Enterotoxigenic Escherichia coli (ETEC) strains producing K88 (F4) or F18 fimbriae and enterotoxins are the predominant cause of pig postweaning diarrhea (PWD). We recently identified neutralizing epitopes of fimbriae K88 and F18, heat-labile toxin (LT), heat-stable toxins type I (STa) and type II (STb), and Shiga toxin 2e (Stx2e). In this study, we explored a novel epitope- and structure-based vaccinology platform, multiepitope fusion antigen (MEFA), for PWD vaccine development. By using an epitope substitution LT toxoid, which lacks enterotoxicity but retains immunogenicity, as the backbone to present neutralizing epitopes of two ETEC fimbriae and four toxins, we generated PWD fimbria-toxin MEFA to mimic epitope native antigenicity. We then examined MEFA protein immunogenicity and evaluated MEFA application in PWD vaccine development. Mice subcutaneously immunized with PWD MEFA protein developed strong IgG responses to K88, F18, LT, and STb and moderate responses to the toxins Stx2e and STa. Importantly, MEFA-induced antibodies inhibited adherence of K88 or F18 fimbrial bacteria to pig intestinal cells and also neutralized LT, STa, STb, and Stx2e toxicity. These results indicated that PWD fimbria-toxin MEFA induced neutralizing antibodies against an unprecedent two fimbriae and four toxins and strongly suggested a potential application of this MEFA protein in developing a broadly protective PWD vaccine.

中文翻译：

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肠毒素原性大肠杆菌的新型抗原决定簇和结构疫苗辅助的菌毛毒素多表位融合抗原在猪断奶后腹泻多价疫苗开发中的应用。

产肠毒素大肠杆菌（ETEC）产生K88（F4）或F18菌毛和肠毒素的菌株是猪断奶后腹泻（PWD）的主要原因。我们最近发现了中和性菌毛K88和F18，热不稳定毒素（LT），热稳定毒素I型（STa）和II型（STb）和志贺毒素2e（Stx2e）的表位。在这项研究中，我们探索了用于PWD疫苗开发的新型基于表位和结构的疫苗学平台，多表位融合抗原（MEFA）。通过使用缺乏肠毒性但保留免疫原性的抗原决定簇替代LT类毒素作为呈现两个ETEC菌毛和四种毒素的中和抗原决定簇的骨架，我们生成了PWD菌毛毒素MEFA以模仿抗原决定簇的天然抗原性。然后，我们检查了MEFA蛋白的免疫原性，并评估了MEFA在PWD疫苗开发中的应用。用PWD MEFA蛋白皮下免疫的小鼠对K88，F18，LT和STb产生了强烈的IgG反应，对毒素Stx2e和STa产生了中度反应。重要的是，MEFA诱导的抗体抑制了K88或F18细菌细菌粘附于猪肠道细胞，并中和了LT，STa，STb和Stx2e毒性。这些结果表明，PWD菌毛毒素MEFA诱导了针对前所未有的两种菌毛和四种毒素的中和抗体，并强烈暗示了该MEFA蛋白在开发广泛保护性PWD疫苗中的潜在应用。和Stx2e毒性。这些结果表明，PWD菌毛毒素MEFA诱导了针对前所未有的两种菌毛和四种毒素的中和抗体，并强烈暗示了该MEFA蛋白在开发广泛保护性PWD疫苗中的潜在应用。和Stx2e毒性。这些结果表明，PWD菌毛毒素MEFA诱导了针对前所未有的两种菌毛和四种毒素的中和抗体，并强烈暗示了该MEFA蛋白在开发广泛保护性PWD疫苗中的潜在应用。