A biomineralized calcium carbonate nanovaccine encapsulating recombinant coat protein elicits robust protective immunity against nervous necrosis virus in grouper.

Recombinant subunit vaccines based on viral coat protein (CP) represent a safe strategy for controlling nervous necrosis virus (NNV) in aquaculture; however, their practical application is limited by the rapid degradation and clearance of free protein antigens under physiological conditions. To address this limitation, we developed a biomineralized CaCO3@CP nanovaccine using a mild co-precipitation method. Physicochemical characterization revealed uniformly spherical nanoparticles, with an increased hydrodynamic diameter and a less negative zeta potential compared with bare CaCO3 nanoparticles. Energy-dispersive X-ray spectroscopy (EDS) further supported the successful incorporation of the protein component into the CaCO3 matrix. In juvenile grouper (Epinephelus coioides), CaCO3@CP induced robust innate and adaptive immune responses. The expression of multiple immune-related genes, including TLRs, cytokines, MHC-Iα, MHC-IIα, CD4, CD8α, and IgM, increased after vaccination and generally peaked at 14 days post-vaccination. Notably, the enhanced expression of MHC-I- and MHC-II-related markers indicated activation of both antigen-presentation pathways and was consistent with a potential enhancement of cross-presentation. NNV-specific serum IgM levels increased markedly and remained elevated for up to 6 weeks. Following lethal NNV challenge, fish vaccinated with CaCO3@CP achieved a cumulative survival rate of 68.89%, corresponding to a relative percentage survival of 64.84%. Moreover, NNV loads in the brain and eye tissues were significantly lower in the CaCO3@CP group than in the control and free-CP groups. Collectively, these findings demonstrate that CaCO3-based biomineralization is a promising and effective antigen-delivery platform for the development of nanovaccines against viral diseases in aquaculture.