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

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

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

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

The chromatography-based ACAM529 purification process results in 250-fold purification factor with respect to Vero HCP.

<p>The chromatography-based ACAM529 purification process results in 250-fold purification factor with respect to Vero HCP.</p

Purity and step yield of ACAM529 (Preparation A) process retains.

a<p>We were unable to determine the amount of Vero DNA in the starting material, as even at high dilutions there was 100% interference of qPCR signal by the sample (dextran sulfate and/or Benzonase®).</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

Chromatography-purified ACAM529 is as immunogenic and protective as cushion-purified ACAM529.

<p>Panel A is a schematic representation of the animal study schedule, long labeled arrows represent viral inoculations (immunizations were performed sc and challenge was intravaginal) short arrows symbolize bleeds, hormone injection (DMPA = depot medroxyprogesterone acetate or Depo-Provera) and the study end day, as indicated. Panel B shows endpoint ELISA titers against a commercially available, purified HSV-2 viral lysate for immunized mice and Panel C depicts survival of animals as a % of the total (n = 15 animals). Mice were immunized either with Mustang® Q (•)- or sucrose cushion (▴)-purified ACAM529 or a placebo (♦)(PBS). Both vaccine preparations elicited similar anti-HSV-2 ELISA titers (Kruskal-Wallis Test P = 0.99) and similar levels of protection against severe virus challenge with wild type HSV-2 strain 333 <b>(</b>Mantel-Cox Test P<0.0001).</p

Virus step yield (PFU) from small-scale screening of anion exchange purification conditions (harvest method, chromatography resins, etc.).

<p>Virus step yield (PFU) from small-scale screening of anion exchange purification conditions (harvest method, chromatography resins, etc.).</p

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