Improved quantification of HIV-1-infected CD4+T cells using an optimised method of intracellular HIV-1 gag p24 antigen detection
Improved quantification of HIV-1-infected CD4+T cells using an optimised method of intracellular HIV-1 gag p24 antigen detection
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DOI：
10.1016/j.jim.2013.03.001
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发表时间：
2013-05-31
影响因子：
2.2
通讯作者：
Dorrell, Lucy
中科院分区：
文献类型：
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作者：
Yang, Hongbing;Yorke, Elisabeth;Dorrell, Lucy
CD8+T细胞在体外抑制HIV-1复制的能力与体内病毒控制密切相关。对HIV-1候选疫苗的特别评估表明，这一免疫学参数是疫苗效力的一个有希望的基准。要大规模分析CD8+T细胞的抗病毒活性，需要一种快速、可靠和经济的方法来准确定量原代CD4+T细胞中的HIV-1感染。用流式细胞术检测细胞内HIV-1p24抗原(p24抗原)就是这样一种方法，但它被认为不如p24抗原ELISA法灵敏和定量。我们报告了使用多聚甲醛/50%甲醇/诺尼特P-40而不是传统的基于多聚甲醛/皂素的方案来固定和渗透艾滋病毒感染细胞，提高了它们在从10(-2)到8×10(-5)的多重感染(MOI)范围内的检测能力，并在测试的最佳MOI(10(-2))下提高了近两倍(p<；0.001)。在培养过程中，感染细胞的频率与p24抗原的释放密切相关，因此证实了它作为生产性感染的一种衡量标准。我们还能够量化代表主要分支的一组HIV-1分离株的感染情况。与酶联免疫吸附试验相比，这里描述的方法快速且经济有效，因此可以作为HIV-1疫苗免疫监测和减少病毒库干预的有用组成部分。(C)2013爱思唯尔B.V.保留所有权利。
The capacity of CD8 + T cells to inhibit HIV-1 replication in vitro strongly correlates with virus control in vivo. Post-hoc evaluations of HIV-1 vaccine candidates suggest that this immunological parameter is a promising benchmark of vaccine efficacy. Large-scale analysis of CD8 + T cell antiviral activity requires a rapid, robust and economical assay for accurate quantification of HIV-1 infection in primary CD4 + T cells. Detection of intracellular HIV-1 p24 antigen (p24 Ag) by flow cytometry is one such method but it is thought to be less sensitive and quantitative than p24 Ag ELISA. We report that fixation and permeabilisation of HIV-infected cells using paraformaldehyde/50% methanol/Nonidet P-40 instead of a conventional paraformaldehyde/saponin-based protocol improved their detection across multiplicities of infection (MOI) ranging from 10(-2) to 8 x 10(-5), and by nearly two-fold (p < 0.001) at the optimal MOI tested (10(-2)). The frequency of infected cells was strongly correlated with p24 Ag release during culture, thus validating its use as a measure of productive infection. We were also able to quantify infection with a panel of HIV-1 isolates representing the major clades. The protocol described here is rapid and cost-effective compared with ELISA and thus could be a useful component of immune monitoring of HIV-1 vaccines and interventions to reduce viral reservoirs. (C) 2013 Elsevier B.V. All rights reserved.