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

Abstract

Mycoplasma pneumoniae (Mp) remains a major cause of community-acquired pneumonia in children and adolescents, and no vaccine against this pathogen is currently available. This study designed and evaluated P1Tx, a novel multi-epitope vaccine candidate derived from the immunodominant regions of the P1 adhesin and the community-acquired respiratory distress syndrome (CARDS) toxin. Immunoinformatics approaches were used to select immunodominant epitopes from the P1 adhesin and the CARDS toxin of Mp , which were assembled into a 263-amino acid chimeric construct designated P1Tx. The construct was expressed in Escherichia coli , and the recombinant protein was characterized for physicochemical properties and predicted structural stability. Its immunoreactivity was assessed using sera from Mp -infected patients. BALB/c mice received three subcutaneous immunizations with P1Tx, and humoral and cellular immune responses were evaluated by antibody endpoint titers and flow cytometric analysis of germinal center B cells and T cell subsets. The functional activity of immune sera was tested by measuring its capacity to block Mp adhesion to human bronchial epithelial cells and to neutralize CARDS toxin-induced cytotoxicity. Protective efficacy was assessed in mice challenged intranasally with the Mp M129 strain, with pulmonary bacterial burden and histopathological changes determined after challenge. P1Tx displayed favorable physicochemical characteristics and structural stability and reacted strongly with sera from Mp -infected patients. Immunized mice developed high-titer, IgG1-dominant antibody responses (endpoint titers up to 1:50,000) accompanied by marked germinal center B cell expansion. Compared with the P1C subunit vaccine, P1Tx elicited substantially stronger cellular immunity, with significant expansion of CD4+ and CD8+ effector and memory T cell populations. Sera from P1Tx-immunized mice inhibited Mp adhesion to bronchial epithelial cells and neutralized CARDS toxin-mediated cytotoxicity in vitro. Following intranasal challenge, P1Tx-immunized mice showed a 98.5% reduction in pulmonary bacterial burden and a 52.4% reduction in lung histopathological scores relative to the PBS group. P1Tx confers protection against Mp infection through complementary mechanisms of adhesion blockade and toxin neutralization, supporting its further development as a vaccine candidate for the prevention of Mp infection.