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Coxsackievirus B3 (CVB3) is a member of the enterovirus genus and linked to several diseases, including myocarditis, which can progress to dilated cardiomyopathy. Despite ongoing preclinical efforts, no clinically approved vaccines against CVB3 are currently available, highlighting the urgent need for effective prophylactic solutions. In this study, a nanovaccine platform based on spider minor ampullate silk protein (MiSp) is introduced. This platform utilizes protein nanoparticles engineered from chimeric proteins that incorporate CVB3 antigenic peptides into customized MiSp, subsequently loaded with all‐trans retinoic acid (RA). These functional nanovaccines are capable of eliciting both mucosal and systemic immune responses following subcutaneous administration and demonstrate significant protective effects against CVB3 infection in mice. This study signifies an approach in peptide‐based parenteral vaccine strategies, utilizing engineered MiSp nanoparticles combined with RA. This methodology represents a promising pathway for preventing enterovirus infections by leveraging the unique immunomodulatory properties of spidroins and RA to combat these pathogens effectively. [ABSTRACT FROM AUTHOR]

Spider silk proteins (spidroins) are particularly attractive due to their excellent biocompatibility. Spider can produce up to seven different types of spidroins, each with unique properties and functions. Spider minor ampullate silk protein (MiSp) might be particularly interesting for biomedical applications, as the constituent silk is mechanically strong and does not super‐contract in water, attributed to its amino acid composition. In this study, we evaluate the potential of recombinant nanoparticles derived from Araneus ventricosus MiSp as a protein delivery carrier. The MiSp‐based nanoparticles were able to serve as an effective delivery system, achieving nearly 100% efficiency in loading the model protein lysozyme, and displayed a sustained release profile at physiological pH. These nanoparticles could significantly improve the delivery efficacy of the model proteins through different administration routes. Furthermore, nanoparticles loaded with model protein antigen lysozyme after subcutaneous or intramuscular administration could enhance antigen‐specific immune responses in mouse models, through a mechanism involving antigen‐depot effects at the injection site, long‐term antigen persistence, and efficient uptake by dendritic cells as well as internalization by lymph nodes. These findings highlight the transnational potential of MiSp‐based nanoparticle system for protein drug and vaccine delivery. [ABSTRACT FROM AUTHOR]