Oral immunization with recombinant L. lactis expressing RGNNV capsid protein protects grouper against RGNNV infection.

Nervous necrosis virus (NNV) is a highly pathogenic positive-sense RNA virus that causes substantial economic losses in global grouper aquaculture industry. Its capsid protein (CP) exhibits strong antigenicity properties and represents a promising immunogen for vaccine development. Among the four major NNV genotypes, Redspotted grouper nervous necrosis virus (RGNNV) is the most prevalent. Lactic Acid Bacteria (LAB) are attractive oral vaccine delivery platforms, capable of inducing both mucosal and systemic immune responses. In this study, Lactococcus lactis (L. lactis) expression system was used with lactose as a screening marker to produce the RGNNV-CP. We successfully constructed the recombinant strain pNZ8149-Usp45-CP/L. lactis NZ3900, and confirmed protein expression by the immunofluorescence assay (IFA) and western blotting. Grouper were orally immunized with this strain and then challenged with RGNNV to assess immune protection. The recombinant L. lactis demonstrated persistent colonization capacity in fish intestinal and enhanced mucosal barrier function, indicating safety for the host. Also, oral vaccination elicited strong neutralizing responses, with serum antibody titers peaking on day 28 post-immunization. Upregulation of immune-related genes in the brain, intestine, and visceral tissues further supported robust immune activation. Vaccinated fish showed reduced viral loads and lower mortality, confirming vaccine's efficacy. These findings highlight pNZ8149-Usp45-CP/L. lactis NZ3900 as a promising antibiotic-free oral vaccine candidate for protecting grouper against RGNNV.