Development of Vaccines against Clostridium difficile Infection

描述(由申请人提供)： 这项提议是对RFA-AI-09-016要求应用程序为包括艰难梭菌在内的选定病原体开发疫苗的回应，艰难梭菌是伪膜性结肠炎的原因，占所有抗生素相关腹泻病例的四分之一。随着最近出现的超强毒力菌株，艰难梭菌感染(CDI)在北美和欧洲的发病率都大幅上升，导致住院时间长，发病率和死亡率高。CDI被认为主要是由外毒素TcdA和TcdB介导的，它们使Rho家族的低分子GTP酶糖基化，导致大量液体分泌，急性炎症和结肠粘膜坏死。对CDI的保护被证明是通过针对这两种关键毒素的系统和粘膜抗体来调节的，尽管已知存在其他毒力属性，这也可能有助于CDI的表现。这项提议的目标是设计一种既针对TcdA又针对TcdB的疫苗，以期激发强大的系统和粘膜免疫，以预防CDI，降低严重性，或消除正在进行的慢性病。我们建议在一个无内毒素的巨大芽孢杆菌系统中开发最近表达的无毒艰难梭菌全毒素蛋白。将对两种免疫原进行评估：由点突变产生的无毒全长艰难梭菌毒素A和B的混合物(指定为aTxAB)，以及包含全长TcdB但其受体结合结构域被TcdA替换的嵌合体蛋白(指定为cTxAB)。CTxAB在跨膜区有一个很小的缺失(97个氨基酸)，使其无毒。初步研究表明，无毒TcdB疫苗诱导了针对多种表位的抗体反应，并产生了比类毒素更强的保护性免疫；cTxAB免疫诱导了针对TcdA和TcdB的抗体和保护性反应。在这个项目中，我们将首先评估这些无毒重组蛋白在肠外免疫后诱导保护性抗体反应的能力，然后用野生型毒素攻击(目标1)。随后将评估几种黏膜免疫方案(口服、鼻腔和舌下免疫)，旨在诱导对野生型毒素系统和粘膜攻击的保护(目标2)。我们将在最近描述的小鼠急性感染模型(AIM 3a)中测试AIMS 1和AIMS 2中开发的各种免疫方案的保护效果，而从小鼠感染研究中得出的最有效的免疫方法将在本实验室开发的慢性CDI仔猪模型(AIM 3b)中进行临床前评估。由于在这个早期阶段，候选疫苗的性质尚不清楚，也不需要佐剂，我们无法形成适当的合作伙伴关系。 摘要简介：艰难梭菌相关性腹泻和肠道炎症性疾病主要由两种分泌性毒素引起。该项目将使用本实验室生产的重组无毒全毒素作为疫苗的基础，以诱导强大的系统和粘膜免疫，以预防艰难梭菌感染，降低严重性，或消除正在进行的慢性病。

DESCRIPTION (provided by applicant): This proposal is in response to RFA-AI-09-016 requesting applications to develop vaccines for selected pathogens including Clostridium difficile, the cause of pseudomembranous colitis, which accounts for a quarter of all cases of antibiotic-associated diarrhea. With the recent emergence of hypervirulent strains, the incidence of C. difficile infection (CDI) has increased significantly in both North America and Europe, causing lengthy hospitalization, substantial morbidity and mortality. CDI is thought to be mainly mediated by exotoxins TcdA and TcdB, which glucosylate low molecular mass GTPases of the Rho family, leading to massive fluid secretion, acute inflammation, and necrosis of the colonic mucosa. Protection against CDI was shown to be mediated through systemic and mucosal antibodies against the 2 key toxins, although other virulence attributes are known to exist which may also contribute to the manifestation of CDI. The goal of this proposal is to design a vaccine that targets both TcdA and TcdB, with a view to elicit strong systemic and mucosal immunity to prevent CDI, reduce the severity, or eliminate an ongoing chronic disease. We propose to exploit the recently expressed atoxic C. difficile holotoxin proteins in an endotoxin-free Bacillus megaterium system. Two immunogens will be evaluated: a mixture of atoxic full-length C. difficile toxin A and B generated by point mutations (designated as aTxAB), and a chimera protein containing full-length TcdB but with its receptor binding domain replaced with that of TcdA (designated as cTxAB). cTxAB has a small deletion (97 amino acids) in transmembrane domain rendering it non-toxic. Preliminary studies showed that atoxic TcdB vaccination induced antibody responses against a wide-spectrum of epitopes and potent protective immunity superior to toxoid; cTxAB immunization induced antibody and protective responses against both TcdA and TcdB. In this project, we will first evaluate the ability of these atoxic recombinant proteins to induce protective antibody responses following parenteral immunization followed by challenge with wild type toxins (Aim 1). This will be followed by evaluating several regimens of mucosal immunizations (oral, intranasal and sublingual) designed to induce protection against systemic and mucosal challenges with wild type toxins (Aim 2). We will test the protective efficacy of the various immunization regimens developed in Aims 1 and 2 in the recently described mouse acute infection model (Aim 3a), and the most efficient immunization method resulting from the mouse infection studies will undergo preclinical evaluation in the chronic piglet model of CDI developed in this laboratory (Aim 3b). Because at this early stage the nature of the candidate vaccine is unknown, nor is the adjuvant required, we are not in a position to form a suitable partnership. Abstract Narrative: Clostridium difficile-associated diarrhea and enteric inflammatory diseases are caused primarily by two secretory toxins. This project will use recombinant atoxic holotoxins produced in this lab as a basis for a vaccine to elicit strong systemic and mucosal immunity to prevent C. difficile infection, reduce the severity, or eliminate an ongoing chronic disease.