Intratracheal Delivery of a CTB-Fused MPN162 Subunit Vaccine Enhances Antibody Responses and Protects Mice Against Mycoplasma pneumoniae

Background: Mycoplasma pneumoniae is a major cause of respiratory infections in children and adolescents. Increasing macrolide resistance and the absence of a commercially available vaccine highlight the need for effective preventive strategies. Our preliminary antigen-screening work identified MPN162 as a potential vaccine antigen, although its immunogenicity alone was limited. This study evaluated whether fusion with cholera toxin B subunit (CTB) and intratracheal delivery could improve MPN162-induced protection. Methods: A recombinant CTB-MPN162 fusion protein, designated CTB-162, was expressed in Escherichia coli. BALB/c mice were immunized with MPN162 or CTB-162 by intratracheal or subcutaneous administration. Antigen-specific serum IgG and IgA responses were measured by ELISA. Following M. pneumoniae challenge, pulmonary bacterial DNA burden was quantified by digital PCR, and lung inflammatory cytokines were measured. The short-term safety of a single high dose of CTB-162 was assessed using body weight, serum biochemical and inflammatory markers, and histopathology. Results: Intratracheal administration induced significantly higher anti-MPN162 IgG and IgA responses than subcutaneous injection and resulted in a lower pulmonary bacterial DNA burden after challenge. CTB-162 elicited stronger IgG responses than MPN162 alone, while the 5 μg dose induced the highest IgA response among the tested groups. Following challenge, the 5 μg CTB-162 dose reduced the pulmonary M. pneumoniae DNA burden by more than three orders of magnitude compared with the PBS group and significantly decreased pulmonary IL-6, IL-1β, and TNF-α levels. The 1 μg dose also provided significant protection. A single 15 μg dose caused no detectable short-term systemic toxicity. Conclusions: CTB fusion and intratracheal delivery enhanced the immunogenicity and protective efficacy of MPN162 in mice. CTB-162 is therefore a promising mucosal subunit vaccine candidate for further preclinical development.