A second type of adaptive immune system has been discovered very recently in the extant jawless vertebrates. Lymphocytes in the lamprey and hagfish undergo somatic recombinatorial assembly of modular leucine-rich-repeat (LRR) genetic units to generate a potential variable lymphocyte receptor (VLR) repertoire estimated to be as large as the antibody repertoire in mammals; i.e., >1014. The antigen-specific VLRs that are generated in response to immunization could have many practical uses similar to those established for antibodies in a variety of diagnostic tests, protein purification, inhibition of intracellular proteins, pathogen identification, and pathogen inhibition. Toward the realization of this potential, the experiments in this proposal are designed to characterize functional and structural properties of the VLR-mediated adaptive immune system in the sea lamprey. The primary aims are to (i) define the immunization requirements, kinetics, and memory potential in the lamprey cellular and humoral immune responses to particulate antigens (Bacillus anthracis exosporium, Salmonella typhimurium, and human erythrocytes) and to soluble antigens (BcIA anthrax protein and its C-terminal domain), (ii) characterize the molecular nature of soluble VLRs in experiments designed to test the hypothesis that VLRs are released from individual lymphocytes as preformed multivalent protein complexes, (iii) identify individual antigen-specific VLR-bearing lymphocytes and isolate them in order to characterize the antigen-binding affinity and structure of their VLR products, and (iv) examine the potential of the VLR adaptive immune system for protective immunity against a model pathogen, S. typhimurium. These experiments involve VLR-bearing lymphocyte identification and isolation by fluorescence activated cell sorting for culture, electron microscopic analysis, generation of cDNA libraries, biochemical analysis of VLRs separated by chromatographic methods including affinity columns and fast protein liquid chromatography (FPLC), VLR cDNA sequencing and expression of recombinant VLR protein, mutation of VLR cystines in VLR transduced cell lines to examine their potential role in disulfide linkage to form the VLR multimers, and analysis of VLR antigen-binding affinity by surface plasmon resonance. In summary, it has recently been learned that jawless fish possess an adaptive immune system that features the production by their lymphocytes of a very large spectrum of antigen-specific receptors that differ structurally from our antibodies. Since these variable lymphocyte receptors (VLR) are as diverse as our antibodies and may have a relatively stable physical structure, they could prove very useful in detecting and inhibiting hazardous pathogens, like Bacillus anthracis. The experiments proposed here will define biological and structural features of the VLRs in order to explore their potential for health-related uses. ? ? ?
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