The Construction, Immunogenicity And Protective Efficacy Of Tuberculosis Ag85A And Ag85B DNA Vaccine

Objective To construct DNA vaccines based on Ag85A, Ag85B genes of M. tuberculosis, and to evaluate their immunogenicity and protective efficacy.Methods Ag85A and Ag85B genes were amplified by PCR and then inserted into eukaryotic expression vector pVAXl after they were digested by restriction endonuclease Nhe I and BamH I . The recombinant plasmids were identified with PCR, restriction endonuclease and DNA sequencing. The plasmids were extracted and purified by Qiagen kits. C57BL/6 mice were grouped randomly, injected three times at 3-week intervals with the saline(A), pVAXl(B), Ag85A-pVAXl(D), Ag85B-pVAXl(E), and injected intradermally with BCG (C) a single time. The antigen-specific antibodies and antibody isotypes were determined with ELISA. Six mice in each group were infected by tail intravenous injection with M.tuberculosis H37Rv at the fourth week after the third immune. The changes of body weight were observed. Their lungs were taken and observed their pathological changes, detected cytokines, and performed mycobacterial cultures the fourth week after infection.Results 1041bp Ag85A and 978bp Ag85B DNA fragments could be observed in the products of amplification and the products of digestion from the recombinant plasmid by the electrophoresis. The results of DNA sequencing also proved that Ag85A and Ag85B DNA vaccines were constructed successfully. Ag85A and Ag85B antigen-specific antibody increased significantly in corresponding groups, and the level of antibody isotypes of IgG2b was much higher than IgGl and IgG2a. The body weight of the mice was observed after the infection. The weight in the mice of group A and group B began to decline at the third week. The weight in the mice of group C increased except for a little slim down at the third week. The weight in the mice of group D beganto decrease at the fourth week. The weight in the mice of group E started to decrease at the second week and then did not change. The stimulating rates of spleen lymphacytes from group D, E increased significantly compared to that from group A, B. The pathological results from lungs showed that the lesion in the lungs from group C and D was less than that from group A, B and E. We could see severe hyperemia in the lungs from group A, B and E. Histopathological results showed the lungs from group A had serious hyperemia, exudate in 70~80% of alveolar spaces and sparse lymphocytic infiltration around capillaries. The group B had exudates in part of alveolar spaces and hyperplasia in alveolar walls and a little lymphocyte infiltration. The main pathological changes of group C were charactered by lymphocyte infiltration and forming granulomas. The group D was similar to that seen in the group C. The group E exhibited thick alveolar wall and lymphocyte infiltration, but did not form granulomas. Lung tissue necrosis was not observed in any mouse. The number of viable bacteria in the lung showed group C were least, subsequently group E, D, B, A.Conclusions M. tuberculosis Ag85A and Ag85B DNA vaccines that were used to immunize mice by the intramuscular injection, could induce specific humoral immune reaction and cell-mediated immune responses. The protective efficacy of Ag85A DNA vaccine against M. tuberculosis challenge was stronger than that of Ag85B DNA vaccine. But they did not show stronger protection than BCG. It is worth to study further on these DNA vaccines.