Dynamic Evaluation Of BCG Immune Mechanism From Systems Vaccinology

The molecular mechanism of vaccination in RNA and protein could be analyzed by systems vaccinology,which predict the immune effect effectively.The neuro-immune interaction is vital mechanism for the maintenance of homeostasis.The interaction between immune system and nerve system through peptide hormones,neurotransmitter and cytokines constitute the defensive system to ensure that timely and accurately response the stimulation from internal and external.From this standpoint,the development and assessment of vaccine can also use neuro-immune interaction,which can be more comprehensive understanding of the regulatory mechanism of vaccine.Bacillus Calmettee-Guerin(BCG),the only currently available vaccine against Tuberculosis(TB),is one of important means that prevention of TB.However,the limitations of BCG vaccination have been gradually found with expansion of vaccination population and inoculation time.There is an urgent need for an improved TB vaccine.The first step in TB vaccine development is understands defense mechanism of BCG and give a comprehensive evaluation.In this study,BCG were grown in Middlebrook 7H9 broth supplemented with10% Middlebrook OADC Enrichment to successfully gained BCG that in line with the provisions of the Chinese Pharmacopoeia.C57BL/6 mice were immunized by subcutaneous injection with 5×105cfu BCG at the base of the tail and in both footpads.The peripheral blood,hypothalamus,spleen and draining lymph nodes from mice vaccine with BCG were collected at different time points.The specific antibodies in serum of vaccine mice were detected by ELISA at every week.The level of lymphocyte subsets in spleen and draining lymph nodes were detected by Flow Cytometry.The level of Leptin and cytokines of helper T cells in hypothalamus,spleen and draining lymph node were detected by RT-PCR,and then analysis the relation among the each index.The research of nerve and immune systems of vaccine mice can provide the experimental basis for the deeper understanding of immune mechanism after BCG vaccination.The experimental results are as follows:1.BCG-specific IgG antibody was determined by ELISA.Absorbance was detected with a microplate reader at 492 nm.The antibody titer level curve was drawn with wavelength as vertical axis and sampling time as abscissa.The group of mice vaccinated with BCG were shown continued increases in BCG specific IgG,especially at the first week until the fifth week maintain at 0.4,which demonstrated vaccination with BCG induce a high level of antibody titer at a long period.2.The effect of BCG vaccination on CD4+ and CD8+ T cells in spleen and lymph nodes were dectected by Flow Cytometry.After vaccination,draining lymph nodes were significantly bigger than injuction with PBS.The percentage of lymph nodes T cells significantly decrease,which demonstrated the counts of B cells or other immune cells significantly increase.Moreover,the ration of CD4+ to CD8+ was not change between the group of PBS and BCG,which demonstrated BCG vaccination induced both of CD4+ and CD8+ T cellular immune response in draining lymph nodes and mainly with CD4+ cellular immune response in spleen.3.The level of Treg was detected by Flow Cytometry that the percentage of Treg in draining lymph nodes after BCG vaccination was significantly increased.However,Tregs was only significantly higher at the first week in spleen,which indicated vaccination induced different immune effect and different bacterial load between spleen and draining lymph nodes.4.The expression of leptin and leptin receptor were detected by RT-PCR.The expression of leptin and leptin receptor in draining lymph nodes were same with hypothalamus which significantly higher after vaccination.The expression of leptin in spleen was significantly lower at every week after vaccination with the expression of leptin receptor has no change.The results demonstrate that draining lymph nodes is the main response organ after vaccination,which regulate interaction with hypothalamus.5.The expression of IL-2 and IFN-?in hypothalamus,spleen and draining lymph nodes were detected after BCG vaccination,which is Th1 cells cytokines.The results indicated vaccination induced response of IFN-?in spleen and draining lymph nodes,which negative regulation with hypothalamus.6.The expression of IL-4 and IL-10 in hypothalamus,spleen and draining lymph nodes were detected after BCG vaccination,which is Th2 cells cytokines.The results indicated vaccination induced response of IL-4 in draining lymph nodes.7.The expression of IL-17 A in hypothalamus,spleen and draining lymph nodes were detected after BCG vaccination,which is Th17 cells cytokines.The results indicated vaccination induced response of IL-17 A in draining lymph nodes,which regulation with hypothalamus.8.The expression of IL-1?,IL-6 and TNF-? in hypothalamus,spleen and draining lymph nodes were detected after BCG vaccination,which is inflammatory cytokines.The results indicated vaccination induced inflammatory reaction between spleen and draining lymph nodes in varying degree and time,which regulation with hypothalamus.In summary,this research detects the vaccinated mice from neuro-immune interaction.Through the detection of specific antibody in serum,T lymphocyte subset,Treg cells,leptin and several cytokines,verifing the influence of BCG to neuro-immune interaction,which laid the foundation of the further research of the development of TB vaccine in the future.