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Asian seabass (Lates calcarifer) holds significant economic value in fish farming in the Asia-Pacific region. Vibriosis caused by Vibrio harveyi (Vh) is a severe infectious disease affecting intensive farming of this species, for which prevention strategies by vaccination have been developed. This study investigated an alternative approach to injectable vaccination to prevent vibriosis in Asian seabass juveniles. The strategy begins with an immersion prime vaccination with a heat-inactivated Vh vaccine, followed by two oral booster doses administered at 14- and 28-days post-vaccination (dpv). Expression of five immune genes TNFα , IL1β , CD4, CD8 , and IgM in the head kidney and spleen, along with investigation of anti- Vh antibody response (IgM) in both systemic and mucosal systems, was conducted on a weekly basis. The efficacy of the vaccines was assessed by a laboratory challenge test at 43 dpv. The results showed that the immunized fish displayed higher levels of mRNA transcripts of the immune genes after the immersion prime and the first oral booster dose compared to the control group. The expression levels peaked at 14 and 28 dpv and then declined to baseline at 35 and 42 dpv. Serum specific IgM antibodies were detected as early as 7 dpv (the first time point investigated) and exhibited a steady increase, reaching the first peak at 21 dpv, and a second peak at 35 dpv. Although the antibody levels gradually declined over subsequent weeks, they remained significantly higher than the control group throughout the experiment. A similar antibody response pattern was also observed in the mucosal compartment. The laboratory challenge test demonstrated high protection by injection with 1.65 × 104 CFU/fish, with a relative percent of survival (RPS) of 72.22 ± 7.86 %. In conclusion, our findings highlight the potential of an immersion prime-oral booster vaccination strategy as a promising approach for preventing vibriosis in Asian seabass. [Display omitted] • We report the immersion prime-oral booster vaccination approach for Lates calcarifer. • Fish vaccinated with heat-killed V. harveyi mounted specific antibodies. • Upregulation of immune genes was observed in vaccinated fish. • The relative percent survival of this vaccination approach was 72.22 % ± 7.86 %. • This vaccination approach may offer farmers a convenient way to immunize their fish. [ABSTRACT FROM AUTHOR]