Combination Clostridium Difficile Toxin and Adhesin Vaccine

描述（由申请人提供）：艰难梭菌是一种芽孢形成的革兰氏阳性厌氧杆菌，是工业化国家院内腹泻和结肠炎的主要原因，仅在美国每年就有超过30万例艰难梭菌相关腹泻（CDAD）报告。广谱抗生素使用、住院、高龄和合并症增加了罹患CDAD的风险。产生两种强效艰难梭菌毒素（毒素A和毒素B）引起症状。对人类的研究表明，预防疾病和复发主要与针对毒素A的血清IgG反应相关，而与毒素b的相关性较弱。目前没有有效预防艰难梭菌疾病的疫苗，通过患者隔离和实施手部卫生和接触预防措施来预防CDAD的措施取得了不同的成功，而且往往是有限的。我们提出将毒素a或毒素B的无毒受体结合域（RBD）与艰难梭菌鞭毛蛋白FliC和FliD融合，制备重组艰难梭菌蛋白疫苗。毒素A和毒素B的RBD已被证明在免疫小鼠中诱导中和抗体。艰难梭菌FliD和flc参与粘附和肠道定植，flc是一种有效的toll样受体（TLR） 5配体。tlr是一类模式识别受体，可识别细菌、真菌和病毒共有的结构成分。当tlr与它们的配体如鞭毛蛋白结合时，可以触发先天反应，并促进适应性的发展

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.