In Silico Design of a Multi-Epitope Vaccine Against Nsclc Targeting T-Cell Exhaustion and Metastasis: Insights From Rna-Seq and Molecular Dynamics Simulation Analysis Publisher



Alishvandi A ; Aram C ; Barancheshemeh M ; Shekari T ; Firuzpour F ; Yazdi MH
Source: Network Modeling Analysis in Health Informatics and Bioinformatics Published:2026

Abstract

Non-small cell lung cancer (NSCLC) represents 85% of lung cancer cases, with immune evasion via checkpoints like PD-L1 and LAG3, and metastasis driven by vimentin, posing major therapeutic challenges. This study aimed to design a multi-epitope vaccine targeting these proteins to reverse T-cell exhaustion and inhibit metastasis, integrating RNA-seq insights for rational target selection. Protein sequences were retrieved from UniProt. RNA-seq data from TCGA (LUAD/LUSC) were analyzed using R packages (TCGAbiolinks, edgeR, limma) to identify differentially expressed genes, PPI networks via STRING/Cytoscape, and immune correlations with TIMER2.0. Epitopes (CTL, HTL, B-cell) were predicted using IEDB, NetCTL, ABCpred, and BepiPred, screened for antigenicity (VaxiJen), allergenicity (AllerTOP), toxicity (ToxinPred), and physicochemical properties (ProtParam). The vaccine was assembled with PADRE/hBD-3 adjuvants and linkers, modeled via SOPMA/Robetta/AlphaFold, refined with GalaxyRefine, docked to TLR1/2 using ClusPro, and simulated dynamically with GROMACS (100 ns). Immune responses were evaluated via C-IMMSIM, codon-optimized with JCat, cloned into pET28a(+), and population coverage assessed with IEDB. RNA-seq revealed upregulated targets linked to EMT, IL2/STAT5, and mTORC1 pathways, with strong immune correlations (e.g., Tregs, CD8 + T-cells, CAFs). Selected epitopes formed a 506-residue construct (antigenicity 0.855, non-allergenic/non-toxic). Secondary structure: 63% coils; tertiary model validated (93% favored Ramachandran regions). Docking showed strong TLR interactions (-1126.9/-913.7 kcal/mol energies). MD confirmed stability (RMSD < 1 nm, Rg ~ 3.1–3.3 nm). Simulations indicated robust IFN-γ, B/T-cell memory responses. Codon optimization yielded CAI = 1.0, GC = 52.34%. Global coverage: 100%. This in silico vaccine demonstrates structural stability, immune potency, and broad applicability, offering a promising approach to combat NSCLC. Experimental validation is essential for clinical translation. © The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature 2026.