治疗用乙型肝炎腺病毒注射液TC007食蟹猴免疫原性、免疫毒性和生物分布研究

Immunogenicity, immunotoxicity, and biodistribution in cynomolgus monkey with hepatitis B adenovirus injection TC007

目的重复给予食蟹猴治疗用乙型肝炎腺病毒注射液(TC007),观察其免疫原性、免疫毒性、生物分布。方法普通级食蟹猴随机分为溶媒对照组,TC007低、中、高剂量(1.0×10~9、1.0×10^(10)、1.0×10^(11 )VP/只)组,Ad5-Null(1.0×10^(11 )VP/只,空载对照)组,每组10只,背部肩胛区sc给药,每周给药1次,共7次,停药恢复28 d。应用流式细胞仪进行外周血中淋巴细胞分群检测,检测经TC007刺激后分泌干扰素-γ(IFN-γ)的特异T细胞水平;免疫组化检测肝脏IFN-γ、肿瘤坏死因子-α(TNF-α)、白细胞介素-2(IL-2)表达;试剂盒法测定血清中抗Ad5抗体的中和活性,补体成分C3、C4及免疫复合物(CIC)水平;给药期及恢复期结束,进行多个组织取材,HE染色后进行病理学阅片,TaqMan探针qPCR法进行Ad5分布检测。结果与溶媒对照组比较,各组动物血浆中CD3^+CD8^+T细胞、CD4^+/CD8^+、CD3^-CD56^+NK细胞、CD3^+CD56^+NKT细胞、巨噬细胞CD45^+CD14^+百分率均未见明显异常;Ad5-Null组特异T细胞均未见明显改变,TC007各剂量组针对HBV 3种抗原(Core、Env、Pol)分泌IFN-γ的特异性T细胞水平显著增加(P<0.05、0.01),停药恢复末期(d68),效应值仍略高;TC007各剂量组和Ad5-Null组食蟹猴肝脏组织中IFN-γ、TNF-α、IL-2均高于溶媒对照组,且呈剂量相关性,但TC007各剂量组与Ad5-Null组比较未见明显差异;TC007各剂量、Ad5-Null组动物均产生抗Ad5抗体,39/40例具有中和活性;TC007各剂量组补体C3、C4未见明显异常,高剂量组CIC轻微升高(P<0.05)。Ad5主要分布于给药局部,在腋下淋巴结有少量分布,其他组织未见分布。TC007及Ad5-Null组均可见给药局部轻微至轻度皮下组织炎性细胞浸润,停药恢复28 d,病变可见明显恢复,其他组织未见病理学改变。结论TC007在食蟹猴体内具有一定免疫原性,抗Ad5抗体具有中和活性,高剂量组可见轻微免疫复合物形成,未见免疫器官损伤,生物分布主要集中于给药部位,可产生特异性T细胞。

Objective In this study, 6-week repeated sc injection to cynomolgus monkey with TC007 to assess the immunogenicity, immunotoxicity, and vivo biodistribution.Methods Ordinary grade cynomolgus monkeys were randomly divided into solvent control group, TC007 low, medium, and high doses (1.0×109, 1.0×1010, 1.0×1011 VP/rat) group, Ad5-Null (1.0×1011 VP/rat, no-load control) group, 10 monkeys in each group, in the back scapular region was sc given once a week for 7 weeks, and the withdrawal was resumed for 28 d. Flow cytometry was used to detect the lymphocyte population in peripheral blood and the level of IFN-γ- secreting T cells stimulated by TC007; immunohistochemistry was used to detect the expression of IFN-γ/TNF-α/IL-2 in liver; kit method was used to determine the neutralization activity of anti-Ad5 antibody in serum and the levels of complement components C3, C4 and immune complex (CIC). At the end of convalescence, multiple tissue samples were taken, histopathological examination was performed after HE staining, and the distribution of Ad5 was detected by TaqMan probe qPCR.Results Compared with solvent control group, the percentage of CD3+ CD8+ T cells, CD4+/CD8+, CD3-CD56+ NK cells, CD3+ CD56+ NKT cells and macrophage CD45+CD14+ in plasma of all groups were not significantly abnormal; the specific T cells of Ad5-Null group were not significantly changed, and the level of specific T cells targeted the three antigens of HBV (Core, Env, and Pol) secreting IFN-γ increased significantly in TC007 group, and the effect remained slightly higher after 4 weeks of withdrawal and recovery. The levels of IFN-γ/TNF-α/IL-2 in liver tissues of cynomolgus monkeys in TC007 and Ad5-Null groups were higher than those in the solvent control group in a dose-dependent manner; But there was no significant difference between TC007 and Ad5-Null groups. Anti-Ad5 antibodies were produced in all the animals in Ad 5-Null group, and 39 of 40 cases had neutralization activity. Complement C3 and C4 were not abnormal in TC007 different dose groups, but CIC was slightly elevated in high dose group (P<0.05). Ad5 was mainly distributed in the local administration, but there was a little distribution in the axillary lymph nodes with no distribution in other tissues. In TC007 and Ad5-Null groups, mild subcutaneous inflammatory cell infiltration was stopped for 28 days, and the pathological changes were obviously recovered. No pathological changes were found in other tissues. Conclusion TC007 has a certain immunogenicity in cynomolgus monkeys and anti-Ad5 antibody has neutralizing activity. In high-dose group, slight immunocomplex formation can be found. There is no damage to immune organs. The biodistribution is mainly concentrated at the site of administration. Specific T cells can be found in high dose group.