结核复合抗原疫苗不同抗原组合对小鼠免疫功能的影响

目的 探讨在辅以卡介苗非甲基化的胞嘧啶鸟嘌呤二核苷酸重复序列(CpG)佐剂和铝佐剂的情况下,结核病复合抗原疫苗中抗原85b(Ag85b)、融合蛋白早期滤液蛋白10(CFP-10)和相对分子质量为6000的早期分泌抗原靶蛋白(ESAT-6)及热休克蛋白X(HspX)不同组合对小鼠免疫功能的影响.方法 将48只BALB/c小鼠按抗原组合分为8组:(1)A组:Ag85b+CFP-10/ESAT-6+HspX+佐剂,(2)B组:CFP-10/ESAT-6+HspX+佐剂,(3)C组:Ag85b+HspX+佐剂,(4)D组:Ag85b+CFP-10/ESAT-6+佐剂,(5)E组:Ag85b+佐剂,(6)F组:CFP-10/ESAT-6+佐剂,(7)G组:HspX+佐剂,(8)对照组:生理盐水.每组6只,皮下免疫,共免疫3次,间隔2周,末次免疫后1周处死小鼠.酶联免疫斑点实验和淋巴细胞增殖实验检测小鼠脾淋巴细胞分泌γ-干扰素和白细胞介素-4及抗原特异性淋巴细胞增殖情况.酶联免疫吸附实验检测小鼠血清中IgG、IgG1和IgG2a抗体滴度.结果 E组γ-干扰素的斑点数[中位数(四分位间距),122.8个(78.4～184.4个)]明显高于C组和对照组[14.3个(6.5～14.6个)和0.5个(0.5～1.3个)],差异均有统计学意义(u值均为0.0,均P＜0.01);D组白细胞介素-4的斑点数[173.5个(7 8.8～233.4个)和132.8个(50.3～159.4个)]明显多于对照组[0.5个(0.5～1.3个)和5.3个(2.9～6.5个)],差异有统计学意义(u值均为0.0,均P＜0.01);A、C、D和E组的刺激指数(2.42±0.50、2.18±0.37、2.86±0.51和2.70±0.15)明显高于对照组(1.11±0.13),差异有统计学意义(F值为20.96,均P＜0.01);HspX激发的抗原特异性IgG、IgG1和IgG2a抗体稀释倍数对数值(3.90～5.21)均明显高于Ag85b(3.30～4.51)和CFP-10/ESAT-6蛋白(3.10～4.05),差异均有统计学意义(F值为43.8～70.4,均P＜0.01).结论 在辅以佐剂的情况下,不同抗原组合对小鼠免疫功能的影响有差别,3种抗原同时免疫并未获得最好的免疫效果,提示抗原间的相互作用可能对免疫效果产生影响.

Abstract：

Objective To study the immune function of mice immunized by different combinations of antigen 85b ( Ag85b), fusion protein culture filtered protein 10 ( CFP-10), early secreted antigenic target 6 kDa protein (ESAT-6) and heat shock protein X (Hsp X) with combined adjuvants of Bacille CalmetteGuerin (BCG) CpG and aluminum.Methods According to antigen combinations, 48 BALB/c mice were divided into 8 groups: ( 1 ) group A: Ag85b + CFP-10/ESAT-6 + HspX + adjuvant; (2) group B: CFP-10/ESAT-6 + HspX + adjuvant; ( 3 ) group C: Ag85b + HspX + adjuvant; (4) group D: Ag85b + CFP-10/ESAT-6 + adjuvant; (5) group E: Ag85b + adjuvant; (6) group F: CFP-10/ESAT-6 +adjuvant; (7)group G: HspX + adjuvants; (8) control group: saline (6 mice per group).The mice were subcutaneously immunized 3 times.One week after the third subcutaneous immunization, spleens were collected for enzymelinked immunospot (ELISPOT) assay to detect IFN-γ and IL-4 secretion, and for the lymphocyte proliferation assay to observe antigen-specific lymphocyte proliferation.Serum samples were separated for enzyme-linked immunosorbent assay (ELISA) to detect the titers of antigen-specific IgG, IgG1 and IgG2a antibodies.Results The amount of IFN-γ spots in Group E [median ( quartile), 122.8 (78.4 - 184.4 )]was significantly more than that in group C [14.3 (6.5 - 14.6)] and the control group [0.5 (0.5 - 1.3)](u =0.0, P ＜ 0.01 ).The amount of IL-4 spots in Group D stimulated with Ag85b and CFP-10/ESAT-6 [173.5 (78.8 -233.4), 132.8 ( 50.3 - 159.4)] were significantly more than those in the control group [0.5 (0.5 - 1.3 ), 5.3 ( 2.9 - 6.5 )] ( u = 0.0, P ＜ 0.01 ).The level of stimulation index of lymphocyte proliferation in Group A, C, D, E(2.42 ±0.50, 2.18 ±0.37, 2.86 ±0.51, 2.70 ±0.15) was significantly higher than that of the control group ( 1.11 ± 0.13 ) ( F= 20.96, P ＜ 0.01 ).The level of antigen-specific IgG, IgG1 , IgG2a antibody titers induced by Hsp X [lg( antibody dilution degree), 3.90 -5.21] was significantly higher than those induced by Ag85b (3.30-4.51 ) and CFP-10/ESAT-6 (3.10 -4.05) ( F = 63.8 - 70.4, P ＜ 0.01 ) .Conclusions With the use of adjuvants, different antigen combinations showed different influences on the immune function in mice.A combination of 3 antigens did not elicit the best immune effect, suggesting that the interaction among antigens may affect their immunity.

The effect of various antigen compounds of tuberculosis composite antigen vaccine on the immune function in mice

Objective To study the immune function of mice immunized by different combinations of antigen 85b ( Ag85b), fusion protein culture filtered protein 10 ( CFP-10), early secreted antigenic target 6 kDa protein (ESAT-6) and heat shock protein X (Hsp X) with combined adjuvants of Bacille CalmetteGuerin (BCG) CpG and aluminum.Methods According to antigen combinations, 48 BALB/c mice were divided into 8 groups: ( 1 ) group A: Ag85b + CFP-10/ESAT-6 + HspX + adjuvant; (2) group B: CFP-10/ESAT-6 + HspX + adjuvant; ( 3 ) group C: Ag85b + HspX + adjuvant; (4) group D: Ag85b + CFP-10/ESAT-6 + adjuvant; (5) group E: Ag85b + adjuvant; (6) group F: CFP-10/ESAT-6 +adjuvant; (7)group G: HspX + adjuvants; (8) control group: saline (6 mice per group).The mice were subcutaneously immunized 3 times.One week after the third subcutaneous immunization, spleens were collected for enzymelinked immunospot (ELISPOT) assay to detect IFN-γ and IL-4 secretion, and for the lymphocyte proliferation assay to observe antigen-specific lymphocyte proliferation.Serum samples were separated for enzyme-linked immunosorbent assay (ELISA) to detect the titers of antigen-specific IgG, IgG1 and IgG2a antibodies.Results The amount of IFN-γ spots in Group E [median ( quartile), 122.8 (78.4 - 184.4 )]was significantly more than that in group C [14.3 (6.5 - 14.6)] and the control group [0.5 (0.5 - 1.3)](u =0.0, P ＜ 0.01 ).The amount of IL-4 spots in Group D stimulated with Ag85b and CFP-10/ESAT-6 [173.5 (78.8 -233.4), 132.8 ( 50.3 - 159.4)] were significantly more than those in the control group [0.5 (0.5 - 1.3 ), 5.3 ( 2.9 - 6.5 )] ( u = 0.0, P ＜ 0.01 ).The level of stimulation index of lymphocyte proliferation in Group A, C, D, E(2.42 ±0.50, 2.18 ±0.37, 2.86 ±0.51, 2.70 ±0.15) was significantly higher than that of the control group ( 1.11 ± 0.13 ) ( F= 20.96, P ＜ 0.01 ).The level of antigen-specific IgG, IgG1 , IgG2a antibody titers induced by Hsp X [lg( antibody dilution degree), 3.90 -5.21] was significantly higher than those induced by Ag85b (3.30-4.51 ) and CFP-10/ESAT-6 (3.10 -4.05) ( F = 63.8 - 70.4, P ＜ 0.01 ) .Conclusions With the use of adjuvants, different antigen combinations showed different influences on the immune function in mice.A combination of 3 antigens did not elicit the best immune effect, suggesting that the interaction among antigens may affect their immunity.