Wkly. cognate peptides as competitors for VlsE binding by patient serum antibodies. IR6 epitopes (by the C6 peptide) were found to dominate the response to invariable segments. IR6 (C6)-specific antibodies were detected in 78% of the serum specimens, whereas <40% of patients generated antibodies that bound the N- or C-terminal domain name and <12% of patients responded to either IR2 or IR4. Interestingly, 15 of 37 patients generated IgG antibodies that reacted with C6 but not with VlsE. Conversely, IgM responses were frequent for VlsE but not for invariable segments. A representative number of the serum specimens (= 8) that contained IgG antibodies reacting with both C6 and VlsE was assessed in competition experiments, using C6 as a competitor. Only half of these specimens contained IgG antibodies whose binding to VlsE could be inhibited >50% by competition with the added C6 peptide. The median percent inhibition was 45.5%. These findings indicate that IR6 epitopes are largely concealed from the VlsE molecular surface and that full-length VlsE-based diagnosis likely detects antibodies to conformational and/or variable region epitopes. Contamination with the spirochete causes the multisystem disease known as Lyme borreliosis. The diagnosis of Lyme borreliosis is made by a combination of clinical observations and laboratory assessments. In areas where Lyme disease is usually endemic, the presence of erythema migrans (EM), an expanding annular skin rash, is considered sufficient to diagnose early Lyme disease. When patients present with later manifestations of Lyme disease that are not specific, reliable laboratory assessments are necessary to support the diagnosis (1). Ideally, contamination would be confirmed by culture or PCR detection of in skin biopsy or blood specimens. In practice, these invasive or time-consuming methods are KU-60019 not sensitive enough for a negative result to rule out contamination. Spirochete recovery from 2- to 4-mm skin biopsy samples of an EM lesion can be achieved, on average, for only 40 to 50% of untreated patients (1). Antibody detection is thus the most frequently used laboratory test to assist in the diagnosis of Lyme disease. The variable surface protein VlsE is an immunogenic molecule of that engages in antigenic variation. Two invariable domains, one at the amino and the other at the carboxyl terminus, together encompass approximately one-half of KU-60019 the molecule’s length. Antigenic variation occurs through gene conversion events that involve regions within the central domain name (12). This domain name contains six variable regions and six invariable regions (IRs), named IR1 to IR6. The six IRs remain unchanged during antigenic variation, and available sequence data indicate that they are conserved among sensu lato genospecies and strains (4, 14). The carboxyl- and amino-terminal domains of VlsE also remain invariant as contamination proceeds (13). In previous studies, the antibody responses to the IRs of VlsE in different host species were examined. Infected humans, monkeys, dogs, and mice either responded to IR6 and not to the other IRs or responded more vigorously to IR6 (7). Some individuals generated responses to peptides C2 and C4 (which comprise IR2 and IR4, respectively). In these studies, a limited selection of serum specimens from Lyme borreliosis patients was tested for immunoglobulin G (IgG) responses only. As with IR6, the C-terminal domain name (Ct peptide) of VlsE was also immunodominant in these animal species (5), but this region’s antigenicity was not as conserved as that of IR6 (5). Thus far, a systematic study of the relative contributions of the IRs and invariable domains of VlsE to the overall antigenicity of this protein has not been performed. In particular, the IgM response to invariable segments and the antigenicity of the N-terminal domain name of VlsE have never been assessed. Over the last 5 years, both the full-length VlsE molecule and the IR6 portion (the synthetic peptide C6) have emerged as diagnostic antigens in enzyme-linked immunosorbent assay (ELISA) assessments that are comparatively sensitive and specific (2). We hypothesized that this antigenicity of VlsE was largely concentrated on that of IR6, to the exclusion of other IRs KU-60019 and invariable domains of the molecule. This KU-60019 result could occur either because other invariant segments are simply not antigenic or Thbs4 because their epitopes are not accessible for antibody binding around the VlsE molecule. To test these possibilities, we set out to evaluate the antigenicity of VlsE in comparison to that of invariant segments known to be immunogenic in some individuals (7) and uncovered around the VlsE surface, per available X-ray crystallography (3) and immunoprecipitation (6) data. We chose to test IR2, IR4, and IR6, represented by peptides C2, C4, and C6, respectively. Both the carboxyl and amino termini of VlsE, represented by peptide Ct and the triad of overlapping.