Proteomics- and immunoinformatics-based design of a multi-epitope vaccine ZZ1 against Glaesserella parasuis

BACKGROUND: Glaesserella parasuis (G. parasuis) is a prevalent opportunistic pathogen of the porcine upper respiratory tract, causing substantial economic losses to the global swine industry. Current commercial vaccines exhibit suboptimal heterologous cross-protection and inherent biosafety concerns, highlighting the need for a safe, broadly effective multi-serotype vaccine. RESULTS: This study targeted outer membrane proteins (OMPs) with robust cross-serotype immunogenicity from virulent G. parasuis strains. Comparative proteomic analysis of five strains identified highly abundant, co-expressed OMPs, which were screened based on antigenicity and physicochemical properties. Epitope prediction for helper T lymphocytes (HTL), cytotoxic T lymphocytes (CTL), and B-cells was performed on selected OMPs. Immunodominant epitopes were concatenated using flexible linkers and fused with the TLR2 agonist phenol-soluble modulin α4 (PSMα4) to engineer a multi-epitope vaccine, designated ZZ1. Screening yielded eight conserved OMPs with high antigenicity, hydrophilicity, and thermostability, from which 6 HTL, 7 CTL, and 11 B-cell epitopes were predicted. Immunoinformatic evaluations revealed that ZZ1 possesses a high antigenicity score of 1.018 (threshold: 0.4) and is non-allergenic and non-toxic. Following successful expression, in vivo trials demonstrated that recombinant ZZ1 elicited robust, specific IgG antibody responses. Challenge tests in piglets revealed 100% protective efficacy against G. parasuis serotype 4, and 80% against serotypes 5 and 13. CONCLUSIONS: These findings indicate that ZZ1 is a promising candidate capable of conferring broad cross-protection against multiple prevalent G. parasuis serotypes, offering an innovative strategy for next-generation subunit vaccine development.