Studies on the Preparation Method of Influenza Vaccine Liposome and the Effect of Particle Size on Immunogenicity
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摘要:
目的 采用薄膜分散法和冻融冻干法制备四价流感疫苗脂质体冻干粉，通过考察其粒径、包封率和免疫原性，筛选最佳制备方法；使用最佳制备方法制备不同粒径流感疫苗脂质体，比较包封率和免疫原性，筛选最优脂质体粒径。 方法 采用Lowry法测定脂质体包封率；腹腔免疫小鼠，通过脾淋巴增殖实验以刺激指数（SI）为指标进行细胞免疫原性研究；采用血凝抑制实验以免后与免前抗体滴度比（HI）为指标，评价体液免疫原性。 结果 冻融冻干法是制备流感疫苗脂质体冻干粉的最佳方法，以最佳方法制备出平均粒径为3.7 µm、2.0 µm、1.0 µm、0.45 µm的4种不同粒径脂质体冻干粉，在一个免疫周期内，各实验组能显著刺激脾淋巴细胞增殖，产生细胞免疫，且3.7 µm组与其他各组比较差异有统计学意义（P < 0.05）；各免疫组免后与免前抗体滴度比始终≥4，且3.7 µm组高于其他组，表明其能产生较强的体液免疫。 结论 冻融冻干法制备的四价流感疫苗脂质体能产生较好的免疫原性，其中以粒径3.7 µm的流感疫苗脂质体免疫增强效果最佳。 Abstract:Objective （1） To prepare the tetravalent influenza vaccine liposome lyophilized powder with the film dispersion method and freeze-thaw lyophilization method, and to screen the best preparation method by investigating its particle size, entrapment efficiency and immunogenicity. （2） To prepare the influenza vaccine liposomes with different particle sizes by using the best preparation method, and to screen the best particle size by comparing the encapsulation efficiency and immunogenicity. Methods Lowry method was used to determine liposome entrapment rate. At given days of mice abdominal immunization, the cellular immunogenicity was conducted by spleen lymphocyte proliferation assay with stimulation index （SI） as indexes. The hemagglutination inhibition test was used to evaluate the humoral immunogenicity after the mice abdominal immunization. Results Freeze-thaw and freeze-drying method was the best method to prepare influenza vaccine liposome freeze-dried powder. Four kinds of liposome freeze-dried powder with average particle size of 3.7 µm, 2.0 µm, 1.0 µm and 0.45 µm were prepared by the best method. In one immune cycle, each experimental group could significantly stimulate the proliferation of spleen lymphocytes and produce the cellular immunity. There was significant difference between 3.7 µm group and other groups（P < 0.05）. The titer ratio of antibody after and before the immunization was always ≥4, and the 3.7 µm group was higher than the other groups, indicating that it could produce the strong humoral immunity. Conclusion The tetravalent influenza vaccine liposomes prepared by freeze-thaw freeze-dried method can produce the strong immunogenicity, and the liposomes with the particle size of 3.7µm have the best immuno-enhancement effect. -
Key words:
- Liposomes /
- Influenza vaccine /
- Particle size /
- Immunogenicity
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表 1 2种制备方法脂质体的平均粒径和包封率
Table 1. Average particle size and encapsulation efficiency of liposomes prepared by the two methods
制备方法 平均粒径（µm） 包封率（%） 薄膜分散法 3.71 82.43 冻融冻干法 2.82 87.48 
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表 2 制备方法筛选时的ConA SI（n = 5，
$ \bar x \pm s $ ）Table 2. ConA SI during preparation method screening （n = 5，
$ \bar x \pm s $ ）制备方法 SI值 薄膜分散法 1.2512 ± 0.0340*# 冻融冻干法 1.3386 ± 0.0136** 疫苗原液组 1.1681 ± 0.1377*# PBS阴性对照组 0.9503 ± 0.0621## 与PBS阴性对照组比较，*P < 0.05，**P < 0.01；与冻融冻干法组比较，#P < 0.05，##P < 0.01。
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表 3 制备方法筛选时HI值（n = 5）
Table 3. HI value at the time of preparation method screening （n = 5）
制备方法 HI值 薄膜分散法 5.50 冻融冻干法 5.75 疫苗原液组 4.10 PBS阴性对照组 - 注：免后免前抗体滴度比 > 4表明具有免疫效应。
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表 4 不同粒径脂质体的包封率（%）
Table 4. Encapsulation efficiency of liposomes with different particle sizes
粒径大小 3.7 µm 2.0 µm 1.0 µm 0.45 µm 包封率 92.92 89.16 88.25 86.35
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表 5 7 d、14 d、28 d时ConA SI（n = 5，
$ \bar x \pm s $ ）Table 5. ConA SI at 7，14，28 d （n = 5，
$ \bar x \pm s $ ）分组 SI值 7 d 14 d 28 d 3.7 µm 1.3386 ± 0.0136**# 1.1739 ± 0.0771**# 1.1722 ± 0.1420**# 2.0 µm 1.3128 ± 0.0522**# 1.1675 ± 0.0764**# 1.1209 ± 0.1218**# 1.0 µm 1.1417 ± 0.1470* 1.1341 ± 0.1214* 1.0979 ± 0.1120* 0.45 µm 1.0816 ± 0.0594* 1.0550 ± 0.0330* 1.0618 ± 0.0492* 原液 1.1681 ± 0.1377* 1.0418 ± 0.0910* 1.0421 ± 0.0413* PBS 0.9503 ± 0.0621# 0.8898 ± 0.1129# 0.8414 ± 0.0159# 与PBS阴性对照组比较, *P < 0.05，**P < 0.01；与原液组比较,#P < 0.05，##P < 0.01。
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表 6 7 d、14 d、28 d时抗体滴度比（n = 5）
Table 6. Antibody titer ratio at 7，14，28 d （n = 5）
分组 HI值 7 d 14 d 28 d 3.7 µm 5.25 5.54 7.84 2.0 µm 5.09 5.47 7.55 1.0 µm 4.72 5.26 7.11 0.45 µm 4.60 5.05 6.44 原液 4.36 5.15 6.88 PBS - - - 注：免后免前抗体滴度比 > 4表明具有免疫效应。
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