Growth of RGΔM2-2 versus parental virus in Vero cells.

<p>(A) Vero cells were infected at an MOI of 0.001 and incubated at 37°C. Samples harvested at the times indicated were titrated on Vero cells.</p

Attenuation and immunogenicity in cotton rats.

<p>(A) Groups of cotton rats were infected as indicated. (B) The degree of attenuation as amount of virus present in the lungs of animals in (A) was determined. ****p < 0.0001 by unpaired t test. (C) Immunogenicity study in cotton rats. Animals were immunized as indicated. (D) Serum neutralization titers were determined. (E) Protective efficacy as amount of challenge virus present in the lungs of immunized animals in (C).</p

IM boost immunization in NHP.

<p>(A) AGMs were infected as indicated. (B) Complement-dependent serum neutralizing titers, PRNT60. (C) Protective efficacy as determined from BAL samples after challenge.</p

IN vs IM immunization in NHP.

<p>(A) AGMs were infected as indicated. (B) Serum F-binding antibodies were determined by ELISA. (C) Complement-dependent serum neutralizing titers were measure by PRNT60. (D) Protective efficacy determined as virus titers from BAL samples after challenge.</p

CX3CR1 Is Expressed in Differentiated Human Ciliated Airway Cells and Co-Localizes with Respiratory Syncytial Virus on Cilia in a G Protein-Dependent Manner

<div><p>Respiratory syncytial virus (RSV) is the principal cause of bronchiolitis in infants and a significant healthcare problem. The RSV Glycoprotein (G) mediates attachment of the virus to the cell membrane, which facilitates interaction of the RSV Fusion (F) protein with nucleolin, thereby triggering fusion of the viral and cellular membranes. However, a host protein ligand for G has not yet been identified. Here we show that CX3CR1 is expressed in the motile cilia of differentiated human airway epithelial (HAE) cells, and that CX3CR1 co-localizes with RSV particles. Upon infection, the distribution of CX3CR1 in these cells is significantly altered. Complete or partial deletion of RSV G results in viruses binding at least 72-fold less efficiently to cells, and reduces virus replication. Moreover, an antibody targeting an epitope near the G protein’s CX3CR1-binding motif significantly inhibits binding of the virus to airway cells. Given previously published evidence of the interaction of G with CX3CR1 in human lymphocytes, these findings suggest a role for G in the interaction of RSV with ciliated lung cells. This interpretation is consistent with past studies showing a protective benefit in immunizing against G in animal models of RSV infection, and would support targeting the CX3CR1-G protein interaction for prophylaxis or therapy. CX3CR1 expression in lung epithelial cells may also have implications for other respiratory diseases such as asthma.</p></div

CX3CR1 in differentiated HAE cells interacts with RSV.

<p>Differentiated HAE cells, grown as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130517#pone.0130517.g001" target="_blank">Fig 1</a>, were incubated with RSV and imaged by immunofluorescence and confocal microscopy. (<b>a</b>) Binding experiment meant to visualize viral particles in association with HAE cells. Cultures were incubated 2h with RSV, then fixed and processed. RSV virions appear in green, β-tubulin is shown in red, and CX3CR1 is colored purple. Two regions of interest, representative of other RSV-bound cells in this image, are outlined with white squares. The top one is shown in expanded views in <b>b</b>, and the other one is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130517#pone.0130517.s002" target="_blank">S2 Fig</a>. Red, green, and purple images corresponding to β-tubulin, RSV F, and CX3CR1 respectively, are shown individually. Also shown is a merged image combining the fluorescence channels for RSV F, CX3CR1, and motile cilia. (<b>c</b>) Infected HAE cells were incubated for 3 days after infection and imaged using the same antibodies and fluorophores as in <b>a</b> and <b>b</b> but pseudo-colored differently: β-tubulin is in blue, RSV F in green, and CX3CR1 is shown in red. The two images are xy planes of the same sample separated along the z axis by 3.7 μm. The bottom image shows cilia and apical cell body, including some purple color indicative of colocalized tubulin and CX3CR1 immunofluorescence. The top image crosses the plane of the nuclei, below the cilia. Only infected cells (green) are surrounded by red-colored CX3CR1-positive circular features. Uninfected cells in the same sample and cells from uninfected control samples do not show these CX3CR1-containing structures (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130517#pone.0130517.g001" target="_blank">Fig 1C</a>). (<b>d</b>) Confocal immunofluorescence of HAE cells grown, differentiated, and infected for 3 days with RSV strain MSA1. Both <i>en face</i> (xy) and side (xz and yz) views are shown in this image. The xy plane of the <i>en face</i> view mostly cuts through the cilia of the cells, above the cell body. Nuclei are shown in blue using DAPI and, as in panel <b>a</b>, anti-RSV F protein is in green, anti-β-tubulin in red, and anti-CX3CR1 in purple. As in panel <b>c</b>, large ovoid structures positive for CX3CR1 immunofluorescence are seen in the side views and located near the nuclei of infected cells.</p

CX3CR1 is expressed in human airway epithelial cells and is localized to the motile cilia.

<p>HAE cells were grown in an air-liquid interface (ALI) culture system until differentiation was achieved, and imaged by confocal immunofluorescence. (<b>a</b>) Anti-β-tubulin (green) and anti-CX3CR1 (red) immunodetection of the cells seen <i>en face</i> are shown individually and merged (scale bar 10 μm). An xz plane image (XZ Merge) further illustrates the localization of CX3CR1 in motile cilia (scale bar 5 μm). Nuclei, stained with DAPI, are rendered in blue. (<b>b</b>) Immunostaining with isotype control antibodies confirms the specificity of the immunostaining. The merge image and XZ merge image are in planes that cross nuclei to confirm the presence of cells and the absence of non-specific immunostaining. (<b>c</b>) Confocal immunofluorescence images of differentiated HAE cells. Cells were either infected with RSV strain MSA1 (top and bottom rows), or mock infected (middle row). Images were acquired either two hours (left two columns), 1 day (middle two columns), or 3 days (right two columns) after infection or mock infection. CX3CR1 immunofluorescence is shown in (purple) (top two rows), or using an isotype negative control (bottom row). The cells were also stained for nuclei using DAPI (blue). Two representative images are shown for each condition tested. The 2-hour time point images are of an xy plane above the nuclei, intersecting the motile cilia. CX3CR1 immunofluorescence is clearly detected in the cilia of both infected and uninfected cells but is absent in the isotype control samples. The 1-day and 3-day time points are in an xy plane crossing the nuclei, 4.3 μm below the motile cilia.</p

Antibody to G protein inhibits RSV binding to HAE cells.

<p>Wild type RSV was incubated with a negative control antibody or with 131-2G for 1h, and added to differentiated HAE cells for 2 h. (<b>a</b>) Fluorescence micrograph of cells fixed and immunostained against RSV F incubated with the indicated antibody. (<b>b</b>) Quantification of immunofluorescence intensity in a by counting pixels in moderate (40–100) or high (101–255) brightness ranges. Incubation with 131-2G significantly reduces immunofluorescence detection of RSV (<i>P</i> = 0.0001 by two-tailed t-test).</p

Attenuation and immunogenicity in African green monkeys (AGMs).

Monkeys were inoculated via the combined intranasal (IN) and intratracheal (IT) route with 106 PFU of each virus in a 1 ml inoculum per site (total dose = 2x106 PFU per AGM). Nasopharyngeal swabs were taken on days 1 through 10 and on days 12 and 14, and tracheal lavages were performed every other day through day 14. Virus shedding in nasopharyngeal swabs (A) and tracheal lavage (B) were measured using an RSV immunoplaque assay on Vero cells. The lower limits of virus detection were 0.7 log10 (A) and 1.0 log10 PFU per mL (B), respectively (dashed lines). (C) Sera were collected on days 21 and 28 post-immunization, and RSV-neutralizing titers, plotted as 1/log2 were determined using a 60% plaque reduction neutralization test (PRNT60) in presence of complement. The lower limit of detection was 3.3 log2 (dashed line) (C). Statistical analyses were performed by 2-way ANOVA with Tukey’s post-hoc analysis (GraphPad Prism, version 9, GraphPad software, San Diego, California, USA).</p

Genome map of ΔNS2-L19F-4M vaccine candidate.

Genes encoding separate mRNAs are indicated by boxes shown in the 3´-to-5´order. The positions of the deletion of the NS2 gene, the deletion of codon 1313 in the L gene, and the missense mutation at codon 1314 in the L gene are shown. Also shown are the positions of the “line 19” F mutations I79M, K191R, T357K and N371Y (named by F protein amino acid position preceded by the A2 assignment and followed by the “line 19” assignment).</p