The relapsing fever agent Borrelia hermsii evades the immune response of its human host be multiphasic antigenic variation. When a borrelia gains entry into a mammal, the spirochetes multiply in the blood. During this proliferation, linear plasmids bearing variable antigen (Vmp) genes spontaneously recombine. the interplasmidic recombinations are nonreciprocal and undirectional; they possibly are initiated by breaks in a plasmid with an expressed vmp gene. The breaks are repaired by hitherto silent vmp genes on other plasmids; the repair is fostered by upstream and downstream homology blocks on both plasmids. Once the expression site at the telomere of a plasmid is effectively """"""""converted"""""""" by the incoming vmp gene, the serotype of the cell changes. In the meantime the infected person mounts an immune response to the initial serotype. The critical effector of clearance of borreliae from the blood are immunoglobulins that specifically bind Vmp proteins. The new serotype comes to predominate in the blood, because the existing anti-Vmp antibodies do not recognize the Vmp of the new serotype, and consequently, the patient suffers a relapse of illness. For our continuing studies of the molecular basis of this antigenic variation in B. hermsii and the immunology of relapsing fever we propose these three lines on inquiry over the next five years: (1) Further definition of the recombination between linear plasmids bearing silent and expressed vmp genes: (a) Analyze the sequences of silent and expressed vmp genes of other switches and other serotypes. (b) Determine the basis of non-switching phenotype in the spontaneous mutant serotype C. (c) Examine products of recombination between B. hermsii sequences cloned in Escherichia coli. (d) Ascertain copy numbers of expressed vmp genes and silent vmp genes relative to each other and to single-copy chromosomal genes. (e) Identify sites of potential strand breakage and recombination on the expression and silent linear plasmids. (2) Further biochemical and immunological characterization of Vmp proteins: (a) Determine the deduced sequence of other Vmp proteins. (b) Identify regions of the proteins bound by protective and non-protective antibodies to Vmp7 and Vmp21. (c) Identify T-cell epitopes of Vmp7 and Vmp21. (d) Develop and characterize modified Vmp proteins. (3) Begin development of systems for delivering marker DNA into borreliae and for creating mutants: (a) Further define conditions for efficient transfer of DNA into borrelia cells. (b) Assess expression of foreign antibiotic resistance and other marker genes in borreliae. (c) Complete sequence of 24 kb expression plasmid of B hermsii. (d) Construction of recombinant linear plasmids for transfer into borreliae.
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