Klebsiella pneumoniae (Kp) is a clinically prevalent opportunistic pathogen, with escalating antimicrobial resistance posing substantial challenges. Current vaccine strategies are constrained by the complexity of serotype diversity and the weak immunogenicity of candidate antigens, hindering effective protection. The construction of a multiepitope vaccine against K. pneumoniae, designated HBc-KpEpi, based on hepatitis B core antigen (HBc) virus-like particles, aims to overcome the weak immunogenicity of epitope peptides and to validate its ability to induce specific immune responses and protective efficacy.

After the multi-epitope vaccine HBc-KpEpi was expressed and purified, its purity and self-assembly status were characterized by SDS-PAGE, gel filtration chromatography, and transmission electron microscopy. Its safety was evaluated using cytotoxicity assay, hemolysis assay, and detection of serum biochemical indicators. Female BALB/c mice (6 to 8 weeks old, weighing 18 to 20 g) were randomly divided into the following groups: PBS control group, KpEpi group, and HBc-KpEpi group(n=10). Each group received intranasal immunization with PBS, KpEpi, or HBc-KpEpi (30 μg/mouse) on days 0, 14, and 21, respectively. In 7 d after the final immunization, the mice were challenged with a lethal dose (6×10⁶ CFU) or a sublethal dose (5×10⁵ CFU) of the clinical strain YBQ via intratracheal instillation. Detection indicators included survival rate and changes in body weight, bacterial colonization in lung tissue, serum inflammatory cytokines (IL-6, TNF-α, and IL-1β), lung histopathological changes, titers of serum specific antibodies (IgG, IgG1, IgG2a), and proportions of splenic CD4⁺ T cell subsets (Th1, Th2, Th17). Meanwhile, the ability of the vaccine to induce the differentiation and maturation of mouse bone marrowderived dendritic cells (BMDCs) was evaluated through in vitro experiments.

The HBc-KpEpi protein was successfully prepared with high purity and self-assembled into regular particles. The survival rates in the HBc-KpEpi group were significantly higher than those in the PBS group (P < 0.05). The bacterial colonization in lung tissue was significantly lower in the HBc-KpEpi group than that of the PBS and KpEpi groups (P < 0.01). The HBc-KpEpi group exhibited significantly lower IL-6 level than the PBS group (P < 0.0001), with the levels of TNF-α and IL-1β showing downward trends. Lung histopathology showed that the alveolar structure remained relatively intact with markedly reduced inflammatory cell infiltration in the HBc-KpEpi group. After immunization, high titers of specific IgG antibodies were induced in the HBc-KpEpi group, with IgG1 as predominant subtype. Flow cytometry detection showed that the proportions of Th2 and Th17 cells in CD4⁺ T cells were significantly higher in the HBc-KpEpi group than in the KpEpi group (P < 0.05). In vitro experiments indicated that HBc-KpEpi significantly promoted the expression of MHC-Ⅱ, CD40, CD80, and CD86 on the surface of BMDCs (P < 0.0001)

The HBc-KpEpi vaccine significantly enhances the immunogenicity of epitope peptides, inducing cellular immune responses dominated by Th2 and Th17 as well as high titers of specific antibodies, and effectively reduce bacterial load, alleviate inflammatory damage, and improve the survival rate of mice.

To construct an engineered milk-derived exosome (mExos) vector for efficient messenger RNA (mRNA) delivery to the respiratory tract by systematically screening cationic modification materials.

mExos were isolated via ultracentrifugation, followed by electroporation-mediated loading of firefly luciferase (Fluc) or enhanced green fluorescent protein (EGFP) mRNA. Chitosan (CS), polyethyleneimine (PEI), poly (beta-amino ester) (PBAE), cationic lipid (DOTAP), and protamine were used to modify mRNA-loaded mExos. Dynamic light scattering, nanoparticle tracking analysis, flow cytometry and luciferase reporter assay were employed to evaluate particle size, zeta potential, colloidal stability, cellular uptake, transfection efficiency, and cytotoxicity. Fifty female BALB/c mice (6 to 8 weeks, about 20 g) received intranasal administration of dye-labeled or Fluc-mRNA-loaded formulations. In vivo distribution and pulmonary transfection kinetics were monitored by IVIS imaging, while histopathology and serum biochemistry of major organs were analyzed at 24 h post-administration.

Cationic-modified mExos were successfully constructed. CS-modified mExos (CS-mExos) demonstrated surface charge reversal while maintaining optimal size and stability (PDI unchanged within 48 h). In vitro, CS-mExos enhanced cellular uptake in 16HBE and A549 cells by 2.56- fold (P<0.001) and 4.56 -fold (P<0.001), respectively, and increased Fluc-mRNA transfection efficiency by 28.9-fold (P<0.001) and 26.5-fold (P<0.001) versus unmodified mExos, with >95% cell viability. In vivo imaging revealed CS-mExos specifically accumulated in the lungs with sustained retention >72 h, mediating 9.5-fold higher peak Fluc-mRNA expression at 6 h post-administration. Histopathology and serum biochemistry confirmed no significant organ damage or abnormal hepatic/renal indices.

Systematic screening identified chitosan as the optimal cationic material for engineering milk exosomes. The developed CS-mExos vector enables efficient targeted mRNA delivery to the respiratory tract with enhanced pulmonary transfection and favorable biosafety.

To establish a high-throughput screening system to obtain key Staphylococcus aureus (S. aureus) secretory proteins which required for S. aureus survival in macrophages.

Based on our validated eukaryotic expression vector library of S. aureus secretory proteins, DNA transfection was used to obtain an RAW264.7 macrophage array expressing S. aureus secretory proteins. After the RAW264.7 cells were infected with S. aureus, the extracellular bacteria were removed to observe the intracellular surviving situation of S. aureus. Finally, the screening results were validated by the overexpression and knockout S. aureus of corresponding secretory proteins.

The optimal transfection dose (1.0 μg/well) of plasmids for RAW264.7, multiplicity of infection (MOI, 1.0), and infection time (4 h after removing extracellular bacteria of S. aureus) were established respectively. To validate the screening results, the corresponding overexpression and knockout strains were constructed. And hypothetical protein and Serine protease E were found to promote the survival of intracellular S. aureus.

We successfully construct a screening system for key secreted secretory proteins which required for S. aureus surviving in macrophages, which may advance the study of the intracellular surviving mechanism of S. aureus.

To design and prepare mRNA-peptide-poloxamine ternary complex（mRNA-PPTC）nanoparticles and explore their physicochemical properties, in vitro/vivo delivery efficiency, and induction of specific antibody production in immunized mice.

The mRNAs coding firefly luciferase（F-luc）, enhanced green fluorescent protein（EGFP）and SARS-CoV-2 receptor binding domain（RBD）were prepared through in vitro transcription. The size, polydispersity（PDI）and Zeta potential of the mRNA-PPTC nanoparticles were studied with dynamic light scattering, and their morphology was observed with transmission electron microscopy（TEM）. After F-luc reporter gene was loaded in the mRNA-PPTC nanoparticles, the obtained F-luc mRNA-PPTC nanoparticles were used to transfect human bronchial epithelial cell line 16HBE, mouse dendritic cell line DC2.4 and mouse macrophage cell line RAW264.7. The transfection efficiency and cell viability were observed with luminescence reporter assay. Fluorescence microscopy was employed to observe the expression of EGFP in EGFP mRNA-PPTC nanoparticles-transfected 16HBE cells. In vivo imaging system was adopted to investigate the delivery efficiency through the intranasal administration in mice. Then, RBD mRNA was used as antigen for vaccine preparation, the levels of specific IgG in the serum and sIgA in bronchoalveolar lavage fluid（BALF）were detected with ELISA after the mice were immunized with RBD mRNA-PPTC nanoparticles.

Our mRNA-PPTC nanoparticles were successfully assembled, as weak cationic nanoparticles in sphere shape, in a diameter of about 100 nm, and with low PDI. The obtained mRNA-PPTC could effectively transfect 16HBE, DC2.4 and RAW264.7 cells（vs controls, P<0.01）with low cytotoxicity. The mRNA-PPTC nanoparticles effectively expressed the reporter gene F-luc in the nose and lung of mice immunized though an intranasal route（vs controls, P<0.01）. High titers of antigen-specific IgG and sIgA were found in the serum and BALF from the mice in 28 d after intranasal immunization with RBD mRNA-PPTC（vs controls, P<0.01, P<0.05）.

Our prepared mRNA-PPTC have good capacity to delivery mRNA in vitro without significant cytotoxicity and in vivo at respiratory mucosal site, and can induce high levels of specific serum IgG and BALF sIgA by intranasal inoculation.