Induction of BBB permeability after treatment with LBNSE-GM-CSF.

<p>Mice were treated with medium or 10⁷ FFU LBNSE-GM-CSF at 0 (A) or 4 (B) dpi. BBB permeability was determined by uptake of sodium fluorescein at day 6 post treatment. Data are given as mean values + SEM. Asterisks indicate significant differences between the indicated experimental groups: <i>*, p<0.05; **, p<0.01.</i></p

Recruitment/activation of DCs in the lymph node and periphery blood of mice treated with LBNSE-GM-CSF.

<p>BALB/c mice were infected with 10 IMLD₅₀ DRV and treated at 0 (A) or 4 (B) dpi with medium, 10⁷ FFU live or inactivated LBNSE-GM-CSF. At days 3, 6, and 9 post treatment, single cell suspensions were prepared from inguinal lymph node or peripheral blood and stained with antibodies against surface markers of DCs (CD11c+). The stained cells were analyzed by flow cytometry. All data are from n = 3 mice in each group and presented as mean values ± standard errors (SE). Asterisk indicates significant differences between the indicated experimental groups as calculated by one-way ANOVA: <i>*, p<0.05; **, p<0.01.</i></p

Virus titers in the brain of mice infected im with DRV (10 IMLD₅₀) and treated with rRABVs at 4 dpi.

<p>*rRABV (GM-CSF), recombinant RABV expressing GM-CSF.</p>†<p>Virus titer in the brain (FFU/g tissue).</p><p>- No virus was detected.</p

Differentiation of inflammatory cells infiltrated into CNS by flow cytomeric analyses.

<p>BALB/c mice were infected i.m. with 10 IMLD₅₀ DRV and were treated at either 0 or 4 dpi with medium, 10⁷ FFU live or inactivated LBNSE-GM-CSF. Leukocytes from CNS were recovered by Percoll centrifugation and stained with cell surface markers CD11b (Microglia/macrophage), Ly6G (Neutrophils) and CD3 (T cells). The stained cells were analyzed by flow cytometry. Representative flow cytometric plots of infiltrated cells in mouse brain at day 6 post treatment for each of the treatment groups (A). The absolute numbers of indicated inflammatory cells in the brain were presented for mice treated at day 0 (B) or 4 (C) dpi, respectively. All data are from n = 3 mice per group. Asterisk indicates significant differences between the indicated experimental groups as calculated by one-way ANOVA: <i>*, p<0.05; **, p<0.01.</i></p

Protective efficacy of recombinant RABVs administrated after infection i.m. with DRV.

<p>ICR mice (group of 10) at age of 4–6 weeks were infected i.m. with 10 IMLD₅₀ DRV, and treated with 10⁷ FFU LBNSE-GM-CSF by intracerebral (A), intramuscular (B), intradermal (C) or intranasal (D) route at different time point post infection, or treated with live or UV-inactivated LBNSE-GM-CSF at day 4 post infection (E). Infected and treated mice were observed daily for 20 days and survivorship was recorded and analyzed. Asterisk indicates significant differences between the indicated experimental groups as calculated by Log-rank test: <i>*, p<0.05;</i> **, <i>p<0.01;</i> ***, <i>p<0.001.</i></p

Recruitment/activation of B cells in the CNS and the periphery and the production of VNA in mice treated with LBNSE-GM-CSF.

<p>BALB/c mice were infected with 10 IMLD₅₀ DRV and treated at 0 (A, C) or 4 (B, D) dpi with medium, 10⁷ FFU live or inactivated LBNSE-GM-CSF. Brain, lymph node and peripheral blood were harvested at days 3, 6, and 9 post treatments. Leukocytes recovered from CNS by Percoll centrifugation or signal-cell suspensions prepared from the lymph node or peripheral blood were stained with antibodies against B cell markers (CD40 and CD19). The stained cells were analyzed by flow cytometry. The number of B cells in the brain and the percentage of mature B cells in the lymph node or blood were presented in mice treated at 0 (A) or 4 (B) dpi. Blood samples were collected at days 6 and 9 post treatment and VNA titers determined by RFFIT (C for VNA in mice treated at 0 and D at 4 dpi). All data are from n = 3 mice in each group and presented as mean values + standard errors (SE). Asterisk indicates significant differences between the indicated experimental groups as calculated by one-way ANOVA: <i>*, p<0.05; **, p<0.01.</i></p

Concentration of chemokines and cytokines in the brain of mice treated with LBNSE-GM-CSF.

<p>BALB/c mice were infected with 10 IMLD₅₀ DRV and treated at either 0 (A) or 4 (B) dpi with medium, 10⁷ FFU live or UV-inactivated LBNSE-GM-CSF, brain samples were collected at days 3, 6, and 9 post treatment. Total RNA was extracted from the brain tissue and mRNA of chemokines and cytokines was analyzed by qRT-PCR. The mRNA copy number was normalized to the housekeeping gene GAPDH. Levels of gene expression in a test sample are presented as the fold change over that detected in sham-infected controls. Data represent the average from three independent experiments. Asterisk indicates significant differences between the indicated experimental groups as calculated by one-way ANOVA: <i>*, p<0.05;</i> **, <i>p<0.01.</i></p

Enhancement of BBB permeability induced by MCP-1 improves protection in mice treated with UV-inactivated rRABV.

<p>Mice were treated with medium or MCP-1 (25 µg) by ic, BBB permeability was determined by uptake of sodium fluorescein at day 2 post treatment (A). ICR mice (group of 10) at age of 4–6 weeks were infected i.m. with 10 IMLD₅₀ DRV, and then treated with medium, MCP-1 (25 µg), 10⁷ FFU LBNSE-GM-CSF, UV-inactivated LBNSE-GM-CSF (equivalent to 10⁷ FFU) or a mixture of MCP-1 (25 µg) and UV-inactivated LBNSE-GM-CSF (equivalent to 10⁷ FFU) by ic at day 4 post infection. The mice were observed for 20 days post infection, and survivorship was recorded and analyzed. Asterisk indicates significant differences between the indicated experimental groups as calculated by one-way ANOVA or Log-rank test: <i>*, p<0.05; **, p<0.01.</i></p

PIV5 antibodies in humans.

<p>45 human serum samples were obtained from 18–50 year old healthy individuals. (A) Comparison of anti-PIV5 and anti-MuV antibody levels. ELISA was performed on plates coated with purified PIV5 or purified MuV with sera serially diluted. PIV5 or Mumps virus specific ELISA OD₄₅₀ values were shown at 320-fold dilution for each human serum sample. (B) Titers of neutralizing antibody against PIV5 in human sera. Data for the antibody titers were the average value of duplicate wells and presented for each human sample. The white column indicates that the PIV5 nAb titer is less than 10, the limit of detection. The black column indicates that the nAb titer is equal to or higher than 10.</p

Titers of anti-PIV5 antibodies in dogs without PIV5 exposure.

<p>Eight PIV5 naïve dogs were immunized with one dose of 8×10⁷ PFU of PIV5 or rPIV5-H3 viruses by intranasal route. The dogs were divided into two groups: PIV5-infected dogs and rPIV5-H3-infected dogs. Blood samples were collected at 0 and 21 days post infection for ELISA (A) and virus neutralization antibody (nAb) assay (B). The grey columns indicate that the PIV5 nAb titer is less than 10, the limit of detection in this assay. The black columns indicate that the nAb titer is equal to or higher than 10.</p