Article Figures & Data
Figures
- FIG 1
Viral load in plasma and CSF of two cohorts of SIV-infected ART-treated macaques. Two cohorts of SIV-infected pig-tailed macaques were treated with similar ART regimens (listed above the horizontal green line). (A and B) Macaques in cohort A1 were suppressed for more than 500 days and were treated with LRAs (ingenol-B, orange; ingenol-B plus suberoylanilide hydroxamic acid [SAHA], purple). (C and D) Macaques in cohort A2 followed similar protocol as those in cohort A1 but were suppressed for 100 to 400 days and treated with LRAs (ingenol plus SAHA in purple) in the last 60 days before euthanasia (Table 1). Median values for each group of animals are depicted in red for plasma (A and C) and dark blue for CSF (B and D). Analyses of samples with values below the limit of detection for the SIV qPCR assay (100 SIV RNA copies/ml) were repeated using ddPCR.
- FIG 2
Analyses of viral decay in plasma and CSF. The line for each cohort represents the curve best fitting the geometric means of longitudinal viral loads (symbols) for that cohort in plasma (A) and CSF (B). Values in the insets show half-lives (t1/2) and R2 values for phase (ph) 1 and phase 2 in the curves. Cohort A0 represents data previously published (34).
- FIG 3
Quantitation of latently infected BrMΦ in ART-treated macaques by MΦ-QVOA. (A and B) Quantitation of infected BrMΦ from two groups of ART-treated macaques. In panel B, wells containing <50 SIV RNA copies/ml were also considered positive, and IUPM (infectious units per million cells) values were calculated accordingly. Boxes in panels A and B indicate values under the limit of detection, i.e., BrMΦ samples that did not present positive well results by qPCR. (C) Comparison between the numbers of SIV-infected BrMΦ isolated from animals that were not given ART (−ART) and the numbers isolated from animals that were treated with ART and showed full suppression (+ART; PmA13 not included). The horizontal black line represents the median IUPM values. The MΦ-QVOA results from SIV-infected animals without ART were previously reported (35). Significance was determined by Mann-Whitney nonparametric t test; a P of <0.05 was considered significant. (D) Numbers of SIV RNA copies in the supernatants of QVOA-positive wells separated according to the pattern of replication (<50 copies/ml, >50 copies/ml without spread, or replicative).
- FIG 4
Virus from BrMΦ QVOA is replication competent in PBMCs. The graph depicts the increased levels of SIV RNA in supernatants of PBMC cultures that were subjected to spinoculation with 100 µl of supernatants collected from BrMΦ QVOA wells. Different wells from each of the five analyzed macaques are depicted in the same color, and each line represents the result of one supernatant transfer. The first time point depicts the number of SIV RNA copies contained in the 100-µl volume of the BrMΦ QVOA well that was used for spinoculation. The other time points show the numbers of total supernatant copies measured at 5 to 7 days and 10 to 14 days postspinoculation.
- FIG 5
In situ hybridization (ISH) for SIV RNA in brain sections. Data are representative of ISH results of analysis of SIV RNA in brain (occipital cortex) of macaque PmA13 (top row) and Mn3, which represents a previously published SIV-infected macaque that discontinued ART and had SIV rebound (lower row) (36); magnification, ×40.
Tables
- TABLE 1
Description of macaque cohorts, the antiretroviral therapy regimen, and levels of CD4+ T cells, monocytes, plasma viral load, and CSF viral load at the terminal time pointa
Cohort and animal identifier ART (no. of days) Cell counts (no. of cells/µl blood) Viral load (no. of SIV RNA copies/ml) Comment CD4+ T cells Monocytes Plasma CSF A1 PmA11 615 474 1,092 <10 <10 NA PmA12 616 1,074 485 <10 <10 Treated with LRAs after 550 dpi PmA13 605 320 1,346 1,800 18,000 Treated with LRAs after 550 dpi A2 PmA21 394 280 340 <10 <10 NA PmA22 391 324 2,310 <10 <10 Treated with LRAs after 242 dpi PmA23 345 331 410 <10 <10 Treated with LRAs after 249 dpi PmA24 182 291 350 <10 <10 NA PmA25 182 790 310 <10 <10 NA ↵ a The antiretroviral therapy (ART) regiment for both cohort A1 and cohort A2 consisted of tenofovir, darunavir, integrase inhibitor L000870812, and ritonavir. LRA, latency reverse agents; dpi, days postinfection; NA, not applicable.
- TABLE 2
SIV DNA and RNA levels in two brain regions (basal ganglia and parietal cortex) at terminal point and number of latently infected BrMΦ for each macaquea
Cohort and animal identifier No. of SIV DNA copies per 106 cells No. of SIV RNA copies per µg RNA Brain MΦ-QVOA IUPM Basal ganglia Parietal cortex Basal ganglia Parietal cortex Without wells (<50 copies/ml) With wells (<50 copies/ml) A1 PmA11 <10 <10 <10 <10 0.147 0.917 PmA12 <10 <10 <10 <10 0.036 0.230 PmA13 <10 64 <10 <10 0.036 0.110 A2 PmA21 <10 <10 <10 <10 1.445 1.445 PmA22 <10 <10 <10 <10 0.177 1.309 PmA23 <10 <10 <10 <10 7.362 7.362 PmA24 <10 <10 <10 <10 <0.021 <0.021 PmA25 <10 <10 <10 <10 0.613 0.613 ↵ a QVOA values are presented and represent the presence or absence of positive wells with fewer than 50 SIV RNA copies/ml of supernatant. Values reflect maximum likelihood estimates of infection frequency in IUPM (infectious units per million cells). Given the resolution of the assay, the 95% confidence interval is typically 0.2 to 4 times the reported value.
- TABLE 3
Quantitation of infected CD4+ T cells in the MΦ-QVOA using detection of TCRβ RNA, as described by Avalos et al. (35)
Cohort and animal identifier TCRβ in MΦ-QVOA (no. of RNA copies/105 cells) % CD3+ T cells in MΦ-QVOA by TCRβ % CD4+ T cells in blood CD3+ T cells % CD4+ T cells in MΦ-QVOA CD4+ T cell IUPM by PBMC QVOA No. of infected CD4+ T cells per 106 QVOA cells A1 PmA11 3,871 0.97 50.9 0.50 1.3 0.0065 PmA12 0 0 60.2 0 0.30 0 PmA13 20,428 5.11 35.1 1.80 0.40 0.0072 A2 PmA21 0 0 55.2 0 0.05 0 PmA22 0 0 56.8 0 0.07 0 PmA23 0 0 56.4 0 0.05 0 PmA24 0 0 45.8 0 0.01 0 PmA25 0 0 48.9 0 0.06 0 - TABLE 4
SIV DNA and RNA levels in BrMΦ isolated from basal ganglia and parietal cortex of SIV-infected ART-treated macaquesa
Cohort and animal identifier SIV DNA (no. of copies per 106 BrMΦ) SIV RNA (no. of copies per 106 BrMΦ) CSF (no. of SIV RNA copies/ml) Basal ganglia (no. of SIV RNA copies/µg total RNA) MΦ-QVOA (no. of infected BrMΦ per million cells) A1 PmA11 20 <5 <10 <10 0.92 PmA12 9 <5 <10 <10 0.23 PmA13 119 33,984 18,000 <10 0.11 A2 PmA21 40 <5 <10 <10 1.45 PmA22 60 <5 <10 <10 1.31 PmA23 1,606 230 <10 <10 7.36 PmA24 57 4,652 <10 <10 <0.02 PmA25 67 1,263 <10 <10 0.61 ↵ a SIV RNA levels in CSF and basal ganglia and no. of infected BrMΦ evaluated at necropsy are also presented for comparison.
- TABLE 5
Number of SIV RNA-positive foci per gram of brain tissue estimated by in situ hybridization
Cohort and animal identifier Area analysed (μm2) No. of positive foci per analysed field Density of SIV+ cells per gram of tissue A1 PmA11 1.41 × 108 0, 1, 0, 0, 0, 0, 0 202 PmA12 2.54 × 108 0, 0, 0, 0, 0, 0, 0 <102 PmA13 1.16 × 108 1, 1, 1, 1, 1, 3, 1 2,220
