Measuring the Inducible, Replication-Competent HIV Reservoir Using an Ultra-Sensitive p24 Readout, the Digital ELISA Viral Outgrowth Assay
Measuring the Inducible, Replication-Competent HIV Reservoir Using an Ultra-Sensitive p24 Readout, the Digital ELISA Viral Outgrowth Assay
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DOI：
10.3389/fimmu.2020.01971
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发表时间：
2020-08-06
影响因子：
7.3
通讯作者：
Archin, Nancie M.
中科院分区：
文献类型：
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作者：
Stuelke, Erin L.;James, Katherine S.;Archin, Nancie M.
定量可诱导的HIV库提供了对人类和动物模型中静止HIV感染细胞频率的估计，并有助于确定潜伏期逆转剂（LRA）的疗效。定量病毒生长试验（QVOA）用于测量可诱导、可复制的HIV，并估计储存库大小。然而，传统的QVOA是时间和劳动密集型的，并且需要大量的淋巴细胞。考虑到在治疗策略中对储层大小和LRA活性进行可再现和准确评估的重要性，简化QVOA的工作具有高度优先性。我们开发了一种改良的QVOA，数字ELISA病毒生长或DEVO测定，具有超灵敏的p24读数，能够对HIV p24蛋白进行毫微微克检测，与传统ELISA的皮克限制相反。对于每个DEVO测定，将来自病毒血症、ART治疗的HIV +参与者的8-12 x 10（6）个静息CD 4 + T细胞以有限稀释度铺板，并用PHA、IL-2和未感染的同种异体照射PBMC最大程度地刺激。来自未感染供体的CD 8耗尽的PHA母细胞或HIV允许细胞（例如，Molt 4/CCR 5）添加到培养物中，并允许病毒扩增8-12天。在第8天通过在第12天确认的Simoa（单分子阵列，超灵敏p24测定）测量来自培养物上清液的HIV p24，并使用最大似然法计算每百万CD 4 + T细胞的感染单位（IUPM）。在进行的所有DEVO试验中，早在刺激后8天就在培养物上清液中检测到HIV p24。重要的是，第8天的DEVO IUPM值与第15天获得的传统QVOA IUPM值相当或更高。有趣的是，在添加或不添加同种异体CD 8耗尽的靶PHA母细胞或传统上用于扩增病毒的HIV容许细胞的情况下，DEVO IUPM值相似。与标准QVOA相比，DEVO检测试剂盒使用更少的静息CD 4 + T细胞，并在更短的时间内提供储库大小评估。该检测提供了一个新的平台，以量化有限的细胞可用性期间的复制能力的HIV。其他潜在的应用包括评估LRA活性，并在潜伏期清除试验期间测量感染细胞的清除率。
Quantifying the inducible HIV reservoir provides an estimate of the frequency of quiescent HIV-infected cells in humans as well as in animal models, and can help ascertain the efficacy of latency reversing agents (LRAs). The quantitative viral outgrowth assay (QVOA) is used to measure inducible, replication competent HIV and generate estimations of reservoir size. However, traditional QVOA is time and labor intensive and requires large amounts of lymphocytes. Given the importance of reproducible and accurate assessment of both reservoir size and LRA activity in cure strategies, efforts to streamline the QVOA are of high priority. We developed a modified QVOA, the Digital ELISA Viral Outgrowth or DEVO assay, with ultra-sensitive p24 readout, capable of femtogram detection of HIV p24 protein in contrast to the picogram limitations of traditional ELISA. For each DEVO assay, 8-12 x 10(6)resting CD4 + T cells from aviremic, ART-treated HIV + participants are plated in limiting dilution and maximally stimulated with PHA, IL-2 and uninfected allogeneic irradiated PBMC. CD8-depleted PHA blasts from an uninfected donor or HIV-permissive cells (e.g., Molt4/CCR5) are added to the cultures and virus allowed to amplify for 8-12 days. HIV p24 from culture supernatant is measured at day 8 by Simoa (single molecule array, ultra-sensitive p24 assay) confirmed at day 12, and infectious units per million CD4 + T cells (IUPM) are calculated using the maximum likelihood method. In all DEVO assays performed, HIV p24 was detected in the supernatant of cultures as early as 8 days post stimulation. Importantly, DEVO IUPM values at day 8 were comparable or higher than traditional QVOA IUPM values obtained at day 15. Interestingly, DEVO IUPM values were similar with or without the addition of allogeneic CD8-depleted target PHA blasts or HIV permissive cells traditionally used to expand virus. The DEVO assay uses fewer resting CD4 + T cells and provides an assessment of reservoir size in less time than standard QVOA. This assay offers a new platform to quantify replication competent HIV during limited cell availability. Other potential applications include evaluating LRA activity, and measuring clearance of infected cells during latency clearance assays.