Construction Of Recombinant BCG Vaccine And Its Immunoprotection Effect Evaluation

Tuberculosis（TB）is a chronic infectious disease caused by Mycobacterium tuberculosis（Mtb）.Mycobacterium tuberculosis mainly invades the human lung via respiratory transmission.So far,the live attenuated Bacillus Calmette-Guerin（BCG）has been used as only vaccine in the clinical against tuberculosis,but the protective efficacy of BCG is not inconsistent,and the immune effect of BCG vaccine on adults is not clear.Repeating vaccination can not improve the efficacy of protection against TB.On the contrary,it is possible to trigger latent infection.In order to generate a better vaccine,in this study,we targeted Ag85 family genes（Ag85A,Ag85B,Ag85C,Ag85D）was linked to the p MV261 shuttle vector to generate recombinant BCG（r BCG）.The immune protective effect of r BCG was evaluated in mouse model compared with BCG.In this experiment,the genomic DNA of Mtb stored in the laboratory was used as template,and the Ag85A,Ag85B,Ag85C and Ag85D genes were amplified by PCR respectively,each gene was individually cloned into p MV261 vector by In-Fusion Cloning method.The shuttle plasmids with targeting gene were confirmed by sequencing,restriction enzyme digestion and PCR.The four recombinant plasmids were transformed into BCG competent cells by electroporation,and the expression of Ag85A,Ag85B,Ag85C and Ag85D protein in recombinant BCG was examined by Western Blot.The four r BCG vaccine strains were successfully constructed and named as r BCG/Ag85A,r BCG/Ag85B,r BCG/Ag85C and r BCG/Ag85D.Finally,the protective effect of r BCG vaccine strains was evaluated in mouse model.C57BL/6 mice were random Ly divided into 6 groups:1)PBS control group;2)BCG group;3)r BCG/Ag85A group;4)r BCG/Ag85B group;5)r BCG/Ag85C group and 6)r BCG/Ag85D group,each group contains 12 mice.The mice were subcutaneously immunized with PBS and each candidate strain of 10⁶CFU/mice on neck back.After 4 weeks of immunization,6 mice in each group were randomly selected and scarified after eyeball blood sampling.The level of immunoglobulin G（Ig G）and immunoglobulin M（Ig M）in serum of mice was detected by mouse immunoglobulin assay kit.The level of Ig G and Ig M in BCG,r BCG/Ag85A,r BCG/Ag85B,r BCG/Ag85C,r BCG/Ag85D group were increased comparison with control group,it suggests those live attenuated vaccine can enhance humoral immune response,especially,the level of Ig M in r BCG/Ag85D group was significantly higher than that in BCG group.This results suggest that immunization with r BCG/Ag85D is beneficial to improve humoral immunity in mice at the early stage of infection.The IFN-γsecreting lymphocytes in mice spleen were evaluated by ELISpot,and the results showed that the number of IFN-γsecreting splenic lymphocytes under the stimulation of purified protein derivative（PPD）was significantly higher in r BCG/Ag85B group than that in BCG group（P<0 05）.The proportion of IFN-γproducing CD4⁺and CD8⁺T cells in total CD4⁺and CD8⁺T cells was also analyzed through flow cytometry.The data showed that the percentage of IFN-γexpressing CD4⁺and CD8⁺T lymphocytes in mice immunized with r BCG/Ag85B or r BCG/Ag85C was significantly elevated compared with BCG group（P<0.01）.The results showed that r BCG/Ag85B and r BCG/Ag85C could induce a better cellular immune response comparison to others.After 4 weeks of immunization,the mice of each group were infected intravenously with 10⁶CFU of Mtb H37Rv strain via tail vein.The weight and mental state of the mice were monitored every a week after infection.The mice were scarified after 4 weeks of infection.The lungs and spleens of the mice were collected aseptically.The bacterial burden was measured in organs and Histopathological sections of organs were observed by H&E staining.The results showed that the weight of mice immunized with r BCG/Ag85B or r BCG/Ag85C steadily increased compared with BCG group and PBS control group after infection.Compared with BCG group and PBS control group,the CFU of lung（spleen）decreased 0.9～1.1log₁₀(1.1～1.4 log₁₀)and 2.4～2.7 log₁₀(2.2～2.5 log₁₀)respectively.The H&E staining showed only a small amount of immune cells infiltration and mild inflammatory lesion were observed in lung tissue of mice immunized with r BCG/Ag85B or r BCG/Ag85C.The CFU of lung in r BCG/Ag85B or r BCG/Ag85C group was decreased and the lung injury rates were 9%and 8%,respectively,however BCG and PBS control groups showed were higher 37%and 22%,respectively.The results showed that r BCG/Ag85B and r BCG/Ag85C could generate better immune response than BCG group.In this study,four recombinant BCG vaccine strains were successfully generated,the results has shown r BCG/Ag85B and r BCG/Ag85C strains could induce stronger humoral and cellular immune responses in mice.It can effectively reduce bacterial burden in the lung and spleen of mice after challenge with H37Rv strain,and has shown better protective effect in mice challenged with Mtb compared with control.It provides important evident to support the recombinant BCG vaccine may be used to generate best vaccine in future.