14 research outputs found
Accumulation of influenza vRNPs at the apical membrane of polarized Caco-2/TC7 cells.
Accumulation of influenza vRNPs at the apical membrane of polarized Caco-2/TC7 cells.</p
Fig 1 -
Polarization of Caco-2/TC7 cells grown on Cytodex 3 microcarrier beads. A. Schematic representation of the microtubule cytoskeleton organization in non-polarized versus polarized epithelial cells. In non-polarized cells the microtubules (in blue) grow from the centrosomes (pink cylinders) located near the nucleus and the Golgi apparatus towards the plasma membrane, whereas in polarized cells microtubules grow towards the basal pole. As a consequence, in non-polarized cells the molecular motors that move cargoes towards the plasma membrane are kinesins, the (+) end motors, whereas dynein, the (-) end motor, is moving cargoes towards the apical membrane in polarized cells. B. Cellular morphology as observed with brightfield microscopy. A 10x objective was used (NA = 0.3, WD = 550mm). A bead cross-section is shown in the inset to illustrate the columnar morphology of Caco-2/TC7 cells at 14 days post-seeding on the beads. Scale bars: 100 μm. C. Confocal imaging upon immunostaining for the PALS1 apical membrane marker. A 40x objective (NA = 1.25, WD = 335 μm) was used. Blue and green colour: DAPI and PALS1 immunostaining, respectively. Step size: 0.3 μm, pixel size: 0.4 μm, speed: 400 Hz. Scale bar: 25 μm. D. Confocal imaging upon immunostaining for the ZO-1 marker of tight junctions labelled with ATTO647n. A 93x Leica glycerol objective (NA = 1.3, WD = 300 μm) was used. Blue colour: ZO-1 immunostaining. Step size: 0.18 μm, pixel size: 61 nm, speed: 600 Hz. Scale bar: 10 μm. E. Electron microscopy of an ultrathin section. Caco-2/TC7 cells grown on Cytodex 3 beads were fixed with glutaraldehyde, post-fixed with osmium tetroxide, dehydrated and resin-embedded for transmission electron microscopy. A representative ultrathin section going through the center of the Cytodex 3 beads is shown. Endoplasmic and Golgi apparatus membranes, as well as a mitochondrion and a tight junction, are indicated by arrows. Scale bar: 1 μm.</p
Fig 2 -
Influenza A virus infection of polarized Caco-2/TC7 cells. A. 3D-reconstructed z-stack of a Cytodex 3 microcarrier bead obtained by confocal imaging with a 40x objective (NA = 1.25, WD = 335 μm), segmented for DAPI-stained nuclei. The number of nuclei was determined using the Imaris software for cell segmentation. Scale bar: 40μm. B. 3D-reconstructed z-stack of half a Cytodex 3 microcarrier bead obtained by confocal imaging with a 40x objective (NA = 1.25, WD = 335 μm) upon immunostaining for the viral NP. Caco-2/TC7 cells grown on Cytodex 3 beads were infected at a MOI of 10 PFU/cell with the A/WSN/33 virus. At 8 hours post-infection (hpi) they were fixed with 4% paraformaldehyde and stained with a mix of antibodies specific for the viral NP or the cellular PALS1 protein, and DAPI for nuclear staining. Blue, red and green colour: DAPI, NP and PALS1 immunostaining, respectively. Step size: 0.3 μm, pixel size: 0.4 μm, speed: 400 Hz. Scale bar: 20 μm. C. Production of infectious IAV particles. Caco-2/TC7 cells grown on Cytodex 3 beads were infected at an estimated MOI of 0.001 PFU/cell with the A/WSN/33 virus, in the presence or absence of 0.1 μM of the viral inhibitor baloxavir. The supernatants were collected at 24, 48 and 72 hpi and titrated by plaque assay. The data are represented as the mean +/- SD of four independent experiments. The dashed line represents the limit of detection (L.O.D.) of 250 PFU/mL.</p
Fig 4 -
Influenza virus-induced arrays of endoplasmic reticulum structures parallel to the apical membrane of polarized Caco-2/TC7 cells. A. Caco-2/TC7 cells grown on Cytodex 3 beads were infected at an estimated MOI of 10 PFU/cell with the A/WSN/33 virus or mock-infected. At 15 hpi they were fixed with glutaraldehyde, post-fixed with osmium tetroxide, dehydrated and resin-embedded for transmission electron microscopy. Representative ultrathin sections going through the center of the Cytodex 3 beads are shown. The lower panels represent a higher magnification of the region defined by the dotted box within the upper panels. Endoplasmic and Golgi apparatus membranes, as well as mitochondrion and tight junctions, and, in the case of infected cells, extracellular virions, are indicated by arrows. Scale bar: 1μm. Scale bar for insets: 0.5 μm. B-C. Distribution of the length of endoplasmic reticulum (ER) structures as observed in IAV-infected versus mock-infected cells. Images in the.ser format were opened using the TIA reader plugin of Image J to determine the pixel size. Free-hand lines were drawn on apically located ER-like structures and their length was measured. Two independent experiments were performed. Five cells were used for each condition, 108 and 127 ER-like structures were measured for mock-treated and IAV-infected samples respectively. Significance was tested with an unpaired t test using GraphPad Prism. *** p-value<0.001.</p
NILV elicit as frequent blood CSP-specific T cells as RAS after 3 injections.
<p>BALB/c mice (n = 6/group) were immunized 3 times by IP injections of NILV. They were primed by administration of NILV particles encoding Py CSP and pseudotyped with VSV-G IND at the dose of 100 or 1500 ng p24/mouse. They were boosted 2 months later with 1500 ng p24 of NILV particles pseudotyped with VSV-G NJ, and boosted again 5 months later with 1500 ng p24 of NILV particles pseudotyped with the glycoprotein from Cocal virus. Additionally, mice (n = 6) from the same batch were immunized 3 times by IV injection with RAS at monthly intervals (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g003" target="_blank"><b>Figure 3A</b></a>). The frequency of S9I-specific blood CD8+ cells was followed over time by S9I/K<sup>d</sup> tetramer staining after NILV (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g003" target="_blank"><b>Figure 3B</b></a>) and RAS immunizations (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g003" target="_blank"><b>Figure 3C</b></a>). Data from individual mice and means are shown. The Y-axis uses a logarithmic scale.</p
The protective efficacy of NILV was confirmed in a second independent study.
<p>Groups of BALB/c mice (n = 8/group) were immunized 3 times with NILV by intraperitoneal injections (in red) or not (in black). They were challenged with 500 spz injected IV one month after the last immunization. The % of parasitized red blood cells was followed every other day from day 5 to day 16 post-challenge by Giemsa-stained blood smears. Individual parasitemia are shown (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g007" target="_blank"><b>Figure 7A</b></a>) as well as means + SD (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g007" target="_blank"><b>Figure 7B</b></a>) and parasitemia at day 10 post-challenge (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g007" target="_blank"><b>Figure 7C</b></a>). Among the NILV-immunized mice (red circles), fully (•) <i>versus</i> partially (○) protected animals were further distinguished (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g007" target="_blank"><b>Figure 7D</b></a>). The Mann-Whitney test was used to compare NILV and naive and the Kruskal-Wallis test followed by a Dunn’s post-test were used to compare fully, partially and naive. The gross morphology of spleens and livers from NILV-immunized and naive mice at necropsy were compared 3 weeks post-challenge (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g007" target="_blank"><b>Figure 7E</b></a>).</p
S1 Protocols -
The life cycle of influenza A viruses (IAV), and notably intracellular trafficking of the viral genome, depends on multiple interactions with the cellular cytoskeleton and endomembrane system. A limitation of the conventional cellular models used for mechanistic study and subcellular imaging of IAV infection is that they are cultured in two dimensions (2D) under non-polarizing conditions, and therefore they do not recapitulate the intracellular organization of the polarized respiratory epithelial cells naturally targeted by IAVs. To overcome this limitation, we developed an IAV-infection assay in a 3D cell culture system which allows imaging along the baso-lateral axis of polarized cells, with subcellular resolution. Here we describe a protocol to grow polarized monolayers of Caco2-TC7 cells on static Cytodex-3 microcarrier beads, infect them with IAV, and subsequently perform immunostaining and confocal imaging, or electron microscopy, on polarized IAV-infected cells. This method can be extended to other pathogens that infect human polarized epithelial cells.</div
NILV are as immunogenic as ILV when 10-times more particles are injected.
<p>BALB/c mice (n = 5/group) were immunized by IM injection with various doses (expressed as TU/mouse) of lentiviral vector particles encoding Py CSP, either NILV (□) or ILV (▪). Ten days later, specific cellular immune responses were assessed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g002" target="_blank"><b>Figure 2A</b></a>). The frequency of S9I-specific blood CD8+ cells was assessed by S9I/K<sup>d</sup> tetramer staining, and the frequency of IFNg secreting splenocytes in response to overnight restimulation with S9I or I10L peptides was measured by IFNg elispot assay (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g002" target="_blank"><b>Figure 2B</b></a>). Means + SD are shown. BALB/c mice (n = 3/group) were IM immunized with NILV (□) or ILV (▪) at the dose of 5E+08 or 5E+07 TU/mouse respectively (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g002" target="_blank"><b>Figure 2C</b></a>). The frequency of S9I-specific blood CD8+ cells was followed over time by tetramer staining (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g002" target="_blank"><b>Figure 2D</b></a>). At day 24 post-immunization, spleen cellular response was analyzed by S9I/K<sup>d</sup> tetramer staining (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g002" target="_blank"><b>Figure 2E</b></a>), by IFNg elispot in response to S9I and I10L peptides (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g002" target="_blank"><b>Figure 2F</b></a>), and by intracellular staining of 3 cytokines, IFNg, IL2 and TNFa, in response to S9I (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g002" target="_blank"><b>Figure 2G</b></a>). Cells secreting individual (green), 2 (blue) or 3 (red) cytokines are shown. Anti-(QGPGAP)<sub>2</sub>-specific IgG at day 21 post-immunization were quantified by ELISA and expressed as titers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g002" target="_blank"><b>Figure 2H</b></a>). Medians + range are shown.</p
Protection is associated with CSP-specific CD8+ T cells responses.
<p>Immune correlates of protection against malaria were studied by plotting day 28 post-challenge immunity and day 9 post-challenge parasitemia as X and Y variables and using the Spearman test (the r<sub>s</sub> and p values are shown) and linear regression (r<sup>2</sup> is shown) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g006" target="_blank"><b>Figure 6A</b></a>). Immune responses in challenged mice were compared 28 days post-challenged between the vaccine candidates and their level of protection (fully (•) or partially (○) protected NILV immunized animals in red) by S9I/K<sup>d</sup> tetramer staining and IFNg elispot assay with splenocytes and liver cells and elisa (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g006" target="_blank"><b>Figure 6B</b></a>). Means and SD are shown. The Kruskal-Wallis test was used to compare 3 or 4 groups, followed by a Dunn’s post-test.</p
Fig 3 -
Accumulation of influenza vRNPs at the apical membrane of polarized Caco-2/TC7 cells. A. Caco-2/TC7 cells grown on Cytodex 3 beads (upper panels) or in standard 2D conditions (lower panels) were infected at an estimated MOI of 10 PFU/cell with the A/WSN/33 virus. At 8 hpi, they were fixed with 4% paraformaldehyde, stained with an antibody specific for the viral NP (red colour) and with DAPI for nuclear staining (blue color), and imaged using a confocal microscope with a 40x objective (NA = 1.25, WD = 335 μm). Upper panels: an optical slice going through the center of a representative Cytodex 3 bead is shown. Step size: 0.3 μm, pixel size: 0.4 μm, speed: 400 Hz Scale bar: 25 μm. Inset scale bar: 10 μm. Lower panels: cells representative of the non-polarized state are shown. Step size: 0.3 μm, pixel size: 0.3 μm, speed: 400 Hz. Scale bar: 10 μm. White arrows indicate an accumulation of NP signal at the apical (upper panels) or plasma membrane (lower panels). B. Percentage of cells showing NP signal accumulation at the plasma/apical membrane in non-polarized versus polarized Caco-2/TC7 cells, at 8 hpi with the A/WSN/33 virus at a MOI of 10 PFU/cell. Three independent experiments were performed, in total 634 non-polarized and 703 polarized cells were randomly selected and inspected visually for NP signal accumulation at the plasma/apical membrane, as indicated by white arrows in panel A. Significance was tested with an unpaired t test after log10 transformation of the data, using GraphPad Prism. ** p-value = 0.009. C. Caco-2/TC7 cells grown on Cytodex 3 beads were infected at an estimated MOI of 10 PFU/cell with the A/WSN/33 virus. At 8 hpi they were fixed with 4% paraformaldehyde, co-stained with antibodies specific for the viral NP (red colour) and the cellular RAB11A protein (cyan colour), and imaged using a confocal microscope with a glycerol 93x objective (NA = 1.3, WD = 300 μm). Step size: 0.18 μm, pixel size: 85.7 nm, speed: 600 Hz. Images were deconvolved using the Huygens software. Scale bar: 10 μm. Inset scale bar: 5 μm. The yellow line corresponds to the intensity plot shown in panel D. D. Fluorescence intensity profile for NP (red colour) and RAB11A (cyan) along the yellow line drawn in Fig 3C (overlay inset). The X axis indicates the distance in μm from the proximal (close to the cell center) extremity of the line.</p