Nanoparticle-based antigen favors high level of humoral immune responses and increases antigenicity of highly glycosylated protein.

Nanoparticles (NPs) have shown great potential as advanced vaccines and immunotherapy platforms due to their size and polyvalent epitopes. However, the immune responses elicited by glycosylated antigens conjugated to NPs with variable antigen densities remain poorly understood. In this study, we analyzed the glycosylation profile of the CD2v ectodomain of African swine fever virus, identifying 16 potential N-glycosylation sites among 192 residues, which markedly reduced its antigenicity. To enhance the antigenicity of CD2v, we designed three protein-based NPs derived from Ferritin, porcine circovirus type 2 (PCV2) capsid protein, and AP205 coat protein. Antigens were conjugated to the NP surfaces using the SpyTag/SpyCatcher system. Immunoassays demonstrated that these NPs rapidly induced higher levels of antigen-specific IgG compared to unconjugated antigens, with the effect being most pronounced for the highly glycosylated CD2v. All NPs significantly promoted IgG2a production, indicating a Th1-biased immune response. Furthermore, PCV2 NP reached the popliteal lymph node within 5 min post-injection and persisted for 21 days, extensively interacting with B cells. These findings provide the potential of tailoring NP design to enhance antigenicity, providing a foundation for developing glycosylated antigen-targeted vaccines and immunotherapies.