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（由应用程序提供）：艾滋病毒治疗研究的主要障碍是缺乏可靠的高吞吐量测定法来评估水库的特征和大小。需要区分感染复制能力和复制缺陷HIV的细胞的需要，使这一挑战更加复杂。目的：我们建议通过计算能够表达HIV GAG+和Down调节CD4的HIV DNA的细胞数来区分有缺陷的PREFIRES和有缺陷的Provirus。我们通过适应一种称为FAST（光纤阵列扫描技术）的新技术来实现高吞吐量检测，以检测表达HIV GAG并调节CD4（GAG+CD4DIM细胞）的稀有细胞。 FAST是一种经过验证的技术，已被用于以高通量方式检测稀有肿瘤细胞（例如，一分钟内有2000万个正常细胞中的〜1个肿瘤细胞）。我们的目标是确定是否可以使用快速来监测针对HIV储层的疗法。方法：我们首先将快速测定与O'Doherty博士开发的FACS分类测定法进行比较，以测试我们的快速测定是否可以专门测量真正的GAG+CD4DIM细胞。在FACS测定中，对GAG+CD4DIM细胞进行分类，并通过测量分类群体中的临时DNA来确定真实阳性的数量。通过仅在GAG+CD4DIM中而不是在Gagneg细胞中显示HIV DNA的富集来证明特异性。在AIM 1中，我们通过将FAST的特异性与基线患者中FACS分类量化的数量进行比较来测试识别GAG+CD4DIM的特异性。 FAST通过可视化内部CD4的特征点状染色来提供额外的特异性。在AIM 2中，我们在刺激否定性选择的CD4+ T细胞后将其快速与FACS进行了比较。在AIMS 1和2中，我们将确定GAG+CD4DIM细胞表型是否通过在限制稀释pCR后与分类的细胞进行测序，将复制与有缺陷的HIV区分开。我们预计，超突变病毒不会表达插科打gog，或任何具有大量缺失的艾滋病毒蛋白和病毒蛋白和病毒蛋白会被禁用以表达堵嘴和/或负责CD4调节的蛋白质。通过选择GAG+CD4DIM，我们希望消除大多数有缺陷的原病毒。在AIM 3中，我们将储层的快速测量与QVOA和集成水平相关联。与公共卫生的意义和相关性：高通量定量评估将允许对靶向储层的多种疗法进行更快速的评估。我们的初步数据向我们表明，在AIM 2中，表达HIV GAG蛋白和向下调节CD4的能力将是复制能力的密切相关性。此外，我们设想该测定的实用性将延伸到Reserver测量之外，因为它可以探索以确定旨在诱导HIV蛋白表达的多种候选疗法的剂量反应，效率和动力学。