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

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

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

Gammaherpesvirus infection and malignant disease in rhesus macaques experimentally infected with SIV or SHIV

<div><p>Human gammaherpesviruses are associated with malignancies in HIV infected individuals; in macaques used in non-human primate models of HIV infection, gammaherpesvirus infections also occur. Limited data on prevalence and tumorigenicity of macaque gammaherpesviruses, mostly cross-sectional analyses of small series, are available. We comprehensively examine all three-rhesus macaque gammaherpesviruses -Rhesus rhadinovirus (RRV), Rhesus Lymphocryptovirus (RLCV) and Retroperitoneal Fibromatosis Herpesvirus (RFHV) in macaques experimentally infected with Simian Immunodeficiency Virus or Simian Human Immunodeficiency Virus (SIV/SHIV) in studies spanning 15 years at the AIDS and Cancer Virus Program of the Frederick National Laboratory for Cancer Research. We evaluated 18 animals with malignancies (16 lymphomas, one fibrosarcoma and one carcinoma) and 32 controls. We developed real time quantitative PCR assays for each gammaherpesvirus DNA viral load (VL) in malignant and non-tumor tissues; we also characterized the tumors using immunohistochemistry and <i>in situ</i> hybridization. Furthermore, we retrospectively quantified gammaherpesvirus DNA VL and SIV/SHIV RNA VL in longitudinally-collected PBMCs and plasma, respectively. One or more gammaherpesviruses were detected in 17 tumors; generally, one was predominant, and the relevant DNA VL in the tumor was very high compared to surrounding tissues. RLCV was predominant in tumors resembling diffuse large B cell lymphomas; in a Burkitt-like lymphoma, RRV was predominant; and in the fibrosarcoma, RFHV was predominant. Median RRV and RLCV PBMC DNA VL were significantly higher in cases than controls; SIV/SHIV VL and RLCV VL were independently associated with cancer. Local regressions showed that longitudinal VL patterns in cases and controls, from SIV infection to necropsy, differed for each gammaherpesvirus: while RFHV VL increased only slightly in all animals, RLCV and RRV VL increased significantly and continued to increase steeply in cases; in controls, VL flattened. In conclusion, the data suggest that gammaherpesviruses may play a significant role in tumorogenesis in macaques infected with immunodeficiency viruses.</p></div

Insertion of genetic barcode into SIVmac239.

<p>(A) A 34 base cassette (yellow) bearing a stretch of 10 random bases was inserted between <i>vpx</i> and <i>vpr</i> of wild-type SIVmac239 to generate the genetically barcoded virus SIVmac239M. The MluI restriction site used is outlined in black, and the sequences of the barcode flanking regions are colored in blue and green to depict the two possible insertion orientations. (B) Representative sequences in single stock aliquots depicting the bimodal distribution of authentic barcodes (green) versus barcodes containing PCR error (red).</p

Pairing of relative viral loads and time of reactivation.

<p>Approximate day of reactivation of cells harboring clonotypes was estimated in study 2 animals following interruption of long-duration therapy for animals DEJX (A), DFGV (B), DEJW (C), H090 (D), DEPI (E), and H105 (F). Total viral load at the time point selected is comprised of the relative proportions of clonotypes determined by sequencing analysis. Each individual clonotype’s growth rate was estimated during the maximum exponential phase of the total viral load curve, and the slope of the growth of each clonotype was extended below the limit of detection to estimate the approximate time of reactivation. The grey line represents the plasma concentration of one viral copy in the total plasma, and the tick marks represent the theoretical reactivations based on calculated reactivation rate for each animal.</p

Plasma viral loads, PBMC CA-vRNA and CA-vDNA in animals in study 2.

<p>(A) 6 rhesus macaques, DEJX (red), DFGV (yellow), DEJW (blue), H090 (purple), DEPI (light green), and H105 (dark green) were infected with SIVmac239M and cART was initiated on day 4 post-infection. Pairs of macaques were removed from therapy on days 305, 374, and 482 post-infection. Viral RNA copies were measured in plasma collected up to 550 days. Bars over the viral load data indicate duration of therapy for each animal. (B-C) Cell-associated viral DNA (B) and RNA (C) was measured in PBMCs over the duration of cART therapy in animals infected with SIVmac239M. Measurements obtained are shown overlaying the detected plasma viral loads. Open symbols represent measurements that were below the limit of detection.</p

SIVmac239M <i>in vitro</i> and <i>in vivo</i> replication.

<p>(A) CD8+ T-cell-depleted PBMCs infected with either SIVmac239 (teal) or SIVmac239M (maroon) were monitored over 13 days and samples collected were assayed by ELISA specific for reverse transcriptase (RT) over time. (B) Infectivity was tested <i>in vivo</i> in two rhesus macaques, ZK37 (light blue) and ZK56 (purple) following intravenous infection. Viral RNA copies were measured in plasma over 100 days post infection with a lower limit of detection of 15 copies/mL.</p