The construction of recombinant DNA vaccine pVAX-ROP27-IL-2-IFN-γ and its immune enhancement effect against Eimeria tenella.

Eimeria tenella (E. tenella) is a highly pathogenic and widely distributed species responsible for significant economic losses in the global poultry industry. The rhoptry protein 27 (EtROP27) DNA vaccine of E. tenella provides certain immune protection against E. tenella infection. Notably, vaccination triggers a sharp increase in the expression of chicken-derived cytokines chIL-2 and chIFN-γ. This study combines chIL-2 and chIFN-γ with the EtROP27 DNA vaccine to investigate the immunomodulatory effects. Specific-pathogen-free (SPF) chickens were immunized intramuscularly, and successful in vivo expression was confirmed by Reverse Transcription Polymerase Chain Reaction and Western blot. Immunoprotective assays, histopathology, enzyme-linked immunosorbent assay (ELISA), quantitative polymerase chain reaction (qPCR), and immunohistochemical staining (IHC) were employed to investigate the immunomodulatory effects and mechanisms of chIL-2 and chIFN-γ in the context of the EtROP27 DNA vaccine. The results showed that pVAX-ROP27, pVAX-ROP27-IL-2, pVAX-ROP27-IFN-γ, and pVAX-ROP27-IL-2-IFN-γ DNA vaccines could be expressed in vivo. All DNA vaccines effectively induced both cellular and humoral immune responses, significantly reduced oocyst shedding, alleviated cecal lesions, and improved weight gain. In addition, the pVAX-ROP27-IL-2-IFN-γ group exhibited the strongest protective efficacy, with an anticoccidial index (ACI) of 183.51, accompanied by the highest levels of IL-2, IFN-γ, IgY, and IgA, while inflammatory cytokine expression was notably reduced. IHC analysis further confirmed that the immune group enhanced the activation of T and B cells, with the most pronounced effect observed in the pVAX-ROP27-IL-2-IFN-γ group. These results indicate that chIL-2 and chIFN-γ have an immune-enhancing effect on the EtROP27 DNA vaccine, and their binding effect is more pronounced.