Abstract
Enteroinvasive Escherichia coli is a gram-negative pathogen closely related to Shigella, and it is one of the leading causes of bacillary dysentery acquired in South Asia and worldwide. The emergence of multidrug-resistant serotypes has severely limited treatment options, with an urgent requirement for vaccines. This study applied immunoinformatics to design a multiepitope vaccine candidate targeting the O96:H19 strain. Here, four invasion plasmid antigens (IpaA, IpaB, IpaC, and IpaD) were taken as targets. Designed vaccine construct integrates three 9-mer PAP (possessing both MHC & B cell inducing properties) linked by proteasomal and lysosomally cleavable spacers. 7-mer TLR4 agonist (RS-04) fused with both its terminals. Computational analysis of the 69-mer vaccine construct predicted strong antigenicity, non-allergenicity and optimum solubility. In Ramachandran's plot, 100% of the residues were located in the most favourable regions. Molecular docking revealed a high affinity of the construct towards the human TLR4 model. Additionally, a 100 ns all-atom molecular dynamics simulation further confirmed the TLR4-vaccine complex formation through 14 H-bonds, 131 non-bonded contacts, and five salt bridges. Post-simulation (100 ns) molecular interaction maps identified specific interactions of TLR4 agonists with the TLR4 model. Immunosimulation for 365 days was associated with rising titers of IgG and IgM antibodies, as well as pro-inflammatory cytokine responses. To express the construct in E.coli expression system, the vaccine sequence was reverse-translated. The codon-optimized sequence was recombined into a modified pET vector using an in-silico method. Hence, laboratory validation was required to assess the real-time efficacy of the EIEC and Shigella cross-protective multiepitope vaccine. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40203-026-00618-3.