Enhanced Immunity of Hybrid Grouper (Epinephelus sp.) Against Viral Nervous Necrosis Using a Recombinant Protein Nanovaccine from Chlorella Vulgaris

Hybrid Grouper (Epinephelus sp.) is an important export commodity in marine aquaculture, yet Viral Nervous Necrosis (VNN) remains a major threat. Nanovaccines are a type of vaccine developed by combining vaccine ingredients with silver nanoparticles and recombinant protein from Chlorella vulgaris. These formulations are designed to effectively boost the body's immune system response to prevent disease infection and destroy pathogenic agents. This study evaluated the efficacy of a recombinant nanovaccine containing Chlorella vulgaris protein with chitosan-AgNPs, using a factorial randomized design of eight treatments and three replicates. Treatments included healthy fish (K–), VNN-infected fish (K+), and fish vaccinated with 33, 66, or 112 μL of nanovaccine, with or without subsequent VNN infection. Parameters assessed were hematology, relative CD4/CD8 T-cell levels, antibody titers, and survival. Results indicated that administering the recombinant nanovaccine significantly enhanced the immune response, evident in increased erythrocyte (2.67×10⁶ cells/mm³), leukocyte (1.71×10⁵ cells/mm³), and hemoglobin (7.68 g/dL) levels, as well as higher lymphocytes (85.2%), monocytes (16.8%), neutrophils (17.4%), basophils (0.20%), relative CD4 (0.67%) and CD8 (2.33%) T-cell levels, and survival (88.8%). The ANOVA results indicated that several parameters exhibited statistically significant differences among treatments (p<0.05). The highest immune response occurred in fish vaccinated with a 33 μL dose. These findings highlight the potential of recombinant nanovaccines containing C. vulgaris protein as a preventive strategy against VNN in Hybrid Grouper.