A broadly protective subunit vaccine against enterotoxigenic Escherichia coli (ETEC) associated diarrhea

腹泻是5岁以下儿童死亡的第二大原因， 公共卫生问题。产肠毒素大肠杆菌（ETEC）是腹泻最常见的细菌原因， 导致2.8亿至4亿例5岁以下儿童的疟疾病例<5 years and 100 million more cases in children > 每年造成15万到30万人死亡。ETEC通常也会引起旅行者腹泻。 虽然改善卫生条件是最终的解决办法，但对许多人来说，接种疫苗将是最好的预防措施 几十年 ETEC的关键毒力因子是细菌粘附素和肠毒素。粘附素介导细菌 附着于宿主受体并在小肠中定殖。肠毒素、不耐热毒素（LT）和热- 稳定毒素（STa），破坏肠道液体稳态，引起液体分泌过多和腹泻。疫苗 阻止细菌定植和中和毒素肠毒性可有效预防ETEC腹泻， 但迄今为止，还没有研制出安全有效的ETEC疫苗。 开发ETEC疫苗具有挑战性，因为异质性ETEC菌株表达至少23 粘附素和两种不同的毒素作为产生任何一种或两种粘附素和毒素（LT或LT）的ETEC菌株， STa）引起腹泻，理想情况下，有效的ETEC疫苗应保护免受所有粘附素和两种毒素的侵害。 但是，对23种粘附素的保护是不可行的，对2种毒素的保护是非常困难的。此外，本发明还 缺乏合适的动物模型来明确评估ETEC候选疫苗的效力。 在本研究中，我们将开发一种安全和广泛保护的ETEC疫苗。我们将应用多表位 融合抗原（MEFA）方法，以产生粘附素MEFA，从而诱导针对7种关键粘附素的抗体 与80%的ETEC腹泻病例（由具有已知粘附素的ETEC菌株引起）相关，并融合LT 类毒素和STa类毒素用于安全的类毒素融合以诱导中和两种毒素的抗体。粘附素 MEFA和类毒素融合物将组合（共同施用）或进一步融合在一起（作为CFA-类毒素 MEFA）以诱导广泛保护性抗粘附素和抗毒素抗体。然后我们将使用一只新的小猪， 兔双重攻毒模型，以评估亚单位候选疫苗对ETEC腹泻的有效性。 我们的初步研究表明，LT-STa类毒素融合，粘附素MEFA和粘附素-类毒素融合诱导 中和两种毒素和7种粘附素的抗体，保护猪免受ETEC腹泻， 猪是评价ETEC疫苗效力的理想动物模型。 广泛保护的亚单位ETEC疫苗将使数百万儿童和旅行者受益。创新 本研究开发的方法可普遍应用于多价疫苗开发， 其它疾病

Project Summary: Diarrhea is the second leading cause of death in children <5 years, and remains a major public health problem. Enterotoxigenic Escherichia coli (ETEC), the most common bacterial cause of diarrhea, causes 280 to 400 million diarrheal cases in children <5 years and 100 million more cases in children >5 years annually, resulting in 150,000 to 300,000 deaths annually. ETEC also commonly cause travellers’ diarrhea. While improved sanitation is the eventual solution, vaccination will be the best preventive measure for many decades. Key virulence factors of ETEC are bacterial adhesins and enterotoxins. Adhesins mediate bacterial attachment to host receptors and colonization in small intestines. Enterotoxins, heat-labile toxin (LT) and heat- stable toxin (STa), disrupt intestinal fluid homeostasis to cause fluid hyper-secretion and diarrhea. A vaccine preventing bacteria colonization and neutralizing toxin enterotoxicity could effectively prevent ETEC diarrhea, but until now, a safe and effective ETEC vaccine has not been developed. Developing ETEC vaccines is challenging since the heterogeneous ETEC strains express at least 23 adhesins and 2 distinct toxins. As ETEC strains producing any one or two adhesins and either toxin (LT or STa) cause diarrhea, ideally, an effective ETEC vaccine should protect against all adhesins and both toxins. But protecting against 23 adhesins is not feasible and protecting against 2 toxins is very difficult. Additionally, suitable animal models to unambiguously assess efficacy of ETEC vaccine candidates have been lacking. In this study, we will develop a safe and broadly protective ETEC vaccine. We will apply a multiepitope fusion antigen (MEFA) method to create an adhesin MEFA to induce antibodies against the 7 key adhesins associates with 80% of ETEC diarrhea cases (caused by ETEC strains with known adhesins), and fuse an LT toxoid and a STa toxoid for a safe toxoid fusion to induce antibodies neutralizing both toxins. The adhesin MEFA and the toxoid fusion will be combined (co-administration) or further fused together (as a CFA-toxoid MEFA) to induce broadly protective anti-adhesin and antitoxin antibodies. We will then use a novel piglet and rabbit dual-challenge model to assess efficacy of a subunit vaccine candidate against ETEC diarrhea. Our preliminary studies show LT-STa toxoid fusions, adhesin MEFA and adhesin-toxoid fusion induce antibodies neutralizing both toxins and 7 adhesins and protecting pigs against ETEC diarrhea, and rabbits and pigs are ideal animal models for ETEC vaccine efficacy assessment. A broadly protective subunit ETEC vaccine will benefit millions of children and travelers. The innovative approaches developed in this research can be generally applied to multivalent vaccine development against other diseases.