Reverse vaccinology and immunoinformatics approaches for multi-epitope vaccine design against Klebsiella pneumoniae reveal a novel vaccine target protein

Klebsiella pneumoniae (K. pneumoniae), a Gram-negative pathogen, is a leading cause of hospital-acquired infections in Sudan and worldwide. The emergence of multidrug-resistant (MDR) strains has severely limited treatment options, underscoring the urgent need for an effective vaccine. In this study, we employed reverse vaccinology and immunoinformatics to design a novel multi-epitope vaccine targeting the hypervirulent NUBRI-K strain. Two conserved, non-host homologous iron acquisition proteins, IucA/IucC and FyuA, were prioritized as targets. The vaccine construct integrates six B-cell, six cytotoxic T lymphocyte (CTL), and six helper T lymphocyte (HTL) epitopes, linked by optimized spacers and fused to a β-defensin adjuvant. Computational analyses confirmed strong antigenicity (1.0429), non-allergenicity, and favorable solubility (0.477). Molecular docking revealed high-affinity binding to Toll-like receptor 4 (TLR4) (-278.22 kcal/mol), stabilized by eight hydrogen bonds and two salt bridges. Structural validation showed that 91 % of residues were located in favored regions of the Ramachandran plot. Additionally, CABSflex 2.0 dynamics analysis confirmed stable vaccine-TLR4 interactions, with minimal residue-level fluctuations (RMSF <1.5 Å), indicating conformational stability of the complex. In silico immune simulations predicted potent humoral and cellular responses, including elevated IgG/IgM titers, T-cell proliferation, and IFN-γ secretion. The construct was further optimized for mammalian expression, achieving an ideal GC content (48.27 %) and a codon adaptation index (CAI) of 1.0, facilitating efficient in silico cloning into the pcDNA3 vector. By targeting conserved iron acquisition systems, this vaccine candidate presents a promising strategy to combat antibiotic-resistant K. pneumoniae while minimizing selective pressure. Future in vitro and in vivo studies are warranted to validate its immunogenicity and protective efficacy.