Generation of Novel Human Monoclonals for Lung Disease

 描述（由申请人提供）：囊性纤维化是美国最常见的致死性遗传性疾病，其特征是气道慢性感染炎症。通过基于培养的方法和基于非培养的方法（16 s测序）鉴定的最常见微生物是铜绿假单胞菌和金黄色葡萄球菌。除了这些细菌，真菌感染烟曲霉菌也很常见。虽然定植是常见的-侵袭性感染菌血症或真菌血症是罕见的。这与铜绿假单胞菌和沙门氏菌的急性感染相反。金黄色葡萄球菌在非CF人群中，无论是社区获得性感染还是医院感染。长期以来，人们已经认识到，CF患者中存在强的适应性免疫应答，其特征在于对铜绿假单胞菌的多克隆IgG应答以及T细胞增殖应答。我们已经确定了一个强大的B细胞反应，在肺和支气管刷CF患者。我们将检验这些粘膜B细胞编码病原体特异性免疫应答的假设，其目的如下：特异性目的1。从CF肺中制备一组人单克隆抗体。B细胞将从临床支气管镜检查或接受移植的CF患者中获得。B细胞将用Epstein巴尔病毒（EBV）转化，然后与骨髓瘤融合伴侣融合。然后，我们将表征IG同种型为如前所述的体细胞超突变的程度。具体目标2。检测人源单克隆抗体的抗原特异性。我们首先检测了临床微生物学实验室从患者配对BAL样本中获得的病原体抗体，并检测了铜绿假单胞菌、嗜麦芽窄食单胞菌、沙门氏菌的实验室菌株的一般反应性。金黄色葡萄球菌（MSSA和MRSA）和A.烟熏。将通过ELSIA和Western印迹分析评估交叉反应性。将在肺部感染的体内动物中使用血小板减少小鼠模型测试对铜绿假单胞菌有反应的克隆。

 DESCRIPTION (provided by applicant): Cystic Fibrosis is the most common lethal genetic disease in the US and is characterized by chronic infection inflammation in the airway. The most common organisms identified by both culture-based methods as well as non-culture based methods (16s sequencing) are Pseudomonas aeruginosa and Staphylococcus aureus. In addition to these bacteria, fungal infection with Aspergillus fumigatus is also common. Although colonization is common - invasive infection with bacteremia or fungemia is rare. This is in contrast to acute infections with P. aeruginosa and S. aureus in non-CF populations whether they are community acquired or nosocomial infections. It has long been recognized that there is strong adaptive immune response in patients with CF characterized by polyclonal IgG responses as well as T-cell proliferative responses to P. aeruginosa. We have identified a strong B-cell response in the lungs and bronchial brushes of CF patients. We will test the hypothesis that these mucosal B-cells encode pathogen specific immune responses with the following Aims: Specific Aim 1. Create a panel of human monoclonal antibodies from the CF lung. B-cells will be obtained from clinical bronchoscopies or from CF patients undergoing transplant. B-cells will be transformed with Epstein Barr Virus (EBV) and then fused to a myeloma fusion partner. We will then characterize Ig isotype as the extent of somatic hyper-mutation as previously described. Specific Aim 2. Test the antigen specificity of the human monoclonal antibodies. We first test the antibodies against the pathogens obtained in the clinical microbiology lab from the paired BAL sample of the patient as well as test for general reactivity against lab strains of P. aeruginosa, Stenotrophomonas maltophilia, S. aureus (both MSSA and MRSA), and A. fumigatus. Cross-reactivity will be assessed by both ELSIA and Western blot analysis. Clones that react against P. aeruginosa will be tested in an in vivo animal of pulmonary infection using the neutropenic mouse model.