Rpf-Toxo: A Preliminary Computationally Designed Dense Granule Antigen-Based Multi-Epitope Vaccine Against Toxoplasma Gondii Publisher Pubmed



Safari M H ; Hosseini S A ; Ghiabi S ; Siamian D ; Majidiani H ; Irannejad H ; Asghari A
Source: Veterinary Medicine and Science Published:2026

Abstract

Background: Toxoplasma gondii is a protozoan parasite of medical and veterinary importance causing abortion. While current therapies are limited to address the chronic phase of infection, effective prophylactic vaccines are warranted. Objectives: In this study, we applied a rational vaccine design approach centred around immunodominant dense granule antigens (GRAs). Methods: In silico epitope mapping was performed on six GRAs (GRA15, GRA60, GRA76, GRA83, GRA-α and GRA-β). Using web servers, B-cell, cytotoxic T-lymphocyte and helper T-lymphocyte epitopes were predicted and then filtered for antigenicity, solubility and allergenicity. We designed four vaccine constructs by fusing predicted epitopes with specific linkers and adjuvants (RS-09, RpfE/50S ribosomal protein of Mycobacterium tuberculosis, and human interferon gamma [IFN-γ]). Constructs were subjected to rigorous evaluation, which included physicochemical evaluation, 3D structural prediction and refinement, toll-like receptor-4 (TLR-4) docking, immune simulation and codon adaptation. Results: All candidates exhibited high antigenicity (VaxiJen scores > 0.9) and solubility (> 0.6). Rpf-Toxo emerged optimal, with non-allergenic, antigenic (0.9148), soluble (0.658), stable (instability index: 35.41) and hydrophilic (grand average of hydropathicity: −0.641) properties. Docking revealed strong TLR-4 binding activity (affinity: −20.6 kcal/mol; Kd: 7.3e-16 M) and immune simulation predicted robust responses, including antibody titers > 170,000, Th1-skewed IFN-γ (380,000 ng/mL) and memory cell activation; however, these in silico predictions need experimental validation. Codon optimization enhanced expression (CAI: 1.00; GC: 65.63%), and in silico cloning indicated compatibility with pET28a(+). Conclusion: These computational predictions require future experimental validation through in vitro and in vivo studies to confirm safety and protective efficacy. © 2026 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.