1 INTRODUCTION

Since the identification of human immunodeficiency virus 1 (HIV-1) as the pathogen responsible for acquired immunodeficiency syndrome (AIDS) in the early 1980s, treatment options have come a long way. Nonetheless, while AIDS is currently treatable with anti-retroviral therapy (ART), HIV-1 infection cannot be cured at present. Once a patient has been infected with HIV-1, the virus forms a latent reservoir that is kept quiescent by a cocktail of different anti-retroviral agents.

HIV-1 preferentially infects CD4⁺ T cells, dendritic cells and monocytes/macrophages, which is mediated through cellular entry via CXCR4 (by so-called X4-tropic strains), CCR5 (by R5-tropic strains), or a combination thereof (by X4R5-tropic strains). After cellular infection and reverse transcription, the viral genome integrates into the host genome, where it can give rise to new viral particles, but also forms a latent reservoir [1]. Several different techniques are currently in use to diagnose and monitor HIV-1 infection. These techniques allow for the measurement of (cell-free) viral genetic material via (digital droplet [dd]) PCR or the Tat/rev induced limiting dilution assay (TILDA), the presence of HIV-1 virions or anti-HIV-1 antibodies via ELISA, or the quantification of replication-competent proviruses through limiting dilutions of isolated CD4⁺ T cells from HIV-1 individuals in a quantitative viral outgrowth assay (QVOA) [2, 3] (Figure 1). These techniques only detect either (cell-free) viral genomic material, viral proteins, virions or integrated viral DNA, and do not provide information about tropism, the percentage of infected cells, or whether translation of viral genomic material is currently ongoing. Furthermore, with the exception of TILDA, these bulk measurements are not suitable to detect latently infected cells.

Recent advances in flow cytometry have enabled the detection of transcriptionally active virus in HIV-1 infected cells by using fluorescent RNA probes targeting HIV-1 RNA, that is, Gagpol mRNA. As this resulted in high false positive rates that made this approach unsuitable to detect low frequencies of infected cells, the addition of a Gag or p24 protein staining was included [4-6]. This concomitant measurement of viral RNA and viral protein is also known as HIV-1 flow cytometry-based fluorescence in situ hybridization (Flow-FISH). While RNA-mediated assessments such as TILDA allow for the detection of low frequencies of infected calls and latently infected cells [7], it does not provide information on a single cell basis. In contrast, due to the single cell approach, Flow-FISH can be used to analyze which cell population(s) are infected in a patient, and detect ongoing HIV-1 transcription in single cells harboring integrated viral genomic information (Figure 1). Furthermore, Flow-FISH analysis is highly customizable and allows for the assessment of other cell markers of interest. Of note, results obtained with HIV-1 Flow-FISH correlate with plasma viral load, intracellular HIV-1 RNA and pro-viral DNA quantified by conventional (diagnostic) methods in CD4⁺ T cells from both ART-treated and untreated HIV-1 infected patients [5].

中文翻译：

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使用 Flow-FISH 解决 HIV-1 潜伏期：寻找、表征和靶向 HIV-1 感染细胞

1 介绍

自从 1980 年代初期人类免疫缺陷病毒 1 (HIV-1) 被确定为导致获得性免疫缺陷综合征 (AIDS) 的病原体以来，治疗选择已经取得了长足的进步。尽管如此，虽然艾滋病目前可以通过抗逆转录病毒疗法 (ART) 进行治疗，但 HIV-1 感染目前无法治愈。一旦患者感染了 HIV-1，病毒就会形成一个潜伏的储存库，通过混合不同的抗逆转录病毒药物保持静止。

HIV-1 优先感染 CD4 ⁺ T 细胞、树突细胞和单核细胞/巨噬细胞，这是通过 CXCR4（通过所谓的 X4 型菌株）、CCR5（通过 R5 型菌株）或其组合进入细胞介导的。由 X4R5 热带菌株）。在细胞感染和逆转录后，病毒基因组整合到宿主基因组中，在那里它可以产生新的病毒颗粒，但也形成一个潜在的储存库 [ 1 ]。目前正在使用几种不同的技术来诊断和监测 HIV-1 感染。这些技术允许通过（数字液滴 [dd]）PCR 或Tat / rev测量（无细胞）病毒遗传物质诱导有限稀释试验 (TILDA)，通过 ELISA 检测 HIV-1 病毒粒子或抗 HIV-1 抗体的存在，或通过有限稀释来自 HIV-1 个体的分离的 CD4 ⁺ T 细胞定量有限稀释有复制能力的原病毒病毒生长试验 (QVOA) [ 2, 3 ]（图 1）。这些技术仅检测（无细胞）病毒基因组材料、病毒蛋白、病毒粒子或整合的病毒 DNA，并且不提供有关趋向性、受感染细胞的百分比或病毒基因组材料的翻译当前是否正在进行的信息。此外，除 TILDA 外，这些批量测量不适用于检测潜伏感染的细胞。

流式细胞术的最新进展已经能够通过使用针对 HIV-1 RNA（即Gagpol mRNA）的荧光 RNA 探针来检测 HIV-1 感染细胞中的转录活性病毒。由于这会导致高假阳性率，使这种方法不适合检测低频率的感染细胞，因此加入了 Gag 或 p24 蛋白染色 [ 4-6 ]。这种病毒 RNA 和病毒蛋白的伴随测量也称为基于 HIV-1 流式细胞术的荧光原位杂交 (Flow-FISH)。虽然 TILDA 等 RNA 介导的评估允许检测低频率的感染调用和潜伏感染细胞 [ 7]]，它不提供基于单个单元格的信息。相比之下，由于采用单细胞方法，Flow-FISH 可用于分析患者体内感染了哪些细胞群，并检测包含整合病毒基因组信息的单细胞中正在进行的 HIV-1 转录（图 1）。此外，Flow-FISH 分析是高度可定制的，并允许评估其他感兴趣的细胞标记。值得注意的是，使用 HIV-1 Flow-FISH 获得的结果与血浆病毒载量、细胞内 HIV-1 RNA 和前病毒 DNA 相关，通过常规（诊断）方法对来自 ART 治疗和未治疗 HIV-1 的CD4 ⁺ T 细胞进行量化感染患者[ 5 ]。