Sequential nanoimmersion and hydrogel-based multivalent vaccination induce durable multilayered immunity against four...

Bacterial infections caused by Flavobacterium oreochromis, Aeromonas veronii, Streptococcus agalactiae, and Edwardsiella tarda represent major threats to Nile tilapia aquaculture. This study evaluated a multistage mucosal vaccination strategy combining sequential nanoemulsion immersion priming with oral hydrogel-based boosters against these four pathogens in Oreochromis niloticus. Vaccination was associated with significant enhancement of both mucosal and systemic humoral immunity, as evidenced by elevated pathogen-specific IgM levels in gills, skin mucus, intestine, and serum across three successive challenge rounds. Immune-related gene expression analysis revealed significant upregulation of ighm, ighd, and ight in key immune tissues, consistent with broad activation of B cell-mediated responses. Label-free quantitative proteomic profiling demonstrated extensive immune remodeling in vaccinated fish, characterized by increased abundance of antigen-presentation molecules, complement factors, lysozyme, serpins, and mucosal defense-associated enzymes. Intestinal microbiome analysis revealed that vaccination reshaped microbial community composition toward a more stable and pathogen-resistant structure, with markedly reduced colonization by all four target pathogens. These immunological and microbial changes were associated with significantly higher survival rates under both immersion and intraperitoneal challenge conditions. Taken together, these findings suggest that multistage sequential vaccination may induce broad-spectrum, durable, multilayered protection in Nile tilapia through synergistic enhancement of humoral immunity, immunoglobulin gene expression, proteomic remodeling, and microbiome stabilization, providing a promising framework for sustainable disease management in intensive aquaculture.