Combination Clostridium Difficile Toxin and Adhesin Vaccine

描述（由申请人提供）：艰难梭菌是一种形成芽孢的革兰氏阳性厌氧杆菌，是工业化国家医院内腹泻和结肠炎的主要原因，有超过30万例艰难梭菌感染病例。仅在美国每年就有艰难梭菌相关性腹泻（CDAD）的报道。广谱抗生素使用、住院、高龄和合并症增加了获得CDAD的风险。症状是由两种强有力的C.艰难梭菌毒素（毒素A和毒素B）。对人类的研究表明，对疾病和复发的保护主要与针对毒素A的血清IgG应答的存在相关，而与毒素B的相关性较低。没有有效预防C.艰难梭菌病目前是商业上可获得的，并且通过患者隔离和实施手部卫生和接触预防措施来预防CDAD的措施已经取得了可变的并且通常有限的成功。 我们建议开发一种重组C.通过将毒素A或毒素B的无毒受体结合结构域（RBD）与艰难梭菌融合而制备艰难梭菌蛋白疫苗。艰难鞭毛蛋白FliC和FliD。毒素A和毒素B的RBD已显示在免疫小鼠中诱导中和抗体。梭艰难梭菌FliD和FliC参与粘附和肠道定殖，并且FliC是有效的Toll样受体（TLR）5配体。TLR是模式识别受体家族，其识别细菌、真菌和病毒共有的结构组分。当TLR与其配体如鞭毛蛋白结合时，可以触发先天性反应以及促进适应性免疫的发展。 免疫力已经用鞭毛蛋白作为抗原或佐剂测试了几种有希望的实验疫苗。抗鞭毛蛋白免疫应答是否能预防C.艰难梭菌定植以及通过TLR信号传导的激活是否在人类对艰难梭菌的应答中起重要作用。艰难梭菌毒素疫苗我们假设，鞭毛蛋白和毒素在疫苗中的掺入可以提供针对定植以及疾病进展的保护。关键的里程碑将是解决使用毒素和鞭毛蛋白的组合疫苗是否可以在接种疫苗的小鼠中表现出强大的免疫原性，从而产生毒素中和抗体，这是疫苗效力的相关因素，以及可以防止定植的抗鞭毛抗体。由于艰难梭菌伊萨一种粘膜病原体，因此将靶向粘膜表面的几种免疫途径如直肠内、鼻内和经皮免疫与在粘膜佐剂如热不稳定肠毒素LT（r192g）存在下的肠胃外免疫进行比较。最有希望的疫苗将在挑战和保护研究中进行评估。针对多种C.艰难梭菌菌株在小鼠和细菌感染的仓鼠模型中进行评估。艰难定植和保护免受CDAD。

DESCRIPTION (provided by applicant): Clostridium difficile is a spore-forming Gram-positive anaerobic bacillus, and is the leading cause of nosocomial diarrhea and colitis in the industrialized world with more than 300,000 cases of C. difficile- associated diarrhea (CDAD) reported each year in the United States alone. Broad spectrum antibiotic usage, hospitalization, advanced age and comorbidities increase the risk for acquiring CDAD. Symptoms result from the production of two potent C. difficile toxins (toxin A and toxin B). Studies with humans have shown that protection against disease and relapse correlates predominantly with the presence of serum IgG responses directed against toxin A and less strongly with toxin B. No vaccine effective at preventing C. difficile disease is currently commercially available, and measures to prevent CDAD through patient isolation and implementation of hand-hygiene and contact precautions have had variable and often limited success. We propose to develop a recombinant C. difficile protein vaccine by fusing the non-toxic receptor binding domain (RBD) of toxin A or toxin B with C. difficile flagellar proteins, FliC and FliD. The RBD of toxin A and toxin B have been shown to induce neutralizing antibodies in immunized mice. The C. difficile FliD and FliC are involved in adherence and gut colonization, and FliC is a potent Toll-like receptor (TLR) 5 ligand. TLRs are a family of pattern recognition receptors that recognize structural components shared by bacteria, fungi and viruses. TLRs when bound to their ligands such as flagellin can trigger innate responses as well as facilitate in the development of adaptive immunity. Several promising experimental vaccines have been tested with flagellin either as an antigen or as an adjuvant. It still remains to be determined whether anti-flagellin immune responses can prevent C. difficile colonization and whether activation through TLR signaling plays a significant role in human responses against a C. difficile toxin vaccine. We hypothesize that the incorporation of flagellar proteins and toxins in a vaccine could provide protection against colonization as well as disease progression. Key milestones will be to address whether the combination vaccine using toxins and flagellar proteins can exhibit robust immunogenicity in vaccinated mice, resulting in the production of toxin neutralizing antibodies, a correlate of vaccine efficacy, and anti-flagellar antibodies that can prevent colonization. Since C difficile isa mucosal pathogen, several routes of immunization that target the mucosal surface such as intra- rectal, intranasal and transcutaneous will be compared to parenteral immunization in the presence of mucosal adjuvants such as heat labile enterotoxin, LT (r192g). The most promising vaccines will then be evaluated in challenge and protection studies. Challenge studies against multiple C. difficile strains in the mouse and the hamster model of bacterial infection will be performed to evaluate C. difficile colonization and protection against CDAD.