Monoclonal antibodies (mAbs) against BBA64, DbpA, and the anti-Bartonella protein were obtained from Barbara J. B. Johnson (CDC, Fort Collins, CO, USA). Anti-BBA64 and -DbpA were generated by recombinant protein immunization, and the anti-Bartonella mAb was generated by whole cell lysate immunization utilizing standard procedures for generating hybridomas in mice (Mbow et al., 2002). The anti-Bartonella mAb was reactive to a 70-kDa band on immunoblot against a rodent-derived Bartonella isolate. Anti-Rev and -OspC (B5) mAbs were generated by tick-bite inoculation of mice and have been described previously (Gilmore and Mbow, 1998; Mbow et al., 1999, 2002). Anti-OspA mAb H5332 was provided by Alan Barbour, UC-Irvine.

Immunofluorescent staining of cultured B. burgdorferi was performed as follows. B. burgdorferi low passage, infectious, clonal strain B31-A3 (Elias et al., 2002) was grown in complete Barbour-Stoenner-Kelly (BSK-II) medium at 34°C in capped tubes. Cultures were grown to mid-to-late logarithmic stage (approx. 5 × 10⁷–1 × 10⁸ organisms/ml), and 2 × 10⁶ bacteria were spun onto Cytospin microscope slides using a Shandon Cytospin 4 (Thermo Electron Corporation, Waltham, MA, USA). After centrifugation, slides were air dried, incubated in blocking solution (2% bovine serum albumin in phosphate buffered saline pH 7.5 [BSA–PBS]) for 1 h at room temperature (rt), and then incubated with the specific mAb (1:75 dilution) for 1 h Slides were washed (3× with PBS) and stained with goat anti-mouse IgG Alexafluor 594 (Molecular Probes, Eugene, OR, USA) at a 1:75 dilution for 45 m. Slides were washed (3× with PBS) and probed with FITC-conjugated goat anti-Borrelia burgdorferi IgG (KPL, Gaithersburg, MD, USA) at a 1:75 dilution for 45 m. Slides were washed (3× with PBS), air dried, and overlaid with Prolong AntiFade (Molecular Probes) and coverslips prior to imaging by epifluorescent microscopy.

Immunoblotting was performed as follows. B. burgdorferi were collected from culture and washed in PBS prior to gel fractionation. Cell lysates were denatured by boiling for 10 min in Laemmli buffer containing 5% 2-mercaptoethanol and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 10–20% gradient gel followed by transfer to nitrocellulose filters according to standard procedures. Immunoblots were incubated with the appropriate mAb (1:5000 dilution) for 1 h, washed with PBS (3× at 5 min), followed by incubation with alkaline phosphatase conjugated goat anti-mouse IgG (1:2000) for 1 h. After PBS wash, the blot was developed colorimetrically with NBT (nitro-blue tetrazolium chloride) and BCIP (5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt) substrate.

Human umbilical vein endothelial cells (HUVEC) and human neuroglial cells (H4) were obtained from American Type Culture Collection (Manassas, VA, USA; ATCC catalog numbers CRL-1730 and HTB-178 respectively). HUVECs were grown in F12-K medium supplemented with 0.1 mg/ml heparin, 0.03 mg/ml endothelial cell growth supplement, and 10% fetal bovine serum. The H4 cells were grown in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% fetal bovine serum. Both cell lines were grown at 37°C in the presence of 5% CO₂ in a humidified environment. Cells were harvested from confluent monolayers with 0.05% trypsin – 10 mM EDTA and enumerated on a hemocytometer cell counter.

HUVEC and H4 cells were seeded at a concentration of 3 × 10⁵ cells/chamber on Lab-Tek II CC chamber slide system (Nalge Nunc International, Rochester, NY, USA), and allowed to adhere overnight. Prior to addition to cells, individual B. burgdorferi aliquots, at a multiplicity of infection (MOI) of 40 (1.2 × 10⁷), were preincubated (35°C shaking at 150 rpm for 30 m) with each anti-B. burgdorferi mAb or control in 0.5 ml volume. Following incubation with the appropriate antibody, B. burgdorferi were added to each cell well. The antibody concentration used was pre-determined empirically by a series of 10-fold dilutions for each mAb to ascertain the highest dilution of antibody that produced significant reduction in cell binding. Anti-BBA64, -DbpA, and -OspA were used at 1:500, 1:5000, and 1:5000 respectively. The coincubation experiments were performed in duplicate using these dilutions. Additionally, three control B. burgdorferi pre-cell incubation treatments were tested: (i) mAb suspension buffer only (50% glycerol/1× PBS); (ii) a non-Borrelia-specific mAb (anti-Bartonella antibody); and (iii) no antibody (with 1× PBS added to volume).

Following overnight coincubation of treated B. burgdorferi with human cells, slide chambers were gently washed 3× with PBS to remove non-adherent bacteria. Cells with associated bacteria were fixed on the slide chamber with 4% paraformaldehyde for 45 m at rt. Fixed cells were washed followed by blocking with 2% BSA-PBS for 1 h. Mammalian cells were stained with Alexa 594 Phalloidin (1:200 dilution; Molecular Probes) for 10 m at rt. Samples were washed, then cells were permeabilized with 2% BSA-PBS supplemented with 0.5% Triton X-100 for 45 m at rt. B. burgdorferi were stained with FITC-conjugated goat anti-B. burgdorferi (1:75 dilution) for 1 h.

Samples were visualized on a LSM 5 Pascal confocal microscope (Carl Zeiss, USA). Micrograph images were acquired at 100× and 250× magnification. B. burgdorferi associated with cells were enumerated microscopically. Counts were made for (1) total human cells/field, and (2) Borrelia associated with cells/field, with 30 individual fields observed and counted from each slide. From the sum of the 30 fields, the average number of B. burgdorferi associated per cell was determined. The percent reduction in cell attachment was calculated by the difference between the antibody-treated B. burgdorferi and the control treatment. Assays were performed in duplicate. Comparisons of the data were performed using a two-sample t-test. Differences were considered significant if p-value was ≤0.05.

HUVEC or H4 cells were grown to confluence in a 150 cm² cell culture flask, trypsinized, and enumerated, from which 2 × 10⁵ cells were seeded in six well cell culture plates and were allowed to adhere for 17 h at 37°C with 5% CO₂. Log-phase B. burgdorferi cultures were centrifuged for 10 m at 9300 × g, with aliquots resuspended in F-12K or DMEM cell culture media and added to the HUVECs, H4 cells, or media alone at a MOI of 40 (7.8 × 10⁷ bacteria). Plates were incubated at 34°C with 5% CO₂. Cell wells were inoculated concurrently in triplicate and harvested for RNA isolation at 2-, 4-, 8-, 24-, and 48-h post-inoculation. At the appropriate time, cell culture media was removed, and the plate was washed 3× with 5 ml PBS to remove non-adherent bacteria. Cells with adherent bacteria were removed with cell scrapers in 300 μl RNAlater (Ambion, Austin, TX, USA). Media from the cell-free Borrelia wells were centrifuged at 9300 × g for 5 min, washed 2× with 5 ml PBS, and resuspended in 300 μl RNAlater. RNAlater samples were stored overnight at 4°C and then at −20°C until the RNA was purified. Prior to RNA extraction, the samples were centrifuged at 16,000 × g for 10 m, the RNAlater was decanted, followed by total RNA extraction using RNAqueous RNA isolation kit (Ambion). Contaminating DNA was removed using the TURBO DNA-free kit (Ambion). RNA samples were tested for contaminating DNA by PCR prior to cDNA synthesis. Total RNA was quantified by spectrophotometry and RNA integrity was analyzed using an Agilent 2100 Bioanalyzer with Agilent RNA Pico Reagents (Agilent Technologies, Boulder CO).

Purified total RNA (100 ng) isolated from the B. burgdorferi-associated cells was used to synthesize cDNA using SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and random decamers. Real-time PCR was performed with TaqMan Universal PCR Master Mix (Applied Biosystems) using flaB, bba64, dbpA, and ospA TaqMan probe and primers described previously (Livengood et al., 2008). Real-time PCR reactions, performed in triplicate per sample, contained 1 μl cDNA, 1 μM 5′ primer, 1 μM 3′ primer, 0.1 μM probe, and 1× TaqMan Universal PCR Master mixture in a total volume of 12.5 μl. Amplification parameters consisted of 1 cycle at 95°C for 10 m and 50 cycles of 95°C for 30 s and 60°C for 1 m, with data collection after each cycle in a BioRad iCycler (BioRad, Hercules, CA, USA). The iCycler software determined crossing threshold (C_T) values. B. burgdorferi gene expression during coincubation with cells was determined relative to borrelial gene expression in cell-free tissue culture media using the 2^−ΔΔCT method (Livak and Schmittgen, 2001), and was normalized against the constitutively expressed flaB gene.

Cultured B. burgdorferi was assessed for the production of OspA, DbpA, BBA64, OspC, and RevA prior to antibody treatment and coincubation with tissue culture cells. Analysis of cultured organisms by IFA demonstrated in vitro production of the individual proteins (Figure 1A). Immunoblot analysis of cultured borrelial lysates also showed protein production (Figure 1B).

Borrelia burgdorferi were preincubated with the mAbs for 30 m prior to placement on HUVEC and H4 cells. Following the antibody incubation, a sample of the spirochetes were visualized under darkfield microscopy to determine viability. B. burgdorferi treated with each mAb were motile indicating that the antibody treatment was not borreliacidal.

An experiment was performed to find the least concentrated mAb solution that produced significant differences (p < 0.05) in cell binding from the no antibody control treatment. We considered that higher dilutions of mAb would provide less chance for steric hindrance that could lead to non-specific blocking. The optimal dilutions were determined to be 5 × 10⁻³ for the anti-OspA and -DbpA mAbs, and 5 × 10⁻² for the anti-BBA64 antibody. Anti-RevA and anti-OspC mAbs, at all dilutions tested, demonstrated no significant inhibition of binding to HUVEC or H4 cells when compared to the no antibody control (data not shown).

Additionally, there were no significant differences between numbers of B. burgdorferi attached to host cells following each of the control treatments, i.e., Borrelia preincubated with antibody buffer only, Borrelia preincubated with non-specific antibody, and Borrelia preincubated with only PBS (data not shown). Therefore, the control data reported (as “without” or “no antibody”) in the text reflect counts obtained with the non-specific anti-Bartonella antibody pretreatment control.

Table 1 shows associated B. burgdorferi/100 cells with and without specific mAb treatment and the calculated reduction in B. burgdorferi cellular attachment. B. burgdorferi treated with anti-OspA, -DbpA, and -BBA64 antibodies demonstrated significant decreases in HUVEC and H4 cell binding when compared to Borrelia treated with the control. (As mentioned above, treatment with anti-OspC and anti-RevA had no effect on binding). The reduction in attachment between antibody treatments in separate experiments ranged from 30.6 to 55.2% for both HUVEC and H4 cells. The average B. burgdorferi/100 cells counted from 30 fields in each experiment were significantly different between antibody-treated Borrelia and controls (p < 0.001). An additive effect in attachment reduction when all three antibodies were combined was not observed. Figure 2 shows images of representative fields of B. burgdorferi binding to HUVECs and H4 cells respectively, with and without antibody treatment, allowing a visual inspection of the diminished attachment.

Antibodies directed against OspA, DbpA, and BBA64 attenuated the ability of B. burgdorferi to bind to host cells in vitro suggesting that these surface proteins may play a role in establishing infection. Therefore, we hypothesized that the genes encoding these proteins may be upregulated during cellular interaction. We employed qRT-PCR to monitor B. burgdorferi gene expression temporally throughout the incubation period with the tissue culture cells. We found that in both H4 and HUVEC interactions, dbpA, bba64, and ospA expression were generally elevated compared to B. burgdorferi incubation in cell-free environment.

dbpA expression was the highest of the three genes in both HUVEC and H4 incubations. At 2-h post-inoculation, gene expression was approximately 150-fold higher in HUVEC than in cell-free media (Figure 3A). dbpA expression continued to be elevated at approximately 20-fold at 4-, 8-, 24-, and 48-h post-inoculation in HUVECs compared to the control. dbpA also was upregulated in H4 cells throughout the incubation period, with a peak around 60-fold at 48 h (Figure 3A).

bba64 expression measured at 2- to 8-h post-inoculation with HUVECs was between approximately five- to sevenfold higher than in cell-free media (Figure 3B). At 24-h post-inoculation, expression was detected but was not significantly increased compared to B. burgdorferi in cell-free media. However, by 48 h, expression had decreased below the cell-free level. In H4 cells, bba64 expression was detected early, but not at significantly higher levels than in cell-free organisms. However, expression increased several-fold by 8-h post-inoculation, and remained elevated compared with the cell-free control at 48-h post-inoculation (Figure 3B).

ospA expression was detectable and slightly elevated in HUVECs in early time points post-inoculation when compared to expression in cell-free media. At 2 h into the incubation period, an approximate threefold increase in expression was observed. At 4-h post-inoculation, the expression level had decreased from 2 h, although a significant increase from cell-free expression was detected at 8 h. Later in the incubation, at 24 and 48 h, ospA expression had decreased to levels below that in cell-free media. A similar pattern was observed in H4 cells where there was no significant difference between expression in cell-free conditions, except at 8 h incubation whereby there was an approximate twofold increase (Figure 3C).