Development of Vaccines against Clostridium difficile Infection

描述（由申请人提供）： 该提案是对RFA-AI-09-016的回应，该提案要求申请开发针对选定病原体的疫苗，包括艰难梭菌，这是伪膜性结肠炎的原因，占所有腹泻相关腹泻病例的四分之一。随着近年来超强毒株的出现，C.在北美和欧洲，艰难梭菌感染（CDI）显著增加，导致住院时间长、发病率和死亡率高。CDI被认为主要由外毒素TcdA和TcdB介导，其使Rho家族的低分子量GTP酶葡糖基化，导致大量液体分泌、急性炎症和结肠粘膜坏死。对CDI的保护作用是通过针对2种关键毒素的全身和粘膜抗体介导的，尽管已知存在其他毒力属性，也可能导致CDI的表现。该提案的目标是设计一种靶向TcdA和TcdB的疫苗，以期引发强的全身和粘膜免疫，以预防CDI，降低严重程度或消除正在进行的慢性疾病。我们建议利用最近表达的无毒C。艰难梭菌全毒素蛋白在无内毒素巨大芽孢杆菌系统中的应用。将评价两种免疫原：无毒全长C.由点突变产生的艰难梭菌毒素A和B（命名为aTxAB），以及含有全长TcdB但其受体结合结构域被TcdA的受体结合结构域取代的嵌合体蛋白（命名为cTxAB）。cTxAB在跨膜结构域中具有小的缺失（97个氨基酸），使其无毒。初步研究表明，无毒TcdB疫苗接种诱导的抗体应答针对广谱表位和有效的保护性免疫上级类毒素; cTxAB免疫诱导的抗体和保护性应答针对TcdA和TcdB。在本项目中，我们将首先评估这些无毒重组蛋白在胃肠外免疫后诱导保护性抗体应答的能力，然后用野生型毒素进行攻击（目的1）。随后将评价几种粘膜免疫方案（口服、鼻内和舌下），旨在诱导针对野生型毒素全身和粘膜攻击的保护作用（目的2）。我们将在最近描述的小鼠急性感染模型（目的3a）中测试目的1和2中开发的各种免疫方案的保护效力，并且将在本实验室开发的慢性CDI仔猪模型（目的3b）中对小鼠感染研究产生的最有效免疫方法进行临床前评价。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，我们无法形成合适的伙伴关系。 摘要叙述：艰难梭菌相关性腹泻和肠道炎性疾病主要由两种分泌毒素引起。本计画将利用本实验室所生产的重组无毒全毒素作为疫苗的基础，以激发强烈的全身及黏膜免疫力来预防C。艰难的感染，降低严重程度，或消除正在进行的慢性疾病。

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.