Zhu Chengru, Ruiz-Perez Fernando, Yang Zhuolu, Mao Ying, Hackethal Veronica L, Greco Karla M, Choy Wendy, Davis Katherine, Butterton Joan R, Boedeker Edgar C
Center for Vaccine Development, University of Maryland School of Medicine, 685 West Baltimore Street, Baltimore, MD 21201, USA.
Vaccine. 2006 May 1;24(18):3821-31. doi: 10.1016/j.vaccine.2005.07.024. Epub 2005 Jul 25.
In this report, we describe the use of an attenuated regulatory mutant of a rabbit enteropathogenic Escherichia coli (rEPEC) as a live vaccine vector to deliver heterologous protein antigens using two dedicated transport systems, a Salmonella autotransporter and the E. coli hemolysin apparatus. We previously reported that an isogeneic ler (LEE encoded regulator) mutant of rEPEC O103:H2 is attenuated and immunogenic in rabbits. We first evaluated the Salmonella autotransporter MisL containing the immunodominant B-cell epitope of the circumsporozoite protein from Plasmodium falciparum, (NANP)8, fused to the C-terminal translocator domain under the control of the constitutive Tac17 promoter. The rEPEC ler mutant was able to express and to translocate the (NANP)8 passenger peptide to the bacterial surface. We next investigated the delivery of Shiga toxin B subunit (Stx1B) from human enterohemorrhagic E. coli by the rEPEC ler mutant via the MisL autotransporter or the E. coli hemolysin secretion apparatus. The autotransporter and hemolysin plasmids expressed similar levels of Stx1B (30-40 ng/ml/OD600). Only 6% of Stx1B was found in the autotransporter supernatants; the rest was cell-associated, with a small fraction of the Stx1B surface-exposed as determined by immunofluorescence. In contrast, 88% of Stx1B was secreted into culture supernatants by the hemolysin secretion system. In an in vivo study, no significant protection was observed in rabbits inoculated with the ler mutant harboring the Stx1B-autotransporter plasmid following experimental challenge with RDEC-H19A, the prototype rEPEC containing an Stx-converting phage. In contrast, rabbits inoculated with the rEPEC ler mutant containing the Stx1B-hemolysin fusion were partially protected from RDEC-H19A infection as demonstrated by decreased weight loss (p<0.008) when compared to rabbits inoculated with the parent ler mutant. Our results suggest that attenuated rEPEC are capable of serving as vaccine vectors to express heterologous protein antigens from different cellular locations and deliver these antigens to the intestinal mucosa. With this system, secreted proteins may be more effective than cell-associated antigens in generating protection.
在本报告中，我们描述了使用兔肠道致病性大肠杆菌（rEPEC）的减毒调节突变体作为活疫苗载体，通过两种专用运输系统——沙门氏菌自转运体和大肠杆菌溶血素装置来递送异源蛋白抗原。我们之前报道过，rEPEC O103:H2的同基因ler（LEE编码调节因子）突变体在兔体内是减毒且具有免疫原性的。我们首先评估了含有恶性疟原虫环子孢子蛋白免疫显性B细胞表位（NANP）8的沙门氏菌自转运体MisL，其与C末端转运结构域融合，并受组成型Tac17启动子控制。rEPEC ler突变体能够表达（NANP）8乘客肽并将其转运至细菌表面。接下来，我们研究了rEPEC ler突变体通过MisL自转运体或大肠杆菌溶血素分泌装置递送来自人肠出血性大肠杆菌的志贺毒素B亚基（Stx1B）的情况。自转运体和溶血素质粒表达的Stx1B水平相似（30 - 40 ng/ml/OD600）。在自转运体上清液中仅发现6%的Stx1B；其余与细胞相关，通过免疫荧光测定，有一小部分Stx1B暴露于表面。相比之下，88%的Stx1B通过溶血素分泌系统分泌到培养上清液中。在一项体内研究中，用携带Stx1B - 自转运体质粒的ler突变体接种的兔子在受到含有Stx转换噬菌体的原型rEPEC即RDEC - H19A实验性攻击后，未观察到显著的保护作用。相比之下，与接种亲本ler突变体的兔子相比，接种含有Stx1B - 溶血素融合体的rEPEC ler突变体的兔子体重减轻减少（p<0.008），这表明它们受到部分保护，免受RDEC - H19A感染。我们的结果表明，减毒的rEPEC能够作为疫苗载体，从不同细胞位置表达异源蛋白抗原并将这些抗原递送至肠黏膜。使用该系统，分泌蛋白在产生保护作用方面可能比与细胞相关的抗原更有效。
