Low-passage (<10) virulent B. burgdorferi sensu stricto isolate 297 was cultured once in modified Barbour-Stoenner-Kelly medium (BSK) (4, 6) at 32°C 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 30% glycerol (Sigma Chemical Co., St. Louis, Mo.), sealed, and stored at −70°C. When needed, a frozen sample of spirochetes was thawed and used to inoculate fresh BSK. The number of spirochetes was determined using a Petroff-Hausser counting chamber under dark-field microscopy.

B. burgdorferi 297 organisms were grown in 1 liter of BSK to log phase, pelleted by centrifugation (10,000 × g, 15°C, 10 min), and washed three times with phosphate-buffered saline (PBS; pH 7.4). The washed pellet was resuspended in 1% formalin, incubated at 32°C for 30 min with periodic mixing, then washed three times by centrifugation (12,000 × g, 10°C, 15 min), and resuspended in PBS. The borrelial protein concentration was determined by using a commercially available kit (Sigma). To make the vaccine, B. burgdorferi 297 organisms were suspended in 10 ml of a 1% suspension of aluminum hydroxide (Imject alum; Pierce, Rockford, Ill.) at a concentration of 250 μg of borrelial protein per ml.

Hamsters were mildly anesthetized with ether contained in a nose-and-mouth cup and vaccinated intramuscularly in each hind thigh with 0.2 ml of the vaccine suspension containing 50 μg of borrelial protein in alum. Nonvaccinated hamsters were included as controls.

Hybridoma cell line 14-4-4s (ATCC HB-32) secretes murine monoclonal antibody (MAb) that recognizes a cell surface marker on hamster B lymphocytes (25, 26, 31, 49, 50) and has been shown to successfully separate hamster T and B lymphocytes (25, 26, 50). Hybridoma 14-4-4s was grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% bovine calf serum (HyClone Laboratories, Inc., Logan, Utah) at 37°C in a humidified atmosphere of 7.5% CO₂. After 7 to 10 days, the culture supernatant was collected following centrifugation at 500 × g for 10 min at 4°C, dispensed into 12-ml aliquots, and frozen at −20°C until used. Subsequently, some of the aliquots were thawed and used at a final dilution of 1:10. For T- and B-lymphocyte panning and enrichment procedures, 100 μg of unconjugated goat anti-mouse immunoglobulin, heavy and light chain specific (Organon Teknika Corporation, Durham, N.C.), in coupling buffer (15 mM Na₂CO₃, 35 mM NaHCO₃ [pH 9.6]) was used to coat 100- by 20-mm tissue culture dishes (Corning Glass Works, Corning, N.Y.). Dishes were maintained overnight at 4°C and washed four times with PBS before the addition of suspensions of lymph node cells that had been incubated with MAb 14-4-4s. T- and B-lymphocyte preparations were stained before and after panning for the presence of B lymphocytes. Cell preparations were stained with a phycoerythrin-conjugated goat anti-hamster immunoglobulin specific for both heavy and light chains (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) and were analyzed by flow cytometry for the presence of B lymphocytes. Phycoerythrin-conjugated goat and rat immunoglobulins were used as isotype controls.

Nonvaccinated hamsters were mildly anesthetized with ether contained in a nose-and-mouth cup and injected intraperitoneally with 5 ml of 3% aged thioglycolate medium (Sigma) in PBS. Three days after injection, hamsters were euthanized by inhalation of CO₂. Twenty milliliters of cold Hanks balanced salt solution was injected intraperitoneally, the peritoneal cavity was massaged for 1 min, and the peritoneal exudate cells were recovered by aspiration with a syringe. The peritoneal exudate suspensions from several animals were pooled and centrifuged at 500 × g for 10 min at 4°C. The supernatants were decanted, and the cells were resuspended in DMEM supplemented with 10% heat-inactivated (56°C for 30 min) bovine calf serum (HyClone) and 5 × 10⁻⁵ M 2-mercaptoethanol (Sigma). Following resuspension, the cells were poured over 100- by 22-mm polystyrene tissue culture dishes (Corning Glass Works) and incubated at 37°C in an atmosphere of 7.5% CO₂ for 4 h. After incubation, the tissue culture dishes were gently rinsed twice with 10 ml of warm Hanks balanced salt solution to remove nonadherent cells; 5 ml of cold, nonenzymatic cell dissociation solution (Sigma) was then added to the dish, which was incubated at 4°C for 30 min. Macrophages were subsequently removed by vigorously tapping and gently scraping the inside of the tissue culture dish with a sterile rubber policeman. Recovered macrophage suspensions were pooled and centrifuged at 500 × g for 10 min at 4°C. After centrifugation, the supernatant was decanted and the macrophages were resuspended in cold PBS. Macrophage viability was determined by trypan blue exclusion. Giemsa stained smears of the isolated cells showed a homogeneous population of macrophages with no other types of leukocytes visible.

Isolation and enrichment of T lymphocytes with MAb 14-4-4s was performed by procedures described previously (25, 26, 49, 50). Briefly, T lymphocytes were isolated from the inguinal lymph nodes of hamsters 7, 14, or 21 days after vaccination with formalin-inactivated B. burgdorferi 297 and from nonvaccinated hamsters. 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 DMEM containing 10% heat-inactivated bovine calf serum and 5 × 10⁻⁵ M 2-mercaptoethanol. The cells were then pelleted by centrifugation (500 × g, 10 min, 4°C), the supernatant was decanted, and the cells were resuspended in PBS at a concentration of 2 × 10⁷ cells per ml. MAb 14-4-4s in DMEM was added to the cell suspensions at a final dilution of 1:10 and incubated for 30 min at 4°C with periodic mixing. After incubation, cells were washed twice by centrifugation (500 × g, 10 min, 4°C) and resuspended in DMEM. Cell suspensions were then poured over 100- by 20-mm tissue culture dishes coated with 100 μg of unconjugated goat anti-mouse immunoglobulin in coupling buffer and incubated for 60 min at 4°C. Nonadherent cells were collected by gently rinsing the tissue culture dishes twice with 10 ml of cold DMEM. The cell suspensions from several tissue cultures dishes were aspirated, pooled, and centrifuged at 500 × g for 10 min at 4°C. The supernatant was decanted; the pellet was resuspended in DMEM, poured over another set of immunoglobulin-coated plates, and incubated for 60 min at 4°C. This process was repeated three times. Following the last panning cycle, nonadherent T lymphocytes were washed twice by centrifugation with PBS (500 × g, 10 min, 4°C) and resuspended in PBS. Cell viability was determined by trypan blue (Sigma) exclusion. Cells obtained using this method were shown to contain >95% T lymphocytes by flow cytometry (11, 23).

Cells remaining on the goat anti-mouse immunoglobulin-coated tissue culture dishes were rinsed twice with 10 ml of cold DMEM to remove any residual nonadherent cells. Five milliliters of cold, nonenzymatic cell dissociation solution was added to the dish, which was incubated at 4°C for 30 min. B lymphocytes were then removed by vigorously tapping and gently scraping the inside of the tissue culture dish with a sterile rubber policeman. Recovered B lymphocytes were pooled and centrifuged at 500 × g for 10 min at 4°C. After centrifugation, the supernatant was decanted and the B lymphocytes were resuspended in cold PBS. B-lymphocyte viability was determined by trypan blue exclusion. Cells obtained by this method were shown to contain 96 to 99% B lymphocytes by flow cytometry.

One-hundred-microliter samples containing 10⁶ lymph node cells were stained before and after panning with MAb 14-4-4s for the presence of B lymphocytes or CD4⁺ T lymphocytes. Cell preparations were stained 1:100 for 15 min at 4°C with a phycoerythrin-conjugated goat anti-hamster immunoglobulin, specific for both heavy and light chains (Boehringer Mannheim Biochemicals). CD4⁺ T lymphocytes were stained with a phycoerythrin-conjugated rat anti-mouse CD4 (L3T4) antibody (1:100; Boehringer Mannheim Biochemicals) for 15 min at 4°C. Samples were then washed twice with PBS by centrifugation, fixed with 1% paraformaldehyde (Sigma), and kept in the dark until analyzed by flow cytometry. Controls used included phycoerythrin-conjugated rat and goat antibodies and unstained T- and B-cell preparations. All samples were analyzed by using a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, Calif.). Cells were detected by forward scatter, side scatter, and phycoerythrin fluorescence. Five thousand events were acquired for each sample and were analyzed by histogram profiles of phycoerythrin fluorescence by using LYSYS II software. Gates were drawn using control preparations of unstained samples or cells stained with the isotype control antibodies. The percentage of B lymphocytes present in the cellular suspensions was determined by the percent shift in the phycoerythrin fluorescence of the stained cells.

Macrophages (5 × 10⁶) and 2 × 10⁸ B. burgdorferi 297 organisms were cocultured with 5 × 10⁶ T and 5 × 10⁶ B cells from vaccinated or nonvaccinated hamsters in DMEM in sterile flat-bottom multiwell tissue culture plates (Becton Dickinson, Lincoln Park, N.J.) at 37°C with 7.5% CO₂ for 10 days. At days 4, 8, and 10 after cultivation, 0.5-ml samples of the supernatants were collected and centrifuged at 12,000 rpm for 10 min to remove spirochetes and other cellular debris. The supernatants were then stored at −70°C until used. In other experiments, 5 × 10⁶, 2 × 10⁶, 1 × 10⁶, or 5 × 10⁵ T cells from hamsters vaccinated for 7 or 21 days were added to the suspensions of macrophages and B. burgdorferi containing T and B cells from hamsters vaccinated for 14 days. Controls included naive macrophages and B. burgdorferi alone, naive macrophages and B. burgdorferi with naive or immune T cells, naive macrophages and B. burgdorferi with naive or immune B cells, naive or immune B or T cells alone, and naive macrophages with naive or immune T and B cells.

B. burgdorferi 297 spirochetes were grown in 1 liter of BSK to log phase, pelleted by centrifugation (10,000 × 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 (12,000 × g, 10°C, 15 min) and resuspended in PBS. The borrelial protein content was measured by using a bicinchoninic acid assay (Sigma), and the spirochetes were suspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. One hundred twenty micrograms of B. burgdorferi 297 lysate was loaded on a preparative 12% acrylamide gel, and the proteins were resolved by electrophoresis at 18 mA for 5.5 h. The proteins were transferred onto a nitrocellulose membrane for 1 h at 15 V, using a semidry blotting apparatus (Bio-Rad, Hercules, Calif.). The nitrocellulose membrane was incubated for 2 h in 3% milk dissolved in Tris-buffered saline with 0.05% Tween 20 (TBS-T; pH 7.4) to block nonspecific reactivity and then washed 2 times each with TBS-T and double-distilled H₂O. The membrane was then incubated 1 h with a 1:5 dilution of the supernatant (diluted in TBS-T) collected from the suspension of macrophages and B. burgdorferi containing T and B cells from hamsters vaccinated for 14 days. Subsequently, the membrane was washed two times with TBS-T and incubated 1 h with a 1:1,000 dilution of an alkaline phosphatase-labeled goat anti-hamster immunoglobulin G (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) in TBS-T. This was followed by three washes with TBS-T. Antibody binding was detected by the addition of bromochloroindolyl phosphate-nitroblue tetrazolium for 3 min.

OspA was purified as previously described (28). 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 PBS, and lysed by sonication. Lysed organisms were mixed with Triton X-100, diluted with PBS, and centrifuged again to remove insoluble materials. The supernatant was mixed with a slurry of glutathione-Sepharose beads (Pharmacia) 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 SDS-PAGE and Western immunoblotting.

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. Swollen beads were centrifuged (13 min, 1,500 rpm), 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 10 times. The loss of OspA antibodies was confirmed by Western immunoblotting, and the treated supernatants were then tested for borreliacidal activity by using the borreliacidal antibody assay.

We next determined that T cells obtained from hamsters 7 or 21 days after vaccination could inhibit the in vitro production of borreliacidal antibody (Table ​(Table2).2). When suspensions of macrophages with B. burgdorferi containing T and B cells from hamsters 14 days after vaccination were coincubated with 5 × 10⁶, 2 × 10⁶, 1 × 10⁶, or 5 × 10⁵ T cells from hamsters 7 or 21 days after vaccination, little borreliacidal activity (titer of less than 20) was detected in the supernatants of the suspensions. By contrast, considerable borreliacidal antibody (titer of 1,280) was produced in cultures containing macrophages and B. burgdorferi coincubated with T and B cells from hamsters 14 days after vaccination. When these experiments were repeated thrice, T cells obtained from hamsters 7 or 21 days after vaccination inhibited the production of borreliacidal antibody when cocultured with macrophages and T and B cells obtained from hamsters 14 days after vaccination.