Oral vaccination via virus-like particles encapsulated in Lactococcus lactis.

Nervous necrosis virus (NNV) causes severe disease in marine fish, leading to high mortality and significant economic losses in aquaculture. Effective oral vaccines are urgently needed for large-scale, labour-efficient immunization of farmed fish. In this study, NNV capsid protein was successfully expressed in Lactococcus lactis (L. lactis) and evaluated as a vaccine candidate in Asian seabass (Lates calcarifer) fingerlings. The capsid protein assembled into virus-like particles (VLPs), structurally similar to those produced in E. coli. Intraperitoneal injection of purified VLPs elicited strong humoral immunity, with NNV-specific IgM titers four-fold higher than oral delivery, despite a tenfold lower antigen dose. However, encapsulation of VLPs in live or heat-inactivated L. lactis cells failed to induce protective immunity through oral delivery, likely due to poor antigen release. In contrast, sodium hypochlorite-inactivated L. lactis preserved VLP encapsulation, solubility and structural integrity, and oral vaccination with these cells induced approximately two-fold higher antibody and neutralizing titers than the purified VLPs. Challenge studies demonstrated a significant ∼2.5-log reduction in brain viral load 7 days post-challenge. Overall, these findings highlight hypochlorite-inactivated L. lactis as a promising oral vaccine platform, offering a sustainable strategy for aquaculture immunization against NNV.