In <i>vitro</i> virus suppression assay of TCR-transduced cell lines.

<p>Flow cytometry analyses of mixed cultures consisting of effector CD8⁺ T-cell lines and a target autologous CD4⁺ T-cell clone that was untreated or exposed to either wild-type SIV_mac239 or SIV_myr- are presented. Effectors are labeled above each column and targets are labeled at the left of each row. The effector CD8⁺ T cells in the co-cultures were stained with CellTrace Violet® and excluded from the analysis so that only the target cells were counted.</p

Flow cytometry analysis of transduced T cells.

<p>A, analysis of the TCR-transduced EZP cell lines for CM9 peptide/MHC tetramer and SL8 peptide/MHC tetramer is presented with that of the untransduced CD8⁺ control cell line from recipient animal EZP. B, tetramer analysis of two SIV-specific CTL clones isolated from donor animal DAJ is presented above tetramer-sorted TCR transduced CD8⁺ cell lines. The DAJ SL8–42 clone is the TCR gene donor for the SL8–42 TCR EZP cell line.</p

Diagram of TCR expressing retroviral vector and the mature TCR chains.

<p>The MSGV1 murine retroviral vectors sequences are displayed as black boxes and lines while the TCR expression cassette is in white. The fine structure of the TCR chain fusion cassette is presented below the vector with the different MamuA*01-restricted TCRs molecularly-cloned from DAJ T-cell clones that were inserted into the vectors indicated above the cassette. The sequences separating the TCR genes, the furin recognition sequence, KAKR, the S-G-S-G spacer, and the P2A fowl pox self-cleaving peptide, are shaded gray. The furin cleavage site and the P2A self-cleavage site are indicated below the cassette with arrows. The mature α and β chains produced by this vector are displayed at the bottom of the figure.</p

Infected CD4 Elimination (ICE) inversely correlates with plasma SIV RNA levels in SIV-infected rhesus macaques.

<p>Red dots represent LTNP/EC (n = 11). Blue dots represent progressors (n = 11). Statistical analysis was performed by the Spearman rank method.</p

SIV-specific CD8⁺ T cells from LTNP/EC mediate greater lysis of SIV-infected CD4⁺ T-cell targets compared with progressors.

<p>GrB target cell activity (<b>A</b>) and infected CD4 elimination (ICE) (<b>B</b>) are shown for LTNP/EC (n = 10, GrB target cell activity; n = 11, ICE) and progressors (n = 11). Horizontal bars represent the median values. <b>C.</b> Correlation between ICE and GrB target cell activity (n = 22) was determined by the Spearman rank method. Red, blue and cyan dots represent LTNP/EC, progressors and one SIV-uninfected animal, respectively.</p

SIV-specific CD8⁺ T cells of LTNP/EC mediate greater per-cell killing of SIV-infected targets than those of progressors, which is not simply due to higher true E∶T ratios.

<p><b>A.</b> The true effector to target (E∶T) ratios, determined by measurements of IFN-γ-secreting CD8⁺ T-cell effectors and p27-expressing CD4⁺ T-cell targets, respectively, as described in the Methods and shown in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003195#ppat.1003195.s001" target="_blank">Figure S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003195#ppat.1003195.s002" target="_blank">Table S1</a>, were compared between LTNP/EC (n = 11) and progressors (n = 11). Horizontal bars represent the median values. <b>B, C.</b> GrB target cell activity (<b>B</b>) or ICE (<b>C</b>) responses plotted against the true E∶T ratios are shown for LTNP/EC (n = 10, GrB target cell activity; n = 11, ICE) and progressors (n = 11). GrB target cell activity is shown after subtraction of background. The response curves were analyzed by regressing ICE and GrB on log true E∶T ratios using analysis of covariance. The standard two-tailed t test from regression analysis was used to compare estimated GrB target cell activity and ICE of LTNP/EC with that of progressors at the 5.8 E∶T ratio, the median of the combined E∶T ranges of both groups.</p

Characteristics of macaques and prediction of SIV control.

<p>ICE, Infected CD4⁺ T cell Elimination (%). LTNP/EC, Long-Term Nonprogressor/Elite Controllers. SP, Slow Progressor. N/A, Not Applicable. PBMC from more than one time point were used in the animals marked with an asterisk.</p

SIV-specific CD8⁺ T cell cytotoxicity measured by granzyme B delivery or Infected CD4 Elimination (ICE).

<p><b>A.</b> The top panels show granzyme B (GrB) target cell activity representative of a “high responder”. The bottom panels show GrB target cell activity representative of a “low responder”. Values indicate percentages of targets with increased fluorescence due to GrB substrate cleavage. Background GrB target cell activity measured in response to uninfected targets (left column) was subtracted from responses measured against infected targets (right column) to determine net GrB target cell activity (red values). <b>B.</b> ICE values calculated based on p27 expression (sum of the upper quadrants) as described in the Methods, are shown in red for the same “high responder” (78.8%, top row) and “low responder” (22.3%, bottom row) as shown in A. Quadrant values indicate percentages of gated targets. In all experiments, CD4⁺ T cell lines were used as targets. CD8⁺ T cells that had been stimulated with SIV-infected targets for 6 days were used as effectors. GrB target cell activity and ICE were calculated after 1 hour of incubation of effectors and plated at an E∶T ratio of 25∶1.</p

Genetically-barcoded SIV facilitates enumeration of rebound variants and estimation of reactivation rates in nonhuman primates following interruption of suppressive antiretroviral therapy

<div><p>HIV and SIV infection dynamics are commonly investigated by measuring plasma viral loads. However, this total viral load value represents the sum of many individual infection events, which are difficult to independently track using conventional sequencing approaches. To overcome this challenge, we generated a genetically tagged virus stock (SIVmac239M) with a 34-base genetic barcode inserted between the <i>vpx</i> and <i>vpr</i> accessory genes of the infectious molecular clone SIVmac239. Next-generation sequencing of the virus stock identified at least 9,336 individual barcodes, or clonotypes, with an average genetic distance of 7 bases between any two barcodes. <i>In vitro</i> infection of rhesus CD4+ T cells and <i>in vivo</i> infection of rhesus macaques revealed levels of viral replication of SIVmac239M comparable to parental SIVmac239. After intravenous inoculation of 2.2x10⁵ infectious units of SIVmac239M, an average of 1,247 barcodes were identified during acute infection in 26 infected rhesus macaques. Of the barcodes identified in the stock, at least 85.6% actively replicated in at least one animal, and on average each barcode was found in 5 monkeys. Four infected animals were treated with combination antiretroviral therapy (cART) for 82 days starting on day 6 post-infection (study 1). Plasma viremia was reduced from >10⁶ to <15 vRNA copies/mL by the time treatment was interrupted. Virus rapidly rebounded following treatment interruption and between 87 and 136 distinct clonotypes were detected in plasma at peak rebound viremia. This study confirmed that SIVmac239M viremia could be successfully curtailed with cART, and that upon cART discontinuation, rebounding viral variants could be identified and quantified. An additional 6 animals infected with SIVmac239M were treated with cART beginning on day 4 post-infection for 305, 374, or 482 days (study 2). Upon treatment interruption, between 4 and 8 distinct viral clonotypes were detected in each animal at peak rebound viremia. The relative proportions of the rebounding viral clonotypes, spanning a range of 5 logs, were largely preserved over time for each animal. The viral growth rate during recrudescence and the relative abundance of each rebounding clonotype were used to estimate the average frequency of reactivation per animal. Using these parameters, reactivation frequencies were calculated and ranged from 0.33–0.70 events per day, likely representing reactivation from long-lived latently infected cells. The use of SIVmac239M therefore provides a powerful tool to investigate SIV latency and the frequency of viral reactivation after treatment interruption.</p></div

Evaluation of clonotypes found in stock and monkeys.

<p>(A) Each individual clonotype was plotted by its rank order in the stock versus the number of animals in which it was found. A linear correlation was generated with an R² value of 0.77. Density of clonotypes at any single point are colored using a log scale heat map where red points depict 2 logs of clonotypes and dark blue points represent single clonotypes. (B) The number of barcodes was plotted against the number of monkeys in which the barcodes were found. Of the 9,336 total stock barcodes, 7,991 were found systemically in at least one animal, and 1,345 were not found in any of the 26 animals. (C) The mean relative frequency of each individual barcode (grey circles) was plotted against the number of monkeys in which the barcode was found. The relative frequencies of the barcodes demonstrate the comparative homogeneity of all clonotypes across all animals.</p