Designing a multi-epitope vaccine using Toxoplasma ROP5, ROP7, and SAG1 epitopes and immunogenicity evaluation against acute and chronic toxoplasmosis in BABL/c mice.

This study designed and evaluated a multi-epitope DNA vaccine targeting Toxoplasma gondii immunodominant antigens-ROP5, ROP7, and SAG1-to assess its protective efficacy against acute and chronic toxoplasmosis in BALB/c mice. A bioengineered multi-epitope vaccine construct (MEVC) was synthesized by integrating computationally predicted B- and T-cell epitopes using SAPGTP linkers to ensure conformational stability and epitope accessibility. In silico analyses confirmed the MEVC's antigenicity (VaxiJen score: 0.96), non-allergenicity, solubility (GRAVY index: 0.45), and physicochemical stability (instability index: 32.14; aliphatic index: 78.3), supporting its suitability for immunization. The codon-optimized sequence (753 bp; 253 amino acids) was cloned into the pcDNA3.1(+) plasmid and amplified in Escherichia coli TOP10 cells. Thirty-six female BALB/c mice (6-8 weeks) were divided into three groups (n = 12/group) and immunized intramuscularly with 100 μg MEVC, empty vector, or phosphate-buffered saline (PBS) at weeks 0, 2, and 4. Post-immunization, mice were challenged with acute (2 × 103 RH strain tachyzoites, intraperitoneal) or chronic (10 PRU strain cysts, oral) infection. Molecular docking simulations demonstrated high-affinity binding of the MEVC to murine toll-like receptor 4 via hydrogen bonds and hydrophobic interactions, suggesting adjuvant-like immunogenicity. In vitro expression in HEK-293 cells confirmed protein synthesis, with Western blot detecting a 26 kDa immunoreactive band. MEVC-immunized mice exhibited significantly elevated anti-Toxoplasma IgG titers (1:12,800), dominated by IgG2a isotypes (P < 0.05), and robust IFN-γ production, indicative of Th1-polarized immunity. IL-4 levels remained low, confirming minimal Th2 skewing. Vaccination reduced cerebral cyst burden by 76 % (P < 0.01) in chronic infection, yet survival post-acute challenge extended only two days compared to controls. These results demonstrate partial protection against toxoplasmosis, with the MEVC eliciting cellular and humoral responses effective against chronic infection but limited efficacy in acute settings.