Multi-epitope vaccine design against Clostridioides difficile using the ABC-type transport system protein

<i>Clostridioides difficile</i> is a major cause of antibiotic-associated nosocomial diarrhoea with increasing global incidence and nearly one million annual cases reported in India. Therefore, it is of interest to design a multi-epitope vaccine design against <i>Clostridioides</i> difficile the ABC-type transport system protein. Hence, B-cell, CTL and HTL epitopes were predicted using BepiPred-2.0, NetCTL 1.2 and NetMHCIIpan 4.0. Thus, a 66-amino-acid epitope construct with AAY and GPGPG spacers for the TLR3 adjuvant to enhance immunogenicity was reported for further consideration. The construct was validated for antigenicity, non-allergenicity and physicochemical stability. Structural modelling, molecular docking with TLR3 (PDB ID: 2A0Z) and 100-ns molecular dynamics simulations showed excellent structural stability (RMSD ~0.1 Å) and receptor binding affinity, while immune simulations via C-ImmSim indicated strong B- and T-cell responses with elevated IFN-γ and IL-2 levels, supporting its potential as a safe, effective, and targeted prophylactic against <i>C. difficile</i> infections.