以白喉毒素无毒变异体蛋白CRM197和破伤风类毒素为载体的Y群脑膜炎球菌多糖结合物对小鼠的免疫原性

Immunogenicity of group Y meningococcal polysaccharide-protein conjugate vaccine using avirulent mutant CRM197 of diphtheria toxoid and tetanus toxoid as carriers in mice

目的探讨以天然白喉毒素无毒变异体蛋白CRM197和破伤风类毒素(tetanus toxoid,TT)为载体的Y群脑膜炎球菌多糖(group Y meningococcal polysaccharide,MenYPS)结合蛋白对小鼠的免疫原性。方法将MenYPS用1,6-己二酰肼(1,6-adipic acid dihydrazide,ADH)进行衍生,制备MenYps-ADH衍生物,将MenYPS-ADH衍生物在碳二亚胺[N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride,EDAC]的作用下,分别与CRM197和TT共价结合,制备3批MenYPS-CRM197和MenYPS-TT结合物,采用琼脂双扩散法检测结合物中多糖抗原的血清型特异性。经BALB/c小鼠大腿内侧皮下注射制备的衍生物及结合物,2.5μg/只,0、14、28 d各免疫1次,分别于第7、21和35天经小鼠眼眶采血,分离血清,ELISA法检测小鼠血清GMT。参考《中国生物制品规程》(2000版)进行无菌试验及异常毒性试验。结果制备的3批MenYPS-CRM197和MenYPS-TT结合物可与MenYPS诊断血清产生沉淀线。MenYPSTT和MenYPS-CRM197免疫小鼠3次后,均产生较高的滴度,MenYPS-TT和MenYPS-CRM197结合物第2、3次免疫小鼠后产生的GMT均明显高于MenYPS-ADH衍生物,差异有统计学意义(P均<0.05),而两种结合物GMT差异无统计学意义(P均>0.05);MenYPS-TT和MenYPS-CRM197结合物第2次免疫后产生的GMT均明显高于第1次免疫后,第3次免疫后明显高于第2次免疫后,差异均有统计学意义(P均<0.01)。无菌试验符合《中国生物制品规程》(2000版)相关要求;异常毒性试验中,豚鼠和小鼠观察7 d后,均无异常反应,体重增加,无死亡。结论 MenYPS与CRM197和TT的结合物均能诱导BALB/c小鼠产生较高的抗体水平,且产生了免疫记忆。

Objective To investigate the immunogenicity of group Y meningococcal polysaccharide （MenYPS）-protein conjugate vaccine using avirulent mutant CRM197 of natural diphtheria toxoid and tetanus toxoid （TT） as carriers in mice. Methods MenYPS was derived with 1,6-adipic acid dihydrazide （ADH） to prepare derivative MenYPS-ADH which was covalently bound to CRM197 and Tr in mediation of N-（3-dimethylaminoprophyl）-N＇-ethylcarbodiimide hydrochloride （EDAC） to prepare three batches of MenYPS-CRM197 and MenYPS-TT conjugates respectively. The serogroup-specificity of polysaccharide antigen in the conjugates was determined by double immunodiffusion test in agar. BALB/c mice were injected s.c. with the prepared derivates and conjugates, 2. 5 μg for each, on days 0, 14 and 28separately. Serum samples were collected on days 7, 29 and 35, and determined for GMTs of antibodies. Sterility and abnormal toxicity tests were performed according to the Chinese Requirements for Biologics （2000 edition）. Results The prepared three batches of MenYPS- CRM197 and MenYPS-TT conjugates formed precipitation lines with diagnostic serum for MenYPS, both of which induced high antibody titer in mice. However, the GMTs of antibodies in sera of mice after immunization with the 2nd and the 3rd doses of MenYPS-Tr and MenYPS-CRM197 showed no significant difference （each P 〉 0.05）, both of which were