A群奈瑟氏脑膜炎球菌荚膜多糖-破伤风类毒素结合疫苗的制备及其免疫原性研究

采用溴化氰(CNBr)活化多糖,以无水己二酸二肼(ADH)作为连接剂,1乙基13(3二甲基氨基丙基)碳化二亚胺(EDAC)为偶联剂制备A群奈瑟氏脑膜炎球菌荚膜多糖(GAMP)与破伤风类毒素(TT)的结合物,经皮下免疫NIH小鼠,用ELISA检测小鼠血清中抗GAMP及抗载体蛋白的IgG抗体水平。用补体介导的体外杀菌试验检测血清中GAMP抗体的杀菌活性。结果显示,实验中制备的多糖衍生物和多糖蛋白质结合物都具有GAMP抗原特异活性。结合物免疫小鼠后可诱生比多糖单独免疫更高水平的GAMP血清IgG抗体,并能形成免疫记忆,产生再次应答。结合物免疫小鼠所诱生的血清GAMP抗体较之多糖组具有更强的体外杀菌活性。表明此方法制备的结合物可获得优于多糖的、稳定的特异免疫原性。

Preparation, characterization and immunogenicity in mice for conjugate vaccine of Neisseria meningococcal serogroup A polysaccharide-tetanus toxoid

Cyanogen Bromide (CNBr) was used to activate the hydroxyls of polysaccharide and Adipic Aciddihydrazide (ADH) was used as a linker, with 1-ethyl-3-(3-dimethylamino-propyl) Carbodiimide (EDAC) as coupling reagent, conjugate of GAMP combined with tetanus toxoid (TT) was prepared. The physicochemical property, antigen specificity and immunogenicity of the conjugate were investigated. Solution of 5 μg polysaccharide alone or conjugate containing 5 μg polysaccharide, were injected into NIH female mice subcutaneously. Serum antibodies against GAMP and TT were measured by enzyme-linked immunosorbent assay (ELISA). The bactericidal activity in serum was measured by complement-dependent bactericidal experiment. The results showed that all of polysaccharide, polysaccharide derivatives and conjugate had the specific antigenicity of GAMP. On the other hand, the mice immunized with polysaccharide alone can not elicit high level serum antibodies to GAMP, but the mice immunized with conjugate induces a higher level serum antibody to GAMP and gives rise to a immunologic memory as well. The complement-dependent bactericidal experiment showed that serum GAMP antibodies induced by the conjugate had stronger bactericidal activity. The prepared conjugates all have specific immunogenicity which is better than those of the polysaccharide alone.