Assessment of Immunogenicity of Multi-Epitope DNA Vaccine Encoding CDPK3, ROP22 & MIC8 of Toxoplasma gondii Adjuvanted with IL-12 against Acute and Chronic Toxoplasmosis in BALB/c Mice
-
Esmaeil Abasi
,
Shahram Khadem vatan
,
Saeedeh Shojaee
,
Mehdi Mohebali
,
Masoud Foroutan
,
Hojjat Zeraati
,
Hossein Keshavarz Valian
Abstract
Background: 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.
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20. Zhao HG, Huang FY, Guo JL, Tan GH. Evaluation on the immune response induced by DNA vaccine encoding MIC8 co-immunized with IL-12 genetic adjuvant against Toxoplasma gondii infection. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2013;31(4):284–9.
21. Zhang NZ, Gao Q, Wang M, et al. Immunization With a DNA Vaccine Cocktail Encoding TgPF, TgROP16, TgROP18, TgMIC6, and TgCDPK3 Genes Protects Mice Against Chronic Toxoplasmosis. Front Immunol. 2018;9:1505.
22. Nayeri T, Sarvi S, Daryani A. Effective factors in the pathogenesis of Toxoplasma gondii. Heliyon. 2024;10(10):e31558.
23. Vogel FR. Improving vaccine performance with adjuvants. Clin Infect Dis. 2000;30 Suppl 3:S266–70.
24. Khosroshahi KH, Ghaffarifar F, Sharifi Z, et al. Comparing the effect of IL-12 genetic adjuvant and alum non-genetic adjuvant on the efficiency of the cocktail DNA vaccine containing plasmids encoding SAG-1 and ROP-2 of Toxoplasma gondii. Parasitol Res. 2012;111(1):403–11.
25. Zhu YC, He Y, Liu JF, Chen J. Adjuvantic cytokine IL-33 improves the protective immunity of cocktailed DNA vaccine of ROP5 and ROP18 against Toxoplasma gondii infection in mice. Parasite. 2020;27:26.
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27. Foroutan M, Ghaffarifar F, Sharifi Z, Dalimi A, Pirestani M. Bioinformatics analysis of ROP8 protein to improve vaccine design against Toxoplasma gondii. Infect Genet Evol. 2018;62:193–204.
28. Froger A, Hall JE. Transformation of plasmid DNA into E. coli using the heat shock method. J Vis Exp. 2007(6):253.
29. Selseleh M, Keshavarz H, Mohebali M, et al. Production and evaluation of Toxoplasma gondii recombinant GRA7 for serodiagnosis of human infections. Korean J Parasitol. 2012;50(3):233–8.
30. Hao R, Li X, Wang N, et al. Optimized plasmid extraction through controlled temperature, prolonged alkaline lysis, and gentle mixing. J Chromatogr B Analyt Technol Biomed Life Sci. 2025;1262:124646.
31. Abasi E, Shahabi S, Golkar M, Khezri P, Mohammadzadeh Hajipirloo H. Evaluation of immunogenic Effect of Toxoplasma gondii Recombinant SAG-1 Antigen with Propranolol as Adjuvant in BALB/c Mice. Adv Pharm Bull. 2019;9(4):632–9.
32. Mohammadzadeh Hajipirloo H, Bozorgomd A, Shahabi S, Hazrati Tappeh K, Karamati SA. Evaluation of Alum-Naltrexone Adjuvant Activity, on Efficacy of Anti-Leishmania Immunization with Autoclaved Leishmania major (MRHO/IR/75/ER) Antigens in BALB/c mice. Iran J Parasitol. 2014;9(3):311–8.
33. Voller A, Bartlett A, Bidwell DE. Enzyme immunoassays with special reference to ELISA techniques. J Clin Pathol. 1978;31(6):507–20.
34. Facciolà A, Visalli G, Laganà A, Di Pietro A. An Overview of Vaccine Adjuvants: Current Evidence and Future Perspectives. Vaccines (Basel). 2022;10: 819(5).
35. Dautu G, Munyaka B, Carmen G, et al. Toxoplasma gondii: DNA vaccination with genes encoding antigens MIC2, M2AP, AMA1 and BAG1 and evaluation of their immunogenic potential. Exp Parasitol. 2007;116(3):273–82.
36. Xue M, He S, Cui Y, Yao Y, Wang H. Evaluation of the immune response elicited by multi-antigenic DNA vaccine expressing SAG1, ROP2 and GRA2 against Toxoplasma gondii. Parasitol Int. 2008;57(4):424–9.
2. Tenter AM, Heckeroth AR, Weiss LM. Toxoplasma gondii: from animals to humans. Int J Parasitol. 2000;30(12-13):1217–58.
3. Salimi M, Keshavarz-Valian H, Mohebali M, Geravand M, Adabi M, Shojaee S. Electrochemical immunosensor based on carbon nanofibers and gold nanoparticles for detecting anti-Toxoplasma gondii IgG antibodies. Mikrochim Acta. 2023;190(9):367.
4. Shojaee S, Teimouri A, Keshavarz H, Azami SJ, Nouri S. The relation of secondary sex ratio and miscarriage history with Toxoplasma gondii infection. BMC Infect Dis. 2018;18(1):307.
5. Abdi J, Shojaee S, Mirzaee A, Keshavarz H. Seroprevalence of Toxoplasmosis in Pregnant Women Ilam Province, Iran. Iran J Parasitol. 2008;3:34–7.
6. Teimouri A, Goudarzi F, Goudarzi K, et al. Toxoplasma gondii Infection in Immunocompromised Patients in Iran (2013-2022): A Systematic Review and Meta-Analysis. Iran J Parasitol. 2022;17(4):443–57.
7. Liu Q, Singla LD, Zhou H. Vaccines against Toxoplasma gondii: status, challenges and future directions. Hum Vaccin Immunother. 2012;8(9):1305–8.
8. Alipour A, Shojaee S, Mohebali M, Tehranidoost M, Abdi Masoleh F, Keshavarz H. Toxoplasma infection in schizophrenia patients: a comparative study with control group. Iran J Parasitol. 2011;6(2):31–7.
9. Khademvatan S, Saki J, Khajeddin N, et al. Toxoplasma gondii Exposure and the Risk of Schizophrenia. Jundishapur J Microbiol. 2014;7(11):e12776.
10. Shahra M, Keshavarz H, Sahraeian MA, et al. Associations between Toxoplasma gondii Infection and Multiple Sclerosis: A Case-Control Seroprevalence Study. Iran J Parasitol. 2023;18(2):165–71.
11. Mahmoudvand H, Ziaali N, Aghaei I, et al. The possible association between Toxoplasma gondii infection and risk of anxiety and cognitive disorders in BALB/c mice. Pathog Glob Health. 2015;109(8):369–76.
12. Mahmoudvand H, Sheibani V, Shojaee S, et al. Toxoplasma gondii Infection Potentiates Cognitive Impairments of Alzheimer's Disease in the BALB/c Mice. J Parasitol. 2016;102(6):629–35.
13. Rahbari AH, Keshavarz H, Shojaee S, Mohebali M, Rezaeian M. IgG avidity ELISA test for diagnosis of acute toxoplasmosis in humans. Korean J Parasitol. 2012;50(2):99–102.
14. Ali-Heydari S, Keshavarz H, Shojaee S, Mohebali M. Diagnosis of antigenic markers of acute toxoplasmosis by IgG avidity immunoblotting. Parasite. 2013;20:18.
15. Teimouri A, Mahmoudi S, Behkar A, et al. Current and Emerging Techniques for Diagnosis of Toxoplasmosis in Pregnancy: A Narrative Review. Iran J Parasitol. 2024;19(4):384–96.
16. Khorshidvand Z, Shirian S, Amiri H, Zamani A, Maghsood AH. Immunomodulatory chitosan nanoparticles for Toxoplasma gondii infection: Novel application of chitosan in complex propranolol-hydrochloride as an adjuvant in vaccine delivery. Int J Biol Macromol. 2023;253(Pt 8):127228.
17. Ayazian Mavi S, Modarressi MH, Mohebali M, et al. Assessment of the immunogenicity and protective efficiency of a novel dual-promoter DNA vaccine, harboring SAG1 and GRA7 genes, from RH strain of Toxoplasma gondii in BALB/c mice. Infect Drug Resist. 2019;12:2519–30.
18. Billker O, Lourido S, Sibley LD. Calcium-dependent signaling and kinases in apicomplexan parasites. Cell Host Microbe. 2009;5(6):612–22.
19. Zhang Z, Li Y, Xie Q, et al. The Molecular Characterization and Immunity Identification of Rhoptry Protein 22 of Toxoplasma gondii as a DNA Vaccine Candidate Against Toxoplasmosis. J Eukaryot Microbiol. 2019;66(1):147–57.
20. Zhao HG, Huang FY, Guo JL, Tan GH. Evaluation on the immune response induced by DNA vaccine encoding MIC8 co-immunized with IL-12 genetic adjuvant against Toxoplasma gondii infection. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2013;31(4):284–9.
21. Zhang NZ, Gao Q, Wang M, et al. Immunization With a DNA Vaccine Cocktail Encoding TgPF, TgROP16, TgROP18, TgMIC6, and TgCDPK3 Genes Protects Mice Against Chronic Toxoplasmosis. Front Immunol. 2018;9:1505.
22. Nayeri T, Sarvi S, Daryani A. Effective factors in the pathogenesis of Toxoplasma gondii. Heliyon. 2024;10(10):e31558.
23. Vogel FR. Improving vaccine performance with adjuvants. Clin Infect Dis. 2000;30 Suppl 3:S266–70.
24. Khosroshahi KH, Ghaffarifar F, Sharifi Z, et al. Comparing the effect of IL-12 genetic adjuvant and alum non-genetic adjuvant on the efficiency of the cocktail DNA vaccine containing plasmids encoding SAG-1 and ROP-2 of Toxoplasma gondii. Parasitol Res. 2012;111(1):403–11.
25. Zhu YC, He Y, Liu JF, Chen J. Adjuvantic cytokine IL-33 improves the protective immunity of cocktailed DNA vaccine of ROP5 and ROP18 against Toxoplasma gondii infection in mice. Parasite. 2020;27:26.
26. Sher A, Collazzo C, Scanga C, Jankovic D, Yap G, Aliberti J. Induction and regulation of IL-12-dependent host resistance to Toxoplasma gondii. Immunol Res. 2003;27(2-3):521–8.
27. Foroutan M, Ghaffarifar F, Sharifi Z, Dalimi A, Pirestani M. Bioinformatics analysis of ROP8 protein to improve vaccine design against Toxoplasma gondii. Infect Genet Evol. 2018;62:193–204.
28. Froger A, Hall JE. Transformation of plasmid DNA into E. coli using the heat shock method. J Vis Exp. 2007(6):253.
29. Selseleh M, Keshavarz H, Mohebali M, et al. Production and evaluation of Toxoplasma gondii recombinant GRA7 for serodiagnosis of human infections. Korean J Parasitol. 2012;50(3):233–8.
30. Hao R, Li X, Wang N, et al. Optimized plasmid extraction through controlled temperature, prolonged alkaline lysis, and gentle mixing. J Chromatogr B Analyt Technol Biomed Life Sci. 2025;1262:124646.
31. Abasi E, Shahabi S, Golkar M, Khezri P, Mohammadzadeh Hajipirloo H. Evaluation of immunogenic Effect of Toxoplasma gondii Recombinant SAG-1 Antigen with Propranolol as Adjuvant in BALB/c Mice. Adv Pharm Bull. 2019;9(4):632–9.
32. Mohammadzadeh Hajipirloo H, Bozorgomd A, Shahabi S, Hazrati Tappeh K, Karamati SA. Evaluation of Alum-Naltrexone Adjuvant Activity, on Efficacy of Anti-Leishmania Immunization with Autoclaved Leishmania major (MRHO/IR/75/ER) Antigens in BALB/c mice. Iran J Parasitol. 2014;9(3):311–8.
33. Voller A, Bartlett A, Bidwell DE. Enzyme immunoassays with special reference to ELISA techniques. J Clin Pathol. 1978;31(6):507–20.
34. Facciolà A, Visalli G, Laganà A, Di Pietro A. An Overview of Vaccine Adjuvants: Current Evidence and Future Perspectives. Vaccines (Basel). 2022;10: 819(5).
35. Dautu G, Munyaka B, Carmen G, et al. Toxoplasma gondii: DNA vaccination with genes encoding antigens MIC2, M2AP, AMA1 and BAG1 and evaluation of their immunogenic potential. Exp Parasitol. 2007;116(3):273–82.
36. Xue M, He S, Cui Y, Yao Y, Wang H. Evaluation of the immune response elicited by multi-antigenic DNA vaccine expressing SAG1, ROP2 and GRA2 against Toxoplasma gondii. Parasitol Int. 2008;57(4):424–9.
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| Issue | Vol 21 No 1 (2026) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijpa.v21i1.21629 | |
| Keywords | ||
| Toxoplasma gondii DNA vaccine Immunology | ||
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How to Cite
1.
Abasi E, Khadem vatan S, Shojaee S, Mohebali M, Foroutan M, Zeraati H, Keshavarz Valian H. Assessment of Immunogenicity of Multi-Epitope DNA Vaccine Encoding CDPK3, ROP22 & MIC8 of Toxoplasma gondii Adjuvanted with IL-12 against Acute and Chronic Toxoplasmosis in BALB/c Mice. Iran J Parasitol. 2026;21(1):11-22.
