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Lyme disease, which is caused by the spirochete Borrelia burgdorferi, affects multiple tissues and organs in humans and in animals (19). In untreated individuals, Lyme disease spirochetes can persist for extended periods, even in the presence of a vigorous and persistent immune response (18). Chronic infection may be achieved through a variety of mechanisms, including limited exposure of antigenic targets (6), seclusion of the organisms into immune-privileged sites (14), local and/or systemic suppression of harmful immune responses (8), and antigenic variation (23).

Antigenic variation is an effective strategy evolved by pathogenic microorganisms to evade the host immune system (4). Antigens such as the variant surface glycoprotein of African trypanosomes (5, 22), the variable major protein of the spirochete Borrelia hermsii (13, 20), and the variable surface antigen (VlsE) of B. burgdorferi (23) contain both invariable and variable domains. Antigenic variation affects only the variable domains. Even within variable domains, short invariable regions may be present. The invariable domains and regions are important in maintaining the functional structure of the molecule (4). The variable domains are highly immunogenic and serve as the major target of the host immune response (4, 5, 13, 20, 22, 23). Invariable portions of the variant surface glycoprotein and variable major protein antigens have not been found to be antigenic during natural infections, although antibodies directed to these conserved sequences may be produced by immunization (1, 3, 7).

Among molecules that undergo antigenic variation, VlsE is unusual in that more than 75% of its primary structure is invariable. This invariable portion of the molecule is composed of two domains at the amino and carboxyl termini, respectively, which together encompass approximately half of the molecule's length, and six small invariable regions (IR₁ to IR₆) that are interspersed within the central variable domain (Fig. 1) (11, 23). Any such invariable portion could be explored as a possible target for a protective immune response and vaccine development.

View larger version (10K): [in this window] [in a new window]   FIG. 1.   Diagrammatic illustration of the VlsE structure. VlsE consists of two invariable domains at the amino and carboxyl termini and a variable one at the center. The variable domain contains six variable regions, VRI to VRVI, and six invariable ones, IR1 to IR6. The sequences of the invariable regions are based on those of the variable domain of VlsE expressed by strain IP90 of B. garinii (11).

To avoid immune responses harmful to the organism, invariable portions may be (i) not exposed on the surface of the molecule, (ii) exposed on the surface of the molecule but not at that of the spirochete, or (iii) nonantigenic, either because of an intrinsic lack of antigenicity in a given host species or because other regions of the molecule are immunodominant. We have already demonstrated that one of the invariable regions of VlsE, namely IR₆, is strongly antigenic, exposed at the molecule's surface but not accessible to antibody at the surface of the spirochete in vitro (11).

In the present study, we investigated the exposure, at the surface both of the VlsE molecule and of the spirochete, of four additional invariable regions, IR₂ to IR₅. To address this issue, we generated antibodies to these four regions by immunizing rabbits with synthetic peptides conjugated to keyhole limpet hemocyanin (KLH). Rabbit antisera were used in immunoprecipitation experiments with native VlsE to determine exposure of invariable regions at the VlsE surface. Exposure of IR₂ to IR₅ at the spirochete's surface was assessed by indirect immunofluorescence and by antibody-dependent, complement-mediated killing (ADCK) assays using the rabbit antipeptide antibodies.

Borrelia garinii strain IP90 (low passage) was obtained from the Centers for Disease Control and Prevention (Fort Collins, Colo.). Spirochetes were cultivated in Barbour-Stoenner-Kelly (BSK-H) medium supplemented with 10% rabbit or human serum (Sigma Chemical Co., St. Louis, Mo.), as described previously (16).

Reactivity of rabbit antipeptide antibody with the VlsE protein. To generate antibody to invariable regions of VlsE, peptides were prepared using the fluorenylmethoxycarbonyl synthesis protocol (2) according to the sequences listed in Fig. 1. The synthetic peptides C₂, C₃, C₄, and C₅ represented the sequences of IR₂, IR₃, IR₄, and IR₅, respectively. A cysteine residue was included at the NH₂ terminus of each synthetic peptide and used as a conjugation site when KLH was used as a carrier. Conjugation of the synthetic peptides to KLH was performed by the N-succinimidyl maleimide carboxylate method. The maleimide reagent was from Molecular Probes (Eugene, Oreg.), and the protocol suggested by the manufacturer was followed. The synthetic peptides were also conjugated to biotin and used as peptide-based enzyme-linked immunosorbent assay (ELISA) antigens. Six-month-old New Zealand White rabbits were given three injections at biweekly intervals of 200 µg of peptide-KLH conjugate emulsified with Freund's complete (first injection) or incomplete adjuvant (remaining injections). Ten days after the last injection, the antibody reactivity was determined by peptide-based ELISA and immunoblotting.

View larger version (20K): [in this window] [in a new window]   FIG. 2.   ELISA titers of rabbit antisera to synthetic peptides. Rabbit antipeptide antisera were serially diluted fourfold and reacted with the corresponding peptide bound to an ELISA plate, following the procedure described in the text. Titer was defined as the highest serum dilution at which the ELISA OD was larger than the mean OD value of the preimmune sera of all of the rabbits plus 3 standard deviations.

View larger version (53K): [in this window] [in a new window]   FIG. 3.   Reactivity of rabbit antipeptide antibodies with VlsE. A whole-cell lysate of B. garinii IP90 spirochetes was separated by SDS-PAGE and transferred onto nitrocellulose. The blot was allowed to react with preimmune or rabbit antipeptide antiserum. MW, molecular weight (values are in thousands).

IR₂, IR₄, and IR₅ but not IR₃ are exposed at the surface of VlsE. Immunoprecipitation was used to determine if invariable regions are exposed at the VlsE surface. Immunoprecipitation was conducted at 4°C. Approximately 2.5 × 10¹⁰ IP90 spirochetes harvested at stationary growth phase were washed twice with PBS and extracted in 7.5 ml of solubilization buffer (50 mM Tris-HCl, 1% Triton X-100, 1 mM EDTA [pH 7.6]) for 30 min. The mixture was centrifuged at 13,000 × g for 30 min, and the supernatant was collected. Each sample of 1.5 ml of this supernatant was mixed with 30 µl of preimmune or immune rabbit serum and incubated for 30 min. Fifty microliters of drained ImmunoPure immobilized protein G beads (Pierce) preequilibrated in solubilization buffer was then added and incubated for an additional 30 min. The beads were washed twice with excess volumes of this buffer by centrifugation at 3,000 × g for 20 min and resuspended in 150 µl of nonreducing SDS-PAGE sample buffer (125 mM Tris-HCl, 3% SDS, and 20% glycerol [pH 6.8]). The suspension was incubated at room temperature for 30 min and then centrifuged at 16,000 × g for 30 min. Ten microliters of supernatant was loaded onto each of 10 lanes of a SDS-12% acrylamide minigel. Separated proteins were electrotransferred to nitrocellulose in Towbin transfer buffer (21). The blot was then processed as described above and was developed with rabbit anti-C₆ antiserum. The sequence of the synthetic peptide C₆ was based on IR₆ (11). This antiserum reacted strongly with VlsE. Rabbit antisera to C₂, C₄, and C₅ were able to precipitate VlsE, indicating that IR₂, IR₄, and IR₅ were exposed at the surface of this antigen (Fig. 4). In contrast, VlsE was not precipitated with anti-C₃ antibody. Preimmune serum was also negative.

View larger version (54K): [in this window] [in a new window]   FIG. 4.   Exposure of IR2, IR4, and IR5 at the VlsE surface. VlsE from B. garinii strain IP90 spirochetes was extracted with solubilization buffer and immunoprecipitated with rabbit antipeptide antiserum or preimmune serum and protein G-agarose. Solubilized immunoprecipitates were separated by SDS-PAGE and blotted onto nitrocellulose. VlsE was visualized with rabbit anti-C6 antiserum and goat anti-rabbit IgG-peroxidase conjugate. In addition to VlsE (approximately 39.5 kDa), precipitated rabbit IgG is visible at the top of each lane. MW, molecular weight (values are in thousands).

IR₂, IR₄, and IR₅ are not accessible to antibody at the surface of intact spirochetes. To assess if IR₂, IR₄, and IR₅ are exposed at the spirochete's surface, immunofluorescence experiments were conducted. Both unfixed and fixed spirochetes were used in this study. For immunofluorescence with unfixed spirochetes, approximately 10⁸ IP90 spirochetes that were harvested from 1.0 ml of stationary-phase culture (10% rabbit serum-BSK) by centrifugation at 4,000 × g for 20 min were gently resuspended in 100 µl of PBS supplemented with 0.2 µl of preimmune or immune rabbit serum and incubated for 1 h at room temperature. B. burgdorferi-infected mouse antiserum and monkey anti-OspA (outer surface protein A) antiserum were used as positive controls. Mice were infected with spirochetes of the B31 strain by tick inoculation. Anti-OspA antiserum was raised by immunization with the OspA vaccine (17). After two washes by centrifugation at 4,000 × g for 8 min with excess volumes of PBS, the spirochetes were resuspended in 100 µl of PBS containing 2 µg of goat anti-rabbit (Pierce), -monkey, or -mouse (Kirkegaard & Perry) IgG antibody conjugated to fluorescein and incubated for an additional 1 h at room temperature. Spirochetes were washed three times with PBS and resuspended in 500 µl of PBS. Approximately 5 µl of spirochete suspension was applied to a microscope slide, glass covered, and observed under a fluorescence microscope. For immunofluorescence with fixed spirochetes, IP90 spirochetes grown in 10% human serum-BSK medium were harvested by centrifugation and washed once with PBS. Spirochetes were fixed in acetone for 30 min and recovered by centrifugation. Fixed organisms were resuspended in PBS containing 5% human serum and incubated at 37°C for 30 min. After one wash with PBS-human serum, spirochetes were processed as for immunofluorescence for unfixed spirochetes, except for the following modifications: PBS was replaced with PBS-human serum, rabbit serum was diluted at 1:1,000 instead of 1:500, and goat anti-rabbit IgG fluorescein conjugate (Sigma) was absorbed with human serum protein and diluted at 1:150 instead of 1:50. None of the antipeptide antibodies were able to make unfixed spirochetes visible under a fluorescence microscope, while the control mouse anti-B. burgdorferi antibody labeled the bacteria under the same conditions (Fig. 5). Like the mouse antiserum, monkey anti-OspA antibody lit up both fixed and unfixed spirochetes (data not shown), evidence that our gentle treatment did not significantly remove outer surface proteins from the spirochete's surface. This indicates that the invariable regions that are exposed at the VlsE surface are inaccessible to antibody on the intact spirochete. As expected, IR₃ also was inaccessible to antibody at the spirochetal surface (Fig. 5). In contrast, acetone-fixed spirochetes were readily labeled with anti-C₂, -C₄, and -C₅ antibodies (Fig. 5). Anti-C₃ antibody was essentially negative, although spotted labeling on some of the organisms was visible. Preimmune rabbit serum was negative (Fig. 5).

View larger version (29K): [in this window] [in a new window]   FIG. 5.   IR2 to IR5 are not accessible to antibody at the surface of spirochetes. B. garinii strain IP90 spirochetes either were fixed with acetone or left unfixed. Unfixed spirochetes were resuspended in PBS containing preimmune or immune rabbit antipeptide antiserum or positive control mouse antiserum, while fixed spirochetes were incubated in PBS-human serum supplemented with the same antiserum samples. Sensitized spirochetes were probed with goat anti-rabbit or -mouse IgG-fluorescein conjugate.