SIV RNA levels in genital tract tissues at 11 weeks post infection.

b<p>log10 vRNA copies per ug tissue RNA.</p

Highlighter plots of SGA derived <i>env</i> sequences from macaque 36338.

<p>(A) Highlighter plot of SIV <i>env</i> nucleotide sequences from blood plasma (red), epididymis (purple), axillary lymph node (green), prostate (yellow), seminal vesicle (blue) and testis (orange) in male macaque 36338, 11 weeks post infection with SIVmac251. Individual nucleotide polymorphisms are indicated by green (adenine), red (thymine), yellow (guanine), or blue (cytosine) ticks and are in comparison to master sequence 1BP11_01. Gaps are indicated in gray. (B) Highlighter plot of SIV env amino acid sequences from tissues.</p

Highlighter plots of SGA derived env sequences from macaque 36199.

<p>(A) Highlighter plot of SIV <i>env</i> nucleotide sequences from blood plasma (red), epididymis (purple), axillary lymph node (green), prostate (yellow), seminal vesicle (blue) and testis (orange) in male macaque 36199. Sequences from blood plasma and seminal plasma are from 5, 8, 9, and 11 weeks post-infection. Sequences from epididymis, axillary lymph node, prostate, and testis were taken 11 weeks post-infection with SIVmac251. Individual nucleotide polymorphisms are indicated by green (adenine), red (thymine), yellow (guanine), or blue (cytosine) ticks and are in comparison to master sequence 4BP05_01. Gaps are indicated in gray. (B) Highlighter plot of SIV <i>env</i> amino acid sequences from tissues.</p

Neighbor joining tree of SGA derived <i>env</i> sequences from macaque 36199.

<p>Midpoint rooted Neighbor-joining tree of the <i>env</i> sequences from SIVmac251 infected animal 36199, 11 weeks post-infection. Bar represents 0.001 nucleotide substitutions per site.</p

SIV RNA levels in blood plasma, seminal plasma and semen cells.

a<p>log10 vRNA copies per ml blood plasma and seminal plasma.</p>b<p>log10 vRNA copies per ug cellular RNA.</p

Neighbor Joining tree of SGA derived <i>env</i> sequences from macaque 36338.

<p>Midpoint rooted Neighbor-joining trees of the <i>env</i> sequences from SIVmac251 infected animal 36338 at 11 weeks post infection. Bar represents 0.001 nucleotide substitutions per site.</p

SIVRNA localizes with immune clusters in brain and spleen.

(A) Genes enriched in TEM and TCM clusters in control and SIV brain (p.adj B) UMAP of immune clusters in brain and vRNA expression in clusters in control and SIV. (C) UMAP of immune clusters in spleen and vRNA expression in clusters in control and SIV. Acute 251 (n = 4) cohort assessed. (TIF)</p

CSF CCR5+ CD8 T cell frequencies do not decrease during acute SIV infection.

shows % CD8 T cells, % CD28- CD95+ CD8 T cells, �R5+ CD8 T cells, and % CD69+ CD8 T cells in CSF in Chronic 251 cohort (n = 6). Significant differences by one-tailed Wilcoxon matched-pairs signed rank test, *, p (TIFF)</p

T cell effector molecular programs induced within the SIV-Infected brain.

Differential gene expression (DGE) analysis of the immune clusters across conditions was performed using functions from Seurat; selection threshold of (adjusted p-value  0.25) based on Benjamini-Hochberg correction. (A) Heat map of DGE genes in controls (C) versus SIV for each immune cluster. (B) Venn diagram shows shared interferon stimulated genes upregulated post SIV across brain and spleen CD4 T cell and monocyte/macrophage immune clusters. (C) Chord plot show pathways and corresponding genes enriched in SIV versus control CD4 TCM cell cluster in brain. (D) Venn diagram shows shared genes downregulated post SIV in brain and spleen CD4 T cell clusters. We used the monocle3 based workflow to estimate lineage differentiation between the cell populations based on the experimental conditions. We extracted the subsets of identified cell types from our integrated Seurat object and further inferred the trajectory graphs. Using the defined root node (TCM), we chose lineages based on the shortest path connecting the root node and the leaf node. After establishing different lineages, we implemented a differential gene test to find genes that changed as a function of pseudotime based on a combination of Moran’s statistic and q-value and visualized using heatmaps and individual gene trajectory plots. (E) Heatmap (Lineage 3) shows changes in gene expression in lineage comprising of T cells. Along this trajectory was induction of genes associated with cell cycle progression (TK1, MKI67, EIF1, S100A10, S100A4), immune cell activation and differentiation (ZEB2, KLF2, CD52) [41], cytotoxic function (PFN1, GZMB, GZMH, NKG7, and CST7). Canonical TCM genes, such as IL7R and LTB, were downregulated in this lineage. (F) shows expression levels genes of select genes from heat map (ZEB2, LTB, GZMB) along pseudo-time as a function of infection. Schematics were generated using BioRender.</p

Decrease in vRNA in brain during antiretroviral therapy.

(A) Chronic 251 cohort (n = 6) assessed. (B) Kinetics of plasma (red lines) and CSF (blue lines) viral suppression and rebound (vRNA copies/mL fluid, measured by RT-qPCR) over the course of ART initiation and interruption. Green bars indicate periods of ART with FTC, TDF, and DTG. Horizontal dashed line indicates limit of detection (15 vRNA copies/ml). (C) Concentration of ARVs (ng/mL) in plasma and CSF quantified by LC-MS. (D) Concentration of ARVs (ng/mg) in PFC and colonic tissue. (E) shows active phosphorylated forms of TFV and FTC. Spearman correlation, two-tailed p value shown. Sampling was performed 2–4 weeks post ART initiation with last ARV dose administered 9–12 hours prior to necropsy, FTC = emtricitabine, TDF = tenofovir disoproxil fumarate, DTG = dolutegravir, Gray shaded area represents lower limit of quantification of assay. (F) SIV vRNA (G) SIV vDNA (copies/10^6 cells) in brain region (RT-qPCR on post-mortem punch biopsies from specified regions. Gray shaded area represents viral loads below threshold of detection. Significant differences by two tailed Wilcoxon matched-pairs signed rank test, * p< 0.05 in C-E. Schematics were generated using BioRender.</p