A FAST Assay to Quantify HIV Reservoirs

 描述(由申请人提供)：艾滋病毒治愈研究中的一个主要障碍是缺乏可靠的高通量分析来评估宿主的特征和大小。由于需要区分感染有复制能力和复制缺陷的艾滋病毒的细胞，这一挑战变得更加复杂。目的：我们建议通过计算含有HIV DNA的能够表达HIV Gag+和下调CD4的细胞的数量来区分复制能力和缺陷前病毒。我们采用一种名为FAST(光纤阵列扫描技术)的新技术来检测罕见的表达HIV Gag并下调CD4(Gag+CD4dim细胞)的细胞，从而实现高通量检测。FAST是一项成熟的技术，已经被用来以高通量的方式检测罕见的肿瘤细胞(例如，一分钟内2000万个正常细胞中有1个肿瘤细胞)。我们的目标是确定FAST是否可以用于监测针对HIV宿主的治疗。方法：我们首先将FAST方法与O‘Doherty博士开发的FACS分选方法进行比较，以测试我们的FAST方法是否能特异性地测量真实的GAG+CD4dim细胞。在FACS检测中，对GAG+CD4dim细胞进行分选，并通过测量分选群体中的前病毒DNA来确定真阳性的数量。特异性只在GAG+CD4dim细胞中表现出来，而不是在Gagneg细胞中。在目标1中，我们测试FAST识别GAG+CD4dim的特异性，方法是将其与基线时FACS分选结果进行比较。FAST通过可视化内化的CD4的特征点状染色提供了额外的特异性。在目标2中，我们将刺激阴性选择的CD4+T细胞后的速度与FACS进行比较。在目标1和目标2中，我们将通过有限稀释PCR后对分选细胞中的前病毒进行测序来确定Gag+CD4dim细胞表型是否能区分复制能力和缺陷HIV。我们预计，超突变的前病毒将不表达GAG或任何HIV蛋白，大量缺失的前病毒将被禁止表达GAG和/或负责CD4下调的蛋白质。因此，通过选择GAG+CD4dim，我们希望消除大多数有缺陷的前病毒。在目标3中，我们将油藏的快速测量与QVOA和集成水平相关联。对公众健康的意义和相关性：高通量定量分析将允许更快速地评估针对水库的多种治疗方法。我们的初步数据向我们表明，在刺激时表达HIV Gag蛋白和下调CD4的能力将与复制能力有很强的相关性。此外，我们预计该分析的用途将扩展到水库测量之外，因为它可以被用来确定旨在诱导HIV蛋白表达的多种候选疗法的剂量反应、有效性和动力学。

 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.