An Attenuated E.coli Vaccine for Enterotoxigenic E.coli (ETEC)

描述（由申请人提供）：产肠毒素大肠杆菌（ETEC）是婴儿和旅行者腹泻的主要原因之一。尽管全世界的ETEC感染数量众多且严重，但目前尚无针对高危人群的许可疫苗。我们通过删除或修改WT O157:H7肠出血性大肠杆菌（EHEC）分离株的主要毒力决定因素，开发了一种减毒大肠杆菌活疫苗株。我们现在建议使用该菌株作为表达ETEC抗原的载体。我们假设，通过将ETEC关键毒力决定因素（粘附素和毒素成分）在减毒的、无创的活疫苗株中传递到粘膜免疫系统，可以产生有效的全身和局部免疫反应，从而保护ETEC免受攻击。我们将使用已建立的小鼠鼻内免疫和病原体攻击模型来确定我们的疫苗株的安全性、免疫原性和保护功效。我们的第一个目标是制备我们的减毒粘附/清除肠出血性大肠杆菌（EHEC）疫苗的衍生物，该疫苗可有效表达两种关键的ETEC抗原，CFA/I定植因子和热不稳定肠毒素（LT）的成分。我们的第二个目标是通过监测免疫小鼠的不良反应来确定减毒疫苗株的安全免疫剂量。我们的第三个目标是测量鼻内接种疫苗后的血清和粘膜免疫反应，以确定保护和反应原性的相关性。我们的第四个目标是使用已建立的肺内攻击模型确定疫苗结构免疫产生的保护效力水平，我们之前曾使用该模型确定其他ETEC疫苗结构的效力。为了达到这些目的，将通过SDS-PAGE、免疫印迹和细菌凝集来证明疫苗构建物对ETEC抗原的表达。我们将通过对接种疫苗的动物进行全血细胞计数和血液化学检查，记录任何不良临床症状、尸检时的大体和组织学变化，从而评估疫苗结构的安全性。我们将通过ELISA、细胞因子反应和功能测定来评估接种小鼠的全身和局部免疫反应水平。我们将通过鼻内注射致死剂量的野生型ETEC菌株，以及通过测量接种小鼠肺部的ETEC细菌清除率来确定保护效果。开发针对ETEC毒株的安全有效的疫苗应该对发展中国家的婴儿和国际旅行者具有重大的公共卫生效益。公共卫生相关性：产肠毒素大肠杆菌（ETEC）是引起世界范围发病率和死亡率的重要细菌病原体。ETEC感染是发展中国家婴儿和五岁以下儿童死亡的重要原因。ETEC也是前往世界高危地区的旅行者腹泻的主要原因。尽管人们对ETEC的毒力因素了解得很清楚，而且在过去几十年里，在志愿者试验和实地研究中测试了几种可能的ETEC疫苗，但目前还没有安全有效的疫苗可用于高危人群。开发安全有效的ETEC减毒活疫苗对发展中国家的婴儿和国际旅行者具有重大的公共卫生意义。

DESCRIPTION (provided by applicant): Enterotoxigenic Escherichia coli (ETEC) are among the primary causes of infantile and traveler's diarrhea. Despite the number and severity of ETEC infections worldwide, at present no licensed vaccine is available for at-risk individuals. We have developed a live attenuated E. coli vaccine strain by deleting or modifying major virulence determinants of a WT O157:H7 Enterohemorrhagic E.coli (EHEC) isolate. We now propose to use this strain as a vector to express ETEC antigens. We hypothesize that by delivering critical ETEC virulence determinants (adhesins and toxin components) to the mucosal immune system in an attenuated, non-invasive live vaccine strain, effective systemic and local immune responses can be generated which will protect against ETEC challenge. We will use an established mouse intranasal model of immunization and pathogen challenge to determine safety and immunogenicity and protective efficacy of our vaccine strains. Our first aim is to prepare derivatives of our attenuated attaching/effacing enterohemorrhagic E. coli (EHEC) vaccine that efficiently express components of two critical ETEC antigens, the CFA/I colonization factor and the heat-labile enterotoxin (LT). Our second aim is to determine a safe immunizing dose of the attenuated vaccine strains by monitoring immunized mice for adverse effects. Our third aim is to measure serum and mucosal immune responses following intranasal immunization with the vaccine constructs in order to determine the correlates of protection, and also of reactogenicity. Our fourth aim is to determine the level of protective efficacy resulting from immunization with the vaccine constructs using an established intra-pulmonary challenge model which we have previously used to determine the efficacy of other ETEC vaccine constructs. To achieve these ends, the expression of ETEC antigens by the vaccine constructs will be demonstrated by SDS-PAGE, immunoblotting, and bacterial agglutination. We will assess the safety of the vaccine constructs by noting any adverse clinical signs, and gross and histological changes at necropsy and by performing complete blood cell counts and blood chemistry in the vaccinated animals. We will assess the level of both systemic and local immune responses in the vaccinated mice by ELISA, cytokine responses and functional assays. We will determine protective efficacy by intranasal challenge with a lethal dose of the wild-type ETEC strain, as well as by measuring ETEC bacterial clearance from the lungs of the vaccinated mice. Development of safe and effective vaccines directed against ETEC strains should have significant public health benefit for infants in developing countries and for international travelers. PUBLIC HEALTH RELEVANCE: Enterotoxigenic E. coli (ETEC) are important bacterial pathogens causing worldwide morbidity and mortality. ETEC infections are important causes of death in infants and children under the age of five years in developing countries. ETEC are also the leading cause of diarrhea in travelers to high-risk areas of the world. Despite the fact that virulence factors of ETEC are well understood, and although in the last few decades there have been several potential ETEC vaccines tested in volunteer trials and field studies, no safe and effective vaccine is yet available for at-risk individuals. The development of safe and effective, live attenuated ETEC vaccines should have great public health significance for infants in developing countries and for international travelers.