Immunoinformatics-Guided Design, Immunological Evaluation, and Macrophage Transcriptomic Profiling of a Multi-Epitope...

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease in ruminants and is difficult to control once persistent infection is established. We designed a MAP multi-epitope vaccine candidate by screening conserved proteins and assembling predicted B-cell, MHC class I-restricted cytotoxic T lymphocyte (CTL), and MHC class II-restricted helper T lymphocyte (HTL) epitopes into a recombinant construct. The construct was examined by physicochemical analysis, structural modeling, molecular docking, and immune simulation, then expressed in Escherichia coli and tested in BALB/c mice as a recombinant protein without external adjuvant or as an exploratory MYY-containing formulation, alongside PBS and inactivated-MAP controls. RAW264.7 macrophages were stimulated with the purified recombinant protein for RNA sequencing. Seven proteins were retained as antigen sources, and the final construct contained seven HTL epitopes, five B-cell epitopes, and ten CTL epitopes. Immunized mice produced antigen-specific IgG, IgG1, and IgG2a, and antigen-restimulated splenocytes secreted higher levels of IFN-γ, IL-2, TNF-α, and IL-17. Vaccine-induced antibodies recognized both the recombinant protein and components present in MAP lysate while showing limited reactivity toward the bovine tuberculosis diagnostic antigen PPD, and sequence analysis predicted no substantial similarity between the construct and the diagnostic antigens ESAT-6, CFP-10, MPB70, and MPB83, supporting potential DIVA compatibility. In macrophages, recombinant-protein stimulation changed gene-expression profiles and enriched pathways related to cytokine-cytokine receptor interaction, NF-κB, TNF, IL-17, Toll-like receptor, and NOD-like receptor signaling. The construct was immunogenic in mice; the exploratory MYY-containing formulation showed numerically higher immune-response readouts in this experimental setting, although these differences were not statistically significant. This adjuvant-associated observation requires further validation, and protection against MAP infection still needs to be tested in challenge experiments and target animals.