Intranasal immunization with a P1 adhesin C-terminal-flagellin fusion protein elicits robust systemic and mucosal immunity and protects against Mycoplasma pneumoniae infection in a mouse model.

Mycoplasma pneumoniae (M. pneumoniae) is a major cause of community-acquired pneumonia. Rising macrolide resistance and limited preventive strategies highlight the urgent need for effective vaccines. A vaccine eliciting robust mucosal immunity is essential to block bacterial colonization and transmission. Here, we generated a recombinant fusion protein, KFD-P1C, by genetically fusing the immunodominant C-terminal region of the P1 adhesin (P1C) to KFD, a truncated non-pathogenic Escherichia coli K12 flagellin (KF) retaining TLR5 agonistic activity while exhibiting reduced intrinsic antigenicity. BALB/c mice were intranasally immunized three times at 3-week intervals with P1C alone, a P1C + KFD admixture, or the KFD-P1C fusion protein. Both KFD-P1C and P1C + KFD elicited significantly stronger systemic and mucosal immune responses than P1C alone and significantly reduced pulmonary inflammation following M. pneumoniae challenge. Although no statistically significant differences were observed between the KFD-P1C and P1C + KFD groups, the fusion protein consistently showed a trend toward higher antibody responses, stronger antigen-specific T cell responses, faster bacterial clearance, and lower histopathological scores. Notably, KFD-P1C induced humoral and antigen-specific T cell responses that persisted for at least nine months after immunization. Collectively, these findings demonstrate that KFD-P1C elicits robust, long-lasting systemic and mucosal immune responses and provides protection against M. pneumoniae comparable to that achieved by the antigen-adjuvant admixture, while offering the practical advantages of a single-component vaccine design.