Kuzmichev Yury V, Veenhuis Rebecca T, Pohlmeyer Christopher W, Garliss Caroline C, Walker-Sperling Victoria Ek, Blankson Joel N
Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA.
Center for AIDS Research, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
J Virus Erad. 2017 Apr 1;3(2):85-89. doi: 10.1016/S2055-6640(20)30292-2.
Latently infected resting CD4 T cells represent a major barrier to HIV-1 eradication efforts. The standard assays used for measuring this reservoir induce activation of resting CD4 T cells with either phytohaemagglutinin (PHA) with irradiated feeder cells, or with anti-CD3 antibodies. We designed a study to compare the sensitivity of a new assay (based on the stimulation of CD4 T cells with anti-CD3 and anti-CD28 coated microbeads) with that of the traditional PHA- and feeder-based viral outgrowth assay.
Resting CD4 T cells from 10 HIV-1-infected patients on suppressive combination antiretroviral therapy (cART) regimens were cultured in the traditional PHA/feeders viral outgrowth assay and the new CD3/CD28 bead-based assay. Flow cytometry was used to assess the kinetics of activation of resting CD4 T cells in the two different assays.
There was no significant difference in the sensitivity of the two assays. The median frequency of latently infected cells was 0.83 infectious units per million (IUPM) for the PHA/feeders assay and 0.54 IUPM with the CD3/CD28 bead-based assay. However, while virus was obtained from all 10 patients with the traditional PHA/feeders outgrowth assay, no virus was obtained from two of 10 patients with the novel anti-CD3/CD28 bead-based viral outgrowth assay (IUPM < 0.02).
The new CD3/CD28 bead-based assay has comparable sensitivity to the PHA/feeders assay and does not require the addition of feeders, making it a simpler and less labour-intensive alternative to the standard PHA/feeders-based assay.
潜伏感染的静息CD4 T细胞是根除HIV-1努力的主要障碍。用于测量该病毒库的标准检测方法会用植物血凝素（PHA）加辐照饲养细胞或抗CD3抗体诱导静息CD4 T细胞活化。我们设计了一项研究，比较一种新检测方法（基于用抗CD3和抗CD28包被的微珠刺激CD4 T细胞）与传统的基于PHA和饲养细胞的病毒增殖检测方法的敏感性。
对10名接受抑制性联合抗逆转录病毒疗法（cART）方案治疗的HIV-1感染患者的静息CD4 T细胞进行传统的PHA/饲养细胞病毒增殖检测和新的基于CD3/CD28微珠的检测。采用流式细胞术评估两种不同检测方法中静息CD4 T细胞的活化动力学。
两种检测方法的敏感性无显著差异。PHA/饲养细胞检测中潜伏感染细胞的中位频率为每百万0.83个感染单位（IUPM），基于CD3/CD28微珠的检测为0.54 IUPM。然而，虽然在传统的PHA/饲养细胞增殖检测中从所有10名患者中都获得了病毒，但在新的基于抗CD3/CD28微珠的病毒增殖检测中，10名患者中有两名未获得病毒（IUPM < 0.02）。
新的基于CD3/CD28微珠的检测方法与PHA/饲养细胞检测方法具有相当的敏感性，且不需要添加饲养细胞，使其成为标准的基于PHA/饲养细胞检测方法的一种更简单、劳动强度更低的替代方法。
