Innovative Use of Gram-positive Enhancer Matrix Particles and Affinity Peptides in a Vaccine Against Coxsackievirus B3

Figure 1. Design of the two vaccine structures based on GEM particles: GEM-PA-VP1 (A) and GEM-Fc-VP1 (B). (A) VP1-PA fusion protein on the surface of GEM particles via the mediation of PA. (B) VP1-Fc fusion protein on the surface of GEM particles via the mediation of FcSP polypeptide.

Figure 2. Construction of recombinant plasmids and their subsequent transfection and expression validation in cells. (A) PCR specific amplification the fragment of VP1, PA-VP1 and Fc-VP1. (B) Identification of the recombinant plasmids by double enzyme digestion. (C) Expression of fusion proteins in CHO cells confirmed using immunofluorescence analysis. (D) Western blotting analysis of the fusion proteins. (E) SDS–PAGE analysis of the purified fusion proteins.

Figure 3. Display of recombinant proteins PA-VP1 and Fc-VP1 on GEM particles. (A) Transmission electron microscopy images of GEM, GEM-PA-VP1, and GEM-Fc-VP1 particle vaccines. (B, C) The Size and Zeta potential of GEM-PA-VP1, and GEM-Fc-VP1 particles detected by DLS and ELS methods using a zetasizer.

Figure 4. Characterization of GEM-PA-VP1 and GEM-Fc-VP1 particles. (A) Measurement of antigen loading efficiency on GEM particles. (B) Antigen release from GEM-PA-VP1 and GEM-Fc-VP1 was assessed by incubating in PBS for 0-168 h and protein content was quantified using a BCA assay. (C) Toxicity of GEM-PA-VP1 and GEM-Fc-VP1 particles to RAW264.7. (D, E) Expression of TNF-α and IL-6 in RAW264.7 after stimulated for 24 h

Figure 5. Effects of the GEM-PA-VP1 and GEM-Fc-VP1 vaccines on IgG levels, subtypes, neutralizing antibodies and SIgA in immunized mice. (A)Spleen index of of immunized mice. (B) Dynamics of IgG in serum samples from of immunized mice at 14, 28, and 42 days post immunization. (C, D) Detection of IgG2a, and IgG1 in serum samples of immunized mice (E) The ratio of IgG2a to IgG1. (F) Dynamics of neutralizing antibodies in serum samples of immunized mice at 14, 28, and 42 days post immunization. (G) SIgA levels in bronchoalveolar lavage fluid of immunized mice at 42 days post immunization.

Figure 6. Detection of GEM-PA-VP1 and GEM-Fc-VP1 vaccines on cytokine secretion by mouse splenocytes. Splenocytes were harvested from the mice 42 days after immunization and stimulated with VP-1-specific antigen in vitro. The 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.

Figure 7. Enhanced immunoprotection against CVB3 infection by GEM-PA-VP1 and GEM-Fc-VP1 immunization. (A) The changes in average body weight of each group. (B, C) Detection of the serum myocarditis inflammation indexes CK and CK-MB enzyme in each group. (D) Hematoxylin and eosin stained myocardial tissue sections of mice (E) The survival rate of mice within 14 days post-CVB3 infection. (F-H) Detection of TNF-α, IL-1β and IL-6 in myocardial tissues