Grass carp Meteorin-like acts as a molecular adjuvant for a DNA vaccine against GCRV infection by enhancing MHC I/CD8-associated responses

Grass carp reovirus (GCRV) causes severe hemorrhagic disease and poses a major threat to grass carp aquaculture. Meteorin-like (Metrnl) is a multifunctional cytokine involved in immune regulation, but its adjuvant potential in fish remains poorly understood. This study evaluated whether grass carp Metrnl could serve as a molecular adjuvant for a DNA vaccine encoding the GCRV-II VP35 protein (pcDNA3.1-s11). Fish were divided into four groups: PBS, pcDNA3.1 vector, VP35 alone, and VP35 + Metrnl. Immunofluorescence microscopy confirmed local expression of VP35 and Metrnl at the injection site, which persisted for at least five weeks. Co-injection with Metrnl significantly increased white blood cell count and lymphocyte proportion. Furthermore, serum IgM antibody levels and IgM mRNA expression in the head kidney, spleen, and intestine were markedly elevated in the VP35 + Metrnl group. Serum enzyme activities (ACP, AKP, LZM, and T-AOC) were also significantly increased following co-vaccination, indicating enhanced antioxidant capacity and non-specific immune defense. Expression levels of immune-related genes such as TLR22, IFN-I, and Mx1 in the head kidney, spleen, and intestine were substantially upregulated. Importantly, Metrnl was associated with upregulated expression of MHC I and CD8, suggesting a potential role in MHC I/CD8-related antigen presentation of the VP35 vaccine, following the booster immunization, it also moderately enhanced MHC II and CD4 expression, further supporting humoral and helper T-cell responses. Protective efficacy testing showed that the relative percent survival (RPS) in the VP35 + Metrnl group reached 70%, higher than the 56.67% observed with the DNA vaccine alone, together with reduced viral VP56 gene expression after GCRV challenge. These results demonstrate that grass carp Metrnl acts as a potent molecular adjuvant that enhances the immunogenicity and protective efficacy of the DNA vaccine, providing a safe and efficient new strategy for green prevention and control of grass carp hemorrhagic disease.