Percentage of apoptotic M-hMPV-treated moDCs.

<p>After 24 h of stimulation, M-hMPV-treated moDCs were labeled by DiOC6(3) and propidium iodide prior analysis by flow cytometry. Data are representative of one out of two independent experiments.</p

M-hMPV activated-DCs present antigen to T cells which induce the production of IFN-γ.

<p>moDCs were treated for 24 h with LPS 10 ng/mL and M-hMPV protein 0.344, 0.172, 0.086, 0.034 nM. Cells were harvested after treatment, washed and cultured for 5 days with allogeneic purified T-cells (2×10⁵/well) at a DC/T ratio ranging between 1∶5 and 1∶40. The amount of IFN-γ in the cell-free supernatants of the co-culture was measured by ELISA. Data are represented as means of three experiments ± S.D.</p

M-hMPV treated-MDMs release low level of IFN-α and IFN-β.

<p>MDMs were treated with elution buffer, LPS 10 ng/mL, M-hMPV 0.172 and 0.0172 nM. Supernatants were collected after 24 h and tested for IFN-α and IFN-β production by ELISA. Bar graph represents concentrations values expressed in pg/mL. Data are represented as means of three experiments ± S.D.</p

MoDCs and MDMs treated with M-hMPV protein produce high level of cytokines and chemokines respectively.

<p>(A) Supernatants of cells treated for 24 h with elution buffer, LPS 10 ng/mL (positive control) and M-hMPV 0.172, 0.086, 0.034, 0.0172 nM were assayed for the level of IL-8, IL-6, TNF, IL-12p70, IL-1β and IL-10 cytokines by CBA. (B) Supernatant of MDMs treated with elution buffer, LPS 10 ng/mL (positive control), M-hMPV 0.172 and 0.0172 nM were assayed for MIP-1β, RANTES and TNF production by CBA. Bar graph represents concentrations values expressed in ng/mL. Data are represented as means of three experiments ± S.D.</p

MoDCs express the CD1a molecule while MDMs express the CD14 molecule.

<p>Monocytes were differentiated during 5 days in complete RPMI medium containing 40 ng/mL GM- CSF and 250 U/mL IL-4 for moDCs and 40 ng/mL M-CSF for MDMs. Phenotype of cells were analyzed on day 5 for the expression of CD14, CD1a and HLA-DR. Data represent histograms with isotype control (bold line) and monocyte-differentiated cells (filled profile).</p

M-hMPV protein binds APCs and is internalized.

<p>(A) Binding experiment. APCs were incubated with increased concentrations of M-hMPV-Fluorescein protein for 20 min at 4°C, washed and then analyzed by FACS. Data are shown as mean fluorescence intensity. (B) Competition experiment. APCs were incubated with M-hMPV-Fluorescein protein 4.1 nM for 20 min at 4°C, washed before addition of increased concentration of unlabeled M-hMPV protein for 20 min at 4°C. Results are shown as % of mean fluorescent intensity of M-hMPV-Fluorescein protein binding. (C) Internalization experiment. Cells were incubated with 1.3 nM M-hMPV-Fluorescein protein during 20 min at 4°C, washed and incubated at different time points at 37°C. Cells were then washed and analyzed. Data are shown as mean fluorescence intensity. (D) Confocal microscopy. Cells were incubated with 0.17 nM M-hMPV protein 30 min at 4°C and then washed and incubated or not at 37°C for 30 min. Observation was realized by confocal microscopy. Data represent one out of two experiments.</p

M-hMPV protein induces maturation of moDCs.

<p>moDCs were treated with M-hMPV protein 0.172 nM (gray filled) and 0.0172 nM (green line), LPS 10 ng/mL (black line), M-hMPV 0.172 nM+polymyxin (orange line), M-hMPV heat treated at 100°C–20 min (pink dotted line) or untreated (iDCs) (gray line) and the phenotype was analyzed after 24 h of treatment. Cells were stained with labeled CD80, CD83, CD86 and CD40 antibodies. Data were acquired by a FACScan and analyzed by CellQuest Pro software. Histograms represent mean fluorescence intensity. Data shown are representative of one out of five independent experiments.</p

M-hMPV bacterial recombinant protein is successfully produced.

<p>(A) SDS-PAGE analysis of M-hMPV recombinant protein was performed after purification by Ni-NTA resin. (B) M-hMPV protein was analyzed by western blot using M-hMPV 1 µg/mL monoclonal antibodies and HCV monoclonal antibody for the negative control together with species-specific secondary antibody goat anti-mouse IgG H+L labeled with alkaline phosphatase.</p

Design of the experimental procedures.

<p>Design of the experimental procedures.</p

Kaplan Meier survival curves of guinea pigs after EBOV infection, according to treatment with anti-EBOV IgGs.

<p>A: Survival of guinea pigs having received the anti-EBOV IgGs (n = 10) compared to guinea pigs receiving the non-immune DKO IgGs (n = 5, *p = 0.0424, using a Log Rank test). B: Survival of guinea pigs having received one (DO) or two (D0 and D3) doses of polyclonal anti-EBOV IgGs.</p