W135群脑膜炎球菌结合物的制备及其在小鼠体内的免疫原性

Preparation of group W135 meningococcal conjugate and its immunogenicity in mice

目的比较不同活化试剂、不同载体蛋白质及不同免疫程序对W135群脑膜炎球菌结合物在小鼠体内的免疫原性的影响。方法分别采用溴化氰(CNBr)和1-氰基-4-二甲基氨基吡啶-四氟硼酸盐(CDAP)为活化试剂,以己二酸二酰肼(ADH)为连接臂制备W135群脑膜炎球菌多糖(group W135 meningococcal polysaccharide,PSW135)衍生物;以碳二亚胺(EDAC)作为缩合剂,将多糖衍生物分别与破伤风类毒素(tetanus toxoid,TT)、白喉类毒素(diphtheria toxoid,DT)和重组铜绿假单胞菌外毒素A(recombinant Pseudomonas aeruginosa exotoxin A,r EPA)3种蛋白质共价结合,制备6种PSW135蛋白质结合物;比较4针免疫程序(0、2、4、8周)和3针免疫程序(0、4、8周)对结合物在小鼠体内免疫原性的影响。结果两种活化试剂均能对多糖进行有效活化,且不会破坏多糖的抗原性;制备的6种结合物在小鼠体内均能产生良好的免疫原性,且具有显著的剂次加强效应;采用间隔2周的3针免疫程序和2.5μg/只的免疫剂量,不同类型的活化试剂和载体蛋白质对结合物的免疫原性无显著影响,但以DT为载体蛋白质的结合物的免疫加强效应要弱于以TT和r EPA为载体的结合物;延长免疫间隔时间可显著提高结合物在小鼠体内的免疫原性。结论初步建立了适用于W135群脑膜炎球菌结合疫苗的结合工艺,并为结合物在小鼠体内的免疫原性研究提供了一种新的候选免疫程序。

Objective To compare the influences of various activating reagents, carrier proteins and immune schedules on immunogenicity of group W135 meningococcal conjugate in mice. Methods Group W135 meningococcal polysaccharide derivative was prepared using CNBr and CDAP as activating agents respectively and ADH as linking arm, and conjugated to tetanus toxoid（TT）, diphtheria toxoid（DT）and recombinant Pseudomonas aeruginosa exotoxin A（r EPA）using EDAC as condensation reagent respectively to obtain six kinds of conjugates, of which the immunogenicity in mice by four（at weeks 0, 2, 4 and 6） and three（at weeks 0, 4 and 8） dose immune schedules were compared. Results Both the activating agents effectively activated the polysaccharide without any damage to antigenicity. All the six kinds of conjugates showed good antigenicity in mice in a dose-dependent manner. Various activating agents and carrier proteins showed no significant influence on the immunogenicity of conjugates inoculated at a dosage of 2. 5 μg by three-dose schedule. However, the enhance effect of conjugate using DT as carrier protein was weaker than those using TT and r EPA.The increase of time interval of inoculation enhanced the immunogenicity of conjugates in mice significantly. Conclusion The conjugation procedure for group W135 meningococcal conjugate was preliminarily developed, which provided a novel alternative immune schedule for study on immunogenicity of the conjugates in mice.