Growth kinetics of RVΔP-LCMV/GPC and RVΔP in BHK-P cells.

<p>BHK-P cells were inoculated with RVΔP-LCMV/GPC or RVΔP at an MOI of 0.01. Culture supernatants were harvested at 1, 3, 5, and 7 days post inoculation, and virus titers were determined using BHK-P cells. The data were expressed as mean ± SE of 4 independent experiments. Asterisks indicate a significant difference (p < 0.05).</p

Confirmation of RV-N and LCMV-GP expression.

<p>BHK-P cells were inoculated with RVΔP-LCMV/GPC (A–C) or RVΔP (D–F) at an MOI of 0.1 and incubated at 33°C for 48 h. Cells were stained with anti-RV N mAb or the anti-LCMV-GP1 mAb. Original magnification, 400×. (G–I) RV-N and the LCMV-GPC were detected in Neuro-2a cells inoculated with RVΔP-LCMV/GPC at an MOI of 8 at 48 h post inoculation). Original magnification, 200×. (J) GPC (70 KDa) and GP1 (40 KDa) proteins were detected in BHK-P cells infected with RVΔP-LCMV/GPC by western blot using the anti-LCMV-GP1 mAb. (K–L) The cultured medium (Sup) of cells infected with RVΔP-LCMV/GPC or RVΔP were PEG precipitated (PEG ppt) and purified (Purified) by ultracentrifugation and stained with the anti LCMV-GP1 Mab (K) or the anti-RV G mAb (L). The lysates of mock-inoculated Vero cells and LCMV-WE -inoculated Vero cells were used as negative and positive controls, respectively (K). Lysates of mock-inoculated Neuro-2a cells and RV HEP-Flury strain-infected Neuro-2a cells were used as negative and positive controls, respectively (L).</p

Survival curves and NAb titers in mice infected with LCMV-WE with or without CD8+ T cell depletion.

<p>(A) RVΔP-LCMV/GPC inoculation, challenge, and blood collection schedules in C57BL/6 mice. Mice were inoculated with RVΔP-LCMV/GPC twice at 3-week intervals. Mice were injected with anti-CD8+ T cell antibody (n = 6) or Isotype control (n = 6) 4 days and 7 days after the last RVΔP-LCMV/GPC inoculation. Seven days after the last inoculation, mice were intracranially infected with 10 PFU of LCMV-WE. Mouse serum was collected 1 day before the first RV inoculation (Pre) and second RV inoculation (1st), and the serum of the surviving mice was collected on the last day of the 3-week observation (Post). (B) The survival curve of mice injected with CD8+ T cell antibody or isotype control. The titers of anti-LCMV (C) and anti-RV (D) NAb in mice with and without CD8+ T-cell depletion.</p

Survival curves in mice inoculated with adenovirus vectors before LCMV-WE infection.

<p>(A) The schedules of immunization with adenovirus and LCMV challenge in C57BL/6 mice. Mice were inoculated twice with Ax-LCMV/GPC or Ax-empty at 1-week intervals. One week after the last inoculation of adenovirus, mice were challenged with 10 PFU of LCMV-WE and observed for 2 weeks. (B) The survival curve of mice inoculated with Ax-LCMV/GPC or Ax-empty. Asterisks indicate a significant difference (p < 0.001).</p

Survival curves and antibody titers in mice inoculated with RV vectors before LCMV-WE infection.

<p>(A) RV inoculation, challenge, and blood collection schedules in C57BL/6 mice. Mice were inoculated twice with RVΔP-LCMV/GPC, RVΔP, UV-irradiated RVΔP-LCMV/GPC, or PBS (mock-inoculated control) at 1-week intervals. One week after the last inoculation of RVs, mice were challenged with 10 PFU of LCMV-WE and observed for 2 weeks. Serum was collected from mice inoculated with RVs 1 day before the first RV inoculation (Pre) and second RV inoculation (1st) and at the LCMV challenge (2nd). Serum was collected from surviving mice on the last day of the 2-week observation period (Post). (B) The survival curve of mice inoculated with RVΔP-LCMV/GPC, RVΔP, UV-irradiated RVΔP-LCMV/GPC or PBS. Asterisks indicate a significant difference (p < 0.001). The titers of IgG antibody against the LCMV-GPC (C) and NAb against LCMV (D) and RV (E) in serum of mice inoculated with RVΔP-LCMV/GPC (n = 17), RVΔP (n = 13), UV-irradiated RVΔP-LCMV/GPC (n = 10), or PBS (n = 10).</p

Schematic diagrams of the recombinant RVs genome constructs.

<p>Recombinant HEP-Flury (rHEP) has a complete genome of RV HEP-Flury strain (upper), RVΔP lacks the RV-P gene (middle). RVΔP-LCMV/GPC harbors the LCMV-GPC gene after the RV-N gene of the genome.</p

Safety profile of RVΔP-LCMV/GPC.

<p>Suckling mice (4-day old) were inoculated with RVΔP-LCMV/GPC (Orange), RVΔP (Green), or rHEP (Pink) by intracerebral inoculation and observed for 3 weeks. Euthanasia was performed when the mouse was considered to have reached a moribund stage. Number of mice in each group was 16.</p

Growth kinetics of RVΔP-LCMV/GPC and RVΔP in BHK-P cells.

<p>BHK-P cells were inoculated with RVΔP-LCMV/GPC or RVΔP at an MOI of 0.01. Culture supernatants were harvested at 1, 3, 5, and 7 days post inoculation, and virus titers were determined using BHK-P cells. The data were expressed as mean ± SE of 4 independent experiments. Asterisks indicate a significant difference (p < 0.05).</p