Serious infection due to Borrelia burgdorferi and the disseminated infection characteristic of the disease possess unique treatment problems. The wide and still increasing incidence of Lyme borreliosis as well as the problems in treatment call for effective prevention strategies by active immunization. Vaccination experiments were done to determine if active immunization of gerbils with recombinant OspA and pC protects against infection with strains of B. burgdorferi. Gerbils were vaccinated with recombinant OspA and pC (20 kDa protein) and challenged four weeks later with a clone (derived from B. burgdorferi strain PKo) which expresses an abundant amount of pC but only little OspA. Non-immunized gerbils challenged with the same B. burgdorferi strain were used as controls. Both groups of immunized gerbils developed antibodies against the recombinant vaccines. The pC vaccinated group was protected against infection, whereas the OspA vaccinated group showed signs of infection. The non-vaccinated group developed generalised infection. These results show that pC should be considered as a further vaccine candidate and probably needs to be combined with OspA for an efficient vaccine against B. burgdorferi.

Western blots (WBs; immunoblots) are a widely used tool for the serodiagnosis of Lyme borreliosis, but so far, no defined criteria for performance, analysis, and interpretation have been established in Europe. For the current study WBs were produced with strains PKa2 (Borrelia burgdorferi sensu stricto), PKo (Borrelia afzelii), and PBi (Borrelia garinii). To improve resolution we used gels of 17 cm in length. In a first step, 13 immunodominant proteins were identified with monoclonal antibodies. Then, the apparent molecular masses of all visually distinguishable bands were determined densitometrically. Approximately 40 bands of between 14 and 100 kDa were differentiated for each strain. From a study with 330 serum samples (from 189 patients with Lyme borreliosis and 141 controls), all observed bands were documented. To establish criteria for a positive WB result, the discriminating ability of a series of band combinations (interpretation rules) were evaluated separately for each strain (for immunoglobulin G [IgG] WB, >40 combinations; for IgM WB, >15 combinations). The following interpretation criteria resulting in specificities of greater than 96% were recommended: for IgG WB, at least one band of p83/100, p58, p56, OspC, p21, and p17a for PKa2; at least two bands of p83/100, p58, p43, p39, p30, OspC, p21, p17, and p14 for PKo; and at least one band of p83/100, p39, OspC, p21, and p17b for PBi; for IgM WB, at least one band of p39, OspC, and p17a or a strong p41 band for PKa2; at least one band of p39, OspC, and p17 or a strong p41 band for PKo; and at least one band of p39 and OspC or a strong p41 band for PBi. The overall sensitivity was the highest for PKo WB, followed by PBi and PKa2 WB, in decreasing order. Standardization of WB assays is necessary for comparison of results from different laboratories.

A recombinant immunoblot was developed for detection of IgM and IgG antibodies in patients with Lyme borreliosis. The recombinant antigens were the chromosomal-encoded Borrelia burgdorferi proteins p100, the flagellin and an internal flagellin fragment thereof as well as the plasmid-encoded outer surface proteins A (OspA) and C (OspC). A panel of 144 sera from patients with Lyme borreliosis (erythema migrans, n = 31; neuroborreliosis state II, n = 60; Lyme arthritis, n = 24 and acrodermatitis chronica atrophicans, n = 19) have been investigated and the results have been compared to the immunofluorescence absorption test (IFA-ABS) and to two different enzyme-linked immunosorbent assays [the flagellin ELISA and a newly developed ELISA (OGP-ELISA)]. The two ELISAs were comparable in sensitivity, whereas the IFA-ABS was less sensitive for IgM antibody but equally sensitive for IgG antibody detection. Immunoblot analysis revealed that IgG antibodies are mainly reactive with p100 and the internal flagellin fragment (sensitivity 51% and 32%, respectively) and rarely with OspC (14%). All patients with late Lyme borreliosis had IgG antibodies against the p100. IgM antibodies were predominantly directed against OspC (43%) and in a lower extent against the internal flagellin fragment and p100 (15% and 13%, respectively). The complete flagellin was not useful due to a high number of unspecific reactions with control sera and the OspA was only exceptionally reactive in Lyme borreliosis patients. The sensitivity of IgM antibody detection could be increased in cases with early Lyme borreliosis from 46% to 65% when the OspC blot was performed in addition to the flagellin ELISA, or from 56% to 65% when performed in addition to the OGP-ELISA. The recombinant blot is, therefore, a valuable diagnostic test to increase sensitivity of early antibody detection and is regarded as a valuable confirmatory test also in late disease.

No abstract

Borrelia burgdorferi sensu lato, the etiological agent of Lyme borreliosis, has been divided into three genospecies: B. burgdorferi sensu stricto (OspA-type 1), B. afzelii (OspA-type 2) and B. garinii (OspA-type 3-7). Whereas in Europe B. afzelii (OspA-type 2) is predominant among human skin isolates and B. garinii (OspA-type 3-7) among human CSF isolates, some previous serological studies suggested that Lyme arthritis is also associated with B. burgdorferi sensu stricto in Europe. In the present study we designed ospA type-specific PCRs and identified four different ospA types associated with Lyme arthritis. Our study group consisted of 20 patients with positive serology (ELISA and immunoblotting) and clinical criteria for Lyme arthritis. B. burgdorferi DNA was detected in 13 patients and in none of 10 control patients from synovial fluid. We identified ospA-type 1 (26.6%), ospA-type 2 (33.3%), ospA-type 4 (6.6%) and ospA-type 5 (33.3%). Our conclusion is that in Europe B. burgdorferi sensu lato strains causing Lyme arthritis are considerably heterogeneous and that there is no prevalence of certain genospecies or OspA-types among this strains.

We have previously described the use of the following recombinant antigens for serodiagnostic immunoblots: p83/100, p39, OspC and p41 (flagellin) internal fragment [Wilske et al. (1993) Med Microbiol Immunol 182:255-270; Rossler et al. (1997) J Clin Microbiol 35:2752-2758]. In our currently used immunoblot p83/100 is derived from strain PKo (Borrelia afzelii), p39 (BmpA) and OspC from strains PKa2 (B. hurgdorferi sensu stricto), PKo and PBi (B. garinii), respectively; the p41 (flagellin) internal fragments were cloned from strains PKo and PBi. In this study we describe the use of two additional recombinantly expressed highly immunogenic proteins Osp 7 (derived from PKo) and p58 (derived from PBi). A clinically well-defined panel of sera from 147 Lyme borreliosis patients and 139 controls previously tested by a standardized whole cell lysate immunoblot [Hauser et al. (1997) J Clin Microbiol 35:1433-1444] was investigated in the recombinant immunoblot without (old recombinant immunoblot) and with Ospl7 and p58 (new recombinant immunoblot) for IgG antibodies. The sensitivity of the recombinant IgG immunoblot for diagnosis of stage II and stage III could be significantly improved by addition of Osp17 and p58 without loss of specificity. With the exception of sera from patients with erythema migrans the diagnostic sensitivity is comparable to the whole cell lysate IgG immunoblot. The main advantage of the recombinant immunoblot is the easy identification of diagnostic bands, whereas the identification of bands in the whole cell lysate immunoblot is difficult. The recombinant immunoblot is especially suitable where large series of sera need to be investigated.

Western blotting (WB; immunoblotting) is a widely used tool for the serodiagnosis of Lyme borreliosis (LB), but so far, no generally accepted criteria for performance and interpretation have been established in Europe. The current study was preceeded by a detailed analysis of WB with whole-cell lysates of three species ofBorrelia burgdorferi sensu lato (U. Hauser, G. Lehnert, R. Lobentanzer, and B. Wilske, J. Clin. Microbiol. 35:1433–1444, 1997). In that study, interpretation criteria for a positive WB result were developed with the data for 330 serum samples (from patients with LB in different stages [n = 189] and from a control group [n = 141]) originating mostly from southern Germany. In the present work, the interpretation criteria for strains PKo (Borrelia afzelii) and PBi (Borrelia garinii) developed in the previous study were reevaluated with 224 serum samples (from patients with LB in different stages [n = 97] and from a control group [n = 127]) originating from throughout Europe that were provided by the European Union Concerted Action on Lyme Borreliosis (EUCALB). De novo criteria were developed on the basis of the reactivities of the EUCALB sera and were evaluated with the data for the samples from southern Germany. Comparison of all results led to the following recommendations: For WB for immunoglobulin G (IgG), at least two bands among p83/100, p58, p43, p39, p30, OspC, p21, p17, and p14 for PKo and at least one band among p83/100, p39, p30, OspC, p21, and p17b for PBi; for WB for IgM, at least one band among p39, OspC, and p17 or a strong p41 band for PKo and at least one band among p39 and OspC or a strong p41 band for PBi. WB with PKo was the most sensitive, and this strain is recommended for use in WB for the serodiagnosis of LB throughout Europe.

The complete coding regions of the chromosomally encoded p83/100 protein of four Borrelia garinii strains and one Borrelia burgdorferi sensu stricto strain have been amplified by the polymerase chain reaction (PCR), cloned and sequenced. From alignment studies with the deduced amino acid sequences presented here, and five other published p83/100 sequences, the most heterologous region of the p83/100 molecule was identified to be located between amino acid position 390-540. To study the structure of this heterogeneous region, and internal fragment of the p83/100 genes from 11 additional B. burgdorferi sensu lato strains was amplified by PCR. The PCR products were analyzed by DNA sequencing and restriction enzyme analysis. These internal p83/100 fragments varied in size and sequence. Cluster analysis of internal p83/100 fragments, as well as restriction enzyme analysis, revealed three major groups in accordance with grouping into the three species causing Lyme disease. Strains within the same species (six B. burgdorferi sensu stricto and six B. afzelii strains) showed similar p83/100 partial structures. Nevertheless, nine B. garinii strains showed more sequence variations and could be further divided into two major subgroups. One group is represented by OspA serotype 4 strains, the other more heterogeneous group is represented by OspA serotypes 3, 5, 6 and 7 strains. Phenotypic analysis with four p83/100-specific monoclonal antibodies revealed four distinct reactivity patterns. Antibody L100 1B4 recognized a common epitope of B. burgdorferi sensu stricto and B. afzelii. Antibodies L100 17D3 and L100 18B4 were reactive with an epitope shared by strains of all three species. The broadest reactivity was shown by L100 18B4 which, in contrast to L100 17D3, additionally recognized the relapsing fever borreliae B. turicatae and B. hermsii. L100 8B8 detected a subgroup of the B. burgdorferi sensu stricto strains. Since comparison of the p83/100 molecule with sequences from protein databases showed similarities with characteristics of eukaryotic cell structures, the p83/100 might mimic these structures and may, therefore, be involved in the immune escape mechanism of the pathogenic agent of Lyme disease.