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*
Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520; and
SmithKline Beecham Biologicals, Rixensart, Belgium
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In North America, B. burgdorferi are maintained in a complex enzootic cycle involving wild rodents and I. scapularis ticks (4). Experimental laboratory models with mice have been developed to study the elaborate B. burgdorferi life cycle and provide insight into the pathogenesis of Lyme disease (5, 6). Mice infected with B. burgdorferi by intradermal experimental syringe challenge or via tick transmission develop an initial cutaneous infection that remains localized to the skin for up to 1 wk (7). Spirochetes then disseminate to distant tissues, and arthritis and carditis become apparent at 2–3 wk (7). Disease resolves in immunocompetent mice over several weeks, but the mice remain persistently infected (7); in SCID mice, disease and infection continue indefinitely (5). The spirochete undergoes major alterations in antigenic composition as it migrates from the arthropod vector to the mammalian host, and these changes may aid in immune evasion or pathogenesis (4). As one example, B. burgdorferi express outer surface protein (Osp)3 A in the tick gut, and then down-regulate OspA and up-regulate OspC during engorgement and early infection within mammals (8, 9, 10, 11). During mammalian infection, decorin binding protein A and BBK32 (a fibronectin binding protein) appear to have a role in adherence of B. burgdorferi to the extracellular matrix, and several other Ags are preferentially produced by the spirochete in the vertebrate (12, 13). In addition, B. burgdorferi undergo antigenic variation involving specific loci such as the vls gene cluster, and this may also contribute to immune evasion and spirochete persistence (14).
Recombinant OspA elicits protective immunity in mice and humans and serves as a Lyme disease vaccine (15, 16). OspA is a transmission-blocking vaccine that affects spirochetes in the arthropod (8). OspA Abs enter the tick during the feeding process and destroy spirochetes in the gut—before B. burgdorferi have had the opportunity to down-regulate OspA (8, 17). During murine infection, OspA generally is not expressed (9, 18, 19). Then, when larval ticks engorge on B. burgdorferi-infected mice, the spirochetes once again up-regulate OspA during migration to the tick (19). Indeed, the postinfection administration of OspA Abs to B. burgdorferi-infected mice has little or no effect on the murine infection, but such Abs are able to interfere with the establishment of B. burgdorferi infection within the feeding tick (17, 20). The temporal and tissue-specific expression of OspA in the gut of the tick suggests that OspA may have a function in the vector, and we have recently shown that OspA mediates the attachment of B. burgdorferi to I. scapularis in vitro (21). In our study, recombinant nonlipidated OspA from B. burgdorferi bound to the I. scapularis gut tissue and this attachment was partially abrogated by site-directed mutagenesis of OspA aa 229–247 (21).
Here we examine spirochete-tick adherence in vivo by using the differential protective capacity of OspA antisera from selected Borrelia genospecies. It has been shown that antigenic diversity in OspA among the B. burgdorferi sensu lato genospecies can result in a lack of cross-protection by OspA antisera (22, 23). For instance, OspA antisera against a B. burgdorferi sensu stricto isolate may not necessarily protect mice against challenge with a B. afzelii or B. garinii isolate and vice versa (22, 23). Protective OspA Abs prominently bind to epitopes within the carboxyl terminal half of OspA, and antigenic diversity within this region of OspA influences the ability of protective OspA Abs to kill B. burgdorferi (24, 25). For example, active immunization of mice with OspA from B. burgdorferi N40 or passive immunization with OspA-N40 mAb C3.78 is protective against B. burgdorferi N40 but not B. burgdorferi 25015 (26). This lack of cross protection is attributable to several amino acid differences in the carboxyl terminus of OspA between B. burgdorferi 25015 and B. burgdorferi N40 that alter the ability of protective B. burgdorferi OspA N40 Abs to bind to B. burgdorferi 25015 (26). Therefore, we now have used OspA mAb C3.78 and OspA antisera from three B. burgdorferi sensu lato genospecies, B. burgdorferi sensu stricto (isolate N40), B. afzelii (isolate ACA-1), and B. garinii (isolate ZQ-1), to differentiate the roles of in vitro characterized borreliacidal and nonborreliacidal OspA Abs in spirochete-vector interactions and to investigate the importance of OspA in B. burgdorferi-tick adherence in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 
A), M5
(VF236–237 to GS), M6
(TIT242–244 to ANA; Ref. 21).
Mutant OspAs together with wild-type OspA and an OspA (M3) with a
mutation in another region of the protein
(EVFK100–103 to AVFA) 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
Immunofluorescence. For staining of cultured spirochetes, B. burgdorferi N40 (107 spirochetes/ml) were suspended in PBS and 10-µl aliquots were placed on sialylated glass slides (PGC Scientific, Gaithersburg, MD) and allowed to air dry. Each slide then was fixed with acetone for 5 min, washed three times with PBS, and incubated in PBS-T with 5% FCS (blocking buffer) for 30 min at room temperature. The samples were then labeled with OspA antisera at a dilution of 1/100 in blocking buffer, washed, and subsequently incubated for 1 h with FITC-labeled goat anti-rabbit IgG (Sigma). The samples were washed three times with PBS-T, mounted in glycerol, and examined with a Zeiss Axioskop fluorescence microscope (Carl Zeiss, Thornwood, NY).
[3H]Adenine incorporation and bactericidal assay. Abs were tested for their bactericidal activity against B. burgdorferi N40 both by dark-field microscopy and by monitoring the uptake of [3H]adenine (31, 32). Spirochetes (5 x 106/ml) were incubated in rabbit sera (Sigma) with selected OspA or control Abs (1/25 dilution) for 24 h at 37°C, after which the percentage of viable spirochetes was determined by dark-field microscopy. Fifty-microliter aliquots were plated in 96-well plates with 200 µl of BSK/20% rabbit serum/[3H]adenine (5 µCi/well; ICN Biomedical), for 48 h at 33°C. Incorporation of [3H]adenine by dividing spirochetes increases in a linear fashion over 48 h with a spirochete density of 1 x 104 to 1 x 106/ml (31). In addition, for visual detection of spirochete viability, aliquots (10 µl) were assessed for spirochete motility and refractivity in five random fields by dark-field microscopy. Spirochetes were considered killed when complete loss of motility and refractivity was observed. Control samples were treated with PBS or control nonbactericidal B. burgdorferi N40 Abs such as OspF or ErpT antisera. In addition, 100-µl samples were removed from each tube, inoculated into 5 ml of BSK medium, and incubated at 33°C for 5 days. The B. burgdorferi then were counted, and these results were compared with the initial viability by dark-field microscopy in a double-blind manner. Bactericidal assays also were performed with different concentrations of OspA mAb C3.78.
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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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A) and recognized recombinant
OspA-N40 in ELISA to a dilution of at least 1/12,500 (Fig. 1B). 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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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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A and Table II). In contrast, OspA-ACA-1 or OspA-ZQ-1
antisera-treated samples had fewer spirochetes associated with the gut
(Fig. 3A and Table II). Gut diverticula were devoid of
spirochetes in samples treated with OspA-N40 antisera or mAb C3.78
(Fig. 3A 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. 3B).
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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 A), M5
(VF236–237 to GS), and M6
(TIT242–244 to ANA) (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 (EVFK100–103 to AVFA) 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Antisera against OspA-ACA-1 and OspA-ZQ-1 were borreliacidal in vitro
against B. afzelii ACA-1 and B. garinii ZQ-1,
respectively (data not shown), but unable to kill B.
burgdorferi N40 (Table I
). As expected, OspA-N40 antisera were
borreliacidal against B. burgdorferi N40. These results are
consistent with prior reports on the genospecies-specific borreliacidal
activity of OspA antisera, both in vitro and by in vivo passive
immunization studies (22, 26). In general, diversity in
the carboxyl terminus of OspA among B. burgdorferi sensu
lato contributes to the inability of specific borreliacidal OspA Abs to
bind diverse OspAs and the lack of cross-protective immunity. The
selective borreliacidal capacities of these OspA antisera provide a
framework for investigation of in vivo effects of such antisera on the
interaction of B. burgdorferi N40 with I.
scapularis.
Our in vivo studies examine the ability of nonborreliacidal OspA antisera to prevent effective tick colonization by B. burgdorferi. To assess the effect of host OspA Abs on B. burgdorferi entry to ticks, Abs were administered to B. burgdorferi-infected mice. OspA Abs transferred after initiation of infection do not protect from infection with B. burgdorferi, in part because the spirochetes down-regulate ospA in the host (9, 18). The influence of OspA Abs becomes apparent when the spirochetes express OspA as they reenter the tick (8). We used SCID mice in this study so that acquired humoral or cellular immune responses to B. burgdorferi during the course of infection could not influence B. burgdorferi within the vector. Our in vivo experiments demonstrate that OspA antisera effectively prevent B. burgdorferi from associating with the I. scapularis gut, even those that do not kill spirochetes. Viable, motile B. burgdorferi were found in ticks exposed to OspA-ACA-1 or OspA-ZQ-1 antisera during engorgement, showing that these antisera did not hinder B. burgdorferi from entering the vector. However, the OspA-ACA-1 or OspA-ZQ-1 antisera diminished the ability of the spirochetes to associate with the gut of the engorged ticks. In addition, markedly fewer B. burgdorferi persisted through the molt from nymph to adult ticks, further demonstrating that these antisera interfered with the ability of B. burgdorferi to establish infection within the arthropod.
OspA-ACA-1 and OspA-ZQ-1 antisera may affect B. burgdorferi in several ways. Epitopes on OspA that bind borreliacidal Abs vs those that bind tick gut epithelium are likely to be different. Mapping studies have shown that protective OspA mAbs, such as LA-2 and C3.78 bind to conformational epitopes within the carboxyl terminus of OspA (25, 35). At least one of the epitopes that facilitates OspA-tick interactions is encoded within residues 229–247 (21) and we now have found that this region is not important for the binding of mAb C3.78, as these Abs bind to OspAs with mutations within residues 229–247. Nonborreliacidal OspA Abs may bind directly bind to the tick gut binding region, or alternatively, these Abs may bind to several epitopes of OspA on the B. burgdorferi surface and steric hindrance might then interfere with OspA binding to tick gut.
The mechanisms by which B. burgdorferi interact with the vector, and the influence of spirochete-tick interactions on the pathogenesis of infection remain to be fully explored. Certainly within the mammalian host, B. burgdorferi have different Ags that may serve similar functions. For example, B. burgdorferi decorin binding protein and fibronectin binding protein (BBK32) both facilitate the adherence of the spirochete to the extracellular matrix, albeit via different ligands (12, 13). Several B. burgdorferi ligands are probably involved in spirochete-tick gut interactions, and B. burgdorferi Ags that are preferentially expressed during the period of tick engorgement, such as OspC, BBK32, and BBK50 among others, may serve functions in the tick as well as the host. Ags differentially expressed during the blood meal help B. burgdorferi colonize the tick salivary glands (transiently) during the migration from the tick gut to the vertebrate host (33). Characterization of the I. scapularis ligands that interact with OspA and other B. burgdorferi Ags should increase our understanding of vector-B. burgdorferi interactions and suggest alternative protective strategies to interfere with pathogen transmission. Immunization of mice with the vector ligand for OspA or specific OspA peptides, could, theoretically at least, elicit Abs to prevent tick colonization by B. burgdorferi, and therefore interrupt the spirochete life cycle. Our current studies show that nonborreliacidal OspA Abs can block the adherence of B. burgdorferi to the tick gut and demonstrate the importance of OspA in spirochete-arthropod interactions in vivo.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Erol Fikrig, 608 Laboratory of Clinical Investigation, Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8031. E-mail address: erol.fikrig{at}yale.edu
3 Abbreviations used in this paper: Osp, outer surface protein; Erp, OspE-related protein.
Received for publication January 11, 2001. Accepted for publication April 10, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 S. Felek, R. Greene, and Y. Rikihisa Transcriptional Analysis of p30 Major Outer Membrane Protein Genes of Ehrlichia canis in Naturally Infected Ticks and Sequence Analysis of p30-10 of E. canis from Diverse Geographic Regions J. Clin. Microbiol., February 1, 2003; 41(2): 886 - 888. [Abstract] [Full Text] [PDF]
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F. T. Liang, F. K. Nelson, and E. Fikrig DNA Microarray Assessment of Putative Borrelia burgdorferi Lipoprotein Genes Infect. Immun., June 1, 2002; 70(6): 3300 - 3303. [Abstract] [Full Text] [PDF]
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A. F. Elias, P. E. Stewart, D. Grimm, M. J. Caimano, C. H. Eggers, K. Tilly, J. L. Bono, D. R. Akins, J. D. Radolf, T. G. Schwan, et al. Clonal Polymorphism of Borrelia burgdorferi Strain B31 MI: Implications for Mutagenesis in an Infectious Strain Background Infect. Immun., April 1, 2002; 70(4): 2139 - 2150. [Abstract] [Full Text] [PDF]
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