Production of borreliacidal antibody to outer surface protein A in vitro and modulation by interleukin-4

Abstract

Borreliacidal antibody production is one of several parameters for establishing the effectiveness of Borrelia burgdorferi vaccines. The production of borreliacidal antibody was studied in vitro by culturing immune lymph node cells with macrophages and B. burgdorferi. We showed that borreliacidal antibody, directed primarily against outer surface protein A (OspA), was readily produced by lymph node cells obtained from C3H/HeJ mice vaccinated with formalin-inactivated B. burgdorferi in aluminum hydroxide, but not recombinant OspA. Anti-OspA borreliacidal antibody was detected in supernatants of cultures of lymph node cells obtained on day 7 after vaccination, peaked on day 17, and rapidly declined. The borreliacidal activity was attributable to immunoglobulin G1 (IgG1), IgG2a, and IgG2b antibodies. When lymph node cells were treated with interleukin-4 (IL-4), production of borreliacidal antibody was inhibited but was unaffected by treatment with anti-IL-4 antibodies. These results suggest that other cytokines, but not IL-4, are mainly responsible for production of the secondary borreliacidal antibody response.

FIG. 1

Detection of borreliacidal antibody in supernatants of cultures of lymph node cells cultured with macrophages and B. burgdorferi obtained from C3H/HeJ mice on the indicated days after vaccination with B. burgdorferi contained in aluminum hydroxide.

FIG. 2

Detection of borreliacidal antibody in supernatants obtained from cultures of lymph node cells from 17-day-vaccinated (■) and nonvaccinated (□) mice cultured with macrophages and B. burgdorferi. Immune lymph node cells were also cultured with macrophages (●) or B. burgdorferi (▴) alone. Titers of borreliacidal antibody did not vary by more than 1 dilution in replicate assays. In most cases (90%) the same titer was obtained.

FIG. 3

Immunoblots of B. burgdorferi incubated with supernatant obtained from cultures of lymph node cells obtained from 17-day-vaccinated C3H/HeJ mice before and after absorption with rOspA. Borreliacidal antibody titers are also listed before and after absorption with rOspA.

FIG. 4

Percentage of B. burgdorferi in supernatants obtained from immune (solid bar), lymph node cells treated with anti-mouse IgG (checkered bar), IgG2a (horizontally striped bar), IgG2b (cross-hatched bar), or IgG3 (vertically striped bar) compared to those obtained from nonimmune lymph node cells (open bar). Error bars represent standard errors of the means calculated from replicate assays.

FIG. 5

Borreliacidal antibody response of lymph node cells obtained from 17-day-vaccinated mice cultured with macrophages and B. burgdorferi and treated with 0.01 (horizontal striped bar), 0.1 (checkered bar), or 1.0 (vertically striped bar) μg of rIL-4 10 min or 4 days (cross-hatched bar) (1 μg of rIL-4) after cultivation. Control cultures (solid bar) were treated with an equivalent volume of sterile PBS. Titers of borreliacidal antibody did not vary by more than 1 dilution in replicate assays. In most cases (90%) the same titer was obtained.

FIG. 6

Borreliacidal antibody response of lymph node cells obtained from 17-day-vaccinated mice cultured with macrophages and B. burgdorferi and treated with anti-murine IL-4 10 min (checkered bar) or 4 days (cross-hatched bar) after cultivation. Control cultures (solid bar) were treated with a non-isotype-specific antibody. Titers of borreliacidal antibody did not vary by more than one dilution in replicate assays. In most cases (90%) the same titer was obtained.