Wisconsin State Laboratory ofHygiene' and Departments ofMedical .... Bio-Rad Laboratories, Richmond, Calif.). ... Rabbit antisera to Syrian hamster IgGl,.
INFECTION AND IMMUNITY, JUlY 1992, p. 2677-2682 0019-9567/92/072677-06$02.00/0 Copyright © 1992, American Society for Microbiology
Vol. 60, No. 7
Immunoglobulin G2 Confers Protection against Borrelia burgdorferi Infection in LSH Hamsters J. L. SCHMITZ,"12t R. F. SCHELL, 12,3* S. M. CALLISTER,3'4 S. D. LOVRICH,2 S. P. DAY," AND J. E. COE6 Wisconsin State Laboratory of Hygiene' and Departments of Medical Microbiology and Immunology, 2 Bacteriology,3 and Preventive Medicine, University of Wisconsin-Madison, Madison, Wisconsin 53706; Gundersen Medical Foundation, La Crosse, Wisconsin 546014; and Rocky Mountain Laboratories, 5
5
National Institutes of Health, Hamilton, Montana 598406 Received 14 January 1992/Accepted 14 April 1992
We showed that immune serum and its immunoglobulin fractions, specifically immunoglobulin G2 (IgG2), could confer complete protection to irradiated hamsters challenged with the Lyme disease spirochete. Immune serum and its immunoglobulin fractions also killed Borrelia burgdorferi in vitro. Depletion of complement in vivo abrogated the ability of IgG2 to confer complete protection against B. burgdorferi. Furthermore, the majority of antibody reactivity directed against B. burgdorfieri was found in the IgG2 fraction. These findings demonstrate that IgG2 plays an important role in acquired resistance against infection with B. burgdorfieri. Additional studies are needed to determine the mechanism(s) by which B. burgdorferi evades host defenses despite the development of an effective borreliacidal antibody response. A picture of antibody-mediated resistance to infection with Borrelia burgdorfen in experimental animals is beginning to emerge. We (21, 22) and others (7, 9-12, 18) have shown that serum and antibody can confer to animals protection against infection with the Lyme disease spirochete. Serum obtained from immunocompetent hamsters infected with B. burgdorfen could confer complete protection to irradiated hamsters challenged with the same isolate (21, 22). Borreliacidal activity was present 7 days after infection, peaked at weeks 3 to 5, and thereafter decreased (22). Depletion of complement abrogated the ability of immune serum to confer complete protection to irradiated hamsters challenged with B. burgdorfen. These results demonstrate that complement-dependent antibody is involved in protection against infection with B. burgdorferi. In the present report, we defined the role of specific immunoglobulins responsible for conferring protection against infection with the Lyme disease spirochete. Sera from immunocompetent hamsters infected with B. burgdorferi were fractionated, and purified immunoglobulins were used to passively immunize irradiated hamsters against challenge with B. burgdorfien. Our results demonstrate that immunoglobulin G2 (IgG2) antibodies are important for protecting hamsters against infection with B. burgdorferi.
and has been maintained by passage in modified BarbourStoenner-Kelly medium (BSK) (1) and hamsters (9-13, 20). The hamster-passed spirochetes were grown in BSK at 35°C for 5 days. The suspension of B. burgdorferi was adjusted with fresh BSK to contain approximately 107 organisms per ml. Samples of 1 ml were dispensed in vials, which were then sealed and stored in liquid nitrogen until use.
Preparation of B. burgdorferi for infection of hamsters. A frozen vial containing a suspension of B. burgdorferi was thawed and used to inoculate fresh BSK. The culture was grown for 5 days at 31°C and diluted with BSK to contain 5 x 106 organisms per ml. The number of spirochetes per milliliter was quantitated by dark-field microscopy. Hamsters were injected subcutaneously in each hind paw with 0.2 ml of this suspension. Irradiation of hamsters. Groups of hamsters were exposed to 600 rads of gamma radiation with a cobalt-60 irradiator (Picker Corp., Cleveland, Ohio). Hamsters survived this level of radiation without reconstitution with normal bone marrow cells. Preparation of hamster serum. Groups of five or more LSH hamsters were injected subcutaneously in each hind paw with 0.2 ml of BSK containing 106 viable B. burgdorfen organisms. At 1, 3, 5, 7, 10, and 52 weeks after infection, hamsters were mildly anesthetized by inhalation of ether contained in a nose-and-mouth cup and bled by intracardiac puncture. The blood was allowed to clot, and the serum was separated by centrifugation at 500 x g, pooled, divided into 1-ml amounts, and frozen at -20°C until use. Concomitantly, pooled normal hamster serum was obtained from
MATERIALS AND METHODS
Animals. Inbred LSH/Ss Lak (hereafter referred to as LSH) hamsters, 6 to 8 weeks old, were obtained from Charles River Breeding Laboratories, Inc. (Wilmington, Mass.). Hamsters weighing 60 to 100 g were housed three or four per cage at an ambient temperature of 21°C. Organism. B. burgdorferi 297 was obtained from Russell C. Johnson (University of Minnesota, Minneapolis). The strain was originally isolated from human spinal fluid (25)
noninfected normal hamsters. Fractionation of hamster serum by gel permeation chromatography. Pooled serum from hamsters infected with B. burgdorferi for 1, 3, 5, 7, 10, and 52 weeks was fractionated on a column of Bio-Gel Al.5m (1.6 by 100 cm; 200/400 mesh; Bio-Rad Laboratories, Richmond, Calif.). One milliliter of serum was diluted with 1.5 ml of 55 mM Tris-0.1 M NaCl (pH 8.0) and applied to the column. The sample was eluted with the Tris buffer by gravitational flow at 0.11 ml/min and
Corresponding author. t Present address: Clinical Microbiology and Immunology Laboratory, University of North Carolina Hospitals, Chapel Hill, NC 29514-2751. *
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4°C. Fractions of 1 ml were collected, and the optical density at 280 nm was determined. Beginning with fractions 40 and 41, consecutive 1-ml fractions were pooled and tested for the presence of IgM and IgG antibodies. Dot blot for the detection of IgM and IgG. Pooled fractions (8 ,ul) or purified hamster IgG (Rockland Inc., Gilbertsville, Pa.) was spotted onto nitrocellulose paper and air dried for 1 h at 25°C. The nitrocellulose was blocked with 3% nonfat dry milk for 1 h at 25°C. After being rinsed with distilled water, the nitrocellulose was incubated with rabbit anti-hamster IgG or IgM contained in 20 ml of 15 mM Tris-0.5 M NaCl-0.05% Tween 20 buffer overnight at 25°C. After incubation, the nitrocellulose was washed three times with the same buffer. Goat anti-rabbit IgG-peroxidase was added, and the mixture was incubated for 1 h at 25°C and then washed three times with the same buffer. The substrate 4-chloro-1-naphthol was added, and the strips were incubated for 20 min. Color development was stopped by rinsing with distilled water. Purification of hamster IgGl and IgG2 from immune serum. Fractionation of immune serum into IgGl and IgG2 fractions was performed as previously described (5). Hamster serum was eluted from a column of staphylococcal protein A linked to Sepharose CL-4B (Pharmacia Fine Chemicals, Piscataway, N.J.) with an acid gradient. Appropriate fractions were pooled, and the quantities of IgM, IgGl, and IgG2 were determined. Quantitation of immunoglobulins. A radial gel diffusion assay was used to quantitate IgGl, IgG2, and IgM in the pooled fractions. Rabbit antisera to Syrian hamster IgGl, IgG2, and IgM were used in this assays and prepared as previously described (2-4). Purified hamster immunoglobulins were used as standards. Passive transfer of resistance. Three or four irradiated hamsters per group were injected intravenously in the sublingual vein with 0.4 ml of saline, normal or immune serum, and its fractions. Fractions containing IgGl and IgG2 were diluted to contain levels of IgGl (0.42 mg/ml) and IgG2 (2.96 mg/ml) present in pooled week-3 immune serum. At 4 to 6 h after administration of serum or immunoglobulins, hamsters were irradiated and injected in each hind paw with 106 B.
burgdorferi organisms. Assessment of arthritis. Swelling of the hind paws of irradiated hamsters was used to evaluate the inflammatory response to infection (8). The size of each hind paw was measured with a plethysmograph (Buxco Electronics, Sharon, Conn.) on days 1 to 14 after challenge with B. burgdorferi. Measurements were obtained by lightly anesthetizing the hamsters, carefully dipping a hind paw into a column of mercury up to the ankle, and recording the amount (milliliters) of mercury displaced. If mercury remained on the paws of hamsters, it was carefully removed with a mercury collector (Scientific Products, Chicago, Ill). Mean plethysmograph values were obtained from three or four hamsters (six or eight paws) and used as an index of the severity of arthritic swelling. Mercury displacement was standardized with a volume calibrator. In some experiments, the severity of hind paw swelling was measured with a
caliper. Isolation of B. burgdorferi from tissues. Sixteen days after
infection, hamsters were killed by inhalation of carbon dioxide. The spleen and urinary bladder were removed aseptically and homogenized separately with 1 ml of BSK in a sterile petri dish. The tissue suspensions were inoculated into 4 ml of BSK and incubated at 31°C. Cultures were monitored weekly for spirochetes by dark-field microscopy.
INFECT. IMMUN.
Complement depletion in vivo. Lyophilized cobra venom factor (CVF) from Naja Haje (Diamedix, Inc., Miami, Fla.) was reconstituted with sterile distilled water. Hamsters were injected intraperitoneally with 0.2 ml (20 U) of CVF every 3 days beginning on day -3 for a total of five injections (days -3, 0, 3, 6, and 9). This quantity of CVF has been shown to depress C3 levels in hamsters for 6 days (26). In vitro killing of B. burgdorferi. Immunoglobulin fractions from the gel permeation column were diluted 1:2 in BSK and sterilized by filtration through 0.2-,um-pore-size syringe filters (Gelman Sciences Inc., Ann Arbor, Mich.). Subsequently, a 3-day-old culture of B. burgdorfeyi was diluted to approximately 2 x 105 organisms per ml, and 50-pu aliquots were added to 1.5-ml microcentrifuge vials containing 100 p. of the diluted fractions. Sterile guinea pig serum (50 p.l) with a complement activity of -210 50% hemolytic complement units per ml was added. The growth control consisted of elution buffer diluted 1:2 with BSK and contained complement. Normal serum and complement also did not inhibit the growth of B. burgdorferi. All assay mixtures were mixed by hand and incubated at 31°C for 6 h. One milliliter of BSK was added to all vials, which were then incubated for 4 to 7 days at 31°C. After incubation, 10-,ul aliquots from control and test vials in triplicate were removed and the total number of live spirochetes was determined. Twenty-five random fields of each 10-p. aliquot were read at x400 by dark-field microscopy. Live spirochetes were defined as those that were motile (14, 16). Percent growth inhibition was determined as follows: [(number of motile borreliae in growth control - number of motile borreliae in fraction sample)/number of motile borreliae in growth control] x 100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting. A culture of B. burgdorfen was grown at 31°C for 3 to 5 days. The spirochetes were centrifuged at 10,000 x g and washed two times in sterile phosphate-buffered saline (PBS). The protein concentration of the resulting suspension was determined with a commercially available kit (Bio-Rad). The spirochete suspension was boiled in sample buffer for 5 min; then, 150 p.g of total protein was loaded onto a 10% polyacrylamide gel (4% stacking gel without comb). Two gels were run simultaneously in an SE600 electrophoresis unit (Hoefer Scientific Instruments, San Francisco, Calif.) at 55 mA for 3 h with the buffer system of Laemmli (15). After electrophoresis, proteins were transferred to nitrocellulose at 300 mA for 3 h under conditions similar to those used by Towbin et al. (27). The nitrocellulose was cut into strips, which were then blocked for 20 min at 22°C in PBS-0.3% Tween 20. The strips were incubated with pooled hamster serum diluted 1:60 or monoclonal antibodies to OspA (H5332) or the flagellin protein (H9724) for 1 h at 22°C. The monoclonal antibodies were obtained from Alan Barbour, University of Texas, San Antonio. The strips were washed three times with PBS-0.05% Tween 20. Peroxidase-labeled anti-hamster IgG (heavy and light chains; Kirkegard and Perry Laboratories Inc., Gaithersburg, Md.) diluted 1:750 in PBS-0.05% Tween 20 or anti-mouse IgG (Organon Teknika Corp., Westchester, Pa.) diluted 1:10,000 in PBS were added to the strips, which were then incubated for 30 min at 22°C. The strips were washed as described above and developed with the TMB membrane peroxidase substrate system (Kirkegard and Perry). The developed strips were washed with distilled water and photographed. Statistical analysis. The plethysmograph values obtained from irradiated hamsters were tested by analysis of variance.
IgG2 PROTECTION AGAINST LYME DISEASE
VOL. 60, 1992
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The Fisher least-significant-difference test (24) 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.
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algG 40
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FIG. 1. (A) Elution profile (A280) of immune hamster serum fractionated on a gel permeation chromatography column. (B) Dot blot of fractions from the gel permeation column with rabbit anti-hamster IgG and IgM antibodies.
RESULTS Onset of borreliacidal IgM and IgG antibodies in hamsters after infection with B. burgdorfieri. Sera were collected 1, 3, 5, 7, 10, and 52 weeks after infection of immunocompetent hamsters with B. burgdorfeni to obtain IgM and IgG antibodies. Figure 1A and B shows the results of fractionation of pooled week-3 immune serum. A similar elution profile was obtained when the other serum pools were fractionated. The column fractions were diluted 1:2 with BSK and filter sterilized. Subsequently, 50 ,ul of complement and 104 B. burgdorferi organisms were added to each vial. Borreliacidal activity was detected in fractions (42 to 52) of week-1 immune serum containing IgM (Fig. 2A). No killing of B. burgdorferi organisms or transfer of passive protection was detected with fractions of immune serum containing IgM after this time. At week 3 after infection, borreliacidal activity was found in IgG fractions (76 to 86) (Fig. 2B). Similarly, borreliacidal activity was detected in IgG fractions (70 to 88) of sera collected 5, 7, 10 and 52 weeks after infection (Fig. 2C, D, E, and F). Slight borreliacidal activity (
