A Multi-Epitope Vaccine Candidate with Predicted Broad Genotype Coverage Against Toxoplasma gondii Designed by Reverse Vaccinology and Immunoinformatics Strategies

Background Toxoplasma gondii ( T. gondii ) is a globally prevalent zoonotic pathogen with major veterinary importance, causing significant livestock reproductive losses and facilitating zoonotic transmission to humans. Despite extensive efforts, there is no available efficient vaccine except for Toxovax, which is only approved for sheep and suffers from some limitations. The parasite’s complex multistage life cycle and strain diversity present major obstacles for vaccine development. Traditional vaccine strategies, including killed, attenuated, and subunit vaccines, provide only limited protection, in addition to posing potential translational drawbacks. Multi-epitope vaccine (MEV) approaches have emerged as a promising alternative that combines conserved immunodominant epitopes, enabling a robust immune response. Our study aimed to computationally design and evaluate a novel MEV candidate capable of eliciting predicted broad genotypic coverage against T. gondii. It thus, serves as a One Health candidate intended for veterinary disease control, which consequently serves the public health by reducing foodborne zoonotic transmission risk and translational potential for cross-species experimental validation. Methods Eighteen T. gondii proteins expressed in different life stages of multiple genotypes were screened for antigenicity, allergenicity, and epitope conservancy. Selected proteins were analyzed to predict MHC-I, MHC-II, and B-cell epitopes using stringent thresholds. Followed by evaluating the candidate epitopes for their cytokine induction potential and theoretical population coverage. Selected epitopes were then assembled into constructs and assessed for physicochemical properties, secondary and tertiary structure stability, immune response simulation, and molecular docking to TLR2 and TLR4. The candidate construct was subjected to complex stability analysis through molecular dynamics (MD) simulation. Codon optimization and in silico cloning were then performed for potential E. Coli expression. Results Forty-three conserved and non-allergenic epitopes were selected and constituted different constructs. Out of which, MEV-2A demonstrated high predicted antigenicity, 100 % hypothetical worldwide population coverage, Th1-skewed cytokine induction, high stability, and computational binding affinity towards TLR2 and TLR4, suggesting that MEV-2A is a promising candidate for further validation. Conclusion MEV-2A is a computationally designed and optimized multi-epitope vaccine targeting multi-stages and multi-genotypes of T. gondii, aimed to serve as a One Health candidate for livestock disease control and human zoonotic risk reduction , which warrants further experimental in vitro and in vivo validations. Clinical trial number: not applicable.