Combination Clostridium Difficile Toxin and Adhesin Vaccine

描述(由申请人提供)：艰难梭菌是一种革兰氏阳性厌氧芽胞杆菌，是工业化国家医院内腹泻和结肠炎的主要原因，仅在美国每年就报告了30多万例艰难梭菌相关性腹泻(CDAD)病例。广谱抗生素的使用、住院、高龄和合并疾病会增加患CDAD的风险。症状是由产生两种强有力的艰难梭菌毒素(毒素A和毒素B)引起的。对人类的研究表明，对疾病和复发的预防主要与针对A毒素的血清免疫球蛋白反应的存在有关，与B毒素的相关性较小。目前还没有有效预防艰难梭菌疾病的疫苗可供商业使用，通过隔离患者和实施手卫生和接触预防措施来预防CDAD的措施取得了各种不同的成功，而且往往有限。我们建议将A、B毒素的无毒受体结合区(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已被证明能在免疫小鼠中诱导中和抗体。艰难梭菌fld和flic参与黏附和肠道定植，flic是一种有效的Toll样受体(TLR)5配体。TLRs是一个模式识别受体家族，可以识别细菌、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，可以触发先天性反应，并促进适应性的发展 豁免权。几种有希望的实验性疫苗已经用鞭毛蛋白作为抗原或佐剂进行了测试。抗鞭毛蛋白免疫反应是否可以阻止艰难梭菌的定植，以及通过TLR信号的激活是否在人类对艰难梭菌毒素疫苗的反应中发挥重要作用，仍有待确定。我们假设，在疫苗中加入鞭毛蛋白和毒素可以提供防止定植和疾病进展的保护。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能够在接种的小鼠中显示出强大的免疫原性，从而产生毒素中和抗体、疫苗效力的相关性以及可以防止定植的抗鞭毛抗体。由于艰难梭菌是一种粘膜病原体，针对粘膜表面的几种免疫途径，如直肠内、鼻腔和经皮免疫，将与存在粘膜佐剂(如不耐热肠毒素LT(R192g))的非肠道免疫进行比较。然后，最有希望的疫苗将在挑战和保护研究中进行评估。在小鼠和仓鼠的细菌感染模型中，将进行对多种艰难梭菌菌株的挑战研究，以评估艰难梭菌的定植和对CDAD的保护。

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.