Generation of Novel Human Monoclonals for Lung Disease

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 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.