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

广泛地说，具有极端广度和效力的艾滋病毒中和抗体(BNAbs)代表着有希望的选择 艾滋病毒/艾滋病治疗、功能性治疗和/或预防。然而，所有的bNab表位类都显示出明显的差距 在报道艾滋病毒携带者人群中的病毒环境多样性方面。因此，预防试验的经验 无法预测的疗效限制。此外，治疗试验无意中招募了窝藏 血浆和/或组织隔间中的抗Nab变异体。如RFA-AI-22-022中所述，这些对象具有 临床获益的可能性很小，因此对疗效和临床效用的估计是负面的。有一个 对创新的临床检测的需求尚未得到满足，这种检测方法可以快速和前瞻性地检测出对bNab耐药的艾滋病毒变异株 潜在的治疗/治愈试验参与者；以及在预防中突破性感染的耐药性特征 审判。新的测试还必须满足测试三个bNab类组合的额外复杂性，这 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 密集的、技术复杂的，涉及假病毒的生产和测试，并需要RNA或DNA测序 这可能是不可靠的，这取决于受试者和样本。一种有希望的方法，以加快和 扩大中和抵抗力测试是基于抗体-靶结合的直接、定量检测 在受试者的病毒种群中。其基本原理是中和源于病毒粒子的结合； 耐药变异体本质上免疫反应弱(或不)。因此，一定部分的bNab未结合病毒 在试验bNab治疗期间，人群中的病毒将预示病毒的生长/逃逸。此外，bNab-病毒粒子结合 可以快速而容易地量化。然而，这种方法需要具有量化敏感性的技术 血浆样本或培养液中的bNab结合(敏感)和非结合(耐药)病毒组分，其最低限度 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，这些 可以通过共聚焦荧光相关光谱(FCS)技术来检测抗体来满足需求 在单个病毒粒子水平上结合。在FCS中使用荧光标记的bNAbs，我们可以同时计数，在一个 多重检测，样本群体中与一种或多种bNab类型或 完全避免被认出。此外，同时与多种bNab类型结合的病毒粒子可以通过交叉计数- 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、 血液或培养样本的多重临床检测：1)确定亚型病毒的存在和比例 人类样本中被三重类组合中的一个或多个bNAb覆盖(或忽略)的群体 从而可靠而敏感地识别携带有反弹风险的抗药性病毒的艾滋病毒携带者 在bNab治疗下或3)在预防试验中表征突破性感染的性质。其影响 该项目的重点是提高bNab耐药性检测能力，以支持众多bNab临床试验 活动，从筛选志愿者到为治疗或治愈量身定做特定对象的bNab组合。

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.