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One of the recently identified Borrelia burgdorferi immunogens, BBK07, is characterized for its expression in the spirochete infection cycle and evaluated for its potential use as a serodiagnostic marker for Lyme disease. We show that the BBK07 gene is expressed at extremely low levels in vitro and in ticks but is dramatically induced by spirochetes once introduced into the host and is highly expressed throughout mammalian infection. In contrast, the expression of BBK12, a paralog of BBK07 with 87% amino acid identity, although expressed in vitro, remained undetectable in vivo throughout murine infection and in ticks. BBK07 is localized in the outer membrane, and the amino-terminal domain of the antigen is exposed on the microbial surface. A truncated BBK07 protein representing the amino-terminal domain is able to effectively detect antibodies to B. burgdorferi, both in experimentally infected mice and in humans. Further characterization of the immunodominant antigens of B. burgdorferi, such as BBK07, could contribute to the development of novel serodiagnostic markers for detection of Lyme disease.
Since the identification of Borrelia burgdorferi as the causative agent of Lyme disease (LD) over 25 years ago, the number of reported cases of LD has increased steadily (4, 49). In some U.S. counties, the incidence is more than 500 cases per 100,000 individuals, and more than 20,000 cases in the United States are diagnosed each year (4). Difficulties in diagnosis have long complicated the treatment of LD, as the bite of an infected tick may go unnoticed by the patient, and the clinical manifestations of LD can significantly vary among diagnosed patients (47). Common symptoms, such as fever, malaise, and arthritis, can resemble those caused by other conditions, further complicating diagnosis. Antibiotic therapy is highly effective, especially if administered in the early stages of LD; however, serious complications can result from false diagnoses and inappropriate treatment (9, 17, 40, 50, 51). There is no commercially available vaccine for human LD, so the development of accurate, sensitive laboratory diagnostics is an important goal of LD research.
While many laboratory methods have been used to assess B. burgdorferi infection, direct detection of the bacterium is difficult, due to the low pathogen load in clinical samples (2, 24). Likewise, the extremely slow growth of B. burgdorferi, the high cost, and the labor-intensive procedure needed to culture this bacterium have limited the effectiveness of culture as a diagnostic tool (34, 46). PCR detection is possible (44), but not widely used for diagnosis, due primarily to low sensitivity in tissues, such as cerebrospinal fluid and blood (2). Instead, the primary means used to detect B. burgdorferi exposure is serodiagnosis (2). Immunodetection has been performed using whole-cell antigens, as well as recombinant proteins or peptide fragments (2). Whole-cell lysate provides a wide variety of antigens for detection, but is difficult to standardize due to variations in protein expression by culture growth phase (42). False-positive results are also an issue, as antibodies against other bacteria can cross-react with conserved B. burgdorferi proteins (5, 13, 21, 29).
To reduce cross-reactivity, several recombinant B. burgdorferi antigens and various fragments thereof have been evaluated as serodiagnostic markers for LD, including OspC (35), BmpA (45), VlsE (27), BBK32 (22), L25 (33), P37 (31), and DbpA (20). OspC is exposed on the B. burgdorferi surface, is produced during early infection, and is highly immunogenic (1, 13, 16, 35). A peptide fragment termed pepC10, containing a conserved immunogenic epitope, has been developed for serodiagnosis (32). BmpA, another surface-exposed protein, has also been studied for use in diagnosis (10, 45). Though immunogenic, significant protein sequence heterogeneity exists among B. burgdorferi isolates, constituting several serotypes, which limit the effectiveness of both OspC (14) and BmpA as serodiagnostic markers (43). VlsE is a dominant surface-exposed antigen of B. burgdorferi, a lipoprotein that undergoes antigenic variation by genetic recombination with silent vls cassettes (53). Expressed throughout late infection, VlsE and C6, a conserved peptide fragment of VlsE, have been evaluated as serodiagnostic markers for LD (15, 27, 28). These studies suggest that while the use of recombinant proteins can reduce cross-reactivity, thereby enhancing specificity, the use of only select antigens can reduce the sensitivity of the diagnostic test (30). A promising sensitivity in such tests was reported by Bacon et al. (3). Using kinetic enzyme-linked immunosorbent assay (ELISA), the combined detection of immunoglobulin M (IgM) against pepC10 and IgG against C6 provided 78% sensitivity in all tested samples. While assays using only recombinant antigens show promise, the identification and inclusion of more immunodominant antigens could improve the sensitivity of these tests.
In an effort to more completely catalogue antigens produced during infection, a recent study by Barbour et al. used synthetic protein arrays to test the immunogenicity of the majority of B. burgdorferi open reading frames (6). Though most open reading frames were not measurably immunogenic, they identified several novel antigens, including BBK07 and BBK12, putative lipoproteins from the linear plasmid lp36. These proteins are extremely similar in sequence, though BBK07 is slightly larger than BBK12 (250 and 232 amino acids, respectively) (18). The genes are members of paralogous family 59, and their products are 87% identical in their overlapping amino acid sequences. While both BBK07 and BBK12 were identified as immunogens and potential antigenic markers, a detailed characterization of their expression and the resulting immune response was not explored. We sought to characterize the expression, surface localization, and immune response against BBK07 to further evaluate its inclusion as a diagnostic marker to improve the accuracy and sensitivity of LD serodiagnosis.
Isolate A3, a clonal derivative of Borrelia burgdorferi B31 M1 and a generous gift from Patricia Rosa, was used throughout the study. Bacteria were grown in BSK-II media at 34°C. The Ixodes scapularis ticks used in this study were maintained in the laboratory as described previously (36). C3H/HeN mice were purchased from the National Cancer Institute. All animal procedures were performed in compliance with the guidelines and with the approval of the Institutional Animal Care and Use Committee. Unless otherwise stated, a single intradermal needle inoculation of 105 B. burgdorferi cells was used to infect each mouse. For generation of immunized serum, each mouse was injected with 100 μg of B. burgdorferi sonicate (five mice/group) intradermally. As injections with lysed spirochetes were performed without an adjuvant, all booster injections were performed at weekly intervals for 9 weeks. Polyclonal antibodies against truncated BBK07 protein representing the amino-terminal region of the mature protein (BBK07N) were obtained by injecting mice intradermally with recombinant protein (10 μg/animal) emulsified in complete Freund's adjuvant once, and twice in incomplete Freund's adjuvant (Sigma) at 10-day intervals. Serum samples were collected and pooled 10 days after final injection (12).
The recombinant protein fragment, BBK07N, containing the amino-terminal part of the protein and excluding the signal peptide, amino acids 18 to 142, was fused to an N-terminal six-histidine tag for purification on the pET302/NT-His Champion vector (Invitrogen). The following oligonucleotide primers were used to construct the expression vector: forward primer (5′ AAT CTA GAA TGT GGC ATG TAG ACA ATC CCA TTG 3′ [XbaI site italicized]) and reverse primer (5′ CCG GGA TCC ATT ACA TCT TTA GTC CAT TCT T 3′ [BamHI site italicized]). Purification was performed using commercial cell lysis buffer (FastBreak; Promega) and MagneHis nickel particles (Promega) according to the manufacturer's instructions. Recombinant VlsE was a generous gift from Fang Ting Liang from Louisiana State University. Recombinant BmpA (37), Lp6.6 (26), OspC (38), and BbCRASP-2 (12) were purified as detailed previously.
Infected ticks (three ticks per time point), as well as infected mouse skin, hearts, and tibiotarsal joints (three mice per time point) were homogenized using mortar and pestle under liquid nitrogen, and total RNA was extracted using TRIzol reagent (Invitrogen). RNA from log-phase, in vitro-grown B. burgdorferi (107 spirochetes/ml) was also isolated using TRIzol reagent (Invitrogen). The purified RNA was treated with DNase I (NEB) to reduce DNA contamination. One microgram of total RNA from each sample was used to synthesize cDNA by using AffinityScript first-strand cDNA synthesis (Stratagene). Quantitative reverse transcriptase PCR (qRT-PCR) analysis was performed on 50 μg of each cDNA by using iQ SYBR green supermix (Bio-Rad). To help protect against DNA contamination, cDNA was compared to an equal concentration of template RNA to measure the contribution of DNA to the final results. Standard curves for the flaB and BBK07 genes were generated using B. burgdorferi genomic DNA purified by the DNeasy blood and tissue kit (Qiagen). The primers used for quantitative PCR were as follows: 5′ TTC AAT CAG GTA ACG GCA CA, GAC GCT TGA GAC CCT GAA AG 3′ for flaB; 5′ CCT ATT TCA AGG GCG TGA GC, TAT GGC CAT TGC TGC ATT CT 3′ for the BBK07 gene; and 5′ GCT GAA AAT TCG GTA AGC GTT T, TAA GTT CGC TGC ATA CAC CTT CA 3′ for the BBK12 gene.
Proteinase K accessibility assays were performed as described previously (8), with the following modifications. B. burgdorferi cells (1 × 109) were gently washed three times in 1 ml of phosphate-buffered saline (PBS; pH 7.4) and collected by centrifugation at 4,000 × g for 5 min. Washed spirochetes were then gently resuspended in 100 μl of PBS and split into two equal 50-μl volumes. One aliquot received 10 μg of proteinase K (Sigma) in PBS, while the other aliquot received an equal volume of PBS without proteinase K. Both aliquots were incubated for 20 min at room temperature and then washed three times with 1 ml PBS with 1 mM phenylmethylsulfonylfluoride (Sigma) to stop proteinase K activity. After washing, the spirochetes were resuspended in PBS and used for immunoblot analysis.
Polyclonal BBK07N antisera were used for immunoblotting at a 1:2,000 dilution ratio in 5% skim milk. FlaB and OspA antisera were used as described previously (52). For ELISA, antigens (100 to 200 ng/well) were coated on PolySorp immunoplates (Nunc) in 50 mM carbonate-bicarbonate buffer (pH 9.6; Sigma). All other murine sera were diluted to 1:5,000 in 1% bovine serum albumin in TBS-T (50 mM Tris, 150 mM NaCl, 0.05% Tween 20 [pH 7.5]). Thirty-five serum samples from humans with a clinical history of LD, collected from the CDC Lyme patient serum panel, were used in the ELISA. The infected serum samples were collected from patients with clinical symptoms associated with either early or disseminated phases of LD. The intervals of serum sample collection from patients ranged from 2 weeks to 13 years following onset of disease. Five serum samples from normal individuals residing in areas where LD is not endemic were collected from the CDC, while additional serum samples from 20 individuals that tested negative for B. burgdorferi infection were provided by Marylou Breitentein at Yale University. The 25 control serum samples were used to define the cutoff value for each antigen (the mean plus 2 standard deviations) (39). Human serum samples were diluted to 1:1,000 in 1% bovine serum albumin in TBS-T. Secondary antibodies against IgG, conjugated to horseradish peroxidase, were used with the following dilutions: goat anti-mouse, 1:10,000; goat anti-human, 1:5,000 (KPL). All steps were carried out either for 1 hour at 25°C or overnight at 4°C. Immunoblots were developed on HyBlot CL film (Denville) using the ECL detection reagent (GE Healthcare). ELISA results were quantified using SureBlue TMB microwell peroxidase substrate and TMB stop solution (KPL).
Results are expressed as the mean ± the standard error of the mean (SEM). The significance of the difference between the mean values of the groups was evaluated by the two-tailed Student t test.
The paralogous gene products BBK07 and BBK12 have recently been identified as potential immunogens of B. burgdorferi (6). The genes are highly homologous, with 87% amino acid identity in their overlapping sequences (18). Due to the nearly identical sequences of BBK07 and BBK12, it is unclear if the host immune response is directed against either or both genes. To ascertain their individual expression patterns, we developed two sets of oligonucleotide primer pairs targeting variable regions of each gene, which specifically amplified either the BBK07 or BBK12 gene with low cross-reactivity, as confirmed by the DNA sequencing of the corresponding amplicons (data not shown). These primers were then used to determine the relative expression levels of each gene in cultured spirochetes or infected host tissue by qRT-PCR analysis. While both genes were transcribed at relatively low levels in vitro, only the BBK07 gene was detectable in vivo, as shown in infected mouse dermis 1 week after inoculation (Fig. (Fig.1A).1A). Strikingly, the transcriptional level of the BBK07 gene is more than 100-fold higher in the infected host tissue than in vitro.
Because of the relatively high transcription level of the BBK07 gene in the infected host tissue, we then studied the expression of the BBK07 gene in the B. burgdorferi life cycle, covering the first 4 weeks of murine infection. Total RNA was isolated from experimentally infected tick and mouse tissues to generate cDNA representative of important stages in the B. burgdorferi life cycle: transmission from infected ticks, murine infection, acquisition by naïve ticks, and persistence through the tick molt. While the BBK07 gene was consistently expressed in multiple murine tissues during the first 4 weeks of murine infection, the transcription of the BBK07 gene was dramatically reduced below the limit of detection in all tested stages of ticks (Fig. (Fig.1B).1B). The same RNA samples did not contain detectable quantities of BBK12 transcripts in any tissues or at any time point examined (data not shown).
Since BBK07 is annotated as a lipoprotein, which might be exposed on the spirochete surface, we next assessed the surface localization of BBK07. Expressing a full-length or truncated protein representing the carboxy-terminal half of BBK07 proved difficult in Escherichia coli; however, an amino-terminal fragment could be purified in sufficient quantities and be used for further experimentation. This fragment contained the amino terminus through the first half of the mature protein, referred to as BBK07N (Fig. (Fig.2A,2A, upper panel). Specific antiserum was generated by immunizing mice with BBK07N and adjuvant. In agreement with a previous study showing the immunogenicity of in vitro-translated BBK07 (6), BBK07N also evoked a robust immune response, and BBK07N antiserum recognized both purified BBK07N and native BBK07 from B. burgdorferi lysate (Fig. (Fig.2A,2A, lower panel).
To test the surface exposure of BBK07, a proteinase K accessibility assay was performed (12). Intact B. burgdorferi was incubated with and without proteinase K and probed with FlaB, OspA, or BBK07N antiserum. FlaB, a known subsurface protein, was not degraded, but both the surface protein OspA and BBK07 were significantly degraded, suggesting that the amino-terminal region of BBK07 is surface exposed (Fig. (Fig.2B2B).
Because qRT-PCR analysis indicated a dramatic induction of the BBK07 gene in vivo during early infection, we next assessed kinetics of BBK07 antibody development in the host over the first 9 weeks of B. burgdorferi infection. As qRT-PCR analysis indicated minor expression of the BBK07 gene in vitro, we also assessed, in parallel, BBK07 antibody development in mice immunized with sonicated spirochetes, in order to test whether BBK07 could differentiate infected hosts from ones vaccinated with killed pathogens. To accomplish this, groups of mice (five animals/group) were needle inoculated with a single B. burgdorferi inoculum (105 cells/mice). In parallel, another group of mice (five animals/group) were injected with sonicated B. burgdorferi (100 μg/mice) at 7-day intervals for a total of 9 weeks. Serum samples were collected and pooled weekly. Equal amounts of B. burgdorferi lysate or BBK07N were used to detect specific antibodies present in each serum sample by ELISA (Fig. (Fig.3A).3A). As a negative control, antibody development against the B. burgdorferi antigen Lp6.6, which is abundant in vitro but known to be downregulated during murine infection, was also measured (26). As expected, antibodies against B. burgdorferi lysate, but not against Lp6.6, were detected in infected mice (7, 26). BBK07 provoked a robust antibody development that was detectable after 1 week and remained elevated throughout the infection. In contrast, the mice immunized weekly with lysed spirochetes produced a low BBK07 antibody response that did not increase over the course of the experiment, while Lp6.6 provoked a robust antibody response (Fig. (Fig.3B).3B). In order to confirm that the immunogenicity of BBK07 is not confined to needle-borne artificial murine infection, groups of naïve mice were infected by tick bite. Serum samples were collected after 4 weeks of infection and tested by ELISA (Fig. (Fig.3C).3C). The serum samples contained a similar response against both lysate and BBK07, indicating that the antibody response against BBK07 does not depend on the route of infection.
The robust and specific immune response provoked by BBK07 led us to investigate a possible diagnostic use of BBK07N. Using mouse serum samples collected 2 weeks after infection, we compared the immunogenicity of BBK07N to several other immunogenic B. burgdorferi antigens, such as VlsE, OspC, BmpA, and BbCRASP-2. As controls, B. burgdorferi lysate and Lp6.6 were also included in the assay. To measure the relative immunogenicity levels of each antigen, equal amounts of proteins and lysate were used in an ELISA, probed with the infected mouse serum (Fig. (Fig.4).4). Due to the high antibody titers detected by BBK07N and VlsE, which quickly reached the upper detection limit of the assay, the reaction was stopped shortly (1 min) after the addition of chromogenic substrate. As expected, naïve serum samples had low reactivity to all antigens. Among all antigens tested, BBK07N reflected the most robust immune response, proving to be more sensitive than several of the antigens currently used in LD diagnosis.
To further investigate a diagnostic use of BBK07N, human serum samples from patients diagnosed with LD and healthy human serum samples were used in an ELISA. Wells were coated with recombinant BBK07N, BmpA, OspC, or B. burgdorferi lysate and probed with human serum followed by the detection antibody. We did not have enough recombinant VlsE in our possession, and therefore, VlsE was excluded from the assay. The panel of control serum samples was used to define the cutoff value for each antigen, representing the 95th percentile absorbance value. Samples with an absorbance higher than the cutoff value were considered positive. B. burgdorferi lysate displayed the highest sensitivity of the antigens tested but had the highest cutoff value due to low specificity. Due to its higher specificity, the recombinant antigen BBK07N (12 out of 35 total samples [34%]) was of comparable diagnostic accuracy to that of B. burgdorferi lysate (15 out of 35 [43%]) when detecting an antibody response in the infected sera (Fig. (Fig.5).5). In contrast, lower sensitivities were observed using BmpA (7 out of 35 [20%]) and OspC (3 out of 35 [9%]). These data suggest that BBK07N could be developed into a diagnostic tool for evaluating human LD patients.
The identification and characterization of in vivo antigens of B. burgdorferi is central to the improvement of current laboratory diagnostics for LD. A previous study identified BBK07 and BBK12 as novel immunogenic antigens of B. burgdorferi (6). We further extend the observation and establish that the BBK07 gene, but not the highly similar paralogous member BBK12, was expressed at relatively high levels in vivo. We show that a recombinant protein representing the amino-terminal region of BBK07 was able to provoke a specific antibody response against the native protein, providing antibodies that were then used to demonstrate the surface exposure of the amino-terminal region of BBK07. The recombinant protein could, accordingly, detect a specific antibody response to active infection with B. burgdorferi. As BBK07 had negligible expression in vitro, we show that this antigen could be useful in discriminating antibody development during active infection versus hosts vaccinated with killed pathogen preparations. The detected antibody response during infection was more robust than that detected by several currently used serodiagnostic antigens (2, 3). Finally, using a human serum panel with diagnosed LD, we show that BBK07 is a possible marker for the laboratory diagnosis of LD.
Because of the low numbers of B. burgdorferi cells present during disease, diagnosis of LD has principally relied on immunological methods (2). Serodiagnosis is more sensitive than is direct detection, but current serodiagnosis methodologies have been responsible for incidences of under- and overdiagnoses (9, 17, 40, 50, 51). False negatives can result from tests with low sensitivity, and test sensitivity can be improved by increasing the number of immunoreactive antigens tested. Because B. burgdorferi can be grown in vitro, many tests include whole B. burgdorferi cells or lysate, which provides an extensive set of antigens (2). However, the sensitivity gained by using cultured cells comes with a price, as some antigens of B. burgdorferi are conserved among other bacterial pathogens (13, 29). Antibodies against conserved antigens, such as flagellin and bacterial heat shock proteins, are naturally present in many uninfected individuals, increasing the chance of a false-positive result (5, 13, 21, 29). Perhaps most importantly, the use of B. burgdorferi cells makes standardization of the tests more difficult and, as a result, the outcome of tests more subjective. For example, B. burgdorferi antigen expression can vary by growth phase, and extended periods of in vitro culture can cause the loss of plasmids, some of which contain important antigens (41, 42). While in vitro-grown B. burgdorferi cells may increase sensitivity by providing a wide array of antigens to detect an immune response, the decreased diagnostic specificity and standardization limit its effectiveness in serodiagnosis.
The use of recombinant proteins in diagnosis can eliminate cross-reactive epitopes and can ease standardization by reducing batch-to-batch variation. This increase in specificity need not come at the cost of sensitivity if the immunodominant antigens of B. burgdorferi are identified and characterized. Our data completely support a previous study showing that BBK07 is highly immunogenic during LD (6). However, plasmid lp36, which contained the BBK07 locus, could be lost during in vitro growth (23), and our data showing extremely low in vitro expression of BBK07 suggest that BBK07 is underrepresented in tests using in vitro-grown B. burgdorferi (6). The inclusion of BBK07 as a diagnostic marker could increase serodiagnostic sensitivity in human patients while maintaining the high specificity afforded by recombinant antigen tests. The low in vitro expression of the BBK07 gene and undetectable immune response against sonicated borrelial cells suggest additional use of BBK07 in animal LD diagnosis. An animal LD vaccine that utilizes killed in vitro-grown B. burgdorferi is commercially available (11, 25). The presence of BBK07 antibodies could serve as an indicator of active infection, as BBK07 reactivity is unlikely in vaccinated animals immunized with cultured organisms (19). Thus, BBK07 could differentiate between infected and vaccinated animals. Sequence analysis also indicates that Borrelia garinii and Borrelia afzelii, the most prevalent causative agents of LD in Europe and Asia, lack an ortholog to BBK07 and, therefore, BBK07 reactivity could be used to discriminate human LD caused by B. burgdorferi from that caused by other strains. BBK07 sequence is highly conserved in major infectious isolates of B. burgdorferi (6). Accordingly, BBK07 antibody generated using the infectious B31 isolate used in the current study also recognized the native protein in infectious B. burgdorferi isolate 297, a human cerebrospinal fluid isolate (48), without detectable cross-reactivity (data not shown).
Compared to the robust BBK07-specific antibody development in our murine experiments, the reactivity of the human serum against BBK07 was low. The lower specific antibody titers in these serum samples could be the result of a number of factors, including long-term storage of the serum samples. However, compared to the human antibody response against B. burgdorferi lysate, the BBK07 antigen was sensitive enough to detect specific antibodies in many patients, with a diagnostic accuracy similar to that of the lysate. Taken together, the current results highlight BBK07 as a promising antigenic marker of LD and suggest its inclusion as a serodiagnostic agent in order to improve the sensitivity of current laboratory testing.
This work was supported by Public Health Service grants AR055323 and AI080615 from the National Institutes of Health.
We are grateful to Martin Schriefer for access to the CDC Lyme serum panel collected from patients diagnosed with Lyme disease and normal healthy individuals from areas where Lyme disease is not endemic. We are also thankful to Marylou Breitentein and Fang Ting Liang for providing reagents and Xinyue Zhang and Bridgett Duarte for help with the study.
Published ahead of print on 23 September 2009.