Nature Medicine: doi: /nm.4411
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1 Supplementary Figure 1. Plasma viral load decay characteristics in each of 4 monkey cohorts studied after prolonged ART. A. Decay in 5 SIV infected animals treated ART for weeks. B, C. Decay in additional SIV infected animals treated with the ART drugs listed. D. Decay in 4 RT-SHIV+ animals treated with efavirenz (RFV), emtricitibine (FTC), and tenofovir (TFV).
2 Supplementary Figure 2. Comparison of qrt-pcr and RNAscope (A) and qpcr and DNAscope (B). Black symbols represent measures prior to ART and colored symbols represent measures during ART.
3
4 Supplementary Figure 3. Representative images from brain, kidney, liver, and lymph node using 35S SIV RNA ISH (red arrows) and SIV RNAscope (black arrows) to identify vrna+ cells. Supplementary Figure 4. Representative image of lymph node, DNA ISH using DNAscope. Red signal located over the nucleus of the cell identifies those cells that are vdna+ (black arrows).
5 Supplementary Figure 5. Change in peripheral blood CD4 T cells with ART in HIV-infected Ugandan patients. Supplementary Figure 6. Change in plasma Viral Load with ART in HIV-infected Ugandan patients.
6 Organ Average SIV RNA+ Cells/Gram Organ weight as a percentage of total weight (%) Total Cells/Organ Heart 7.54 x % 2.89 x 10 5 Brain 2.16 x % 5.58 x 10 4 Kidney (x2) 1.42 x % 1.29 x 10 6 Liver 1.88 x % 5.24 x 10 5 Gut 5.57 x % 1.86 x 10 8 Lungs (x2) 3.10 x % 5.26 x 10 6 LN 8.73 x % 7.68 x 10 7 Spleen 1.53 x % 1.19 x 10 6 Supplementary Table 1. Total estimated frequency of vrna+ cells in each organ system. Estimated vrna+ cells/gram of tissue from the animal sampled in acute infection were determined from a series of 5 sections from each organ and the average weight of each organ was estimated from the total weight of the animal using published necropsy literature to determine what average percent of the total body weight in a particular(1). Note the animal weight at necropsy was 8800 grams(1).
7 A. P568 (Peak Viremia) R096 (Early Chronic) P533 (Late Chronic) Brain 2.16 x x x 10 4 Heart ND ND 1.95 x 10 3 Lung 1.88 x x x 10 5 Liver 6.99 x x x 10 4 Kidney 3.10 x x x 10 2 Spleen 1.76 x x x 10 5 Axillary LN 1.53 x x x 10 6 Mesenteric LN 3.42 x x x 10 6 Inguinal LN 1.11 x x x 10 6 Colonic LN 5.10 x x x 10 6 Jejunum 5.82 x x x 10 6 Duodenum 2.05 x x x 10 4 Ileum 7.54 x x x 10 5 Ascending Colon 1.09 x x x 10 5 Transverse Colon 2.13 x x x 10 4 Rectum ND 9.17 x x 10 6 Prostate 1.42 x 10 4 ND ND Seminal Vesicle 9.74 x x 10 3 B. R365 R366 R367 R406 R410 Brain ND 1.80 x 10 3 ND 7.7 x 10 3 Heart ND ND ND 3.93 x 10 2 Lung 9.04 x x x x x 10 3 Liver ND 6.55 x 10 2 ND 1.67 x x 10 2 Kidney ND 5.38 x 10 2 ND 5.48 x x 10 2 Spleen ND 4.76 x x x 10 5 Axillary LN ND ND ND 6.69 x x 10 3 Mesenteric LN 1.14 x 10 4 ND ND 7.64 x x 10 4 Inguinal LN ND ND ND 1.10 x x 10 4 Colonic LN 1.46 x 10 4 ND ND ND 1.05 x 10 5 Jejunum ND ND ND 2.93 x x 10 5 Duodenum 2.98 x x x x x 10 4 Ileum 1.02 x 10 4 ND 1.11 x x x 10 4 Ascending Colon 2.47 x x x x x 10 5 Transverse Colon 2.11 x x x x x 10 5 Rectum 1.53 x 10 4 ND 3.42 x 10 5 Prostate * * * * * Seminal Vesicle * * * * * C Cerebrum ND 1.1 x 10 3 ND ND Cerebellum 3.7 x 10 2 ND ND ND Basal Ganglion ND ND Bone Marrow 2.5 x x 10 5 ND 7.9 x 10 4 Lungs 2.3 x x x x 10 5
8 Liver 4.8 x x 10 4 ND 3.1 x 10 4 Thymus 9.1 x x 10 5 ND 4.4 x 10 4 Spleen 2.5 x x x x 10 6 Axillary LN 4.0 x 10 5 ND 7.0 x x 10 6 Iliac LN 5.3 x x 10 6 ND 4.4 x 10 6 Mesenteric LN 2.9 x x x x 10 6 Inguinal LN 6.3 x x x x 10 6 Jejunum 5.9 x x x x 10 6 Ileum 1.5 x x x x 10 5 Colon 6.7 x x x x 10 5 D Cerebrum 1.1 x x x x 10 2 Cerebellum ND Spinal Cord ND Bone Marrow ND ND ND ND Thymus ND ND ND ND Heart 1.8 x 10 2 Lungs 1.2 x x x 10 2 ND Liver 1.9 x 10 2 ND ND ND Kidney ND Spleen 3.3 x x x 10 2 ND Cervical LN ND Axillary LN 1.3 x x 10 2 ND Iliac LN 3.5 x x x 10 2 ND Inguinal LN ND 3.2 x x 10 3 ND Mesenteric LN 1.1 x x x 10 3 ND Jejunum ND ND 3.6 x 10 2 ND Duodenum ND Ileum 8.8 x 10 2 ND 2.8 x x 10 2 Colon ND ND ND Supplementary Tables 2 A-D: Tables providing raw data on frequency of vrna+ cell/gram for each animal in each organ studied. Table 2A = 3 animals infected with SIV mac251 and sacrificed in acute, early and late chronic infection without ART, table 2B = macaques infected with SIV mac251 after 22 weeks of ART, Table 2C = 4 macaques infected with RT-SHIV and sacrificed after 26 weeks of infection without ART, and Table 1D = 5 macaques infected with RT-SHIV and sacrificed after 26 weeks of ART. ND, not detected. Cells in the table that are blank means the tissue was not available, * means the animal was female.
9 A. Tenofovir (TFV) TFV (ng/ml) IC TFV-DP (foml/10^6 cells) Ratio to PBMC ID Plasma PBMC LN GALT RALT LN GALT RALT DC1G * DCCP DCT DCLI DCEA DCEW B. Emtricitabine (FTC) FTC (ng/ml) IC FTC-TP (fmol/10^6 cells) Ratio to PBMC ID Plasma PBMC LN GALT RALT LN GALT RALT DC1G * DCCP DCT DCLI DCEA DCEW C. Darunavir (DRV) DRV (ng/ml) IC DRV (fmol/10^6 cells) Ratio to PBMC ID Plasma PBMC LN GALT RALT LN GALT RALT DC1G * BLQ BLQ BLQ 6531 NA NA NA DCCP BLQ BLQ NA NA DCT BLQ BLQ 1007 NA NA 4.47 DCLI BLQ BLQ 1722 NA NA DCEA BLQ BLQ 406 NA NA 9.89 DCEW BLQ BLQ NA NA Supplementary Table 3: Plasma and intracellular ARV drug concentrations. Samples where concentrations were below the limit of quantitation are noted as BLQ; ratios that could not be calculated are noted as NA, not available.
10 vrna+ cells Virus-producing cells Supplementary Table 4: Direct comparison of frequency of vrna+ cells in multiple sections from a LN of a suppressed macaque to the frequency of virus producing cells using ISH TSA/ELF. 1. Fremming BD, Benson RE, Young RJ. Weights of organs in sixty-six male Macaca mulatta monkeys. J Appl Physiol. 1955;8(2): PubMed PMID:
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