V. Preac‐Mursic

A total of 136 Borrelia burgdorferi sensu latu strains from various biological sources (ticks, human skin, and cerebrospinal fluid) and geographical sources (Europe and North America) were investigated by Western blot (immunoblot) with eight monoclonal antibodies against different epitopes of the outer surface protein A (OspA). On the basis of the differential reactivities of these monoclonal antibodies, seven OspA serotypes were defined. As determined by 16S rRNA sequence analysis, these serotypes correlated well with recently delineated genospecies: serotype 1 corresponds to B. burgdorferi sensu strictu, serotype 2 corresponds to group VS461, and serotypes 3 to 7 correspond to Borrelia garinii sp. nov. (G. Baranton, D. Postic, I. Saint Girons, P. Boerlin, J.-C. Piffaretti, M. Assous, and P. A. D. Grimont, Int. J. Syst. Bacteriol. 42:378-383, 1992). Antigenic differences were confirmed by partial sequence analysis of OspA of representatives of each serotype. Comparative sequence analysis suggested that serotype 5 OspA resulted from genetic recombination of serotype 4 and 6 ospA genes. Serotype 2 (group VS461) was most prevalent among European skin isolates (49 of 62 isolates). Among all B. garinii strains included in this study, serotype 6 was most frequently found in ticks and only rarely in human skin and cerebrospinal fluid, whereas serotypes 4 and 5 were isolated from patients but never from ticks. Our data suggest different pathogenic potentials and organotropisms of distinct OspA serotypes and raise the question of true antigenic variation among B. garinii strains.

Borrelia burgdorferi strains (six isolates from North America and 28 isolates from Europe) were analyzed by physicochemical and immunological methods. By SDS-PAGE, all Borrelia burgdorferi strains tested had two major proteins with constant molecular weights of 60 and 41 kDa and one, two, or three variable low molecular weight proteins (OspA = 30-32 kDa, OspB = 34-36 kDa, pC = 21-22 kDa). All combinations--except OspB alone or OspB/pC--were observed. Borrelia burgdorferi strains were different from relapsing fever borreliae by strong reactivity with OspA- and/or pC-specific polyclonal antibodies, whereas relapsing fever borreliae were only weakly reactive. Among 25 Borrelia burgdorferi isolates, seven different serotypes of Borrelia burgdorferi were defined according to their reactivity in the Western blot with three monoclonal OspA-specific antibodies (H5332, H3TS, and LA5), four OspA- or OspB-specific polyclonal antibodies, and 12 polyclonal antibodies against whole borreliae. Antigenic differences between European CSF and skin isolates were observed, all skin isolates (n = 11) belonging to serotype 2 in contrast to only two out of seven CSF isolates. CSF isolates were antigenically heterogenous (serotypes 1, 2, 3, 4, and 5). Serotypes 6 and 7 were represented by two tick isolates, and the other European tick isolates are not yet fully characterized. Antigenic differences between European and North American strains may play a role in differences in the clinical picture of Lyme borreliosis.

The gene of the immunodominant major protein pC of Borrelia burgdorferi was previously cloned and sequenced (R. Fuchs, S. Jauris, F. Lottspeich, V. Preac-Mursic, B. Wilske, and E. Soutschek, Mol. Microbiol. 6:503-509, 1992). pC is abundantly expressed on the outer surface of B. burgdorferi, as demonstrated by immunoelectron microscopy with monoclonal antibody L22 1F8. Accordingly, pC is renamed OspC, by analogy to the outer surface proteins OspA and OspB. Western immunoblot analysis of 45 B. burgdorferi isolates with monoclonal antibodies revealed that OspC is immunologically heterogeneous. Partial sequence analysis of the ospC gene confirmed the protein heterogeneity at the genetic level. We found that the degree of identity between the ospC partial sequences of five strains representing different OspA serotypes was only 63.3 to 85.4%. Immunological heterogeneity was also observed among representatives of the three newly designated genospecies of B. burgdorferi sensu lato, B. burgdorferi sensu stricto, B. garinii, and group VS461. Heterogeneity was confirmed for B. garinii at the genetic level. The ospC gene was also cloned from strains that did not express OspC, and antibody-reactive OspC was expressed in Escherichia coli. In addition, OspC-expressing variants were obtained from a nonexpressing strain by plating single colonies on solid medium. These findings confirm that the ospC gene is also present in nonexpressing strains. Because OspC is an immunodominant protein for the early immune response in Lyme borreliosis and was effective as a vaccine in an animal model, the immunological and molecular polymorphisms of ospC and OspC have important implications for the development of diagnostic reagents and vaccines.