DbpA, but not OspA, is expressed by Borrelia burgdorferi during spirochetemia and is a target for protective antibodies

Abstract

DbpA is a target for antibodies that protect mice against infection by cultured Borrelia burgdorferi. Infected mice exhibit early and sustained humoral responses to DbpA and DbpB, suggesting that these proteins are expressed in vivo. Many antigens expressed in mammals by B. burgdorferi are repressed in vitro at lower growth temperatures, and we have now extended these observations to include DbpA and DbpB. To confirm that the protective antigen DbpA is expressed in vivo and to address the question of its accessibility to antibodies during infection, we examined B. burgdorferi in blood samples from mice following cutaneous inoculation. B. burgdorferi was visualized by dark-field microscopy in plasma samples from spirochetemic mice, and an indirect immunofluorescence assay showed that these spirochetes were DbpA positive and OspA negative. We developed an ex vivo borreliacidal assay to show that hyperimmune antiserum against DbpA, but not OspA, killed these plasma-derived spirochetes, demonstrating that DbpA is accessible to antibodies during this phase of infection. Blood transferred from spirochetemic donor mice readily established B. burgdorferi infection in naive recipient mice or mice hyperimmunized with OspA, while mice hyperimmunized with DbpA showed significant protection against challenge with host-adapted spirochetes. Antiserum from persistently infected mice had borreliacidal activity against both cultured and plasma-derived spirochetes, and adsorption of this serum with DbpA substantially depleted this killing activity. Our observations show that immunization with DbpA blocks B. burgdorferi dissemination from the site of cutaneous inoculation and suggest that DbpA antibodies may contribute to control of persistent infection.

FIG. 1

In vitro expression of DbpA and DbpB is regulated by temperature. (A) Immunoblot detection of proteins expressed by B. burgdorferi grown at 37°C using antibodies against DbpB (lane 1), DbpA (lane 2), OspC (lane 3), OspA (lane 4), and FlaB (lane 5). (B and C) B. burgdorferi growing at 23°C was further passaged at this temperature (lanes 1) or at higher growth temperatures (lanes 2 and 3). Likewise, cultures growing at 37°C (lanes 6) were passaged at lower temperatures (lanes 4 and 5). Protein expression was evaluated by immunoblotting with a cocktail of the five antibodies (B) or by SDS-PAGE and Coomassie blue staining (C).

FIG. 2

Plasma-derived, uncultured B. burgdorferi express DbpA, but not OspA. B. burgdorferi B31 from in vitro cultures (grown in BSKII medium) or recovered from plasma samples from spirochetemic mice were incubated first with 1:50 dilutions of antisera from B31-infected mice or hyperimmune antiserum against OspA_B31 or (His)₆DbpA_B31 and then with phycoerythrin-conjugated goat anti-mouse antibodies. Wet-mount slides of unfixed spirochetes were examined at ×1,000 magnification by fluorescence microscopy under UV light and by dark-field microscopy.