In the current grant we propose to expand our studies of genetically engineered antibody molecules, focusing our attention on the goal of providing more effective immunity to the fetus and neonate. In one series of experiments we will identify and produce antibodies with the optimal combination of functional properties to provide protection at the mucosal surface following oral administration or transport from the mother via the placenta or mammary gland. Human IgA is the antibody normally found in the exocrine secretions. IgA, domain exchange proteins between human IgA and IgG, and site directed mutants will be characterized with respect to their stability, half-life, and biodistribution when administered either orally or intravenously. They will be characterized with respect to their ability to bind and be transported by the epithelial receptors. They also will be assessed for their ability to activate the complement cascade and to bind to Fc receptors specific for either IgG or IgA on phagocytes. We will also attempt to develop a cell which expresses antibody with attached secretory component (SC). One goal will be to produce recombinant antibodies which provide treatment or protection against enteric infections, a significant health problem world wide. We will assay the most promising recombinant antibodies in the suckling mouse model for enterotoxigenic Escherichia coli (ETEC) using antibodies specific for the F41 antigen. Antibodies to F4l have proven efficacy and recombinant antibodies administered orally to pups or intravenously to dams will be assessed for their ability to prevent lethality and to decrease colonization. In vitro assays of adherence, agglutination and complement mediated cytotoxicity will address the mechanism(s) of protection. If recombinant antibodies are to be broadly applied, an inexpensive expression system must be available and we will focus our attention on the development of the transgenic chicken as such an expression system. The chicken is inexpensive to maintain and targets large amounts of antibody to the egg yolk. Intravenous injection of iodinated antibodies into laying hens will be used to determine which antibodies will be transported to the yolk. Vectors tested for Ig expression using chicken lymphoid cell lines will be transfected into Stage X blastoderm cells and these transfected cells used to make chimeric embryos. The resulting chickens will be tested for the presence of chimeric lg in their serum and in the eggs of laying hens.
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