Exploiting antibody catalysis for treating Cryptococcosis

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Patients with advanced HIV infection are at risk for cryptococcosis, a devastating fungal infection that affects primarily the central nervous system in the form of a difficult to treat meningoencephalitis. Numerous studies over the past two decades have shown that antibody (Ab)-mediated immunity can make a critical contribution to host defense against Cryptococcus neoformans. Monoclonal antibodies (mAbs) are promising therapeutic reagents for cryptococcosis. This fungus is unusual among pathogenic fungi in that it has a large polysaccharide capsule that releases copious amounts of polysaccharide antigen into infected tissues, where it can interfere with the immune response and thus facilitate the persistence of infection. Tissue antigens are implicated in the pathogenesis of immune reconstitution inflammatory syndrome, which often complicates the rebound in immunity associated with antiretroviral therapy in patients with concurrent HIV and cryptococcal infection. Currently, there is no therapy to remove polysaccharide from tissues. Our group discovered that some mAbs to the C. neoformans capsule have catalytic activity that degrades the capsular polysaccharide. This is an exciting finding because it raises the possibility of developing a new type of therapy for cryptococcosis based on making cryptococcal cells vulnerable to the immune system by removing their capsule and degrading tissue antigen deposits. This application proposes experiments to ascertain the contribution of Ab-mediated catalysis to Ab-mediated protection with the goal of developing a new therapeutic strategy for patients with AIDS-associated cryptococcosis based on clearance of tissue antigen deposits. Tissue clearance of cryptococcal antigen will be done in both normal and immunodeficient mice to ascertain the contribution of inflammation to this effect and mimic tissue responses in patients with AIDS. Our approach is to evaluate a large existing collection of mAbs to identify the most effective catalytic Ab and explore the mechanisms by which Ab- mediated catalysis affects cryptococcal cells. We will use site directed mutagenesis of the Ab-binding site to map the catalytic domain and to generate variants with null catalytic activity that will allow us to discriminate between classical Ab functions from those resulting from catalytic activity. Catalytic antibodies will be studied in mouse models to ascertain their capacity to remove polysaccharide from tissue. Three aims are proposed: 1) To establish the effect of Ab-mediated catalysis on the capsule of CN; 2.To identify the site of Ab-mediated catalysis and generate mAbs with and without catalytic activity; and 3) To establish the consequences of Ab- mediated GXM hydrolysis.. We anticipate that at the completion of this work we will be in a position to deploy a new type of antibody therapy for patients with AIDS-related cryptococcosis.