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<p>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.&#xa0;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&#xa0;ns). Immune responses were evaluated via C-IMMSIM, codon-optimized with JCat, cloned into pET28a( +), and population coverage assessed with IEDB.&#xa0;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&#xa0;kcal/mol energies). MD confirmed stability (RMSD &lt; 1&#xa0;nm, Rg ~ 3.1–3.3&#xa0;nm). Simulations indicated robust IFN-γ, B/T-cell memory responses. Codon optimization yielded CAI = 1.0, GC = 52.34%. Global coverage: 100%.&#xa0;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.</p>

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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

  • 期刊论文

  • 发表日期：

    2026年1月13日

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  • Ali Alishvandi,
  • Cena Aram,
  • Maryam Barancheshemeh,
  • Tara Shekari,
  • Faezeh Firuzpour,
  • Mohammad Hossein Yazdi

摘要

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.