In Silico Development of a Chimeric Multi-Epitope Vaccine Targeting Helcococcus kunzii: Coupling Subtractive...

Background: Helcococcus kunzii, 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 H. kunzii, and its rising resistance to conventional antibiotics presents a growing public health concern. Objectives: In this study, we employed an integrated subtractive proteomics and immunoinformatics pipeline to design a multi-epitope subunit vaccine (MEV) candidate against H. kunzii. Initially, pan-proteome analysis identified non-redundant, essential, non-homologous, and virulent proteins suitable for therapeutic targeting. Methods/Results: 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 E. coli K12 host employing the pET-28a(+) vector suggested high translational efficiency and suitability for bacterial expression. Conclusions: 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 H. kunzii-associated infections.