两种ACYW135群脑膜炎球菌多糖-CRM197结合疫苗的制备及质量与免疫原性分析

Preparation, quality and immunogenicity research of serogroup A,C,Y,W135 meningococcal polysaccharide-CRM197 conjugate vaccines with two conjugating modes

目的 对比2种结合方式的以白喉毒素无毒突变体197(cross-reacting material 197,CRM197)蛋白为载体制备的ACYW135群脑膜炎球菌结合疫苗的质量与免疫原性。方法 方法一以CRM197蛋白羧基为结合位点,用1-氰基-4-(二甲氨基)吡啶四氟硼酸盐(1-cyano-4-dimethylaminopyridinium tetrafluoroborate, CDAP)分别将A、C、Y、W135群脑膜炎球菌多糖羟基活化为氰基,以己二酸二酰肼(adipic acid dihydrazide, ADH)作为连接剂,最后通过4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐[4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl morpholinium chloride, DMTMM]与CRM197反应得到单价结合物;方法二用CRM197蛋白氨基为结合位点,用高碘酸钠分别氧化A、C、Y、W135群脑膜炎球菌多糖羟基为醛基,加入CRM197蛋白生成席夫碱,最后通过氰基硼氢化纳还原,得到单价结合物。对2种结合方式制备的结合物原液进行理化检测,合格后再分别将2种路线的单价结合物按比例与冻干辅料混合后经冻干制成2种ACYW135群脑膜炎球菌结合疫苗(ACYW135-CRM197),分别免疫NIH小鼠后,用ELISA检测小鼠血清中抗体并进行统计学分析。结果 2种结合方式制备的ACYW135群脑膜炎球菌结合疫苗,游离多糖含量均<20.00%,K_D值在0.2以前的洗脱液多糖回收率均>80.00%,但以CRM197蛋白羧基为结合位点的ACYW135群脑膜炎球菌单价结合物原液中游离多糖含量(3.20%~11.50%)均低于以氨基为结合位点的相应结合物原液中的游离多糖(8.35%~17.42%);疫苗成品的平均相对分子质量,以CRM197蛋白羧基为结合位点的ACYW135群脑膜炎球菌结合疫苗更大。2种结合疫苗免疫小鼠后其体内产生的抗体经检定后统计分析,A群、C群多糖免疫原性差异均无统计学意义(P=0.080,P=0.176),W群、Y群多糖免疫原性差异均有统计学意义(P=0.015,P=0.027)。结论 以CRM197蛋白载体羧基为结合位点的ACYW135群脑膜炎球菌结合疫苗在质量和免疫原性方面均不差于传统的以CRM197蛋白载体氨基为结合位点的ACYW135群脑膜炎球菌结合疫苗。

Objective The quality and immunogenicity analysis of ACYW135 meningococcal conjugate vaccine with cross-reacting material 197(CRM197) protein as the carrier were compared for two different conjugating modes. Methods The first route, carboxyl group of CRM197 as the conjugated point, hydroxyl group of serogroup A,C,Y and W135 meninococcal polysaccharide would be activated and transformed to cyano group with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate(i.e. CDAP),and adipic acid dihydrazide be introduced as the linker. Then, the monovalent conjugates were obtained by the reaction of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl morpholinium chloride(DMTMM) with CRM197. And amino group of CRM197 used as the binding site, and the hydroxyl group of polysaccharide for mencoccal serogroup A,C,Y and W135 was oxidized with sodium periodate to be transformed into the aldehyde group, and the CRM197 be added to produce the Schiffbase, and then the monovalent conjugate collected by the reduction of sodium cyanoborohydride. Physical and chemical items were detected for the conjugate stock solution prepared by the two different conjugating methods, and then the univalent conjugate of the two routes was prepared with lyophilized excipients in proportion as the regulation and lyophilized to obtain two kinds of ACYW135 group meningococcal conjugate vaccines(ACYW135-CRM197) by the other route. Tetravalent conjugate vaccine immunized in mice, serum antibodies of the mice were determined by ELISA kit, and statistical analysis were performed to evaluate immunogenicity of the two kind of conjugate vaccines. Results The free polysaccharide content of ACYW135 groupmeningococcal conjugate vaccines prepared by the two conjugation methods were less than 20.00%,and the polysaccharide recovery rates of eluate before K_D value 0.2 went beyond 80.00%. However, the content of free polysaccharides in the stock solutions of serogroup A,C,Y,W135 meningococcal monovalent conjugates with carboxyl group of CRM197 protein as the conjugating site(3.20%-11.50%) were relatively lower than that of the corresponding conjugates activited with amino group as the binding site(8.35%-17.42%). Vaccine average relative molecular mass of products with carboxyl group of CRM197 as the binding site of ACYW135 meningococcal vaccine were higher than that activited with amino group ones. The antibodies immunized in mice after conjugate vaccination, there were no significant difference in the immunogenicity by ELISA of polysaccharide between group A-conjugates(P=0.080) or group C-conjugates(P=0.176),however, there were statistically significant difference between group W135-conjugates(P=0.015) or group Y-conjugates(P=0.027). Conclusion The quality and immunogenicity of ACYW135 group meningococcal conjugate vaccines activited by the carboxyl group as the binding site of CRM197 were not inferior to that of the activation amino group as the binding site.