Figure 5.

Vlp15/16 and Vlp18 inhibit complement activation independently from binding to FH and protect serum sensitive B. garinii from complement-mediated lysis. Assessment of the inhibitory capacity of Vmp’s on complement activation by an ELISA-based assay. Microtiter plates immobilized with IgM (CP) (A), mannan (LP) (B), and LPS (AP) (C) were incubated with NHS pre-incubated with the purified proteins or BSA (10 µg each). Formation of the MAC was detected by a monoclonal anti-C5b-9 antibody. Dose-dependent inactivation of the AP (D). Microtiter plates immobilized with LPS and after blocking, wells were incubated with NHS pre-incubated with increasing concentrations of the borrelial proteins or BSA. Formation of the MAC was detected by a monoclonal anti-C5b-9 antibody. Binding of recombinant borrelial proteins to FH (E), C3b (F), C5 (G), and Factor B (FB) (H). Microtiter plates were coated with recombinant proteins, incubated with purified FH, C3b, and FB, respectively, and antigen–antibody complexes were detected using specific antisera. All experiments were performed at least three times, with each individual test carried out in triplicate. *p ≤ 0.033; ***p ≤ .0002; ****p ≤ 0.0001, one-way ANOVA with Bonferroni post-hoc test (confidence interval = 95%). n.s., no statistical significance; NC, negative control. Protection of serum sensitive B. garinii by Vlp15/16 and Vlp18 (I). NHS pre-incubated with 4 µM of the respective proteins was added to 1 × 10⁸ spirochetes and viability and motility of borrelial cells were determined at 0, 1, 2, 3, 4, 5, and 6 h of incubation. At least three independent experiments were conducted and ± SEM were calculated.