DataSheet_2_Reliable Estimation of CD8 T Cell Inhibition of In Vitro HIV-1 Replication.docx

The HIV-1 viral inhibition assay (VIA) measures CD8 T cell-mediated inhibition of HIV replication in CD4 T cells and is increasingly used for clinical testing of HIV vaccines and immunotherapies. The VIA has multiple sources of variability arising from in vitro HIV infection and co-culture of two T cell populations. Here, we describe multiple modifications to a 7-day VIA protocol, the most impactful being the introduction of independent replicate cultures for both HIV infected-CD4 (HIV-CD4) and HIV-CD4:CD8 T cell cultures. Virus inhibition was quantified using a ratio of weighted averages of p24+ cells in replicate cultures and the corresponding 95% confidence interval. An Excel template is provided to facilitate calculations. Virus inhibition was higher in people living with HIV suppressed on antiretroviral therapy (n=14, mean: 40.0%, median: 43.8%, range: 8.2 to 73.3%; p < 0.0001, two-tailed, exact Mann-Whitney test) compared to HIV-seronegative donors (n = 21, mean: -13.7%, median: -14.4%, range: -49.9 to 20.9%) and was stable over time (n = 6, mean %COV 9.4%, range 0.9 to 17.3%). Cross-sectional data were used to define 8% inhibition as the threshold to confidently detect specific CD8 T cell activity and determine the minimum number of culture replicates and p24+ cells needed to have 90% statistical power to detect this threshold. Last, we note that, in HIV seronegative donors, the addition of CD8 T cells to HIV infected CD4 T cells consistently increased HIV replication, though the level of increase varied markedly between donors. This co-culture effect may contribute to the weak correlations observed between CD8 T cell VIA and other measures of HIV-specific CD8 T cell function.

HIV-1病毒抑制试验（HIV-1 viral inhibition assay, VIA）可检测CD8阳性T细胞（CD8 T cell）介导的CD4阳性T细胞（CD4 T cell）内HIV复制抑制效果，目前已愈发广泛地应用于HIV疫苗与免疫治疗的临床检测。该试验存在多种可变来源，均源于体外HIV感染过程以及两种T细胞群体的共培养操作。本研究对为期7天的VIA实验方案进行了多项优化，其中最具影响力的改进为：为HIV感染的CD4阳性T细胞（HIV-CD4）以及HIV-CD4与CD8阳性T细胞共培养体系均设置独立重复培养孔。病毒抑制活性通过重复培养孔中p24阳性细胞（p24+ cells）的加权平均值之比，结合对应的95%置信区间进行量化。本研究附带Excel计算模板，以简化数据分析流程。接受抗逆转录病毒治疗且病毒得到抑制的HIV感染者（n=14，平均值：40.0%，中位数：43.8%，范围：8.2%~73.3%）的病毒抑制活性，显著高于HIV血清阴性供体（HIV-seronegative donors，n=21，平均值：-13.7%，中位数：-14.4%，范围：-49.9%~20.9%），两组差异具有统计学意义（双侧精确曼-惠特尼检验（two-tailed, exact Mann-Whitney test），p<0.0001）；且该病毒抑制活性在随访中保持稳定（n=6，平均变异系数%COV为9.4%，范围0.9%~17.3%）。本研究利用横断面数据（cross-sectional data），将8%的病毒抑制率设定为可可靠检测特异性CD8阳性T细胞活性的阈值，并明确了达到90%统计效力以检测该阈值所需的最小培养重复孔数与p24阳性细胞数。最后，本研究观察到：在HIV血清阴性供体中，向HIV感染的CD4阳性T细胞中加入CD8阳性T细胞，会持续提升HIV的复制水平，不过不同供体间的提升幅度差异显著。这种共培养效应或许可以解释CD8阳性T细胞VIA检测结果与其他HIV特异性CD8阳性T细胞功能检测指标之间相关性较弱的现象。