POLYCLONAL ANTIBODY LIBRARIES FOR C PARVUM THERAPY

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The goal of this project is to develop a safe and effective drug for the treatment or prevention of opportunistic infection with the parasite Cryptosporidium parvum (C. parvum). In AIDS patients, this parasite causes severe chronic diarrhea with fluid and weight loss, and often death, and there is currently no known effective treatment. We propose to develop polyclonal antibody libraries to be used as passive immunotherapy for Cryptosporidiosis (C. parvum infection). Polyclonal antibody libraries combine the advantages of targeting multiple antigenic determinants (low likelihood of antigen escape variants' and efficient mediation of effector functions) with the advantages of using monoclonal antibodies (unlimited supply of standardized reagents and the availability of the genetic material for desired manipulations). The specific aims of this proposal are to generate polyclonal libraries of human and mouse-human chimeric IgA and IgG antibodies specific for C. parvum and to test the efficacy of the polyclonal antibody libraries in vitro, s well as in vivo in an animal model of cyrptosporidiosis. To generate the polyclonal antibody libraries, Fab phage display libraries will first be created by recombinant Dna techniques from mice immunized with various developmental stages of C. parvum, and from humans with high antibody levels to C. parvum. The Fab phage display libraries will be positively selected for reactivity to C. parvum developmental stages and negatively selected to remove cross-reactivities to human and mouse components. The heavy and light chain variable region gene pairs of the selected Fab phage display libraries will be transferred in bulk to mammalian expression vectors to express polyclonal libraries of intact IgA and IgG antibodies that are all human, all-mouse, or mouse-human chimeras. The IgA polyclonal libraries will also be converted to secretory IgA (sIgA) libraries by in vitro association with secretory component. All the libraries will be evaluated, at various stages of production, for efficacy against C. parvum by in vitro assays including ELISA, immunofluorescence, immunoblot analysis, and inhibition of C. parvum attachment to and invasion of epithelial cells. The IgG, IgA, and sIgA libraries will also be tested for efficacy when administered orally and parenterally to C. parvum- infected mice in a severe combined immunodeficiency (SCID) mouse model of cryptosporidiosis.