新型ACYW135群脑膜炎球菌-白喉毒素无毒变异体结合疫苗的制备及其免疫原性评价

Preparation and immunogenicity evaluation of a novel ACYW135 group meningococcal-cross-reacting material 197 conjugate vaccine

目的 制备新型ACYW135群脑膜炎球菌-白喉毒素无毒变异体(cross-reacting material 197,CRM197)结合疫苗,并评价其免疫原性,以期为流行性脑脊髓膜炎的防控奠定基础。方法 用1-氰基-4-(二甲氨基)吡啶四氟硼酸盐(1-cyano-4-dimethylamino pyridinium tetrafluoroborate,CDAP)分别将A、C、Y、W135群脑膜炎球菌多糖羟基活化为氰基,并以己二酸二酰肼(adipic acid dihydrazide,ADH)作为连接剂与活化多糖通过氰酸酯连接,生成裸露酰肼基的多糖-ADH衍生物,再通过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生成酰胺键偶联,得到各单价结合物原液,并进行相关检测。将单价结合物原液按一定比例混合后,分别加入3种冻干保护剂(蔗糖溶液、乳糖溶液及蔗糖+甘露醇溶液),经冻干,制备3种ACYW135-CRM197结合疫苗。将制备的疫苗于0、14 d经雄性NIH小鼠背部皮下注射,0.2 mL/只,每组10只,并于初次免疫后21 d,经小鼠颌下静脉采血,分离血清,ELISA法检测特异性抗体水平。结果 A、C、Y、W135-CRM197单价结合物原液的检测结果均符合质量标准。ACYW135-CRM197疫苗成品可与各群多糖抗血清形成明显沉淀线;平均分子量均在1 680 000~5 230 000范围内。3种ACYW135-CRM197结合疫苗的抗A、C、Y、W135群多糖IgG抗体阳转率均为100%;ACYW135-CRM197(蔗糖)、ACYW135-CRM197(乳糖)和ACYW135-CRM197(蔗糖+甘露醇)组比较,差异均无统计学意义(t=0.249~1.157,P均> 0.05)。结论 本研究制备了新型ACYW135-CRM197结合疫苗,且该疫苗在小鼠体内具有良好的免疫原性。

Objective To prepare a novel ACYW135 group meningococcal-cross-reacting material 197(CRM197)conjugate vaccine and evaluate its immunogenicity,so as to lay a foundation for the prevention and control of epidemic cerebrospinal meningitis.Methods Group A,C,Y and W135 meningococcal polysaccharide hydroxyl was activated into cyano group with1-cyano-4-dimethylamino pyridinium tetrafluoroborate(CDAP),respectively.Adipic acid dihydrazide(ADH)was used as a linker to connect one end of the activated polysaccharide by cyanate ester to generate polysaccharide-ADH derivative with exposed hydrazide groups.The monovalent conjugate vaccines were obtained through amide bond coupling between 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl morpholinium chloride(DMTMM)and CRM197.Then the monovalent conjugate was mixed with different lyophilized excipients(sucrose solution,lactose solution,and sucrose + mannitol solution)in proportion respectively and lyophilized to prepare three ACYW135-CRM197 meningococcal conjugate vaccines.The male NIH mice were subcutaneously immunized with the vaccines at 0 and 14 d,with 0.2 mL/mouse,10 mice in each group.At 21 d after the initial immunization,the blood samples were collected from submandibular vein of the mice,and the serum was separated and detected for the specific antibody level by ELISA.Results The test results of A,C,Y and W135-CRM197 monovalent conjugate all met the requirements of the quality standards.ACYW135-CRM197 vaccine product formed obvious precipitation line with each group of polysaccharide antiserum.The average molecular mass was all in the range of 1 680 000-5 230 000.The seroconversion rates of IgG antibodies against A,C,Y,W135 polysaccharide of three ACYW135-CRM197 conjugate vaccines were all 100%.There was no significant difference in polysaccharide antibody level among ACYW135-CRM197(sucrose),ACYW135-CRM197(lactose)and ACYW135-CRM197(sucrose + mannitol)groups(t = 0.249-1.157,each P >0.05).Conclusion In this study,a novel ACYW135-CRM197 conjugate vaccine was prepared and had good immunogenicity in mice.