Treatment with cytokines is moderately effective against mouse and human infection with Cryptococcus neoformans. However, systemic delivery of cytokines can be problematic for several reasons. Cytokines act in a local manner. Furthermore, they are rapidly degraded and side effects often limit their use. To overcome these limitations, we propose to construct and test several antibody-cytokine fusion proteins against C. neoformans infection with the goal of developing novel prophylactic and therapeutic vaccination strategies. For these fusion proteins, we will use an antibody specific for the outer capsule of C. neoformans, targeting cytokines directly to the site(s) of infection, while improving cytokine stability;thereby increasing the effective dose while decreasing systemic toxicities. There are currently no effective vaccines in use against C. neoformans. By linking cytokines to C. neoformans antigens, antibody-cytokine fusion proteins may be effective in generating protective immunity. Therefore, to test the efficacy of antibody-cytokine fusion proteins in preventing and treating C. neoformans infection, we propose: 1. To construct, produce and characterize antibody-cytokine fusion proteins specific for C. neoformans capsule. Antibody-cytokine fusion proteins consisting of the hinge-flexible human IgG3 against cryptococcal capsular polysaccharide genetically fused to either interleukin-2 (IL-2), granulocyte- macrophage colony stimulating factor (GM-CSF) or IL-12 will be produced recombinantly. Fusion proteins will be characterized for binding to C. neoformans as well as their functionality as cytokines and antibodies. 2. To determine the potential of antibody-cytokine fusion proteins to generate protective immunity to C. neoformans. Heat-killed C. neoformans or capsular polysaccharide will be opsonized with antibody- cytokine fusion proteins described above and administered to mice. Alternatively, dendritic cells will be loaded ex vivo with these immune complexes and delivered to mice. Host antibody and cellular immune responses specific for C. neoformans will be analyzed and protective efficacy of vaccination determined in mouse models of pulmonary and disseminated infection. 3. To determine the efficacy of cytokines genetically fused to antibody specific for C. neoformans capsule in cryptococcal infection. Antibody-cytokine fusion proteins will be given to mice with new or established infection to determine whether they can directly protect against disseminated cryptococcal infection. Host immune responses will be defined and correlated with efficacy. Mortality from cryptococcal meningitis is 10-20% indicating the need for better therapies. The population at risk for cryptococcal disease continues to grow due to increased survival of persons infected with HIV, new HIV infections, and the continued expansion of the number of persons with immunocompromise due to immuno- suppressive therapies for organ transplantation and rheumatic diseases as well as chemotherapy for cancer. The proposed studies have relevance beyond cryptococcal infection, since a significant long-term objective is to design antibody-cytokine fusion proteins for use in other types of infections.
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