检测。

Abstract TB remains a major source of mortality in young children in endemic areas, particularly in areas where there is a high rate of HIV infection. These children are difficult to diagnose with existing assays, and it is believed that many of the >230,000 children who died because of TB in 2017 could have been saved with better diagnostics. A highly promising biomarker for diagnosing TB is lipoarabinomannan (LAM), a major mycobacterial surface glyolipid that accumulates at different concentrations in the great majority of actively infected TB patients. Our lab has recently made major contributions towards understanding the diversity of the antigenic properties of LAM and the complexity of the humoral immune rsponse against this antigen. One surprising discovery resulting from our work is that the urinary form of TB LAM (uLAM) is antigenically distinct from the bacterially associated form (ManLAM), and we have identified novel combinations of monoclonal antibodies that allow the sensitive and specific detection of uLAM. This has recently led to the production of a new lateral flow assay (the Fujifilm SILVAMP TB LAM assay) that has a significantly increased sensitivity for uLAM over that of the commercial assay. However, despite this improvement, the sensitivity and robustness of this assay is still not sufficient to meet the WHO requirements for an optimal assay. This proposal will build on our previous work to further improve the affinities and specificities of our existing reagents, and identify new reagents with increased sensitivity and accuracy for uLAM. To better understand the nature of uLAM and provide standardized samples for future assays we will solate large volumes (~ 2L) of urine from positive patients with a range of uLAM levels, and freeze away multiple aliquots of these samples for future use. A selected set of these urines will be used to purify high concentrations of uLAM from multiple patients, and the structural and immunological properties of these antigens will be compared. We have engineered forms of existing capture and detection reagents that have improved affinities/avidities for ManLAM, and the sensitivity of these reagents will be compared to those of existing reagents and lateral flow assays against several cohorts of pediatric urine samples that meet the requirements of this RFA (<5 years old, >20% HIV+). We will screen memory B cell populations from selected patients to identify new mabs with high affinities and/or specificities for uLAM, and select yeast display libraries generated by randomization of the key CDR regions of our lead detection reagent for variants with higher affinities and specificites than the parental antibody. The binding properties of these new mAbs will be characterized, and their efficacy in detecting uLAM in the pediatric samples will be tested. Antibody combinations with demonstrably better sensitivities than existing reagents will be transferred to our commercial collaborators at Fujifilm and other companies for incorporation and testing in their platforms. The overall goals of this study are to develop an improved LAM detection assay that meets or exceeds the Target Product Profile (TPP) proposed by the WHO for high priority biomarker-based, non-sputum-based, POC tests for TB detection.