Data_Sheet_3_Improving HIV Outgrowth by Optimizing Cell-Culture Conditions and Supplementing With all-trans Retinoic Acid.doc

The persistence of replication-competent HIV reservoirs in people living with HIV (PLWH) receiving antiretroviral therapy (ART) is a barrier to cure. Therefore, their accurate quantification is essential for evaluating the efficacy of new therapeutic interventions and orienting the decision to interrupt ART. Quantitative viral outgrowth assays (QVOAs) represent the “gold standard” for measuring the size of replication-competent HIV reservoirs. However, they require large numbers of cells and are technically challenging. This justifies the need for the development of novel simplified methods adapted for small biological samples. Herein, we sought to simplify the viral outgrowth procedure (VOP) by (i) using memory CD4+ T-cells, documented to be enriched in HIV reservoirs (ii) optimizing cell-culture conditions, and (iii) supplementing with all-trans retinoic acid (ATRA), a positive regulator of HIV replication. Memory CD4+ T-cells were sorted from the peripheral blood of ART-treated (HIV+ART; n = 14) and untreated (HIV+; n = 5) PLWH. The VOP was first performed with one original replicate of 1 × 106 cells/well in 48-well plates. Cells were stimulated via CD3/CD28 for 3 days, washed to remove residual CD3/CD28 Abs, split every 3 days for optimal cell density, and cultured in the presence or the absence of ATRA for 12 days. Soluble and intracellular HIV-p24 levels were quantified by ELISA and flow cytometry, respectively. Optimal cell-culture density achieved by splitting improved HIV outgrowth detection. ATRA promoted superior/accelerated detection of replication-competent HIV in all HIV+ART individuals tested, including those with low/undetectable viral outgrowth in the absence of ATRA. Finally, this VOP was used to design a simplified ATRA-based QVOA by including 4 and 6 original replicates of 1 × 106 cells/well in 48-well plates and 2 × 105 cells/well in 96-well plates, respectively. Consistently, the number of infectious units per million cells (IUPM) was significantly increased in the presence of ATRA. In conclusion, we demonstrate that memory CD4+ T-cell splitting for optimal density in culture and ATRA supplementation significantly improved the efficacy of HIV outgrowth in a simplified ATRA-based QVOA performed in the absence of feeder/target cells or indicator cell lines.

接受抗逆转录病毒治疗（antiretroviral therapy, ART）的艾滋病病毒感染者（People Living with HIV, PLWH）体内，具有复制能力的HIV储存库持续存留，这是阻碍艾滋病治愈的核心难题之一。因此，对该储存库进行精准定量，对于评估新型治疗干预策略的疗效、指导ART中断决策均至关重要。定量病毒生长测定法（Quantitative viral outgrowth assays, QVOAs）堪称衡量具有复制能力的HIV储存库规模的“金标准”，但该方法需耗费大量细胞，且技术门槛较高，这推动了适配小型生物样本的新型简化检测方法的开发需求。本研究旨在简化病毒生长程序（viral outgrowth procedure, VOP），具体通过以下三项策略实现：其一，采用已被证实富含HIV储存库的记忆CD4+ T细胞；其二，优化细胞培养条件；其三，添加全反式维甲酸（all-trans retinoic acid, ATRA）——一种可正向调控HIV复制的小分子物质。我们从接受ART治疗的PLWH（HIV+ART组，样本量n=14）及未接受ART治疗的PLWH（HIV+组，样本量n=5）的外周血中分选得到记忆CD4+ T细胞。首先按照原始方案开展VOP：在48孔板中以每孔1×10^6个细胞设置1个复孔，用CD3/CD28抗体刺激细胞3天，随后洗涤去除残留的CD3/CD28抗体，每3天进行一次传代以维持最佳细胞密度，并在添加或不添加ATRA的条件下培养12天。分别通过酶联免疫吸附试验（ELISA）与流式细胞术定量检测可溶性及细胞内HIV-p24抗原水平。通过传代维持最佳细胞密度，可显著提升HIV生长检测的检出效能；ATRA可促进所有受试HIV+ART组个体中具有复制能力的HIV的检出，且可加速检测进程，即使是那些在无ATRA条件下病毒生长检出水平较低或无法检出的个体亦是如此。最后，基于该优化后的VOP方案，我们设计了一款基于ATRA的简化版QVOA：分别在48孔板中设置4个、6个原始复孔（每孔1×10^6个细胞），以及在96孔板中设置每孔2×10^5个细胞的复孔。一致性结果显示，在添加ATRA的条件下，每百万细胞感染单位（infectious units per million cells, IUPM）数值显著升高。综上，本研究证实，通过传代维持培养细胞的最佳密度，并添加ATRA，可显著提升HIV生长检测的效能；所构建的基于ATRA的简化版QVOA，无需饲养细胞/靶细胞或指示细胞系即可完成检测。