An Albumin-Binding Domain Peptide Confers Enhanced Immunoprotection Against Viral Myocarditis by CVB3 VP1 Vaccine

Figures

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  Figure 1. Albumin-binding ability of ABD-VP1vaccine. (A) SDS-PAGE and (B) western blot analysis of the recombinant ABD-VP1 and VP1 proteins. (C) The binding abilities of ABD-VP1 and VP1 proteins to albumins were detected by ELISA assays. (D) Serum complex of ABD-VP1 with albumin was detected by ELISA assays. (E) The serum half-lives of ABD-VP1 and VP1 vaccines. VP1 and ABD-VP1 proteins were injected subcutaneously into mice, sera were taken at indicated time points for VP1 amount detection by ELISA assays. The level of serum VP1 protein at 5 min after injection was uniformly quantified as 100%. Individual experiment was repeated 3 times. *P < 0.05; **P < 0.01; ***P < 0.001.

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  Figure 2. The bio-distributions of ABD-VP1 and VP1 vaccines. Mice were subcutaneously given mCherry-fused ABD-VP1 or mCherry-fused VP1 vaccine, and the vaccine distributions in (A) dLNs, (B) kidneys, (C) hearts, (D) livers, (E) lungs, (F) spleens were analyzed by the bioluminescence imaging assays at the indicated time points. Each group contained 6 mice. *P < 0.05; **P < 0.01; ***P < 0.001.

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  Figure 3. ABD-VP1-induced local immune responses in the dLNs. (A) Schematic diagram of the immunization procedure. (B) FACS analysis of DC recruitment and maturation in the dLNs of immunized mice. (C) Statistical analysis of (B). (D) Mean fluorescence intensity (MFI) of MHC II, CD86 or CD80 molecule on CD11c+ DC in the dLNs. (E) FACS analysis of IFN-γ+ CD8+ and IFN-γ+ CD4+ cells in the dLNs (unstimulated background has been subtracted). (F) Statistical analysis of (E). Each group contained 8 mice. *P < 0.05; **P < 0.01; ***P < 0.001.

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  Figure 4. ABD-VP1-induced splenic T cell immune responses. (A) CVB3-specific T cell proliferation in the spleens of immunized mice. (B) CVB3-specific CTL activity in the spleens. (C) Percentages of IFN-γ+ CD8+ and IFN-γ+ CD4+ cells in the spleens (unstimulated background has been subtracted). (D) Statistical analysis of (C). (E) Levels of serum neutralizing antibody in immunized mice. Each group contained 6 mice. *P < 0.05; **P < 0.01; ***P < 0.001.

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  Figure 5. Enhanced immunoprotection against viral myocarditis induced by ABD-VP1 vaccine. Mice were challenged with 3LD50 CVB3 2 weeks post the final immunization, and myocarditis severity was evaluated 7 days later by (A) body weight loss, (B) cardiac function and (C) histomorphological changes of heart tissues. (D) Statistical analysis of (C). (E) Meanwhile, viral loads in the hearts were also detected. (F) A 28-day survival rate was observed after 5LD50 CVB3 infection. *P < 0.05 for VP1 versus ABD-VP1. Each group contained 8 mice. For histomorphological observation, each group contained 6 mice. For survival rate evaluation, each group contained 15 mice. *P < 0.05; **P < 0.01; ***P < 0.001.

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  Figure 6. Adoptive transfer of memory T cells derived from ABD-VP1-immunized mice protected naive mice against CVB3 challenge. (A) Timeline of memory T cell detection and transfer. (B) Percentages of CD44hi CD62hi memory T cells in spleens and dLNs of immunized mice were detected 8 weeks after the final immunization. (C) Statistical analysis of (B). (D) Naive mice were adoptively transferred with memory T cells derived from ABD-VP1-immunized mice at the day of CVB3 (3LD50) infection, and the heart histomorphological changes were observed 7 days later. (E) Statistical analysis of (D). Each group contained 6 mice. *P < 0.05; **P < 0.01; ***P < 0.001.

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  Figure 7. Schematic representation of protection conferred by the dLNs-targeting ABD-VP1 vaccine against CVB3-induced viral myocarditis. By forming complexes with albumins, ABD-VP1 vaccine possessed a longer serum half-life and the dLNs-targeting ability. It efficiently recruited and promoted the maturation of DCs, and elicited more robust CVB3-specific T cell immune responses as well as a higher level of serum neutralizing antibodies. Accordingly, ABD-VP1 vaccine provided enhanced immunoprotection against CVB3-induced myocarditis compared with the original VP1 vaccine.