Detection Assays for Virion Susceptibility to HIV Broadly Neutralizing Antibodies in Plasma and Culture Fluids

血浆和培养液中病毒体对 HIV 广泛中和抗体敏感性的检测分析

• 批准号: 10675310
• 负责人: Anthony L DeVico
• 金额: $ 52.33万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10675310
• 关键词: Antibodies; Antibody Therapy; Binding; Biological Assay; Blood Tests; Certification; Clinical; Clinical Laboratory Improvement Amendments; Clinical Trials; Coculture Techniques; Combined Modality Therapy; Complex; Consumption; DNA sequencing; Dedications; Detection; Enrollment; Epitopes; Exhibits; Fluorescence; Goals; HIV; HIV resistance; HIV/AIDS; Human; In Vitro; Label; Laboratories; Liquid substance; Methods; Nature; Participant; Performance; Peripheral Blood Mononuclear Cell; Persons; Phase; Plasma; Population; Predictive Value; Predisposition; Preparation; Prevention; Prevention trial; Procedures; Production; Reproducibility; Resistance; Sampling; Sensitivity and Specificity; Signal Transduction; Specific qualifier value; Specificity; Specimen; Spectrum Analysis; Standardization; System; Technology; Testing; Time; Tissues; Transference; Translating; Translations; Variant; Viral; Virion; Virus; Virus Latency; antibody detection; breakthrough infection; detection assay; detection method; experience; experimental study; immunoreactivity; innovation; instrument; multiplex assay; neutralizing antibody; next generation; pressure; prospective; prototype; rapid test; research clinical testing; screening; stem; tool; transcriptome sequencing; treatment trial; viral detection; volunteer

Broadly HIV-neutralizing antibodies (bnAbs) with extreme breadth and potency represent promising options for HIV/AIDS treatment, functional cure and/or prevention. However, all bnAb epitope classes exhibit distinct gaps in covering viral Env diversity in populations of people living with HIV. Consequently, prevention trials experience unpredictably restricted efficacy. Further, treatment trials inadvertently enroll infected participants harboring bnAb-resistant variants in plasma and/or tissue compartments. As noted in RFA-AI-22-022, such subjects have little chance of clinical benefit, and therefore negatively skew estimates of efficacy and clinical utility. There is an unmet need for innovative clinical assays that rapidly and prospectively detect bnAb- resistant HIV variants in potential therapy/cure trial participants; and characterize resistance in breakthrough infections in prevention trials. New tests must also meet the additional complexities of testing triple bnAb class combinations, which seems imminent. Current screening approaches do not meet these needs as they are time consuming, labor intensive, technically complex, involve pseudovirus production and testing, and require RNA or DNA sequencing which may be unreliable, depending on the subject and sample. A promising approach toward expediting and expanding neutralization resistance testing is based on direct, quantitative detection of antibody-target binding within a subject’s virus population. The underlying rationale is that neutralization stems from virion binding; resistant variants are by nature poorly (or not) immunoreactive. Thus, a certain fraction of bnAb-unbound virus in a population will presage viral outgrowth/escape during treatment by the test bnAb. Also, bnAb-virion binding can be rapidly and readily quantified. However, this approach demands technology with the sensitivity to quantify bnAb-bound (sensitive) and unbound (resistant) virus fractions in plasma samples or culture fluids, with minimal target perturbations. To date, such technology has not come forward. Our ongoing studies now show that these demands may be met by confocal fluorescence correlation spectroscopy (FCS) techniques for detecting antibody binding at the single virion level. Using fluorescent labeled bnAbs in FCS, we can simultaneously count, in one multiplex assay, the numbers of virions in a sample population that are bound by one or more bnAb types or avoid recognition altogether. Further, virions concurrently bound to multiple bnAb types can be counted by cross- correlated signals. Our goal for this R61/R33 project is to translate this technology into a rapid, economical, multiplex clinical test for blood or culture samples that: 1) determines the presence and proportions of virus sub- populations in a human sample that are covered (or ignored) by one or more bnAbs in a triple class combination and thus reliably and sensitively 2) identifies people living with HIV who harbor resistant viruses risking rebound under bnAb treatment or 3) characterizes the nature of breakthrough infections in prevention trials. The impact of this project will be to advance bnAb resistance detection capacities to support numerous bnAb clinical trial activities, from screening volunteers to tailoring subject-specific bnAb combinations for treatment or cure.

广泛的HIV中和抗体（bnAb）具有极高的广度和效力，代表了治疗HIV感染的有希望的选择。 艾滋病毒/艾滋病治疗、功能性治愈和/或预防。然而，所有bnAb表位类别都表现出不同的缺口， 在覆盖艾滋病毒感染者群体中病毒Env多样性方面。因此，预防性试验的经验 无法预测的有限功效。此外，治疗试验无意中招募了携带病毒的受感染参与者， 血浆和/或组织隔室中的bnAb抗性变体。如RFA-AI-22-022所述，此类受试者 临床获益的可能性很小，因此对疗效和临床效用的估计产生负面影响。有一个 对创新的临床检测方法的需求尚未得到满足，这些方法可以快速、前瞻性地检测HIV耐bnAb变异体， 潜在的治疗/治愈试验参与者;并描述预防中突破性感染的耐药性 审判新的测试还必须满足测试三重bnAb类组合的额外复杂性， 似乎迫在眉睫。目前的筛查方法不能满足这些需求，因为它们耗时、费力， 密集、技术复杂，涉及假病毒生产和测试，并需要RNA或DNA测序 这取决于受试者和样本，可能是不可靠的。一种有希望的方法， 扩大中和抗性测试基于抗体-靶结合的直接定量检测 在受试者的病毒群体中。基本原理是中和作用源于病毒体结合; 抗性变体本质上免疫反应性差（或没有）。因此，一定比例的bnAb未结合的病毒 在群体中的感染将预示在测试bnAb治疗期间病毒生长/逃逸。此外，bnAb-病毒体结合 可以快速且容易地量化。然而，这种方法要求技术具有量化的灵敏度， 血浆样本或培养液中bnAb结合（敏感）和未结合（耐药）病毒组分， 目标扰动到目前为止，这种技术还没有出现。我们正在进行的研究表明， 共聚焦荧光相关光谱（FCS）技术可以满足检测抗体的需求 在单个病毒体水平上结合。在FCS中使用荧光标记的bnAb，我们可以同时计数，在一个 多重测定，样品群体中被一种或多种bnAb类型结合的病毒体的数量，或 完全避免承认。此外，同时结合多种bnAb类型的病毒体可以通过交叉免疫计数来计数。 相关信号我们在R61/R33项目中的目标是将这项技术转化为一种快速、经济、 血液或培养样本的多重临床检测：1）确定病毒亚群的存在和比例 人类样本中被三重分类组合中的一种或多种bnAb覆盖（或忽略）的群体 从而可靠而灵敏地2）识别出携带有耐药性病毒的艾滋病毒感染者， 或3）表征预防试验中突破性感染的性质。的影响 该项目的主要目标是提高bnAb耐药性检测能力，以支持众多的bnAb临床试验 活动，从筛选志愿者到定制用于治疗或治愈的受试者特异性bnAb组合。