Tehran University of Medical Sciences Iranian Journal of Parasitology 1735-7020 21 01 2026 05 19 11 22 esmaeil abasi Urmia medical university Shahram Khadem vatan Department of Medical Parasitology and Mycology Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran Saeedeh Shojaee Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Mehdi Mohebali Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Masoud Foroutan Department of Basic Medical Sciences, Faculty of Medicine, Abadan University of Medical Sciences, Abadan, Iran Hossein Keshavarz valian Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2026 02 11 Abstract Background: In this study, a non-allergenic, immunogenic recombinant DNA plasmid encoding CDPK3, MIC8, and ROP22 epitopes was evaluated as a DNA vaccine against Toxoplasma gondii in BALB/c mice, used in combination with IL-12 as a genetic adjuvant, to assess protection against both acute and chronic toxoplasmosis.   Methods: BALB/c mice were immunized three times at two-week intervals with the DNA vaccine combined with IL-12. Humoral immune responses were measured by total IgG, IgG1, and IgG2a levels, while cellular responses were evaluated through interferon-γ production and lymphocyte proliferation assays. Protective efficacy was assessed using challenge models with T. gondii: the virulent RH strain for acute infection survival and the non-virulent Tehran strain for chronic infection, based on reduction of brain tissue cysts. Results: Vaccinated mice exhibited strong antigen-specific immunity, with elevated total IgG, IgG1, and IgG2a titers. Enhanced IFN-γ production and lymphocyte proliferation confirmed cellular activation. These responses correlated with reduced brain cyst counts after infection with the avirulent Tehran strain and prolonged survival with decreased parasite burden following virulent RH strain challenge, indicating improved protection. Conclusion: Co-delivery of IL-12 enhanced both the immunogenicity and protective efficacy of the CDPK3–MIC8–ROP22 multi-epitope DNA vaccine by inducing a Th1-biased response that conferred protection against acute and chronic T. gondii infection. These findings support this vaccination strategy as a promising approach for toxoplasmosis control and highlight the need for further evaluation in diverse animal models and clinical settings to confirm safety, efficacy, and long-term immunity. https://ijpa.tums.ac.ir/index.php/ijpa/article/view/4884 https://ijpa.tums.ac.ir/index.php/ijpa/article/download/4884/1474