Department of Biology, Shahed University, Tehran-Qom Express Way, 3319118651, Tehran, Iran.
Department of Biology, Imam Hussein University, Tehran, Iran.
Curr Microbiol. 2021 Sep;78(9):3464-3473. doi: 10.1007/s00284-021-02601-x. Epub 2021 Jul 15.
Enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of diarrhea in children globally, and thus suitable vaccines are desired. Antigen display on lactic acid bacteria is a reliable approach for efficient oral vaccination and preventing bowel diseases. To develop an oral vaccine against ETEC, the gene of the binding domain from heat-labile toxin (LTB), a key ETEC virulence factor, was codon-optimized and cloned into a construct containing a signal peptide and an anchor for display on L. lactis. Bioinformatics analysis showed a codon adaptation index of 0.95 for the codon-optimized gene. Cell surface expression of LTB was confirmed by transmission electron microscopy and blotting. White New Zealand rabbits were immunized per os (PO) with the recombinant L. lactis, and the antibody titers were assayed with ELISA. In vitro neutralization assay was performed using mouse adrenal tumor cells and rabbit ileal loop test was performed as the in vivo assay. ELISA results indicated that oral administration of the engineered L. lactis elicited a significant production of IgA in the intestine. In vitro neutralization assay showed that the effect of the toxin could be neutralized with 500 µg/ml of IgG isolated from the oral vaccine group. Furthermore, the dose of ETEC causing fluid accumulation in the ileal loop test showed a tenfold increase in rabbits immunized with either recombinant L. lactis or LTB protein compared to other groups. Our results imply that recombinant L. lactis could potentially be an effective live oral vaccine against ETEC toxicity.
肠产毒性大肠杆菌（ETEC）是全球儿童腹泻的主要原因之一，因此需要合适的疫苗。抗原在乳酸菌上的展示是一种有效的口服疫苗接种和预防肠道疾病的可靠方法。为了开发针对 ETEC 的口服疫苗，将不耐热毒素（LTB）的结合结构域基因进行密码子优化，并克隆到包含信号肽和用于在乳链菌上展示的锚的构建体中。该基因的密码子适应性指数为 0.95。通过透射电子显微镜和印迹证实了 LTB 的细胞表面表达。通过口服（PO）用重组乳链菌对新西兰白兔进行免疫，并用 ELISA 测定抗体滴度。使用小鼠肾上腺肿瘤细胞进行体外中和测定，并进行兔回肠环试验作为体内测定。ELISA 结果表明，口服工程化乳链菌可在肠道中引起显著的 IgA 产生。体外中和试验表明，用从口服疫苗组分离的 500µg/ml IgG 可中和毒素的作用。此外，在回肠环试验中，与其他组相比，用重组乳链菌或 LTB 蛋白免疫的兔子引起液体积聚的 ETEC 剂量增加了十倍。我们的结果表明，重组乳链菌可能是一种有效的针对 ETEC 毒性的活口服疫苗。
