Research On Immune Protective Efficacy And Safety Of A New Type Of Tuberculosis Vaccine

Objective: In order to investigate the immune protective efficacy and safety of the new type of tuberculosis vaccine called fusion strains(B/R strains), that used Bacillus Calmette Guerin(BCG) and Mycobacterium tuberculosis H37 Ra strains(H37Ra) for Parents Source merged by Protoplast fusion technique.Methods: In order to study the immune protective efficacy and safety of B/R strains, experiment was divided into two parts :animal level experiment and cell level experiment.(1) animal level experiment: 204 C57BL/6 mice were randomly divided into saline group(36 mice), BCG group(56 mice)、B/R group(56 mice) and H37 Ra group(56 mice), they were intradermal vaccinated used sterile saline 0.1ml, BCG bacteria,B/R strain and H37 Ra strain suspension,each take 0.1ml(containing viable count of 1.0×106), a:After inoculation, observe basic survival status of each group of mice and record their weight on the third, Sixth, ninth, twelfth, fifteenth and eighteenth weeks. b:After 2, 3, 6, 9, 12 weeks of inoculation, randomly selecte 4 mice from each BCG group,B/R group and H37 Ra group;The spleen and lung of mice were isolated to make tissue slurryand suitable dilutions of the homogenates were plated on Lowenstein-Jensen medium and put them in a 37℃incubator. The numbers of CFU were counted after 20 days incubation. c:On the third, Sixth, ninth, twelfth, fifteenth and eighteenth weeks. selected 6 mice randomly from each saline group,BCG group,B/R group, H37 Ra group, these mice were killed by cervical vertebra dislocation and weighted.On the third, sixth, ninth week before the mice were killed, take eyeball blood and separate the plasma,ues ELISA method measure interleukin-2(IL-2) and interferon-γ(IFN-γ) level in mouse plasma; The dead mouse opened the chest and abdominal cavity,remove the lung,liver and spleen, put them in Formaldehyde Solution in 48 hours to produce histology and observe the variations(after removing the spleens weighed and record). then calculate the spleen coefficient for each group of mice.(2) cell level experiment: a: Cultured mouse macrophages RAW264.7 strain,Select the cells in good condition and were infected with BCG,B/R, H37 Ra strain bacteria suspension infection, use annexin V-FITC / PI double staining method to detect the infection after 6, 12, 24 hours of RAW264.7 macrophages strains of apoptosis rate and analysis comparison in the differences between the groups.b: to collect the C57BL/6 mouse peritoneal macrophages, and cultured for 24 hours in vitro, then the cells were infected with BCG, B/R, H37 Ra strain bacteria suspension, after 0, 48 and 96 hours, respectively lysed macrophages with 1% Triton X-100, then get cracking liquid diluted 1000 times, take amount of diluted liquid inoculation to L-J medium culture.count the CFU after 20 days, analysis the difference among the three groups.Results:(1) After 3, 6, 9, 12,15,18 weeks of inoculation,there was no mice died, the living condition of the four group mice were generally good, the four group mice survival rate were 100%; B/R group mice of body weight compared with saline group were not statistically significant(P>0.05).(2) Organ colony count experiment in mice found that B/R strain,BCG and H37 Ra strain can colonized in mice spleen and lungs, the capability of B/R strain colonize in mice spleen and lung were enhanced than BCG,reduced than H37 Ra strains, the difference was statistically significant( P < 0.05).(3) After3, 6, 9weeks of inoculation,the levels of IL-2 and IFN-γ in mice blood, B/R strain group compared with BCG were significantly increased, but lower than H37 Ra strain group. the difference was statistically significant(P<0.05).(4) After inoculation,the lungs of BCG group, H37 Ra strain group and B/R strain group mice were all exhibited varying degrees of inflammatory cell infiltration,Partial airway epithelial depigmentation,Widened alveolar septum etal. Livers all showed varying degrees of liver sinusoidal expansion congestion,Portal area of inflammatory cell infiltration, section hepatocyte edema.the changes degree of BCG group and H37 Ra group were similar,B/R group changes was lighter than them. After inoculation,the spleen of BCG group, H37 Ra strain group and B/R strain group mice all showed varying degrees of red pulp expansion congestion, polymorphonuclear cells and macrophages increased,white pulp ratio were elevated, lymphoid nodules volume increased etal.The changes of B/R strain group was similar with H37 Ra strain group,BCG group changes was lighter than them.(5) After3,6,9,12 weeks of inoculation, mice spleen coefficient,B/R strain group compared with saline group were significantly increased the difference was statistically significant(P<0.05). mice spleen coefficient of B/R strain group compared with and H37 Ra group and BCG group the difference was not statistically significant(P>0.05).(6) Apoptosis assay showed that BCG, B/R and H37 Ra strain could increase the apoptosis rate of macrophage cell line RAW264.7.The ability of B/R strain induced macrophage apoptosis was no different with BCG, but enhanced compared with H37 Ra strain. the difference was statistically significant(P<0.05).(7) Macrophage proliferation experiment found that BCG, B/R strain, H37 Ra strains can survive and reproduce in murine peritoneal macrophages, there was no difference of proliferation in mouse peritoneal macrophages between B/R strain and BCG, but the ability of B/R strain proliferation in mouse peritoneal macrophages was lower than H37 Ra strains,the difference was statistically significant(P<0.05).Conclusions: 1. B/R strain as a tuberculosis vaccine strain used in C57 BL / 6 mice will not not affect the general growth of mice and survival time.2.B/R strain can colonize and grow at least three month in C57BL/6 mice, compared with BCG its colonization forces becomes stronger, but weaker than H37 Ra strains.3.B/R strain induce the cell immune levels in C57BL/6 mice compared with BCG is significantly enhanced, but weaker than H37 Ra strain.4.cellular level experiments show that the virulence of B/R strains was no significant difference with BCG,but compared with H37 Ra strain the virulence is reduced.