小鼠重复给予CpG 684-狂犬疫苗安全性评价

Safety Evaluation on Repeated Dose of CpG 684-Rabies Vaccine in Mice

目的对小鼠重复给予CpG684-狂犬疫苗，考察该新型疫苗的非临床安全性，为临床设计人用剂量及临床毒副反应的监测提供参考依据。方法使用BALB／c小鼠，分为5组：溶媒对照组、狂犬疫苗对照组、（2．5μg CpG684＋1／4狂犬疫苗）·只“组、（5μg CpG684＋1／4狂犬疫苗）·只“组和（15μgCpG684＋1／4狂犬疫苗）·只。组。肌肉注射给药，在第0、3、7、14、28、42天各给药1次，实验期间观察动物的临床症状、注射部位刺激性，测定体重、摄食量，进行抗狂犬病毒抗体和抗体亚型IgG2a检测；末次给药后1d、3周分别进行血液学和血清生化测定，分别对每组部分动物进行剖检、脏器称取质量，并采集组织样本进行组织病理学检查。结果狂犬疫苗本身和CpG684-狂犬疫苗各剂量在第4～6次给药后使小鼠产生严重过敏反应。各剂量CpG684-狂犬疫苗在末次给药后引起雌、雄小鼠单核细胞数量增加及雌性小鼠血小板减少，高剂量CpG684-狂犬疫苗还引起雄性小鼠血小板减少；高剂量CpG684-狂犬疫苗引起雌、雄小鼠血清总胆固醇（CHO）增加；狂犬疫苗和中、高剂量CpG684-狂犬疫苗引起小鼠血清碱性磷酸酶（ALP）水平降低，以上改变在停药后3周均恢复。单独狂犬疫苗和各剂量CpG684-狂犬疫苗均引起雌、雄小鼠血清球蛋白增加、A／G比值降低，停药后3周未恢复。给予单独狂犬疫苗和各剂量CpG684-狂犬疫苗均引起小鼠脾脏质量增加，停药后3周有-定的恢复。与疫苗相关的组织病理学改变为脾脏白髓增生和白髓生发中心易染体巨噬细胞数目增多，以及注射肌肉局部显著炎症反应和纤维组织增生，停药后3周除高剂量组外，其他组均能较好恢复。结论狂犬疫苗和CpG684．狂犬疫苗引起小鼠严重过敏反应、血清球蛋白和脾脏质量增加、ALP降低，CpG684-狂犬疫苗引起小鼠血小板减少、单核细胞数量和血清CHO增加。与疫苗相关的组织病理学改变为脾脏白髓增生和白髓生发中心易染体巨噬细胞数目增多，以及注射肌肉局部显著炎症反应和纤维组织增生。停药后3周上述变化多数能较好恢复。

OBJECTIVE To investigate nonclinical safety of the novel CpG 684-rabies vaccine through repeated dose to BALB/c mice, and provide reference for the close level design and side effects monitoring in clinical trials. METHODS BALB/c mice were used and divided into five treatment groups：vehicle control, rabies vaccine control, （2. 5μg CpG 684 ＋ 1/4 rabies vaccine） · mouse-1, （5 μg CpG 684 ＋ 1/4 rabies vaccine） · mouse-1 and （15 μg CpG 684 ＋ 1/4 rabies vaccine） · mouse-j treated groups. Animals were dosed by intramuscular injection on days 0, 3, 7, 14, 28 and 42. During the study, each animal was observed for clinical signs and injection site irritations. Body weights and food consumption were measured. Serum anti-rabies IgG antibody and IgG2a antibody were determined. Hematology and serum chemistry examinations were conducted respectively 1 day and 3 weeks post the last dose. A full necropsy was then conducted on animals, and tissues were weighed and processed for microscopic examination. RE- SULTS Rabies vaccine alone and all dose levels of CpG 684-rabies vaccine caused severe allergic reactions after the fourth, fifth or sixth dose in mice. After the last administration, each dose of CpG 684-rabies vaccine caused the increase in monocyte in both sexes ofmice and the decrease in platelet in female mice, and high dose of CpG 684-rabies vaccine also induced the decrease in platelet in male mice. High dose of CpG 684-rabies vaccine led to the increase in serum CHO in both sexes of mice. Rabies vaccine alone and middle and high doses of CpG 684-rabies vaccine induced the decrease in serum ALP in mice. All the above changes recovered three weeks post the last dose. Rabies vaccine alone and all dose levels of CpG 684-rabies vaccine caused the increase in serum globulin and the decrease in A/G ratio in male and female mice, which did not recover 3 weeks post the last dose. Rabies vaccine alone and all dose levels of CpG 684-rabies vaccine induced the increase in spleen weight, which recovered to some degree 3 weeks post the last dose. Treatment-related microscopic findings were cell hyperplasia in white pulp of spleen and the increase in tingible body macrophages in germinal center of white pulp, and the marked inflammatory reaction and hyperplasia of fibrous tissue in injection site muscle, which basically vanished at the end of the recovery period except the high dose group. CONCLUSION Rabies vaccine alone and CpG 684- rabies vaccine cause severe allergic reactions, the increases in serum globulin and spleen weight and the decrease in serum ALP. CpG 684-rabies vaccine induces the decrease in platelet and the increases in monocyte and serum CHO. Treatment-related microscopic find- ings are cell hyperplasia in white pulp of spleen and the increase in tingible body macrophages in germinal center of white pulp, and the marked inflammatory reaction and hyperplasia of fibrous tissue in injection site muscle. Most of the changes recover 3 weeks post the last dose.