African swine fever virus (ASFV) remains a serious global threat to the swine industry due to its high mortality rate and the lack of a widely effective vaccine. In this study, we developed an attenuated Salmonella Typhimurium-based delivery platform, JOL2500, to deliver constructs encoding selected ASFV antigens, including the structural proteins p54 and p72, and virulence- or immune-associated proteins 9GL, HA/CD2v, and C-type lectin. The expression of these recombinant antigens was confirmed in vitro using SDS-PAGE, RT-PCR, and indirect immunofluorescence assay (IFA) in RAW 264.7 cells. The immunogenicity of the recombinant constructs was then evaluated in BALB/c mice following intramuscular immunization. Compared with the vector control group, vaccinated mice showed increased antigen-specific systemic antibody responses, including IgM, IgG1, and IgG2a, as well as enhanced mucosal secretory IgA responses. In addition, splenocytes from immunized mice showed increased proliferation and metabolic activity after antigen simulation. Cytokine analysis revealed a pro-inflammatory and Th1-associated cytokine profile, marked by elevated levels of IFN-γ, TNF-α, and IL-17. Flow cytometry further demonstrated immunization-associated shifts in splenocyte immune-cell populations. Overall, these findings demonstrate that Salmonellamediated delivery of multiple ASFV antigens can induce broad humoral, mucosal, and cellular immune responses in mice. Although protective efficacy against ASFV infection still needs to be confirmed in future studies, this work provides a promising proof of concept for developing safe, practical, and cost-effective ASFV vaccine candidates.

Abstract
INTRODUCTION
MATERIALS AND METHODS
    Ethical approval and animal care
    Bacterial strains, plasmids, and growth conditions
    Design and construction of ASFV vaccine candidates
    In vitro characterization: Expression and cytotoxicity
    Animal immunization and sample collection
    Evaluation of humoral and cellular immune responses
    Histopathology and tissue localization of the deliverystrain
    Data presentation and analysis
RESULTS
    In vitro expression of recombinant ASFV antigens
    Tissue localization and histopathological findings inthe vector control group
    Systemic and mucosal antibody responses followingimmunization with recombinant ASFV antigen constructs
    Antigen-specific splenocyte proliferative responsesafter immunization
    ASFV antigen stimulation induced distinct cytokineexpression profiles
    MTT assay showed increased metabolic activity inantigen-stimulated splenocytes
    Flow cytometry showed immunization-associatedshifts in splenocyte immune-cell populations
DISCUSSION
ACKNOWLEDGEMENTS
DECLARATION OF INTEREST
REFERENCES

• Maryum Tanveer(Department of Veterinary Public Health, College of Veterinary Medicine, Jeonbuk National University, Iksan, 54596, Republic of Korea)
• Suyeon Park(Department of Veterinary Public Health, College of Veterinary Medicine, Jeonbuk National University, Iksan, 54596, Republic of Korea)
• Gayeon Won(Department of Veterinary Public Health, College of Veterinary Medicine, Jeonbuk National University, Iksan, 54596, Republic of Korea) Corresponding author
• John Hwa Lee(Department of Veterinary Public Health, College of Veterinary Medicine, Jeonbuk National University, Iksan, 54596, Republic of Korea) Corresponding author