论文部分内容阅读
目的比较原位共沉淀法(以下简称原位法)和直接吸附法制备的乙型肝炎疫苗的免疫效果。方法采用原位法和直接吸附法制备乙型肝炎疫苗。将小鼠随机分为5组,即原位法疫苗组、直接吸附法(本所佐剂)疫苗组、直接吸附法(进口佐剂)疫苗组、佐剂对照组(铝佐剂+PBS)和空白对照组(PBS),分别于0、4、8周经小鼠腓肠肌免疫,0.1 ml/只,按照《中国药典》三部(2010版)进行疫苗吸附完全性试验及疫苗体外相对效力检测。于初免后第1、2、4、8、12、16周,经小鼠尾动脉采血,分离血清,采用ELISA法测定血清中Anti-HBs水平;于初免后第4、8、12周,各组分别取5只小鼠,经尾动脉采血,分离血清,采用流式细胞术,检测血清中T淋巴细胞亚群分布;于初免后第12周,各组分别取5只小鼠,无菌取脾,分离得到脾单个核细胞(mononuclear cell,MNC),采用酶联免疫斑点试验(enzymelinked immunospot assay,ELISPOT)法检测血清中HBsAg特异性IFNγ分泌水平。结果原位法疫苗组、直接吸附法(本所佐剂)疫苗组、直接吸附法(进口佐剂)疫苗组HBsAg吸附效率分别为95%、95%和96%,疫苗体外相对效力分别为2.2、2.1和2.3,均符合《中国药典》三部(2010版)要求。各疫苗组Anti-HBs阳转率均达到100%,随接种剂次的增加,Anti-HBs水平逐渐升高,于初免后第12周达到高峰,第16周出现下降;初免后第8周,原位法疫苗组Anti-HBs GMT高于直接吸附法(本所佐剂)和直接吸附法(进口佐剂)疫苗组,差异均有统计学意义(P<0.05),初免后第4、12、16周,差异无统计学意义(P>0.05)。初免后第4、8、12周,各组小鼠血清中CD3+CD4+T细胞水平均未见明显改变,第12周,各疫苗组CD3+CD8+T细胞水平均高于免疫前,差异有统计学意义(P<0.01)。各疫苗组分泌IFNγ的斑点形成细胞(spot forming cell,SFC)数明显高于铝佐剂对照组和空白对照组;原位法疫苗组与直接吸附法(本所佐剂)和直接吸附法(进口佐剂)疫苗组相比,SFC数差异无统计学意义(P>0.05)。结论两种方法制备的乙型肝炎疫苗均具有较好的免疫效果。
Objective To compare the immune effects of Hepatitis B vaccine prepared by in situ coprecipitation (hereinafter referred to as in situ method) and direct adsorption method. Methods Hepatitis B vaccine was prepared by in situ method and direct adsorption method. The mice were randomly divided into 5 groups: the vaccine in situ method, the direct adsorption method (in-house adjuvant) vaccine group, the direct adsorption method (imported adjuvant) vaccine group and the adjuvant control group (aluminum adjuvant + PBS) And control group (PBS) were immunized with gastrocnemius muscle of mice at 0, 4, and 8 weeks respectively, 0.1 ml per mouse. The vaccine was completely tested according to the Chinese Pharmacopoeia (2010 edition) and the relative potency of the vaccine was tested in vitro . At the first, second, fourth, eighth, twelfth, and sixteenth weeks after initial immunization, serum was collected from the tail artery of the mice and the serum levels of Anti-HBs were measured by ELISA. , 5 mice were taken from each group, the blood was collected via caudal artery and the serum was separated. The distribution of T lymphocyte subsets in serum was detected by flow cytometry. At the 12th week after initial immunization, 5 mice The spleen mononuclear cells (MNCs) were isolated by aseptic spleen transplantation. The level of HBsAg-specific IFNγ secretion in serum was detected by enzyme linked immunospot assay (ELISPOT). Results The efficiency of HBsAg adsorption in the vaccine group of in situ method, direct adsorption method (the adjuvanted adjuvant) vaccine group and direct adsorption method (imported adjuvant) vaccine group were 95%, 95% and 96%, respectively. The relative in vitro efficacy of the vaccine was 2.2 , 2.1 and 2.3, all in line with the “Chinese Pharmacopoeia” three (2010 version) requirements. Anti-HBs positive rate reached 100% in each vaccine group. Anti-HBs level increased gradually with the inoculation times, peaked at the twelfth week after the initial immunization and decreased at the 16th week. (P <0.05). The anti-HBs GMT of in-situ vaccine group was higher than that of the direct adsorption method (direct injection method) and direct adsorption method (imported adjuvant) vaccine group 4,12,16 weeks, the difference was not statistically significant (P> 0.05). At the 4th, 8th and 12th week after primary immunization, the levels of CD3 + CD4 + T cells in serum of the mice in each group did not change significantly. At the 12th week, the levels of CD3 + CD8 + T cells in each vaccine group were higher than those before immunization, The difference was statistically significant (P <0.01). The numbers of spot forming cells (SFCs) secreting IFNγ in each vaccine group were significantly higher than those in aluminum adjuvant control group and blank control group. In situ vaccination group was compared with direct adsorption method (direct injection method) and direct adsorption method Import adjuvant) vaccine group compared to the SFC number difference was not statistically significant (P> 0.05). Conclusion Both Hepatitis B vaccines prepared by the two methods have good immunogenicity.