弓形虫复合黏膜疫苗鼻内免疫小鼠抵抗弓形虫感染作用的观察

目的 观察弓形虫复合黏膜疫苗鼻内免疫小鼠诱导的肠黏膜和系统免疫应答及其抗弓形虫感染作用。方法 BALB/c小鼠52只随机分为两组（每组26只），免疫组小鼠用弓形虫复合黏膜疫苗（每毫升含可溶性速殖子抗原1 mg， 霍乱毒素50 μg） 20 μl/只滴鼻免疫2次，间隔2周；对照组用等剂量PBS滴鼻。末次免疫后14 d，各组处死6只，摘眼球取血（0.5～1 ml）；取直肠内粪便（4～5粒），ELISA测定血清IgG和粪IgA抗体；分别计数脾组织、派伊尔集合淋巴结（PP）和肠上皮淋巴细胞（IEL），免疫细胞化学法检测各组织中CD4⁺、CD8⁺ T细胞亚群水平。用RH株弓形虫速殖子（4×10⁴个/只）灌胃攻击感染各组其余小鼠，30 d后颈椎脱位处死，计数肝、脑组织速殖子虫荷。 结果 免疫后14 d，免疫组小鼠血清IgG和粪IgA抗体水平（分别为0.224和0.371），显著高于对照组（分别为0.041和0.037）（P<0.05），脾、PP和IEL中T淋巴细胞与对照组相比明显增生（P<0.01），其中脾、PP中CD4⁺、CD8⁺ T淋巴细胞增殖显著（P<0.05），IEL中以CD8⁺ T细胞为主，增殖显著（P<0.01），CD4⁺/CD8⁺ 比值降低（P<0.05）。攻击后30 d，免疫组小鼠存活率（85.0%）显著高于对照组（45.0%）（P<0.05）。免疫组肝、脑组织速殖子数比对照组分别减少86.3%、86.7%，两组差异有统计学意义（P<0.05）。 结论 弓形虫复合黏膜疫苗鼻内免疫小鼠，能有效诱导黏膜和系统免疫应答，小鼠存活率显著提高，肝、脑组织虫荷显著降低。

Intranasal Immunization with Mucosal Complex Vaccine Protects Mice Against Toxoplasma gondii

OBJECTIVE: To study the mucosal and systemic immune response after intranasal immunization with mucosal complex vaccine for Toxoplasma gondii, and to observe the protective effect on mice. METHODS: The mucosal complex vaccine was made of soluble tachyzoite antigen (STAg) and cholera toxin (CT), which were mixed and dissolved in PBS (1 ml PBS containing 1 mg STAg and 50 microg CT). Fifty-two BALB/c mice were randomly divided into two groups: immunized group and control. Mice were intranasally immunized with 20 microl mucosal complex vaccine (20 microg STAg and l microg CT) per mouse twice at an interval of two weeks, while the control mice were given PBS solution instead. Six mice of each group were killed by dislocation of cervical vertebra on day 14 after the last immunization. The specific IgG antibodies in serum and IgA in feces were detected by ELISA. Lymphocytes in spleen, Peyer's patches (PP) and intestinal intraepithelial lymphocyte (IEL) were isolated and counted. Percentage of CD4+ and CD8+ T cells was determined by immunocytochemistry. Other mice were challenged intragastrically each with 4 x 10(4) tachyzoites of RH strain Toxoplasma gondii on day 14 after the last immunization. Their health condition was observed and the number of tachyzoites in liver and brain was determined microscopically on the 30 th day after challenge. RESULTS: IgG antibodies in serum and IgA antibodies in feces of immunized mice were higher than the control (P < 0.05). Lymphocytes in spleen, PP and IEL significantly increased after immunization (P < 0.01). The CD4+ and CD8+ T cells were both higher than that of the control (P < 0.05) in spleen and PP. The number of CD8+ T cells in IEL increased significantly (P < 0.01), and the ratio of CD4+ and CD8+ T cells was reversed with significance (P < 0.05). On the day 30 after challenge, the survival rate of immunized mice was higher than that of control (P < 0.05), while the tachyzoite load in liver and brain was significantly smaller respectively. CONCLUSION: Intranasal inoculation with mucosal complex vaccine effectively induces the mucosal and systemic immune response, and protects mice against Toxoplasma gondii.