Designing a Multi-Epitope Vaccine Candidate Against Rhodococcus equi Based on the Bioinformatics Technique.

Rhodococcus equi (R. equi) primarily induces fatal pulmonary and extrapulmonary pyogenic granulomatous infections in foals, imposing substantial economic burdens on the equine industry. The emergence and spread of multidrug-resistant (MDR) R. equi have led to a therapeutic impasse in clinical settings. Although vaccination is a proven strategy against MDR pathogens, no commercial vaccine is currently available for R. equi. In this study, we employed a bioinformatics approach to systematically identify and prioritize antigenic epitopes derived from R. equi for multi-epitope vaccine design. Using ABCPred, NetMHCpan EL, and IEDB servers, 27 MHC-I and 9 MHC-II epitopes were selected from five previously validated R. equi vaccine candidates: ABC transporter, PBD2, NlpC/P60, Esterase, and M23. These epitopes were coupled with distinct peptide linkers to construct six multi-epitope vaccine constructs, designated V1-V6. The physicochemical properties, antigenicity, immunogenicity, and toxicity of the six vaccine constructs were analyzed, and the V3 and V4 constructs were ultimately selected. Using the HDOCK and Gromacs tools, the intermolecular interactions, binding affinity, and thermal stability of the V3 and V4 constructs with the equine MHC molecules EQCA-I and EQCA-II were evaluated. The results confirm that V3 and V4 exhibit strong binding affinity to EQCA-I and EQCA-II, with stable conformations following binding, indicating theoretical potential to induce humoral and cellular immunity in foals. Recombinant plasmids for V3 and V4 were constructed, and the V3 and V4 proteins were successfully prepared, confirming the feasibility of prokaryotic expression for these vaccine constructs. Immunization assays in SPF BALB/c mice showed that the multi-epitope vaccines elicited robust antigen-specific IgG antibody responses, reflecting preliminary humoral immunogenicity. However, these murine data have translational limitations, as they cannot fully represent equine immune responses. The findings establish a crucial theoretical foundation for the advancement of vaccines targeting R. equi while offering a reference for the design of vaccines against other drug-resistant microbial pathogens.