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人体对海藻酸铜绿假单胞菌无效免疫反应的分子和细胞基础，并开发免疫干预措施，提供介导海藻酸盐特异性感染抗性的效应物。从慢性感染囊性纤维化（CF）患者分离的铜绿假单胞菌菌株过度表达海藻酸盐。从目前的资助中产生的结果表明，藻酸盐在许多临床非黏液分离株上的表达水平足够高，特别是在体内感染期间，从而允许将藻酸盐作为这些菌株的靶向疫苗。藻酸盐是免疫抵抗和清除的有效抗原靶点，但几乎所有的天然人体免疫反应都产生不能介导细胞杀伤和免疫的抗体。疫苗诱导的抗体是无害的和保护性的，但未结合的海藻酸盐对人的免疫原性很差。这项应用的目的包括进一步开发海藻酸盐结合疫苗，重点是利用铜绿假单胞菌鞭毛作为载体蛋白，提高人单克隆抗体（Mab）对海藻酸盐的亲和力，以评估其保护潜力。鞭毛将通过巯基键与海藻聚甘露醛酸或铜绿假单胞菌海藻酸盐结合，在小鼠和家兔中评估疫苗的免疫原性，并在小鼠急性肺炎模型和转基因CF小鼠慢性肺部感染模型中使用非粘液性铜绿假单胞菌菌株评估疫苗的保护活性。目前可用的海藻酸盐人单克隆抗体可介导对黏液样和非黏液样菌株的拮抗活性，并可保护小鼠免受非黏液样菌株引起的肺炎。它们的亲和力将通过噬菌体展示和容易出错的PCR进一步提高。所得单克隆抗体将在体外对铜绿假单胞菌黏液和非黏液菌株进行抑声活性评估。体内研究将包括预防、被动保护非黏液样菌株引起的急性肺炎，以及使用非黏液样菌株的转基因CF小鼠的慢性口咽定植和肺部感染。这些单抗还将被评估其在转基因CF小鼠中调节已建立的粘液样铜绿假单抗感染和病理的能力。这些目标将导致CF中铜绿假单胞菌感染的免疫治疗的进一步发展，并评估海藻酸盐作为非黏液性和黏液性铜绿假单胞菌感染的抗原靶点。

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.