Generation of Novel Human Monoclonals for Lung Disease

 描述(申请人提供)：囊性纤维化是美国最常见的致命性遗传病，以呼吸道慢性感染炎症为特征。以培养为基础的方法以及非基于培养的方法(16S测序)鉴定的最常见的微生物是铜绿假单胞菌和金黄色葡萄球菌。除了这些细菌，真菌感染烟曲霉也很常见。虽然定植很常见，但侵袭性感染合并菌血症或真菌血症的情况很少见。这与非社区获得性感染或医院感染人群中的铜绿假单胞菌和金黄色葡萄球菌的急性感染形成对比。长期以来，人们已经认识到，以多克隆免疫球蛋白G反应和T细胞对铜绿假单胞菌的增殖反应为特征的CF患者存在较强的适应性免疫反应。我们已经在CF患者的肺和支气管刷中发现了强烈的B细胞反应。我们将验证这样的假设，即这些粘膜B细胞编码病原体特异性免疫反应，目的如下：1.从肺组织中产生一组人的单抗。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细胞将与EB病毒(EBV)转化，然后与骨髓瘤融合伙伴融合。然后，我们将如前所述，将Ig同种异型定性为体细胞超突变的程度。特异性目的2.检测人源单抗的抗原特异性。我们首先测试临床微生物实验室从患者的配对BAL样本中获得的针对病原体的抗体，以及对实验室铜绿假单胞菌、嗜麦芽窄食单胞菌、金黄色葡萄球菌(MSSA和MRSA)和烟曲霉菌的一般反应性。交叉反应将通过ELSIA和Western印迹分析进行评估。对铜绿假单胞菌起反应的克隆将在肺部感染的活体动物中使用中性粒细胞减少的小鼠模型进行测试。

 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.