Evaluation of the cross-immunogenicity and protective efficacy of the multi-epitope antigen Tzi from Glasserella parasuis in mouse model.

Glässer's disease is an important swine respiratory infectious disease caused by Glaesserella parasuis (G. parasuis), which leads to considerable economic losses in the swine industry. At present, the primary method for preventing Glässer's disease is the use of inactivated vaccines. However, inactivated vaccines provide limited protection against infections caused by heterologous serotypes of G. parasuis, thereby limiting their overall efficacy. To develop a G. parasuis vaccine with enhanced cross-protective efficacy, this study utilized bioinformatics approaches to predict and analyze B-cell (B lymphocyte), cytotoxic T lymphocyte (CTL), and helper T lymphocyte (Th) epitopes of G. parasuis antigenic proteins, including The Pili Subunit A (PilA), Iron donor protein CyaY protein, ABC transporter substrate-binding protein A (HbpA), and Cytolethal distending toxin subunit B (CtdB). These epitopes were connected via linkers and cleavage sites, and an intrastrand adjuvant was added to construct the multi-epitope antigen Tzi. The results of molecular docking and immune system simulation indicated that the multi-epitope protein Tzi has the potential to activate immune responses. Western blotting results showed that Tzi could interact with mouse antisera against 6 serotypes (4, 5, 10, 12, 14 and non-typeable) of G. parasuis. In vitro antibacterial assays demonstrated that mouse antisera against Tzi exhibited bactericidal activity against six serotypes of G. parasuis. Serum levels of antibodies, Interleukin-4 (IL-4), and Interferon gamma (IFN-γ) were significantly higher in Tzi-immunized mice than in the adjuvant group. Similarly, the concentrations of Interleukin-2 (IL-2), IL-4, Interleukin-12 subunit alpha (IL-12A), and IFN-γ were markedly higher in both splenic and pulmonary tissues compared to the adjuvant group. Mice immunized with Tzi were challenged with serotypes 4, 5, and 12 of G. parasuis, and the survival rates were 50 %, 62.5 %, and 87.5 %, respectively. Moreover, compared with the adjuvant group, the clinical symptoms and lung tissue lesions in Tzi-immunized mice were significantly alleviated. Overall, the multi-epitope protein Tzi exhibited cross-immunogenicity, effectively induced both humoral and cellular immune responses in mice, and offered robust cross-protection against G. parasuis serotypes 4, 5, and 12, thereby indicating its potential as a promising candidate for a subunit vaccine against G. parasuis.