In Silico Optimization of GAD65 ₁₁₄–₁₂₂ Autoantigen for Potential Type 1 Diabetes Antigen-Specific Immunotherapy

Type 1 diabetes (T1D) is mediated by autoreactive T cells targeting pancreatic β-cell antigens, with CD8+ T cells specific for islet autoantigens playing a central role. CD8+ T cell reactivity to glutamic acid decarboxylase 65 (GAD65) in HLA-A*02:01 individuals has been reported to focus on the immunogenic region around residues 114-122 (VMNILLQYV). Here, we design GAD65₁₁₄₋₁₂₂ mimotopes with enhanced human leukocyte antigen (HLA) binding as potential T1D vaccine candidates. Using all-atom molecular dynamics (MD) simulation and free energy perturbation (FEP), we evaluate single, double, and swap mutations on HLA-A*02:01-GAD65₁₁₄₋₁₂₂ binding. Our results identify positions 3 and 7 as key sites for affinity enhancement. Position 3 favors negatively charged residues aspartic acid (N3D) and glutamic acid (N3E) over native asparagine (ASN), suggesting favorable electrostatic interactions, while position 7 prefers hydrophobic residues methionine (Q7M) and isoleucine (Q7I) over native glutamine (GLN), enhancing binding stability. Double mutations at both positions 3 and 7 display an overall additive or even synergistic effect, with N3D_Q7M, N3D_Q7I, N3E_Q7M, and N3E_Q7I double mutants identified as strong candidates for further experimental validation of T cell activation. This work highlights key insights for optimizing antigen-based vaccine design and optimization for T1D.