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. 2023 Dec 13;15(726):eadi9867.
doi: 10.1126/scitranslmed.adi9867. Epub 2023 Dec 13.

Lymphoid tissues contribute to plasma viral clonotypes early after antiretroviral therapy interruption in SIV-infected rhesus macaques

Antonio Solis-Leal  1 , Nongthombam Boby  1 , Suvadip Mallick  1 , Yilun Cheng  2 , Fei Wu  1 , Grey De La Torre  1 , Jason Dufour  3 , Xavier Alvarez  1 , Vinay Shivanna  1 , Yaozhong Liu  4 , Christine M Fennessey  5 , Jeffrey D Lifson  5 , Qingsheng Li  2 , Brandon F Keele  5 , Binhua Ling  1
Affiliations

Lymphoid tissues contribute to plasma viral clonotypes early after antiretroviral therapy interruption in SIV-infected rhesus macaques

Antonio Solis-Leal et al. Sci Transl Med. .

Abstract

The rebound-competent viral reservoir, composed of a virus that is able to persist during antiretroviral therapy (ART) and mediate reactivation of systemic viral replication and rebound viremia after ART interruption (ATI), remains the biggest obstacle to treating HIV infection. A better understanding of the cellular and tissue origins and the dynamics of viral populations that initiate rebound upon ATI could help develop therapeutic strategies for reducing the rebound-competent viral reservoir. In this study, barcoded simian immunodeficiency virus (SIV), SIVmac239M, was used to infect rhesus macaques to enable monitoring of viral barcode clonotypes contributing to virus detectable in plasma after ATI. Blood and tissues from secondary lymphoid organs (spleen, mesenteric lymph nodes, and inguinal lymph nodes) and from the colon, ileum, lung, liver, and brain were analyzed using viral barcode sequencing, intact proviral DNA assay, single-cell RNA sequencing, and combined CODEX and RNAscope in situ hybridization. Four of seven animals had viral barcodes detectable by deep sequencing of plasma at necropsy, although plasma viral RNA remained below 22 copies per milliliter. Among the tissues studied, mesenteric lymph nodes, inguinal lymph nodes, and spleen contained viral barcodes detected in plasma. CD4+ T cells were the main cell type harboring viral RNA after ATI. Furthermore, T cell zones in lymphoid tissues showed higher viral RNA abundance than B cell zones for most animals. These findings are consistent with lymphoid tissues contributing to the virus present in plasma early after ATI.

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Conflict of interest statement

Competing interests: J.D.L. has served as a compensated advisor for Gilead Sciences. The other authors declare that they have no competing interests.

Figures

Fig. 1.
Fig. 1.. Experimental design and dynamics of SIV plasma viral load.
The limit of detection of this assay was 81 copies/ml (red dashed line), and the samples from 28, 32, 36, and 40 weeks postinfection (wpi), as well as samples collected at necropsy, were further tested by a more sensitive qRT-PCR assay with a limit of detection of 22 copies/ml (blue dashed line). These time points are marked with an asterisk (*). Plasma viral loads were undetectable at the necropsy time point during ATI-2.
Fig. 2.
Fig. 2.. Detection of SIVmac239M barcodes in plasma during acute and chronic phases of SIV infection, ART, and ATIs.
(A) Shown are animals in which barcodes were detectable in plasma during ATI-2 (group 1). (B) Shown are animals in which no viral barcodes were detected in plasma during ATI-2 (group 2). Cyan lines represent clonotypes found at ATI-1. Red lines represent clonotypes found at ATI-2. Black lines represent clonotypes found at ATI-1 and ATI-2.
Fig. 3.
Fig. 3.. Longitudinal assessment of barcode clonotype numbers and the size of the viral reservoir in PBMCs.
(A) Percentage of SIVmac239M barcodes in DNA from PBMCs at different stages normalized to the viral rebound at ATI-1 in plasma in at least 1% of the total barcodes detected. No barcodes were detected in samples on or below the black dashed line. (B) Viral reservoir sizes in PBMCs (copies/106 cells) are shown at the indicated time points in both groups of animals.
Fig. 4.
Fig. 4.. Tissue viral clonotype numbers were higher in secondary lymphoid tissues at ATI-2.
(A and B) Shown is the number of clonotypes in each tissue from CA-DNA (A) and CA-RNA (B) samples. Horizontal bars indicate the mean values of MesLN (44.0, 8.7; DNA, RNA), IngLN (39.3, 5.1), spleen (11.3, 6.1), ileum (3.1, 0.0), colon (3.9, 1.7), lung (1.1, 1.0), liver (0.1, 0.0), and brain (0.0, 0.1). (C) CA-DNA mean values for each tissue paired with their respective CA-RNA mean value showed a statistically significant positive Spearman’s correlation (P = 0.0131).
Fig. 5.
Fig. 5.. SIVmac239M barcode detection in CA-DNA from nine tissues corresponds with plasma barcode detection at ATI-1 and ATI-2 in group 1.
(A) Shown are the clonotypes found in each animal. The barcodes from plasma viral RNA at ATI-1 and ATI-2 were included as references related to rebound. Barcodes associated with ATI-1 are shown with a cyan line. Barcodes associated with ATI-2 are shown with a red line. Barcodes associated with ATI-1 and ATI-2 are shown with a black line. (B) The 11 barcodes detected in plasma during ATI-2 are represented by the letters A to K, and the viral clonotypes are divided by the animals in which they were detected as follows: DJ01 (red, A and B), EM77 (blue, C to F), EP15 (gold, G and H), and KT12 (purple, I to K). (C) Shown is the tissue distribution of the total proportion of the ATI-2 detected plasma viral clonotypes. The colors of the dots indicate the animal ID. Data are presented as means ± SD.
Fig. 6.
Fig. 6.. Group 1 shows a larger SIV intact provirus reservoir in CD4+ T cells in the MesLNs and spleen at ATI-2 as compared with group 2.
(A) Shown are the copy numbers of intact provirus per 1 × 106 CD4+ T cells in MesLNs and spleen. (B) Shown are the copy numbers of intact provirus per 1 × 106 CD11b+ cells in MesLNs and spleen. Data are presented as means ± SD. Data were analyzed using nonparametric Mann-Whitney tests; *P < 0.05.
Fig. 7.
Fig. 7.. Distribution of detected SIV RNA–positive cells in MesLN and spleen samples at ATI-2.
Slides were fully scanned to detect and quantify the presence of viral RNA in cells. (A) Shown are representative images of the MesLNs and spleens from animal DJ01 in group 1 and animal LC31 in group 2. Viral RNA is shown in red and positive cells are indicated by red arrows. (B) Shown is a comparison of the proportion of viral RNA-positive cell numbers in the MesLNs and spleens of all animals. The horizontal bars indicate mean values. Data were analyzed by unpaired t test. *P < 0.05. (C) Shown are representative Comb-CODEX-RNAscope images of MesLNs from animals DJ01 and LC31. (D) Voronoi plots of the Comb-CODEX-RNAscope show the viral RNA (red) distribution in the B cell zones (marked by CD20 in green) and T cell zones (marked by CD3 in blue).
Fig. 8.
Fig. 8.. Gene dysregulation was observed in MesLNs isolated from group 1 animals at ATI-2.
(A) Shown are Louvain clusters at 0.1 resolution. (B) Shown is a dot plot of the genes frequently dysregulated across multiple Louvain clusters in group 1 animals. (C) Shown are DEGs repeated across different Louvain clusters at 2.0 resolution with the genes H2AC20, COX7C, and KCNQ5 shown with red bars. (D) Shown is the differential expression of H2AC20 and COX7C in the two groups.
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