Alloimmunization to the Rhesus blood group system through transfusion or pregnancy can limit the effectiveness of transfusions, can lead to clinically-significant transfusion reactions, and can cause hemolytic disease of the newborn. In order to design specific and effective methods for the prevention and management of the problems associated with Rh alloimmunization, it is first necessary to characterize the anti-Rh immune response at the molecular level. To this end, the proposed research will utilize a number of novel cellular and recombinant approaches to examine the repertoire of variable regions genes used by anti-Rh alloantibodies as well as the structural heterogeneity of their antigenic binding sites. Specifically, we will express human anti-Rh alloantibodies in vitro from the B-cells of sensitized patients by first expanding the B-cells with crosslinked anti- CD40 and IL-4, and then screening for anti-Rh producing clones prior to cell transformation with EBV or somatic cell hybridization. In parallel, filamentous phage immunoglobulin display libraries will be created from the initial populations of B-cells, as well as from expanded and transformed clones, to rescue cells that are unstable or refractory to immortilization. This combined approach will provide both the expressed immunoglobulins and the cDNAs that encode them. This will enable the identification of immunodominant epitopes on Rh antigens and conserved DNA sequences on anti- Rh antibodies. Finally, we will isolate peptides that inhibit anti-Rh binding to RBCs in vitro through the analysis of hypervariable loops of blocking (anti-idiotypic) antibodies and the screening of random peptide libraries with anti-Rh antibodies. Collectively, these studies will provide an understanding of the nature of the human immune response to Rh antigens on a molecular genetic level. This will facilitate t he design of therapeutically useful inhibitors that modulate anti-Rh/Rh antigen interactions in vivo. It will also lead to the development of genetic vaccines that modulate anti-Rh/Rh antigen interactions in vivo. It will also lead to the development of genetic vaccines that induce T-cell responses that down-regulate athe production of anti-Rh alloantibodies. In addition, the in vitro production of monoclonal anti-Rh antibodies/recombinant Fab fragments and the structural analogs of their antigenic epitopes will provide useful biochemical and serological reagents to increase our understanding of the biochemistry and immunology of this important blood group system.
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