(A) Diagrammatic illustration of the VlsE sequence showing the location of the peptides representing the protein’s three dominant epitopes, which were examined for immunoreactivity in this study. These included VlsE₂₁ (aa 21–44; N-terminal epitope), VlsE₂₇₄ (aa 274–298; IR6 epitope), and VlsE₃₃₆ (aa 336–349; C-terminal epitope) of the VlsE protein from B. burgdorferi B31. (B) Spatial position of the three sequences in the crystal structure of the VlsE protein. A ribbon diagram (left) and an orthographic molecular surface representation (right) of VlsE monomer are depicted using the visual molecular dynamics molecular graphics program, based on National Center for Biotechnology Information’s three-dimensional structure database coordinates. Parts of the protein that were missing from the three-dimensional structure database coordinates are represented as dashed lines in the ribbon diagram. The bottom of the figure represents the membrane proximal region.

Ab response to B. burgdorferi (whole-cell lysate), full-length VlsE, and individual VlsE epitopes. Mean levels of IgG reactivity to B. burgdorferi (whole-cell lysate) (A), IgG to full-length recombinant VlsE protein (B), IgG to VlsE₂₇₄ (C), C6 Ab (D), IgG to VlsE₂₁ (E), and IgG to VlsE₃₃₆ (F), as measured by ELISA, in healthy controls (HC) (n = 30) and in patients with single EM (n = 18), multiple EM (n = 17), early neurologic (n = 16), late neurologic (n = 16), antibiotic-responsive arthritis (n = 12), and antibiotic-refractory arthritis (n = 11) manifestations of Lyme disease. Error bars represent the SEM. Levels of statistical significance for differences in Ab reactivity are as follows. (A) Healthy versus multiple EM, early neurologic, late neurologic, responsive arthritis, and refractory arthritis: p < 0.001, p < 0.0001, p < 0.0001, p < 0.0001, and p < 0.0001, respectively; single EM versus early neurologic, late neurologic, responsive arthritis, and refractory arthritis: p < 0.01, p < 0.01, p < 0.0001, and p < 0.0001, respectively. (B) Healthy versus single EM, multiple EM, early neurologic, late neurologic, responsive arthritis, and refractory arthritis: p < 0.01, p < 0.001, p < 0.0001, p < 0.0001, p < 0.0001, and p < 0.0001, respectively; single EM versus early neurologic, late neurologic, responsive arthritis, and refractory arthritis: p < 0.01 for each comparison. (C and D) Healthy versus individual Lyme disease subgroups: p < 0.0001 for each; single EM versus other Lyme disease subgroups: p < 0.05 for each. (E) Healthy versus late neurologic, responsive arthritis, and refractory arthritis: p < 0.01, p < 0.0001, and p < 0.0001, respectively; single EM versus late neurologic, responsive arthritis, and refractory arthritis: p < 0.01, p < 0.0001, and p < 0.0001; multiple EM versus late neurologic, responsive arthritis, and refractory arthritis: p < 0.01, p < 0.0001, and p < 0.0001, respectively; early neurologic versus late neurologic, responsive arthritis, and refractory arthritis: p < 0.05, p < 0.001, and p < 0.0001, respectively. (F) Healthy versus late neurologic, responsive arthritis, and refractory arthritis: p < 0.01, p < 0.001, and p < 0.0001, respectively; single EM versus late neurologic, responsive arthritis, and refractory arthritis: p < 0.05, p < 0.01, and p < 0.001; multiple EM versus late neurologic, responsive arthritis, and refractory arthritis: p < 0.05, p < 0.01, and p < 0.001, respectively; early neurologic versus responsive arthritis and refractory arthritis: p < 0.05 and p < 0.01, respectively. Differences for other group comparisons were not statistically significant.

Ab response to VlsE_mp. (A and B) A single contiguous molecule containing only the membrane-proximal region of VlsE and its associated VlsE₂₁ and VlsE₃₃₆ epitopes was cloned and expressed as a C3b fusion protein: the expected—and mass spectrometry confirmed—sequence (A) and the gel electrophoresis (denaturing) profile (B) of the expressed protein that was used for subsequent Ab assays. (C) Mean IgG Ab reactivity to VlsE_mp, as measured by ELISA, in healthy controls (HC) (n = 30) and in patients with single EM (n = 18), multiple EM (n = 17), early neurologic (n = 16), late neurologic (n = 16), responsive arthritis (n = 12), and refractory arthritis (n = 11) manifestations of Lyme disease. Error bars represent the SEM. Levels of statistical significance for differences in Ab reactivity to VlsE_mp are as follows. Healthy versus early neurologic, late neurologic, responsive arthritis, and refractory arthritis: p < 0.05, p < 0.0001, p < 0.0001, and p < 0.0001, respectively; single EM versus late neurologic, responsive arthritis, and refractory arthritis: p < 0.001, p < 0.0001, and p < 0.0001, respectively; multiple EM versus late neurologic, responsive arthritis, and refractory arthritis: p < 0.01, p < 0.0001, and p < 0.0001, respectively; early neurologic versus late neurologic, responsive arthritis, and refractory arthritis: p < 0.05, p < 0.01, and p < 0.01, respectively. Differences for other group comparisons were not statistically significant.

IgG subclass Ab responses to the IR6 and the membrane-proximal epitopes, as represented by VlsE₂₇₄ and VlsE_mp. Mean levels of IgG1, IgG2, IgG3, and IgG4 Ab reactivity to VlsE₂₇₄ (A) and VlsE_mp (B) in healthy controls (n = 30) and in Lyme disease patients found to be positive for total IgG reactivity to each peptide (n = 86 for VlsE₂₇₄, n = 36 for VlsE_mp). In comparison with the healthy control group, IgG1, IgG2, and IgG3 reactivities to each peptide were significantly increased in the Lyme disease patient group (p < 0.0001, p < 0.01, and p < 0.0001, respectively for both peptides). Error bars represent the SEM.

Accessibility of major VlsE immunogenic regions to Abs. (A) Assessment of binding of Abs to partially denatured VlsE protein by immunoblotting. IgG Abs to VlsE274, VlsE21, and VlsE336 bound to the localized VlsE on membrane, as did the control Ab to full-length recombinant VlsE. IgG from preimmunization sera showed no binding (representative image shown). (B) Immunofluorescence assessment of binding of IgG Abs to fixed or unfixed B. burgdorferi B31 cells. Staining of B. burgdorferi spirochetes with IgG Abs against VlsE21, VlsE274, and VlsE336 was only observed if the cells were fixed. The anti-B. burgdorferi (whole-cell lysate) and anti-VlsE (full-length) IgG Abs stained both fixed and unfixed cells. IgG from preimmunization sera did not stain cells (representative images shown).