The Indian rhesus macaques used in this study were housed at Bioqual, Inc., according to standards and guidelines as set forth in the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC), following approval by the Institutional Animal Care and Use Committee (IACUC). Eight SIVmac251-infected macaques in the chronic phase of the infection were enrolled in this study starting from June 2010. The baseline viro-immunological conditions of the study subjects and their antiretroviral treatments preceding April 2012 have been described in detail in a recent publication . Macaques in the late chronic phase of the infection displaying antiretrovirally suppressed viremia (i.e. < 50 copies/mL) were kept under highly-intensified ART (H-iART; consisting of tenofovir, emtricitabine, raltegravir, ritonavir-boosted darunavir and maraviroc) and auranofin for three months. Two macaques were similarly kept under H-iART in the absence of auranofin to serve as controls. Additional H-iART cycles (mean duration: 31 days) were administered to animals P177 and P252 in order to decrease viral reservoir replenishment at viral rebound. Auranofin (Prometheus Laboratories, San Diego, CA) was administered by the oral route twice daily with food (0.4 mg/kg/day). BSO (Sigma-Aldrich, St.Louis, MO, USA) was administered to three macaques intraperitoneally at 450 mg/kg every eight hours for a total of five administrations (the details of the timing and its rationale are given in the Results section). All animals were dosed subcutaneously with tenofovir, and emtricitabine, and orally (with food) with raltegravir, ritonavir-boosted darunavir, and maraviroc. Drug dosages were: tenofovir, 30 mg/kg/day; emtricitabine, 50 mg/kg/day; raltegravir, 100 mg bid; darunavir, 375 mg bid (for macaques starting from viral loads lower than 105 viral RNA copies/mL) or 700 mg bid (for macaques starting from viral loads higher than 105 viral RNA copies/mL); ritonavir 50 mg bid; maraviroc 100 mg bid. Tenofovir and emtricitabine were kindly provided by Gilead Sciences (Foster City, CA). Raltegravir, DRV/r and MRV were purchased from the manufacturers.
Pilot treatments of macaques P255 and P252 were conducted in 2010/early 2011. Treatments of macaques P157, P177, P185, P188, and 4416 were conducted simultaneously in 2011. An additional SIVmac251-infected animal (4890) was treated in 2012 in order to ensure reproducibility of the results.
Macaques P157 and P177 were subjected to CD8+ cell depletion after therapy suspension in order to study the contribution of CD8+ cells to post-therapy viral load control. The depletion was performed with the previously validated antibody cm-T807 ([25, 44] kindly provided by the NIH Nonhuman Primate Reagent Resource center). The antibody was administered on four different occasions (days 0, 3, 7 and 10) at 10 mg/kg subcutaneously for the first administration and at 5 mg/kg intravenously for the three remaining administrations.
Quantitative assay for SIVmac251 viral RNA levels
For measurement of plasma SIVmac251 RNA levels, a quantitative TaqMan RNA reverse transcription-PCR (RT-PCR) assay (Applied Biosystems, Foster City, CA, USA) was used, which targets a conserved region of the gag transcripts. The samples were then amplified according to a method previously described in [12, 45]. The sensitivity of the method is two copies per run, which results in a detection limit as low as 40 RNA copies/mL in our routine analyses. This method and its validation data are described extensively in Ref. . For the SIV real-time NASBA assay, macaque plasma was clarified by centrifugation at 2300 × g for 3 mins. The clarified plasma was either lysed directly (0.1 mL) in lysis buffer (bioMerieux, Durham, NC, USA) or further centrifuged to pellet virus from a higher volume (0.5–1 mL) by ultracentrifugation at 49 100 × g for 60 min. The virus pellet was then lysed in 1 mL lysis buffer. A fixed amount of Q calibrator RNA (105 copies or 104 copies) was added to the lysed sample and the nucleic acid was extracted using acidified silica as described previously . The quantitative range of the assay was determined by the concentration of the calibrator added . For samples expected to have a viral load >104 copies/mL, 105 copies of calibrator RNA were used. However, for lower loads, 104 copies of Q calibrator RNA were used.
The SIV real-time amplification final reaction volume was 20 μl, containing 5 μl of nucleic acid extract and 40 mM Tris, pH 8.5; 12 mM MgCl2; 90 mM KCl; 5 mM dithiothriotol; 1 mM each dATP, dCTP, dGTP, dTTP; 2.0 mM each ATP, CTP, UTP; 1.5 mM GTP; 0.5 mM ITP; 0.1 ìg/ìl BSA; 1.5 M sorbitol; 0.08 units RNase H; 32 units T7 RNA polymerase; 6.4 units avian myeloblastosis virus reverse transcriptase (AMV-RT); 0.01 μM SIV WT molecular beacon probe; 0.1 μM SIV Q molecular beacon probe; 0.2 μM each of the two amplification oligonucleotides, and 15% DMSO. The amplification oligonucleotides used are as follows—P1: 5′-AATTCTAATACGACTCACTATAGGGCACCAGATGACGCAGACAGTATTA-3′; P2: 5′-CTCCGTCTTGTCAGGGAAGAAAGCA-3′. The SIV WT molecular beacon has the fluorophore FAM linked to the 5′ end and a quencher linked to the 3′ end; 5′-FAM-CGATGCATGTAGTATGGGCAGCAAATGAAGCATCG-DABCYL-3′. The SIV Q molecular beacon has the fluorophore 6-ROX linked to the 5′ end and a quencher linked to the 3′ end; 5′-6-ROX-CGATGCGTTGAAGTGCAGTAGTGATGGCATCG-DABCYL-3′. All reagents, except for enzymes and BSA, were mixed and preincubated at 65°C for 2 min. The reaction mixture was then cooled for 2 min at 41°C and the enzymes and BSA were added. Samples were mixed and placed in the NucliSENS EasyQ Analyzer (bioMerieux, Durham, NC, USA) and isothermal amplification took place at 41°C for 90 min. The NucliSENS EasyQ Analyzer took measurements throughout the amplification reaction, resulting in two fluorescence recovery curves. An algorithm was then applied that uses the kinetics of fluorescence recovery from WT and Q RNA to calculate the SIV RNA copy number in the plasma samples. The SIV RNA load was expressed as viral RNA copies per ml plasma. The inter-assay coefficient of variation of the technique was 19%, well within the limits of the typical inter-assay variability of SIV RNA detection techniques [e.g.[12, 48].
Quantitative assay for SIVmac251 proviral DNA
For proviral DNA detection, DNA was extracted with the phenol-chloroform method. Quantification was performed by amplifying a region of the gag gene by real time PCR in a 7700 Sequence Detection System (Applied Biosystems). Details and validations of the technique are extensively described in [12, 45].
Immunofluorescent staining and flow-cytometric analysis
Hematological analyses were performed by IDEXX (IDEXX Preclinical Research, North Grafton, MA). For calculation of absolute CD4+ and CD8+ T-cell numbers, whole blood was stained with anti-CD3-fluorescein isothiocyanate (FITC)/anti-CD4-phycoerythrin (PE)/anti-CD8-peridinin chlorophyll α protein (PerCP)/anti-CD28-allophycocyanin (APC), and anti-CD2-FITC/anti-CD20-PE, and red blood cells were lysed using lysing reagent (Beckman Coulter, Inc., Fullerton, CA, USA). Samples were run on a FACSCalibur (BD Biosciences, San Jose, CA, USA).
Staining for naïve (TN: CD28+CD95-), central memory (TCM: CD28+CD95+), and effector memory (TEM: CD28-CD95+) T-cells was performed on PBMCs isolated from total blood as described in . The mAbs used (BD Biosciences) were: anti-CD3 (APC-Cy7), anti-CD4 (Per-CP), anti-CD8 (Pe-Cy7), anti-CD20 (APC), anti-CD28 (FITC) and anti-CD95 (PE). Six-parameter flow-cytometric analysis was performed on a FACS Canto II instrument (BD Biosciences). The absolute numbers of TN (CD95-CD28+), TCM (CD95+CD28+) and TEM (CD95+CD28-) memory CD4+ T-cells were deduced from percentage values of parent cells.
Detection of neutralizing antibodies
Sera from different time points during the study were assayed in the TZM-bl assay system  against a neutralization-sensitive (SIVmac251.6) and a neutralization-resistant (SIVmac251.30) pseudotyped virus. Virus pseudotyped with Murine Leukemia Virus Env (SVA-MLV) was included to assess non-SIV-specific neutralizing activity in the sera. Briefly, serial dilutions of sera from the indicated time points were pre-incubated with virus (~150,000 relative light unit equivalents) for 1 hr at 37°C. Following addition of TZM-bl target cells, the cultures were incubated for 48 hours, then lysed and assayed for luciferase activity. Neutralization titers are the sample dilution at which relative luminescence units (RLU) were reduced by 50% compared to RLU in virus control wells after subtraction of background RLU in cell control wells. In several instances, ID50 endpoint titers against SIVmac251.6 were not achieved in the dilution series employed in the assays. Thus, an 80% neutralization titer (ID80) was calculated to expose the relative potency of the neutralizing antibody response against this virus.
IFN-γ ELISpot assay
Specific immune responses were detected by measuring gamma interferon (IFN-γ) secretion of macaque PBMCs stimulated with SIVmac239 Gag peptides (125 overlapping 15-mer peptides, obtained through the AIDS Research and Reference Reagent Program, National Institutes of Health [NIH]) in an enzyme-linked immunospot (ELISpot) assay. The peptides were resuspended according to the manufacturer’s instructions and divided in two pools (pool 1: peptides 1–63; pool 2: peptides 64–125). The assay was performed with the ELISpotPRO for monkey interferon-γ kit (Mabtech AB, Nacka Strand, Sweden) according to the manufacturer’s instructions. Briefly, 1.5 × 105 Ficoll isolated macaque PBMCs were added to 96 well plates pre-coated with an anti-human/monkey IFN-γ antibody (MAb GZ-4). Cells were resuspended in RPMI 1640 + 10% FBS with or without 5 μg/mL of each peptide pool, or concanavalin as a positive control. Triplicate wells were employed for each experimental condition. After 48 hours incubation at 37°C with 5% CO2, the cells were rinsed from the plates, and a biotinylated anti-human/monkey IFN-γ antibody (MAb 7-B6-1; Mabtech) was added to the wells. The plates were then washed with PBS and incubated with the substrate solution (BCIP/NBT-plus). Spot forming cells (SFC) were counted by using an automated reader (Immunospot Reader, CTL analyzers, LLC, Cleveland, OH). Data were expressed as average numbers of SFC/106 cells after subtracting the average number of background spots detected in the negative controls.
Statistical analyses and mathematical simulations
The inter-assay coefficient of variation of the SIV real time NASBA technique was calculated in accordance with a standard procedure [e.g. see Ref. . Two different control samples (containing ≈ 104.7 and ≈ 102.7 copies of SIV RNA copies/mL, respectively) were tested in three different runs per sample. Two coefficients of variation were calculated from the results of these runs and the overall coefficient of variation was calculated as the mean of the two. Differences between variables were calculated using parametric tests (Student’s t-test for comparisons between two groups, and ANOVA followed by the Student-Newman-Keuls post-test for multiple comparisons). An appropriate transformation was applied to restore normality, where necessary. Repeated-measures tests were used when analyzing matched observations. Trends were analyzed by regression analysis, followed by the extra sum-of-squares F post-test. Calculations were conducted using the software GraphPad Prism 5.00.288 (GraphPad Software, Inc., San Diego, CA, USA).
The viral set point was calculated using the area-under-curve (AUC) method using the GraphPad (v.5) software. To ensure consistency in the results obtained, we considered the post-therapy viral load values in the same time interval as that of the available pre-therapy values. The time frames analyzed in the different macaques were largely comparable, since the standard deviation of the periods considered accounted for only 15% of the mean value. To ensure accuracy in the analyses, the starting value employed for calculation of the post-therapy vial load set point was selected according to the following criteria:
For animals that had received auranofin, and had therefore experienced the previously published acute-infection-like condition , we started the follow-up immediately after the acute-infection-like peak in viremia. This choice mimics calculations of the viral set point in the natural course of the infection, discarding the acute infection phase as non-representative of the equilibrium reached thereafter. The initial value adopted for our viral load set point calculations was the minimum in the curve after the initial peak.
For animals that had the initial viral load peak abated artificially by H-iART, the period under drugs was not taken into account, and the viral set point calculations were started after therapy withdrawal.
For animals that had not received auranofin and for which there was no clear initial peak, follow-up was started when viremia plateaued. This allowed discarding the initial values and providing a sensible comparison with the data from the auranofin-treated animals. We considered that the plateau was reached when viral load reached values above 80% of the asymptote of the one-phase association curve describing the viral rebound and obtained by non-linear regression analysis option embedded in the GraphPad software. Additional file 5 provides an illustrated example of the method adopted.
For each of the cases presented in 1), 2) and 3), the input values were analyzed using the “area under curve” option in the GraphPad software. Peaks accounting for less than 10% of the AUC were automatically discarded by the default option of the software adopted. To allow an overall comparison of the results obtained, results were normalized by dividing by the length of the temporal window.
Multivariate analysis was used to study the contribution of independent predictors to the post-therapy viral load set point and the difference between the pre- and post-therapy viral load set points points (Δ viral set point). The analysis was conducted with the IBM SPSS software (v. 21, Armonk, NY, USA), using a type-I regression parameter, and the post-therapy viral set point and the Δ viral set point as dependent variables. The choice of the potentially predicting parameters was based on literature analysis. Possibly independent predictors were considered to be the number of drugs simultaneously employed, the total duration of therapy (i.e. the number of days during which macaques were exposed to drugs), the number of therapeutic cycles and the pre-therapy CD4 nadir [12, 49]. A “therapy” was considered to be a sequence of therapeutic cycles wherein the distance between neighboring treatments did not exceed 90 days. Partial correlation analysis was conducted using SPSS, starting from the same parameters, and controlling by the total duration of therapy.
For a discussion of mathematical methods and numerical tools employed in the numerical simulations, see the “Statistical and biomathematical analyses” paragraph in the “Materials and methods” section and Text S1 in Ref. . See also Ref. . Briefly, for our simulations we adjusted for a macaque model the system of ordinary differential equations (system 4) introduced in . In this system, a random step function is used to simulate the periods in which there is activation of resting latently infected CD4+ T-cells. The activation function used in the simulations was generated with the RANDLIB package of the Scilab 5.3.3 software (freely available at http://www.scilab.org). This function is shown in the figure of Additional file 6. Further information on mathematical modeling is given in the text of Additional file 6 while the starting data employed for the numerical simulations are shown in Additional file 7.