目的 对大鼠重复给予重组九价人乳头瘤病毒疫苗(9vHPV疫苗),考察该创新疫苗的非临床安全性和免疫原性,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Wistar大鼠,设4个主试验组:阴性对照组、佐剂对照组、HPV疫苗低剂量组(每只1/2剂)和高剂量组(每只2剂),每组各设1个卫星组。于第0、2、4、6周分别肌肉注射给予重组九价HPV疫苗,试验期间对所有动物观察临床症状和注射部位刺激性,对主试验组动物测定体重、摄食量、体温,末次给药结束及恢复期结束进行眼科检查、尿检查、血液学、血液凝固、血清生化、外周血T淋巴细胞亚群测定,并分别进行剖检、脏器称重,并采集组织样本进行组织病理学检查。卫星组动物进行HPV结合抗体、中和抗体和抗核抗体测定。结果 大鼠对低、高剂量HPV疫苗含有的9种抗原均能产生高水平的结合抗体和具有较强活性的中和抗体。低、高剂量HPV疫苗和佐剂引起大鼠外周血中性粒细胞和嗜酸性粒细胞增加,在末次免疫后3周基本恢复。佐剂和低、高剂量疫苗均引起大鼠血清ALB轻度降低、球蛋白增加和A/G比值降低。与给予佐剂及疫苗相关的大体病理学改变为双侧注射部位肌肉白色斑块,相关的组织病理学改变为注射部位肌肉肌纤维变性坏死、混合炎性细胞浸润及纤维组织增生,坐骨神经鞘周围混合细胞浸润及纤维组织增生,恢复期结束时部分病变恢复。本疫苗未对其他评价指标产生明显影响。结论 九价HPV疫苗对Wistar大鼠有良好的免疫原性。低、高剂量HPV疫苗和佐剂引起大鼠外周血中性粒细胞和嗜酸性粒细胞增加、血清球蛋白增加和注射部位刺激性病理改变。在本试验剂量下,九价HPV疫苗对Wistar大鼠有较好的安全性,无明显毒性剂量为每只2剂。

OBJECTIVE To investigate nonclinical safety and immunogenicity of the recombinant nine-valent human papillomavirus vaccine(9vHPV vaccine) through repeated dose to rats, and provide reference for the dose level design and side effects monitoring in clinical trials. METHODS Wistar rats were used and divided into four main toxicity groups: negative control, adjuvant control, low dose HPV vaccine group(1/2 dose·rat^-1) and high dose HPV vaccine group(2 dose·rat^-1). Each group had a satellite group. Animals were dosed by intramuscular injection in weeks 0, 2, 4 and 6 respectively. During the study, all the animals were observed for clinical signs and injection site irritations. For main toxicity groups, body weights, food consumption and body temperature were measured, and ophthalmologic examination, urinalysis, hematology, coagulation, serum chemistry examinations and T lymphocyte subset assay in peripheral blood were done at terminal and recovery necropsy respectively. A full necropsy was then conducted on animals, and tissues were weighed and processed for microscopic examination. Serum anti-HPV binding antibody and neutralizing antibody and antinuclear antibody were determined for satellite animals. RESULTS Rats produced high levels of anti-HPV binding antibody and neutralizing antibody with strong activity to the nine antigens contained in the low and high dose HPV vaccines. The low and high dose vaccine and the adjuvant caused the increases in peripheral blood neutrophils and eosinophils, which basically recovered three weeks post the last dose. Both the adjuvant and the two dose levels of vaccine led to the slight decrease in serum ALB, increase in globulin and the decrease in A/G ratio in treated rats. Adjuvant and vaccine-related gross necropsy changes were white spots in bilateral injection site muscles. Treatment-related microscopic findings were the degeneration and necrosis of muscle fibers, mixed inflammatory cell infiltration and hyperplasia of fibrous tissue in injection site muscles, and the mixed inflammatory cell infiltration and hyperplasia of fibrous tissue around the sciatic nerve sheath, which partially recovered at the end of the recovery period. The HPV vaccine did not have obvious impact on other end points determined. CONCLUSION The low and high dose vaccine and the adjuvant can cause the following changes in rats: the increases in peripheral blood neutrophils and eosinophils, the increase in serum globulin and the pathological irritation changes at injection sites. The 9vHPV vaccine has a good safety profile in Wistar rats under the dose levels of this study, with the no observed adverse effect level of 2 dose·rat^-1.