Escherichia coli O157:H7 and Shigella flexneri are the predominant diarrhoeal pathogens and those strains producing Shiga toxins cause life-threatening sequelae including hemolytic uremic syndrome (HUS) upon their entry into the host. Intimate adherence of E. coli O157 and invasion of S. flexneri in the host intestinal epithelial cells is mainly mediated by Intimin and IpaB proteins, respectively. In this study, we have synthesized chimera of immunodominant regions of Intimin (eae) and IpaB (ipaB) designated as EI and expressed it in Lactococcus lactis (LL-EI) to develop a combinatorial oral vaccine candidate. Immune parameters and protective efficacy of orally administered LL-EI were assessed in the murine model. Significant EI-specific serum IgG, IgA, and fecal IgA antibody titer were observed in the LL-EI group. Considerable increase in EI-specific splenocyte proliferation and a concurrent upregulation of both Th1 and Th2 cytokines was observed in LL-EI immunized mice. Flow cytometry analysis also revealed a significant increase in CD4 and CD8 cell counts in LL-EI immunized group compared to PBS, LL control group. In vitro studies using LL-EI immunized mice sera showed substantial protection against bacterial adhesion and invasion caused by E. coli O157 and Shigella flexneri¸ respectively. LL-EI immunized group challenged with E. coli O157 ceased fecal shedding within 6 days, and mice challenged with S. flexneri showed 93% survival with minimal bacterial load in the lungs. Our results indicate that LL-EI immunization elicits systemic, mucosal and cell-mediated immune responses, and can be a promising candidate for oral vaccine development against these pathogens.

大肠杆菌O157：H7和弗氏志贺氏菌是主要的腹泻病原体，那些产生志贺毒素的菌株在进入宿主后会导致危及生命的后遗症，包括溶血性尿毒症综合征（HUS）。的亲密粘附大肠杆菌O157和侵袭弗氏志贺菌在宿主肠上皮细胞主要是由紧密粘附和IPAB蛋白介导的分别。在这项研究中，我们合成了命名为EI的Intimin（eae）和IpaB（ipaB）免疫优势区域的嵌合体，并在乳酸乳球菌中表达（LL-EI）开发一种组合口服疫苗候选物。在鼠模型中评估了口服LL-EI的免疫参数和保护功效。在LL-EI组中观察到了EI特异性血清IgG，IgA和粪便IgA抗体的显着效价。在LL-EI免疫小鼠中，观察到EI特异性脾细胞增殖显着增加，同时Th1和Th2细胞因子同时上调。流式细胞仪分析还显示，与PBS，LL对照组相比，LL-EI免疫组的CD4和CD8细胞计数显着增加。 使用LL-EI免疫的小鼠血清进行的体外研究表明，对大肠杆菌O157和弗氏志贺氏菌引起的细菌粘附和入侵具有实质性保护作用分别。用大肠杆菌O157攻击的LL-EI免疫组在6天内停止了粪便脱落，用弗氏链球菌攻击的小鼠表现出93％的存活率，并且肺中的细菌负荷最小。我们的结果表明，LL-EI免疫引发全身，粘膜和细胞介导的免疫反应，并且可以作为针对这些病原体的口服疫苗开发的有前途的候选者。