Multivalent Oral Vaccine against Enterotoxigenic Escherichia coli and Enteric Fevers

由产肠毒素大肠杆菌(ETEC)、志贺氏菌和伤寒沙门氏菌引起的肠道疾病 它们是NIAID B类优先药物，每年总共影响全球4亿人。 目前，还没有针对ETEC或志贺氏菌病的疫苗。伤寒疫苗已经存在。一个负担得起的，有效的， 针对这三种生物的口服多价疫苗将具有巨大的公共卫生重要性，并且 在旅行者和军事人员中有很大的商业市场。我们的长期目标是创造一个 稳定的口服ETEC、志贺氏菌病和伤寒疫苗。开始实现目标的过程 为此，我们使用获得许可的Ty21a伤寒疫苗表达了宋内氏志贺氏菌的O抗原，以 生产一种名为TyOraSs的候选疫苗，该疫苗正在开发中。主要毒力决定因素 ETEC的致病因子是定植因子抗原(CFAs或粘附素)和两种肠毒素，即不耐热毒素(LT)和 耐热毒素(StA)。一种有效的ETEC疫苗应该能诱导出阻断细菌附着的抗体。 和/或中和毒素。在动物模型中，抗CFA抗体和毒素具有协同保护作用。它 我们团队的成员已经证明，代表7个独立的 CFA、ETEC粘附素和两种毒素可以融合为单一蛋白(这里称为Mefa T)来诱导 阻断异种ETEC株与人结肠癌细胞黏附的交叉保护性抗体 在体外，并中和所有ETEC菌株中的两种毒素。在这个项目中，我们将稳定地表达这些多个 粘附素和类毒素均稳定作为全毒素结构的CFA-类毒素融合盒抗原 在Ty21a中进行免疫原性和免疫保护效果的评价 小猪和兔子挑战模特。具体来说，我们将，1)产生和鉴定疫苗株(S) 表达染色体整合的ETEC多表位融合抗原(MEFA)的遗传优化Ty21a LT-sta类毒素(命名为Ty21a-ETEC Mefa-T)细胞内或分泌形式，2)表现 对ETEC和伤寒沙门氏菌的免疫原性，通过小鼠的粘膜免疫，以及3)建立保护性 在兔定植模型和哺乳仔猪致死性感染模型中对ETEC的抗感染效果。我们的建议 是独一无二的，因为我们在构建多价ETEC融合抗原方面的专业知识，使用 Ty21a作为表达异源抗原的平台，以及与动物模型的能力 毫不含糊地评估疫苗的保护效果。一种独立的ETEC伤寒疫苗将有 重大影响，但我们的目标是将此项目的成功作为开发 ETEC、伤寒和志贺氏菌病多价疫苗。在第二阶段中，我们将生成一个三元组 病原体疫苗、TyOraSs-ETEC疫苗或两种双价疫苗；产生 疫苗候选株(S)，用于生产符合cGMPs和泡沫干燥疫苗 产品(S)；进行必要的临床前研究；设计临床方案；并准备IND。

Enteric diseases caused by enterotoxigenic E. coli (ETEC) strains, Shigella spp, and Salmonella Typhi, all of which are NIAID Category B priority agents, collectively affect > 400 million people annually worldwide. Currently, there is no vaccine against ETEC or shigellosis. A typhoid vaccine exists. An affordable, effective, oral multivalent vaccine against all 3 organisms would have enormous public health importance, and a substantial commercial market among travelers and military personnel. Our long-term goal is to create a stable, orally administered vaccine against ETEC, shigellosis and typhoid. To begin the process of achieving this goal we have used the licensed Ty21a typhoid vaccine to express the O-antigen of Shigella sonnei to produce a vaccine candidate called TyOraSs, which is under development. The major virulence determinants of ETEC are the colonization factor antigens (CFAs or adhesins) and two enterotoxins, the heat-labile (LT) and heat-stable toxin (STa). An effective ETEC vaccine should induce antibodies that block bacterial attachment and/or to neutralize the toxins. In animal models antibodies to CFA and toxin are synergistically protective. It has been shown by members of our team that a multi-epitope fusion antigen (MEFA) representing 7 separate CFA ETEC adhesins and two toxins can be fused as a single protein (designated here as MEFA+T) to induce cross-protective antibodies that blocks adherence of heterogeneous ETEC strains to human colon cancer cells in vitro, and neutralizes two toxins in all ETEC strains. In this project we will stably express these multiple adhesins and the toxoid form of both toxins stably as a holotoxin structured CFA-toxoid fusion cassette antigen in Ty21a, and assess immunogenicity and protective efficacy of our Ty21a-ETEC vaccine using the suckling piglet and rabbit challenge models. Specifically we will, 1) Generate and characterize vaccine strain(s) of genetically optimized Ty21a expressing chromosomally integrated, ETEC multi-epitope fusion antigen (MEFA) + toxoid LT-STa (designated Ty21a-ETEC MEFA-T) either intra-cellularly or in secreted form, 2) Demonstrate immunogenicity against ETEC and S. Typhi, by mucosal immunization of mice, and 3) Establish protective efficacy against ETEC in the rabbit colonization model and suckling piglet lethal infection model. Our proposal is unique because of our expertise at construction of multivalent ETEC fusion antigens, experience with using Ty21a as a platform for expressing heterologous antigens, and capabilities with animal models to unambiguously assess vaccine protective efficacy. A stand-alone ETEC-typhoid vaccine would have substantial impact, however our aim to use success in this project as a foundation for the development of a multivalent vaccine against ETEC, typhoid, and shigellosis. In Phase II we will generate a single triple pathogen vaccine, TyOraSs-ETEC vaccine, or two bivalent vaccines; generate a master cell bank of the vaccine candidate strain(s) for manufacturing in compliance with cGMPs and as a foam-dried vaccine product(s); conduct required pre-clinical studies; design a clinical protocol; and prepare an IND.