Toward a Vaccine Against Multidrug-Resistant Elizabethkingia anophelis: Comprehensive Immunoinformatics-Based Design of a Multi-Epitope Vaccine and mRNA Construct.

Elizabethkingia anophelis is a multidrug-resistant opportunistic pathogen associated with severe neonatal meningitis, sepsis, and hospital outbreaks, with high mortality rates and limited treatment options. In this study, a hybrid multi-epitope vaccine (MEV) was designed using an extensive immunoinformatics approach targeting four key outer membrane and secretion-associated proteins. Highly antigenic B-cell and T-cell (MHC-I and MHC-II) epitopes were predicted, rigorously screened for antigenicity, allergenicity, toxicity, and cytokine induction potential, and selected based on binding affinity and population coverage. The final MEV construct incorporated eight MHC-I, eight MHC-II, and eight B-cell epitopes linked with appropriate linkers, adjuvanted with Human Beta Defensin-3, and tagged with a 6 × His sequence. Population coverage analysis revealed 99.38% global coverage. Structural modeling using AlphaFold2 followed by refinement yielded a high-quality 3D model (98.0% Ramachandran favored residues, ERRAT 98.621, ProSA Z-score - 4.68). Molecular docking demonstrated strong binding of the MEV to TLR-2, while 200 ns molecular dynamics simulations confirmed the stability of the vaccine-receptor complex. Immune simulations predicted robust Th1-biased responses, high antibody production (IgG class switching), and generation of long-term memory B and T cells. Codon optimization for E. coli K12 (CAI 0.982) and in silico cloning into pET-28a( +) vector supported efficient recombinant expression, while mRNA secondary structure analysis indicated high stability. This computationally designed MEV offers a promising preventive strategy against E. anophelis infections. Further experimental validation is warranted to confirm its immunogenicity and protective efficacy.