Integrative Immunoinformatics and Molecular Dynamic Simulation for Multi-epitope Vaccine Design targeting Tregs against House Dust Mite Allergy.

• Structural and immunological functional analysis of 6 epitopes from HDM major allergens. • Epitopes Prediction and conservancy analysis of Derp1 and Derp2 allergens. • Novel multi epitope-based vaccine construct with Tregitope targeting Tregs. • MD simulation of TMEV-vaccine shows high binding affinity with MHC-II immune receptors. House Dust Mite (HDM) is a key contributor to asthma and allergic rhinitis, driven by Th2-dominant immune responses and elevated IgE levels. Although allergen-specific immunotherapy (AIT) can modify disease progression, its wider clinical application is restricted by IgE-mediated hypersensitivity. In this study we demonstrate, the successful fusion of a regulatory T-cell binding epitope with the principal allergenic epitopes of Der p 1 and Der p 2 which is a novel Tregitope-fused multi-epitope vaccine (TMEV) integrating immune tolerance and epitope-specific activation, designed through an advanced computational workflow. Predicted CD4⁺ T-cell epitopes were screened for antigenicity, safety, solubility, and sequence conservation, and assembled using GPGPG and EAAAK linkers for optimal immune presentation. Structural modelling, molecular docking, and 100 ns molecular dynamics simulations demonstrated stable binding to HLA-DR molecules (−894.3l; RMSD 1–3 Å). The 158-residue construct (16.17 kDa) showed high antigenicity (VaxiJen 0.937), favorable solubility (0.70), and strong structural integrity, with 94.3% residues in favored Ramachandran regions. Codon optimization predicted efficient expression in E. coli and mammalian hosts (CAI 0.995 and 0.92). The novelty of this work lies in fusing Tregitope-mediated immune regulation with a rationally selected multi-epitope construct to minimize allergic reactivity while enhancing immune tolerance. TMEV thus represents a promising vaccine candidate for effective HDM-specific immunotherapy.