Immunodominant proteins are variable in molecular and antigenic structure among different genospecies of Borrelia burgdorferi sensu lato. We have recently developed an immunoblot using five recombinant antigens: the chromosomal-encoded B. burgdorferi proteins p100, the flagellin and an internal flagellin fragment thereof, and the plasmid-encoded outersurface proteins A (OspA) and C (OspC). In the present study the same antigens (derived from strain PKo, genospecies B. afzelii) were compared with the homologous recombinant proteins from strain B31 (genospecies B. burgdorferi sensu stricto) and with OspA, OspC and the internal flagellin fragment from strain PBi (genospecies B. garinii). Patients with neuroborreliosis (n = 28) and patients with acrodermatitis chronica atrophicans (n = 20) were investigated in the IgG immunoblot; the IgM immunoblot was performed only in patients with neuroborreliosis. There was a small increase in the detection rate of OspA-specific IgG or IgM antibodies using the different variants of recombinant OspA; however, OspA remained an insensitive antigen for antibody detection in Lyme borreliosis. The same was true to OspC-specific IgG antibodies. The sensitivity of OspC, which is the immunodominant antigen for IgM antibody detection, could not be increased using recombinant antigens derived from different strains. However, some sera which were negative in the recombinant immunoblot reacted with OspC in the conventional immunoblot using B. burgdorferi whole cell lysate as antigen. The most unexpected finding was the high degree of immunological heterogeneity of the internal flagellin fragments: IgG antibodies were detected in 18 of 48 patients using B31 fragments, in 25 of 48 using PKo fragments, in 23 of 48 using PBi fragments versus 33 of 48 when the three recombinant proteins were combined. PKo-derived fragments were more sensitive for antibody detection in patients with acrodermatitis chronica atrophicans, B31- and PBi-derived fragments for antibody detection in patients with neuroborreliosis. This is in agreement with the fact that isolates from patients with neuroborreliosis are predominantly belonging to the genospecies B. burgdorferi sensu stricto and B. garinii. For detection of IgM antibodies in sera from patients with neuroborreliosis, recombinant internal fragments derived from strains B31 and PBi were more sensitive than the PKo-derived fragment. The best discrimination between neuroborreliosis sera and control sera was achieved when the IgM blot was performed using recombinant internal flagellin fragments derived from strains PKo and PBi and OspC derived from B31 or PKo.
The genes coding for the outer surface protein A (OspA) of 19 different Borrelia burgdorferi strains belonging to the seven OspA-serotypes 1-7, previously described [Wilske et al. (1993) J Clin Microbiol, 31: 340-350], have been investigated. B. burgdorferi sensu lato strains were chosen from various biological sources (ticks, human skin and cerebrospinal fluid) as well as different geographical origins (Germany, Slovenia, Austria, United States). The open reading frames of all ospA genes consist of 819-825 nucleotides corresponding to proteins of approximately 30 kDa. The ospA sequences obtained in this study and previous published studies were compared with the results from OspA serotyping with monoclonal antibodies. The classification into the seven OspA serotypes could be confirmed on a genetic basis (ospA genotypes 1-7) for all strains analyzed so far (n = 29). In addition, one strain without OspA expression could be assigned to ospA genotype 2. Genetic stability could be proven for the ospA gene of B. burgdorferi strain PWudI after inocculation and reisolation from a gerbil. However, we found evidence for intragenic recombination by cluster analysis of ospA sequence data. Accordance of ospA genotype 1 strains with B. burgdorferi sensu stricto and ospA genotype 2 strains with B. afzelii, as well as the ospA genotype strains 3-7 with B. garinii was confirmed by pulsed-field gel electrophoresis of MluI-digested genomic DNA. B. garinii is not only more heterogeneous in respect to the OspA-encoding genes, but shows moreover major subgroups formed by genotypes 4, 5 and 6 and genotypes 3 and 7, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Borrelia burgdorferi sensu lato, the etiological agent of Lyme borreliosis is considerably heterogeneous in Europe. Since the outer surface proteins OspA and OspC are the most promising candidates for a Borrelia vaccine the immunological heterogeneity of these proteins was investigated. By immunological analysis with monoclonal antibodies and sequence analysis of PCR amplified OspA and OspC at least seven and 16 different types, respectively, were found. Whereas skin isolates (n = 68) were quite homogeneous (84% belonged to OspA-serotype 2 or Borrelia afzelii), isolates from human cerebrospinal fluid and from ticks (n = 43 and n = 90 respectively) were highly heterogeneous in their OspA-serotypes with prevalence of the Borrelia garinii associated types (about 70%). OspA-type 4 was often found among isolates from cerebrospinal fluid (28%). In ticks type 4 OspA has not been detected by culture so far. However, as reported in a previous study, type 4 OspA could be detected in ticks by the highly sensitive PCR technique.
It has been shown by analysis with monoclonal and polyclonal antibodies that outer surface protein C (OspC) of Borrelia burgdorferi sensu lato is highly heterogeneous. To determine if the heterogeneity has a genetic basis, the genes of 18 different B. burgdorferi sensu lato strains have been amplified by PCR, cloned, and sequenced. The ospC genes could be amplified from all strains tested, even from two strains which did not express OspC in detectable amounts. Among the 18 strains, 16 significantly different types of ospC sequences have been found. The sequence identities of the deduced amino acid sequences of different ospC genotypes range between 62 and 80% (determined without the leader peptide). The sequences correspond to one of the 13 OspC types distinguishable by analysis with monoclonal antibodies (B.
Molecular analyses of the genes encoding OspC, a major immunodominant protein of Borrelia burgdorferi sensu lato, revealed a considerable degree of heterogeneity. In the present study, we investigated whether a similar heterogeneity of the OspC phenotype can be shown by analysis with monoclonal antibodies (MAbs). Thirteen OspC-specific MAbs (L22 MAbs) were produced by immunizing mice with either different combinations of whole-cell antigens or recombinantly expressed OspCs cloned from strains belonging to different Borrelia spp. Ten of them differed in their reactivities with various strains. Western blot (immunoblot) analyses of 38 B. burgdorferi sensu lato strains resulted in 13 different reactivity patterns. These 13 different patterns were observed among only six different OspA serotypes, indicating that OspC is more heterogeneous than OspA. Patterns 1 to 4 were present only in B. burgdorferi sensu stricto, patterns 5 to 7 were present only in Borrelia afzelii, and patterns 9 to 13 were present only in Borrelia garinii. Pattern 8 was observed among B. afzelii and B. garinii strains but not among B. burgdorferi sensu stricto strains. One L22 MAb (2B8) recognized a common OspC-specific epitope of all 38 B. burgdorferi sensu lato strains analyzed, and another one (22C11) recognized a common epitope of OspC from both B. afzelii and B. garinii and was not reactive with OspC from B. burgdorferi sensu stricto. Western blot and sequence analysis of truncated OspCs located the 22C11 epitope as well as a species-specific sequence motif between amino acids 20 and 35. Other broadly reactive L22 MAbs were 10D3, 1F8, and 7G5. Some L22 MAbs (1C3, 1C3, 12E5, 1B11, 1F10, and 6C8) bound to epitopes present only in a few strains. Relapsing fever borreliae (Borrelia hermsii, Borrelia turicatae, and Borrelia duttoni) were nonreactive, with the following exception: three L22 MAbs (2B8, 6C4, and 10C5) recognized an abundantly expressed 20-kDa-range protein of B. turicatae. Because OspC is an immunodominant protein during the early immune response in Lyme borreliosis and has been shown to be effective as a vaccine in an animal model, our findings have important implications for the development of diagnostic reagents as well as vaccine research.
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