Immunogenicity and protective efficacy of Bacillus anthracis poly-γ-D-glutamic acid capsule covalently coupled to a protein carrier using a novel triazine-based conjugation strategy
Immunogenicity and protective efficacy of Bacillus anthracis poly-γ-D-glutamic acid capsule covalently coupled to a protein carrier using a novel triazine-based conjugation strategy
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DOI：
10.1074/jbc.m509432200
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发表时间：
2006-02-24
影响因子：
4.8
通讯作者：
Caulfield, M
中科院分区：
文献类型：
--
作者：
Joyce, J;Cook, J;Caulfield, M
炭疽芽孢杆菌的荚膜多肽由独特的聚谷氨酸聚合物组成，其中D-谷氨酸单体通过γ-肽基键连接。该胶囊的免疫原性差，开发用于疫苗开发的聚合物的努力集中在通过与免疫刺激蛋白载体的化学偶联来增加其固有的免疫原性。通常的策略是使用基于马来酰亚胺的缩合试剂来活化游离的α-羧基，尽管有报道称这种化学反应可能导致断链。我们已经将高分子量胶囊纯化至> 95%的均一性，并且已经证明聚合物含有> 99%的聚-γ-D-谷氨酸。通过圆二色性和多角度激光光散射评估的聚合物的主要结构是无序的，在近中性pH值。我们研究了各种活化化学的影响，我们证明了在水性条件下的碳二亚胺处理的结果在显着裂解的α-肽基键，而断裂显着减少在非水极性溶剂中，虽然不希望的侧链修饰仍然观察到。使用基于三嗪的试剂4-（4，6-二甲氧基（1，3，5）三嗪-2-基）-4-甲基吗啉鎓氯化物开发了活化化学，其允许α-羰基的受控和可再现的衍生化。在两锅反应方案中，活化的胶囊用巯基反应性异双功能部分衍生化，随后偶联到硫醇化载体蛋白。该缀合物在小鼠中引起非常高的胶囊特异性免疫滴度。更重要的是，用结合胶囊免疫的小鼠表现出良好的保护作用，抵抗来自毒性B的致死性攻击。炭疽菌株在两种感染模型中的表达。我们还表明，第一次，用碳二亚胺处理胶囊显着减少识别胶囊特异性抗血清同时与试剂诱导的聚合物质量的减少。数据表明，对于疫苗开发，保持聚合物的高质量可能是重要的。
The capsular polypeptide of Bacillus anthracis is composed of a unique polyglutamic acid polymer in which D-glutamate monomers are joined by gamma-peptidyl bonds. The capsule is poorly immunogenic, and efforts at exploiting the polymer for vaccine development have focused on increasing its inherent immunogenicity through chemical coupling to immune-stimulating protein carriers. The usual strategy has employed carbodiimide-based condensing reagents for activation of free alpha-carboxyl groups, despite reports that this chemistry may lead to chain scission. We have purified the high molecular mass capsule to > 95% homogeneity and have demonstrated that the polymer contains > 99% poly-gamma-D-glutamic acid. The predominant structure of the polymer as assessed by circular dichroism and multiangle laser light scattering was unordered at near-neutral pH. We investigated the effects of various activation chemistries, and we demonstrated that carbodiimide treatment under aqueous conditions results in significant cleavage of the alpha-peptidyl bond, whereas scission is significantly reduced in nonaqueous polar solvents, although undesired side chain modification was still observed. An activation chemistry was developed using the triazine-based reagent 4-(4,6-dimethoxy (1,3,5) triazin-2-yl)-4-methylmorpholinium chloride, which allowed for controlled and reproducible derivatization of alpha-carbonyls. In a two-pot reaction scheme, activated capsule was derivatized with a sulfhydryl-reactive heterobifunctional moiety and was subsequently coupled to thiolated carrier protein. This conjugate elicited very high capsule-specific immune titers in mice. More importantly, mice immunized with conjugated capsule exhibited good protection against lethal challenge from a virulent B. anthracis strain in two models of infection. We also showed, for the first time, that treatment of capsule with carbodiimide significantly reduced recognition by capsule-specific antisera concurrent with the reagent-induced reduction of polymer mass. The data suggested that for vaccine development, maintenance of the high mass of the polymer may be important.