The escalating threat of antimicrobial resistance poses significant clinical challenges, particularly in cases of co-infections involving Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA), which synergistically increase disease severity and complicate therapeutic interventions. A bivalent nanovaccine, MntC-rePO@LS, was constructed by co-displaying the SA antigen Manganese transporter C (MntC) and the PA recombinant PcrV-OprI fusion antigen (rePO) based on self-assembling lumazine synthase (LS) nanoparticles. We hypothesized that this vaccine would provide superior immunoprotection against both single and co-infections with SA and PA compared to monomeric or physically mixed formulations.

The MntC-rePO@LS bivalent nanoparticles were prepared using genetic engineering and the SpyTag/SpyCatcher system. Female BALB/c mice (6 to 8 weeks old, weighing 18 to 20 g) were randomly divided into 6 groups: PBS, LS (8.57 μg LS), rePO (9.43 μg rePO), MntC (10 μg MntC), physical mixture (8.57 μg LS, 10 μg MntC, and 9.43 μg rePO), and MntC-rePO@LS (10.86 μg rePO-ST and 18.57 μg MntC@LS) groups, with 5 animals in each group. The mice from the above groups were immunized intramuscularly with corresponding agents dissolved in PBS buffer on days 0, 7, and 14. The vaccine was characterized by SDS-PAGE, size-exclusion chromatography, dynamic light scattering, and transmission electron microscopy (TEM). In vitro and in vivo safety of the vaccine was assessed through hemolysis assay, cytotoxicity test, body weight monitoring, histopathological examination, and hematological and serum biochemical analyses. The serum samples were collected on day 7 after the final immunization, and the titers and subtypes of anti-MntC and anti-rePO IgG were evaluated by indirect ELISA to evaluate the immunogenicity of the vaccine. Protective efficacy was assessed in mouse pneumonia models of single PA, single SA, and PA/SA co-infections by monitoring body weight and infection scores post-challenge, and by quantifying lung bacterial loads and observing histopathological changes at 24 h post-challenge.

We successfully constructed homogeneous MntC-rePO@LS nanoparticles with a hydrodynamic diameter of approximately 65. 69 nm. The vaccine exhibited no significant hemolysis or cytotoxicity at concentrations ranging from 10 to 200 μg/mL. The immunized mice demonstrated normal body weight gain, with no pathological damage observed in major organs or at the injection site, and all hematological and biochemical parameters remained within normal ranges. Immunogenicity studies revealed that the MntCrePO@LS group induced significantly higher anti-MntC and anti-rePO specific IgG titers at all 3 time points tested (day 0, 7, and 14) compared to monomer and physical mixture groups (P < 0.05), with a predominant IgG2b subtype response. In the models of single PA, single SA, and co-infections, the MntC-rePO@LS group exhibited faster body weight recovery, lower infection scores, significantly reduced bacterial loads in the lungs (P < 0.05), and markedly attenuated lung histopathological damage compared to all control groups.

The successfully constructed MntC-rePO@LS bivalent nanovaccine demonstrates favorable safety and immunogenicity, providing significant immune protection against single and co-infections with SA and PA. These findings validate its potential as a candidate vaccine to address antimicrobial resistance and polymicrobial infections.