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（BNABS）具有极端的广度和效力，代表了有希望的选择 艾滋病毒/艾滋病治疗，功能治愈和/或预防。但是，所有BNAB表位类别都暴露了不同的差距 在涵盖艾滋病毒患者人群中的病毒环境中。因此，预防试验经验 效率不足以预测。此外，治疗试验无意中招募了受感染的参与者 血浆和/或组织室中的BNAB抗性变体。如RFA-AI-22-022中所述，此类主题具有 临床益处的机会很小，因此对有效性和临床实用性的负面估计负有偏见。有一个 未满足的创新临床评估需要快速和前瞻性检测抗BNAB的HIV变体 潜在的治疗/治疗试验参与者；并表征预防突破性感染中的抗药性 试验。新测试还必须满足测试三重BNAB类组合的其他复杂性，这 似乎迫在眉睫。当前的筛查方法不满足这些需求，因为它们耗时，劳动力 密集，技术上复杂，涉及假病毒的生产和测试，需要RNA或DNA测序 这可能是不可靠的，具体取决于主题和样本。一种有前途的加快和 扩展中和抗性测试基于直接的，定量检测抗体 - 靶标结合 在受试者的病毒人群中。基本的理由是病毒结合的中和步骤。 抗性变体本质上是不良的免疫反应性的。这是一定一部分BNAB unbound病毒 在人群中，在测试BNAB治疗期间，将预示病毒的生长/逃生。另外，BNAB视觉结合 可以迅速而容易量化。但是，这种方法需要技术以量化 血浆样品或培养液中的BNAB结合（敏感）和未结合的（抗性）病毒分数，最少 目标扰动。迄今为止，这种技术还没有挺身而出。我们正在进行的研究现在表明这些 通过共聚焦荧光相关光谱（FCS）技术来检测抗体，可以满足需求 在单个病毒素水平上结合。使用FCS中使用荧光标记的BNAB，我们可以轻松地计数 多重分析，样本群体中受一种或多种BNAB类型或多种束缚的病毒数量或 避免完全识别。此外，可以通过交叉来计数与多种BNAB类型结合的病毒 相关信号。我们这个R61/R33项目的目标是将这项技术转化为快速，经济的， 血液或培养样品的多重临床测试：1）确定病毒亚的存在和比例 人类样本中的种群被三类组合中的一个或多个BNAB覆盖（或忽略） 因此可靠，敏感2）确定患有艾滋病毒的人避免了抗反弹的风险 在BNAB治疗或3）预防试验中的突破性感染的性质。影响 该项目将是提高BNAB抵抗检测能力，以支持众多BNAB临床试验 活动，从筛查志愿者到调整特定主题的BNAB组合进行治疗或治愈。