Soluble recombinant enterovirus 71 VP1 fused to truncated newcastle disease virus nucleoprotein elicits immune responses in mice

Fig. 1. Purification of SP fraction of the NPt- VP1198-297 proteins. The purification was done using a HisTrap HP column as described in the Materials and Methods. (a) Aliquots of each purification step were electrophoresed on a 12% SDS-PAGE and stained. (b) A replicate gel was transferred onto membrane and probed with an anti-His antibody. M: pre-stained protein molecular weight markers

Fig. 2. Purification and detection of the IB of the NPt-VP1t protein. The purification was carried out under denaturing conditions using 8 M urea and a HisTrap HP column, as described in the Materials and Methods. (a) Aliquots of each purification step were electrophoresed on a 12% SDS-PAGE and stained with Coomassie Brilliant Blue R-250. (b) A replicate gel was transferred onto PVDF membrane and probed with (b) rabbit anti-VP1 antibody and (b) rabbit anti-NDV antibody

Fig. 3. Mice body weight after immunization with recombinant vaccine. Weight of the mice was for weight every two weeks and recorded

Fig. 4. Total anti-VP1 IgG antibodies in mice sera following immunization with soluble and insoluble recombinant NPt-VP1t proteins. Mice (n = 5 per group) were immunized at weeks 0, 4, and 8 (arrows). Sera were collected at the indicated time points and antibody levels were determined by indirect ELISA. Bars represent mean ± SD. The dashed horizontal line represents the background cut-off value (O.D. = 0.1). Control = PBS; Soluble Protein ; Insoluble Protein. Statistical differences compared with the control group are indicated (p < 0.01 = **, p < 0.001 = ***; one-way ANOVA with Tukey’s post hoc test)

Fig. 5. Splenocytes activation of control mice after treatment with ConA stimulator and VP1 antigen. (A) splenocytes before treatment; (B) ConA-treated splenocytes; (C) VP1-treated splenocytes. (D) Untreated splenocytes after 72 h incubation. Arrow indicates splenocyte clumping after treatment. Magnification = 100X

Fig. 6. Splenocytes activation soluble protein immunized mice after treatment with ConA stimulator and VP1 antigen. (A) splenocytes before treatment; (B) ConA-treated splenocytes; (C) VP1-treated splenocytes. (D) Untreated splenocytes after 72 h incubation. Arrow indicates splenocyte clumping after treatment. Magnification = 100X

Fig. 7. Splenocytes activation insoluble protein immunized mice after treatment with ConA stimulator and VP1 antigen. (A) splenocytes before treatment; (B) ConA-treated splenocytes; (C) VP1-treated splenocytes. (D) Untreated splenocytes after 72 h incubation. Arrow indicates splenocyte clumping after treatment. Magnification = 100X

Fig. 8. T-cell proliferation of splenocytes from soluble protein-immunized, insoluble protein-immunized,and control mice (n = 5) at week 9 post-immunization. Cells were stimulated with 3 µg/mL VP1 protein or Concanavalin A (ConA, positive control), with unstimulated wells as background controls. Proliferation was measured using a BrdU ELISA, and stimulation index (S.I.) was calculated as the ratio of stimulated to unstimulated OD490 values. Bars show mean ± SD; symbols indicate individual values. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test; p < 0.05 was considered significant

Fig. 9. Cytokine levels in mice sera at week 10 post-immunization. Bars represent mean ± SD for control (C), soluble protein (SP), and insoluble protein (IB) groups. SP induced significantly higher IFN-γ, IL-2, IL-4, IL-6, and IL-10 than C and IB (p < 0.05), except TNF-α, which showed no significant differences. Significance was determined by one-way ANOVA with Tukey’s post hoc test