Evaluation of the effects of different vaccinations in muscles with DNA vaccine against Mycobacterium tuberculosis delivered by electroporation
Objective To evaluate the effects of different vaccinations in muscles with DNA vac-cine against Mycobacterium tuberculosis delivered by electroporation and to obtain an optimum immune strate-gy.Methods Forty female BALB/c mice were randomly divided into four groups and immunized intramus-cularly by electroporation with saline , 50 μg of Ag85A DNA, 100 μg of Ag85A DNA and mixed Ag85A DNA （ first time 10 μg of DNA, second time 100 μg of DNA and third time 10 μg of DNA） for three times with two weeks interval , respectively .At the second and sixth weeks after the final immunization , five mice from each group were sacrificed each time .The levels of IFN-γand IL-4 in supernatants of splenic lympho-cyte culture were measured by enzyme-linked immunosorbent assay （ELISA）.The ratios of CD4＋T cells ex-pressing IFN-γ（ Th1） and IL-4 （ Th2） in whole blood samples were detected by flow cytometry .The copies of Ag85A DNA in the positions of intramuscular injection were measured by quantitative RT-PCR.The levels of specific IgG antibodies response to Ag85A in serum samples from mice before immunization （pre-immune serum samples ） and 10 days after each immunization were detected by ELISA .Results Two weeks after the final immunization , the IFN-γlevels in the supernatants of splenocyte culture from mice immunized with mixed Ag85A DNA [（703.66±394.74） pg/ml] and 50 μg of Ag85A DNA [（648.60±439.41） pg/ml] were significantly higher than those in saline treated group [（70.58±108.76） pg/ml, t=3.975 and 3.629, P=0.0004 and 0.0010] and 100μg of Ag85A DNA immunized group [（86.08±135.73） pg/ml, t=3.878 and 3.532, P=0.0005 and 0.0013].No differences with the levels of IFN-γwere observed between mixed Ag85A DNA immunized group and 50 μg of Ag85A DNA immunized group （t=0.3457, P=0.7318）.The ratios of Th1/Th2 cells in whole blood samples react to Ag 85A protein from mice in mixed Ag85A DNA trea-ted group （3.82±1.09） were significantly higher than those in saline treated group （0.37±0.11）, 50μg of Ag85A DNA treated group （1.20±0.80） and 100 μg of Ag85A DNA treated group （1.10±0.60） （t=2.980, 2.260 and 2.352, P=0.0058, 0.0315 and 0.0257）.The copies of Ag85A DNA in intramuscular injection positions of mice from 100 μg of Ag85A DNA treated group [（963.40±892.79） copies] were sig-nificantly higher than those in 50 μg of Ag85A DNA treated group [（270.90±398.18） copies] and mixed Ag85A DNA treated group [（205.80±136.95） copies] （t=2.639 and 2.887, P=0.0144 and 0.0081）. Six weeks after the final immunization , IFN-γlevels in the supernatants of splenocyte culture from mice im-munized with mixed Ag85A DNA [（238.43±258.90）pg/ml）] were higher than those in saline treated group [（0±0） pg/ml], 50 μg of DNA treated group [（83.14±135.08） pg/ml] and 100 μg Ag85A DNA treated group [（163.83 ±207.13） pg/ml], but there were no significant differences between them （ t=1.497, 0.9750 and 0.4684, P=0.1442, 0.3369 and 0.6427）.The ratios of Th1/Th2 cells in whole blood samples react to Ag85A protein from mice immunized with mixed Ag85A DNA （4.67±5.05） were signifi-cantly higher than those immunized saline （0.77±0.19）, 50μg of Ag85A DNA （1.23±0.74） and 100 μg of Ag85A DNA （0.51±0.49） （t=3.199, 2.971 and 3.610, P=0.0033, 0.0059 and 0.0011）.The cop-ies of Ag85 A DNA in the positions of intramuscular injection showed no significant differences among all groups .Conclusion The results of this study suggested that lower doses of DNA immunization delivered by electroporation could improve immune responses and produce better immune effects .The optimum immune strategy was mixed immunization of DNA by electroporation .After the second immunization , the levels of specific IgG antibodies react to Ag85A protein in serum samples from mice immunized with 50 μg of Ag85A DNA , 100μg of Ag85A DNA and mixed Ag85A DNA were obviously increased than those of each group af-ter the first immunization （F=7.17,P=0.0111）, but there was no significant difference between them .Ex-cept the 50 μg Ag85A DNA treated group, the levels of specific IgG antibodies response to Ag 85A in serum samples from mice immunized with 100 μg of Ag85A DNA and mixed Ag85A DNA were increased after the third immunization as compared with those of each group after the second immunization , but no significant difference was found between them . 
 
Key words: 
Mycobacterium tuberculosis; DNA vaccine; Electroporation; Immune strategy