Innovative Use of Gram-positive Enhancer Matrix Particles and Affinity Peptides in a Vaccine Against Coxsackievirus B3
Description
Figure 1. Design of two vaccine structures based on GEM particles: GEM -PA-VP1 (A) and GEM-Fc-VP1 (B) (A) The VP1-PA fusion protein was displayed on the surface of GEM particle via the mediation of PA. (B) The VP1-Fc fusion protein was displayed on the surface of GEM particles via the mediation of FcSP polypeptide. Figure 2. The construction of recombinant plasmids and their subsequent transfection and expression validation in cells (A) shows the results of double digestion of the recombinant plasmids pcDNA3.1-PA-VP1 and pcDNA3.1-Fc-VP1 with KpnI and XhoI restriction enzymes. (B)Expression and localization of PA-VP1 and Fc-VP1 fusion proteins in CHO cells, as observed by immunofluorescence staining post-transfection. (C)Expression of fusion proteins in CHO cells confirmed using via immunofluorescence analysis. Figure 3. The expression, purification, and display of recombinant proteins PA-VP1 and Fc-VP1 on GEM particles (A) SDS-PAGE analysis of the fusion proteins. (B)Western Blotting analysis results. (C) Transmission electron microscopy images of GEM, GEM-PA-VP1, and GEM-Fc-VP1 particle vaccines. Figure 4. The effects of GEM-PA -VP1 and GEM-Fc -VP1 vaccines on IgG antibody levels, their subtypes, and neutralizing antibodies in mice Data are expressed as mean ± standard deviation and were analyzed using one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). ELISA measured various indices in mouse serum on days 14, 28, and 42 post-initial, booster, and final immunizations, respectively. (A) Spleen index; (B) the distribution of IgG (C) and (D) the distribution of IgG2a and IgG1 (E) ratio of IgG2a to IgG1 (F) the levels of neutralizing antibodies in serum. Figure 5. The impact of GEM-PA-VP1 and GEM-Fc-VP1 vaccines on cytokine secretion by mouse spleen cells Ten days post-immunization, spleens were harvested and stimulated in vitro with AP-1 specific antigen. Cytokine levels of IL-4 (A), IFN-γ (B), TNF-α (C), IL-2 (D), IL-6 (F), and IL-10 (E) were measured using ELISA. Data, derived from three parallel repeats per sample, are presented as mean ± SD. **P < 0.01; *P < 0.05. Figure 6. Two weeks post-final immunization, mice were challenged with CVB3 (104 TCID50). Seven days later, (A) weight loss, (B) and (C) myocardial enzyme activities CK and CK-MB, (D) survival rate 28 days post-CVB3 infection, and (E) myocardial tissue sections were assessed. Each group included 8 mice for weight and cardiac function evaluation, 6 mice for histological examination, and 15 mice for survival rate assessment. *P < 0.05; **P < 0.01; ***P < 0.001.