| Objective To prepare DNA vaccine, encoding the mycolyl-transferase Ag85A, by which BCG priming is boosted. To access the immunoprotection of’BCG prime-Ag85A DNA booster’regimen, and to analysis the T cell response after immunization, aim at investigate the relationship between immunogenicity and immunoprotection.Method A subunit DNA vaccine was constructed by eukaryotic expression vector pVAXl expressing mycobacterial antigen Ag85A and prepared endotoxin-free. Mice were vaccinated with PBS, BCG,’BCG prime-Ag85A DNA booster’regimen and Ag85A DNA.10mice per group were aerosol challenged by Mtb, H37Rv8weeks after primary immunization, and5mice per group were sacrificed10weeks after primary immunization for T cell response analysis. At1and5weeks post-infection the mycobacteria burden and cytokine level including IFN-y, TNF-α, IL-2, IL-12, IL-4, IL-6, IL-10and TGF-β in spleen and lung were determined, and the spleen and lung of mice infected5weeks were embedded in paraffin, sectioned and for H&E stain and immunohistochemisty assay. At10weeks after immunization, the single cell suspensions of lung and spleen were prepared for ELISPOT assay of IFN-y secretion and intracellular flow cytometry of IFN-γ, TNF-a and IL-2production and CD44, CCR7and CD62L expression.Result The Ag85A gene was successfully inserted in pVAX1plasmid, and sequencing result was consistent with the one published on GenBank. There was no significant difference in CFU counts in lungs between groups at1week after challenge, and in spleens no Mtb was detected then. At5weeks post-infection, BCG-Ag85A DNA group had the lowest CFU counts in both spleen and lung, and compared with PBS group BCG vaccination resulted a significant decrease in CFU counts, while the difference of CFU counts between Ag85A DNA group and PBS, BCG groups was not statistically significant. BCG-Ag85A DNA vaccination also resulted in slightest pathological damage. There was no significant difference in cytokine level at1week after infection, while at5weeks after infection the expression of IFN-y, IL-2, IL-12, IL-10and TGF-P was highest in Ag85A DNA group, followed by BCG group and BCG-Ag85A DNA group lowest. At10weeks post primary immunization,’BCG prime-Ag85A DNA booster’regimen induced more PPD-specific IFN-y producing cells than PBS and Ag85A DNA, while with Ag85A peptide pool stimulation’BCG prime-Ag85A DNA booster’regimen induced more IFN-y producing cells than PBS only. When studied by flow cytometry, mice vaccinated with ’BCG prime-Ag85A DNA booster’regimen had significantly increased percentages of IFN-γ+TNF-α+IL-2+, TNF-a+IL-2+, IFN-γ+IL-2+and IL-2+CD4T cells, while BCG vaccinated mice had more IFN-y+and IFN-γ+TNF-α+CD4T cells, and the T cell response Ag85A DNA induced was much lower. Further studied iMFI for each cytokine of these responding cells, BCG-Ag85A DNA group had highest iMFI for IL-2. All cytokine producing cells had a phenotype of CD44high, and TNF-a producing cells were predominant in CD62Lhigh cells, while both CD62Lhigh and CD62Llow cells produced IFN-y and IL-2. Nearly all cells except for part of TNF-a producing cells were restricted primarily to CCR7low cells. Moreover,’BCG prime-Ag85A DNA booster’regimen also induced higher CD8T cell response.Conclusion’BCG prime-Ag85A DNA booster’regimen provided better protection than BCG, that mice exhibited reduced bacillary load and reduced pathology. It induced stronger CD4and CD8T cell response, and IFN-y+TNF-a+IL-2+multifunctional CD4T cells and IL-2producing cells were increased. Therefore’BCG prime-Ag85A DNA booster’regimen protected better. However, both BCG and’BCG prime-Ag85A DNA booster’regimen didn’t induced enough TCM, that might be why BCG failed to provide longtime protection. |
