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. 2021 Jul 27;13(8):1472. doi: 10.3390/v13081472

Figure 3.

Figure 3

Comparison of the effects of different doses of morphine, buprenorphine and methadone during early HIV-1 infection of PBMC. (A) Experimental approach to assess in vitro HIV-1 infection and replication. PBMC were incubated with opioids for 72 h and after infection and washes, drugs were withdrawn. Aliquots were taken at the indicated timepoints, and HIV-1 replication was measured by quantifying nanoluciferase enzymatic activity in the supernatants. Chemiluminescence signals were quantified using a luminometer. (B) Representative nanoluciferase experiment done with PBMC from one donor. We cultured PBMC for up to 72 h with PHA/IL-2 and morphine (1 or 100 µM), buprenorphine (2 or 20 nM), or vehicle control. In these experiments, the vehicle control consisted of R10 medium supplemented with PHA, IL-2 and 1.25% v/v of methanol. The viability, assessed by trypan-blue staining or Guava Muse® analyzer, was similar across different conditions (Supplementary Figure S1A). We infected them with sNLuc HIV-1 and opioids were washed off. Luminescence signals were determined with supernatants taken at 1 and 3 days after HIV-1 infection. Y-axis denotes relative light units (RLU); number next to Y-axis indicates the Multiplicity of Infection (MOI) of the HIV-1 reporter virus used to infect the PBMC. Each treatment is represented as a different symbol (specified in the X-axis); open and close symbols denote the lowest and highest dose for each drug, respectively. We did a total of three technical replicates for MOI of 0.1 and 0.5, and 6 for MOI 0.2. (C) Summary plots showing the effect of opioids on nanoluciferase activity measured in supernatants taken at 1 and 3 days after HIV-1 infection. The MOI here was 0.2; we did six technical replicates per condition. Each donor is represented by a different color; the number of different donors per treatment is indicated with an “n” above the plots. X-axis indicates different treatments and concentrations. Y-axis shows the ratio of luciferase activity measured in opioid-treated cultures over luciferase activity measured in vehicle-treated cultures. Plots indicate median (vertical lines in the middle), interquartile range (boxes), and min to max range (whiskers). p-values were two-tailed and calculated using the Wilcoxon Signed Rank Test, comparing the median obtained in each treatment with a theoretical median value of 1. *: p < 0.05; **: p < 0.01.