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Infect Immun. Oct 2000; 68(10): 5496–5501.
PMCID: PMC101497
Production of Borreliacidal Antibody to Outer Surface Protein A In Vitro and Modulation by Interleukin-4
Erik L. Munson,1,2 Brian K. Du Chateau,1,3 Dean A. Jobe,4 Steven D. Lovrich,4 Steven M. Callister,4,5 and Ronald F. Schell1,2,3*
Wisconsin State Laboratory of Hygiene1 and Departments of Medical Microbiology and Immunology2 and Bacteriology,3 University of Wisconsin, Madison, Wisconsin 53706, and Microbiology Research Laboratory4 and Department of Infectious Diseases,5 Gundersen Lutheran Medical Center, La Crosse, Wisconsin 54601
Editor: R. N. Moore
*Corresponding author. Mailing address: Wisconsin State Laboratory of Hygiene, University of Wisconsin, 465 Henry Mall, Madison, WI 53706. Phone: (608) 262-3634. Fax: (608) 265-3451. E-mail: RFSchell/at/Facstaff.wisc.edu.
Present address: Path Lab, Incorporated, Portsmouth, NH 03801.
Received March 21, 2000; Revisions requested May 12, 2000; Accepted June 30, 2000.
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.
Infection with Borrelia burgdorferi, the etiologic agent of Lyme borreliosis, induces a protracted, yet vigorous, humoral immune response (6, 8). Most of the anti-B. burgdorferi antibodies involved in this response are important for confirming the clinical diagnosis of Lyme borreliosis (6, 8, 9), while they play a minor role in protecting the host against infection. Furthermore, several of these anti-B. burgdorferi antibodies, especially those directed against outer surface protein A (OspA), OspB, OspC, and the 39-kDa periplasmic protein, have a unique dual function. These antibodies are highly specific for the serodiagnostic identification of infection with B. burgdorferi, and they can kill B. burgdorferi in the presence of complement (2, 20, 2427). Induction of borreliacidal antibodies is helpful in evaluating the potential of B. burgdorferi vaccines (5, 11, 22, 29).
Recently, clinical trials of two Lyme borreliosis vaccines containing OspA demonstrated that they could protect humans from becoming infected with B. burgdorferi (28, 32). A major concern, however, is the duration of protection afforded by the anti-OspA borreliacidal antibody response. Previously we showed (22) that vaccination with recombinant OspA (rOspA) induced only a short-lived protective borreliacidal antibody response, even after a booster vaccination. Similarly, OspA borreliacidal antibody waned rapidly in hamsters by week 10 of vaccination (22). Thus rOspA or other B. burgdorferi antigens that induce borreliacidal antibodies must be capable of maintaining sustained high levels of borreliacidal antibodies. This would reduce the number of vaccinations required for induction of borreliacidal antibody and lessen the potential for developing adverse side effects that may resemble arthritis (7). Recently, we showed that severe destructive arthritis could be elicited in vaccinated animals challenged with B. burgdorferi only during periods when levels of borreliacidal antibody were low (17).
In order to improve the production and maintenance of borreliacidal antibody, more needs to be known about the immunologic events following vaccination with B. burgdorferi or its components. Interleukin-4 (IL-4) has been shown to regulate B-lymphocyte growth and differentiation (23). Moreover, IL-4 is necessary to generate and sustain some secondary antibody responses (10, 13). In this study we developed an in vitro culture system to study the induction of borreliacidal antibody and effects of IL-4. C3H/HeJ mice were vaccinated with rOspA or B. burgdorferi in the presence or absence of aluminum hydroxide. Lymph node cells from vaccinated mice were then cultured with macrophages and B. burgdorferi in the presence or absence of IL-4. Our results show that treatment of lymph node cells capable of producing B. burgdorferi antibody with IL-4 inhibited the anti-OspA borreliacidal antibody response.
Mice.
Eight-to-twelve-week-old inbred male C3H/HeJ mice were obtained from our breeding colony located at the Wisconsin State Laboratory of Hygiene. Mice weighing 20 to 30 g were housed four per cage at an ambient temperature of 21°C. Food and acidified water were provided ad libitum.
Organism.
B. burgdorferi sensu stricto isolate 297 was originally isolated from human spinal fluid (31). Low-passage (fewer than six passages) organism was cultured once in modified Barbour-Stoenner-Kelly (BSK) medium (3) containing screened lots of bovine serum albumin (4) to a concentration of 5 × 107 spirochetes per ml. Five hundred-microliter samples were then dispensed into 1.5-ml screw-cap tubes (Sarstedt, Newton, N.C.) containing 500 μl of BSK supplemented with 10% glycerol (Sigma Chemical Co., St. Louis, Mo.), sealed, and stored at −70°C. When necessary, a frozen suspension of spirochetes was thawed and used to inoculate fresh BSK medium. Spirochetes were viewed by dark-field microscopy and enumerated using a Petroff-Hausser counting chamber.
Preparation of vaccines.
OspA was purified as described previously (19). Briefly, transformed Escherichia coli containing the B. burgdorferi ospA gene was grown in 2× tryptone yeast extract broth containing ampicillin at 37°C for 12 h. Cultures were then diluted with fresh broth and incubated for an additional hour. Isopropyl-β-d-thiogalactopyranoside was added, and cultures were incubated for 5 h. Subsequently, bacteria were pelleted by centrifugation, resuspended in phosphate-buffered saline (PBS) (pH 7.4), and lysed by sonication. Lysed organisms were mixed with Triton X-100, diluted with PBS, and centrifuged again to remove insoluble material. The supernatant was mixed with a slurry of glutathione-Sepharose beads (Pharmacia, Piscataway, N.J.) and washed with ice-cold PBS. Fusion proteins were eluted by mixing beads with Tris-HCl containing reduced glutathione and collected after centrifugation. The elution procedure was repeated four times, and the fractions were analyzed for purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblotting. OspA (60 μg) was then mixed with 0.5 ml of 1% aluminum hydroxide (Reheis, Berkeley Heights, N.J.).
A whole-cell vaccine was also prepared. B. burgdorferi organisms were grown in 1 liter of BSK medium for 6 days, pelleted by centrifugation (15,000 × g, 15°C, 10 min), and washed three times with PBS. The washed pellet was resuspended in 1% formalin, incubated at 32°C for 30 min with periodic mixing, washed three times by centrifugation with PBS (18,000 × g, 10°C, 15 min), and resuspended in PBS. Subsequently, the formalin-inactivated spirochetes were mixed in a volume of a 1% suspension of aluminum hydroxide to yield 4 × 106 spirochetes/ml.
Vaccination of mice.
One hundred twenty-five mice were mildly anesthetized with methoxyflurane contained in a mouth-and-nose cup and vaccinated subcutaneously in the inguinal region with 0.25 ml (~106 B. burgdorferi organisms) of the formalin-inactivated vaccine preparation. The suspension contained approximately 100 μg of borrelial protein. Another fifty mice were vaccinated subcutaneously in the inguinal region with 0.25 ml of OspA (30 μg) with or without aluminum hydroxide. Nonvaccinated mice were injected with BSK medium or aluminum hydroxide alone.
Recovery of macrophages.
Five to ten nonvaccinated mice per experimental protocol were mildly anesthetized with methoxyflurane contained in a mouth-and-nose cup and injected intraperitoneally with 2 ml of 3% thioglycollate in PBS. Four days after injection, mice were euthanatized by CO2 asphyxiation, and 8 ml of cold Hanks' balanced salt solution (Sigma) was injected intraperitoneally. The peritoneal cavity was massaged for ~1 min, and the exudate cells were recovered by aspiration with a syringe. The suspension of peritoneal exudate cells was centrifuged at 500 × g for 10 min at 4°C. The supernatant was decanted, and the cells were resuspended in Dulbecco's modified Eagle's medium (DMEM) (Sigma) that was free of antimicrobial agents but supplemented with 10% heat-inactivated (56°C, 45 min) fetal bovine serum (HyClone Laboratories, Logan, Utah), 5 × 10−5 M 2-mercaptoethanol (Sigma), and l-glutamine (2.92 mg/mL; Sigma). Aliquots of the cell suspension were then poured over polystyrene tissue culture dishes (100 by 20 mm; Corning Glass Works, Corning, N.Y.) and incubated at 37°C in a humidified atmosphere of 5.0% CO2 for 4 to 6 h. After incubation, nonadherent cells were aspirated from the tissue culture dishes. The dishes were then gently rinsed twice with 8-ml portions of warm Hanks' balanced salt solution to further eliminate nonadherent cells. Five milliliters of cold, nonenzymatic cell lifter (Sigma) was added to each tissue culture dish and incubated at 4°C for 30 min. Macrophages were detached from the surface of the dishes by vigorously tapping and gently scraping the inside of the tissue culture dishes with a sterile rubber policeman. Suspensions of macrophages from several tissue culture dishes were aspirated, pooled, and centrifuged at 500 × g for 10 min at 4°C. The supernatant was decanted, and the pellet was resuspended in 1 ml of DMEM. Cell viability of leukocytes was determined by trypan blue dye exclusion. The preparations of macrophages obtained by this method were 98% free of lymphocyte contamination. Giemsa-stained smears of lymphocytes showed a homogenous population of lymphocytes with no other types of leukocytes visible.
Isolation of lymph node cells.
Mice were euthanatized by CO2 inhalation at 7, 14, 17, 21, 28, 35, and 42 days after vaccination with formalin-inactivated B. burgdorferi or rOspA. Inguinal lymph nodes were removed from vaccinated and nonvaccinated mice, and each was placed into cold DMEM. Single-cell suspensions of lymph node cells were prepared by teasing apart the lymph nodes with forceps and pressing them through a sterile stainless steel 60-mesh screen into antimicrobial-free cold DMEM supplemented with 10% heat-inactivated fetal bovine serum, l-glutamine, and 2-mercaptoethanol. Lymph node cells were washed twice by centrifugation (500 × g, 4°C, 10 min) with DMEM. Supernatants were decanted and pellets were resuspended in 1 ml of cold DMEM. Cell viability was assessed by trypan blue dye exclusion.
Production of antibody in vitro.
Sterile six-well flat-bottom tissue culture dishes (Becton Dickinson, Lincoln Park, N.J.) were inoculated with lymph node cells (5 × 106) obtained from vaccinated or nonvaccinated mice, macrophage (105), and 106 live B. burgdorferi organisms. DMEM was added to the suspensions of cells to bring the final volume to 3 ml. At days 3, 6, 9, 12, and 15 after cultivation at 37°C in the presence of 5.0% CO2, 1.0-ml samples of the supernatants were removed after gentle agitation and replaced with an equal volume of warm DMEM. In other experiments, recombinant IL-4 (rIL-4) or rat anti-murine IL-4 (R&D Systems, Minneapolis, Minn.) at quantities ranging from 0.01 to 1.0 μg was added to cultures of immune lymph node cells, macrophage, and B. burgdorferi at 10 min and 4 days of incubation. Control cultures were also incubated with a rat isotype nonspecific antibody. Supernatants were collected after centrifugation at 18,000 × g for 8 min to remove spirochetes and other cellular debris. Supernatants were stored at −70°C until used.
Detection of borreliacidal antibody by membrane filtration.
Frozen supernatants were thawed, heat-inactivated (56°C, 30 min), sterilized with a 0.2-μm-pore-size filter (Acrodisk; Gelman Sciences, Ann Arbor, Mich.), and serially twofold diluted (neat to 1:8,192) with fresh BSK medium. One hundred-microliter aliquots of each dilution were transferred to 1.5-ml screw-cap tubes (Sarstedt), and 100 μl of BSK containing 104 B. burgdorferi organisms per ml was added along with 20 μl of sterile guinea pig complement (Sigma). The tubes were then gently shaken and incubated for 3 days at 32°C. Controls included filter-sterilized supernatants obtained from suspensions of nonimmune lymph node cells with macrophages and B. burgdorferi. Other controls included supernatants from nonimmune lymph node cells, macrophages alone, and DMEM.
After incubation, 100 μl of each suspension was removed and placed into individual 1.5-ml screw-cap tubes (Sarstedt). Subsequently, 100 μl of a solution of propidium iodide (1.0 mg/mL; Molecular Probes, Eugene, Oreg.) diluted 1:20 in sterile PBS was added. The suspensions were briefly mixed before being incubated at 56°C for 30 min to permit intercalation of propidium iodide into the spirochetes. One hundred microliters of each sample was then filtered through 0.2-μm-pore-size Nuclepore polycarbonate membrane filters (47-mm diameter; Whatman Nuclepore, Clifton, N.J.) under negative pressure with a single-place sterility test manifold (Millipore Corporation, Bedford, Mass.) attached to a vacuum pump. Membrane filters were washed with ~8 ml of sterile double-distilled H2O (ddH2O), removed from the vacuum apparatus, allowed to dry, and placed onto glass microscope slides. Coverslips were placed on the filters before viewing with a Laborlux S fluorescence microscope (Leitz, Wetzlar, Germany) using a 50× oil immersion objective.
The number of spirochetes on each filter was quantitated by viewing ~30 fields. The borreliacidal antibody titer was defined as the reciprocal of the dilution preceding the dilution at which the number of spirochetes or clumping was equal to that of the control. Generally, individual spirochetes with a few clumps were uniformly distributed throughout the fields on filters of the control supernatants.
Neutralization of borreliacidal activity.
Borreliacidal assays were performed with the following modifications: 20 μl of sheep anti-mouse immunoglobulin G1 (IgG1), IgG2a, IgG2b, and IgG3 (diluted 1:7 in sterile PBS; The Binding Site, Birmingham, United Kingdom) was added to 100-μl aliquots of a 1:64 dilution of heat-inactivated and filter-sterilized supernatant (borreliacidal antibody titer previously determined as 512). Controls received 20 μl of PBS. The assays were then incubated for 4 to 6 h at 37°C. Following incubation, 100 μl of approximately 103 single cells of B. burgdorferi were added to each assay. In addition, 20 μl of complement was added at 10 min, 24 h, and 48 h before completion of a 72-h incubation at 32°C. Subsequently, 100 μl of propidium iodide (1.0 mg/ml) was added, and the assays were filtered through 0.2-μm-pore-size Nuclepore filters. The number of spirochetes and clumps of spirochetes was determined by counting 30 fields using a Laborlux S fluorescence microscope. Assays were also inoculated with fresh BSK to determine the viability of the spirochetes and clumps of spirochetes. If assays contained borreliacidal antibody no growth of B. burgdorferi was detected or growth was inhibited (>10 days) compared to growth of B. burgdorferi in assays containing supernatants from nonimmune cells.
Western immunoblotting.
B. burgdorferi 297 organisms were grown in 1 liter of BSK medium for 6 days, pelleted by centrifugation (15,000 × g, 15°C, 10 min), and washed three times with PBS at pH 7.4. The washed pellet was resuspended in 1% formalin and incubated at 32°C for 30 min with periodic mixing and then washed three times by centrifugation with PBS (18,000 × g, 10°C, 15 min) and resuspended in PBS. The borrelial protein content was determined by using a bicinchoninic acid assay (Sigma). Spirochetes were suspended in SDS-PAGE sample buffer and boiled for ~3 min. One hundred twenty micrograms of B. burgdorferi lysate was loaded onto a preparative 12% acrylamide gel, and the proteins were resolved by overnight electrophoresis at a constant current of ~7 mA with the buffer system of Laemmli (16). The proteins were transferred onto a nitrocellulose membrane for 1 h at 20 V, using a semidry blotting apparatus (Bio-Rad Laboratories, Hercules, Calif.). The nitrocellulose membrane was incubated overnight at 4°C in 5% skim milk dissolved in Tris-buffered saline (TBS) with 0.05% Tween 20 (TBS-T) (pH 7.4) to block nonspecific reactivity, washed two times each with TBS-T and ddH2O, allowed to dry, and finally cut into strips. Strips were then incubated for 1 h with samples of the supernatants (diluted in 5% milk in TBS-T). The strips were washed three times with TBS and subsequently incubated 1 h with a 1:1,000 dilution of an alkaline phosphatase-conjugated goat anti-murine IgG (heavy and light chain-specific; Kirkegaard & Perry Laboratories, Gaithersburg, Md.) in 5% milk in TBS-T. This was followed by four washes with TBS. Strips were developed by the addition of 5-bromo-4-chloro-3-indolylphosphate–nitroblue tetrazolium substrate (Kirkegaard & Perry). Reactions were stopped after two minutes with several large volumes of chilled ddH2O.
Preparation of OspA column.
Three samples of the OspA fusion protein containing >1.29 mg of protein each were combined and dialyzed overnight in coupling buffer (0.1 M NaHCO3, 0.5 M NaCl [pH 8.3]). Following dialysis, 0.8 g of CNBr-activated agarose beads was weighed and soaked for 1 h in coupling buffer. Smaller beads were centrifuged (13 min, 500 × g), and the buffer was drawn off. The dialyzed OspA fractions were then added to the activated beads and rocked gently for 2 h at room temperature. After incubation, beads were washed twice with coupling buffer, and a 100 mM solution of ethanolamine (pH 9) was added for 2 h with rocking. The beads were then washed three times with coupling buffer (pH 4), resuspended in a small amount of PBS, and packed into a 5-ml column. Supernatants containing OspA antibodies were placed onto the column and run through approximately 15 times. The removal of OspA antibodies was confirmed by Western immunoblotting. The treated supernatants were also assayed for borreliacidal activity.
Statistical analysis.
A t test (35) was used to determine significant differences in the titers of borreliacidal antibody among supernatants. In addition, titers that were determined during kinetic studies of in vitro borreliacidal antibody production were tested by analysis of variance, utilizing the Minitab statistical analysis program. The Fisher least-significant-difference test (35) was used to examine pairs of means when a significant F ratio indicated reliable mean differences. The alpha level was set at 0.05 before the experiments were started.
Ability of lymph node cells obtained from rOspA-vaccinated mice or mice vaccinated with formalin-inactivated B. burgdorferi to produce borreliacidal antibody.
Initially, 30 C3H/HeJ mice were vaccinated with 30 μg of rOspA alone or 30 μg of rOspA contained in 1% aluminum hydroxide. Lymph node cells (5 × 106) were collected from mice at days 7, 17, and 21 after vaccination and cultured with 105 macrophages and 106 B. burgdorferi organisms for 9 days. Anti-OspA borreliacidal antibody titers of 32 and 8 were detected in supernatants of lymph node cells at the peak interval (day 17) from mice vaccinated with rOspA in the presence or absence of aluminum hydroxide, respectively. When these studies were repeated similar results were obtained.
Subsequently, 50 C3H/HeJ mice were vaccinated with formalin-inactivated B. burgdorferi contained in 1% aluminum hydroxide. Lymph node cells were obtained from mice at 0, 7, 14, 17, 21, 28, 35, and 42 days after vaccination and cocultured with macrophages and B. burgdorferi (Fig. (Fig.1).1). Borreliacidal antibody was detected in supernatants obtained from cultures of lymph node cells 7, 14, 17, 21, 28, 35, and 42 days after vaccination. Lymph node cells obtained from mice 17 days after vaccination produced the highest level of borreliacidal antibody (titer, 128). Thereafter, the level of borreliacidal antibody rapidly decreased.
FIG. 1
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.
Detection of borreliacidal antibody in cultures of lymph node cells.
Fifty C3H/HeJ mice were vaccinated with formalin-inactivated B. burgdorferi contained in aluminum hydroxide. Lymph node cells were collected from mice 17 days after vaccination and cultured in vitro in the presence or absence of macrophages and/or B. burgdorferi for 15 days. Supernatants were then collected on days 3, 6, 9, 12, and 15, filter sterilized, and incubated with 103 B. burgdorferi and complement. Borreliacidal antibody (titer, 128) was first detected on day 6 of culture of lymph node cells with macrophages and B. burgdorferi (Fig. (Fig.2).2). Peak borreliacidal antibody (titer, 512) was detected with supernatants obtained on days 9 to 15 of culture. By contrast, no borreliacidal antibody was detected in cultures of lymph node cells obtained from vaccinated mice when cocultured with macrophages or B. burgdorferi alone. In addition, no borreliacidal antibody was detected with supernatants from cultures of lymph node cells obtained from nonvaccinated mice and cocultured with macrophages and B. burgdorferi. When these studies were repeated similar results were obtained.
FIG. 2
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 (more ...)
In other studies the peak borreliacidal antibody titers (eight experiments) detected with supernatants from immune lymph node cells obtained 17 days after vaccination ranged from 64 to 1,024. The level of borreliacidal antibody production was dependent upon the number of lymph node cells in the cultures. We also found that production of borreliacidal antibody by lymph node cells was dependent upon the size of the inoculum. Increasing the inoculum from 106 to 107 B. burgdorferi organisms or more decreased or abrogated the borreliacidal antibody response. An inoculum of 107 B. burgdorferi organisms absorbed the borreliacidal antibody produced by the lymph node cells. When 106 B. burgdorferi organisms were used for priming immune cells, borreliacidal antibody was readily detected in the supernatants of the immune lymph node cells.
Contribution of anti-OspA antibody to borreliacidal activity.
rOspA was used to absorb supernatant collected 9 days after lymph node cells from 17-day-vaccinated mice were cocultured with macrophages and B. burgdorferi. Figure Figure33 shows that treatment of supernatant with rOspA prevented detection of the 31-kDa protein by Western immunoblotting. In addition, absorption of the supernatant with rOspA reduced the borreliacidal titer from 64 to 16. The remaining borreliacidal activity is due to anti-Osp C borreliacidal antibody.
FIG. 3
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 (more ...)
Neutralization of anti-OspA borreliacidal antibody.
When B. burgdorferi organisms were incubated with pooled immune supernatant (diluted 1:64) and complement, the spirochetes were lysed (Fig. (Fig.4).4). Treatment of immune supernatant with anti-mouse IgG1 prevented approximately 80% of the lysis of B. burgdorferi. Similarly, treatment of immune supernatant with anti-mouse IgG2a and IgG2b prevented approximately 30 and 40% of the borreliacidal activity, respectively. When immune supernatant was treated with anti-mouse IgG3, the borreliacidal activity of immune supernatant was not affected. In other studies, immune supernatant was treated with anti-mouse IgG2a and IgG2b. Many clumps of B. burgdorferi were present, although viable spirochetes were not recovered. When IgG1 was removed, clumping of B. burgdorferi was eliminated.
FIG. 4
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 (more ...)
Effects of rIL-4 and anti-murine IL-4 on the anti-OspA borreliacidal antibody response.
Lymph node cells from 17-day-vaccinated mice were cocultured with macrophages and B. burgdorferi. At 10 min of cultivation 0.01, 0.1, or 1.0 μg of rIL-4 was added. Figure Figure55 shows that 0.01 μg of rIL-4 inhibited the borreliacidal antibody response 16-fold compared to the nontreated immune lymph node cells. Borreliacidal antibody production was also inhibited by 0.1 and 1.0 μg of rIL-4; however, a four- to eightfold reduction in activity was detected. By contrast, no reduction in borreliacidal antibody production was detected when rIL-4 was added on day 4 of cultivation. Similarly, treatment of OspA borreliacidal antibody producing cells with anti-murine IL-4 at 10 min or day 4 of cultivation had no effect on borreliacidal activity (Fig. (Fig.6).6).
FIG. 5
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 (more ...)
FIG. 6
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 (more ...)
A major step towards prevention of infection with B. burgdorferi has been the development of rOspA vaccines for humans (28, 32) and dogs (33). Vaccination of susceptible individuals in areas where Lyme borreliosis is endemic most likely will reduce the morbidity associated with infection with B. burgdorferi. The vaccines, however, will not entirely eliminate the risk of becoming infected with B. burgdorferi even after repeated vaccinations. One currently available rOspA vaccine was less than 50% effective in preventing infection with B. burgdorferi after two injections and only 76% effective after a third injection (32). Most importantly, the duration of protective immunity and the number of subsequent yearly booster vaccinations required to maintain sustained high levels of protective borreliacidal antibody remains unknown.
The purpose of this investigation was to develop an in vitro system to determine the effects of cytokines on the production of borreliacidal antibody. We showed that borreliacidal antibody, specifically anti-OspA borreliacidal antibody, was readily detected in cultures of lymph node cells obtained from C3H/HeJ mice vaccinated with formalin-inactivated B. burgdorferi in aluminum hydroxide. Anti-OspA borreliacidal antibody was detected on day 7 of vaccination, peaked on day 17, and thereafter rapidly declined. By contrast, vaccination with rOspA with or without aluminum hydroxide induced less anti-OspA borreliacidal antibody. When anti-OspA borreliacidal antibody-producing cells were treated with rIL-4, borreliacidal activity was inhibited. In addition, treatment of anti-OspA borreliacidal antibody-producing cells with anti-murine IL-4 failed to alter the production of cidal antibody.
Our results show that rOspA is a poor immunogen for inducing production of borreliacidal antibody, even when administered with aluminum hydroxide. Although our rOspA was not lipidated, vaccination of mice with rOspA in aluminum hydroxide should have enhanced borreliacidal antibody production. Instead, low titers of borreliacidal antibody were produced by immune lymph node cells cocultured with macrophages and B. burgdorferi. These results confirm and extend our previous findings that humans and hamsters launch a poor anti-OspA borreliacidal antibody response, even after multiple vaccinations with lipidated rOspA (22). By contrast, vaccination of mice with formalin-inactivated B. burgdorferi with aluminum hydroxide induced high levels of borreliacidal antibody. Anti-OspA borreliacidal antibody was detected in cultures of lymph node cells 7 days after vaccination and peaked on day 17 of vaccination. The enhanced production of borreliacidal antibody may be due to better recognition and processing of native OspA on the formalin-inactivated B. burgdorferi. Taken together, these results suggest that a recombinant protein may lose the conformational epitopes necessary to induce high levels of borreliacidal antibody.
Despite production of high levels of anti-OspA borreliacidal antibody (range, 64 to 1,024), the anti-OspA borreliacidal antibody response waned rapidly. Borreliacidal antibody decreased 8- to 16-fold within days after the period (17 days) of maximum production. Once borreliacidal antibody production was down-regulated (21 days after vaccination), immune lymph node cells failed to produce borreliacidal antibody, even in the presence of antigen-presenting cells and borrelial antigen. These in vitro results suggest that the rapid decrease of circulating anti-OspA borreliacidal antibody in serum detected in vaccinated humans and animals (22) is the result of limited production of borreliacidal antibody by immune cells. It is possible that immune lymph node cells become less responsive to or more tolerant of epitopes of OspA that are responsible for the induction of borreliacidal antibody. In support of this argument, we showed previously (22) that production of nonborreliacidal anti-OspA antibody remained elevated for many months after vaccination of humans and hamsters with rOspA, while the level of anti-OspA borreliacidal antibody was undetectable within weeks after vaccination.
This selective down-regulation of borreliacidal antibody production is important because it affects the duration of protection against infection with B. burgdorferi. In this study we attempted to enhance the production of borreliacidal antibody by treatment of immune cells with rIL-4. IL-4 has been shown to enhance B-lymphocyte survival (14, 15, 23), increase the rate of B-lymphocyte proliferation (12, 14), and augment secondary antibody responses (12, 13, 15). Unexpectedly, treatment with rIL-4 inhibited borreliacidal responses. When borreliacidal antibody producing cells were treated immediately (10 min) with various concentrations of rIL-4, borreliacidal activity was greatly inhibited (range, 4- to 16-fold) compared to control lymph node cells. In addition, a treatment delay of 4 days did not affect the production of borreliacidal antibody. Furthermore, treatment of immune cells capable of producing borreliacidal antibody immediately or on day 4 of cultivation with anti-murine IL-4 also did not affect the production of borreliacidal antibody. These combined results suggest that IL-4 plays a minor role in the regulation of the secondary borreliacidal antibody response in vitro.
Our in vitro results, however, show that anti-OspA borreliacidal antibody is due primarily to the production of IgG1 antibody. This antibody is IL-4-dependent (30). It is puzzling why addition of rIL-4 to cultures of immune lymph node cells did not enhance production of borreliacidal antibody. An explanation may be that IL-4 is more involved with initial processing and production of the primary antibody response. Our in vitro antibody system uses immune cells that are restimulated with B. burgdorferi. Although IgG1 anti-OspA borreliacidal antibody is produced, this response may now be IL-4 independent (10).
What then accounts for the production of anti-OspA borreliacidal antibody if IL-4 plays a minor role? We hypothesize that gamma interferon (IFN-γ) is involved. It is interesting that production of borreliacidal antibody occurred only when antigen-processing cells (macrophage) were added to cultures of immune lymph node cells. It is known that IFN-γ mediates apoptosis of T and B lymphocytes in the absence of accessory cells (1, 18, 21, 34). When accessory cells are present, IFN-γ promotes progression of T-lymphocyte activation (18) and antibody isotype switching (34). In addition, we detected lysis of B. burgdorferi when supernatants obtained from immune lymph node cells contained IgG2a antibodies. Production of IgG2a antibody is IFN-γ dependent (23). Additional studies are needed to define the role of IFN-γ, especially if its antagonist, IL-4 (15), does not play a major role in the regulation of secondary borreliacidal antibody responses.
Our results also show that borreliacidal activity occurs by different functions of subclasses of IgG antibody. IgG1 borreliacidal antibody kills B. burgdorferi organisms by clumping, while IgG2a and IgG2b kill individual spirochetes by lysis. Although both activities result in the death of B. burgdorferi cells, enhanced production of clumping antibodies (IgG1) could adversely affect the effectiveness of a vaccine. If rOspA induces primarily IgG1 borreliacidal antibody, the antibody may be effective only when large numbers of B. burgdorferi cells are located at a site of infection. By contrast, IgG2a and IgG2b borreliacidal antibody can kill individual spirochetes in the presence of complement. More studies are needed to determine if rOspA vaccines induce primarily IgG1 antibody. A more effective vaccine would produce primarily IgG2a and IgG2b borreliacidal antibody.
In conclusion, we developed an in vitro system to study the role of cytokines on production or inhibition of protective borreliacidal antibody. The present study shows that IL-4 does not play a major role in production of a protective borreliacidal antibody response generated by lymph node cells in vitro. More studies are needed to determine means to prolong the borreliacidal antibody response for protection against infection with B. burgdorferi.
ACKNOWLEDGMENTS
We thank Jani R. Jensen, Cindy L. Croke, Monica C. Remington, and John A. Christopherson for critical evaluation of the manuscript.
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