A multi-epitope vaccine targeting Mycoplasma pneumoniae P1 adhesin and CARDS toxin confers synergistic protection by adhesion inhibition and toxin neutralization

Mycoplasma pneumoniae (Mp) is a leading cause of community-acquired pneumonia in children and adolescents, yet no commercial vaccine is currently available. This study developed P1Tx, a novel multi-epitope vaccine (MEV) targeting immunodominant epitopes from the P1 adhesin and community-acquired respiratory distress syndrome (CARDS) toxin of Mp. The 263-amino acid chimeric construct was rationally designed using immunoinformatics approaches and expressed in Escherichia coli. P1Tx exhibited favorable physicochemical properties, structural stability, and strong immunoreactivity with sera from Mp-infected patients. In BALB/c mice, three subcutaneous immunizations with P1Tx elicited robust humoral responses, characterized by high-titer IgG1-dominant antibodies, germinal center B cell expansion, and long-lived central memory T cell formation. Functionally, P1Tx immune sera blocked Mp adhesion to human bronchial epithelial cells and neutralized CARDS toxin-induced cytotoxicity. Following intranasal challenge with the Mp M129 strain, P1Tx-immunized mice demonstrated significantly reduced bacterial lung loads and attenuated pulmonary inflammation. Notably, P1Tx induced superior cellular immune activation compared to the P1C subunit vaccine alone, particularly in expanding CD4+ and CD8+ effector and memory T cell populations. These findings demonstrate that P1Tx confers synergistic protection through dual mechanisms of adhesion inhibition and toxin neutralization, supporting its development as a promising vaccine candidate against Mp infection.