A multi species autophagy-inducing nano-vaccine platform: uncovering immunoenhancement properties of PCV2 cap nanoparticles

Cap VLPs (virus-like-particles) are self-assembling nanoparticles (NPs) derived from PCV2 ORF2 with ordered and repetitive antigen display, which have been widely used as a landmark veterinary vaccine in the swine industry. Building upon the pivotal role of virus-activated autophagy in antigen presentation and immune regulation, efforts were made to identify the key molecular elements and mechanisms by which Cap NPs activate autophagy to enhance immunogenicity and expand their application as a vaccine platform. Self-assembled Cap NPs exhibited efficient cell internalization, upregulated various immune regulatory cytokines, particularly TNF-α, and promoted the expression of molecules involved in antigen presentation. Cap NPs induced autophagy by activating the CaMKKβ-AMPK-mTOR pathway and elevating intracellular ROS, which facilitated the processing and degradation of the Cap antigen and led to subsequent activation of TNF-α signaling. Inhibition of either autophagy or TNF-α downregulated antigen presentation. The animal immunization experiment confirmed that Cap NP-induced autophagy and TNF-α signaling promoted humoral and cellular immune responses. To further investigate the key components affecting immunogenicity, truncated Cap peptides were displayed on self-assembled mi3 nanoparticles. Among these, the chimeric mC-9-10 NPs autonomously induced autophagy-TNF-α signaling and stimulated immune responses, suggesting potential adjuvant-like activity. More importantly, Cap NPs were able to enter and activate autophagy-TNF-α signaling in multiple cell lines across different species, making them candidates for a broad-spectrum antigen delivery nano-vaccine platform. Overall, this work not only systematically elucidates the complete molecular mechanisms by which Cap NPs enhance immune responses through cellular internalization, autophagy induction, TNF-α signaling activation, and antigen presentation, but also highlights the great potential of Cap NPs and their functional peptides as multi-species nano-vaccine carriers and novel immune adjuvants. These findings provide a solid theoretical foundation and technical feasibility for the design of next-generation nano-delivery platforms and the development of immune-enhancing tools.