Inhalable epitope vaccine 1022-SpaA V3 establishes nasal mucosal fortress against zoonotic Streptococcus suis

Streptococcus suis serotype 2 (SS2), a zoonotic pathogen, can breach the respiratory barriers and cause systemic infections in both humans and swine. Current vaccines against SS2 are mainly formulated as an injectable product and primarily induce serum IgG antibodies. However, there is an urgent need for inhalable vaccines that induce the mucosal immune response. Leveraging immunoinformatics, we designed a multi-epitope vaccine targeting conserved epitopes from two virulence factors of SS2, SSU05-1022 (IgA1 protease) and SpaA (pilus-associated protein). Four cytotoxic T-lymphocyte (CTL), seven helper T-lymphocyte (HTL), and six B-cell epitopes were selected and fused using AAY/GPGPG/EAAAK linkers, with the L7/L12 ribosomal protein incorporated as an immunostimulatory adjuvant. Molecular docking and dynamics simulations revealed strong binding affinity between the vaccine (1022-SpaA V3 ) and Toll-like receptor 4 (TLR4),and further cellular experiments confirmed that TLR4 mediated the pro-inflammatory cytokine expression induced by the candidate vaccine in macrophages. The recombinant vaccine was subsequently successfully expressed in vitro with high solubility. Intranasal delivery of 60 µg vaccine robustly elevated respiratory mucosal sIgA and IgG in serum, enhanced the proportion of B and CD4⁺T cells in nasal-associated lymphoid tissue (NALT), lungs and spleens, and activated the CD80⁺ dendritic cell/macrophage in NALT and lungs. Challenge experiments in mice indicated that the inhalable vaccine provided significant, albeit strain-dependent, protection against selected SS2 clinical isolates, including the virulent strain SC19, 05ZYH33, CVCC606, and JZLQ022. Thus, these data suggest that the 1022-SpaA V3 vaccine, optimized through epitope prioritization, adjuvant synergy, and mucosal delivery, represents a promising candidate for combating SS2 infections via dual mucosal-systemic immunity.