Viral load following postexposure treatments.

<p>Viral loads were determined by plaque assay from BALB/c mice 8 days postexposure (p.e.) following infection with 18 i.n. ECTV LD₅₀. (A, C, E) viral load in livers, spleens and lungs of mice treated on day 2 p.e. (B, D, E) viral load in livers, spleens and lungs of mice treated on day 3 p.e. Horizontal lines represent the geometric mean of each group. Survival proportions of each group are designated. Asterisk denote for significant reduction in viral load (n = 3 in each treated group) compared to the infected untreated group (n = 6, P<0.05).</p

Influence of Poly(I:C) treatment on viral dissemination evaluated by <i>in-vivo</i> bioluminescence imaging.

<p>BALB/c mice were infected with 2 i.n. ECTV-Luc LD₅₀ and left untreated (n = 5) or treated with poly(I:C) on day 3 p.e. (n = 3). (A) Infected untreated mice 8 days p.e. (B) Infected mice treated on day 3 p.e. with poly(I:C) and imaged on day 8 p.e. (C) Poly(I:C) treated group on day 3 p.e. imaged on day 16 p.e. Bioluminescent images were obtained using an f/stop of 1, binning factor of 4, and acquisition time of 1 sec (A, B) or 40 sec (C). Relative photon flux expression is represented by a pseudocolor heat map. (D) Morbidity, based on weight change (lines, left Y axis) and bioluminescence signal on days 7, 8, 14 and 16 p.e. (bars, right Y axis) of the groups shown in panels A–C (red for infected untreated, black for poly(I:C) treated on day 3 p.e.). Bioluminescent signal intensity as total photon flux (photon/s/cm2/sr), was calculated by region of interest (ROI) analysis on the chest and abdomen area marked by a white box on the right mouse in panel (A). Same ROI was used for all mice examined. Asterisk denote for significant reduction in photon flux (n = 3–5 in each group, P<0.05). Dagger represent dead mice.</p

Effect of p.e. treatments on the spleen of infected mice.

<p>Spleens were dissected 8 days p.e. from treated mice (3 days post treatment) (A–C, G–L), from untreated (D–F) or from un-infected naïve mice (M–O). Left column - hematoxylin and eosin stain (H&E), middle column - CD45 (brown staining of WBC), right column – viral antigens (brown staining ). Serial sections of (A–C) VACV-Lister treatment; (D–F) infected untreated; (G–I) poly(I:C) and VACV-Lister treatment and (J–L) poly(I:C) treatment. Magnification in all images: X40.</p

Elevation of IFN-α levels following Poly(I:C) administration.

<p>IFN-α levels were examined in BALB/c mice sera. (A inset) Sera of uninfected naïve mice before (n = 2) and 24 h after poly(I:C) administration (n = 6). IFN-α in sera of 20 i.n. LD₅₀ ECTV (A) infected untreated mice examined on days 4 (n = 6), 5 (n = 3) or 6 (n = 3) p.e. or 24 hours after treatment with VACV-Lister given on day 3, MVA on day 3, 4 or 5 or placebo treated on days 3, 4 or 5 (n = 3 in all groups). (B) IFN-α in sera of mice 24 hours after treatment with poly(I:C) on days 3, 4 or 5 given alone or in combination with VACV-Lister or MVA (n = 3–5/group).</p

Correlation between IFNγ and viral load.

<p>(A, C, E) Viral loads and IFNγ levels in BALB/c mice infected with 18 i.n. ECTV LD₅₀, untreated (n = 6) or single treated on day 2 or 3 with: poly(I:C) with or without VACV-Lister, CpG-ODNs 1585 and 1826 with or without VACV-Lister and VACV-Lister only (n = 3/group). (B, D, F) Viral loads and IFNγ levels in C57BL/6j mice infected with 2–3 i.n. ECTV LD₅₀, untreated (n = 6) or single treated on day 0 with: poly(I:C) with or without VACV-Lister, VACV-Lister or placebo; day 1: poly(I:C) with or without VACV-Lister or MVA, VACV-Lister or MVA, placebo; day 2: poly(I:C) with or without MVA (n = 3/group), MVA or placebo (n = 3/group). (A, B) spleens; (C, D) livers and (E, F) lungs. Green dots - examined mice from groups in which the survival rate was 50% and above. Red dots - examined mice from groups in which the survival rate was less than 50%, (n = 48 for either BALB/c or C57BL/6j).</p

Morbidity based on weight change following post exposure (p.e.) treatments of BALB/c mice.

<p>Mice were infected with 4–20 i.n. ECTV LD₅₀. (A, B) CPGs treatments with or without VACV-Lister on day 2 (A) and 3 (B) p.e. (C) Poly(I:C) treatments with or without VACV-Lister or VACV-Lister alone on day 2 p.e. (D) Poly(I:C) treatments with or without VACV-Lister or MVA and only vaccines treatments on day 3 p.e. (E, F) Poly(I:C) treatments with or without VACV-Lister or MVA and only MVA on day 4 (E) and 5 (F) p.e. Asterisk denote for significant difference in the area-under-the curve of weight changes along the entire experiment of the treated groups vs. the infected untreated group (* P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001, <i>t</i>-test). Data collection for each treatment (weight change (mean, SE)) is indicated. Morbidity of Infected untreated mice from corresponding relevant experiments is shown. The number of mice succumbed to the infection in each time point is outlined color coded in a box above each graph. Survivals out of the total mice in each group are designated color coded next to the legend.</p

Survival Table – BALB/c mice.

<p>Infected untreated BALB/c mice i.n. infected with ECTV (survived/total (n)): 3/39 (8%) which are the sum of Exp.1 4LD₅₀ (0/6); Exp.2 15LD₅₀ (0/15); Exp.3 5LD₅₀ (2/6); Exp.4 20LD₅₀ (1/12).</p><p>*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 Log-rank (Mantel-Cox test) vs. infected untreated (of the relevant infected untreated group in the same experiments).</p><p>N.D. – not determined; p.e. – postexposure.</p><p>Survival Table – BALB/c mice.</p

Survival Table – C57BL/6j mice.

<p>Infected untreated C57BL/6j mice i.n. infected with ECTV (survived/total (n)): Exp.1 2LD₅₀ (0/3); Exp.2 3LD₅₀ (0/8).</p><p>*P<0.01, **P<0.001, ***P<0.0001 Log-rank (Mantel-Cox test) vs. infected untreated (0/11).</p><p>N.D. – not determined; p.e. – postexposure.</p><p>Survival Table – C57BL/6j mice.</p

Cellular-immune response following p.e. treatment.

<p>BALB/c mice were infected with 4 i.n. ECTV LD₅₀, left untreated or treated on day 3 p.e. and their spleens were photographed (A) and analyzed by flow-cytometry for the distribution and activation (intracellular IFNγ) of various cell populations. (B) Counting of viable lymphocytes was performed under light microscope. (C–H) Number of total and activated cells of the different cell populations: NK (C, D), of CD3⁺ CD4⁺ (E, F) and CD3⁺ CD8⁺ (G, H) cells, respectively.</p