[Immunogenicity and protection of divalent DNA vaccine encoding antigens MPT83 and MPT64 of Mycobacterium tuberculosis].
OBJECTIVE
To investigate the imunogenicity and protective efficacy of the divalent DNA vaccine encoding the antigens MPT83 and MPT64 of Mycobacterium tuberculosis.


METHODS
Two genes of the M. tuberculosis, MPT83 and MPT64, were amplified and cloned into the vector pJW4303. The vector containing the fusion gene DNA-MM and pJW4303 blank vector were transfected into CO57 cells. The expression of DNA-MM in the supernatant was detected by Western blotting. Thirty-six C57BL/6 mice were divided into 3 equal groups to be injected subcutaneously with the vector containing the fusion gene DNA-MM, pJW4303 blank vector, or bacillus of Calmette-Guerin vaccine (BCG) once a week for 3 weeks. Twelve mice were used as non-immunized controls. Blood samples were collected from the mice before and after every immunization to detect the titer of antibody by ELISA. Three weeks after the 3rd immunization 3 mice from each were killed and their spleens were taken out. The splenocytes were cultured and stimulated by the recombinant antigen to detect the concentrations of interferon (IFN)-gamma and interleukin (IL)-4. Six weeks after the 3rd immunization M. tuberculosis of the line H37Rv was injected intravenously into the mice. Eight weeks later, the mice were killed and their lungs and spleens were taken out. The tissues were cultured to calculate the numbers of M. tuberculosis.


RESULTS
Western blotting showed that the CO57 cells transfected with the vector containing the fusion gene expressed DNA-MM. ELISA showed that no antibody titer was detected before immunization in every group. The titer of MPT83 and MPT64 were increased to 1:200, 1:800, and 1:6400 3 weeks after the 1st, 2nd, and 3rd immunization respectively in the divalent group, increased to 1:800 3 weeks after the 3rd immunization in the BCG group, and remained 0 at any time points in the blank vector group. The IFN-gamma titer of the divalent group was 7520 pg/ml, significantly higher than that of the BCG group (6675.6 pg/ml, P < 0.05). The IL-4 concentration was basically the same and at the ng level in all groups. After challenge of H37Rv the bacterial loading in the lung was (2.21 +/- 0.032) x 10(5) in the divalent group, one thousandth of that in the blank vector group, and one tenth of that in the BCG group.


CONCLUSIONS
The divalent vaccine containing the antigens MPT83 and MPT64 effectively heightens the protective efficiency of vaccine against tuberculosis.
Tian
Xia
X
National Laboratory of Protein Engineering and Plant Genetic Engineering, Peking University, Beijing 100871, China.
Cai
Hong
H
Zhu
Yu-xian
YX
D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology*; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL; D014162:Transfection; D014376:Tuberculosis / Q000517:prevention & control; D019444:Vaccines, DNA / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C080865:MPB64 protein, Mycobacterium; C094904:MPB83 protein, Mycobacterium; D008565:Membrane Proteins; D019444:Vaccines, DNA