Abstract:
Coagulation factor replacement therapy for the X-linked bleeding disorder Haemophilia, characterized by a deficiency of coagulation protein factor VIII (FVIII), is severely complicated by antibody (inhibitors) formation. The development of FVIII inhibitors drastically alters the quality of life of the patients and is associated with a tremendous increase in morbidity as well as treatment costs. The ultimate goal of inhibitor control is antibody elimination. Immune tolerance induction (ITI) is the only clinically established approach for developing antigen-specific tolerance to FVIII. This work aims to establish a novel cost-effective strategy to produce FVIII molecules in fusion with cholera toxin B (CTB) subunit at the N terminus using the Bacillus subtilis expression system for oral tolerance, as the current clinical immune tolerance protocols are expensive. Regions of B-Domain Deleted (BDD)-FVIII that have potential epitopes were identified by employing Bepipred linear epitope prediction; 2 or more epitopes in each domain were combined and cDNA encoding these regions were fused with CTB and cloned in the Bacillus subtilis expression vector pHT43 and expression analysis was carried out. The expressed CTB-fused FVIII epitope domains showed strong binding affinity towards the CTB-receptor GM1 ganglioside. To conclude, Bacillus subtilis expressing FVIII molecules might be a promising candidate for exploring for the induction of oral immune tolerance.
摘要:
凝血因子替代疗法治疗X连锁出血性疾病血友病,以凝血蛋白VIII(FVIII)缺乏为特征,抗体(抑制剂)的形成严重复杂化。FVIII抑制剂的开发极大地改变了患者的生活质量,并且与发病率以及治疗成本的极大增加有关。抑制剂控制的最终目标是抗体消除。免疫耐受诱导(ITI)是临床上唯一建立的用于开发对FVIII的抗原特异性耐受的方法。这项工作旨在建立一种新的经济有效的策略,以使用枯草芽孢杆菌表达系统在N端产生与霍乱毒素B(CTB)亚基融合的FVIII分子,用于口服耐受性。因为目前的临床免疫耐受方案是昂贵的。通过采用Beippred线性表位预测来鉴定具有潜在表位的B结构域缺失(BDD)-FVIII的区域;将每个结构域中的2个或更多个表位组合,并将编码这些区域的cDNA与CTB融合并克隆到枯草芽孢杆菌表达载体pHT43中,并进行表达分析。表达的CTB-融合的FVIII表位结构域显示对CTB-受体GM1神经节苷脂的强结合亲和力。最后,表达FVIII分子的枯草芽孢杆菌可能是探索诱导口服免疫耐受的有希望的候选者。