A subunit vaccine for enterotoxigenic E. coli associated traveler's diarrhea

描述(申请人提供)：腹泻大肠杆菌菌株被国家卫生研究所(NIH)列为优先B类病原体。产肠毒素大肠杆菌(ETEC)是引起旅行者腹泻的最常见原因。世界卫生组织(WHO)报告称，ETEC菌株每年导致3.8亿-5亿儿童腹泻病例和4亿多成人旅行者腹泻病例，每年导致约30万-50万人死亡。世卫组织和其他全球和区域卫生项目的当务之急是开发一种针对ETEC相关旅行者腹泻的广泛保护性疫苗。目前，还没有有效的疫苗来预防旅行者的腹泻。新城疫病毒的毒力决定因素 腹泻中的ETEC是细菌粘附素和肠毒素。粘附素介导细菌在小肠的附着和定植。肠毒素破坏宿主上皮细胞的液体动态平衡，导致液体过度分泌和腹泻。一种诱导抗粘附素免疫以阻断ETEC附着和定植，并诱导抗毒素免疫以中和肠道毒性的疫苗可有效预防ETEC旅行者的腹泻。我们的长期目标是了解腹泻疾病的分子发病机制，并开发疾病预防策略。我们的近期目标是开发一种针对ETEC腹泻的广泛保护性疫苗。我们提供的初步数据表明：1)携带7个粘附素代表性表位的ETEC CFA多表位抗原诱导广泛的保护性免疫反应；2)LT和STA类毒素融合抗原诱导针对这两种毒素的中和抗体；3)猪型CFA类毒素融合诱导对异种粘附素和毒素的保护性免疫。这项研究的具体假设是：1)多表位CFA-类毒素抗原将诱导对最流行和最毒力的ETEC株表达的7种粘附素以及LT和STA毒素的广泛免疫反应；2)这种多表位CFA-类毒素抗原诱导的免疫将保护表达这7种粘附素的ETEC株的粘附性，并中和这两种ETEC毒素，并且这种多表位CFA-类毒素融合抗原可以开发成针对ETEC旅行者腹泻的亚单位疫苗。利用创新的策略构建CFA-毒素融合蛋白、小鼠免疫研究和一种新的仔猪挑战研究，提出了以下具体目标来验证我们的假设：1)构建针对7种ETEC粘附素和两种毒素的多表位CFA-毒素抗原；2)评估该多表位CFA-毒素抗原用于ETEC旅行者腹泻亚单位疫苗的候选。这项研究的结果将改变目前ETEC疫苗的开发策略，即将多个灭活或活的大肠杆菌菌株与重组毒素亚单位蛋白混合在一起诱导抗粘附素免疫。在一种产品中混合多种菌株已被证明会导致较低的免疫反应(没有明显的保护作用)，并由于产品中含有过多的体细胞抗原和脂多糖而导致不良反应。表达最流行和毒力最强的ETEC粘附素和这两种毒素的抗原表位作为单一抗原，可以产生安全有效的亚单位疫苗，广泛预防旅行者腹泻。

DESCRIPTION (provided by applicant): Diarrheal E. coli strains are listed the priority category B pathogen by the National Institute of Health (NIH). Enterotoxigenic Escherichia coli (ETEC) are the most common cause of traveler's diarrhea. The World Health Organization (WHO) reported that each year ETEC strains cause 380-500 million diarrhea cases to children and 400 million more cases to adult travelers, which results in approximately 300,000- 500,000 deaths annually. It is a top priority for WHO and other global and regional health programs to develop a broadly protective vaccine against ETEC-associated traveler's diarrhea. Currently, there are no effective vaccines available to protect against traveler's diarrhea. The virulence determinants of ETEC in diarrhea are bacterial adhesins and enterotoxins. Adhesins mediate bacteria attachment and colonization at small intestines. Enterotoxins disrupt fluid homeostasis in host epithelial cells that leads to fluid hyper-secretion and diarrhea. A vaccine inducing anti-adhesin immunity to block ETEC attachment and colonization and also antitoxin immunity to neutralize enterotoxicity can effectively protect against ETEC traveler's diarrhea. Our long-term goal is to understand molecular pathogenesis of diarrhea diseases and to develop disease prevention strategies. Our immediate objective is to develop a broadly protective vaccine against ETEC diarrhea. We here present preliminary data showing that: 1) a multiepitope ETEC CFA antigen carrying representative epitopes of 7 adhesins induced broadly protective immune responses; 2) LT and STa toxoid fusion antigens elicited neutralizing antibodies against both toxins; and 3) a porcine-type CFA-toxoid fusion induced protective immunity against heterogeneous adhesins and also the toxin. The specific hypotheses for this proposed study are that: 1) a multiepitope CFA- toxoid antigen will induce broad immune responses to 7 adhesins expressed by the most prevalent and most virulent ETEC strains and also to both LT and STa toxins; and 2) immunity induced by this multiepitope CFA-toxoid antigen will protect against adherence of ETEC strains expressing these 7 adhesins and also neutralize against both ETEC toxins, and this multiepitope CFA-toxoid fusion antigen can be developed as a subunit vaccine against ETEC traveler's diarrhea. Using innovative strategies to construct a CFA-toxoid fusion, mouse immunization studies and a novel piglet challenge study, the following specific aims are proposed to test our hypothesis: 1) To construct a multiepitope CFA-toxoid antigen for immunity against 7 ETEC adhesins and both toxins; 2) To evaluate this multiepitope CFA-toxin antigen for subunit vaccine candidacy against ETEC traveler's diarrhea. Results from this study will change current ETEC vaccine development strategies, of which multiple killed or live E. coli strains and recombinant toxin subunit protein are mixed together to induce anti-adhesin immunity. Mixing multiple strains in a product has been shown to result in lower immune responses (with no significant protection) and to cause adverse effects due to excessive somatic antigens and LPS included in products. Expressing antigenic epitopes of the most prevalent and virulent ETEC adhesins and both toxins as a single antigen can lead to a safe and effective subunit vaccine for broad protection against traveler's diarrhea.