中试生产的结核DNA疫苗免疫效能研究

目的 以300 L发酵罐中试规模生产的冻干Mtb Ag85A质粒DNA疫苗为基础免疫小鼠，对该疫苗的免疫效能进行研究。 方法 将4~6周龄的BALB/c小鼠20只通过数字表法随机分为2组，一组免疫Mtb Ag85A质粒DNA疫苗（结核DNA疫苗免疫组），另一组免疫PBS作为阴性对照（PBS对照组）；于免疫前采血，肌内注射免疫3次。在第3次免疫后3周摘眼球处死小鼠，收集血清，进行血清特异性抗体检测和细胞因子(IFN-γ、IL-4)检测；同时对每只小鼠取脾细胞进行特异性γ干扰素 (IFN-γ) 分泌检测和脾细胞CD4⁺、CD8⁺百分率检测。 结果 （1）免疫前后免疫组与对照组抗体水平吸光度A值均小于0.2。（2）结核DNA疫苗免疫组血清细胞因子IL-4水平［（146.2±34.3）pg/ml］低于PBS对照组［（177.7±28.1）pg/ml］，差异有统计学意义（t=2.244，P=0.038），而IFN-γ水平［（129.6±159.0）pg/ml］虽然高于PBS对照组［（76.5±21.5）pg/ml］，但差异无统计学意义（t=－1.047，P=0.309）。（3）酶联免疫斑点检测结果表明结核DNA疫苗免疫组脾细胞特异性IFN-γ分泌水平（分泌IFN-γ细胞个数）（103.60±112.14）高于PBS对照组（5.78±5.83），差异有统计学意义（t=36.538，P=0.018）。（4）结核DNA疫苗免疫组CD4⁺细胞百分率均值（21.57%）高于PBS对照组（12.17%），差异有统计学意义（t=3.043，P=0.038）；CD8⁺细胞百分率均值（13.70%）与PBS对照组（10.57%）相比，差异无统计学意义（t=0.847，P=0.445）。 结论 Mtb Ag85A质粒DNA疫苗主要刺激机体Th1型细胞免疫，而对诱导机体产生Th2型细胞免疫应答没有显著的促进作用。

Immune efficacy of tuberculosis DNA vaccine produced by magnifying technology

Objective To study the immunogenicity of Mycobacterial tuberculosis Ag85A DNA vaccine with pilot scale production in mice.Methods Twenty BALB/c mice were randomly divided into 2 groups,and then immunized intramuscularly for 3 times with Mtb Ag85A DNA or PBS respectively.The mice were killed and the whole blood samples were collected at 3 weeks after the last immunization.The specific antibody and cytokines(IL-4) in sera were detected.The Mtb Ag85A specific interferon-γ(IFN-γ) level and the percentages of CD4+ or CD8+ T cell in mouse spleen cells were also detected.Results The data obtained by statistical analyses demonstrated that:(1) The absorbance values of Ag85A-specific antibody in the sera of each group mice before and after immunization were all less than 0.2.(2) The IL-4 level in sera from Ag85A DNA group [(146.2±34.3) pg/ml] was significantly lower than that from PBS group [(177.7±28.1) pg/ml](t=2.244,P=0.038).The IFN-γ level in sera from Ag85A DNA group [(129.6±159.0) pg/ml]was higher than that from PBS group [(76.5±21.5) pg/ml],but there was no significant difference(t=-1.047,P=0.309).(3) The data from the ELISPOT assay for IFN-γ showed that the number of spot-forming spleen cells in Ag85A DNA group(103.60±112.14) was significantly higher than that in PBS group(5.78±5.83)(t=36.538,P=0.018).(4) The percentage(21.57%) of CD4+ T cells in Ag85A DNA group was higher than that(12.17%) in PBS group(t=3.043,P=0.038);however,there was no significant difference on the percentage of CD8+ T cells between the two groups(13.70% vs.10.57%,t=0.847,P=0.445).Conclusion The Th1 cellular immunity was mainly stimulated in the mice immunized by Mtb Ag85A DNA vaccine.But Th2 cellular immune response was not significantly induced in these mice.