Inhibition of Borrelia burgdorferi-Tick Interactions In Vivo by Outer Surface Protein A Antibody

B. burgdorferi and I. scapularis

A low-passage clonal isolate of B. burgdorferi N40 that is infectious to mice was used throughout (27). Spirochetes were cultivated in Barbour-Stoenner-Kelly (BSK) II medium at 33°C. Adult female I. scapularis ticks were collected in Connecticut. The egg mass was laid in the laboratory. Hatched larvae were allowed to feed on uninfected C3H mice to produce pathogen-free nymphs. All tick rearing was performed in an incubator at 26°C in 85% relative humidity and a 12-h light-dark photoperiod cycle.

Antisera and mAb

B. burgdorferi N40 OspA mAb C3.78, which can protect mice from spirochete infection, was purified from a C3.78-producing hybridoma culture supernatant (16). Supernatant (40 ml) was passed over a 0.5-ml protein A column (Bio-Rad Laboratories, Hercules, CA) that then was washed twice with 20 ml of PBS, pH 7.4. The bound IgG was eluted in 1 ml of 0.1 M glycine, pH 3.0. The Ab then was concentrated and desalted with a spin column (Amicon, Beverly, MA), and the protein concentration was determined by using a Bio-Rad protein assay kit (Bio-Rad Laboratories) with BSA (American Bioanalyticals, Natick, MA) as a standard. The concentration of purified C3.78 IgG was 11.25 μg/ml.

Polyclonal rabbit antisera against recombinant OspA from B. afzelii (isolate ACA-1) and B. garinii (isolate ZQ-1) were supplied by SmithKline Beecham Biologicals (Philadelphia, PA; Ref. 22). Rabbit antisera against recombinant OspA-, OspF-, and OspE-related protein (Erp) T from B. burgdorferi N40 have been described previously by our group (16, 28, 29). OspF is encoded by a member of a multigene family (erps) that are differentially expressed throughout the B. burgdorferi life cycle (30). B. burgdorferi erpT is preferentially expressed in vivo, and antiserum directed against ErpT is not borreliacidal (28).

ELISA

Microtiter wells (ICN Biomedical, Costa Mesa, CA) were coated with 0.1 μg of recombinant OspA-N40 and incubated overnight at 4°C. Nonspecific sites then were blocked by PBS with 15% FBS. The wells were incubated with 100 μl of OspA antisera in PBS with 0.05% Tween 20 (PBS-T) at 37°C for 1 h. Abs specific for OspAs were detected with HRP-labeled goat anti-rabbit IgG (Sigma, St. Louis, MO) for 1 h at 37°C. After incubation with tetramethylbenzidine peroxidase substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) for 15 min, the absorbance at 450 nm was measured.

Immunoblot

Wild-type or mutant recombinant OspAs from B. burgdorferi N40 with selected amino acid mutations (designated M4, M5, and M6) in the major tick gut-OspA binding epitope (aa 229–247) were expressed and purified in the nonlipidated form without a fusion partner (21). The amino acid number and sequence in single-letter abbreviation in these mutant OspAs are M4 (K₂₃₁ to ΔA), M5 (VF_236–237 to ΔGS), M6 (TIT_242–244 to ΔANA; Ref. 21). Mutant OspAs together with wild-type OspA and an OspA (M3) with a mutation in another region of the protein (EVFK_100–103 to ΔAVFA) were probed with OspA mAb C3.78 by immunoblot. Two micrograms of recombinant OspA were electrophoresed in a 10% SDS-PAGE, transferred onto nitrocellulose membranes, and then incubated with mAb C3.78 (1/100 dilution). The signal was detected with a goat anti-mouse IgG-conjugated alkaline phosphatase (Sigma).

Ab characterization in vitro

In vivo infection and adherence studies

Pathogen-free NCr immunodeficient mice (NCr-SCID) from the National Institutes of Health (Bethesda, MD) were infected with B. burgdorferi N40 (10⁵ spirochetes/mouse, 3–5 animals/group) by intradermal injection into the back. After 4 wk, mAb C3.78 (2.5 μg) or selected polyclonal antisera were administered to groups of mice (100 μl i.p. and 100 μl s.c.). Twenty-four hours later, 10 I. scapularis nymphs were placed on each mouse. The animals again were treated with mAb or antisera on the next day. The nymphs were allowed to feed to repletion and detach from the mice, which usually occurred at ∼72 h. Guts from each group of nymphs were dissected under a microscope in PBS (20 μl/gut) and examined at 24, 48, 72, and 96 h after tick detachment. Five-microliter aliquots were examined for viable spirochetes under dark-field microscopy. Selected nymphs from each group also were reared in an incubator at 26°C with 85% relative humidity and a 12-h light-dark photoperiod regimen for ∼1 mo until they molted to adults.

Organs from nymphal ticks were prepared for microscopy as described previously (33) by dissection of gut diverticula in PBS (20 μl/gut). The lumen of each gut diverticulum was exposed by a vertical incision with a fine blade so that individual diverticula were separated from each other, and both ends were opened to facilitate outflow of blood. The organs were washed three times under a dissecting microscope until the cessation of visible flow of blood from open diverticula. The isolated organs were placed on sialylated glass slides (PGC Scientific) to enhance attachment, allowed to dry, and fixed with acetone for 5 min. Acetone-fixed slides were rinsed twice with PBS and incubated for 30 min with PBS-T and 5% normal goat serum at room temperature. Organs were incubated with an affinity-purified FITC-labeled goat anti-B. burgdorferi Ab (Kirkegaard & Perry Laboratories) at a dilution of 1/50 in PBS-T with 5% normal goat serum at room temperature for 1 h. The samples were counterstained with propidium iodide (50 μl of a 10-μg/ml solution) for 3 min at room temperature, washed three times with PBS-T, and mounted in glycerol for examination. The tissues were viewed with a Zeiss LSM 510 scanning laser confocal microscope equipped with an argon/krypton laser. The distribution of spirochetes in the gut was determined by scanning the entire organ from end to end and throughout its depth at each point.

Statistical analysis

Results are expressed as the mean ± SE. The significance of the difference between the mean values of the groups was evaluated by Student’s t test or repeated-measure ANOVA with the Fisher protected least significant difference test with Statview software (SAS Institute, Cary, NC).

Characterization of OspA Abs

The borreliacidal activities of OspA antisera and mAb were characterized so that the influence of these Abs on B. burgdorferi-I. scapularis interactions in vivo could be studied. C3.78 is an OspA mAb directed against B. burgdorferi N40 that can protect mice from B. burgdorferi N40 infection (16). We first determined that 0.4 μg/ml of mAb C3.78 was sufficient to kill the vast majority of B. burgdorferi N40 in vitro (Table I⇓). As expected, we then demonstrated that antisera against OspA from B. burgdorferi N40 (OspA-N40), but not antisera against OspA from B. afzelii ACA-1 (OspA-ACA-1) or B. garinii ZQ-1 (OspA-ZQ-1), were able to kill B. burgdorferi N40 (Table I⇓). Similar bactericidal activities of mAb C3.78 and OspA-N40 antisera were detected in our bactericidal assay when heat-inactivated serum was used (data not shown). Therefore, for the purpose of this study, we termed N40 Abs (mAb C3.78 and OspA-N40 antisera) as “borrelicidal” and non-N40 Abs (OspA-ACA-1 and OspA-ZQ-1) were termed as “nonborrelicidal.” Control antisera against OspF-N40 or ErpT-N40 (a B. burgdorferi gene expressed in vivo) did not have significant borreliacidal activity (Table I⇓). However, antisera against OspA-ACA-1 or OspA-ZQ-1 were able to kill B. afzelii and B. garinii at dilutions of 1/3000 and 1/7000, respectively (data not shown). The borreliacidal effect of the Abs studied was determined both visually, by loss of motility, and was confirmed after an extended period of time by incorporation of [³H]adenine into dividing spirochetes.

In contrast to their strain-specific killing capacity, all of the OspA Abs were able to bind to OspA-N40. Each of the OspA antisera strongly stained B. burgdorferi N40 spirochetes as shown by immunofluorescence microscopy (Fig. 1⇓A) and recognized recombinant OspA-N40 in ELISA to a dilution of at least 1/12,500 (Fig. 1⇓B). Therefore, OspA-ACA-1 or OspA-ZQ-1 antisera effectively bound to, but did not kill, B. burgdorferi N40. Moreover, in a standard agglutination assay (34), neither of these OspA antisera resulted in appreciable agglutination of N40 spirochetes (data not shown).

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FIGURE 1.

Recognition of B. burgdorferi N40 and OspA-N40 by diverse OspA Abs. A, Immunofluorescence of B. burgdorferi N40 stained with OspA-N40 (lane 1), OspA-ACA-1 (lane 2), OspA-ZQ-1 (lane 3), or without primary antisera (lane 4). Spirochetes were fixed in acetone and labeled as described in Materials and Methods. All antisera were used at a 1/100 dilution. B, OspA Ab levels by ELISA. Recombinant OspA-N40 was probed with antisera to OspA-N40, OspA-ACA-1, and OspA-ZQ-1 (at 1/100, 1/500, 1/2,500, 1/12,500, and 1/62,500 dilutions). Data shown are OD₄₅₀ (mean ± SE; n = 3).

OspA Abs inhibit association of spirochetes with the tick gut in vivo

We next examined whether B. burgdorferi N40 exposed to different OspA Abs could effectively colonize I. scapularis, particularly in cases where the Abs did not have borreliacidal activity. SCID mice were used in these studies because the animals cannot mount their own humoral response to B. burgdorferi during infection; thus, the effects of the administered Abs can be evaluated without the contribution of any acquired host response to the spirochete. Groups of three SCID mice were challenged with B. burgdorferi N40, and within 4 wk all the mice had developed visible swelling of the tibiotarsal joints (data not shown). Then OspA antisera (N40, ACA-1, or ZQ-1), mAb C3.78, control (OspF or ErpT) antisera, or PBS were administered to groups of animals. Twenty-four hours later, 10 uninfected ticks were allowed to feed to repletion on each mouse, and the engorged ticks were collected. Tick guts were dissected and the luminal contents examined for B. burgdorferi under dark-field microscopy. In ticks that fed on animals treated with OspA-ACA-1 or OspA-ZQ-1 antisera, control antisera, or PBS, viable B. burgdorferi were consistently detected (Fig. 2⇓). For example, control (PBS-treated) slides (5 μl of tick content out of a total volume of 20 μl of tick content) contained 50 ± 8 spirochetes/slide (or 200 spirochetes/tick). In contrast, spirochetes were not detected in guts of ticks that had fed on mice administered OspA-N40 antisera or mAb C3.78 (Fig. 2⇓).

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FIGURE 2.

Detection of B. burgdorferi within ticks after exposure to OspA Abs. Groups of from three to five B. burgdorferi-infected mice were administered selected antisera, mAb, or PBS before tick placement. OspA-ACA-1, OspA-ZQ-1, and OspA-N40 antisera and OspA mAb C3.78 were used. Control groups were treated with ErpT-N40 and OspF-N40 antisera or PBS. Three days after attachment, at least 10 engorged ticks from each group of mice were dissected and examined by dark-field microscope for motile, viable spirochetes. Fifty fields were examined under a ×43 objective, and the results are expressed relative to the B. burgdorferi within ticks that fed on control (PBS) mice (200 ± 32). Data represent the mean ± SE for three experiments. Spirochete numbers in control (PBS), ErpT-N40, OspF-0N40, OspA-ACA-1, and OspA-ZQ-1 groups were similar (ANOVA or Student’s t test, p > 0.5). Spirochete numbers in the OspA-N40 or mAb C3.78 groups were essentially zero (ANOVA or Student’s t test, p < 0.001).

To determine whether specific OspA antisera could interfere with the ability of B. burgdorferi to adhere to the tick gut, we examined Borrelia-tick tissue associations at 24, 48, 72, and 96 h after tick engorgement. The tick gut diverticula were dissected, washed to remove unbound luminal contents, and examined by double-labeled confocal microscopy. B. burgdorferi in the control groups (PBS and OspF and ErpT antisera) were found in close proximity to the lumen of the nymphal tick gut (Fig. 3⇓A and Table II⇓). In contrast, OspA-ACA-1 or OspA-ZQ-1 antisera-treated samples had fewer spirochetes associated with the gut (Fig. 3⇓A and Table II⇓). Gut diverticula were devoid of spirochetes in samples treated with OspA-N40 antisera or mAb C3.78 (Fig. 3⇓A and Table II⇓). We then examined the persistence of the spirochetes in I. scapularis by allowing some groups of ticks to molt to the adult stage. The effects of borreliacidal and nonborreliacidal Abs were maintained at the adult stage (Fig. 3⇓B).

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FIGURE 3.

Distribution of B. burgdorferi within the I. scapularis gut. A, Nymphal ticks were allowed to engorge for 72 h on B. burgdorferi-infected mice that had been treated with selected antisera: OspA-N40, OspA mAb C3.78, OspA-ACA-1, OspA-ZQ-1, or controls (OspF, ErpT, and PBS). The spirochetes (arrows) were stained with an FITC-labeled goat anti-B. burgdorferi Ab (green), and the nuclei of the gut epithelial cells were stained with propidium iodide. Images from both fluorescent chambers were recorded at ×630 magnification and are presented as a single image for clarity. B, Spirochetes (arrows) in adult ticks. Selected engorged nymphal ticks (A) were allowed to molt to the adult stage and then were then examined for B. burgdorferi in a similar fashion. The representative images show that spirochetes did not flourish in the guts of tick that had been exposed as nymphs either to borreliacidal or nonborreliacidal OspA Abs. Images were recorded at ×400.

Bactericidal OspA mAb C3.78 binds to OspAs with mutations in major gut binding epitope

To further differentiate the regions of OspA that are important in tick gut binding from the epitopes that bind to borreliacidal OspA (24, 25), we examined whether mAb C3.78, which we know binds to the borreliacidal epitope, could bind to recombinant OspAs with mutations in the tick gut binding domain, aa 229–247. Protective OspA mAbs such as C3.78 have been shown to bind to a conformational epitope in the carboxyl terminus of OspA and are sufficient to protect mice against B. burgdorferi infection (16, 25). Our previous work demonstrated that OspA aa 229–247 contain an epitope that is important in OspA-gut binding and that mutations in this region greatly reduced OspA-gut interaction (21). Therefore, we expressed several recombinant OspAs with such mutations in this area, including M4 (K₂₃₁ to ΔA), M5 (VF_236–237 to ΔGS), and M6 (TIT_242–244 to ΔANA) (21) and determined whether mAb C3.78 could bind to these OspAs. M5 and M6 have been shown previously to have reduced binding to the tick gut when compared with wild-type OspA (21). Wild-type OspA and a mutant OspA (M3) with an alteration in a different region of the protein (EVFK_100–103 to ΔAVFA) that does not appear to be involved in spirochete-tick gut binding, served as controls. In a standard immunoblot, mAb C3.78 readily bound to wild-type or all the mutant OspAs (M4, M5, and M6) with alterations in the OspA-tick gut binding region (data not shown), suggesting that the important gut binding domain is distinct from the borreliacidal domain.