Evaluation of a hydrogel platform for encapsulated multivalent Vibrio antigen delivery to enhance immune responses and disease protection against vibriosis in Asian seabass (Lates calcarifer).

This study reports the development and evaluation of a novel multivalent oral hydrogel vaccine designed to protect Asian seabass (Lates calcarifer) against vibriosis caused by multiple Vibrio species. The hydrogel formulation, composed of alginate and bentonite, was engineered to encapsulate three Vibrio pathogens (V. harveyi, V. vulnificus, and P. damsela), and subsequently freeze-dried to yield stable, dry hydrogel beads. Although the process achieved a relatively low yield (approximately 10 %), the dry beads maintained their structural integrity, retained antigenic components with 2.4 ± 1.6 × 10 DNA copies of total cell antigens/mg dry beads, and resisted acidic degradation, ensuring antigen preservation during simulated gastric exposure. Physicochemical characterization (FTIR) confirmed that the encapsulation process preserved the structural and functional properties of alginate, bentonite, and bacterial antigens without introducing new chemical bonds. Scanning electron microscopy (SEM) revealed a porous, sponge-like internal architecture that supported antigen entrapment and controlled release. Under simulated gastric conditions (pH 2.0), the hydrogel exhibited remarkable stability for up to 8 h, preventing antigen loss. Upon transitioning to intestinal conditions (pH 7.2), the matrix gradually disintegrated, releasing the antigens in a controlled manner for up to 8 h. Oral vaccination trials demonstrated enhanced immune responses in Asian seabass. Fish receiving the multivalent Vibrio antigen-containing hydrogel vaccine (Hg-mVibrioAg) displayed elevated serum-specific IgM titers against all three Vibrio species, with significantly higher and more sustained antibody levels following a 7-day and 14-day vaccination regimen. Increased lysozyme and bactericidal activity further supported improved innate defense mechanisms. Subsequent pathogen challenge tests confirmed that vaccinated fish, particularly those following the 7-day and 14-day regimens, exhibited significantly higher survival rates and robust protection. Concurrently, immune-related gene expression in the head kidney, peripheral blood leukocytes, gills, and intestines was upregulated, indicating a broad immune activation associated with specific IgM responses. No significant alterations in blood biochemistry or tissue histology were observed, highlighting the vaccine's biocompatibility. Additionally, these findings underscore the potential of this multivalent oral hydrogel vaccine as a promising, safe, and effective prophylactic strategy against Vibrio infections in Asian seabass aquaculture.