Lentiviral vector transduction leads to effective antigen expression both in conventional dendritic cells (cDC) and in plasmacytoid DC (pDC).

<p>(A) Dose-response transduction experiments with eGFP-integrative vectors (TRIP.I eGFP) or eGFP-non integrative vectors (TRIP.NI eGFP), heat-inactivated (HI) or not. On day 6 of differentiation, FL-DC were exposed to vector particles for 48 hours and transduction of CD11c positive cells was assessed by measuring eGFP expression by flow cytometry. Numbers indicate the percentage of CD11c⁺ cells expressing eGFP. Blue gates delineate a DC population expressing high levels of eGFP. (B) Transduction of pDC and cDC by lentiviral vectors. Expression of eGFP by cDC (CD11c⁺ B220⁻) and pDC (CD11c⁺ B220⁺) is shown. Thin lines, control cells; filled profiles, FL-DC transduced with 300 ng/ml of vector particles.</p

Rapid protection against WNV infection conferred by nonintegrative lentiviral vector immunization.

<p>Six mice/group were vaccinated with 100 ng of TRIP.NI E_WNV or 100 ng of TRIP.I E_WNV. A control group of mice inoculated with phosphate-buffered saline (PBS) was included. One week after the vaccination, mice were challenged with 1,000 i.p. LD₅₀s of WNV strain IS-98-ST1. Survival was recorded for 21 days. Data are representative of three independent experiments.</p

Efficient transduction of non-dividing cells with lentiviral vectors defective for integration.

<p>Aphidicolin-treated HeLa cells were transduced with graded doses (1–100 ng of p24 antigen) of eGFP-integrative vectors (TRIP.I eGFP) or eGFP-nonintegrative vectors (TRIP.NI eGFP). FACS analysis of the percentage of GFP positive cells (A) and the MFI (B) at 48 hours post-transduction is shown.</p

Efficient long-term protection against WNV infection by nonintegrative lentiviral vector-based vaccine.

<p>Two months post-immunization with graded doses (1–100 ng of p24 antigen) of TRIP.NI E_WNV or TRIP.I E_WNV particles, groups of mice (6 mice per group) were inoculated with 1,000 i.p. LD₅₀s of WNV strain IS-98-ST1. Survival was recorded for 21 days. Data are representative of three independent experiments.</p

Dengue Virus Activates Membrane TRAIL Relocalization and IFN-α Production by Human Plasmacytoid Dendritic Cells <i>In Vitro</i> and <i>In Vivo</i>

<div><p>Background</p><p>Dengue displays a broad spectrum of clinical manifestations that may vary from asymptomatic to severe and even fatal features. Plasma leakage/hemorrhages can be caused by a cytokine storm induced by monocytes and dendritic cells during dengue virus (DENV) replication. Plasmacytoid dendritic cells (pDCs) are innate immune cells and in response to virus exposure secrete IFN-α and express membrane TRAIL (mTRAIL). We aimed to characterize pDC activation in dengue patients and their function under DENV-2 stimulation in vitro.</p><p>Methods & Findings</p><p>Flow cytometry analysis (FCA) revealed that pDCs of mild dengue patients exhibit significantly higher frequencies of mTRAIL compared to severe cases or healthy controls. Plasma levels of IFN-α and soluble TRAIL are increased in mild compared to severe dengue patients, positively correlating with pDC activation. FCA experiments showed that <i>in vitro</i> exposure to DENV-2 induced mTRAIL expression on pDC. Furthermore, three dimension microscopy highlighted that TRAIL was relocalized from intracellular compartment to plasma membrane. Chloroquine treatment inhibited DENV-2-induced mTRAIL relocalization and IFN-α production by pDC. Endosomal viral degradation blockade by chloroquine allowed viral antigens detection inside pDCs. All those data are in favor of endocytosis pathway activation by DENV-2 in pDC. Coculture of pDC/DENV-2-infected monocytes revealed a dramatic decrease of antigen detection by FCA. This viral antigens reduction in monocytes was also observed after exogenous IFN-α treatment. Thus, pDC effect on viral load reduction was mainly dependent on IFN-α production</p><p>Conclusions</p><p>This investigation characterizes, during DENV-2 infection, activation of pDCs <i>in vivo</i> and their antiviral role <i>in vitro</i>. Thus, we propose TRAIL-expressing pDCs may have an important role in the outcome of disease.</p></div

TRAIL and IFN-α expression on dengue fever (DF) patients.

<p>PBMCs from acute dengue fever patients were analyzed for mTRAIL expression on pDCs gated as CD14⁻, CD3⁻, CD4⁺, CD123⁺ and BDCA-4⁺ (<b>A</b>). (<b>B</b>) Blood pDC percentages and (<b>C</b>) mTRAIL expressing pDCs were assessed for healthy donors, DF and severe DF patients. (<b>D</b>) IFN-α and (<b>E</b>) Soluble TRAIL were analyzed by ELISA in plasma samples from healthy donors, DF and severe DF patients. Each dot represents one individual and median values are shown as blue bars. Data values were submitted to Mann-Whitney statistical test in which * <i>p</i><0.05 and **<i>p</i><0.005.</p

TRAIL localization in DENV-2-activated pDCs by 3D microscopy analysis.

<p>Freshly purified pDCs stimulated with DENV-2 pre-treated or not with chloroquine (Chloro), or mock infected. TRAIL expression was analyzed by flow cytometry or by a 3D microscope. (<b>A</b>) Membrane TRAIL flow cytometry profiles (left column) on pDC stimulated by different stimuli -mock, DENV-2 or DENV-2+Chloro overlaid by unstimulated (grey). Microscopic images from pDC cultured with the mock, DENV-2 or DENV-2+Chloro showing DAPI-colored nucleus, TRAIL staining (green) and overlay. (<b>B</b>) 3D interactive surface plots analysis of 3D microscopic image. Overlay of nucleus (blue), TRAIL (green) and phase contrast (grey) as seen in (<b>C</b>) for different stimuli: DENV-2-stimulated pDC (DENV-2) exhibits membrane TRAIL localization in contrast to DENV-2+Chloro or unstimulated, where TRAIL is detected only intracellularly. (<b>D</b>) Percentage of pDCs expressing intracellular TRAIL only (Intra) or on the membrane (Mb) is shown as percentage of total analyzed cells. Values were submitted to paired t test in which * p<0.05 and *** p<0.0005.</p

3D microscopy of DENV-2 particles in purified plasmacytoid dendritic cells.

<p>Freshly purified pDCs cultured with DENV-2 pre-treated or not with chloroquine (Chloro), or mock infected were stained with anti-DENV (green) and nucleus was colored with DAPI (blue). (<b>A</b>) pDC images (nucleus, virus and overlay) for mock, DENV-2 and chloroquine-treated plus DENV-2. Inhibition of endosomal acidification (chloroquine) allowed easier detection of DENV particles (DENV-2+Chloro at 2 h or 18 h stimulation). 2 h pDC incubation with DENV-2 was sufficient to detect viral proteins in contrast to the overnight (18 h) DENV-2-incubated pDCs when no virus was detected. (<b>B</b>) pDCs cultured with mock, DENV-2 or DENV-2+chloro were observed by 3D microscope. DENV staining (green) was merged with DAPI (blue)-colored nucleus and with phase contrast (grey). DENV particles were co-localized with pDC cell membrane at 2 h stimulation. Chloroquine allowed DENV-2 detection inside pDCs after 18 h of culture whereas DENV-2 alone did not. Panels shown microscopic images analyzed by 3D interactive surface plot. (<b>C</b>) Quantification of PDCs expressing DENV antigens without (DENV-2) and with chloroquine pre-treatment (DENV-2+Chloro) is shown as percentage of total analyzed cells.</p

Demographic information about the study population with dengue fever (DF)¹.

1<p>Study population with 43 patients.</p>2<p><b>DF ± WS</b> dengue fever without or with warning signs; <b>Severe DF</b>, dengue fever with severe clinical manifestations according to WHO criteria <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002257#pntd.0002257-WHOTDR1" target="_blank">[43]</a>.</p>3<p>Number of patients with the available information during hospitalization.</p>4<p>Hemorrhagic manifestations (epistaxis, gengivorrhagia, metrorrhagia, bleeding after coughing).</p>5<p>Postural hypotension with decrease in systolic arterial pressure in 20 mmHg in supine position or systolic arterial pressure <90 mm Hg.</p>6<p>Pleural, pericardial effusion or ascites.</p>7<p>Average ± standard error from minimal recorded platelet, leukocyte/maximal hematocrit counts/ALT or AST values.</p>8<p>Elevated hematocrit (20% during course of illness and recovery; or >45%, men and >41%, women).</p

Purified DENV-2-induced <i>in vitro</i> mTRAIL expression and IFN-α production by purified plasmacytoid dendritic cells.

<p>PBMCs from healthy donors were stimulated overnight with DENV-2, mock or none (unstimulated). (<b>A</b>) mTRAIL expression profile on pDCs gated from PBMCs (overlay) and (<b>B</b>) mTRAIL positive pDCs for three donors induced by mock SNT (orange) or DENV-2 SNT (blue) using unstimulated (grey fill) pDCs as negative control. DENV positive C6/36 cells infected for 48 h with supernatant of DENV-2-infected C6/36 cells (DENV-2 SNT) or ultracentrifuged DENV-2 SNT (DENV-2 UC) as described in M&M and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002257#pntd.0002257.s001" target="_blank">figure S1</a>. (<b>C</b>) DENV antigens/AlexaFluor488 (green) and nucleus/DAPI (blue) of C6/36 cells infected with DENV-2 SNT (left) and UC (right) at the same inocula dilution (10⁻³). (<b>D</b>) DENV positive C6/36 cells by flow cytometry in which cells were infected with SNT (orange) or UC (blue) DENV-2 inocula at different dilutions. PBMCs from healthy donors were stimulated overnight with DENV-2 UC, mock UC or none (unstimulated). (<b>E</b>) mTRAIL expression profile on pDCs gated from PBMCs (overlay) and (<b>F</b>) mTRAIL positive pDCs for four donors induced by mock UC (orange) or DENV-2 UC (blue) using unstimulated (grey fill) pDCs as negative control. Freshly purified pDCs were stimulated overnight with DENV-2 UC, mock UC or not (unstimulated). (<b>G</b>) TRAIL expression induced by different MOIs of DENV-2 UC (blue) using unstimulated cells (grey) as negative control. (<b>H</b>) Purified pDCs positive for mTRAIL expression and (<b>I</b>) IFN-α secretion by unstimulated (grey), mock UC (orange), DENV-2-UC-stimulated pDCs pre-treated (black) or not (blue) with chloroquine, for four donors. Values were submitted to paired t test in which * p<0.05 and ** p<0.005.</p