b型流感嗜血杆菌奈瑟球菌荚D蛋白作为载体的膜多糖C群脑膜炎-蛋白结合物的免疫效果评价  

Evaluation on immune effect of group C meningococcal polysaccharide conjugates using protein D of Haemophilus influenzae type b as protein carrier

作　　者：王丽婵 谭亚军[1] 卫辰[1] 张华捷[1] 马霄[1] WANG Lichan;TAN Yajun;WEI Chen;ZHANG Huajie;MA Xiao(Division of Diphtheria,Tetanus and Pertussis Vaccine and Toxins,National Institutes for Food and Drug Control,NHC Key Laboratory of Research on Quality and Standardization of Biotech Products,NMPA Key Laboratory for Quality Research and Evaluation of Biological Products,Beijing 102629,China)

机构地区：[1]中国食品药品检定研究院百白破疫苗与毒素室国家卫生健康委员会生物技术产品检定方法及其标准化重点实验室国家药品监督管理局生物制品质量研究与评价重点实验室,北京102629

出　　处：《中国生物制品学杂志》2024年第10期1161-1166,共6页Chinese Journal of Biologicals

基　　金：北京市自然科学基金-海淀原始创新联合基金(L222010)。

摘　　要：目的对b型流感嗜血杆菌(Haemophilus influenzae type b,Hib)D蛋白(protein D,PD)作为载体的C群脑膜炎奈瑟球菌荚膜多糖(group C meningococcal polysaccharide,GCMP)-蛋白结合物进行免疫效果评价,以确定PD作为GCMP蛋白载体的可行性。方法采用1-氰基-4-二甲氨基-吡啶四氟硼酸(CDAP)活化法,将重组PD、破伤风类毒素(tetanus toxin,TT)分别与GCMP结合制备结合物;将GCMP及结合物分别经腹部皮下免疫雌性BALB/c小鼠,分为GCMP、GCMP-PD、PD、GCMP-TT、TT及阴性对照(生理盐水)组,每组12只,共免疫3次,分别于每次加强免疫前及末次免疫后7 d,经眼眶采血,分离血清,用于抗体水平检测及血清杀菌力试验(serum bactericidal assay,SBA);同时在末次免疫后7 d取脾脏,进行细胞免疫水平检测。结果蛋白与GCMP结合后能增强抗-GCMP抗体IgG水平,第3次免疫后,与单独GCMP组相比,GCMP-PD和GCMP-TT组小鼠血清抗-GCMP IgG水平均显著升高(F分别为5.294、14.917,P均<0.05),且产生免疫记忆,诱导IgG1产生,但GCMP-PD组低于GCMP-TT组(F=5.828,P<0.05);CMP-PD、PD组小鼠血清抗-PD IgG抗体水平各次免疫后均显著增长,第3次免疫后,GCMP-PD组抗-PD IgG抗体水平显著高于PD组(F=15.426,P<0.01),而GCMP-TT组低于TT组(F=211.207,P<0.001)。SBA结果显示,GCMP-PD组杀菌抗体滴度高于GCMP组(F=11.797,P<0.05),但低于GCMP-TT组(F=8.688,P<0.05)。流式细胞术结果显示,GCMP-PD组Th1、Th2细胞亚群比值与GCMP及GCMP-TT组相比,差异均无统计学意义(F分别为0.073、3.021,P均>0.05)。结论重组PD与GCMP结合后,既能增强多糖疫原性,提高体液免疫反应能力,也能激发细胞免疫反应,同时免疫后血清杀菌效果显著,提示PD具备作为GCMP蛋白载体的可行性。Objective To evaluate the immune effect of group C meningococcal polysaccharide(GCMP)conjugates using protein D(PD)of Haemophilus influenzae type b(Hib)as vector,in order to determine the feasibility of PD protein as GCMP protein vector.Methods The recombinant PD and tetanus toxin(TT)were conjugated to GCMP by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate(CDAP)activation method to prepare conjugates respectively.The female BALB/c mice were subcutaneously immunized with GCMP and GCMP conjugates,which were divided into GCMP,GCMP-PD,PD,GCMP-TT,TT and negative control group(normal saline),with 12 mice in each group for 3 times of immunization.The blood samples were collected from the rim of eye before each booster immunization and 7 d after the last immunization,and the sera were separated for the detection of antibody levels and serum bactericidal assay(SBA).In addition,the spleen samples were taken 7 d after the last immunization for the detection of cellular immune levels.Results The conjugates of protein and GCMP enhanced the level of anti-GCMP IgG.After the third immunization,serum anti-GCMP IgG levels in GCMP-PD and GCMP-TT groups were significantly higher than those in GCMP group(F=5.294 and 14.917,respectively,each P<0.05),and the immune memory was produced to induce IgG1,while the IgG1 levels in GCMP-PD group were lower than those in GCMP-TT group(F=5.828,P<0.05).The levels of anti-PD IgG in serum of GCMP-PD and PD groups increased significantly after each immunization,and the levels of anti-PD IgG in GCMP-PD group were significantly higher than those in PD group after the third immunization(F=15.426,P<0.01),while those in GCMP-TT group were lower than those in TT group(F=211.207,P<0.001).The results of SBA showed that the titers of bactericidal antibody in GCMP-PD group were higher than those in GCMP group(F=11.797,P<0.05),while lower than those in GCMP-TT group(F=8.688,P<0.05).The results of flow cytometry showed that the ratio of Th1 and Th2 cell subsets in GCMP-PD group had no significant differenc

关 键 词：D蛋白 C群脑膜炎奈瑟球菌荚膜多糖 结合物 载体 体液免疫 细胞免疫 

分 类 号：R392-33[医药卫生—免疫学]