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

针对产肠毒素大肠杆菌 (ETEC) 的减毒大肠杆菌疫苗

• 批准号: 7669911
• 负责人: EDGAR C. BOEDEKER
• 金额: $ 18.75万
• 依托单位: UNIVERSITY OF NEW MEXICO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-15 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7669911
• 关键词: Adjuvant; Adverse effects; Age; Agglutination; Animal Model; Animals; Antibodies; Antigens; Area; Attenuated; Attenuated Live Virus Vaccine; Attenuated Vaccines; Autopsy; Bacteria; Bacterial Adhesins; Biological Assay; Blood Cell Count; Blood Chemical Analysis; Body Weight decreased; Breathing; CD4 Positive T Lymphocytes; Cattle; Cause of Death; Cells; Child; Clinical; DNA; Developed Countries; Developing Countries; Development; Diarrhea; Dose; Drainage procedure; Eating; Electron Microscopy; Enterotoxins; Enzyme-Linked Immunosorbent Assay; Escherichia coli EHEC; Escherichia coli Infections; Escherichia coli Vaccines; Eye; Future; Genetic Techniques; Health Benefit; Heating; Helper-Inducer T-Lymphocyte; IgG1; IgG3; Immune response; Immune system; Immunization; Immunoblotting; Immunoglobulin A; Immunoglobulin G; Individual; Infant; International; Intestines; Irrigation; Laboratories; Licensing; Life; Lung; Measures; Metabolic Clearance Rate; Microspheres; Modeling; Molecular Genetics; Monitor; Morbidity - disease rate; Mucosal Immune Responses; Mus; Nose; Operon; Organ; Plasmids; Posture; Property; Public Health; Research Personnel; Risk; Safety; Sampling; Serum; Severities; Shiga Toxin; Shivering; Structure of mucous membrane of nose; Tissues; Toxin; Traveler' s diarrhea; Vaccinated; Vaccination; Vaccine Antigen; Vaccines; Virulence; Virulence Factors; Work; bactericide; base; clinical effect; cytokine; drinking; enterotoxigenic Escherichia coli; field study; high risk; immunogenicity; indexing; infancy; meetings; mortality; mouse model; mutant; oral vaccine; pathogen; prevent; protective efficacy; prototype; public health relevance; response; safety testing; vaccine efficacy; vaccine evaluation; vaccine safety; vector; vector vaccine; volunteer

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.

描述（由申请方提供）：产肠毒素大肠杆菌（ETEC）是婴儿和旅行者腹泻的主要原因。尽管全球ETEC感染的数量和严重程度，但目前没有许可的疫苗可用于高危人群。我们已经开发出一种减毒活大肠杆菌。通过删除或修饰WT O 157：H7肠出血性大肠杆菌（EHEC）分离株的主要毒力决定簇，对大肠杆菌疫苗株进行了重组。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，通过在减毒、非侵入性活疫苗株中向粘膜免疫系统递送关键的ETEC毒力决定因子（粘附素和毒素组分），可以产生有效的全身和局部免疫应答，从而保护免受ETEC攻击。我们将使用已建立的小鼠鼻内免疫和病原体攻毒模型来确定我们的疫苗株的安全性、免疫原性和保护效力。我们的第一个目标是制备我们的减毒的附着/消除肠出血性大肠杆菌的衍生物。大肠杆菌（EHEC）疫苗，其有效表达两种关键ETEC抗原的组分，CFA/I定殖因子和不耐热肠毒素（LT）。我们的第二个目的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。我们的第三个目的是测量用疫苗构建体鼻内免疫后的血清和粘膜免疫应答，以确定保护和反应原性的相关性。我们的第四个目的是使用我们先前用于确定其他ETEC疫苗构建体的有效性的已建立的肺内激发模型，确定疫苗构建体免疫产生的保护性有效性水平。为了实现这些目的，将通过SDS-PAGE、免疫印迹和细菌凝集来证明疫苗构建体表达ETEC抗原。我们将通过观察任何不良临床体征以及尸检时的大体和组织学变化，并通过在接种疫苗的动物中进行全血细胞计数和血液化学来评估疫苗构建体的安全性。我们将通过ELISA、细胞因子应答和功能测定评估接种小鼠的全身和局部免疫应答水平。我们将通过使用致死剂量的野生型ETEC菌株进行鼻内攻毒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力。针对ETEC菌株的安全有效疫苗的开发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益处。公共卫生相关性：肠源性大肠杆菌。大肠杆菌（ETEC）是引起世界范围内发病和死亡的重要细菌病原体。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也是前往世界高风险地区旅行者腹泻的主要原因。尽管ETEC的毒力因子已被充分了解，尽管在过去的几十年里，在志愿者试验和田间研究中测试了几种潜在的ETEC疫苗，但尚未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。