In vivo self-assembled DNA vaccine elicits efficient immune protection against viral myocarditis by targeting draining lymph nodes

In this study, we engineered two recombinant fusion proteins by conjugating the streptococcal albumin-binding domain (ABD) to either the N-terminus (AP) or C-terminus (PA) of protamine (P). These proteins spontaneously assembled with the CVB3 VP1 DNA vaccine (pVP1) into nanovaccines, leveraging ABD's albumin-binding capacity to target draining lymph nodes (dLNs) and enhance immunogenicity and efficacy. All recombinant AP, PA, and P proteins efficiently bound to and protected the pVP1 plasmid, forming nanoparticles with a diameter of 170-200 nm. In vivo distribution assays showed that AP-pVP1 enrichment was approximately 1.8-fold higher than that of PA-pVP1 and P-pVP1. Following intramuscular immunization, the AP-pVP1 group showed the highest levels of CVB3-specific neutralizing serum IgG. The cytotoxicity response was approximately 31% and 90% higher than the PA-pVP1 and P-pVP1 groups, respectively. The percentage of IFN-γ⁺CD8⁺T cells in the AP-pVP1 group was 4.45%, significantly higher than 3.51% in the PA-pVP1 group and 2.84% in the P-pVP1 group. Upon viral challenge, the AP-pVP1 group exhibited superior protection, including the lowest heart viral load, minimal myocardial histomorphological changes, and the highest survival rate. Our study demonstrated that ABD-mediated albumin hijacking enhances the efficacy of the CVB3 DNA vaccine by its dLN-targeting capability. We also found that lymph node targeting efficacy was affected by the position of ABD within the fusion protein. These findings may provide insights into the development of potent CVB3 vaccines. STATEMENT OF SIGNIFICANCE: To enhance the targeted delivery of a protamine-based DNA vaccine carrier to lymph nodes, we designed recombinant proteins by fusing the streptococcal albumin-binding domain (ABD) to protamine. This approach leverages ABD's ability to bind albumin, which naturally traffics to lymph nodes, thereby actively guiding DNA-protamine nanoparticle vaccines to draining lymph nodes. The fused ABD orientation significantly affected targeting efficiency: N-terminal fusion (AP) increased lymph node accumulation more than C-terminal (PA) or unmodified protamine. A DNA vaccine formulated with an AP and a CVB3 VP1-encoding plasmid induced strong neutralizing antibody responses and CD8⁺ T cell activity, thereby improving protection against CVB3 myocarditis. This albumin-mediated targeting strategy provides a feasible platform for enhancing DNA vaccine efficacy via active lymphatic delivery.