The Effect Of Ghosts From Lactobacillus Casei As Delivery Carrier For DNA Vaccines On Immune Response

Compared with traditional vaccines,DNA vaccines have obvious advantages.Because DNA is a biological macromolecule,and it is difficult to be directly taken in by cells.And nucleic acids are easily degraded.Therefore,the"naked"DNA vaccines are difficult to be taken up,expressed and presented by antigen presenting cells.It results in a lower level of immune response,which is restricted its use and promotion.Bacterial ghosts(BGs)are the cell envelopes after lysing bacterial cell walls and removing cytoplasmic contents.They have complete natural surface antigens and can be used not only as a vaccine but also as a good carrier.In this study,we used Lactobacillus casei bacteria ghosts as a packaging carrier to analyze its effect on DNA vaccines immunity.Firstly,the plasmid pCI-EGFP and ghosts loading with plasmid pCI-EGFP transfected RAW264.7 cells and peritoneal macrophages respectively.Untreated macrophages were used as a negative control.The results were observed by fluorescence microscopy and showed that only weak fluorescence was observed after transfection of RAW264.7cells and peritoneal macrophages with plasmids,whereas large amounts of green fluorescence were observed after transfection of RAW264.7 cells and peritoneal macrophages with ghosts loading plasmids.The results indicated that EGFP had been effectively expressed in transfected cells.At the same time,the transfection efficiency was analyzed by flow cytometry.The results showed that after transfection of RAW264.7 cells and peritoneal macrophages with plasmids,the positive cell rates were 1.4%and 1.8%respectively.After transfection of RAW264.7 cells and peritoneal macrophages with ghosts loading plasmids,the positive cell rates were 7.2%and 57.6%respectively.These results indicated that Lactobacillus casei bacteria ghosts can promote macrophages transfection efficiency.Detection of IL-1?,IL-10,TNF-?,iNOS cytokines,and Toll receptor in macrophages by Real time qPCR showed that IL-1?,IL-10,TNF-?,iNOS,TLR2 and TLR9 mRNA levels increased after Lactobacillus casei bacteria ghosts with plasmids stimulating macrophages,however,TLR4 mRNA levels decreased.These results demonstrated that after transfection of macrophages with ghosts loading plasmids could regulate the expression of the relevant immune molecules mRNA significantly.The bone marrow-derived dendritic cells were stimulated with the plasmid pCI-PRV-VP6 and Lactobacillus casei bacteria ghosts loading plasmid pCI-PRV-VP6,respectively.And untreated dendritic cells served as a negative control.Dendritic cell surface marker molecules including MHC-II,CD80 and CD86 were analyzed by flow cytometry.The results showed that the number of mature markers of dendritic cells increased after being stimulated by Lactobacillus casei bacteria ghosts.And these results also suggested that Lactobacillus casei bacteria ghosts could stimulate dendritic cells maturation.The mRNA levels of IL-1?,IL-6,IL-10,TNF-?and IFN-?were detected by Real time qPCR.The results showed that the mRNA levels of these genes were increased significantly,after stimulating dendritic cells with Lactobacillus casei bacteria ghosts loading plasmid.These results demonstrated that the mRNA expression levels up-regulated in dendritic cells significantly,after being stimulated by Lactobacillus casei bacteria ghosts.When dendritic cells and T cells were mixed at a ratio of 1:1 and 1:10,the dendritic cells stimulated by Lactobacillus casei bacteria ghosts loading plasmid could stimulate the proliferation of T cells effectively.These results indicated that the Lactobacillus casei bacteria ghosts could promote dendrites cells maturation and activation.Finally,mice were immunized plasmid pCI-PRV-VP6 and Lactobacillus casei bacteria ghosts loading plasmid pCI-PRV-VP6 respectively.The mice were immunized with gavage,intramuscular injection and intraperitoneal injection.The results were detected by indirect ELISA.The results showed that the serum IgG in the Lactobacillus casei bacteria ghosts loading DNA group was higher than that in the DNA immunized group significantly after being immunized by three routes.These results indicated that the packaging DNA vaccines could improve the humoral immunity effectively.The proliferation of the T cells was measured using MTT.The results showed that the proliferation index of T cells in DNA vaccine packaging group was significantly higher than that in the DNA immunization group with gavage and intraperitoneal injection.The proportion of CD4~+/CD8~+T cells was analyzed by flow cytometry.The results showed that the proportion of CD4~+T cells was significantly increased compared with the DNA immunization group with gavage and intraperitoneal injection.The results showed that immunization with packaging DNA could improve the level of cellular immunity significantly.The levels of cytokines IL-2,IL-4,IL-10 and IFN-?in serum were measured by ELISA.The results showed that the serum cytokines in the Lactobacillus casei bacteria ghosts loading DNA group was higher than that in the DNA immunized group significantly.These results indicated that packaging DNA vaccines could regulate the levels of cytokines.In summary,packaging DNA by Lactobacillus casei bacteria ghosts could enhance transfection efficiency of DNA in macrophages.BGs could promote the maturation and activation of dendritic cells,and further regulate the gene expression level of immune-related factors.Lactobacillus casei bacteria ghosts could improve the level of humoral and cellular immune responses effectively.It provides a new technology platform for the development of a new,safe and effective DNA delivery vehicle,and it also provides a reference for the study of other proteins or drugs using Lactobacillus casei as a delivery vehicle.