Antibody-dependent immune response of olive flounder (Paralichthys olivaceus) induced by inactivated viral hemorrhagic septicemia virus (VHSV) vaccine

In teleosts, vaccine-induced protection is commonly assessed using serum antibody titers; however, these measurements often fail to fully explain protective outcomes due to limited understanding of B cell differentiation and antibody effector functions. In olive flounder (Paralichthys olivaceus), the cellular basis of antibody-mediated immunity remains poorly characterized. In this study, we investigated B cell differentiation, antibody production, and antibody-dependent immune responses post-immunization with an inactivated viral hemorrhagic septicemia virus (VHSV) vaccine. Vaccinated fish showed significantly higher survival rates post-challenge with VHSV than control, along with reduced spleen viral loads at early time points. At 3 days post-immunization (dpi), membrane IgM positive (mIgM⁺) B cells decreased, while total IgM antibody-secreting cells (ASCs) increased. VHSV-specific IgM ASCs were significantly elevated at 7 and 14 dpi. Serum VHSV- and glycoprotein (G)-specific IgM titers increased at 14 and 28 dpi, indicating the induction of antigen-specific humoral immunity. Following viral challenge, VHSV G-specific IgM titers declined rapidly at 1 day post-challenge (dpc), coinciding with reduced viral loads, suggesting early antibody utilization. Consistently, IgM from vaccinated fish showed enhanced binding to VHSV G expressed on infected HINAE cells. In addition, leukocytes exhibited increased cytotoxicity against VHSV-infected HINAE cells pre-incubated with immune serum, supporting the involvement of antibody-dependent cellular cytotoxicity (ADCC)-like activity. Overall, the vaccine induced antigen-specific antibody production through B cell differentiation, contributing to early, multilayered protection via neutralizing and Fc-mediated mechanisms.