Fimbriae mediate the initial adherence of ETEC to the piglet small intestine and play an important role in development of ETEC-driven post-weaning diarrhea (PWD). PWD inflicts huge economic losses on the swine industry each year, making development of alternative treatment and prevention measures for PWD essential. Vaccine candidates that induce anti-fimbriae antibodies that block the initial attachment and colonization of ETEC pathogens with fimbriae are one approach that could help prevent PWD. In this study, we constructed two multi-epitope fusion antigens (MEFAs) that carried, expressed, and displayed representative epitopes of F4, F5, F6, F18 and F41 ETEC fimbriae. These MEFAs used either the F4 major subunit FaeG or the F18 adhesive subunit FedF as a backbone. To assess the potential of these MEFAs as anti-fimbriae vaccine candidates that could help prevent PWD, we generated computational models of the MEFAs, constructed them, and then tested their immunogenicity by using them to immunize mice. Computational modeling showed that all relevant epitopes were exposed on the MEFA surface. We found that co-administration of our MEFAs in mice successfully induced five fimbriae specific antibodies in accordance with the epitopes included in the MEFA constructs. Furthermore, the induced antibodies can significantly inhibit the ability of ETEC strains that express F4, F5, F6, F18, and F41 fimbriae adhere to piglet small intestinal IPEC-1 and IPEC-J2 cells. Our findings indicate that the anti-fimbriae antibodies induced by our FaeG-Fim41a-FanC-FasA and FedF-FasA-Fim41a-FanC fimbriae MEFAs blocked adherence of five ETEC fimbriae, suggesting these multivalent fimbriae MEFAs may be useful for developing broadly protective anti-fimbriae vaccines against PWD caused by ETEC infections.

Importance

Enterotoxigenic Escherichia coli (ETEC)-associated post-weaning diarrhea (PWD) is still a leading disease in recently weaned piglets. Vaccination is considered to be the most ideal and efficacious strategy for preventing PWD. Recently, a commercialized live monovalent F4 oral vaccine and a bivalent F4/F18 oral vaccine have been demonstrated to effectively protect piglets in the F4+ and F18+ ETEC challenge models. However, they will not provide cross-protection against F5+, F6+, or F41+ ETEC-associated PWD cases due to lack of expressing these fimbriae antigens. Thus, a multivalent vaccine containing all ETEC five fimbriae would be more effective in preventing against ETEC-driven PWD. In this study, we designed two fimbriae-targeted MEFAs using the MEFA technology, further study demonstrated co-administrated these MEFAs in mice can induced protective antibodies against five fimbriae expressed by ETEC. These MEFAs could be used as an efficient PWD vaccine candidate, furthermore, MEFA-based structural technology provides an alternative and promising strategy for the development of vaccines against pathogens with heterogeneous virulence factors.

菌毛介导ETEC对仔猪小肠的最初粘附，并在ETEC驱动的断奶后腹泻（PWD）的发生中发挥重要作用。每年，残疾人都会给养猪业造成巨大的经济损失，因此必须开发替代治疗和预防措施。诱导抗菌毛抗体的疫苗候选物可阻止菌毛ETEC病原体的最初附着和定殖，这是一种有助于预防PWD的方法。在这项研究中，我们构建了两个携带，表达和展示F4，F5，F6，F18和F41 ETEC菌毛代表性表位的多表位融合抗原（MEFA）。这些MEFA使用F4主要亚基FaeG或F18粘性亚基FedF作为骨架。为了评估这些MEFA作为可能有助于预防PWD的抗菌毛疫苗候选物的潜力，我们生成了MEFA的计算模型，构建了它们，然后通过使用它们免疫小鼠来测试其免疫原性。计算模型表明，所有相关的表位都暴露在MEFA表面上。我们发现，根据MEFA构建体中包含的表位，在小鼠中共同施用我们的MEFA成功诱导了五种菌毛特异性抗体。此外，诱导的抗体可以显着抑制表达F4，F5，F6，F18和F41菌毛的ETEC菌株粘附于仔猪小肠IPEC-1和IPEC-J2细胞的能力。

重要性

产肠毒素大肠杆菌（ETEC）相关的断奶后腹泻（PWD）仍是最近断奶仔猪的主要疾病。接种疫苗被认为是预防PWD的最理想，最有效的策略。最近，已经证明了商业化的单价F4活疫苗和二价F4 / F18活疫苗可以在F4 +和F18 + ETEC攻击模型中有效保护仔猪。但是，由于缺乏表达这些菌毛抗原，它们不会针对F5 +，F6 +或F41 + ETEC相关的PWD病例提供交叉保护。因此，包含所有ETEC五个菌毛的多价疫苗将更有效地预防ETEC驱动的PWD。在这项研究中，我们使用MEFA技术设计了两种针对菌毛的MEFA，进一步的研究表明，在小鼠中共同施用这些MEFA可以诱导针对ETEC表达的五种菌毛的保护性抗体。这些MEFA可以用作有效的PWD疫苗候选者，此外，基于MEFA的结构技术为开发针对具有异源毒力因子的病原体的疫苗提供了另一种有希望的策略。