Journal: Frontiers in Immunology

Article Title: Lactobacillus casei bacterial ghost-based DNA vaccine enhances the immune protection effect by stimulating macrophages to regulate intestinal mucosal immunity

doi: 10.3389/fimmu.2026.1839510

Figure Lengend Snippet: Characteristics of the L. casei bacteria ghosts carrying plasmids. (A) Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analyses were conducted on L. casei 393 following induction. pPG-2/ L. casei group is control, pPG-2-hocb/ L. casei group prepares bacterial ghosts by inducing lytic gene expression. (B, C) EGFP-VP7 and VP7 protein production were analyzed by western blotting, which resolved EGFP-VP7 as an immunoreactive 62 kD (B) band and VP7 as 37kDa (C) band. M, marker; 1, 293 cells were transfected with pCI-PoRV-VP7; 2, 293 cells were transfected with pCI-neo; 3, Untreated 293 cells. (D, E) RT-qPCR detected EGFP-VP7 (D) and VP7 (E) DNA content to determine the loading capacity and loading stability of L. casei BGs. (F) Intestinal macrophages were isolated and analyzed by flow cytometry for the expression of surface markers CD45 and CD64. Left: CD45; Right: CD64. (G) Peritoneal macrophages were isolated from mice and analyzed by flow cytometry for the expression of CD11b and F4/80. Left: CD11b; Right:F4/80. (H) Proportions of CD45 + CD64 + and CD11b + F4/80 + dual-positive macrophages in the intestinal and peritoneal compartments. PBS was used as the negative control. *Data are presented as mean ± SD, with significance indicated by ** p < 0.01. Statistical significance was determined using one-way ANOVA followed by Dunnett’s post hoc test. n = 3 independent animal-derived biological samples.

Article Snippet: According to a previously described method , recombinant L. casei was cultured in MRS medium supplemented with 10 μg/mL chloramphenicol (MedChemExpress, USA) at 37 °C for 24 h , and then induced with 2% fructose for 36 h . BGs were harvested after OD600 stabilization and centrifuged at 6500 rpm (≈ 5,000 × g) for 20 min .

Techniques: Bacteria, Transmission Assay, Electron Microscopy, Control, Gene Expression, Western Blot, Marker, Transfection, Quantitative RT-PCR, Isolation, Flow Cytometry, Expressing, Negative Control, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Lactobacillus casei bacterial ghost-based DNA vaccine enhances the immune protection effect by stimulating macrophages to regulate intestinal mucosal immunity

doi: 10.3389/fimmu.2026.1839510

Figure Lengend Snippet: L. casei BGs-based DNA vaccines enhanced macrophage phagocytic function. (A) Stimulate Peritoneal macrophages with pCI-PoRV-VP7-loaded L. casei BGs, pCI-PoRV-VP7, L. casei BGs, LPS or PBS, and detect their cell viability using CCK-8 assay. (B) Mice intramuscular or orally immunized with pCI-PoRV-VP7-loaded L. casei BGs, pCI-PoRV-VP7 or L. casei BGs were tested for intestinal macrophage activity using the CCK-8 assay. Control, mice was immunized with PBS. (C) Western blot detection of His-VP7 and EGFP-VP7 protein expression levels in intestinal tissue after oral immunization with VP7-loaded L. casei BGs or VP7. M, marker; 1, Oral pCI-PoRV-VP7 group; 2, Oral pCI-PoRV-VP7-loaded L. casei BGs group; 3, Oral pCI-VP7-EGFP group; 4, Oral pCI-EGFP-PoRV-VP7-loaded L. casei BGs group. (D) Treat peritoneal macrophages with pCI-EGFP-loaded L. casei BGs or pCI-EGFP and observe the green fluorescence intensity during phagocytosis by peritoneal macrophages using a fluorescence microscope. The nuclei were stained with DAPI. (E) Detection of fluorescent protein content in mouse peritoneal macrophage phagocytosis loaded with pCI-EGFP plasmid expression by flow cytometry. (F) Fluorescence observation of intestinal macrophage phagocytosis of glucan in mice by fluorescence microscope after oral and intramuscular immune. PBS was used as the negative control. *Data are presented as mean ± SD, with significance indicated by * p < 0.05; *** p < 0.001. Statistical significance between two groups was determined using the Mann–Whitney U test. For comparisons among multiple groups, one-way ANOVA followed by Dunnett’s post hoc test was used. n = 3 independent animal-derived biological samples.

Article Snippet: According to a previously described method , recombinant L. casei was cultured in MRS medium supplemented with 10 μg/mL chloramphenicol (MedChemExpress, USA) at 37 °C for 24 h , and then induced with 2% fructose for 36 h . BGs were harvested after OD600 stabilization and centrifuged at 6500 rpm (≈ 5,000 × g) for 20 min .

Techniques: Vaccines, CCK-8 Assay, Activity Assay, Control, Western Blot, Expressing, Marker, Fluorescence, Microscopy, Staining, Plasmid Preparation, Flow Cytometry, Negative Control, MANN-WHITNEY, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Lactobacillus casei bacterial ghost-based DNA vaccine enhances the immune protection effect by stimulating macrophages to regulate intestinal mucosal immunity

doi: 10.3389/fimmu.2026.1839510

Figure Lengend Snippet: The expression of cytokine after L. casei BGs-based DNA vaccine treatment. (A) Mice peritoneal macrophages were stimulated with pCI-PoRV-VP7-loaded L. casei BGs, pCI-PoRV-VP7, L. casei BGs, LPS or PBS for 24h, 48h and 72h. IL-2, IL-1β, IL-6, IL-12 and TGF-β expression levels in the supernatant, all of them were determined by ELISA. (B) After intramuscular or oral immunization with pCI-PoRV-VP7-loaded L. casei BGs, pCI-PoRV-VP7 or L. casei BGs, RT-qPCR detection the intestinal macrophage cytokines mRNA expression levels of IL-2, IL-1 β, IL-6, IL-12, and TGF-β. Significance of differences between the PBS, pCI-PoRV-VP7-loaded L. casei BGs, pCI-PoRV-VP7, L. casei BGs, groups is represented as follows: a, p < 0.05; b, p < 0.01; c, p < 0.001; * p < 0.05; ** p < 0.01; *** p < 0.001. Statistical significance between two groups was determined using the Mann–Whitney U test. For comparisons among multiple groups, one-way ANOVA followed by Dunnett’s post hoc test was used. n = 3 independent animal-derived biological samples. PBS was used as the negative control.

Article Snippet: According to a previously described method , recombinant L. casei was cultured in MRS medium supplemented with 10 μg/mL chloramphenicol (MedChemExpress, USA) at 37 °C for 24 h , and then induced with 2% fructose for 36 h . BGs were harvested after OD600 stabilization and centrifuged at 6500 rpm (≈ 5,000 × g) for 20 min .

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, MANN-WHITNEY, Derivative Assay, Negative Control

Journal: Frontiers in Immunology

Article Title: Lactobacillus casei bacterial ghost-based DNA vaccine enhances the immune protection effect by stimulating macrophages to regulate intestinal mucosal immunity

doi: 10.3389/fimmu.2026.1839510

Figure Lengend Snippet: L. casei BGs-based DNA vaccines regulates macrophage polarization through TLR/ MyD88/NF-κB Pathway. (A, B) The relative mRNA expression of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 in peritoneal macrophages stimulated in vitro (A) , as well as in the jejunum of mice immunized by oral or intramuscular injection (B) , detected using RT-qPCR. (C, D) The contents of TLR2, TLR6, TLR8, and TLR9 in peritoneal macrophages stimulated in vitro were determined by Western blot analysis. (E–H) The protein expression of MyD88 and NF-κB in peritoneal macrophages stimulated in vitro (E) , as well as in the jejunum of mice immunized by oral (G) or intramuscular injection (G) , detected using western blot. (I) The relative mRNA expression of MyD88 and NF-κB in the jejunum of mice immunized by oral or intramuscular injection, detected using RT-qPCR. (J, K) The relative mRNA expression of NOS2, TNF-α, IL-10, and Arg-1 in peritoneal macrophages stimulated in vitro (J) , as well as in the jejunum of mice immunized by oral or intramuscular injection (K) , was detected using RT-qPCR, with M1-polarization control cells induced by LPS (50 ng/mL) and M2-polarization control cells induced by IL-4 (40 ng/mL). PBS was used as the negative control. Data are presented as mean ± SD from three independent biological replicates, with significance indicated by * p < 0.05; ** p < 0.01; *** p < 0.001. Statistical significance between two groups was determined using the Mann–Whitney U test. For comparisons among multiple groups, one-way ANOVA followed by Dunnett’s post hoc test was used. n = 3 independent animal-derived biological samples.

Article Snippet: According to a previously described method , recombinant L. casei was cultured in MRS medium supplemented with 10 μg/mL chloramphenicol (MedChemExpress, USA) at 37 °C for 24 h , and then induced with 2% fructose for 36 h . BGs were harvested after OD600 stabilization and centrifuged at 6500 rpm (≈ 5,000 × g) for 20 min .

Techniques: Vaccines, Expressing, In Vitro, Injection, Quantitative RT-PCR, Western Blot, Control, Negative Control, MANN-WHITNEY, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Lactobacillus casei bacterial ghost-based DNA vaccine enhances the immune protection effect by stimulating macrophages to regulate intestinal mucosal immunity

doi: 10.3389/fimmu.2026.1839510

Figure Lengend Snippet: L. casei BGs-based DNA vaccines enhances antigen presentation ability of intestinal macrophages. (A, B) The expression of CD80, CD86, MHC-I and MHC-II in the Intestinal macrophages of mice immunized through oral (A) or intramuscular (B) routes were also determined using flow cytometry. (C) VP7 mRNA expression in the liver, spleen, small intestine and colon tissues of mice immunized through oral or intramuscular routes were analyzed using RT-qPCR. (D) Intestinal tissues were harvested from mice 7 days after oral immunization with naked pCI-EGFP plasmid or bacteria ghost–loaded pCI-EGFP and processed into frozen sections. EGFP expression was visualized by fluorescence microscopy. Scale bar: 100μm. PBS was used as the negative control. Data are presented as mean ± SD, with significance indicated by * p < 0.05; ** p < 0.01; *** p < 0.001.Statistical significance between two groups was determined using the Mann–Whitney U test. For comparisons among multiple groups, one-way ANOVA followed by Dunnett’s post hoc test was used. n = 3 independent animal-derived biological samples.

Article Snippet: According to a previously described method , recombinant L. casei was cultured in MRS medium supplemented with 10 μg/mL chloramphenicol (MedChemExpress, USA) at 37 °C for 24 h , and then induced with 2% fructose for 36 h . BGs were harvested after OD600 stabilization and centrifuged at 6500 rpm (≈ 5,000 × g) for 20 min .

Techniques: Vaccines, Immunopeptidomics, Expressing, Flow Cytometry, Quantitative RT-PCR, Plasmid Preparation, Bacteria, Fluorescence, Microscopy, Negative Control, MANN-WHITNEY, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Lactobacillus casei bacterial ghost-based DNA vaccine enhances the immune protection effect by stimulating macrophages to regulate intestinal mucosal immunity

doi: 10.3389/fimmu.2026.1839510

Figure Lengend Snippet: Effect of L. casei BGs-based DNA vaccines treatment on lymphocyte proliferation. (A, B) CCK-8 method was used to detect lymphocyte proliferation index Lymphoproliferation assay. The effects of different in vitro treatment groups on the proliferation of lymphocytes after co-culture with peritoneal macrophages and spleen cells (A) . Lymphocytes from the mice immunized by the intramuscular (B) , or oral (B) route were prepared at the endpoint and cultured with VP7 protein of final concentration of 0.5 or 5 mg/mL. (C) After mixing macrophages stimulated in vitro with splenic lymphocytes, the CD4 + and CD8 + T lymphocyte populations of mice were analyzed using flow cytometry, and the CD4 + /CD8 + ratio was calculated. (D) After intramuscular and oral immunization of mice, the CD4 + /CD8 + ratio in splenic lymphocytes was detected by flow cytometry. (E) ELISA method was used to detect the secretion levels of cytokines IFN-γ, IL-2, IL-17, IL-6 and IL-22 in lymphocyte mixing experiments. PBS was used as the negative control. Data are presented as mean ± SD from three independent biological replicates, with significance indicated by * p < 0.05; ** p < 0.01; *** p < 0.001.Statistical significance between two groups was determined using the Mann–Whitney U test. For comparisons among multiple groups, one-way ANOVA followed by Dunnett’s post hoc test was used. n = 3 independent animal-derived biological samples.

Article Snippet: According to a previously described method , recombinant L. casei was cultured in MRS medium supplemented with 10 μg/mL chloramphenicol (MedChemExpress, USA) at 37 °C for 24 h , and then induced with 2% fructose for 36 h . BGs were harvested after OD600 stabilization and centrifuged at 6500 rpm (≈ 5,000 × g) for 20 min .

Techniques: Vaccines, CCK-8 Assay, In Vitro, Co-Culture Assay, Cell Culture, Concentration Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Negative Control, MANN-WHITNEY, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Lactobacillus casei bacterial ghost-based DNA vaccine enhances the immune protection effect by stimulating macrophages to regulate intestinal mucosal immunity

doi: 10.3389/fimmu.2026.1839510

Figure Lengend Snippet: Effect of L. casei BGs as a DNA vaccine packaging carrier on immune efficacy. (A) The levels of specific antibodies IgG and IgA in the serum were detected by ELISA at 0, 7, 14, 21, 28, 35, and 42 days in intramuscular and oral immunization. (B) Expression of IgA in the large and small intestine of mice after intramuscular or oral immune. (C) Intestinal morphology of jejunum shown by H&E staining and scanning electron microscope; red arrows indicate sites of intestinal villus damage. (D) Analysis of villus length, crypt depth, and the villus length-to-crypt depth ratio. (E) The TCID50 titers in serum were measured by virus neutralization test at 7 days post-infection in mice immunized via intramuscular or oral route. (F) The viral load (LOG10 copies) in jejunum tissues was measured by qPCR at 7 days post-infection in mice immunized with via intramuscular or oral route. (G) Expression of IgA in the jejunum tissues of mice after intramuscular injection or oral immunization. PBS was used as the negative control. Data are presented as mean ± SD from three independent biological replicates, with significance indicated by a, p < 0.05; b, p < 0.01; c, p < 0.001; * p < 0.05; ** p < 0.01; *** p < 0.001.Statistical significance between two groups was determined using the Mann–Whitney U test. For comparisons among multiple groups, one-way ANOVA followed by Dunnett’s post hoc test was used. n = 3 independent animal-derived biological samples.

Article Snippet: According to a previously described method , recombinant L. casei was cultured in MRS medium supplemented with 10 μg/mL chloramphenicol (MedChemExpress, USA) at 37 °C for 24 h , and then induced with 2% fructose for 36 h . BGs were harvested after OD600 stabilization and centrifuged at 6500 rpm (≈ 5,000 × g) for 20 min .

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Staining, Microscopy, Virus, Neutralization, Infection, Injection, Negative Control, MANN-WHITNEY, Derivative Assay