Ethics statement
This study was approved by the Institutional Review Board of the National Institute of Respiratory Diseases (INER) (registry: DHHS IORG0003948; CONBIOETICA-09-CEI-003-20160427) in Mexico City (project code: B26-09), and was conducted according to the principles of the Declaration of Helsinki. All participants gave written informed consent before blood sample donation.
Patients
This study included two parallel designs, longitudinal and cross-sectional. Longitudinal blood samples from 12 HIV-infected persons with a short history of suppressive ART (21–30 months on ART and viral load 40 copies/mL) were obtained in -month intervals for 2 years. Cross-sectional blood samples from ten additional HIV-infected persons with a long history of suppressive ART (6–15 years) were obtained. All blood samples were obtained and processed at the Center for Research in Infectious Diseases (CIENI) of INER in Mexico City. Plasma and DNA of total CD4+ T cells were stored from each blood sample donation. Plasma viral loads for clinical follow-up were determined using the m2000 system (Abbott, Abbott Park, IL, USA). CD4+ and CD8+ T cell counts were assessed by flow cytometry using the TruCount kit in a FACSCanto II instrument (BD Bioscience, San Jose, CA, USA).
Residual plasma viral load measurement
Viral RNA was extracted from 9 mL of plasma. Plasma was centrifuged at 15,000 rpm for 3 h, at 4 °C. The supernatant was removed and the pellet re-suspended in 140 µL. Viral RNA was purified from the pellet using QIAamp viral RNA mini kit (QIAGEN, Valencia, CA, USA; resuspending in 41 µL of elution buffer) and stored at -80 °C until use. Residual plasma viral load (<40 RNA copies/mL)was determined by real-time PCR (qPCR) with TaqMan Fast Virus One-Step Master Mix (ThermoFisher, Waltham, MA, USA) using an in-house NL4-3-derived RNA standard. NL4-3 RNA was extracted from culture supernatants (QIAamp viral RNA mini kit, QIAGEN) and quantified (m2000 system, Abbott). The standard curve was generated performing two-fold dilutions ranging from 256 to 1 RNA copies. qPCR was performed in triplicate for standard curve points and duplicate for sample points using primers Fwd 5′-GGTCTCTCTGGTTAGACCAGAT-3′ (HXB2 positions: 455–476), Rv 5′-CTGCTAGAGATTTTCCACACTG-3′ (635–614), and the TaqMan probe 5′-6FAM-AGTAGTGTGTGCCCGTCTGTT-TAMRA-3′ (552–572), directed to the viral LTR, in 50 µL reactions containing 20.5 μL of standard or sample RNA, on a 7500 Real Time PCR System instrument (ThermoFisher). qPCR conditions were as follows: 50 °C for 50 min for reverse transcription, 95 °C for 20 s, followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s.
Proviral load measurement from total CD4+ cells
Peripheral blood mononuclear cells (PBMC) were obtained from 16 mL of blood by Ficoll gradient centrifugation. Total CD4+ T cells were purified by negative selection from 50 million PBMC, using EasySep Human CD4+ T Cell kit (StemCell Technologies, Vancouver, Canada), and DNA was purified using iPrep Purelink gDNA Blood kit (ThermoFisher) on an iPrep Nucleic Acid Purification System (ThermoFisher). Proviral load was measured from total CD4+ T cells using TaqMan Gene Expression Master Mix (ThermoFisher). An in-house DNA standard derived from the ACH-2 cell line (obtained through the National Institutes of Health AIDS Reagent Program; catalogue 349) [50], containing two HIV proviral copies per cell, was used [51, 52]. To construct the standard curve, DNA was extracted from 1 × 106 ACH-2 cells (iPrep Purelink gDNA Blood kit; ThermoFisher), and quantified (NanoDrop 1000; Thermo Scientific). The standard curve was obtained from 12 1:3 serial dilution points starting from a DNA stock containing 400 ng, in triplicate. qPCR was performed from 400 ng total CD4+ T cell DNA, using the same primers and probe used for residual plasma viral load quantification (see above). RNase P quantification was used as internal reference (Taqman RNase P Control Reagents kit; ThermoFisher). PCR conditions were as follows: 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 s and 60 °C for 1 min. All values were corrected for CD4+ T cell purity, assessed by multi-color flow cytometry as explained below, and adjusted to 1 × 106 genomes.
Viral LTR methylation analysis method
Bisulfite conversion
Methylation was assessed by bisulfite conversion using 900 ng of total CD4+ T cell DNA, with EpiTect Fast DNA Bisulfite kit (Qiagen), on a QIAcube instrument (Qiagen). Samples were eluted in 16 μL elution buffer. The complete volume of bisulfite-treated DNA was used immediately to perform first-round LTR-Gag PCR in duplicate, as described below. Human Methylated & Non-methylated DNA Set kit (Zymo Reseach, Irvine, CA, USA) was used as control to evaluate the efficiency of bisulfite conversion, according to manufacturer’s specifications, using Platinum Taq DNA polymerase (ThermoFisher). Illumina adaptors for sequencing library construction were added to the primers recommended by the manufacturer (Additional file 5: Table S2).
5′-LTR amplification
The viral 5′-LTR region was amplified using nested PCR, with Platinum Taq DNA polymerase (ThermoFisher), obtaining U3-R amplicons from both bisulfite-treated and untreated samples. Illumina adaptors for sequencing library construction were included as part of the second-round primers (Additional file 5: Table S2). For bisulfite-untreated samples, primers LTR1-20Fw and LTR835-856Rv (Additional file 5: Table S2) were used for the first-round PCR, allowing amplification of an LTR-gag fragment, with the following conditions: 94 °C for 3 min, 35 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min, and a final step of 72 °C for 5 min. For the second-round PCR, primers Nex-LTR213-236Fw and Nex-LTR471-495Rv were used, allowing amplification of the U3-R region of the 5′-LTR, with the following conditions: 94 °C for 3 min, 35 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min, and a final step of 72 °C for5 min. For bisulfite-treated samples, primers LTR1-20TxFw and LTR835-856TxRv were used for the first-round PCR with the following conditions: 94 °C for 3 min, 35 cycles of 94 °C for 30 s, 48 °C for 30 s, and 72 °C for 1 min, and a final step of 72 °in, an5 min. For the second-round PCR, primers Nex-LTR213-236TxFw and Nex-LTR471-495TxRv were used, with the following conditions: 94 °C for 3 min, 35 cycles of 94 °C for 30 s, 47 °C for 30 s, and 72 °C for 1 min, and a final step of 72 °C for5 min.
LTR next generation sequencing
Next generation sequencing (NGS) libraries were constructed from purified U3-R LTR amplicons (both from bisulfite-treated and -untreated DNA), using the amplicon approach with Nextera XT DNA sample preparation and index kits (Illumina, San Diego, CA, USA) (Fig. 1). Unique index combinations were added to each sample for multiplexing. Libraries were normalized to 60 nM and agarose gel-purified (338–440 bp bands, QIAquick gel extraction kit, Qiagen). After normalizing again, libraries were pooled, quantified (Qubit 3.0 Fluorometer; ThermoFisher), and run on a MiSeq instrument (Illumina), using v2 300-cycle pair-end kits (Illumina), according to manufacturer’s instructions.
Read alignment and filtering
Reads were assembled and filtered using USEARCH program version 9.2.64 [53]. The following algorithm was used: (1) fastq R1 and R2 files were fused together, (2) reads were filtered by primer binding site presence (allowing a maximum of two nucleotide difference) and size (209–309 nucleotides for samples, 224–324 nucleotides for the DAPK methylation controls), (3) primer sequences were removed (24 and 25 nucleotides at the 5′ and 3′ end respectively for the samples, 32 and 35 for the controls), (4) reads were filtered by quality (maximum expected error = 1), (5) abundance of each sequence was determined, (6) singlets were eliminated, and (7) reads with abundance < 2% were filtered out. The reads obtained from USEARCH software showed 97.6% (range 95.1–100%) conversion rate of cytosines outside CpG sites for the samples and 100% for the controls, using the software Quma (Quantification Tool for Methylation Analysis) [54].
LTR methylation analysis calculations
Sequencing reads obtained for each time point for each individual were normalized to 100 and aligned to the HXB2 reference with Clustal Omega [55]. A position-specific score matrix (PSSM) was constructed using R and Seqinr [56]. The nine canonical CpG sites in the amplified HIV LTR region were located in the PSSM and the proportion of methylated (CpG), non-methylated (TpG), and mutated sequences was estimated for each one. The PSSM also provided information about additional CpG, CHG, and CHH methylation sites. These data were represented by multiple pie graphs using the program Raw Graphs [57].
We used Quma software [54], optimized for NGS reads analysis, to quantify and evaluate differences in global methylation, and estimated a “CpG Methylation Index” metric, averaging the proportion of methylated sequences for each of the nine canonical HXB2 LTR CpG sites in each sample. The CpG Methylation Index metric ranged from 0 (completely unmethylated) to 100 (fully methylated). Methylation differences between samples of different time-points were assessed using Mann-Whitney U test, using Quma. In order to be more comprehensive in the methylation analysis, we also studied non-canonical methylation patterns along the follow-up period, establishing a measure of methylation on CpG sites different to the canonical ones, present in the HXB2 reference (additional CpG methylation index), and methylation in sites other than CpG (non-CpG methylation index), averaging the proportion of methylated sequences for each relevant position in each category.
Immunophenotyping
A panel of 12 antibodies was selected to identify by flow cytometry CD4+ T lymphocyte subpopulation proportions from each sample time point for all participants included in the study. Antibodies used included the following: Biolegend: HLA-DR BV785 clone L243 (307641), CD45RO BV650 clone UCHL1 (304231), CD3 BV 570 clone UCHT1 (300435), CD95 PECy5 clone DX2 (305610), CCR7 APC Cy7 clone G043H7 (353211), CCR5 BV421 clone J418F1 (359117), PD1 PE clone EH12.2H7 (329906), and CD127 FITC clone A019D5 (351311). BD: CD28 BV711 clone CD28.2 (563131), CD38 AF700 clone HIT2 (560676), CD45 APC clone RUO (340943). Invitrogen: CD4 PE Cy5.5 clone S3.5 (MHCD0418). Life technologies: LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (L34957).
All antibodies were previously titrated using healthy donor CD4+ T lymphocytes. For staining, 1 × 106 CD4+ T cells were resuspended in 2 mL of staining buffer (1× PBS, 2% fetal bovine serum and 0.5% EDTA). Cells were centrifuged at 2000 rpm for 5 min and decanted. Staining was carried out in the remaining volume (approximately 50 μL), adding the anti-CCR7 antibody first. After 10-min incubation,the rest of the antibodies were subsequently added and the tube was incubated for 1 h at room temperature in the dark. Cells were then washed with 3 mL of staining buffer, centrifuged at 2000 rpm for 5 min, decanted, and fixed with 0.3 mL of 1% paraformaldehyde. Cells were acquired on an LSR Fortessa cytometer (BD).
CD4+ T cell subpopulations were defined as follows: Naïve CD4+ T cells (CD3+/CD4+/CD45RO−/CCR7+/CD28+/CD95−), memory CD4+ T cells (CD3+/CD4+/CD28+ −/CD95+), stem cell memory (TSCM: CD3+/CD4+/CD45RO−/CCR7+/CD28+/CD95+), central memory (TCM: CD3+/CD4+/CD45RO+/CCR7+/CD28+), transitional memory (TTM: CD3+/CD4+/CD45RO+/CCR7−/CD28+), effector memory (TEM: CD3+/CD4+/CD45RO+/CCR7−/CD28−), terminal effector (TTE: CD3+/CD4+/CD45RO−/CCR7−/CD28−), and T new (TNEW: CD3+/CD4+/CD45RO−/CCR7−/CD28+) using the program Cytobank [58]. For cell subpopulations with a limited number of captured events, we used a Poisson distribution to establish the minimal threshold for analysis in 100 positive events and 10% variation coefficient. The gating strategy is shown in Additional file 3: Figure S2.
Statistical analysis
Comparisons of clinical and methylation parameters of the longitudinal data were performed for each time-point against month 30 as reference (overall and both for the methylation increase and constant methylation groups), and against the long-term ART group (months 30 and 42 are shown in figures and tables). Medians with interquartile ranges were used for descriptive analysis. Variables from related samples (different time-points form the same participant) were compared using the Wilcoxon test and variables from independent samples were compared using the Mann-Whitney test. A p < 0.05 was considered significant. Statistical analyses were conducted using SPSS version 23.0 and STATA version 14.0.
Exploratory factorial analysis using principal components (PCA) was used as a variable reduction method to describe data structure and summarize the variance in baseline and follow-up clinical variables into fewer dimensions. Indexes were constructed, followed by orthogonal (varimax) rotation. Variables included were age, nadir CD4+ T cell count, pre-ART pVL, delta CD4+ T cell count and % at 48 months, delta CD8+ T cell count and % at 48 months, CD4/CD8 ratio change at 48 months, time on ART, time to achieve undetectable pVL, residual pVL, proviral load, CpG methylation index, number of ART regimen changes, ART backbone (tenofovir + emtricitabine vs. abacavir + lamivudine), ART third drug (non-nucleoside RT inhibitors vs. protease inhibitors), presence of blips, and CD4+ T cell subpopulation proportions: naeoside RT inhibitors vSCM, TCM, TEM, TTM, TNEW, TTE. Six factors showed an eigenvector value higher than 1 (cumulative variance 77%), from which three were selected (cumulative variance 51%) for further presentation and analysis. Factor scores were obtained and graphed. PCA was performed using STATA version 14.0.