Borrelia burgdorferi BBA52 is a potential target for transmission blocking Lyme disease vaccine

Abstract

The surface-exposed antigens of Borrelia burgdorferi represent important targets for induction of protective host immune responses. BBA52 is preferentially expressed by B. burgdorferi in the feeding tick, and a targeted deletion of bba52 interferes with vector-host transitions in vivo. In this study, we demonstrate that BBA52 is an outer membrane surface-exposed protein and that disulfide bridges take part in the homo-oligomeric assembly of native protein. BBA52 antibodies lack detectable borreliacidal activities in vitro. However, active immunization studies demonstrated that BBA52 vaccinated mice were significantly less susceptible to subsequent tick-borne challenge infection. Similarly, passive transfer of BBA52 antibodies in ticks completely blocked B. burgdorferi transmission from feeding ticks to naïve mice. Taken together, these studies highlight the role of BBA52 in spirochete dissemination from ticks to mice and demonstrate the potential of BBA52 antibody-mediated strategy to complement the ongoing efforts to develop vaccines for blocking the transmission of B. burgdorferi.

Figure 1. Characterization of recombinant and native BBA52

(A) Recombinant BBA52. Production of recombinant BBA52 in insect cells using baculovirus expression system (left panel, purified recombinant BBA52, arrow). (B) Immunoblot analysis of recombinant BBA52 (middle panel), or B. burgdorferi lysates (right panel) in the presence or absence of β-mercaptoethanol (β-ME). Note that in addition to monomeric form, native BBA52 exists as higher-order oligomer (arrows) in the absence of β-ME. (C) Native BBA52 exists as oligomeric protein in B. burgdorferi cells. Spirochetes cells were lysed in the presence or absence of N-Ethylmaleimide (NEM) and assessed using BBA52 antibody. The oligomerization property of BBA52 under non-reducing conditions remained unaltered in the presence of NEM, indicating that major fraction of native BBA52 exists in trimeric conformation (arrow). Note that except for trimeric conformation, other relatively less abundant forms of native BBA52 appeared as faint bands (arrowheads). (D) Production of BBA52 in major infectious strains of B. burgdorferi sensu lato. B. burgdorferi cell-lysates prepared from isolates B31, 297 or N40 and B. garinii isolate PBi were immunoblotted with anti-BBA52 antibody generated against B. burgdorferi B31 isolate.

Figure 2. BBA52 is an outer membrane protein and is exposed on the microbial surface via carboxyl terminus

(A) BBA52 is distributed in the outer membrane of cultured spirochetes. B. burgdorferi protoplasmic cylinders (PC) and outer membranes (OM) were separated by sucrose density gradient centrifugation. Equal amounts of protein from two sub-cellular fractions were separated by SDS-PAGE, and immunoblotted with BBA52, OspA and FlaB antiserum. (B) Triton-X-114 phase partitioning of B. burgdorferi proteins. Spirochete lysates were subjected to Triton X-114 phase partitioning of aqueous and detergent phases and immunoblotted using BBA52 antibody or antibodies against known hydrophilic (BBA74) protein. (C) Association of BBA52 with borrelial membranes as assessed by the salt and detergent treatment. B. burgdorferi preparations were treated with indicated PBS, salt, detergent or sonication as described in the text. Supernatant (S) and pellet (P) fractions were separated by centrifugation and assessed by immunoblot analysis using BBA52 or FlaB antibody. (D) BBA52 is exposed on the B. burgdorferi surface via the carboxyl terminus. Viable spirochetes were incubated with (+) or without (–) proteinase K for the removal of protease-sensitive surface proteins and processed for immunoblot analysis using antibodies raised against full-length BBA52 or against a carboxyl terminus peptide of the protein. B. burgdorferi OspA and FlaB antibodies were utilized as controls for surface exposed and sub-surface proteins, respectively.

Figure 3. BBA52 antibodies bind to the surface of B. burgdorferi but lack borreliacidal activities in vitro

(A) BBA52 antibodies bind to the surface of intact unfixed B. burgdorferi (arrow). Spirochetes were immobilized on glass slides and probed with BBA52 or control (GST) antibodies. Antibody against known surface (OspA) and subsurface (Lp6.6) spirochete proteins were used as controls. Spirochete (arrow) loading and antibody labeling was assessed using propidium iodide (PI) and Alexa-488 tagged secondary antibodies, respectively. Images were acquired using a 40x objective lens of a Zeiss confocal microscope. (B) BBA52 antibodies lack borreliacidal activities in culture. Spirochetes were incubated without serum (control) or in the presence of either normal rabbit sera (NRS), rabbit OspA antibodies (OspA), serum collected from 15-day infected mice or peptide antibodies raised against carboxyl terminus of BBA52. The sensitivity of spirochetes to the bactericidal effects of the antibodies was assessed by a re-growth assay after 48 h of antibody incubation and presented as mean ± SEM of viable spirochete number (cells/ml). The numbers of viable spirochetes were significantly reduced in the samples exposed to the OspA or B. burgdorferi antibodies, compared to spirochetes that received no treatment or were incubated with BBA52 antibodies or normal serum (P < 0.002).

Figure 4

Active immunization of mice with recombinant BBA52 induces robust antibody responses and interfere with spirochete transmission from infected ticks to naïve hosts. A) ELISA showing development of high-titer serum antibodies induced in recombinant BBA52-immunized mice. Groups of C3H mice (5 animals/group) were immunized with recombinant BBA52, and two weeks after final immunization, serum was collected and subjected to ELISA. The wells were coated with recombinant BBA52 and probed either with BBA52 antiserum (black bars) or normal mouse serum (NMS, white bars) serially diluted from 1,000 -128,000. Differences between the OD values of wells probed with BBA52 antiserum with respective control wells treated with NMS were significant (P < 0.05). B) Active immunization of mice with recombinant BBA52 significantly interferes with spirochete transmission from infected ticks. Mice (5 animals/group) were immunized with either BBA52 or PBS (control) mixed with an equal amount of adjuvant, as described in Figure 4A. Two weeks after final immunization, B. burgdorferi-infected nymphs were allowed to engorge on naive immunized mice (2 ticks/mouse) and murine skin, heart and bladder samples were assessed for spirochete transmission after 7 days of feeding. Total RNA was isolated from murine samples, and B. burgdorferi flaB was measured using quantitative RT-PCR. Amount of murine β-actin was determined in each sample and used to normalize the quantities of spirochete RNA. The entire animal immunization and challenge studies were independently repeated three times and the bars represent the mean measurements ± SEM of three experiments reflecting similar results. Difference in the spirochete transmission in BBA52 immunized group with the control PBS group is significant (*P < 0.05).

Figure 5. Passive transfer of BBA52 antibodies in ticks interfere with B. burgdorferi transmission to mice

(A) Passive transfer of antibodies raised against a carboxyl-terminal BBA52 peptide does not interfere with spirochete persistence in unfed ticks. Naturally-infected nymphal ticks were microinjected with equal amounts of antibodies against BBA52 peptide (anti-BBA52 Ab) or control antibody (normal rabbit Ab), and spirochete distribution in the gut of unfed ticks were analyzed 5 days after injection. Spirochetes (arrow) were labeled with FITC-labeled goat anti-B. burgdorferi antibody (shown in green), and the nuclei of the gut cells were stained with propidium iodide (shown in red). Images were obtained using a confocal immunofluorescence microscope and presented as merged image for clarity. (B) Quantitative representation of the data shown in Fig. 5A. B. burgdorferi burden in ticks were assessed by qRT-PCR analysis by measuring copies of the B. burgdorferi flaB RNA and normalized against tick β-actin levels. Bars represent the mean ± SEM of four qRT-PCR analyses derived from two independent infection experiments. Spirochete burdens in BBA52 antibody-treated ticks are similar to the control ticks (P > 0.05). (C) BBA52 peptide antibodies blocked transmission of B. burgdorferi from ticks to mice. Naturally infected nymphal ticks were microinjected with antibodies as described in Fig. 5A and placed on naive mice 48 h after injection. Ticks were allowed to fully engorge on mice and the transmission of B. burgdorferi was assessed by measuring copies of the B. burgdorferi flaB gene in the indicated murine tissues 7 days after tick feeding. Amounts of mouse β-actin were determined in each sample and used to normalize the quantities of B. burgdorferi flaB. Bars represent the mean ± SEM of relative tissue levels of B. burgdorferi from two independent animal experiments showing similar results. * Spirochetes were undetectable in the anti-BBA52 Ab group.