Vaccine Research

other Investigation of the Effect of Adjuvant Positioning within a FliC-derived Multi-Epitope Construct on TLR5 Engagement and Epithelial Immune Modulation Vaccine development, efficacy and safety evaluation Vaccine development, efficacy and safety evaluation پژوهشي Research Introduction: Rational vaccine design is a promising strategy for combating bacterial infections. Multi-epitope-based vaccines as a modern platform need to improvement by immunostimulants. Flagellin from Salmonella typhimurium acts as a potent Toll-like receptor 5 (TLR5) ligand. The incorporation of vaccine antigens into different sites, including the N- and C-terminal regions, as well as the hypervariable domains of FliC influences receptor docking and immune signaling. This study evaluates the effect of adjuvant positioning in a FliC-derived construct on TLR5 docking and epithelial IL-8 modulation. Methods: Variable D2 and D3 domains of FliC were substituted with antigenic regions of PapG II and FimH from Escherichia coli, connected via rigid and flexible linkers to generate three constructs differing in adjuvant position (N-terminal, central, and C-terminal). Computational analyses including 3D structure prediction and TLR5 docking were assessed. The optimal construct was expressed in E. coli, purified, and applied to HT-29 cells to measure IL-8 secretion using ELISA. Results: Docking analyses revealed that central placement of FliC in the multi-epitope construct (FlFPFl) provided the highest TLR5 binding affinity and stable interaction. Functional assays confirmed that FlFPFl significantly modulated IL-8 secretion compared to untreated cells (p < 0.05), demonstrating the critical role of adjuvant positioning in epithelial immune activation. Conclusion: This study highlights the importance of adjuvant topology in multi-epitope vaccine design. Based on in silico analyses, structural optimization of flagellin-based constructs derived from S. typhimurium improved receptor-accessible motif exposure and folding stability. Central adjuvant placement enhanced interaction with TLR5 and FlFPFl model showed the most favorable docking performance. Salmonella typhimurium, flagellin, adjuvant, vaccine, docking 7 14 http://vacres.pasteur.ac.ir/browse.php?a_code=A-10-66-2&slc_lang=other&sid=1 Saeide Mirsharifi Yes Department of Microbiology, ShQ.C., Islamic Azad University, Shahr-e Qods, Iran Mohammad Reza Asadi Karam No Department of Molecular Biology, Pasteur Institute of Iran, Tehran, Iran Fatemeh Foroohi No Department of Microbiology, ShQ.C., Islamic Azad University, Shahr-e Qods, Iran Mehri Habibi No Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran. Touraj Rahimi No Department of Plant Production and Genetics, ShQ.C., Islamic Azad University, Shahr-e Qods, Iran