| Tuberculosis(TB)is a chronic infectious disease caused by Mycobacterium tuberculosis(M.tb),which invades the lungs and causes body damage.It is one of the leading infectious diseases that cause human death.However,the BCG vaccine,the only widely accepted and vaccinated TB vaccine in the world,provides successful immune protection for children,but its protection is extremely weak for adults with the largest number of cases.In addition,with the spread of TB drug resistance,it is urgent to develop a TB vaccine that can have a lasting protective effect on the human body and have a preventive effect on different groups of all ages.With the in-depth research on TB vaccine and the continuous progress of technology,more and more TB vaccines have been reasonably designed and put into clinical trials,but so far no new effective TB preventive vaccine has come out,and the global TB epidemic has not been effectively controlled,This is probably due to insufficient immunogenicity and protection efficiency of the vaccine.Therefore,the strategy of optimizing the vaccination pathway and combining it with new adjuvants of the vaccine may enhance the immunogenicity of the vaccine and improve the immune protection and immunotherapeutic effect of the vaccine on tuberculosis.Our group successfully developed ag85 ab DNA vaccine in the previous study,which has shown to be effective against murine tuberculosis.However,given the apparent immunogenicity deficiency of TB DNA vaccines prevalent in large animals and humans,our group immunized ag85 ab DNA vaccine by intramuscular injection combined with electroporation(IM+EP)in the early research and found that the IM+EP method successfully reduced vaccine dosage used,enhanced immunogenicity of the vaccine,and had a good immunotherapeutic effect on the mouse TB model.Considering that M.tb invades the pulmonary mucosa through the respiratory tract and causes infection in the body,the pulmonary mucosa is the first line of defense to prevent M.tb infection,and the lung has a large absorption area and a large number of immune cells,pulmonary mucosal immunity can enhance the local immunity of the lungs and remove pathogenic bacteria.Therefore,this study compared the immunogenicity of ag85 ab DNA vaccine immunized between by IM+EP pathway and pulmonary delivery pathway with the hand-held liquid aerosol lung delivery device and determined which pathway is more advantageous in inducing a more robust and effective immune response in the body and in the lung,laying the foundation for establishing an effective DNA vaccine delivery system.Fifty BALB/c mice were randomly divided into PBS intramuscular injection electroporation group(PBS+EP group),p VAX1 intramuscular injection electroporation group(p VAX1+EP group),ag85 ab intramuscular injection electroporation group(ag85ab+EP group),p VAX1 Pulmonary delivery group(p VAX1+PD group),and ag85 ab Pulmonary delivery group(ag85ab +PD group),with 10 mice in each group.Mice were immunized by intramuscular injection electroporation and pulmonary delivery,once every 2 weeks,for a total of 3 times.6 and4 mice were killed at weeks 6 and 12 after the last immunization,respectively.The changes in the body weight of the mice were recorded.The levels of specific antibodies Ig G,Ig G1,and Ig G2 a in mouse plasma and the levels of specific antibodies Ig A in alveolar lavage fluid of the mice in each group were determined by ELISA.The number of effector lymphocyte spots secreting IFN-γ in the lung and spleen lymphocytes was detected by enzyme-linked immune spot assay(ELISPOT assay).The proportion of Fox P3 regulatory T cells in mouse spleen cells was determined by flow cytometry.The levels of Th1,Th2,and Th3 cytokines in the lung and spleen lymphocyte culture supernatants were detected by the Luminex method.The results showed that at weeks 6 and 12 after the last immunization,the levels of the plasma-specific antibodies Ig G,Ig G1,and Ig G2 a in the ag85 ab +EP group were significantly higher than those in the ag85 ab +PD group(P<0.001).At week 6 after the last immunization,the level of specific antibody Ig A in the alveolar lavage fluid of mice in the ag85 ab +PD group was significantly higher than that in the the ag85 ab +EP group(P<0.001).At week 12 after the last immunization,the level of specific antibody Ig A in the alveolar lavage fluid of mice in the ag85 ab +PD group was significantly higher than that in the PBS +EP group(P<0.05).At week 6 after the last immunization,the number of lung T lymphocyte spots secreting IFN-γ in the ag85 ab +PD group was 2 times higher than that in the ag85ab+EP group(P<0.0001).Compared with week 6 after the last immunization,The number of spleen lymphocyte spots secreting IFN-γsignificantly decreased in the ag85 ab +EP group and ag85 ab +PD group at week 12 after the last immunization(P<0.001,P<0.001).but there was no statistically significant difference between ag85ab+PD group and ag85ab+EP group(P>0.05).At week 6 after the last immunization,the proportion of Fox P3 regulatory T cells in spleen cells of the ag85 ab +PD group and ag85ab+EP group was significantly higher than that of PBS +EP group(P<0.05).At week 12 after the last immunization,the proportion of Fox P3 regulatory T cells in spleen cells of the ag85 ab +PD group and ag85ab+EP group was still significantly higher than that of PBS +EP group and p VAX1 +EP group(P<0.001 or P<0.01).At weeks 6 and 12 after the last immunization,there was no significant difference between the ag85ab+PD group and the ag85 ab +EP group(P>0.05).Among the Th1-type cytokines produced in the supernatant of lung lymphocyte culture of the mice at week 6 after the last immunization,the levels of interferon-γ(IFN-γ),interleukin(IL)-2,granulocyte-macrophage colony-stimulating factor(GM-CSF),active interleukin 12(IL-12P70),IL-18,and IL-27 in the ag85 ab DNA lung delivery group were significantly higher those in the PBS +EP group,the p VAX1 +EP group,and the p VAX1+PD group(P<0.05,P<0.01,P<0.001,P<0.001).The levels of IFN-γ,IL-12P70,IL-18,and IL-27 in the ag85 ab +PD group were significantly higher in the ag85 ab +EP group(P<0.001,P<0.001).It is worth noting that the IFN-γ level in the supernatant of lung lymphocyte culture in the ag85 ab +PD group was 4 times higher than that in the ag85 ab +EP group.Among the Th2-type cytokines produced in the supernatant of lung lymphocyte culture,the levels of IL-4,IL-5,IL-6,IL-9,IL-10,and IL-13 in the ag85 ab DNA +PD group were significantly higher than those in the PBS +EP group,and the p VAX1 +PD group(P<0.05,P<0.01,P<0.01 or P<0.001).The IL-10 level in the ag85 ab +PD group was significantly higher than that in the ag85 ab +EP group(P<0.001).The ratio of Th1-/Th2-type cytokines(IFN-γ/IL-4)in the ag85 ab +PD group was two times higher than that of the ag85 ab +EP group.Among the Th3-type cytokines produced in the supernatant of lung lymphocyte culture,the levels of IL-17 A and IL-22 in the ag85 ab +PD group and ag85 ab +EP group were significantly higher than those in the other group(P < 0.001 or P < 0.0001).There was no statistically significant difference between ag85ab+PD group and ag85ab+EP group(P>0.05).Among the Th1-type cytokines produced in the supernatant of spleen lymphocyte culture of the mice at week 6 after the last immunization,the levels of IFN-γ,IL-2,TNF-α,GM-CSF,IL-12P70,and IL-18 in the ag85 ab +PD group and ag85 ab +EP group were significantly higher those in the PBS +EP group and the p VAX1 +EP group(P<0.05,P<0.01,P<0.001,P<0.001).It is worth noting that IL-2 and TNF-α levels in the ag85 ab +PD group were significantly higher than that in the ag85 ab +EP group(P<0.05).Among the Th2-type cytokines produced in the supernatant of spleen lymphocyte culture of the mice,the levels of IL-4,IL-5,IL-6,IL-10,and IL-13 in ag85 ab +PD group and ag85 ab +EP group were significantly higher than those in PBS +EP group(P<0.05,P<0.01,P<0.001,P<0.001).It should be noted that the IL-6 level in the ag85 ab +PD group was significantly higher than that in the ag85 ab +EP group(P<0.05),while its IL-10 level was significantly lower than that in the ag85 ab +EP group(P<0.001).The ratio of Th1-/Th2-type cytokines(IFN-γ/IL-4)ag85ab+EP group was one time higher than that of the ag85 ab +PD group.Among the Th3-type cytokines produced in the supernatant of spleen lymphocyte culture of mice,the levels of IL-17 A and IL-22 in the ag85ab+PD group were significantly higher than that in the ag85 ab +EP group(P<0.001 or P<0.0001).In conclusion,M.tb ag85 ab DNA vaccine delivered both by the pulmonary delivery pathway and intramuscular injection electroporation pathway can effectively induce effector T lymphocytes from the lung and spleen to produce IFN-γ,but the pulmonary delivery pathway can induce more effector T cells and stronger responses.In addition,immunization by two delivery pathways can induce lung and spleen lymphocytes to produce Th1-and Th3-type cellular immune responses.The immunization by the intramuscular injection electroporation pathway can induce the body to produce higher levels of specific antibodies Ig G,Ig G1,and Ig G2 a.Most importantly,the immunization by pulmonary delivery pathway can induce higher levels of specific antibodies Ig A in the respiratory tract mucosa than by the intramuscular injection electroporation pathway.In summary,DNA vaccine immunized by lung delivery can more effectively stimulate the body to produce stronger cellular immunity,and mucosal immunity(especially in the lung)than by intramuscular injection electroporation pathway,and can significantly enhance the immunogenicity of ag85 ab DNA vaccine,which is a feasible and effective immunization pathway. |
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