Development of anti-CD4-1/CD8β monoclonal antibodies and spatiotemporal immune profiling of response to live attenuated IHNV in rainbow trout (Oncorhynchus mykiss).

Understanding the cellular mechanisms of live attenuated vaccines against infectious hematopoietic necrosis virus (IHNV) has long been hindered by the lack of species-specific immunological reagents in rainbow trout (Oncorhynchus mykiss). To bridge this gap and elucidate the protective basis of a high-efficacy mutant candidate (rIHNVMut), this study first established a core technical platform by developing and validating monoclonal antibodies (mAbs) targeting the T-cell co-receptors CD4-1 and CD8β. Validation via Western blot and immunohistochemistry confirmed the high specificity of these mAbs, enabling the first in situ visualization of T-cell subsets within the melanomacrophage centers of lymphoid tissues. Leveraging these novel tools, we systematically profiled the spatiotemporal immune dynamics induced by rIHNVMut. In contrast to the wild-type strain (IHNVWT), rIHNVMut exhibited restricted replication in the spleen and head kidney, which inversely correlated with a rapid, robust, and synchronized expansion of CD4-1+ and CD8β+ T lymphocytes (flow cytometry). This cellular mobilization was further corroborated by the coordinated upregulation of Th1/CTL-signature genes (IL-2, IFN-γ, Prf1) and a sustained IgM humoral response. Collectively, this study demonstrates that the safety and immunogenicity of rIHNVMut rely on a synergistic activation of cellular and humoral immunity, a mechanism revealed only through the application of the newly developed mAb toolkit.