Plysup B.Lueangyangyuen A.Khrisanapant P.Senapin S.Rattanarojpong T.Somsoros W.Khunrae P.Sangsuriya P.Mahidol University2026-05-302026-05-302025-09-01Agriculture and Natural Resources Vol.59 No.5 (2025)24681458https://repository.li.mahidol.ac.th/handle/123456789/116970Importance of the work: Co-immunization with recombinant S5<inf>196–272</inf> and S6<inf>200–317</inf> proteins enhances protective immunity and provides insights for future TiLV vaccine development. Objectives: To evaluate the vaccine potential of combined S5<inf>196–272</inf> and S6<inf>200–317</inf> compared to individual immunization. Materials and Methods: A sample of Nile tilapia was divided into three main groups: immunized with S5<inf>196–272</inf> and S6<inf>200–317</inf> individually, or co-immunized. Antibody responses were measured weekly using enzyme-linked immunosorbent assay, with virus neutralization being assessed using a methylthiazolyldiphenyl-tetrazolium bromide (MTT) cell viability assay. A viral challenge test was conducted to determine the relative percentage of survival (RPS). Results: Co-immunization of the fish with S5<inf>196–272</inf> and S6<inf>200–317</inf> resulted in a synergistic effect, leading to the higher production of S6<inf>200–317</inf>-specific antibodies than for immunization with S6<inf>200–317</inf> alone. A significant increase in serum antibody levels was observed from 7 d, 21 d, 28 d and 35 d post-co-immunization. In contrast, S5<inf>196–272</inf>-specific antibodies were generated at consistently high levels following both individual and co-immunization. The MTT cell viability assay findings demonstrated that antibodies from the co-immunization group had the highest virus-neutralizing effect (87.22% viability). Furthermore, the viral challenge assay revealed that the co-immunization group had the highest RPS (57.14%), whereas individual immunization provided no protection effect against TiLV infection. Main finding: Co-immunization with S5<inf>196–272</inf> and S6<inf>200–317</inf> induced a synergistic antibody response and provided effective protection against TiLV in Nile tilapia.Agricultural and Biological SciencesArticleSCOPUS10.34044/j.anres.2025.59.5.142-s2.0-1050394705922452316X