Morón-López Sara, Puertas Maria C, Gálvez Cristina, Navarro Jordi, Carrasco Anna, Esteve Maria, Manyé Josep, Crespo Manel, Salgado Maria, Martinez-Picado Javier
AIDS Research Institute IrsiCaixa, Institut d'Investigació en Cièncias de la Salut Germans Trias i Pujol, Universitat Autònoma de Barcelona, Badalona, Spain.
Infectious Diseases Department, Hospital Universitari Vall d'Hebron, Barcelona, Spain.
PLoS One. 2017 Apr 17;12(4):e0175899. doi: 10.1371/journal.pone.0175899. eCollection 2017.
The implementation of successful strategies to achieve an HIV cure has become a priority in HIV research. However, the current location and size of HIV reservoirs is still unknown since there are limited tools to evaluate HIV latency in viral sanctuaries such as gut-associated lymphoid tissue (GALT). As reported in the so called "Boston Patients", despite undetectable levels of proviral HIV-1 DNA in blood and GALT, viral rebound happens in just few months after ART interruption. This fact might imply that current methods are not sensitive enough to detect residual reservoirs. Showing that, it is imperative to improve the detection and quantification of HIV-1 reservoir in tissue samples. Herein, we propose a novel non-enzymatic protocol for purification of Lamina Propria Leukocytes (LPL) from gut biopsies combined to viral HIV DNA (vDNA) quantification by droplet digital PCR (ddPCR) to improve the sensitivity and accuracy of viral reservoir measurements (LPL-vDNA assay).
Endoscopic ileum biopsies were sampled from 12 HIV-1-infected cART-suppressed subjects. We performed a DTT/EDTA-based treatment for epithelial layer removal followed by non-enzymatic disruption of the tissue to obtain lamina propria cell suspension (LP). CD45+ cells were subsequently purified by flow sorting and vDNA was determined by ddPCR.
vDNA quantification levels were significantly higher in purified LPLs (CD45+) than in bulk LPs (p<0.01). The levels of vDNA were higher in ileum samples than in concurrent PBMC from the same individuals (p = 0.002). As a result of the increased sensitivity of this purification method, the Poisson 95% confidence intervals of the vDNA quantification data from LPLs were narrower than that from bulk LPs. Of note, vDNA was unambiguously quantified above the detection limit in 100% of LPL samples, while only in 58% of bulk LPs.
We propose an innovative combined protocol for a more sensitive detection of the HIV reservoir in gut-associated viral sanctuaries, which might be used to evaluate any proposed eradication strategy.
实施成功的策略以实现治愈艾滋病已成为艾滋病研究的一个优先事项。然而，由于评估肠道相关淋巴组织（GALT）等病毒庇护所中艾滋病病毒潜伏状态的工具有限，目前艾滋病病毒储存库的位置和大小仍然未知。正如在所谓的“波士顿患者”中所报道的那样，尽管血液和GALT中前病毒HIV-1 DNA水平检测不到，但在抗逆转录病毒治疗中断后的短短几个月内病毒就会反弹。这一事实可能意味着目前的方法对检测残留储存库不够敏感。由此可见，改进组织样本中HIV-1储存库的检测和定量至关重要。在此，我们提出一种新的非酶法方案，用于从肠道活检组织中纯化固有层白细胞（LPL），并结合液滴数字PCR（ddPCR）对病毒HIV DNA（vDNA）进行定量，以提高病毒储存库测量的灵敏度和准确性（LPL-vDNA检测法）。
从12名接受cART抑制治疗的HIV-1感染受试者中采集内镜下回肠活检组织。我们采用基于二硫苏糖醇/乙二胺四乙酸（DTT/EDTA）的处理方法去除上皮层，随后对组织进行非酶处理以获得固有层细胞悬液（LP）。随后通过流式分选纯化CD45+细胞，并通过ddPCR测定vDNA。
纯化的LPL（CD45+）中的vDNA定量水平显著高于总体LP中的水平（p<0.01）。回肠样本中的vDNA水平高于同一受试者同期的外周血单个核细胞（PBMC）中的水平（p = 0.002）。由于这种纯化方法的灵敏度提高，LPL的vDNA定量数据的泊松95%置信区间比总体LP的更窄。值得注意的是，100%的LPL样本中的vDNA在检测限以上得到明确量化，而总体LP中只有58%。
我们提出了一种创新的联合方案，用于更灵敏地检测肠道相关病毒庇护所中的艾滋病病毒储存库，该方案可用于评估任何提议的根除策略。
