结核分枝杆菌多组分蛋白候选疫苗EPDPA015f和EPDPA015m的免疫作用初步评价

Preliminary immunological evaluation of Mycobacterium tuberculosis multicomponent protein vaccine candidates EPDPA015f and EPDPA015m

目的初步评价新型结核融合型多组分蛋白候选疫苗EPDPA015f和混合型多组分蛋白候选疫苗EPDPA015m的免疫原性和有效性, 为研制结核疫苗提供新的抗原组合。方法将构建和表达的EPDPA015f和EPDPA015m蛋白与铝佐剂混合, 采用皮下多点的方式对6周龄BALB/c小鼠进行3次免疫, 间隔为10 d, 每次50 μg/只。末次免疫10 d后采集血液和脾脏样本。采用酶联免疫吸附试验、多重微球技术、酶联免疫斑点法检测血清抗体滴度、细胞因子分泌水平;采用体外结核分枝杆菌生长抑制试验检测小鼠脾细胞体外抑制结核分枝杆菌生长的能力。多组间比较采用单因素方差分析, 两组间比较采用t检验。结果 EPDPA015f和EPDPA015m均可诱导产生多种高水平的细胞因子和较高滴度的IgG抗体。与佐剂组相比, EPDPA015f组诱导产生Th1(IL-2、TNF-α、IFN-γ)、Th2(IL-4、IL-6、IL-10)、Th17(IL-17)型细胞因子及其他促炎细胞因子(GM-CSF、IL-12)的差异有统计学意义(P均<0.05);EPDPA015f组诱导产生的IgG抗体滴度高达1∶4×10^(6)。体外结核分枝杆菌生长抑制试验结果显示, PBS组、佐剂组、EPDPA015f组和EPDPA015m组的菌落数(lgCFU)分别为3.46±0.11、3.51±0.06、2.98±0.09和3.19±0.08;其中EPDPA015f组的菌落数最少, 与其他组比较差异均有统计学意义(P<0.001,P<0.001和P<0.01)。结论 EPDPA015f引起较为全面且高水平的细胞免疫和体液免疫应答, 并表现出较优的体外分枝杆菌抑制能力, 具有作为预防型疫苗或加强型疫苗的潜力。

Objective To preliminarily evaluate the immunogenicity and efficacy of two novel tuberculosis vaccine candidates(a fusion multicomponent protein EPDPA015f and a mixed multicomponent protein EPDPA015m)and to provide a new antigen combination for the development of tuberculosis vaccines.Methods Recombinant plasmids for the expression of EPDPA015f and EPDPA015m proteins were constructed.Six-week-old BALB/c mice were immunized with EPDPA015f or EPDPA015m in combination with aluminium adjuvant(50μg/mouse)for three times with an interval of 10 d.The mice were sacrificed 10 d after the last immunization to collect blood and spleen samples.Serum antibody titers and cytokine levels were measured by ELISA,Luminex technique and enzyme-linked immunospot assay(ELISPOT).Mycobacterial growth inhibition assay(MGIA)was used to detect the ability of mouse splenocytes to inhibit the growth of Mtb in vitro.One-way analysis of variance and t-test were used for statistical analysis.Results Both EPDPA015f and EPDPA015m could induce the production of various cytokines and IgG antibodies at a high level.The levels of cytokines related to Th1(IL-2,TNF-α,IFN-γ),Th2(IL-4,IL-6,IL-10)and Th17(IL-17)as well as other proinflammatory cytokines(GM-CSF,IL-12)were higher in the EPDPA015f group than in the adjuvant group(P<0.05).The titer of IgG antibody induced by EPDPA015f was as high as 1∶4×10^(6).The results of MGIA showed that the numbers of Mtb(lgCFU)in the PBS,adjuvant,EPDPA015f and EPDPA015m groups were 3.46±0.11,3.51±0.06,2.98±0.09 and 3.19±0.08,respectively.The number of colonies in the EPDPA015f group was the least as compared with that in the other three groups(P<0.001,P<0.001,P<0.01).Conclusions The vaccine candidate EPDPA015f could elicit more comprehensive and high-level cellular and humoral immune responses,and exhibited superior in vitro inhibitory activity against the growth of Mtb.EPDPA015f had the potential to be used as a preventive vaccine or a booster vaccine.