Development of ZnO-Chitosan-Based Nanovaccine with Chlorella vulgaris Recombinant Protein against VNN in Hybrid Grouper (Epinephelus fuscoguttatus × lanceolatus)

Viral Nervous Necrosis (VNN), caused by Nervous Necrosis Virus (NNV), is a major constraint in hybrid grouper (Epinephelus spp.) aquaculture, with larval and juvenile mortality reaching nearly 100%. Effective prophylactic strategies are urgently required to ensure sustainable production. This study aimed to test the hypothesis that recombinant Chlorella vulgaris protein, delivered using ZnO-chitosan nanoparticles, enhances immune response, improves growth, and mitigates VNN-induced tissue damage in hybrid grouper. An in vivo challenge experiment was conducted with juvenile fish assigned to 5 groups: Negative control (K−), positive control (K+), and 3 nanovaccine dosages (P1 = 33 µL, P2 = 66 µL and P3 = 112 µL). Vaccination was administered twice via oral sonde, with a 14-day interval between the primary and booster doses prior to viral challenge. The 66 µL dose (P2) yielded the most consistent protective effects, including significant improvements in body length and weight (p < 0.05), as well as elevated antioxidant enzyme activities (SOD = 3.997 U/mL; CAT = 109.7 U/mL), indicating enhanced oxidative defense. Histological analyses further confirmed reduced tissue damage in vaccinated groups, with P2 exhibiting attenuated vacuolization in the gills. Interestingly, although P2 was superior in growth and antioxidant responses, the 33 µL dose (P1) demonstrated the lowest tissue damage, suggesting a more favorable safety profile. These findings highlight a dose-dependent trade-off, where P2 maximizes immune and growth benefits, while P1 minimizes histopathological alterations. While ZnO-chitosan nanoparticles are generally considered safe at sub-toxic concentrations reported in fish toxicology studies (< 100 mg/L waterborne exposure), further ecotoxicological and bioaccumulation assessments are required to substantiate their long-term environmental safety. Within the scope of this study, the 33 µL dosage represents the most balanced option between efficacy and safety, supporting its potential as a nanovaccine platform for VNN control in hybrid grouper aquaculture.