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

基于尿形式阿拉伯脂甘露聚糖抗原的超灵敏检测，增强 HIV 感染儿童结核病的 POC 检测

• 批准号: 10611413
• 负责人: ABRAHAM PINTER
• 金额: $ 74.73万
• 依托单位: RUTGERS BIOMEDICAL AND HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-14 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10611413
• 关键词: 5 year old; Affinity; Aliquot; Antibodies; Antigenic Diversity; Antigens; Area; Avidity; B-Lymphocytes; Binding; Biological Assay; Biological Markers; CD4 Positive T Lymphocytes; Carbohydrates; Characteristics; Child; Childhood; Collaborations; Collection; Detection; Devices; Diagnosis; Diagnostic; Engineering; Epitopes; Freezing; Future; Generations; Glycolipids; Goals; HIV; HIV Infections; HIV Seronegativity; HIV-1; HIV/TB; Human; Immune; Immunologics; Industrialization; Infection; Kenya; Lateral; Lead; Libraries; Link; Mannosides; Memory B-Lymphocyte; Methods; Monoclonal Antibodies; Mycobacterium tuberculosis; Nature; Patient Selection; Patients; Pediatric cohort; Polysaccharides; Population; Production; Property; Randomized; Reagent; Sampling; Serum; Source; South African; Specificity; Standardization; Structure; Surface; Testing; Tuberculosis; Tuberculosis diagnosis; Urine; Variant; Vertebral column; Work; Xylose; Yeasts; co-infection; cohort; detection assay; detection platform; diagnosis standard; efficacy testing; human monoclonal antibodies; immunoreactivity; improved; lateral flow assay; lipoarabinomannan; man; manufacture; mortality; mycobacterial; novel; patient screening; point of care; screening; sugar; urinary

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.

抽象的 结核病仍然是流行地区幼儿死亡的主要来源，尤其是在有 艾滋病毒感染率高。这些孩子很难诊断现有测定法，据信 在2017年因结核病而死亡的23万名儿童中，许多儿童都可以通过更好的诊断来保存。 诊断结核病的高度有前途的生物标志物是脂肪动物群（LAM），这是主要的分枝杆菌表面 在大多数主动感染的结核病患者中以不同浓度积累的乙醇酸。我们的 实验室最近为理解抗原特性的多样性做出了重大贡献 LAM和对这种抗原的体液免疫反应的复杂性。一个令人惊讶的发现 由我们的工作导致的是，TB LAM（ULAM）的尿形式在抗原上与细菌不同 相关的形式（横山），我们已经确定了单克隆抗体的新型组合，这些组合允许 对ULAM的敏感和特异性检测。最近，这导致了新的侧向流量测定法 （Fujifilm Silvamp TB LAM分析）对ULAM的敏感性显着提高 商业测定法。但是，尽管有这种改善，但该测定的敏感性和鲁棒性仍然不是 足以满足WHO最佳测定法的要求。这项建议将以我们以前的工作为基础 进一步改善我们现有试剂的亲和力和特异性，并确定增加的试剂 ULAM的敏感性和准确性。更好地了解ULAM的性质并提供标准化样本 对于未来的测定 并冻结这些样品的多个等分试样，以备将来使用。选定的这些尿液将用于 纯化来自多名患者的高浓度ULAM，以及 这些抗原将被比较。我们已经设计了现有捕获和检测试剂的形式 对Manlam的亲和力/狂热有改善，将这些试剂的敏感性与那些敏感性进行比较 现有试剂和横向流量测定，针对符合该小儿的几个小儿尿液样本 此RFA的要求（<5岁，> 20％的HIV+）。我们将从选定的 患者识别具有高亲和力和/或特异性的新单元格，并选择酵母显示库 由我们铅检测试剂的关键CDR区域的随机分组而产生 亲和力和特定材料比亲本抗体。这些新mAb的绑定特性将是 表征，将测试它们在小儿样品中检测ULAM的功效。抗体 与现有试剂相比，与现有试剂的敏感性更高的组合将被转移到我们的商业 Fujifilm和其他公司的合作者在其平台中融合和测试。总体目标 这项研究是为了开发符合或超过目标产品概况的改进的LAM检测测定法 （TPP）由WHO提出的用于高优先级生物标志物，基于非估计的POC检测，以进行结核病检测。