A novel vaccine to prevent Clostridium difficile colitis

描述(由申请人提供)： 在退伍军人卫生保健系统中迫切需要新的治疗和预防方法来治疗艰难梭菌(艰难梭菌)感染。这项提议的总体目标是开发表达艰难梭菌毒素抗原的口服活疫苗，这种疫苗将诱导对毒素的保护性粘膜和系统免疫，以预防艰难梭菌相关疾病(CDAD)。为了实现这一点，我们开发的作为抗原传递疫苗载体的减毒大肠杆菌株将被改造成表达艰难梭菌主要毒力决定因素的免疫原性C末端受体结合部分，即A和B毒素(TcdA和TcdB)。为了评估疫苗的有效性，我们将使用最近描述的一种抗生素(头孢哌酮)治疗的CDAD(70)小鼠模型，该模型已在我们的实验室得到验证。为了在我们的大肠杆菌载体菌株中以免疫原性的形式表达保护性毒素抗原，我们将把它们整合到自体转运蛋白的乘客区中，以将它们运送到粘膜免疫系统，就像我们对其他毒素抗原所做的那样。该方案有3个特定目的：第一个特定目的是在我们的减毒表达载体ZCR533(6)中表达艰难梭菌TcdA和TcdB毒素的免疫原性成分作为抗原。毒素的C末端结合区将被表达为与O157：H7大肠杆菌固有的V型分泌系统(28)--自身转运蛋白ESPP的乘客区--的融合，并被引入到中等拷贝数的质粒上。第二个特异性目的是通过小鼠鼻腔免疫和灌胃免疫来确定构建的疫苗的安全性和粘膜免疫原性，然后测定血清和粘膜抗TcdA和/或抗TcdB抗体的结合和中和毒素的能力。第三个具体目标是使用新描述的艰难梭菌挑战的抗生素治疗小鼠模型(70)，确定疫苗构建物在C57/BL6小鼠中的保护效果。我们最近在我们的实验室验证了这个模型。我们的疫苗载体将是ZCR533，这是一种来自O157：H7肠出血性大肠杆菌(EHEC)的减毒大肠杆菌菌株，通过删除其产生志贺毒素的基因并截断其主要粘附素--内膜，使其不再能诱导附着/消失的病变，但仍能诱导抗内毒素抗体(8，75)。我们将使用标准的分子遗传学技术，通过将包含TcdA和TcdB毒素结合域的C末端重复序列表达到多拷贝质粒上的ESPP自动转运蛋白(28)中来制备艰难梭菌疫苗。我们最近的研究表明，这种方法用于针对毒素抗原的免疫是有效的，因为类似大小的志贺毒素B亚单位抗原是免疫原性的，当表达在有机体表面的自身转运蛋白ESPP中或分泌到介质中时，对产生志贺毒素的大肠杆菌(STEC)感染具有保护作用。为了测试疫苗构建的安全性、免疫原性和保护效力，我们将利用小鼠鼻内和胃内免疫模型(12)和我们在实验室验证的新的头孢哌酮治疗CDAD小鼠模型(70)，以应对艰难梭菌的挑战。这些研究可能导致开发一种针对艰难梭菌毒素的新型安全有效疫苗，以预防CDAD。

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.