A novel vaccine to prevent Clostridium difficile colitis

一种预防艰难梭菌结肠炎的新型疫苗

• 批准号: 8668719
• 负责人: EDGAR C. BOEDEKER
• 金额: --
• 依托单位: ALBUQUERQUE VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8668719
• 关键词: Antibiotics; Antibodies; Antigens; Attenuated; Bacterial Adhesins; Binding; C-terminal; Clostridium difficile; Colitis; Development; Disease; Elderly; Engineering; Escherichia coli; Escherichia coli EHEC; Escherichia coli Infections; General Population; Genes; Genetic Techniques; Goals; Healthcare Systems; Hospitals; Immune system; Immunity; Immunization; Incidence; Infection; Intestines; Laboratories; Lead; Lesion; Life; Measurement; Modeling; Molecular Genetics; Mus; Nosocomial Infections; Organism; Patients; Phase; Plasmids; Production; Proliferating; Proteins; Recurrence; Reproduction spores; Risk; Safety; Serum; Shiga Toxin; Surface; System; Systemic disease; Terminal Repeat Sequences; Toxin; United States Department of Veterans Affairs; Vaccines; Veterans; Virulence; immunogenic; immunogenicity; mouse model; novel therapeutics; novel vaccines; oral vaccine; prevent; prophylactic; protective efficacy; public health relevance; receptor binding; safety testing; vaccine efficacy; vector; vector vaccine

DESCRIPTION (provided by applicant): The need for novel therapeutic and prophylactic approaches to Clostridium difficile (C. difficile) infections in the VA Health Care system, is urgent. The overall goal of this proposal is to develop live oral vaccines which express C. difficile toxin antigens which will induce protective mucosal, and systemic, immunity to the toxins in order to prevent Clostridium difficile-associated disease (CDAD). To achieve this, an attenuated strain of E.coli which we have developed as a vaccine vector for antigen delivery will be engineered to express the immunogenic C-terminal receptor binding portions of the major virulence determinants of C.difficile, the A and B toxins (TcdA and TcdB). To evaluate vaccine efficacy we will use a recently described antibiotic (cefoperazone)-treated mouse model of CDAD(70) which we have validated in our laboratory. To express protective toxin antigens in immunogenic form in our E.coli vector strain, we will incorporate them into the passenger domain of autotransporter(28) proteins to deliver them to the mucosal immune system as we have done for other toxin antigens. This proposal has 3 specific aims: The first specific aim is to express immunogenic components of the TcdA and TcdB toxins of C. difficile as antigens in our attenuated E. coli vector ZCR533(6). The C-terminal binding regions of the toxins will be expressed as fusions with the passenger domain of the autotransporter EspP, the intrinsic type V secretion system(28) of O157:H7 E.coli, and introduced on a medium copy number plasmid. The second specific aim is to determine the safety and mucosal immunogenicity of the vaccine constructs by intranasal, and intragastric, immunization of mice, followed by measurement of the binding and toxin neutralizing capacity ofserum and mucosal anti-TcdA and/or anti-TcdB antibodies. The third specific aim is to determine the protective efficacy of the vaccine constructs in C57/Bl6 mice using a newly described antibiotic-treated mouse model(70) for C. difficile challenge. We have recently validated this model in our laboratory. Our vaccine vector will be ZCR533, an attenuated E.coli strain derived from an O157:H7 enterohemorrhagic E.coli (EHEC) by deleting its genes for shiga toxin production and truncating its major adhesin, intimin, such that it can no longer induce attaching /effacing lesions, but can still induce anti-intimin antibody(8,75). We will use standard molecular genetics techniques to prepare C. difficile vaccine constructs by expressing the C terminal repeats comprising the binding domains of the TcdA and TcdB toxins, into the EspP autotransporter(28) on a multicopy plasmid. Our recent studies have indicated the validity of this approach to immunization against toxin antigens, since the similarly-sized shiga toxin B subunit antigen is immunogenic, and protective against shiga toxin producing E.coli (STEC) infection, when expressed in the autotransporter EspP on the surface of the organism, or secreted into the medium. To test the safety, immunogenicity and protective efficacy of the vaccine constructs we will utilize mouse models of intranasal, and intragastric, immunization(12) and a new cefoperazone-treated mouse model (70) of CDAD, which we have validated in our laboratory, for C. difficille challenge. These studies could lead to the development of a new class of safe and effective vaccines directed against C.difficile toxins to prevent CDAD.

描述（由申请人提供）： 需要新的治疗和预防方法，艰难梭菌（C。艰难）感染的VA医疗保健系统，是紧迫的。该提案的总体目标是开发表达C.艰难梭菌毒素抗原，其将诱导对毒素的保护性粘膜和全身免疫，以预防艰难梭菌相关疾病（CDAD）。为了实现这一点，我们已经开发作为抗原递送的疫苗载体的大肠杆菌的减毒株将被工程化以表达艰难梭菌的主要毒力决定簇A和B毒素（TcdA和TcdB）的免疫原性C末端受体结合部分。为了评价疫苗的有效性，我们将使用最近描述的抗生素（头孢哌酮）治疗的CDAD小鼠模型（70），我们已经在我们的实验室验证。为了在我们的大肠杆菌载体菌株中以免疫原性形式表达保护性毒素抗原，我们将它们掺入自转运蛋白（28）的乘客结构域中，以将它们递送到粘膜免疫系统，就像我们对其他毒素抗原所做的那样。 该方案有3个具体目的：第一个具体目的是表达C. difficile作为抗原。coli载体ZCR 533（6）。毒素的C-末端结合区将表达为与自转运蛋白EspP（O 157：H7大肠杆菌的固有V型分泌系统（28））的乘客结构域的融合物，并引入中等拷贝数质粒上。 第二个具体目的是通过鼻内和胃内免疫小鼠，然后测量血清和粘膜抗TcdA和/或抗TcdB抗体的结合和毒素中和能力来确定疫苗构建体的安全性和粘膜免疫原性。 第三个具体目的是使用新描述的C.艰难的挑战我们最近在实验室验证了这个模型。 我们的疫苗载体将是ZCR 533，这是一种减毒大肠杆菌菌株，来源于O 157：H7肠出血性大肠杆菌（EHEC），通过删除其产生滋贺毒素的基因并截短其主要粘附素-我们将使用标准的分子遗传学技术来制备C。通过将包含TcdA和TcdB毒素的结合结构域的C末端重复序列表达到多拷贝质粒上的EspP自转运蛋白（28）中，来构建艰难梭菌疫苗构建体。我们最近的研究已经表明了这种针对毒素抗原的免疫方法的有效性，因为类似大小的滋贺毒素B亚单位抗原是免疫原性的，并且当在生物体表面上的自转运蛋白EspP中表达或分泌到培养基中时，保护免受产滋贺毒素的大肠杆菌（STEC）感染。为了测试疫苗构建体的安全性、免疫原性和保护效力，我们将利用鼻内和胃内免疫的小鼠模型（12）和新的头孢哌酮治疗的CDAD小鼠模型（70），我们已经在我们的实验室中验证了该模型用于C.艰难的挑战这些研究可能导致开发出一类新的安全有效的疫苗，针对艰难梭菌毒素，以预防CDAD。