Department of Diagnostic Medicine/Pathobiology, Kansas State University College of Veterinary Medicine, Manhattan, Kansas, USA.
Department of Veterinary Basic Sciences, University of Nebraska-Lincoln School of Veterinary Medicine & Biomedical Sciences, Lincoln, Nebraska, USA.
Appl Environ Microbiol. 2020 Nov 24;86(24). doi: 10.1128/AEM.00274-20.
Enterotoxigenic (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. ETEC-associated postweaning diarrhea (PWD) causes significant economic losses to swine producers worldwide. Currently, there is no effective prevention against PWD. A vaccine that blocks ETEC fimbriae (K88 and F18) from attaching to host receptors and prevents enterotoxins from stimulating water hypersecretion in pig small intestinal epithelial cells can effectively protect against PWD and significantly improves pig health and well-being. The fimbria-toxin MEFA generated from this study induced neutralizing antibodies against both ETEC fimbriae and all four ETEC toxins, suggesting a great potential of this fimbria-toxin MEFA in PWD vaccine development and further supporting the general application of this novel MEFA vaccinology platform for multivalent vaccine development.
肠产毒性（ETEC）菌株产生 K88（F4）或 F18 菌毛和肠毒素是猪断奶后腹泻（PWD）的主要原因。我们最近鉴定了 K88 和 F18 菌毛、不耐热毒素（LT）、耐热毒素 I 型（STa）和 II 型（STb）以及志贺毒素 2e（Stx2e）的中和表位。在这项研究中，我们探索了一种新的基于表位和结构的疫苗学平台，多表位融合抗原（MEFA），用于 PWD 疫苗的开发。我们使用缺乏肠毒性但保留免疫原性的 LT 类毒素作为骨架，来展示两种 ETEC 菌毛和四种毒素的中和表位，从而产生了模拟表位天然抗原性的 PWD 菌毛-毒素 MEFA。然后，我们研究了 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，并对毒素 Stx2e 和 STa 产生了中等反应。重要的是，MEFA 诱导的抗体抑制了 K88 或 F18 菌毛细菌对猪肠细胞的粘附，并且中和了 LT、STa、STb 和 Stx2e 的毒性。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，并强烈表明该 MEFA 蛋白在开发广泛保护性 PWD 疫苗方面具有潜在应用。肠产毒性大肠杆菌（ETEC）相关的断奶后腹泻（PWD）给全球养猪生产者造成了重大经济损失。目前，尚无有效的 PWD 预防措施。一种能够阻止 ETEC 菌毛（K88 和 F18）与宿主受体结合并防止肠毒素刺激猪小肠上皮细胞过度分泌水分的疫苗，可以有效地预防 PWD，并显著改善猪的健康和福利。本研究中产生的菌毛-毒素 MEFA 诱导了针对两种 ETEC 菌毛和所有四种 ETEC 毒素的中和抗体，表明该菌毛-毒素 MEFA 在 PWD 疫苗开发中具有巨大潜力，并进一步支持将这种新型 MEFA 疫苗学平台广泛应用于多价疫苗开发。
