In Silico Development of a Chimeric Multi-Epitope Vaccine Targeting Helcococcus kunzii: Coupling Subtractive Proteomics and Reverse Vaccinology for Vaccine Target Discovery

<b>Background</b>: <i>Helcococcus kunzii</i>, a facultative anaerobe and Gram-positive coccus, has been documented as a cunning pathogen, mainly in immunocompromised individuals, as evidenced by recent clinical and microbiological reports. It has been associated with a variety of polymicrobial infections, comprising diabetic foot ulcers, prosthetic joint infections, osteomyelitis, endocarditis, and bloodstream infections. Despite its emerging clinical relevance, no licensed vaccine or targeted immunotherapy currently exists for <i>H. kunzii</i>, and its rising resistance to conventional antibiotics presents a growing public health concern. <b>Objectives</b>: In this study, we employed an integrated subtractive proteomics and immunoinformatics pipeline to design a multi-epitope subunit vaccine (MEV) candidate against <i>H. kunzii</i>. Initially, pan-proteome analysis identified non-redundant, essential, non-homologous, and virulent proteins suitable for therapeutic targeting. <b>Methods/Results</b>: From these, two highly conserved and surface-accessible proteins, cell division protein FtsZ and peptidoglycan glycosyltransferase FtsW, were selected as promising vaccine targets. Comprehensive epitope prediction identified nine cytotoxic T-lymphocyte (CTL), five helper T-lymphocyte (HTL), and two linear B-cell (LBL) epitopes, which were rationally assembled into a 397-amino-acid-long chimeric construct. The construct was designed using appropriate linkers and adjuvanted with the cholera toxin B (CTB) subunit (NCBI accession: AND74811.1) to enhance immunogenicity. Molecular docking and dynamics simulations revealed persistent and high-affinity ties amongst the MEV and essential immune receptors, indicating a durable ability to elicit an immune reaction. In silico immune dynamic simulations predicted vigorous B- and T-cell-mediated immune responses. Codon optimization and computer-aided cloning into the <i>E. coli</i> K12 host employing the pET-28a(+) vector suggested high translational efficiency and suitability for bacterial expression. <b>Conclusions</b>: Overall, this computationally designed MEV demonstrates favorable immunological and physicochemical properties, and presents a durable candidate for subsequent in vitro and in vivo validation against <i>H. kunzii</i>-associated infections.