Received 11 January 1996/Returned for modification 7 March 1996/Accepted 3 April 1996

Transcription

1 INFECTION AND IMMUNITY, June 1996, p Vol. 64, No /96/$ Copyright 1996, American Society for Microbiology Outer Surface Protein C (OspC), but Not P39, Is a Protective Immunogen against a Tick-Transmitted Borrelia burgdorferi Challenge: Evidence for a Conformational Protective Epitope in OspC ROBERT D. GILMORE, JR.,* KIMBERLY J. KAPPEL, MARC C. DOLAN, THOMAS R. BURKOT, AND BARBARA J. B. JOHNSON Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado Received 11 January 1996/Returned for modification 7 March 1996/Accepted 3 April 1996 Outbred mice were immunized with the soluble fraction of a crude Escherichia coli lysate containing either recombinant outer surface protein C (OspC) or P39 of Borrelia burgdorferi B31 (low passage). Following seroconversion, the mice were challenged with an infectious dose of B. burgdorferi B31 via the natural transmission mode of tick bite. Three mice immunized with P39 were not protected; however, all 12 of the recombinant OspC-immunized mice were protected from infection as assayed by culture and serology. Although OspC has been shown to be a protective immunogen against challenge with in vitro-cultured borrelia administered by needle, this study is the first to demonstrate OspC effectiveness against tick-borne spirochetes. Following feeding, all ticks still harbored B. burgdorferi, suggesting that the mechanism of protection is not linked to destruction of the infectious spirochete within the tick. In a separate experiment, groups of four mice were immunized with protein fractions from B. burgdorferi B31 purified by preparative gel electrophoresis in an attempt to identify potential protective antigens. Many of these mice developed high-titer-antibody responses against OspC, but curiously the mice were susceptible to B. burgdorferi infection via tick bite. These results suggest that the protective epitope(s) on OspC is heat sensitive/conformational, a finding which has implications in vaccine development. The outer surface proteins (Osps) of Borrelia burgdorferi have been demonstrated to confer full or partial protection in experimental animals against B. burgdorferi infection, the cause of Lyme disease. These antigens are OspA (5, 7, 26, 29), OspB (8, 22), OspC (21, 22), and OspF (18). Additionally, a nonsurface fusion protein with a molecular mass of 110 kda containing a truncated HSP70 (2) has been reported to be protective, and studies have suggested that P39, also known as BmpA, may also be protective (15, 25). OspA has been the subject of the most complete studies as an effective protective immunogen in experimental animals (and under evaluation in humans), although some recent data imply that it may have some limitations as a vaccine candidate. In particular, OspAvaccinated animals are not completely protected against needle-administered challenges of heterologous strains (9, 17, 23). The lack of cross-protection may be attributed to variation of OspA across genospecies and within isolates of B. burgdorferi sensu stricto (17). The selection of escape mutants that arise in vivo (10) may also be a mechanism of vaccine failure. OspA vaccines are effective against tick-transmitted challenge, mainly because of spirochete destruction in the tick on ingestion of bloodmeal containing OspA antibodies (11). Complete clearance of spirochetes in an infected tick may require ingestion of high-titered antibody from the immunized host. The duration of high titers of antibody, therefore, may be a critical determinant of the long-term efficacy of an OspA vaccine. However, an effective boost of the antibody response to OspA in a vaccinee may not occur at the time of tick bite, either because spirochetes do not reach mammalian tissues or because the OspA dose is inadequate. When inoculated intradermally, low numbers of spirochetes do not elicit a detectable anti-ospa response (1, 19a), and OspA-immunized mice are susceptible to infection conferred by transplantation of tissues containing host-adapted B. burgdorferi (1). Although OspA is observed on B. burgdorferi cells in culture and in ticks (24), the level may drop or be masked on introduction and adaptation in the host animal, thus accounting for the lack of a strong antibody response against OspA in Lyme disease patients (except in some cases late in infection) (4). Thus, a protective mechanism of an OspA-based vaccine may be dependent on the duration of the OspA serum antibody level in the host. Meanwhile, OspC levels on the outer surface of tick-borne borrelias increase after tick feeding (24). Therefore, unlike those to OspA and OspB, antibodies to OspC are usually some of the first detected following the onset of a B. burgdorferi infection (4, 6, 13). This observation has become important in Lyme disease diagnosis and may have significant implications for vaccine development. As an alternative vaccine candidate to OspA, OspC has shown promise in its ability to protect against a B. burgdorferi infection in mice and gerbils when cultured organisms are administered by needle (16, 21, 22). In this study, we evaluated the protective properties of OspC, P39, and other immunogens against infection when the challenge inoculum was administered by the natural transmission mode of tick bite. * Corresponding author. Mailing address: P.O. Box 2087, Foothills Campus, DVBID, Centers for Disease Control and Prevention, Fort Collins, CO Phone: (970) or Fax: (970) Electronic mail address: rbg9@cidvbi1.em.cdc.gov. MATERIALS AND METHODS B. burgdorferi cultures. Low-passage strain B31 (passage 6) was obtained from Alan Barbour (University of Texas Health Science Center at San Antonio) and maintained in Barbour-Stoenner-Kelly (BSK II) medium as previously described 2234

2 VOL. 64, 1996 OspC PROTECTS AGAINST TICK-TRANSMITTED B. BURGDORFERI 2235 (14). The development and maintenance of a B31-infected Ixodes scapularis tick colony have been described previously (20). Briefly, the I. scapularis ticks used in these experiments originated from adult ticks that were flagged on Great Island, Mass., in 1984 and The colony was determined to be free of transovarially acquired spirochetes by examining egg batches after oviposition by fluorescent antibody microscopy. This uninfected colony of ticks served as our source for generating infected ticks. B. burgdorferi B31 organisms at the lowest passage (passage 6) were inoculated intradermally into 3- to 6-week-old male mice from a breeding colony of the Institute for Cancer Research (Philadelphia, Pa.) outbred mice maintained at the Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Co. At four weeks postinfestation, mice were assayed for infection by culture of ear biopsy samples (28). Larvae from the uninfected tick colony were allowed to feed to repletion on biopsy-positive mice. Larvae were collected and held in a bioclimatic chamber at 97% relative humidity and 21 C. The infection rate in resultant nymphs was approximately 90%, as determined by dissection of the tick midgut and identification of spirochetes by dark-field microscopy. All infected ticks were maintained by a cycle of infected nymphs transmitting spirochetes to mice, and these mice served to infect subsequent larval ticks. Antibodies. Monoclonal antibody to P39 (H1141) was provided by Tom Schwan (Rocky Mountain Laboratories, Hamilton, Mont.). The monoclonal antibody to OspC (4B8F4) for calibration of immunoblots was provided by Steven Padula (University of Connecticut, Farmington). The monoclonal antibody to OspA (H5332) was provided by Alan Barbour (University of Texas Health Science Center at San Antonio). Mouse polyclonal antibodies to these proteins were generated in this laboratory by standard procedures, as described below. Construction of B. burgdorferi genomic DNA library. Genomic DNA was purified from B. burgdorferi early-log-phase 10-ml cultures grown in BSK II medium to a density of 10 7 organisms per ml as follows. The cells were collected by centrifugation and resuspended in 1 ml of 50 mm Tris-HCl (ph 8.0) 50 mm EDTA. The cell suspension was frozen at 20 C. One hundred microliters of a fresh lysozyme solution (10 mg of lysozyme per ml of 0.25 M Tris-HCl [ph 8.0]) was added to the thawing cell suspension and mixed. The solution was placed on ice for 45 min. A solution (200 l) of 0.5% sodium dodecyl sulfate (SDS), 50 mm Tris-HCl (ph 7.5), 100 mm EDTA, and 1 mg of proteinase K (powder added immediately before use) per ml was added to the suspension, mixed, and heated at 50 C for 60 min. An equal volume of Tris-buffered phenol-chloroform was added and mixed gently to emulsify the mixture. The layers were separated by centrifugation, the aqueous layer was removed, and the phenol-chloroform extraction step was repeated. Approximately 5 ml of sterile distilled water was added to the aqueous layer to prevent SDS precipitation in the next step. The addition of sodium acetate (0.1 volume of a 3 M concentration) was followed by the addition of 2 volumes of 100% ethanol, and the solution was gently mixed. The precipitated nucleic acids were pelleted by centrifugation. The pellet was dried and dissolved in 300 to 500 l of 10 mm Tris 1 mm EDTA (ph 8.0) containing 200 g of RNase A per ml. The DNA was quantitated spectrophotometrically and analyzed on a 0.4% Tris-acetate-EDTA agarose gel. To construct the genomic library, approximately 5 to 10 g of genomic DNA was subjected to EcoRI star activity, which creates random cuts at the site NAATTN, in buffer that contained a low-ionic-strength (no salt), high glycerol concentration ( 5% [vol/vol]) and excess EcoRI enzyme (approximately 30 U). The reaction mixture was incubated at 37 C for 20 min. The resultant DNA was randomly digested into fragments ranging in size from 1 to 10 kb. The digested DNA was ligated into an EcoRI-digested LambdaZap II vector (Stratagene, La Jolla, Calif.) and packaged as phage according to the manufacturer s instructions. Isolation of OspC and P39 antigen genes. Lambda phage from the unamplified genomic library were plated according to standard procedures. Following an incubation period when the plaques were just visible, the plates were overlaid with nitrocellulose filters previously dampened with 1 mm isopropyl-1-thio- -Dgalactopyranoside (IPTG) and allowed to incubate at 37 C overnight. The next day, the filters were removed from the plates and probed with antibodies specific to the protein of interest, either OspC or P39. Mouse polyclonal serum against OspC (see Fig. 2) was used, as was the P39 monoclonal antibody. Immunoscreening of the plaque filters was done according to standard procedures with the primary antisera and secondary anti-immunoglobulin G antibodies conjugated with alkaline phosphatase or horseradish peroxidase. Immunoreactive plaques were detected either colorimetrically or by chemiluminescence (ECL; Amersham, Arlington Heights, Ill.) and plaque purified by standard procedures. Cloned inserts were subcloned from the LambdaZap II vector into the plasmid pbluescript (Stratagene) by the phagemid rescue method of the manufacturer into Escherichia coli XLI Blue MRF or SURE (Stratagene). Expression of recombinant gene products. E. coli clones containing either the OspC or P39 antigen gene were grown in Luria-Bertani broth culture to late log or stationary phase with the addition of 0.5 to 1 mm IPTG during mid-log phase. The cells were pelleted, and an aliquot was resuspended in 2 SDS-polyacrylamide gel electrophoresis (PAGE) sample loading buffer (containing 5% 2-mercaptoethanol and 4% SDS), boiled for 5 min, and loaded onto a 10% polyacrylamide gel for SDS-PAGE protein analysis. Gels were blotted electrophoretically onto Immobilon (Millipore, Bedford, Mass.) polyvinylidene difluoride membranes, and Western blot (immunoblot) analyses were performed according to standard procedures (30). The OspC and P39 antibodies were used to detect recombinant protein expression in the E. coli lysates. Preparative SDS-PAGE. In vitro-cultured B. burgdorferi B31 (low passage) was harvested, and the pellet was homogenized and stored as frozen aliquots as described previously (14). The preparation was mixed with 2 SDS-PAGE sample loading buffer (containing 5% 2-mercaptoethanol and 4% SDS) and boiled for 5 min prior to being loaded onto a preparative SDS-PAGE gel (10% and 12% gels were used) according to standard procedures with the use of a Mini-Protean II (Bio-Rad Laboratories, Hercules, Calif.) gel apparatus. After electrophoresis, the gel was fixed and stained overnight with Coomassie brilliant blue (solubilized in acetic acid and methanol) and destained. The gel was fractionated with a clean scalpel into slices containing proteins with approximate molecular masses of (i) 14, (ii) 14 to 19, (iii) 20 to 23, (iv) 24 to 30 (v) 32 to 38, (vi) 42 to 49, and (vii) 70 kda. Proteins were purified from the gel slices by electroelution in a Bio-Rad model 422 electroeluter. Eluted protein fractions were concentrated and dialyzed by centrifugation in a Centricon 10 (Amicon, Beverly, Mass.) filter apparatus. Immunization of mice. Specific-pathogen-free outbred mice (Institute for Cancer Research) were used for vaccinations. Recombinant E. coli lysates were grown as described above and prepared for mouse immunizations as follows. Cells were pelleted by centrifugation and resuspended in a 0.5 volume of sterile saline. The cell suspension was sonicated on ice to prevent heat denaturation with a Branson (Danbury, Conn.) model 450 sonifier. The suspension was centrifuged to pellet the insoluble cellular debris, and the soluble supernatant containing recombinant protein was collected. An equal volume of the crude sonicated E. coli lysate was mixed with adjuvant (TiterMax, Norcross, Ga.), and this mixture (approximately 50 to 100 l per mouse) was used to inoculate mice subcutaneously according to the manufacturer s directions. The E. coli lysate containing the pbluescript with no insert was also prepared as described above and used as a negative control immunogen. For the preparative gel fraction proteins, groups of four mice were injected intradermally with a saline suspension containing approximately 5 to 10 g of protein precipitated with the adjuvant, aluminum hydroxide. All mice were boosted at 2 and 4 weeks after the initial inoculation and were bled 1 week following each boost. A final boost, if needed, was administered 1 to 2 weeks prior to tick infestation. Mice were bled serially to track seroconversion of individual mice to the immunogen. Seroconversion was assessed by Western immunoblotting against the B. burgdorferi B31 low-passage antigen with Marblot (MarDx Diagnostics, Inc., Carlsbad, Calif.) strips and antisera at 1:100 dilution. B. burgdorferi challenge by tick feeding. Once seroconversion was determined, the mice were challenged with B. burgdorferi B31 by infestation of nymphal ticks harboring the spirochete. Ten ticks were placed on each mouse and allowed to feed to repletion. Three to four days later, engorged ticks were collected and stored at 21 C under an atmosphere of 97% relative humidity. Ear biopsy culture assay. An ear biopsy was performed on each mouse 29 to 30 days after tick feeding to test for the presence of borrelias in the tissue as previously described (28). Cultures were observed microscopically for borrelia growth 1 to 2 weeks later. Cultures were deemed positive if borrelia cells were observed in any field and negative if no borrelia cells were observed in 20 fields. OspA antigen-capture ELISA. Ten to 15 days after feeding to repletion, the ticks were assayed for the presence of B. burgdorferi by OspA antigen-capture enzyme-linked immunosorbent assay (ELISA) as previously described (3). Briefly, replete ticks were homogenized in 50 l of 0.25% Nonidet P-40 prior to the addition of 250 l of 2.5% milk with 0.05% Tween 20 in phosphate-buffered saline. OspA capture was accomplished with monoclonal antibody H5332 with a signal generated by horseradish peroxidase-labelled anti-ospa monoclonal antibody 1-15 (provided by Pfizer Animal Health, Lincoln, Nebr.). Negative controls consisted of uninfected nymphs physiologically identical to the test nymphs. B. burgdorferi-positive ticks were defined as those which generated an absorbance value greater than the mean value plus three standard deviations for the negative control ticks. The positive control was a standard curve for recombinant OspA. Some ticks were crushed in BSK II medium and examined for the presence of live spirochetes 2 to 4 weeks later. RESULTS Cloning and expression of OspC and P39 antigen genes. Lysates of the recombinant E. coli cultures were assayed for expression of the gene products by Western blot. Figure 1 shows the recombinant product for the OspC and P39 clones. The recombinant OspC migrates with an approximate molecular mass of 28 to 30 kda because the OspC gene is expressed as a LacZ fusion protein from the pbluescript vector. The fusion adds about 4 to 5 kda to the OspC product, whose size is of approximately 23 kda. The in-frame fusion junction of the vector and the gene was verified by DNA sequencing of the clone. The fusion junction begins with nucleotide 94 of the open reading frame of the B31 strain OspC gene (19b). There-

3 2236 GILMORE ET AL. INFECT. IMMUN. FIG. 2. Western blots of sera from mice that seroconverted to OspC following inoculation of B. burgdorferi protein fractions purified by denaturing SDS- PAGE. These samples were blotted at a 1:500 dilution against B. burgdorferi B31 low-passage antigens. FIG. 1. Expression of recombinant antigens in E. coli assayed by immunoblot. Lanes: 1, B. burgdorferi B31 low-passage lysate; 2, lysate from E. coli harboring pbluescript with no insert; 3, E. coli lysate expressing OspC (A) or P39 (B). Molecular mass markers (in kilodaltons) are indicated on the left. The OspC and P39 antibodies used are specified in Materials and Methods. fore, the fusion OspC gene is truncated from the first 31 amino acids of the open reading frame, resulting in a construct minus the leader peptide. The P39 gene was expressed as a full-length product and not a fusion protein. DNA sequencing analysis demonstrated that the gene is contained internally on an approximately 3-kb insert (data not shown). Therefore, expression was presumably driven from the borrelial promoter present within the cloned insert. The gene sequence was identified by comparison to the sequence of P39 published by Simpson et al. (27). Immunization of mice and challenge by tick bite. In an initial study, we immunized groups of mice with gel-purified protein fractions of B. burgdorferi from SDS-PAGE preparative gels. The objective of this experiment was to observe whether any fractions contained novel proteins capable of eliciting a protective immune response against B. burgdorferi infection. We found no such protective activity in any fractions. However, we observed that although individual mice demonstrated polyclonal serological reactivity against proteins from their immunization fraction, several mice from each fraction group showed a strong antibody response against OspC. In total, 24 mice seroconverted to OspC. It is unclear why so many mice immunized with gel fractions not in the 20- to 24-kDa range seroconverted to OspC and gave such high-titerantibody responses. Perhaps, small, undetectable amounts of OspC remained stuck throughout the gel during its electrophoretic migration, and because the molecules are such potent immunogens, an antibody response was elicited. Representative immunoreactivity from 11 of these mice is shown in Fig. 2. The antibody response of the mice against OspC was distinct, with reactivity at dilutions of 1:1,000 on Western blots. These serum samples also were immunoblotted against recombinant OspC to ensure that this response was specific for OspC and not a comigrating protein (data not shown). Each of 24 mice inoculated with gel-fractionated proteins which became seroreactive for OspC showed no protection from infection. As shown in Table 1, each of these OspCreactive mice was culture positive, and Western blot serology showed reactivity to a wide range of B. burgdorferi antigens, indicative of infection. However, protection was demonstrated in two mice that had seroconverted to OspA and OspB, respectively, after being immunized with gel fraction preparations. OspA and OspB are known to elicit protection in mice. These mice were tissue culture negative and serology negative (Table 1), thus showing that gel purification did not alter the ability of OspA and OspB to function as protective immunogens. In addition, we found that ticks which had fed on the OspA-reactive mouse were cleared of spirochetes (four negative ticks of four ticks tested), a phenomenon demonstrated by others (11). The failure of our OspC-seropositive mice to be protected led us to see whether we could duplicate the protective properties of OspC demonstrated by others (16, 21, 22) but using a tick bite challenge instead of a needle challenge of cultured borrelia organisms. Purification of the recombinant protein from the E. coli host proteins was not necessary for the mice to FIG. 3. Immunoreactivity of serum samples from mice inoculated with recombinant antigen preparations. In each panel, three representative Western blots demonstrating the seroconversion of all mice in the group to the B. burgdorferi antigen are shown. The mouse sera were blotted against B. burgdorferi B31 low-passage proteins at a 1:100 dilution. (A) OspC; (B) P39. The first lane is a serum standard from a patient with Lyme disease with the sizes of the reactive antigens (in kilodaltons) indicated on the left.

4 VOL. 64, 1996 OspC PROTECTS AGAINST TICK-TRANSMITTED B. BURGDORFERI 2237 TABLE 1. Results of infectivity assays 4 weeks postchallenge Immunogen No. of mouse samples positive/no. tested a Borrelia clearance from fed ticks E. coli with pbluescript 5/5 Negative Recombinant protein OspC 0/12 Negative P39 3/3 Negative Denatured protein b OspC 24/24 Negative OspA 0/1 Positive OspB 0/1 Negative a The results for the two assays, ear biopsy and Western blot, were identical. b Purified from B. burgdorferi lysate. seroconvert against the OspC antigen, as judged by immunoblot (Fig. 3). Twelve outbred mice inoculated with recombinant protein in the form of the soluble portion of sonicated crude E. coli lysates seroconverted against the OspC antigen (Fig. 4). Following seroconversion, the immunized mice were challenged with B. burgdorferi by infestation with ticks harboring the spirochetes. A group of five mice which had been inoculated with the E. coli lysate from cells containing pbluescript with no insert served as the infection control. To determine whether the mice became infected following challenge, direct culture for borrelia cells by ear biopsy was done 4 weeks after tick feeding. In addition, a Western blot of serum from each mouse was performed. The results are shown in Table 1 and Fig. 4. All 12 mice immunized with the recombinant OspC lysate were protected from infection; i.e., no spirochetes were detected in cultures from the ear tissue samples for as long as 3 weeks following biopsy. Also, immunoblots of the mouse serum samples indicated no reactivity to B. burgdorferi antigens other than the immunogen. In contrast, the control mice yielded both cultured spirochetes from the ear biopsies (after 7 days) as well as immunoreactivity to several B. burgdorferi antigens (Fig. 4). A group of three mice were immunized with lysate containing recombinant P39 and were also challenged by tick infestation as described above. In contrast to the recombinant OspCimmunized group, these mice were not protected. Cultures of the ear biopsies were positive for borrelial growth 7 days after inoculation (Table 1), and the serologic profiles for these mice at 2 weeks following challenge indicated several immunoreactive bands to B. burgdorferi proteins (Fig. 4). Presence of borrelia in ticks fed on recombinant OspCimmunized mice. Following feeding on the host, the replete ticks were collected and later analyzed for the presence of borrelia cells. Ten to 15 days following feeding, each tick collected from the recombinant OspC-immunized mice and the pbluescript mice were tested by OspA antigen-capture ELISA. Also, samples from the midguts of two ticks from each mouse were inoculated into BSK II medium. ELISA results from the OspA antigen capture showed high levels of OspA in ticks of both groups, indicative of the presence of spirochetes. No significant differences were found in the proportions of ticks positive by OspA antigen-capture Downloaded from on April 15, 2019 by guest FIG. 4. Immunoblots of sera from immunized mice prechallenge (A) and postchallenge (B) with B. burgdorferi from infected ticks. Twelve mice were immunized with E. coli recombinant (rec.) OspC lysate, and five were inoculated with lysate from E. coli harboring pbluescript with no insert. The seroreactivity postchallenge was assessed 4 weeks after ticks had fed to repletion. The serological profiles of the five control mice in panel B are indicative of infectivity and are identical to the patterns for serum following challenge of the 24 denatured-ospc-immunized mice and the recombinant P39-immunized mice. The positions of size markers (in kilodaltons) are indicated on the right.

5 2238 GILMORE ET AL. INFECT. IMMUN. ELISA that had fed on either the control or the recombinant OspC-immunized mice (chi square, 2.89; df, 1; P, 0.09). A total of 92% (54 of 59) of the ticks that had fed on recombinant OspC-immunized mice and 97% (31 of 32) of the ticks that had fed on control mice had detectable levels of OspA. These results correlate with the approximately 90% infection rate for the tick colony. However, OspA levels in B. burgdorferi-positive ticks in the two groups were significantly different (unpaired Student s t test: t, 3.169; df, 89; P, 0.002); i.e., ticks that had fed on the OspC-immunized mice had a mean of 1,060 pg of OspA per tick (standard deviation, 746; n 59), and ticks that had fed on the control mice had a mean of 1,567 pg of OspA per tick (standard deviation, 698; n 32). Statistically, this is a significant difference between the two groups; however, it is not a biologically relevant difference in that these OspA levels correspond to a large number of spirochetes in all ticks after feeding. There does not seem to be a correlation between the number of borrelia organisms present within a tick and its ability to transmit infection (2a). Moreover, this finding is very different from that observed for ticks fed on OspA-immunized animals, as the borrelia organisms residing in them are completely eradicated (11). Spirochetes were cultured from ticks that had fed on both control mice (10 culture positive of 10 ticks cultured) and recombinant OspC-immunized mice (19 culture-positive ticks of 20 cultured), which showed that these organisms were viable. These results indicate that spirochetes are not lysed within the tick midgut following the intake of the bloodmeal containing OspC antibodies, unlike that observed for OspA. DISCUSSION OspC has been documented to elicit a protective immune response in gerbils and inbred mice either in recombinant form or purified from borrelia cells (16, 21, 22) when the challenge dose of borrelia organisms to these animals was administered by needle inoculation of in vitro-cultured organisms. The experiments in this study also demonstrate that OspC is a protective immunogen, but this study differs significantly from the above-described studies in that the challenge was administered by the natural route of tick transmission. Changes in borrelia structure within the tick vector differ markedly from that seen in in vitro-grown borrelia organisms and may be important in assessing the efficacy of B. burgdorferi antigens as protective immunogens. Schwan et al. (24) showed that prior to the ticks feeding on a host, borrelia organisms within ticks have OspA present on their surfaces, but not OspC. Following tick feeding, the borrelia outer membrane structure alters dramatically in that OspC is expressed on the surface with OspA. Further, evidence suggests that during early infection in the host, B. burgdorferi may produce OspC, but little or no OspA, as seen by the lack of a host antibody response against OspA (4, 19, 24). This finding is both critical and relevant to the evaluation of Lyme disease vaccine candidates, especially in view of the structural changes that occur on the surface of the tick-borne borrelias which do not occur in culture-grown borrelias. It has been demonstrated that OspA-immunized animals exhibit a high antibody titer to OspA and that these animals are effectively protected from B. burgdorferi infection whether the challenge dose is administered with cultured organisms or by tick bite (7, 12, 26). One mechanism responsible for protection against tick-borne infection is eradication of borrelias from the tick midgut during feeding on a host that has a high serum antibody level to OspA (11). Also, since in vitro-cultured borrelias exhibit high levels of OspA on their surfaces, the OspA-immune host can effectively clear these organisms. Thus, these protective mechanisms may depend on the duration of a circulating serum antibody response. Host-adapted spirochetes are not cleared from an OspA-immunized host (1), perhaps because these organisms have little or no surfaceexposed OspA and therefore OspA antibody has no effect. Since OspC is highly expressed on B. burgdorferi transmitted by ticks, the protective immune mechanism afforded by an OspC vaccination is distinct from and may be more advantageous than that of OspA. This study further demonstrates that ticks which have fed on OspC-immunized mice still harbor live spirochetes. That is, we see no evidence of borrelicidal activity by the anti-ospc serum in the feeding ticks. Consequently, the protective mechanism for OspC-induced immunity may be postulated as two possibilities. Either borrelia organisms transmitted to the host animal are disposed of by the host s immune response, or the OspC antibodies in the bloodmeal taken by the tick prevents transmission of the borrelia organisms to the host. Considering the former, experiments are in progress to determine if a high level of host serum OspC antibody is necessary for this protection or whether a vaccinated animal whose serum antibody level has decreased with time mounts a protective response when boosted with a challenge dose of borrelias. It will be interesting to determine if the surface-exposed OspC on infectious borrelias can induce a protective anamnestic response in the host and thus serve to produce long-term immunity. In either case, it appears that the protective immune mechanism for OspC is different from that for OspA-induced immunity. In an earlier and separate experiment, OspC purified from B. burgdorferi lysates failed to protect mice against a ticktransmitted challenge infection, a result opposite that seen by Livey et al. (16) for animals immunized with native OspC from B. burgdorferi. However, the OspC in our experiment was purified by preparative SDS-PAGE, and the antigen was subjected to denaturation by heat, SDS, and 2-mercaptoethanol. Although capable of eliciting a strong anti-ospc response, this antigen preparation did not confer protection from infection. The recombinant form of OspC which was not subjected to denaturation prior to inoculation was protective, thus indicating the strong probability of a conformational protective epitope. The presence of a heat-stable linear protective epitope on OspC seems to be unlikely. Since recombinant OspC was effective in protecting against infection, it is probable that the conformational neutralizing epitope is reproduced during expression in E. coli and could be used to localize the protective epitope. Some studies have provided results suggesting that the P39 antigen may be protective (15, 25). We performed a direct evaluation of the protective effectiveness of recombinant P39 used as an immunogen in outbred mice. Even though the test mice exhibited a high titer of anti-recombinant P39 when challenged by tick bite, they were not protected from infection. We have not proven, however, that native borrelial P39 is not protective. There is the possibility that recombinant P39 is not expressed in its native form in E. coli, and any putative protective conformational epitopes that may exist on native P39 may not be formed by the proper protein folding during expression. In conclusion, we have demonstrated the protection of mice from a tick-transmitted infection of B. burgdorferi when the animals were immunized with recombinant OspC and the failure of OspC derived from denatured borrelial lysates to confer similar protection. Ticks that fed on mice with high levels of serum antibodies against the recombinant OspC still harbored live B. burgdorferi organisms, indicating that the protective response is not mechanistically related to destruction of the

6 VOL. 64, 1996 OspC PROTECTS AGAINST TICK-TRANSMITTED B. BURGDORFERI 2239 borrelias in the tick prior to host transmission. The observation that denatured OspC elicits an antibody response in mice but fails to confer protection, coupled with the demonstrated protective capabilities of native recombinant OspC, suggests that the protective epitope of OspC is conformational in nature. This finding may prove to be significant in studies involving OspC epitope mapping for vaccine development. ACKNOWLEDGMENTS This project was supported by a collaborative research and development agreement with SmithKline Beecham Biologicals, Rixensart, Belgium, and Pfizer Animal Health, Lincoln, Nebr. We acknowledge the contributions of Lamine Mbow for his time in reviewing the manuscript; the Centers for Disease Control and Prevention Lyme disease group including Chris Happ, Bill Golde, and Steve Sviat for their assistance; and Joe Piesman in providing and supervising the maintenance of the tick colonies. 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