Abstract: Objective To study the immunogenicity of different dosages DNA vaccines from Mycobacterium tuberculosis medicated by electroporation.  Methods Twenty female BALB/c mice were immunized intramuscularly with saline, 10 μg Ag85A DNA, 50 μg Ag85A DNA and 100 μg Ag85A DNA for three times at two-week intervals, respectively. Twenty female BALB/c mice were medicated intramuscularly by electroporation with saline, 10 μg Ag85A DNA, 50 μg Ag85A DNA and 100 μg Ag85A DNA for 3 times at two-week intervals, respectively. There were 5 mice each group. Mice were sacrificed at 2 weeks after the final immunization respectively. The levels of IFN-γ and IL-4 in the culture supernatants of splenic lymphocytes were measured with enzyme-linked immunosorbent assay (ELISA). The ratio of CD4⁺ T cells expressing IFN-γ (Th1) and IL-4 (Th2) in whole blood was detected by flow cytometry. Data were expressed as means and standard deviations. Statistical significance between each group was calculated using two factors factorial design ANOVA followed by compared with t test using SAS 9.2 software and a P-value of <0.05 was considered to be statistically significant.  Results At 2 weeks after the final immunization, IFN-γ levels in splenocyte culture supernatant in 50 μg DNA (646.05±342.53) pg/ml and 100 μg DNA intramuscular injection group (738.61±372.68) pg/ml was significantly higher than that in saline (1.73±3.88) pg/ml (t=4.065 and 4.647, all P<0.05) and 10 μg DNA intramuscular injection group (87.83±120.82) pg/ml (t=3.513 and 4.094, all P<0.05); IFN-γ level in splenocyte culture supernatant in 10 μg DNA electroporation group (357.06±105.18) pg/ml was significantly higher than that in saline (t=2.247, P<0.05)，and higher than that in 100 μg DNA electroporation group (86.08±135.73) pg/ml，but the difference was not statistically significant(t=1.706, P>0.05). 50 μg DNA electroporation group (648.60±439.41) pg/ml was significantly higher than that in saline(t=4.081, P<0.05) and 100 μg DNA electroporation group(t=3.539, P<0.05); compared with intramuscular injection group, IFN-γ levels of 10 μg DNA electroporation group increased 3 times (357.06 pg/ml)/(87.83 pg/ml); that of 50 μg DNA electroporation group had no significant change (t=－0.016, P＞0.05); that in 100 μg DNA electroporation group declined 88.35% (t=－4.105, P<0.05). Percentage of Th1 cells secreted IFN-γ in different dosages of intramuscular DNA (10 μg Ag85A DNA：(1.39±0.84)%, 50 μg Ag85A DNA：(1.55±0.33)%, 100 μg Ag85A DNA：(2.13±0.47)%) and DNA electroporation groups (10 μg Ag85A DNA：(1.42±0.47)%, 50 μg Ag85A DNA：(1.88±0.51)%, 100 μg Ag85A DNA：(1.43±0.68)%) were higher than that in saline group (0.65±0.31)% (t=2.002, 2.431, 4.015, 2.084, 3.332 and 2.105, all P<0.05), there has no statistical difference between doages DNA electroporation groups and intramuscular injection groups (t=0.081, 0.901 and －1.91, all P>0.05). Percentage of Th2 cells secreted IL-4 in different dosages of intramuscular DNA (10 μg Ag85A DNA：(1.42±1.18)%, 50 μg Ag85A DNA：(1.14±0.78)%, 100 μg Ag85A DNA：(1.24±0.76)%) and DNA electroporation groups (10 μg Ag85A DNA：(1.19±1.09)%, 50 μg Ag85A DNA：(2.06±0.96)%, 100 μg Ag85A DNA：(1.47±0.65)%) were higher than that in saline electroporation group (4.14±2.55)% (t=－3.392, －3.738, －3.616, －3.676, －2.599 and －3.325, all P<0.05), there was no statistically significant difference between doages DNA electroporation groups and intramuscular injection groups(t=0.284, －1.139 and －0.292, all P>0.05).  Conclusion The results suggest that lower doses of DNA immunization by electroporation could improve immune response, using a small amount of DNA vaccine can produce good immune effect.

Key words: Mycobacterium tuberculosis, Vaccines, DNA, Electroporation, Immunity