Physicochemical analysis of vaccines.

Tularemia is an extremely contagious zoonotic illness resulting from infection with the intracellular bacterium Francisella tularensis. It is transmitted primarily via vector bites particularly from ticks, flies, and mosquitoes and is a severe public health threat. Because of its high virulence, low infective dose, aerosol transmissibility, and potential for mass casualties, F. tularensis is also considered a potential biological warfare agent. Despite its severity, there is presently no licensed vaccine against this pathogen. In the present work, a subtractive proteomics pipeline was implemented to identify potential antigenic targets to prepare a multi-epitope vaccine. Five vaccine constructs were generated through the combination of B-cell, HTL, and CTL epitopes with suitable adjuvants and linkers. Among these, two constructs V1 and V2 were extremely non-allergenic and antigenic. To assess immune receptor engagement, molecular docking was conducted with TLR4 and TLR5, followed by 200 ns molecular dynamics simulations. Vaccine-receptor complexes were analyzed using RMSD, RMSF, radius of gyration (Rg), Dynamic Cross-Correlation Matrix (DCCM), SASA, PCA, H-bond analysis and MMPBSA binding energy calculations, all confirming structural stability and strong binding affinity. In-silico cloning revealed a GC content of 50% and 1.0 codon adaptation index (CAI), suggesting high expression potential in E. coli. Immune simulation further supported the construct`s ability to elicit a robust and long-lasting immunity. These computational findings highlight the potential of the constructed vaccines as effective candidates against F. tularensis, though experimental substantiation is requisite.