Suppression of inhibitor formation against FVIII in a murine model of hemophilia A by oral delivery of antigens bioencapsulated in plant cells
Suppression of inhibitor formation against FVIII in a murine model of hemophilia A by oral delivery of antigens bioencapsulated in plant cells
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DOI：
10.1182/blood-2013-10-528737
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发表时间：
2014-09-04
期刊：
影响因子：
20.3
通讯作者：
Daniell, Henry
中科院分区：
文献类型：
--
作者：
Sherman, Alexandra;Su, Jin;Daniell, Henry
血友病A是由凝血因子VIII（FVIII）缺乏引起的X连锁出血性疾病。为了解决目前替代疗法中抑制性抗体形成的严重并发症，我们创建了表达FVIII抗原、重链（HC）和C2的烟草转质体系，其与经粘膜载体霍乱毒素B亚基融合。霍乱毒素B-HC和霍乱毒素B-C2融合蛋白在新鲜叶片中表达高达80或370 μ g/g，组装成五聚体形式，并与GM 1受体结合。通过免疫染色证实了FVIII抗原通过植物细胞中的生物包封和口服递送至肠道免疫系统的保护作用。在2种不同品系背景的血友病A小鼠中，饲喂HC/C2混合物可显著抑制T辅助细胞应答和FVIII抑制物形成。需要延长口服给药时间以长期控制抑制剂形成。免疫前小鼠中抑制剂滴度的大幅降低表明该方案还可以逆转抑制剂的形成。基因表达和流式细胞术分析显示免疫抑制细胞因子（转化生长因子β和白细胞介素10）上调。连续转移实验证实了主动抑制机制，并显示诱导CD 4（+）CD 25（+）和CD 4（+）CD 25（-）T细胞，有效抑制抗FVIII形成。总之，这些数据支持基于植物细胞的口服耐受性抑制针对FVIII的抑制剂形成。
Hemophilia A is the X-linked bleeding disorder caused by deficiency of coagulation factor VIII (FVIII). To address serious complications of inhibitory antibody formation in current replacement therapy, we created tobacco transplastomic lines expressing FVIII antigens, heavy chain (HC) and C2, fused with the transmucosal carrier, cholera toxin B subunit. Cholera toxin B-HC and cholera toxin B-C2 fusion proteins expressed up to 80 or 370 mu g/g in fresh leaves, assembled into pentameric forms, and bound to GM1 receptors. Protection of FVIII antigen through bioencapsulation in plant cells and oral delivery to the gut immune system was confirmed by immunostaining. Feeding of HC/C2 mixture substantially suppressed T helper cell responses and inhibitor formation against FVIII in mice of 2 different strain backgrounds with hemophilia A. Prolonged oral delivery was required to control inhibitor formation long-term. Substantial reduction of inhibitor titers in preimmune mice demonstrated that the protocol could also reverse inhibitor formation. Gene expression and flow cytometry analyses showed upregulation of immune suppressive cytokines (transforming growth factor beta and interleukin 10). Adoptive transfer experiments confirmed an active suppression mechanism and revealed induction of CD4(+)CD25(+) and CD4(+) CD25(-) T cells that potently suppressed anti-FVIII formation. In sum, these data support plant cell-based oral tolerance for suppression of inhibitor formation against FVIII.