宋内氏痢疾杆菌O-SP-TT结合疫苗的制备及其免疫学特性的研究

Preparation,characterization and immunogenicity of conjugate vaccine composed of the O-specific polysaccharide of S.sonnei and tetanus toxoid

宋内氏痢疾杆菌 (S .Sonnei)的一个重要保护性抗原是菌体抗原 (O抗原 )即细菌细胞壁中的脂多糖 (LPS)成分 ,已证实血清中足够水平的抗LPSIgG抗体即能对该菌提供免疫保护作用。本文选用Westphal热酚法提纯宋内氏痢疾菌LPS ,经酸水解法进一步脱毒处理后得到无毒但却具弱免疫原性的O 特异性多糖 (O SP) ,然后采用化学方法即通过连接剂己二酸二肼 (ADH)将其共价结合到破伤风类毒素 (TT)上 ,共制备得到了三批宋内氏痢疾杆菌O SP TT结合疫苗。经生化和免疫学方法检测证实我们提纯的LPS、OSP及其合成的O SP AH衍生物、O SP TT结合物均具有宋内氏痢疾杆菌O抗原特异性 ,同时其核酸和杂蛋白质含量低 (≤ 2 % ) ,表明纯度较高。同时经小鼠免疫原性试验证实三批结合物免疫小鼠后产生的抗LPSIgG抗体滴度均比单一O SP免疫后产生的要高出 10倍以上 ,且结合物再次注射后存在加强应答。补体介导的体外杀菌力试验表明结合物免疫小鼠后诱导产生的血清抗LPSIgG抗体对宋内氏痢疾杆菌具特异性杀菌活性。本文结果表明宋内氏痢疾杆菌O SP TT结合疫苗可望作为一种有效的侯选痢疾疫苗做进一步的大规模人体观察。

Lipopolysaccharide(LPS) is one of the important protective antigens of Shigellae.It was confirmed that serum IgG LPS specific antibody could confer protective immunity to Shigellosis.In the study,the LPS of Shigella sonnei were firstly extracted by the method of Westphal,then the O SP,which was purified after acid hydrosis of LPS,was covalently coupled to tetanus toxoid(TT)by using adipic acid dihydrazide(ADH)as a spacer in the presence of carbodimide.The resultant O SP TT conjugates elicited high levels of IgG LPS antibodies and booster responses in mice when injected subcutaneously in saline at 2.5 μg dose.Meanwhile,The results showed that the IgG LPS antibodies in serum had the bactericidal activity by the complement dependent bactericidal test in vitro.Hopefully,the O SP TT conjugate of S.sonnei was potentially used as a candidate Shigella sonnei vaccine in a large scale of clinical study.