Preparation and Immunological Efficacy Assessment of an Oral Nanovaccine with CyHV-3 ORF57 Recombinant Protein.

Koi herpesvirus disease (KHVD), caused by Cyprinid herpesvirus 3 (CyHV-3), is highly contagious and lethal, with no specific therapeutics available. Vaccine development is the most promising control strategy. In this study, the structural protein ORF57 of CyHV-3 was used as antigen, and chitosan (CS) combined with carboxymethyl chitosan (NOCMS) served as delivery carriers to construct an oral nanovaccine (CS-NOCMS/CyHV-3 ORF57) via ionotropic crosslinking. The preparation was optimized by single-factor and response surface methodology. The nanovaccine was characterized for physicochemical properties, in vitro release, in vivo retention, and safety. Carp were orally immunized and then challenged with CyHV-3, specific antibodies, non-specific immune parameters, immune-related gene expression, and tissue viral loads were assessed. Under optimal conditions, the encapsulation efficiency reached 71.02%. The nanoparticles exhibited uniform size, good dispersion, prolonged intestinal retention, and satisfactory biosafety. After challenge, the vaccine conferred a relative percent survival of 100%, significantly inhibited viral replication in gill and kidney, markedly elevated serum IgM and intestinal IgT levels, enhanced T-SOD and complement C3 activities, and upregulated expression of IgM, IgT, MHC II, I-IFN, and other immune genes in head kidney and spleen. Histopathological and immunofluorescence analyses confirmed that the nanovaccine effectively reduced tissue damage and viral burden. Collectively, the CS-NOCMS/CyHV-3 ORF57 oral nanovaccine simultaneously activates mucosal, humoral, and cellular immune responses, providing robust protection against CyHV-3 infection. This work offers a promising candidate vaccine and theoretical basis for KHVD prevention and control.