2种14型肺炎球菌多糖-蛋白结合物的制备及免疫原性比较

Preparation and immunogenicity comparison of two conjugates of serotype 14 pneumococcal polysaccharides

目的:以破伤风类毒素(tetanus toxoid,TT)和己二酸二酰肼(adipic acid dihydrazide,ADH)衍生的TT(TTAH)分别作为载体蛋白制备14型肺炎球菌多糖-蛋白结合物,比较2种结合物在小鼠体内的免疫原性。方法:以1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDAC)作为缩合剂,介导TT与ADH反应,制备TTAH衍生物,2,4,6-三硝基苯磺酸(2,4,6-trinitrobenzenesulfonic acid,TNBS)法测定TTAH的-AH衍生率;将14型肺炎球菌多糖(PS14)水解并用1-氰基-4-二甲基氨基吡啶四氟化硼(CDAP)活化后直接与TT或TTAH结合,结合物经Sepharose 4 FF层析柱纯化后收集分配系数(KD)<0.2的样品并除菌过滤作为结合物原液,并对其生化指标、血清学特异性和小鼠免疫原性进行检测。结果:2种结合物原液游离糖含量均在1%以内,多糖回收率分别为PS14-TT:33.80%及PS14-TTAH:29.15%。与A组(多糖组)相比,2种结合物B组(PS14-TT)和C组(PS14-TTAH)能够诱导更高的抗PS14抗体水平(P<0.001)并具有剂次加强效应;与C组相比,B组能够诱导更高的抗PS14抗体水平且差异显著并具有统计学意义(P<0.05);2组结合物免疫3针后C组抗TT抗体水平高于B组,但差异无统计学意义,B组无剂次加强效应,而C组有剂次加强效应。结论:PS14-TT在小鼠体内诱导了较高的抗PS14抗体水平及较低的抗TT抗体水平,是较理想的多糖蛋白结合疫苗。

Objective： To prepare serotype 14 pneumococcal polysaccharide（ PS14） conjugates using tetanus toxoid（ TT） and its derivative（ TTAH） as carrier proteins,and to compare their immunogenicity in mice.Methods： With EDAC as a condensing agent,derivatives were prepared by modification of TT with adipic acid dihydrazide（ ADH）,and AH derivation rate was determined by 2,4,6-trinitrobenzenesulfonic acid（ TNBS） method.The PS14 was hydrolyzed,activated with CDAP,and combined with TT or TTAH. The conjugates were purified with Sepharose 4 FF chromatography,samples（ KD 0. 2） were collected,and aseptic filtration was performed. The biochemical,serological specificity,and immunogenicity were detected in mice. Results： The contents of free PS in the two conjugates were 1%,the PS recoveries of PS14-TT and PS14-TTAHwere 33. 80% and 29. 15% respectively. Compared to PS14,PS14-TT and PS14-TTAHcould induce a significantly higher antibody level（ P〈0. 001）with a dose-enhancing effect. PS14-TT induced a higher anti-PS14 antibody level than PS14-TTAH（ P〈0. 05）,while PS14-TTAHinduced a higher anti-TT antibody level than PS14-TT,but no statistical significance was found.PS14-TTAHhad a dose-enhancing effect,but PS14-TT had no such an effect. Conclusion： PS14-TT induces higher anti-PS14 antibody and lower anti-TT antibody level,so it is the ideal polysaccharide protein conjugate vaccine.