Abstract
Toxoplasma gondii is an obligate intracellular parasite is an important global public health concern; particularly for pregnant women and immunocompromised persons in whom it can cause congenital malformations, fetal loss and life threatening encephalitis. Current antiparasitic therapies are hindered by toxicity, poor tolerability and low success against chronic tissue cysts emphasising the need for new prophylactic vaccines. This study aimed to detect T. gondii in placentas of women with first-trimester miscarriage using Giemsa staining, real-time PCR targeting the B1 gene and in vitro isolation on CHO cell monolayers; and to design a redox-sensitive multi-epitope mRNA vaccine against the immunodominant surface antigen SAG1. An immunoinformatics pipeline predicted Cytotoxic T-lymphocyte )CTL(, Helper T-lymphocyte )HTL( and linear B cell epitopes that were assembled as a construct with AAY, GPGPG and KK linkers (one-linker type of each) fused to the 50S ribosomal protein L7/L12 adjuvant for improved efficacy; sequence was also codon optimized for human expression. Structural validation of the 428 amino acid construct (a) AlphaFold3, (b) Ramachandran analysis scores showing 82.3% favored, ERRAT quality factor=94.62, d ProSA Z score –5.21 In molecular docking analysis, TLR2 had an energy cluster of −1284.6 kcal/mol with stable binding at 100 ns in molecular dynamics. Following LNP encapsulation, the efficiency of in vitro transcription was 95.12%. 100% protection (p < 0.0001) was conferred by the vaccine in a BALB/c mouse model (n=40, 30 µg IM days 0 and 14, challenged with 1×10³ tachyzoites, where all unvaccinated controls had succumbed by day 8). Vaccinated mice exhibited >99% reduction in hepatic and cerebral parasite burdens, robust anti-T. gondii lysate antigen (anti TLA) IgG titers (95.15 µg/mL), and a dominant Th1 polarized cytokine profile (IFN γ 4,785 pg/mL, IL 12A 2,240 pg/mL, IFNA1 912 pg/mL) with low IL 10, alongside marked expansion of CD4⁺ (25.85%) and CD8a⁺ (84.33%) T cells. These findings demonstrate that the rationally designed multi-epitope LNP mRNA vaccine elicits durable, comprehensive protective immunity, representing a promising translational candidate for preventing congenital toxoplasmosis and reducing disease burden in high risk populations.

Key words: Toxoplasma gondii, mRNA vaccine, SAG1, Immunoinformatics, Lipid nanoparticles.