Participant samples
Peripheral blood was obtained from HIV infected donors and processed for peripheral blood mononuclear cells (PBMC) to prepare CD4+ T lymphocytes. The donors had been chronically HIV-infected with baseline viral loads between 40,000 and 500,000 copies HIV RNA/mL plasma before receiving ART and being suppressed below 50 copies for all measurements for at least 1 year. All had > 500 CD4 cells/μL blood at the time of donation, which followed a UCSD Institutional Review Board informed written consent. PBMC were isolated using density gradient centrifugation from whole blood. Total CD4+ T cells were enriched from PBMC by negative selection using Stem Cell Technologies EasySep CD4 Enrichment kit previously described [7].
Modified quantitative viral outgrowth assay (mQVOA) and Induced cell free (cf)-HIV RNA assays
As previously described in detail, enriched CD4+ T cells were serially diluted in a 24-well plate coated with anti-CD3 (Clone SK7) and anti-CD28 (Clone CD28.2) monoclonal antibodies (both from BD Biosciences) [7]. We have elected to measure total negatively selected CD4 lymphocytes rather than just resting cells, since in subjects well suppressed on ART all HIV-infected CD4 cells represent the reservoir that must be addressed with eradication strategies for cure. Six serial threefold dilutions were performed with starting concentrations of 1 × 106 cells/well and with 6 replicates for each dilution. The concentration of raltegravir used throughout the QVOA assay was 250 nG/mL, which is 40 times the human serum adjusted IC95 of 31 nM.
After 2 days of stimulation, 200,000 MOLT-4/CCR5 cells (NIH AIDS Reagents program) were added to each cell culture well (day 0). Cell cultures were split twice weekly; half of cell culture supernatants (500 μL) were collected for analysis at days 3, 5, 7, 10, 12 and 14, except for CARE 197 with collections on days 3, 7, 10 and 14. Of note extending the assay beyond 14 days rarely if ever adds a positive well, as has been shown with the original description of the use of the MOLT-4/CCR5 cells [15]. Supernatants were spun at 300 g for 10 min and frozen at − 80 °C until use. HIV-specific magnetic beads were used to extract cell free-RNA from supernatants (Aptima HIV-1 kit, Hologic Incorporated). Briefly, supernatants were incubated with 400 μl of Target Capture Reagent (TCR, containing HIV-specific magnetic beads) for 7 min at 80 °C, 30 min at 60 °C and 22 min at 25 °C (a gift from Hologic, Inc.). After incubation, the magnetic particles were concentrated using a KingFisher instrument (Life Technologies). Extracted HIV RNA was then detected using One-step RT-PCR (Applied Biosystems) for detection of pol (integrase) [16]. The number of wells positive for HIV RNA was determined using the criteria described in Fig. 1, and the maximum likelihood method was applied to determine Infectious Units Per Million (IUPM) with the Infection Frequency Calculator (http://silicianolab.johnshopkins.edu/) [17].
Statistical analysis
Data were analyzed on a log10 scale. Descriptive analyses including area under the curve (AUC) were used to summarize the data. Replicates without raltegravir were individually compared to the mean plus 5 standard deviations of the 6 replicates performed with raltegravir. The criterion for a positive well assessing amplification on day 14 used the mean and SD for all six wells in that dilution in the presence of raltegravir. A positive well without raltegravir was defined as greater than the mean plus 5 SD in the presence of raltegravir. The criterion for a positive well assessing amplification by AUC without raltegravir used the mean and SD of the area under the curve for the 2 weeks of measurements for all six wells in that dilution in the presence of raltegravir. A positive well without raltegravir was defined as greater than the mean plus 5 SD of the area under the curve in the presence of raltegravir. Statistical analyses were performed using Prism 7.0 and R software (version 3.3.2, http://www.Rproject.org/) [18].