Cloning and molecular characterization of plasmid-encoded antigens of Borrelia burgdorferi

Thirteen independent clones that encode Borrelia burgdorferi antigens utili zing antiserum from infection-immune rabbits were identified, The serum was adsorbed against noninfectious B. burgdorferi B31 to enrich for antibodies directed against either infection-associated antigens of B, burgdorferi B3 1 or proteins preferentially expressed during mammalian infection. The adso rption efficiency of the immune rabbit serum (IRS) was assessed by Western immunoblot analysis with protein lysates derived from infectious and noninf ectious B. burgdorferi B31. The adsorbed IRS was used to screen a B. burgdo rferi expression library to identify immunoreactive phage clones. Clones we re then expressed in Escherichia coli and subsequently analyzed by Western blotting to determine the molecular mass of the recombinant B. burgdorferi antigens, Southern blot analysis of the 13 clones indicated that 10 contain ed sequences unique to infectious B, burgdorferi. Nucleotide sequence analy sis indicated that the 13 clones were composed of 9 distinct genetic loci a nd that all of the genes identified were plasmid encoded. Five of the clone s carried B, burgdorferi genes previously identified, including those encod ing decorin binding proteins A and B (dbpAB), a rev homologue present on th e 9-kb circular plasmid (cp9), a rev homologue from the 32-kb circular plas mid (cp32-6), erpM, and erpX, Additionally, four previously uncharacterized loci with no known homologues were identified. One of these unique clones encoded a 451-amino-acid lipoprotein with 21 consecutive, invariant 9-amino -acid repeats near the amino terminus that we have designated VraA (for "vi rulent strain-associated repetitive antigen A"). Since all the antigens ide ntified are recognized by serum from infection immune rabbits, these antige ns represent potential vaccine candidates and, based on the identification of dbpAB in this screen, may also be involved in pathogenic processes opera tive in Lyme borreliosis.