Autotransporter-Based Antigen Display in Bacterial Ghosts
Autotransporter-Based Antigen Display in Bacterial Ghosts
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DOI：
10.1128/aem.02733-14
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发表时间：
2015-01-01
影响因子：
4.4
通讯作者：
de Gier, Jan-Willem
中科院分区：
文献类型：
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作者：
Hjelm, Anna;Soderstrom, Bill;de Gier, Jan-Willem
细菌菌影是革兰氏阴性细菌的空细胞包膜，可用作抗原递送的载体。通过噬菌体phi X174裂解蛋白E的受控生产产生的细胞包膜中的通道释放细菌细胞质内容物，产生菌影。虽然鬼拥有原始宿主菌株的所有免疫刺激表面特性，但它们不会造成与活疫苗相关的任何感染威胁。最近，我们已经将大肠杆菌自身转运血红蛋白蛋白酶（Hbp）改造成用于在革兰氏阴性菌中高效表面展示异源蛋白HbpD的平台。利用结核分枝杆菌疫苗靶点ESAT 6（6 kDa早期分泌抗原靶点），探索HbpD在修饰大肠杆菌中的应用。大肠杆菌和沙门氏菌的菌影。不同启动子系统的使用使得HbpD-ESAT 6和裂解蛋白E的协同生产成为可能。通过基于CFU确定裂解效率、一组细胞标志物的定位、荧光显微镜、流式细胞术和电子显微镜来监测血影形成。使用蛋白酶可及性测定、荧光显微镜、流式细胞术和（免疫）电子显微镜的组合来监测ESAT 6的Hbp介导的表面展示。在这里，我们表明，HbpD和裂解蛋白E在E.大肠杆菌和沙门氏菌可用于产生在其表面上有效展示抗原的菌膜。该系统有望开发具有最佳内在佐剂活性和将异源抗原暴露于免疫系统的安全且具有成本效益的疫苗。
Bacterial ghosts are empty cell envelopes of Gram-negative bacteria that can be used as vehicles for antigen delivery. Ghosts are generated by releasing the bacterial cytoplasmic contents through a channel in the cell envelope that is created by the controlled production of the bacteriophage phi X174 lysis protein E. While ghosts possess all the immunostimulatory surface properties of the original host strain, they do not pose any of the infectious threats associated with live vaccines. Recently, we have engineered the Escherichia coli autotransporter hemoglobin protease (Hbp) into a platform for the efficient surface display of heterologous proteins in Gram-negative bacteria, HbpD. Using the Mycobacterium tuberculosis vaccine target ESAT6 (early secreted antigenic target of 6 kDa), we have explored the application of HbpD to decorate E. coli and Salmonella ghosts with antigens. The use of different promoter systems enabled the concerted production of HbpD-ESAT6 and lysis protein E. Ghost formation was monitored by determining lysis efficiency based on CFU, the localization of a set of cellular markers, fluorescence microscopy, flow cytometry, and electron microscopy. Hbp-mediated surface display of ESAT6 was monitored using a combination of a protease accessibility assay, fluorescence microscopy, flow cytometry and (immuno-) electron microscopy. Here, we show that the concerted production of HbpD and lysis protein E in E. coli and Salmonella can be used to produce ghosts that efficiently display antigens on their surface. This system holds promise for the development of safe and cost-effective vaccines with optimal intrinsic adjuvant activity and exposure of heterologous antigens to the immune system.