Development of rotavirus-based enterotoxigenic Escherichia coli dual vaccines

项目摘要 腹泻疾病占全球儿童死亡人数的十分之一。轮状病毒与产肠毒素 大肠埃希菌(ETEC)是世界范围内急性胃肠炎和严重腹泻的主要病原， 加在一起，每年大约有300,000人死亡，主要是五岁以下的儿童。当前 轮状病毒疫苗在流行国家的效力有限，而且没有直接的抗病毒药物可用。没有疫苗是 获得ETEC许可。我们的总体目标是更好地了解轮状病毒和ETEC的生物学并使用它 开发治疗干预措施以减轻腹泻和后遗症的信息。 在初步研究中，我们利用了我们实验室开发的改进的RV反向遗传学系统，并 产生了编码人诺如病毒VP1蛋白突起结构域的重组小鼠轮状病毒。 我们发现这种以rv为基础的病毒载体能诱导出强大的抗原特异性血清免疫球蛋白和粪便免疫球蛋白A反应。 在接种的小鼠幼崽中。基于这些数据，我们构建了几个新的重组RV，它们可以表达 ETEC热不稳定和热稳定毒素。我们推测一种或多种新型轮状病毒-ETEC双重疫苗 候选者将在小鼠体内诱导保护性体液免疫反应，并减少病原体负担 以及随后RV和ETEC感染的致病机制。 为了验证这一假设，我们开发了一种高度易处理的小鼠轮状病毒反向遗传学方法，疾病 相关的新生和成年小鼠模型，以及几种创新的初级人类小肠有机化合物 培养，这将为理解免疫原性和保护性提供前所未有的解决方案 我们的候选疫苗的有效性。在目标1中，我们将描述轮状病毒复制的特征并定义免疫学 我们的双联疫苗在新生小鼠中的反应。在目标2中，我们将检测重组RV- ETEC双重疫苗在成年小鼠中保护免疫小鼠免受轮状病毒和ETEC的挑战。总而言之， 我们期望我们的原则证明研究将开始确立房车作为一种创新的活体衰减车的效用 黏膜疫苗平台，编码外来抗原，广泛防御常见肠道病原体。

Project Summary Diarrheal diseases account for 1 in 10 child deaths worldwide. Rotavirus (RV) and enterotoxigenic Escherichia coli (ETEC) are major etiological causes of acute gastroenteritis and severe diarrhea worldwide, together resulting in approximately 300,000 deaths each year, mostly in children under the age of five. Current RV vaccines have limited efficacy in endemic countries and no direct antivirals are available. No vaccine is licensed for ETEC. Our overall objectives are to better understand the biology of RV and ETEC and to use that information to develop therapeutic interventions to alleviate diarrhea and sequelae. In preliminary studies, we utilized an improved RV reverse genetics system developed by our lab and generated a recombinant murine RV that encodes the protruding domain of the human norovirus VP1 protein. We found that this RV-based viral vector induces a robust antigen-specific serum IgG and fecal IgA responses in inoculated mouse pups. Based on these data, we constructed several new recombinant RVs that express ETEC heat-labile and heat-stable toxins. We hypothesize that one or more novel RV-ETEC dual vaccine candidates will induce a protective humoral immune response in mice and reduce pathogen burden and pathogenesis from subsequent RV and ETEC infections. To test this hypothesis, we have developed a highly tractable murine RV reverse genetics method, disease relevant neonatal and adult mouse models, and several innovative primary human small bowel organoid cultures, which will provide an unprecedented resolution of understanding of the immunogenicity and protective efficacy of our vaccine candidates. In Aim 1, we will characterize RV replication and define the immunological responses of our dual vaccines in neonatal mice. In Aim 2, we will examine the efficacy of the recombinant RV- ETEC dual vaccines in protecting immunized mice from RV and ETEC challenges in adult mice. Collectively, we expect our proof-of-principle study to start to establish the utility of RVs as an innovative live-attenuated mucosal vaccine platform to encode foreign antigens and broadly protect against common enteric pathogens.