A FAST Assay to Quantify HIV Reservoirs

 描述（由申请人提供）：HIV治愈研究的一个主要障碍是缺乏可靠的高通量测定来评估储库的特征和大小。这一挑战由于需要区分感染有复制能力和复制缺陷型HIV的细胞而变得更加复杂。目的：我们建议通过计数含有能够表达HIV Gag+和下调CD 4的HIV DNA的细胞数量来区分有复制能力的前病毒和有缺陷的前病毒。我们通过采用一种名为FAST（光纤阵列扫描技术）的新技术来实现高通量检测，以检测表达HIV Gag并下调CD 4的罕见细胞（Gag+ CD 4dim细胞）。FAST是一种已被证实的技术，已被用于以高通量方式检测罕见肿瘤细胞（例如，在一分钟内，2000万个正常细胞中约有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测定与由Dr. O 'Doherty开发的FACS分选测定进行比较，以测试我们的FAST测定是否可以特异性地测量真正的Gag+ CD 4dim细胞。在FACS测定中，对Gag+ CD 4dim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。通过显示HIV DNA仅在Gag+ CD 4dim中富集而不在Gagneg细胞中富集来证明特异性。在目标1中，我们通过将其与基线时患者中通过FACs分选定量的数量进行比较来测试FAST鉴定Gag+ CD 4dim的特异性。FAST通过可视化内化的CD 4的特征性点状染色提供额外的特异性。在目标2中，我们在刺激阴性选择的CD 4 + T细胞后比较FAST和FACS。在目标1和2中，我们将通过对有限稀释PCR后分选细胞的前病毒进行测序，确定Gag+ CD 4dim细胞表型是否区分有复制能力的HIV和有缺陷的HIV。我们预期高度突变的前病毒将不表达Gag或任何HIV蛋白，并且具有大量缺失的前病毒将不能表达Gag和/或负责CD 4下调的蛋白。因此，通过选择Gag+ CD 4dim，我们期望消除大多数有缺陷的前病毒。在目标3中，我们将储层的FAST测量与QVOA和积分水平相关联。对公共卫生的意义和相关性：高通量定量测定将允许更快速地评估靶向储库的多种疗法。我们的初步数据向我们表明，表达HIV Gag蛋白并在Aim 2中刺激后下调CD 4的能力将与复制能力密切相关。此外，我们设想该测定的实用性将扩展到储库测量之外，因为它可以用于确定设计用于诱导HIV蛋白表达的多种候选疗法的剂量反应、功效和动力学。

 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.