A FAST Assay to Quantify HIV Reservoirs

量化 HIV 病毒库的快速检测

• 批准号: 8966489
• 负责人: Una T O'Doherty
• 金额: $ 60.64万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-15 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8966489
• 关键词: Address; Biological Assay; CD4 Positive T Lymphocytes; Cell Count; Cell Separation; Cells; Characteristics; Collaborations; Competence; DNA; Data; Detection; Disabled Persons; Dose; Down-Regulation; Drug Design; Evaluation; Fiber Optics; Frequencies; Gagging; Goals; HIV; Kinetics; Letters; Measurement; Measures; Methods; Modeling; Monitor; Mutate; Normal Cell; Patients; Phenotype; Population; Proteins; Proviruses; Public Health; Research; Scanning; Sorting - Cell Movement; Specificity; Speed; Staining method; Stains; T-Lymphocyte; Techniques; Technology; Testing; Virus; cancer cell; gag Gene Products; high throughput screening; high throughput technology; in vivo; neoplastic cell; new technology; novel; protein expression; public health relevance; response; selective expression; targeted treatment; therapy design; tool

 DESCRIPTION (provided by applicant): A major hurdle in HIV cure research is the lack of robust high throughput assays to assess the character and size of the reservoir. This challenge is compounded by the need to distinguish between cells infected with replication competent and replication defective HIV. Objectives: We propose to distinguish replication competent from defective proviruses by counting the number of cells that contain HIV DNA that are capable of expressing HIV Gag+ and down regulating CD4. We achieve high throughput detection by adapting a new technology, called FAST (Fiber-optic Array Scanning Technology), to detect rare cells that express HIV Gag and down regulate CD4 (Gag+CD4dim cells). FAST is a proven technique that has been utilized to detect rare tumor cells in a high throughput fashion (e.g. ~1 tumor cell in 20 million normal cells in one minute). Our goal is to determine if FAST can be used to monitor therapies that target HIV reservoirs. Method: We first compare the FAST assay to a FACS sorting assay developed by Dr. O'Doherty to test if our FAST assay can specifically measure true Gag+CD4dim cells. In the FACS assay, Gag+CD4dim cells are sorted and the number of true positives is determined by measuring proviral DNA in the sorted population. Specificity is demonstrated by showing enrichment for HIV DNA only in the Gag+CD4dim and not in the Gagneg cells. In Aim 1, we test the specificity of FAST to identify Gag+CD4dim by comparing it to the number quantified by FACs sorting in patients at baseline. FAST provides additional specificity by visualizing the characteristic punctate staining of internalized CD4. In Aim 2, we compare FAST to FACS after stimulating negatively-selected CD4+ T cells. In Aims 1 and 2, we will determine if the Gag+CD4dim cell phenotype distinguishes replication competent from defective HIV by sequencing provirus from the sorted cells after limiting dilution PCR. We expect that hyper mutated proviruses will not express Gag or any HIV proteins and proviruses with massive deletions will either be disabled to express Gag and/or the proteins responsible for CD4 down regulation. Thus, by selecting for Gag+CD4dim we expect to eliminate most proviruses that are defective. In Aim 3, we correlate FAST measures of reservoir with QVOA and integration levels. Significance and relevance to public health: A high-throughput quantitative assay would allow more rapid evaluation of multiple therapies that target reservoirs. Our preliminary data suggest to us that the ability to express HIV Gag proteins and down regulate CD4 upon stimulation as in Aim 2 will be a strong correlate of replication competence. Moreover, we envision the utility of the assay will extend beyond reservoir measurement as it can be exploited to determine the dose response, efficacy and kinetics of multiple candidate therapies that are designed to induce HIV protein expression.

 描述（由申请人提供）：HIV 治疗研究的一个主要障碍是缺乏可靠的高通量测定来评估储存库的特征和大小，这一挑战因需要区分具有复制能力和复制缺陷的细胞而变得更加复杂。 HIV 目标：我们建议通过计数含有能够表达 HIV Gag+ 并下调 CD4 的 HIV DNA 的细胞数量来区分复制能力和缺陷原病毒。我们通过采用称为 FAST 的新技术实现高通量检测。 （光纤阵列扫描技术），用于检测表达 HIV Gag 并下调 CD4 的稀有细胞（Gag+CD4dim 细胞），FAST 是一项经过验证的技术，已用于以高通量方式检测稀有肿瘤细胞（例如 1）。我们的目标是确定 FAST 是否可用于监测针对 HIV 储存库的治疗方法：我们首先将 FAST 检测与 FACS 开发的分选检测进行比较。 O'Doherty 博士测试我们的 FAST 测定是否可以特异性测量真正的 Gag+CD4dim 细胞。在 FACS 测定中，对 Gag+CD4dim 细胞进行分选，并通过测量分选群体中的原病毒 DNA 来确定真阳性的数量。通过仅在 Gag+CD4dim 中而非 Gagneg 细胞中显示 HIV DNA 的富集 在目标 1 中，我们测试了 FAST 识别 Gag+CD4dim 的特异性。通过将其与基线时 FAC 分选量化的数量进行比较，通过可视化内化 CD4 的特征性点状染色，我们将 FAST 与刺激阴性选择的 CD4+ T 细胞后的 FACS 进行比较。 2，我们将通过有限稀释 PCR 后对分选细胞中的原病毒进行测序，确定 Gag+CD4dim 细胞表型是否能够区分复制能力和缺陷型 HIV。超突变原病毒不会表达 Gag 或任何 HIV 蛋白，并且具有大量缺失的原病毒将无法表达 Gag 和/或负责 CD4 下调的蛋白。因此，通过选择 Gag+CD4dim，我们希望消除大多数原病毒。在目标 3 中，我们将储存库的 FAST 测量值与 QVOA 和整合水平相关联：高通量定量分析将允许更快速地评估针对储存库的多种疗法。我们的初步数据表明，如目标 2 所示，表达 HIV Gag 蛋白并在刺激后下调 CD4 的能力将与复制能力密切相关。此外，我们预计该测定的实用性将超出储库测量范围。用于确定旨在诱导 HIV 蛋白表达的多种候选疗法的剂量反应、功效和动力学。