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

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

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

Viral loads in serum at day 3 post-EBOV infection.

<p>A: Levels of circulating EBOV transcripts at day 3 post infection in the serum of each animal was evaluated by RT-qPCR using primers targeting EBOV polymerase gene. The horizontal bars represent the median values. The median D3 virus load was 839,333 following the injection of non-immune IgGs from a DKO pig and 259 following the injection of anti-EBOV antibodies (p = 0.055, using a Mann-Whitney test). The limit of detection of the test was of 180 relative genome copies/ml, and all values under this threshold were considered as negative data (as represented on the graph). B: Correlation between viral load on day 3 and guinea pig survival. Kendall's rank correlation coefficient showed a significant negative correlation (p = 0.0003) between EBOV viral load at day 3 and survival following infection, when considering all pooled data. Non-treated animal’s values are displayed as circles whereas treated animal’s values are displayed as squares.</p