Figure 1.
Expression of VP1 protein in BHK cells.
Primary rabbit anti-FMDV type O-IgG antibody (LVRI) was applied to the cells, which were then incubated with FITC-conjugated goat anti-rabbit-IgG (Sigma). (A) BHK cells transfected with the eukaryotic plasmid pRC/CMV-VP1 show typical cytoplasm immunospecific fluorescence. (B) BHK cells transfected with pRc/CMV alone express no immunofluorescence.
Figure 2.
FMDV-specific humoral response.
(A) Graphic timeline in the study from animal immunization to sera collection. (B) FMDV-specific IgG titers. Asterisks (*) indicate the values of pRC/CMV-VP1- and LASW1-treated group were statistically different than mice immunized with pRC/CMV-VP1 alone (P<0.05). (C) Effects of LASW1 on the level of FMDV-specific IgG isotypes. The levels of IgG isotypes were expressed as the optical density values at 450 nm (OD450) of mice sample. Asterisks (**) indicate that the values of pRC/CMV-VP1- and LASW1-treated group were statistically different from those of mice immunized with pRC/CMV-VP1 alone (P<0.01).
Figure 3.
Induction of anti-FMDV-neutralizing antibodies.
Mean titers ±SD are shown. Asterisks (*) indicate that the values of pRC/CMV-VP1- and LASW1-treated group were statistically different from those of mice immunized with pRC/CMV-VP1 alone (P<0.05).
Figure 4.
Effects of oral LASW1 on T-cell proliferation.
Single splenocyte suspension was isolated from animals 14 days after the booster immunization, plated in triplicate in a 96-well plate and stimulated in vitro for 48 h with 146S antigen, ConA (positive control), BSA (irrelevant control), and medium (negative control). Proliferation was analyzed by the MTS assay and expressed as stimulation index.
Figure 5.
IFN-γ, TNF-α, IL-4, and IL-10 levels as measured by ELISA.
Splenocytes prepared from LASW1 treated or control mice immunized with pRC/CMV-VP1 were cultured with 146S antigen (2 μg/ml). The concentrations of (A) IFN-γ, (B) TNF-α, (C) IL-4 and (D) IL-10 in culture supernatants were measured at 48 h. Bars represent the mean ± SD of triplicate wells. *P<0.05, **P<0.01.
Figure 6.
Concentration of IFN-γ-secreting cells in the mice immunized with pRC/CMV-VP1 with or without oral administration of LASW1.
ELISpot assay was used to determine the number of cells secreting IFN-γ in the spleen. A single-cell suspension of lymphocytes was prepared and stimulated in culture with 146S antigen, PHA as positive control, and BSA as an irrelevant antigen. Results represent mean ± SD of number of IFN-γ producing cells of 3 male mice per group in triplicate wells.