thesummer (due to high water temperature), a 6-month duration ofimmunity should protect groupers against S. iniae infection duringthis critical period.It is well known that vaccination with an oil-adjuvant antigenstimulates the expression of certain immune-related genes [31].This work assessed gene expression in PBLs by RT-qPCR analy-sis, and examined portions of the adaptive and innate immuneresponses. In the peripheral blood of groupers, igm expressionincreased 14 days PPV and peaked at 3 months PSV. This suggeststhat activation of an immune response is complex and time con-suming.Il-1 is a pleiotropic molecule produced by activatedmacrophages and blood monocytes [32]. TNF- has been shownto eliminate pathogens by enhancing various cellular responsesincluding phagocytosis and chemotaxis; it also increases the pro-duction of effector molecules, reactive oxygen species, and nitrogenintermediates [33–35]. Phagocytosis aids in the destruction ofextracellular pathogens. This phenomenon was evident in groupersin which il-1ˇ and tnf-˛ expression were up-regulated after vacci-nation, and phagocytic activity was elevated in the two vaccinatedgroups compared to the control group. These results suggested thatthe inactivated S. iniae vaccine could enhance the innate immuneresponse in groupers, and regulate the expression of immune-related genes. Furthermore, analytical results indicated that PA andPI values were markedly higher in vaccinated groups than in thecontrol group. This finding suggested that the inactivated S. iniaevaccine may have increased the number of phagocytic cells in theperipheral blood, and enhanced non-specific defense mechanisms.Vaccine efficacy is usually evaluated through challenge experi-ments, which are time consuming and require many controls [36].TNF- has been proposed as a biomarker for monitoring fish healthand vaccine efficacy [37,38]. Therefore, this study also demon-strated the feasibility of using transcriptional biosignatures (il-1ˇand tnf-˛ mRNA expression) as indicators of vaccine efficacy, whichcould improve conventional immunological evaluation methodsand allow for more efficient testing of vaccines. The expression ofimmune-related genes after vaccination provides valuable insightinto immune mechanisms against S. iniae in groupers. Further workis necessary to determine the functions of these immune responsegenes during S. iniae infection. As humoral immunity plays animportant role in fish [39], this study also analyzed the fish-specificantibody titer against S. iniae whole cells by agglutination tests.We found that the agglutination titer under laboratory conditionsis higher than that observed in the field trial after vaccination,which may have resulted from fish of different genetic backgroundsresponding differently to vaccination.5. ConclusionsThis work developed an efficacious and safe vaccine againstS. iniae infection in farmed groupers. The vaccine produced adetectable specific antibody that offered protection against S.iniae infection for at least 6 months. Mortality of vaccinatedgroupers remained below 2%, while RPS exceeded 97%, implyingthat groupers treated with the tested vaccine mounted a protec-tive immune response against S. iniae. Moreover, the tested vaccinesignificantly elevated innate and adaptive immune responses, andregulated the expression of immune-related genes