Wen Sharon X, Teel Louise D, Judge Nicole A, O'Brien Alison D
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA.
Proc Natl Acad Sci U S A. 2006 May 2;103(18):7082-7. doi: 10.1073/pnas.0510843103. Epub 2006 Apr 25.
Hemolytic uremic syndrome, the leading cause of kidney failure in children, often follows infection with enterohemorrhagic Escherichia coli and is mediated by the Shiga type toxins, particularly type 2 (Stx2), produced by such strains. The challenge in protecting against this life-threatening syndrome is to stimulate an immune response at the site of infection while also protecting against Shiga intoxication at distal sites such as the kidney. As one approach to meeting this challenge, we sought to develop and characterize a prototypic orally delivered, plant-based vaccine against Stx2, an AB5 toxin. First, we genetically inactivated the Stx2 active A subunit gene and then optimized both subunit genes for expression in plants. The toxoid genes were then transformed into the Nicotiana tabacum (tobacco) cell line NT-1 by Agrobacterium tumefaciens-mediated transformation. Toxoid expression was detected in NT-1 cell extracts, and the assembly of the holotoxoid was confirmed. Finally, mice were immunized by feeding with the toxoid-expressing NT-1 cells or by parenteral immunization followed by oral vaccination (prime-boost strategy). The immunized mice produced Stx2-specific mucosal IgA and Stx2-neutralizing serum IgG. The protective efficacy of these responses was assessed by challenging the immunized mice with E. coli O91:H21 strain B2F1, an isolate that produces an activatable variant of Stx2 (Stx2d) and is lethal to mice. The oral immunization fully protected mice from the challenge. Results of this study demonstrated that a plant-based oral vaccine can confer protection against lethal systemic intoxication.
溶血尿毒综合征是儿童肾衰竭的主要原因，通常继发于肠出血性大肠杆菌感染，由这类菌株产生的志贺样毒素介导，尤其是2型（Stx2）。预防这种危及生命综合征的挑战在于，既要在感染部位刺激免疫反应，又要防止在肾脏等远端部位发生志贺样毒素中毒。作为应对这一挑战的一种方法，我们试图研发并表征一种针对AB5毒素Stx2的原型口服植物疫苗。首先，我们对Stx2活性A亚基基因进行基因失活，然后优化两个亚基基因以便在植物中表达。接着，通过根癌农杆菌介导的转化将类毒素基因导入烟草细胞系NT-1。在NT-1细胞提取物中检测到类毒素表达，并证实了全毒素的组装。最后，通过喂食表达类毒素的NT-1细胞或先进行肠胃外免疫再进行口服疫苗接种（初免-加强策略）对小鼠进行免疫。免疫后的小鼠产生了Stx2特异性黏膜IgA和Stx2中和血清IgG。通过用大肠杆菌O91:H21菌株B2F1攻击免疫后的小鼠来评估这些反应的保护效力，该菌株产生一种可激活的Stx2变体（Stx2d），对小鼠具有致死性。口服免疫完全保护小鼠免受攻击。这项研究的结果表明，一种植物源口服疫苗可以提供针对致死性全身中毒的保护。
