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.

To construct SpyCatcher-mi3 nanoparticle vaccine delivery vectors, evaluate their role in enhancing the immunogenicity of the ovalbumin CD8⁺ T-cell epitope peptide, OVA_257-264, and determine its protective effect in a model which mice were immunized and subcutaneously challenged with E. G7-OVA tumor cells.

SpyCatcher-mi3 proteins were expressed by E.coli and purified by affinity chromatography and anion exchange chromatography sequentially. OVA_257-264-SpyTag peptide was obtained by synthesis. The OVA_257-264-mi3 nanoparticles were produced by the SpyTag/SpyCatcher system. The toxicity of OVA_257-264-mi3 was evaluated using hemolysis assay, CCK-8 assay and mouse experiment. A total of 42 female SPF-grade C57BL/6 mice (6~8 weeks old, 18~20 g) were randomly divided into OVA_257-264-mi3, OVA_257-264, and control groups, with 14 mice in each group. Then the mice in each group were immunized on days 0, 14 and 28. In 14 d after the last immunization, the amounts of spot-forming cells (SFCs, indicating IFN-γ secreting cells in splenic lymphocytes) were determined using ELISpot assay to evaluate their immunogenicity. After the immunized mice were subcutaneously implanted with E.G7-OVA tumor cells, the antitumor effect of the vaccine inprophylactic xenograft tumor model was evaluate by observing tumor volumes with a caliper and tumor growth with MRI.

Both SpyCatcher-mi3 and OVA_257-264-mi3 could be self-assembled to form homogeneous and stable nanoparticles, with an average particle size of about 43.8 and 91.3 nm, respectively. The OVA_257-264-mi3 was safe for in vitro and in vivo toxicity evaluation. The number of IFN-γ secreting cells per 1×10⁶ splenic lymphocytes reached 253 in the OVA_257-264-mi3 group of mice, significantly higher than that in the OVA_257-264 group and the Control group (P<0.05). The tumor volume of mice in the OVA_257-264-mi3 group was about 151.1 mm³ on day 22, which was significantly smaller than that of the OVA_257-264 group and the Control group (P<0.05), and the survival rate during the observation period reached 60%, which was significantly higher than that of the OVA_257-264 groups (P<0.05).

Nanoparticle vaccine OVA_257-264-mi3 is successfully constructed, and it shows enhancing effect on the immunogenicity of the antigen epitope peptide, and exerts protective effect on prophylactic xenograft tumor model, providing a theoretical basis for the research of tumor neoantigen vaccines.

To establish a model of Pseudomonas aeruginosa（PA）infection in mice with open fractures after the femur with intramedullary nailing and to explore the immunoprotective effects of recombinant PcrV-OprI.

PcrV-OprI protein was expressed in E. coli, and then the target protein was purified by affinity chromatography and hydrophobic interaction chromatography. The C57BL/6J mice were immunized with PcrV-OprI protein on Day 0, 14 and 21. After that, the titers of specific IgGs and their subtypes were determined by ELISA. In 14 d after the final vaccination, open femur fractures and intramedullary nailing were performed in mice, which was followed by a challenge with PAO1 strain. The protection of PcrV-OprI was assessed with bacterial colonization, body weight, histology, and X-ray imaging.

The levels of specific antibodies were significantly higher in PcrV-OprI-immunized sera than that of the control group（P<0.05）, and the obtained IgG1 was the predominating subtype. The loss of body weight of the immunized mice was significantly lower than that of the control group（P<0.05）. In addition, a similiar trend was observed in the amount of colonized bacteria in the tissues, bones, and implants（P<0.05）. Moreover, the histological observation suggested that bacteria caused less damage in the PcrV-OprI-immunized mice. At the end of the observation period, the fracture line in immunized mice was clear, indicating gradual healing.

Vaccination of PcrV-OprI is efficient in causing a humoral immune response and shows significant protective effect on PA infection model in mice after open fractures of the femur with intramedullary nailing.