Scholarlink

<p>Cattle are the major reservoir and an important source of environmental dissemination of enterohemorrhagic <i>E. coli</i> (EHEC), a human pathogen responsible for outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) worldwide. Preharvest vaccines are aimed to reduce bacterial carriage and consequently, the impact of this zoonosis. The chimeric antigen EIT has demonstrated to be protective against EHEC in murine models. In a recent study, we proposed a simple and cost-effective approach for a vaccine formulation for cattle, in which EIT was expressed and recovered from the periplasm of the laboratory strain <i>E. coli</i> BL21. As a proof-of-concept, the protective efficacy in mice as well as the induction of humoral immune responses in bovines were verified. In this work, we aimed to improve the production of the recombinant antigen EIT using <i>E. coli</i> fed-batch fermentations. An initial screening was conducted to select the medium that best sustained EIT expression using lactose for heterologous induction. M9 medium supplemented with yeast extract yielded the highest relative levels of EIT and was used for subsequent fermentation in a stirred-tank bioreactor. Thus, EIT concentration and volumetric productivity increased by approximately 600% compared to shake-flask cultivations, while EIT yield relative to biomass and specific productivity increased by approximately 130%. Following thermal permeabilization of cells for antigen recovery, the method yielded, overall, about 350 doses per L of fermented culture. The process was reproducible across three independent fermentations while retaining EIT antigenicity, as assessed by ELISA, and functionality, as it could induce antibodies that inhibited actin pedestal formation in in vitro infection assays. Thus, heterologous EIT expression was successfully transferred to a stirred-tank bioreactor, increasing antigen output and establishing a basis for further development of a scalable process for EHEC vaccine applications.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

Towards scalable production of recombinant EIT as a protective antigen against EHEC

  • 期刊论文

  • 发表日期：

    2026年7月22日

查看全文
  • Laura A Basile,
  • Diego G Noseda,
  • Ingrid Milstein,
  • Mara S Roset,
  • Gabriel Briones

摘要

Cattle are the major reservoir and an important source of environmental dissemination of enterohemorrhagic E. coli (EHEC), a human pathogen responsible for outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) worldwide. Preharvest vaccines are aimed to reduce bacterial carriage and consequently, the impact of this zoonosis. The chimeric antigen EIT has demonstrated to be protective against EHEC in murine models. In a recent study, we proposed a simple and cost-effective approach for a vaccine formulation for cattle, in which EIT was expressed and recovered from the periplasm of the laboratory strain E. coli BL21. As a proof-of-concept, the protective efficacy in mice as well as the induction of humoral immune responses in bovines were verified. In this work, we aimed to improve the production of the recombinant antigen EIT using E. coli fed-batch fermentations. An initial screening was conducted to select the medium that best sustained EIT expression using lactose for heterologous induction. M9 medium supplemented with yeast extract yielded the highest relative levels of EIT and was used for subsequent fermentation in a stirred-tank bioreactor. Thus, EIT concentration and volumetric productivity increased by approximately 600% compared to shake-flask cultivations, while EIT yield relative to biomass and specific productivity increased by approximately 130%. Following thermal permeabilization of cells for antigen recovery, the method yielded, overall, about 350 doses per L of fermented culture. The process was reproducible across three independent fermentations while retaining EIT antigenicity, as assessed by ELISA, and functionality, as it could induce antibodies that inhibited actin pedestal formation in in vitro infection assays. Thus, heterologous EIT expression was successfully transferred to a stirred-tank bioreactor, increasing antigen output and establishing a basis for further development of a scalable process for EHEC vaccine applications.