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Humans produce highly specific borreliacidal antibodies against outer surface protein C (OspC) shortly after infection with Borrelia burgdorferi sensu stricto. We previously demonstrated the epitope recognized by immunoglobulin M (IgM) and IgG OspC borreliacidal antibodies was located within the 50 amino acids nearest the carboxy (C) terminus. In this study, we show the immunodominant epitope is located in the highly conserved region within the seven C-terminal amino acids. Six early Lyme disease sera that contained borreliacidal activity and IgM and/or IgG OspC antibodies were chosen randomly and adsorbed with truncated OspC containing the 16 or 7 amino acids nearest the C terminus. Adsorptions with each truncated protein abrogated the borreliacidal activity completely. In addition, only small concentrations of OspC antibodies remained detectable by enzyme-linked immunosorbent assay and Western blotting. Moreover, borreliacidal OspC antibodies were not induced in laboratory mice or hamsters despite heavy infections with B. burgdorferi spirochetes. These findings confirm that borreliacidal antibodies comprise the majority of the IgM and IgG OspC antibody response in human Lyme disease and that the epitope is located in the highly conserved C terminus. In addition, rodent animal models appear to be inappropriate subjects for assessing the effectiveness of the epitope for serodiagnosis or as a human Lyme disease vaccine.
Lyme disease caused by infection with Borrelia burgdorferi sensu stricto is the most prevalent arthropod-borne human illness in the United States. The bacteria thrive in a variety of diverse niches as they cycle between small mammals, Ixodes scapularis ticks, and other alternative hosts including humans. Their ability to survive appears dependent on the differential expression or repression of genes selected in response to a variety of environmental cues. For example, the spirochetes express outer surface protein A (OspA) in the midgut of resting nymphal and adult ticks but downregulate OspA and upregulate OspC in response to temperature after the tick begins feeding (27). More recently, researchers have demonstrated the ability of the spirochetes to regulate a multitude of genes in the tick (13), at the tick-host interface (14), and in the mammalian host (4) in response to additional environmental signals that likely include components in blood, biochemical reactions, and the host immune response.
Despite the propensity for antigenic variation, however, the complement-dependent borreliacidal (killing) antibody response detected in a human infection is remarkably predictable. Several B. burgdorferi proteins, including OspA, OspB, OspC, DbpA, Oms66, and the 39-kDa periplasmic protein, induce borreliacidal antibodies (7, 9, 10, 17, 19, 26, 28, 31), but the response in early human Lyme disease is specific primarily for OspC (5, 6, 16, 17, 25). In addition, borreliacidal antibodies specific for OspA and OspB may be produced, but the response is almost exclusively seen in patients with Lyme arthritis (7, 10, 26). Moreover, traditional laboratory isolates that express OspA and OspB in vitro can be used to detect OspA and OspB borreliacidal antibodies, because the antibodies can attach to the outer surface proteins (Osps). However, OspC borreliacidal antibodies have not been detected by isolates that also express OspA and OspB, likely because the concurrent expression of these Osps hinders the attachment of OspC antibodies. Detecting OspC borreliacidal antibodies is therefore dependent on isolates such as B. burgdorferi 50772, because the spirochetes lack the plasmid containing ospA and ospB (2), and the absence of the Osps enables borreliacidal OspC antibodies to bind (6, 25). A borreliacidal antibody test that uses each type of isolate reliably provides sensitive and specific serodiagnostic confirmation of Lyme disease (5, 6, 8).
We previously exploited the susceptibility of B. burgdorferi 50772 to OspC borreliacidal antibodies (25) to demonstrate that the human antibody response was specific for the 50 amino acids (aa) nearest the carboxy (C) terminus of OspC (16). In this study, we examined smaller fragments of the OspC C terminus and again used borreliacidal activity specific for B. burgdorferi 50772 to more precisely identify the location of the borreliacidal epitope. We also determined whether the response was induced after the infection of laboratory mice or hamsters. The results confirm that the OspC borreliacidal antibody response is highly conserved in human Lyme disease and provide compelling evidence that the epitope responsible will be an extremely valuable serodiagnostic antigen and may provide the basis for an effective Lyme disease vaccine. Additionally, the results demonstrate that the production of OspC borreliacidal antibodies is unique to human infection.
B. burgdorferi sensu stricto 50772 is a noninfectious isolate that lacks the plasmid containing ospA and ospB (36). B. burgdorferi sensu stricto 297 was isolated originally from human cerebrospinal fluid, and the spirochete expresses OspA, OspB, and OspC. Escherichia coli JM109 (Promega, Madison, Wis.) was used for cloning.
Ten-week-old female C3H/Hej mice (Jackson Laboratories, Bar Harbor, Minn.) or 6-week-old LVG hamsters (Charles River Laboratories, Kingston, N.Y.) were housed three or four per cage at ambient temperature. Food and water were available ad libitum. Animal experiments were reviewed and approved by the Gundersen Lutheran Animal Care and Use Committee (Institutional Animal Care and Use Committees).
Serum samples that contained significant concentrations of immunoglobulin M (IgM) and/or IgG OspC antibodies were obtained from patients with early Lyme disease characterized by single or multiple erythema migrans lesions. In addition, immune sera specific for host-adapted (3) Lyme disease spirochetes were obtained from hamsters and mice. Groups of donor animals were infected by injecting 100-μl volumes of Barbour-Stoenner-Kelly (BSK) medium containing 106 B. burgdorferi 297 cells subcutaneously in the lower back. Five weeks postchallenge, 12 approximately 2- by 2-mm pieces of infected ear tissue were removed from each animal by punch biopsy and implanted subcutaneously into the lower back of a naïve recipient animal. After 15 weeks, the recipient animals were bled to exsanguination and killed by inhalation of CO2, and the bladder, kidney, spleen, heart, and ear tissues were cultured in BSK medium. Cultures were then examined weekly for 3 weeks by dark-field microscopy.
Recombinant OspC and OspC-Dra (50 C-terminal amino acids) were recovered from the previously described (16) E. coli JM109 containing either pX3-22 (OspC) or pX2-Dra (OspC-Dra). A recombinant protein containing the fragment with the final 16 OspC C-terminal amino acids (C16) was constructed using forward primer 5′-AGTTAAAGAACTTACAAGTCCTGTTGTGG-3′, reverse primer 5′-GGTTAAGGTTTTTTTGGACTTTCTG-3′, and pX3-22 (15) as templates. The amplified C16 fragment was precipitated using Pellet paint (Novagen, Madison, Wis.) and ligated (pXT16) into a Pinpoint Xa-1 T-vector (Promega, Madison, Wis.). A fragment containing the final seven C-terminal amino acids (C7) was similarly made by annealing primers 5′-GGCAGAAAGTCCAAAAAAACCTTAACCA-3′ and 5′-GGTTAAGGTTTTTTTGGACTTTCTTTCTTCTGCCAA-3′ and also ligated (pXT7) into a PinPoint Xa-1 T-vector (Promega). Both pX16 and pX7 were then transformed into E. coli JM109, and proper orientations were confirmed by DNA sequencing (BigDye ABI, Foster City, Calif.).
The recombinant OspC or OspC fragments were recovered by culturing the E. coli in a 100-ml volume of 2× tryptone-yeast extract (TY) broth containing 100 μg/ml of ampicillin (Sigma Chemical Co., St. Louis, Mo.) for 12 h at 37°C, diluting tenfold with additional 2× TY broth, and incubating for another hour. Isopropyl-β-d-thiogalactosidase (IPTG) (Sigma) was then added to a final concentration of 0.1 μM, and the cultures were reincubated for 4 h at 37°C. The suspensions were pelleted by centrifugation at 10,000 × g for 15 min at 4°C; resuspended in purification buffer containing 50 mM Tris (pH 8.0), 50 mM NaCl, 2 mM EDTA, and 0.1% Triton X-100; and lysed with a sonicator (model W350; Branson Sonic Power, Danbury, Conn.). Sonicated E. coli cells were pelleted by centrifugation at 10,000 × g for 20 min, and the supernatants were passed over columns containing SoftLink resin (Promega) at a rate of 0.5 ml/min at 4°C. The fusion proteins contained a 16-kDa biotinylated purification tag on the amino terminus of the protein that binds to SoftLink resin. Columns containing protein were then washed with 5 column volumes of purification buffer, and the bound proteins were eluted with purification buffer that also contained 5 mM biotin (Sigma). The proteins were then examined by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with mouse monoclonal antibodies specific for OspC to confirm their purity.
One-milliliter volumes of Tetralink tetrameric avidin resin (Promega) were washed and suspended in 40 ml of phosphate-buffered saline (PBS; pH 7.2) and loaded into separate 10- by 70-mm polypropylene columns. Amounts (0.5 mg) of OspC, OspC-Dra (18), C16, or C7 were solubilized in 1-ml volumes of PBS and passed over separate columns. Complete binding was confirmed by protein assay (Bio-Rad). One-milliliter volumes of sera diluted 1:10 in PBS were then passed over the column four times, and the amounts of antibodies removed were confirmed by using OspC-Dra, C16, or C7 enzyme-linked immunosorbent assays (ELISAs).
An OspC ELISA and ELISAs containing the C16 or C7 OspC fragments were prepared as described previously (16). Serum from a person not exposed to B. burgdorferi was used as a normal control.
Borreliacidal antibodies were detected by a flow cytometric procedure (6, 8). Viable B. burgdorferi 297 or 50772 organisms in logarithmic growth phase were enumerated with a Petroff-Hausser counting chamber and diluted with fresh BSK medium to a concentration of approximately 5 × 105 organisms/ml. Concomitantly, serum samples were diluted 1:20 with BSK and filter sterilized by passage through a 0.2-μm microfuge filter (Costar, Cambridge, Mass.). The filtered serum samples were then transferred to sterile 1.5-ml screw-cap microfuge tubes (Sarstedt, Newton, N.C.) and diluted serially (1:40 to 1:20,480) with BSK. Serum samples were heat inactivated at 56°C for 10 min, and a 100-μl aliquot of the spirochetes and 10 μl of sterile guinea pig serum (50% hemolytic component ≥ 200 units/ml) were added. The assay mixtures were mixed thoroughly and incubated for 16 to 24 h at 35°C.
Following incubation, 100 μl of each assay suspension was transferred to a 12- by 75-mm polystyrene tube (Becton-Dickinson, Franklin Lakes, N.J.) containing 400 μl of PBS and 1 μg of acridine orange (Sigma) per ml. A FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) was then used to detect borreliacidal activity. Spirochetes were isolated by gating (CellQuest software; Becton Dickinson) and analyzed for 1 to 2 min with the flow rate set at low. Borreliacidal antibodies kill the spirochete by inducing a complement cascade that disrupts the outer membrane and causes the membrane to bleb. Borreliacidal antibodies were detected indirectly by monitoring the increased fluorescence intensity that occurs when the acridine orange intercalates into blebbed, nonviable spirochetes. A ≥13% shift in the mean fluorescence intensity compared to that of a normal serum control was considered positive (5, 6). The presence of blebbed, nonmotile B. burgdorferi was then confirmed by dark-field microscopy. The undiluted and adsorbed sera were assayed concurrently to eliminate the variability of titers caused by interassay variation in the concentrations of spirochetes.
Western blotting was performed using standard procedures. Briefly, 225 μg of B. burgdorferi 50772 protein was loaded into the preparative wells of 0.1% sodium dodecyl sulfate-12% polyacrylamide gels, and the proteins were separated by running in an electrophoresis unit (PROTEAN IIxi; Bio-Rad Laboratories, Hercules, Calif.) at 24 mA for 4 h. The proteins were transferred from the gels to polyvinylidene difluoride membrane (Perkin-Elmer Life Sciences Inc., Boston, Mass.) by electrophoresing overnight at 10 V. The polyvinylidene difluoride was cut into strips and blocked with 1% bovine serum albumin in PBS (pH 7.2)-0.1% Tween 20 for 1 h at 22°C. Strips were incubated for 1 h at 22°C with serum diluted to a ratio of 1:100 and washed four times with PBS-0.1% Tween 20. Horseradish peroxidase-labeled anti-human IgM or IgG heavy and light chains (Kirkegaard and Perry Laboratories, Gaithersburg, Md.) were added, and the strips were incubated for 1 h at 22°C. Strips were washed and developed using the TMB membrane peroxidase substrate system (Kirkegaard & Perry).
The early Lyme disease sera contained high concentrations of IgM OspC antibodies, and three sera also contained significant amounts of IgG OspC antibodies (Table (Table1).1). In agreement with previous findings (16), the OspC antibodies were removed almost completely by adsorption with OspC-Dra. More notably, binding to the C16 and C7 OspC fragments also almost completely removed the OspC antibodies detected by ELISA (≥4-fold reduction in titer) or by Western blotting (Fig. (Fig.1).1). In contrast, the reactivity on the Western blots for the other B. burgdorferi proteins appeared unaffected.
We then determined whether removing the OspC antibodies by adsorption to the OspC-Dra, C16, or C7 OspC fragments also affected the borreliacidal activity. To confirm that the decreased activity was not due to nonspecific binding, we passed a Lyme disease serum that contained only OspA and OspB borreliacidal antibodies (7) over the columns prior to passing the Lyme disease sera that contained the OspC borreliacidal antibodies. In addition, we passed the Lyme disease sera over a column that contained just the fusion protein. As reported previously (16), adsorbing the early Lyme disease sera with OspC-Dra also abrogated the borreliacidal activity completely (Table (Table2).2). In addition, the borreliacidal antibodies were removed completely by binding to either the C16 or the C7 OspC fragments. The collective results therefore confirmed that the IgM and IgG OspC borreliacidal antibodies were specific for an epitope contained within the C-terminal 16- and 7-aa fragments of OspC. Moreover, removing the borreliacidal antibodies also removed almost the entire concentration (≥4-fold reduction) of OspC antibodies.
We next determined whether the immune response in laboratory mice or hamsters would mimic the human antibody response after infection with Lyme disease spirochetes. To ensure that the antibodies were specific for proteins expressed in vivo (3), the animals were challenged with B. burgdorferi 297 spirochetes that were adapted to each animal by infecting donor animals with a needle challenge and harvesting spirochete-infected ear tissues to challenge additional naïve animals. The immune sera from the infected-tissue-challenged animals were then harvested, pooled (n = 5), and evaluated for borreliacidal activity and OspC antibodies. Both responses were detected, but the titers of all responses were significantly lower than the titers detected in human immune sera (Table (Table3),3), even though the animals were heavily infected (Table (Table4)4) and produced significant amounts of antibodies against many other proteins (Table (Table3).3). Furthermore, removing the OspC antibodies by adsorbing with the recombinant OspC had no effect on the borreliacidal activity (Table (Table3,3, Fig. Fig.22).
Researchers showed previously (6, 16, 25) that the majority of IgM and IgG OspC antibodies produced after infection with B. burgdorferi sensu stricto were complement-dependent borreliacidal antibodies that killed the spirochetes without the necessity of scavenging by phagocytic cells. Moreover, the OspC borreliacidal antibodies were specific for an epitope(s) contained within the 50 C-terminal amino acids of the protein (16). In this study, we demonstrate that the OspC borreliacidal antibodies are specific for a highly conserved epitope within the seven C-terminal amino acid residues.
This finding is remarkable. As an infected tick begins feeding on a human, OspC is expressed (27) and becomes the major outer membrane protein expressed in early infection, regardless of the Borrelia genospecies infecting the patient (11, 22, 24, 35). Correspondingly, OspC antibodies quickly become the dominant humoral immune response (22, 35); however, OspC ELISAs have lacked accuracy for confirming Lyme disease (12, 20, 24, 30, 33). The shortcomings were explained most often by the heterogeneity of ospC (15, 18, 29, 30), even among isolates within the same genospecies (32). The antibody responses induced by the highly variable regions would have widely variable specificities. Additionally, antibodies induced by other infections may also react to these regions. However, previous studies that characterized the ability of OspC antibodies to kill the spirochetes independent of scavenging by phagocytic cells have provided compelling evidence that the borreliacidal antibodies are specific for a conserved region of the protein. For example, a flow cytometric test that detects borreliacidal OspC antibodies by monitoring the ability of the antibodies to kill only B. burgdorferi sensu stricto 50772 detected the response in >70% of early Lyme disease sera collected from throughout the United States (5).
In this study, we extend these findings by demonstrating that the dominant OspC borreliacidal antibody epitope is located within the surface-exposed (22) seven amino acid residues of the C-terminal end of the protein. More importantly, a BLAST search (1) confirmed the region is highly conserved among infectious B. burgdorferi sensu stricto, B. garinii, and B. afzelii isolates. The results therefore provide explanation for the increased accuracy (5, 6) of the flow cytometric borreliacidal antibody test. Moreover, the findings provide strong evidence that an ELISA comprised of the epitope would correlate directly with the highly specific borreliacidal antibody response. This should provide a sensitive and highly specific serodiagnostic confirmation of Lyme disease without the technical complexity and live organisms necessary to detect borreliacidal activity.
Mathiesen et al. (22) also reported that the OspC antibodies in sera from patients with neuroborreliosis caused by infection with B. garinii were specific predominantly for the identical sequence of 10 C-terminal amino acid residues of the protein. However, an ELISA with a synthetic peptide (21), while more reactive than the rOspC ELISA, detected OspC antibodies in European sera significantly less often (36%) than in sera from U.S. patients (11, 16, 20, 33). The authors noted the distinct absence of IgG OspC antibodies in sera from European patients (34) and suggested the failure of the response to reach maturity was because the epitope induced antibodies in a T-cell-independent fashion (22). However, this is clearly not the case after infection with B. burgdorferi sensu stricto. In this study, we detected high titers of borreliacidal IgG OspC antibodies, and numerous other investigators (5, 7, 11, 20, 25) have also confirmed that IgG OspC antibodies are commonly present in all stages of the illness. It seems unlikely the identical epitope would induce both a T-cell-independent and a T-cell-dependent response.
Rather, we suspect the failure of the OspC antibodies to switch classes in the European Lyme disease patients is more likely due to the predilection of the spirochetes to colonize the central nervous system, which may inhibit the maturation of the antibody response. In support, European patients are often infected with B. garinii or B. afzelii, which commonly cause neuroborreliosis (23). These genospecies are not found in the United States, where the patients are instead infected with B. burgdorferi sensu stricto, which most often colonizes the skin or joints. Additional studies to evaluate this hypothesis remain necessary, but the collective results still highlight the immunodominance of the OspC antibody response specific for the C terminus of the protein and provide support for the development of serodiagnostic procedures to exploit this finding.
In addition, the results provide compelling evidence that the C terminus of OspC is a good human vaccine candidate. However, we failed to detect borreliacidal OspC antibodies in sera from the laboratory mice or hamsters, despite vigorous antibody responses against many other B. burgdorferi proteins. The sera instead contained only small concentrations of OspC antibodies detected by ELISA or Western blotting. More significantly, the OspC antibodies were not borreliacidal antibodies. The findings therefore confirmed a significant difference in the antigenic reactivities of B. burgdorferi sensu stricto spirochetes that infect human or rodent hosts. More importantly, the results cast considerable doubt on the ability of rodent animal models to provide experimental data that accurately predict the ability of vaccination with OspC to provide protection against human infection.
In conclusion, the immunodominant IgM and IgG OspC antibody response in human Lyme disease is specific for the highly conserved seven C-terminal amino acid residues. This finding provides valuable information for future efforts to develop effective serodiagnostic tests and vaccines, but an accurate assessment of their effectiveness cannot be obtained by using laboratory mice or hamsters.
We thank Jamie Griffin for expert technical assistance.
Financial support was provided by the Gundersen Lutheran Medical Foundation, La Crosse, Wisconsin.