Virulence and Immunity to Mucoid P. aeruginosa

描述（由申请人提供）：本申请的长期目标是了解对铜绿假单胞菌藻酸盐的无效人体免疫应答的分子和细胞基础，并开发免疫干预，其将提供介导藻酸盐特异性抗感染的效应物。从慢性感染的囊性纤维化（CF）患者分离的铜绿假单胞菌菌株过度表达藻酸盐。从目前的资助产生的结果表明，藻酸盐在许多临床非粘液分离株上以足够的水平表达，特别是在体内感染期间，以允许将藻酸盐靶向作为这些菌株的疫苗。 藻酸盐是免疫抗性和清除的有效抗原靶标，但实际上所有天然人类免疫应答都产生不能介导调理素杀伤和免疫的抗体。疫苗诱导的抗体是调理剂和保护性的，但未缀合的藻酸盐对人类的免疫原性差。本申请的目的包括进一步开发藻酸盐缀合物疫苗，重点是使用铜绿假单胞菌鞭毛作为载体蛋白，并提高人单克隆抗体（Mab）对藻酸盐的亲和力，以评估其保护潜力。鞭毛将使用硫醇键与海藻聚甘露糖醛酸或铜绿假单胞菌藻酸盐缀合，评价疫苗在小鼠和兔中的免疫原性，以及在鼠急性肺炎模型和转基因CF小鼠中的慢性肺部感染模型中使用非粘液铜绿假单胞菌菌株的保护活性。目前可用的针对藻酸盐的人单抗可以介导针对粘液和非粘液菌株的调理素活性，以及在小鼠中针对由于非粘液菌株引起的肺炎的保护。利用噬菌体展示和易错PCR，它们的亲和力将进一步提高。将在体外评价所得单克隆抗体对铜绿假单胞菌的粘液和非粘液菌株的调理活性。体内研究将包括使用两种非粘液菌株在转基因CF小鼠中预防性、被动保护免受由于非粘液菌株引起的急性肺炎和慢性口咽定植和肺部感染。还将评价Mab调节转基因CF小鼠中已建立的粘液样铜绿假单胞菌感染的感染和病理学的能力。 这些目标将导致CF中铜绿假单胞菌感染的免疫治疗剂的进一步开发，并评估藻酸盐作为非粘液样以及粘液样铜绿假单胞菌感染的抗原靶标。

DESCRIPTION (provided by applicant): The long term goal of this application is to understand the molecular and cellular basis for the ineffective human immune response to Pseudomonas aeruginosa alginate and to develop immunologic interventions that will provide effectors that mediate alginate-specific resistance to infection. Alginate is over-expressed by strains of P. aeruginosa isolated from chronically infected cystic fibrosis (CF) patients. Results generated from current funding indicate that alginate is expressed at sufficient levels on many clinical non-mucoid isolates, particularly during in vivo infection, to allow for targeting of alginate as a vaccine for these strains. Alginate is an effective antigenic target for immune resistance and clearance but virtually all natural human immune responses result in antibody that fails to mediate opsonic killing and immunity. Vaccine induced antibody is opsonic and protective but unconjugated alginate is poorly immunogenic for humans. The aims of this application include further development of alginate-conjugate vaccines, focusing on use of P. aeruginosa flagella as a carrier protein and improvement of the affinity of human monoclonal antibodies (Mab) to alginate for evaluation of their protective potential. Flagella will be conjugated to seaweed polymannuronic acid or P. aeruginosa alginate using thiol-bonds, vaccines evaluated for immunogenicity in mice and rabbits and for protective activity using non-mucoid P. aeruginosa strains in a murine acute pneumonia model and a model of chronic lung infection in transgenic CF mice. Currently available human Mabs to alginate can mediate opsonic activity against both mucoid and non-mucoid strains, and protection in mice against pneumonia due to non-mucoid strains. Their affinities will be further improved using phage display and error-prone PCR. The resultant Mabs will be evaluated for opsonic activity against both mucoid and non-mucoid strains of P. aeruginosa in vitro. In vivo studies will encompass prophylactic, passive protection against acute pneumonia due to non-mucoid strains and chronic oropharyngeal colonization and lung infections in transgenic CF mice using both non-mucoid strains. The Mabs will also be evaluated for their ability to modulate infection and pathology from established mucoid P. aeruginosa infection in transgenic CF mice. These aims will lead to further development of immunotherapeutics for P. aeruginosa infection in CF and evaluate alginate as an antigenic target for non-mucoid, as well as mucoid, P. aeruginosa infections.