1999, 67(9):4407. Infect. Immun. 

Michael A. LovettDeanna C. Moore, David R. Blanco, James N. Miller and Jonathan T. Skare, Denise M. Foley, Santiago R. Hernandez, 

burgdorferiBorreliaPlasmid-Encoded Antigens of

Cloning and Molecular Characterization of

http://iai.asm.org/content/67/9/4407Updated information and services can be found at:

These include:

REFERENCEShttp://iai.asm.org/content/67/9/4407#ref-list-1at:

This article cites 45 articles, 24 of which can be accessed free

CONTENT ALERTS more»articles cite this article),

Receive: RSS Feeds, eTOCs, free email alerts (when new

http://journals.asm.org/site/misc/reprints.xhtmlInformation about commercial reprint orders: http://journals.asm.org/site/subscriptions/To subscribe to to another ASM Journal go to:

on March 22, 2014 by guest

http://iai.asm.org/

Dow

nloaded from

on March 22, 2014 by guest

http://iai.asm.org/

Dow

nloaded from

INFECTION AND IMMUNITY,0019-9567/99/$04.0010

Sept. 1999, p. 4407–4417 Vol. 67, No. 9

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Cloning and Molecular Characterization of Plasmid-EncodedAntigens of Borrelia burgdorferi

JONATHAN T. SKARE,1,2* DENISE M. FOLEY,2† SANTIAGO R. HERNANDEZ,2 DEANNA C. MOORE,1

DAVID R. BLANCO,2 JAMES N. MILLER,2 AND MICHAEL A. LOVETT2,3

Department of Medical Microbiology and Immunology, Texas A&M University Health Science Center, College Station,Texas 77843-1114,1 and Departments of Microbiology and Immunology2 and of Medicine,3

UCLA School of Medicine, Los Angeles, California 90095-1747

Received 16 March 1999/Returned for modification 30 April 1999/Accepted 16 June 1999

Thirteen independent clones that encode Borrelia burgdorferi antigens utilizing antiserum from infection-immune rabbits were identified. The serum was adsorbed against noninfectious B. burgdorferi B31 to enrich forantibodies directed against either infection-associated antigens of B. burgdorferi B31 or proteins preferentiallyexpressed during mammalian infection. The adsorption efficiency of the immune rabbit serum (IRS) wasassessed by Western immunoblot analysis with protein lysates derived from infectious and noninfectious B.burgdorferi B31. The adsorbed IRS was used to screen a B. burgdorferi expression library to identify immuno-reactive phage clones. Clones were then expressed in Escherichia coli and subsequently analyzed by Westernblotting to determine the molecular mass of the recombinant B. burgdorferi antigens. Southern blot analysis ofthe 13 clones indicated that 10 contained sequences unique to infectious B. burgdorferi. Nucleotide sequenceanalysis indicated that the 13 clones were composed of 9 distinct genetic loci and that all of the genes identifiedwere plasmid encoded. Five of the clones carried B. burgdorferi genes previously identified, including thoseencoding decorin binding proteins A and B (dbpAB), a rev homologue present on the 9-kb circular plasmid(cp9), a rev homologue from the 32-kb circular plasmid (cp32-6), erpM, and erpX. Additionally, four previouslyuncharacterized loci with no known homologues were identified. One of these unique clones encoded a451-amino-acid lipoprotein with 21 consecutive, invariant 9-amino-acid repeats near the amino terminus thatwe have designated VraA (for “virulent strain-associated repetitive antigen A”). Since all the antigens iden-tified are recognized by serum from infection immune rabbits, these antigens represent potential vaccinecandidates and, based on the identification of dbpAB in this screen, may also be involved in pathogenicprocesses operative in Lyme borreliosis.

Lyme disease, caused by various strains of Borrelia burgdor-feri sensu lato (referred to as B. burgdorferi throughout thisreport), is a multistage disorder that presents initially as aflu-like illness and, if untreated, can progress to a chronic state,characterized predominantly by arthritic manifestations in af-fected individuals in the United States (19, 29, 41–44). As yet,few virulence determinants have been identified for B. burg-dorferi even though significant differences have been detectedin the plasmid DNA profiles of infectious and noninfectiousisolates of in vitro cultivated B. burgdorferi (37, 50). A signifi-cant amount of work has been devoted to the identification ofplasmid-encoded proteins associated with an infectious pheno-type (10, 31, 32). However, identification and characterizationof infection-associated proteins have been difficult inasmuch asprotein profiles of whole-cell lysates derived from infectiousand noninfectious B. burgdorferi isolates show only subtle vari-ations, even when resolved by two-dimensional polyacrylamidegel electrophoresis (PAGE) (31). This observation suggeststhat the genes encoding infection-associated proteins arepoorly expressed during in vitro cultivation or that the tech-niques used for detection were not sensitive enough to accountfor the slight differences in protein content between the infec-

tious and noninfectious cells. More recently, we (40) and oth-ers (8, 31) have analyzed differences in the antigenic profilesfrom infectious and noninfectious B. burgdorferi to identifyinfection-associated antigens expressed during in vitro cultiva-tion and those expressed preferentially during infection (1, 10,13, 47).

In this report, we describe the use of infection-derived im-mune rabbit serum (IRS) enriched for antibodies directedagainst infection-associated antigens to identify cloned genesencoding these molecules from B. burgdorferi phage l expres-sion libraries. Using this approach, we have identified 13 inde-pendent clones. Nucleotide sequence analysis, in conjunctionwith the recently completed B. burgdorferi genome project (18),indicated that all the clones identified in our screen were plas-mid borne and were restricted to nine genes including thoseencoding the previously described decorin binding proteins Aand B (9, 24–26). Inasmuch as the antigens identified in thisscreen are recognized by serum from infection-immune rab-bits, these molecules may serve as a subset of targets for anti-body-dependent killing and, as such, may have utility as alter-native immunogens to protect against Lyme borreliosis.Additionally, the identification of the decorin binding proteinssuggests that other antigens identified in this screen may pro-vide insight into the pathogenesis of Lyme disease.

MATERIALS AND METHODS

Bacterial strains and plasmids. B. burgdorferi sensu stricto B31 was used in allstudies described in this report. B. burgdorferi was cultured at 32°C in a 1% CO2atmosphere in BSK II medium (4) supplemented with 6% normal rabbit serum(Pel-Freez Biologicals, Rogers, Ark.). Clonal isolates of B. burgdorferi wereisolated by plating agarose overlays containing dilutions of B. burgdorferi as

* Corresponding author. Mailing address: Department of MedicalMicrobiology and Immunology, 407 Reynolds Medical Building, TexasA&M University Health Science Center, College Station, TX 77843-1114. Phone: (409) 845-1376. Fax: (409) 845-3479. E-mail: jskare@tamu.edu.

† Present address: Department of Biological Science, ChapmanUniversity, Orange, CA 92866.

4407

on March 22, 2014 by guest

http://iai.asm.org/

Dow

nloaded from

previously described (32). All infectious isolates used were passaged no morethan seven times in vitro, and they infect both mice and rabbits (16). The B.burgdorferi B31 noninfectious isolate used in this study has been passaged severalhundred times in vitro and has previously been shown to be noninfectious in bothmice and rabbits (16).

Escherichia coli ER1647, BM25.8, and BL21 (lDE3) pLysE were purchasedfrom Novagen Inc., Madison, Wis. Strain ER1647 (F2 fhuA2 D(lacZ)r1 supE44recD1014 trp31 mcrA1272::Tn10 his-1 rpsL104 xyl7 mtl2 metB1 D(mcrC-mrr)102::Tn10 hsdS[rK12

2 mK122]) was used for propagating phage lEXlox (Nova-

gen Inc.). Phage l overlays were plated with 1% Bacto Tryptone, 0.5% yeastextract, and 0.5% NaCl in 0.6% agarose. Strain BM25.8 (F9 [traD36 lacIq

lacZDM15 proAB] supE thi D(lac-proAB) [P1] Cmr r2 m2 limm434 Kanr) wasused for Cre-mediated in vivo excision of plasmid DNA from lEXlox. Therescued plasmids (designated pEX) were isolated from BM25.8 by alkali lysis andwere used to transform competent strain BL21 (lDE3) pLysE. The subsequenttransformants were then used to express genes encoding B. burgdorferi antigens.All E. coli strains were grown in Luria broth at 37°C with aeration or on eitherLuria broth or 23 YT agar at 37°C. E. coli was grown in appropriate antibioticsat the following concentrations: ampicillin, 100 mg/ml; kanamycin, 25 mg/ml; andchloramphenicol, 50 mg/ml.

Serum preparation. Passage 4 B. burgdorferi B31 (4 3 103 cells) was inoculatedintradermally into the shaved backs of New Zealand White rabbits, and afterapproximately 7 to 10 days, lesions similar to erythema migrans (EM) wereobserved (16). Punch biopsy specimens obtained in and around the location ofthe EM lesions contained infectious B. burgdorferi as assessed by growth ofspirochetes in BSK II medium (16). Additional punch biopsies and necropsy ofselected rabbits were conducted over the next 2 months to determine whether theorganisms persisted in the skin and viscera, respectively (16). It should be notedthat the rabbits were not subjected to antibiotic therapy at any stage of theinfection. When organisms were no longer detected in the skin punch biopsyspecimens, the rabbits were challenged with 107 cells of passage 4 B. burgdorferiB31. Previous studies indicated that the absence of B. burgdorferi in punch biopsyspecimens correlated with the systemic clearance of the spirochetes from theviscera (16). The challenged rabbits did not develop EM lesions, and no culture-positive B. burgdorferi was obtained from either skin punch biopsy specimens orviscera. Serum from these infection-immune rabbits was designated IRS.

To reduce the population of antibodies in IRS that recognize common anti-gens between infectious and noninfectious B. burgdorferi and enrich for antibod-ies specific for infection-associated antigens, we adsorbed IRS with noninfectiousB. burgdorferi B31 prepared as described by Gruber and Zingales (23). Briefly,0.5 liter of noninfectious B. burgdorferi B31 (approximately 1.0 3 1011 to 1.5 31011 cells) was grown to late log phase and pelleted at 5,800 3 g for 20 min at 5°C,the supernatant was removed, and the resulting pellet was suspended in an equalvolume of phosphate-buffered saline (PBS) (pH 7.4). The sample was split intotwo 250-ml volumes; half was autoclaved at 22 lb/in2 and 121°C for 1 h, and theother half was incubated with 0.5% (wt/vol) formaldehyde at 37°C for 2 h withvigorous shaking. Each sample was then centrifuged separately at 5,800 3 g for20 min, washed with an equal volume of PBS, and repelleted as described above.The pellets were then resuspended in 100 ml of PBS, combined, and centrifugedas indicated above, and the final pellet was then resuspended in 15 ml of PBS.For adsorption, 1-ml volumes of the above preparation were centrifuged at16,000 3 g for 5 min, the supernatant was decanted, and the pellet was combinedwith 1 ml of IRS. IRS was incubated with the noninfectious B. burgdorferi B31isolate for 8 h or overnight with rocking at 4°C, after which the insoluble B.burgdorferi fraction was pelleted and the subsequent supernatant was transferredto a new tube for readsorption. This process was repeated until the adsorbed IRSlost reactivity with noninfectious B. burgdorferi B31 as determined by Westernimmunoblotting (see below).

Construction of the B. burgdorferi lEXlox library. Chromosomal, linear plas-mid, and circular plasmid DNA was isolated from infectious B. burgdorferi B31passage 4 as previously described (20). Linear and circular plasmid DNA wasseparated on a CsCl density gradient and enriched as previously described (20)or purified with tip 500 Maxi-prep columns from Qiagen Corp. (Chatsworth,Calif.). All three DNA isolates were digested separately with dilutions of ApoI,and DNA fragments, ranging in size from 0.5 to 4 kb, were separated by agarosegel electrophoresis, cut from the gel, purified from the gel with GeneClean (Bio101, San Diego, Calif.), and ligated to phosphatase-treated lEXlox arms thathave EcoRI ends (Novagen). The resulting ligations were packaged in vitro intophage l by using the Gigapack Gold kit from Stratagene Inc. (San Diego, Calif.)and subsequently amplified to obtain phage lysates on the order of 1 3 1010 to5 3 1010 PFU per ml. Phage lysates were maintained and stored as outlined bythe manufacturer.

To determine whether our libraries were representative, we used antibodiesspecific for either endoflagella, OspD, or EppA to screen our B. burgdorferichromosomal, linear plasmid, and circular plasmid libraries, respectively. TheClarke-Carbon formula was used to determine whether each library was repre-sentative. Genes that mapped to the telomere-like ends of the chromosome andlinear plasmids were absent from their respective libraries.

Screening of the B. burgdorferi expression library. Nitrocellulose filters (137mm in diameter) were preincubated in 10 mM isopropyl-b-D-thiogalactopyrano-side (IPTG), dried, and then overlaid separately onto plates (15 by 150 mm)containing approximately 3,000 plaques from each B. burgdorferi phage l library.

After a 4-h incubation at 37°C, the filters were removed and washed with PBS(pH 7.4) to remove excess bound agar and bacteria. The filters were thenprocessed as a conventional Western immunoblot to identify immunoreactiveplaques (see below). Briefly, adsorbed immune rabbit serum (see above andreferences 16 and 40) was incubated with the filters at a dilution of 1:500 in PBScontaining 5% powdered milk and 0.2% Tween 20 (PM-PBS-T). After incuba-tion, the filters were washed with PBS–0.2% Tween 20 (PBS-T) and then incu-bated with a 1:2,500 dilution of goat anti-rabbit immunoglobulin with conjugatedhorseradish peroxidase (HRP) in PM-PBS-T. The filters were then washedextensively with PBS-T, and the specific antibody-antigen interaction was iden-tified with the enhanced chemiluminescence (ECL) system from AmershamCorp. (Arlington Heights, Ill.). Immunoreactive plaques were selected and re-screened as described above to ensure that a clonal isolate was obtained.

Characterization of recombinant infection-associated clones. Plasmids encod-ing B. burgdorferi antigens were isolated as described by Novagen, using E. coliBM25.8 (see above for the genotype) and were designated pEXC, pEXL, orpEXS for those isolated from the chromosomal, linear plasmid, or supercoiledcircular plasmid libraries, respectively. The plasmids were then isolated fromstrain BM25.8 by alkali lysis and used to transform BL21 (lDE3) pLysE. BL21(lDE3) pLysE cells containing individual pEX plasmids were grown to mid-logphase (optical density at 600 nm [OD600] of 0.5) and induced with 0.4 mM IPTGfor 2 h at 37°C. The cells were then pelleted, washed with PBS (pH 7.4), andresuspended in Laemmli final sample buffer (28) to a final concentration of 0.01OD600 unit per ml. Protein from 0.05 to 0.1 OD600 unit of cells was separated bysodium dodecyl sulfate (SDS)-PAGE, transferred to polyvinylidine fluoride (Im-mobilon-P; Millipore Corp., Bedford, Mass.), and processed for immunoblottingas described below.

Immunoblotting. Western immunoblotting was conducted essentially as de-scribed previously (40). Antisera to DbpA and DbpB were generously providedby Betty Guo and Magnus Hook, Institute of Biosciences and Technology, TexasA&M University, Houston, Tex. Antisera specific for OspD and VlsE weregenerous gifts from Steven Norris, University of Texas Health Science Center,Houston, Tex. Monoclonal antibody H9724, specific for B. burgdorferi endofla-gella, was generously provided by Alan Barbour, University of California, Irvine,Calif. The adsorbed serum from infection-immune rabbits was prepared as de-scribed above. Infection-derived mouse serum was obtained by inoculating micewith 104 CFU of low-passage B. burgdorferi B31 intradermally. After 3 months,the mice were bled and sera from six mice were pooled. Human sera weregenerously provided by Alan Steere, Tufts University, and were from patientsthat had documented cases of chronic Lyme borreliosis.

The primary antibodies used were diluted as follows: anti-DbpA, anti-DbpB,and anti-OspD at 1:5,000, anti-EppA (10) at 1:2,000, anti-EF at 1:25, and ad-sorbed IRS at 1:500 to 1:1,500. Both the mouse and human sera were diluted1:1,000. Secondary antibodies or protein A conjugated to HRP were purchasedfrom Amersham Corp. Donkey anti-rabbit immunoglobulin-HRP, sheep anti-mouse immunoglobulin-HRP, and protein A-HRP were used at dilutions of1:5,000, 1:2,500, and 1:1,000, respectively. All blots were developed with the ECLsystem.

Southern blotting. Linear or supercoiled, circular plasmid DNA was digestedto completion with either EcoRI or HindIII, and the DNA was resolved on 0.8%agarose gels, transferred to a positively charged nylon membrane (BoehringerMannheim, Indianapolis, Ind.) via capillary transfer overnight, and then cross-linked to the membrane by exposure to UV light for 5 min. Random primedprobes were prepared by using the Gene Images kit from Amersham. Briefly, thepEX clones (i.e., template) were diluted to 40 ng/ml in sterile H2O, boiled for 5min, and placed immediately in a wet ice bath. A 25-ml volume of the appropriatetemplate (i.e., 1 mg) was then added to a collection of random decamer primers,a nucleotide mix containing dATP, dGTP, dTTP, dCTP, dUTP conjugated tofluorescein, and 5 U of Klenow fragment. The reaction mixture was incubated at37°C for 1 h, and the reaction was continued overnight at room temperature inthe dark. After the overnight transfer and cross-linking indicated above, the blotwas incubated in 10 ml of prehybridization buffer (75 mM sodium citrate–0.75 Msodium chloride [53 SSC], 0.1% SDS, 0.5% Hammersten’s casein [United StatesBiochemical, Cleveland, Ohio], 5% dextran sulfate [Pharmacia Biotech, Piscat-away, N.J.]) for 1 h at 65°C. During the prehybridization step, the probe wasdiluted in 5 ml of hybridization buffer (same as the prehybridization buffer listedabove), boiled for 5 min, and, following the prehybridization step, added imme-diately to the blot and incubated at 65°C for 4 h. After 4 h, the probe wasremoved and stored at 280°C. The blot was rinsed with low-stringency washbuffer (23 SSC, 0.1% SDS) at room temperature followed by two additional15-min washes at 65°C. The blot was then washed twice in high-stringency washbuffer (0.13 SSC, 0.1% SDS) at 65°C for 15 min. The blot was washed in buffer1 (0.1 M Tris HCl [pH 7.5], 0.15 M NaCl) and then incubated overnight at 4°Cin blocking buffer (buffer 1 containing 0.5% Hammersten’s casein). The blot wassubsequently incubated for 1 h in blocking buffer containing a 1:1,000 dilution ofanti-fluorescein conjugated to HRP (Amersham). After 1 h, the blot was washedfive times with buffer 1, developed with the ECL system, and exposed to KodakXAR-5 film for 10 to 30 min.

Nucleotide sequence accession number. The nucleotide sequence of a 2-kbfragment containing the vraA locus (i.e., clone L3) has been deposited in Gen-Bank under accession no. AF019082.

4408 SKARE ET AL. INFECT. IMMUN.

on March 22, 2014 by guest

http://iai.asm.org/

Dow

nloaded from

RESULTS AND DISCUSSION

Preparation of adsorbed immune rabbit serum. To developa reagent that would preferentially identify B. burgdorferi an-tigens that are associated with infectious isolates, we usednoninfectious B. burgdorferi B31 to adsorb serum from immunerabbits that were resistant to challenge with 107 CFU of infec-tious B. burgdorferi B31 passage 4. We then used the adsorbedserum as the primary antibody in a Western blot assay to assessthe clearance of antibodies that recognize common antigens byprobing whole-cell protein lysates from both in vitro-cultivatednoninfectious and infectious (passage 7) B. burgdorferi. Asshown in Fig. 1, after either seven or nine adsorptions, someimmunoreactive bands were still observed in the noninfectiousisolate, although significantly more bands were observed fromthe infectious isolate. After 12 adsorptions against noninfec-tious B. burgdorferi, no immunoreactive bands were observedin the noninfectious B. burgdorferi blot, but a subset of antigensretained strong reactivity in the infectious samples, indicatingthat these are infection-associated antigens (Fig. 1).

The three major infection-associated antigens had molecularmasses of 29, 35, and approximately 40 to 43 kDa. We haveretrospectively determined that the highest-molecular-mass,major-infection associated antigen observed by Western blotanalysis is VlsE (approximately 40 to 43 kDa [Fig. 1]) (refer-ence 51 and data not shown). Although VlsE is a major im-munogen in the rabbit, we did not identify clones encoding thisantigen, suggesting that vlsE and other analogous loci locatednear the telomere-like ends are missing from our chromosomaland linear plasmid libraries. Alternatively, we cannot excludethe possibility that some of our recombinant clones, includingthose carrying vlsE, were toxic when expressed in E. coli andwere subsequently lost during or after the in vivo excision step.The 40- to 43-kDa range observed most probably reflects the

fact that the protein lysate used for immunoblotting was de-rived from a nonclonal population of infectious B. burgdorfericontaining different VlsE variants. The 29-kDa antigen is thepreviously described infection associated lipoprotein, OspD(31). This was determined by screening 20 clones derived frominfected C3H/HeJ mouse skin. Infected tissue was grown inliquid medium and plated to isolate individual colonies. Of the20 clones tested, 4 lacked the 38-kb linear plasmid (lp38), theplasmid known to carry ospD. Additionally, protein lysatesfrom these clones analyzed by Western blotting with antibodyspecific for OspD showed no immunoreactive species. Subse-quent two-dimensional PAGE demonstrated that these cloneslacked a spot that immunoreacted with antibody to OspD (datanot shown). The 35-kDa antigen has not been definitively iden-tified but most probably represents a p35/p37 paralogous fam-ily member (13, 18).

A similar type of analysis was conducted by Carroll andGherardini (8) to identify antigens that were associated withinfectious strains. The profile reported in their study contrastswith what we have observed, perhaps due to the differencespertaining to the sera which were used; that is, the serum usedin this study was acquired after approximately 5 months frominfection-immune rabbits (16, 40), whereas the serum used intheir study was isolated from rabbits at 4 weeks postinfection(8).

Identification and characterization of cloned antigens.Clones identified in our expression library screen were furtheranalyzed to determine the molecular mass of the recombinantantigens. As shown in Fig. 2, the clones identified encodedproteins that were antigenic, ranging in molecular mass fromapproximately 18 to 70 kDa. Sequence analysis indicated thatthe 13 immunoreactive clones encoded nine putative infection-associated antigens and had insert sizes ranging from 302 to4,343 bp (Table 1 gives a summary). Nucleotide sequenceanalysis indicated that the 13 clones remaining were composedof nine distinct genes. Seven of the nine genes identified in ourscreen are carried by plasmids that have been shown by pre-vious investigators (10, 20, 32, 37, 50, 51) and in this study to belost during in vitro cultivation, while the other two, carrying thegenes encoding decorin binding proteins A and B (dbpAB) andthe erpX gene, are located on plasmids common to both infec-tious and noninfectious B. burgdorferi B31 (Fig. 3, erpX anddbpAB panels), suggesting that dbpAB and erpX are preferen-tially expressed in the rabbit. The Southern blot data shown inFig. 3 demonstrates the utility of our screen in terms of iden-tifying loci that are infection associated or expressed in vivo.Therefore, such methodology should be applicable to othersystems that are genetically intractable like B. burgdorferi.

Previous studies have shown a correlation between the lossof a subset of plasmids and a loss of infectivity in animalmodels of infection (37, 50), suggesting that plasmid-bornegenes are required for the infectious phenotype. More re-cently, Norris et al. has shown that one can discriminate be-tween B. burgdorferi of high- and low-infectivity phenotypeswithin a nonclonal population of B. burgdorferi based, in part,on the presence of a 28-kb linear plasmid now designatedlp28-1 (32), which contains the antigenically variable locusvlsE1 (51). Other independent studies have identified antigensexpressed preferentially in vertebrate infection (10, 13, 26, 49),notably ospE or ospE-related proteins (erp genes) (11, 46) andospF homologues (2). This report incorporates an additionalimportant aspect not evaluated in previous studies, i.e., the useof antibodies from an infected and immune animal to identifyantigens either specific for or expressed preferentially in infec-tious isolates of B. burgdorferi. Inasmuch as the rabbit is theonly small-animal model that results in infection-derived im-

FIG. 1. Demonstration that adsorbed IRS is cleared of common antibodiesand contains antibodies specific for infection-associated antigens. IRS was ad-sorbed as described in Materials and Methods and used as a primary antibodyagainst proteins lysates from either passage 5, infectious strain B31 B. burgdorferi(left) or high-passage, noninfectious strain B31 B. burgdorferi (right). The low-passage and high-passage antigen strips contained protein from 2.5 3 107 and1 3 108 whole-cell equivalents, respectively. The numbers listed horizontallyindicate the number of adsorptions against noninfectious strain B31 B. burgdor-feri. Vertical numbers indicate the molecular masses of markers in kilodaltons.

VOL. 67, 1999 B. BURGDORFERI PLASMID-ENCODED ANTIGENS 4409

on March 22, 2014 by guest

http://iai.asm.org/

Dow

nloaded from

munity in the absence of antibiotic treatment to clear theprimary infection and since several studies have indicated theimportance of antibodies in B. burgdorferi clearance (5, 27), thegenes identified by this approach may have significance toprotective immunity. Along these lines, adsorbed immune rab-bit serum preferentially binds to the surface of infectious B.burgdorferi (40) and mediates complement-dependent killingof infectious B. burgdorferi (15). Additionally, the identificationin our screen of decorin binding protein, a known adhesin of B.burgdorferi (24), supports the likelihood that additional anti-gens identified in this screen may be important in the patho-genesis of Lyme borreliosis. The following subheadings pro-vide details on the clones identified.

(i) rev (BBC10 [cp9] and BBM27 [cp32-6]). Nucleotide se-quence data, coupled with the recently completed B. burgdor-feri genome project, indicated redundancy within our screenfor both the rev and dbpAB loci. The rev locus was clonedindependently seven times, and the dbpAB locus was clonedtwice. Of the seven rev clones isolated, six mapped to the 9-kbcircular plasmid (cp9), a previously described B. burgdorferiinfection-associated plasmid (10, 37, 50). Two of the six cp9 revclones were identical and as such have been eliminated fromfurther analyses. Previously, rev had been associated only withcp32 plasmids (21, 36); however, not until the completion ofthe genomic sequence was it revealed that infectious B. burg-dorferi contained three copies of rev that mapped to cp9,cp32-1, and cp32-6 (18, 48). pEX clones S4, S7, and S10 allcontain the rev locus found on the 9-kb circular plasmid (cp9)that is distinct from but homologous to the rev loci located bythe 32-kb circular plasmids. The cp9 Rev protein is 175 amino

acids in length, as opposed to its 160-amino-acid cp32 Revparalogue. cp9 Rev and cp32 Rev are 37% identical and 51%similar at the amino acid level; however, cp9 Rev containsdomains near both the amino and carboxy termini that areunique (data not shown). Whether these domains define animmunodominant infection-associated epitope(s) specific forcp9 Rev is not known. As expected, the Southern blot witheither the S1, S4, S7, or S10 clones as probes indicates that cp9rev is a infection-associated gene enriched for in our circularDNA fractions (Fig. 3, cp9 rev panel).

The Institute for Genomic Research (TIGR) database indi-cates that B. burgdorferi B31 contains seven copies of cp32 andthat two of these plasmids, cp32-1 and cp32-6, contain the revlocus (48). Clone S3 makes an in-frame fusion to the T7 gene10 protein, adding 81 residues from cp32 Rev to 260 aminoacids of the T7 gene 10 protein (Fig. 2), resulting in the for-mation of an approximately 38-kDa recombinant fusion pro-tein. This particular rev clone corresponds to the locus re-ported recently by Gilmore and Mbow (21). Sequence analysisindicated that clone S3 contains the fragment from cp32-6containing rev that is specific to infectious B. burgdorferi (des-ignated BBM27 by TIGR); however, the S3 probe also recog-nizes the rev locus present on cp32-1 (designated BBP27 byTIGR). This was determined by analyzing the nucleotide se-quences of cp32-1 and cp32-6 present in the TIGR databaseand comparing the predicted restriction fragments containingrev with the fragments we visualized in our Southern blotanalyses (Fig. 3, panel S3). The 3.3-kb EcoRI and 5.5-kbHindIII bands observed in the lanes containing DNA fromnoninfectious B. burgdorferi correspond to rev-containing frag-

FIG. 2. Molecular mass of recombinant antigens encoded by expression library clones. Immunoreactive clones were identified from phage l libraries and insertsencoding the antigens excised in vivo as described in Materials and Methods. The clones were then resolved by SDS-PAGE, blotted to a polyvinylidene difluoridemembrane, and incubated with adsorbed immune rabbit serum. Each lane is labeled with the pEX clone designation assigned to each immunoreactive recombinant(see Table 1 for a summary). The asterisk above clone S3 indicates that it contains the rev insert from cp32-6. Vertical numbers indicate the molecular masses of markersin kilodaltons.

4410 SKARE ET AL. INFECT. IMMUN.

on March 22, 2014 by guest

http://iai.asm.org/

Dow

nloaded from

ments from cp32-1, while the lanes containing DNA derivedfrom infectious B. burgdorferi have rev-containing fragmentsfrom cp32-1 as well as the 4.7-kb EcoRI and 3.7-kb HindIII revbands specific for cp32-6. Sequence analysis of clone pEXS3indicated that the nucleotide sequence matches in its entiretythe cp32-6 rev sequence (data not shown). These results, takentogether, indicate (i) that clone S3 encodes cp32-6 rev and (ii)that cp32-1 and cp32-6 are both present in our infectious iso-lates of B. burgdorferi B31 whereas cp32-6 is missing from ournoninfectious B. burgdorferi isolate, indicating that it is aninfection-associated plasmid. Curiously, the cp32-6 rev probedid not recognize the cp9 rev locus; however, in contrast, cp9rev did weakly hybridize with the rev-containing 5.5-kb and3.7-kb HindIII fragments from cp32-1 and cp32-6, respectively(Fig. 3, cp9 rev panel, lane 1).

Although Porcella et al. reported that Rev encoded by B.burgdorferi 297 was processed by a leader peptidase I cleavagesite (36), our studies suggest that Rev is processed by leaderpeptidase II at the lipoprotein consensus sequence VISC forcp9 Rev (residues 19 to 22 of the precursor protein) andVMAC for cp32 Rev (residues 17 to 20 of the precursor pro-tein). More recently, Gilmore and Mbow reported on the cp32rev locus from B. burgdorferi B31 and demonstrated that serumfrom patients with documented Lyme infection recognize theRev protein, suggesting that Rev may have use both as aprotective immunogen and in diagnostic applications (21). Weare currently in the process of testing the efficacy of Rev in thiscontext.

(ii) vraA (BBI16; lp28-4). pEXL3 encoded a novel lipopro-tein antigen, found only in infectious isolates of B. burgdorferiB31 (Fig. 3, vraA panel), that contains 21 consecutive 9-amino-acid repeats near the amino terminus of the mature protein.Based on this characteristic, we have designated this proteinVraA (for “virulent strain-associated repetitive antigen A”).The vraA locus (BBI16 in the TIGR nomenclature [48]) mapsto lp28-4 and belongs to a large paralogous gene family (18,48). However, vraA is distinct from the rest of the paralogousfamily due to the repetitive units (data not shown). Although

we cannot be certain that our screen was exhaustive, it iscurious that none of the other paralogues were identified.Since the proteins within this paralogous gene family havesignificant similarity at their amino and carboxy termini, themost likely choice for the antigenic domain is the repetitivesegment. Along these lines, we have prepared fusion proteinsand antisera to the (i) mature full-length VraA, (ii) amino-terminal half (containing the repeat units), and (iii) carboxy-terminal half (lacking any of the repeat units) and have deter-mined that the repetitive region is the antigenic domain (3).We are currently evaluating whether the repetitive domain ofVraA has any biological activity.

(iii) erp clones: erpM (BBO40; cp32-7) and erpX (BBQ47;lp56). Two of the immunoreactive clones mapped to erp loci:pEXC3 contained erpM and pEXL11 contained erpX. TheerpM locus maps to cp32-7 (designated BBO40 by TIGR [48]),and erpX maps to lp56 (designated BBQ47 by TIGR [48]).Southern blot analysis indicated that the erpM-containingprobe recognized sequences from the low-passage, infectiousB. burgdorferi B31 as well as sequences unique to the high-passage, noninfectious isolate (Fig. 3, erpLM panel). By access-ing the cp32-7 sequence, one can predict which EcoRI andHindIII fragments should hybridize to DNA derived from in-fectious B. burgdorferi when incubated with the 2,804-bppEXC3 probe in a Southern blot assay. An EcoRI restrictiondigest should yield a single 11,532-bp fragment, whereas aHindIII digest should yield 4,675-, 3,744-, 994-, and 111-bpfragments. These predictions are in good agreement with theSouthern profile of DNA derived from infectious B. burgdorferishown in Fig. 3, excluding the weakly reactive 3-kb band in theHindIII digest (Fig. 3, erpLM panel, lane 1) and the 3.5-kbfragment in the EcoRI digest (Fig. 3, erpLM panel, lane 2),which probably reflect hybridizing segments in other cp32 se-quences relative to non-erp sequences found in pEXC3. Whilereverse transcription-PCR (RT-PCR) analysis with erpM prim-ers amplified fragments from both infectious and noninfectiousB. burgdorferi strains, different sizes were detected, suggestingeither that the primers recognize common sequences in several

TABLE 1. Summary of expression clones identified

pEX clone Plasmid DNAa

Position of b:

Insert size (bp) TIGRdesignationc Genetic locus Infection

associateddT7g10 end SP6

end

S1 cp9 7139 5940 1,199 BBC10 reve 1S3 cp32-6 16838 16537 301 BBM27 revf 1S4 cp9 6866 5940 926 BBC10 reve 1S7 cp9 7551 5940 1,611 BBC10 reve 1S10 cp9 6866 6190 676 BBC10 reve 1L3 lp28-4 7076 9712 2,636 BBI16 vraA 1L4 lp54 17068 15035 2,033 BBA24,A25 dbpAB 2L9 lp36 12084 14038 1,954 BBK19 NDg 1L11 lp56 31373 29408 1,965 BBQ47 erpX 2L12 lp38 26454 23852 2,602 BBJ34 ND 1C3 cp32-7 25206 28010 2,804 BBO39,O40 erpLM 6C5 lp36 24866 29209 4,343 BBK45 p37 h 6C8 lp54 17177 15035 2,142 BBA24,A25 dbpAB 2

a Indicates the plasmid location of the insert obtained; designation of plasmids is based on TIGR nomenclature. cp, circular plasmid; lp, linear plasmid.b Indicates the exact nucleotide location of the ApoI site in the B. burgdorferi genome in accordance with the TIGR numbering system for the plasmid indicated; also

indicates the orientation of the insert relative to the T7 and SP6 sequencing primers that flank the EcoRI site of the pEXlox vector.c See reference 48 for details.d Indicates whether the putative candidate is infection associated at the DNA level; 1, DNA fragments are infection associated only; 6, some DNA fragments are

infection associated and some are not; 2, DNA fragments are indistinguishable between infectious and noninfectious isolates.e rev carried by BBC10 on cp9.f rev carried by BBM27 on cp32-6.g ND, no homologues; genes are uncharacterized.h BBK45 is homologous to the previously characterized p37 locus encoded by BBK50.

VOL. 67, 1999 B. BURGDORFERI PLASMID-ENCODED ANTIGENS 4411

on March 22, 2014 by guest

http://iai.asm.org/

Dow

nloaded from

cp32 plasmids or that all or some of the cp32-7 sequences arealso found in the noninfectious isolates (data not shown).Longer exposures of the Southern blot shown in Fig. 3 (erpLMpanel) would seem to suggest the former hypothesis, inasmuchas reactive fragments were also identified in lanes correspond-ing to the noninfectious isolate, albeit to a lesser extent. How-ever, the different restriction pattern observed for DNA frominfectious samples relative to DNA from noninfectious isolates(Fig. 3, erpLM panel, lanes 5 to 8) would suggest either that thecp32-7 sequences have undergone a recombination event orthat the pattern observed may be due to the non-erp sequence

on the pEXC3 insert. Further studies with erpLM alone as aprobe should reconcile these possibilities.

The erpX locus was identified in both the infectious andnoninfectious samples (Fig. 3, panel L11), indicating that it isnot an infection-associated gene. Although the erpX probepreferentially recognizes linear plasmid DNA, DNA from ourcircular plasmid sample also hybridizes to the erpX probe,presumably due to related erp sequences on the cp32 plasmids.Subsequent RT-PCR analysis with erpX-specific probes (datanot shown) indicated that a transcript is synthesized at lowlevels in both infectious and noninfectious B. burgdorferi. This

FIG. 3. Southern blot analysis of expression library clones. Southern blot analyses of plasmid DNA derived from infectious (passage 4) and noninfectious B.burgdorferi B31 are shown. Purified circular and linear plasmid DNA was digested with either HindIII or EcoRI, Southern blotted, and probed with the plasmid clonecontaining the antigen indicated above each frame. The vertical numbers refer to the sizes of markers in kilobase pairs. Arrows indicate bands specific for DNA frominfectious B. burgdorferi that are recognized by the erpLM-containing pEXC3 and BBK45-containing pEXC5 probes. The asterisk in panel p37 indicates that this locusis related to the p35/p37 paralogous gene family (48). Lanes: 1 and 2, circular plasmid DNA from passage 4 infectious B. burgdorferi cut with HindIII and EcoRI,respectively; 3 and 4, linear plasmid DNA from passage 4 infectious B. burgdorferi cut with HindIII and EcoRI, respectively; 5 and 6, circular plasmid DNA fromhigh-passage noninfectious B. burgdorferi cut with HindIII and EcoRI, respectively; 7 and 8, linear plasmid DNA from high-passage noninfectious B. burgdorferi cut withHindIII and EcoRI, respectively.

4412 SKARE ET AL. INFECT. IMMUN.

on March 22, 2014 by guest

http://iai.asm.org/

Dow

nloaded from

observation is consistent with the recent report by Stevenson etal., who detected a transcript in a Northern blot when totalRNA was isolated from B. burgdorferi grown at 35°C, eventhough no ErpX expression was observed (46). We have inde-pendently cloned erpX into an expression vector, overproducedand purified recombinant ErpX, and used this molecule toimmunize rabbits. The polyclonal antiserum obtained recog-nizes nanogram amounts of the recombinant ErpX protein butdoes not detect native ErpX from protein derived from asmany as 3 3 108 infectious or noninfectious B. burgdorferiwhole cells grown at 32 or 37°C (data not shown), suggestingthat the erpX transcript is not translated, that the level of nativeErpX made is well below nanogram amounts, or that ErpX ispreferentially synthesized within the infected host, i.e., therabbit. Further studies are required to reconcile whether trans-lational regulation is operative during in vitro cultivation of B.burgdorferi or whether erpX is significantly upregulated in vivo.

Although the identification of genes like erpX (and dbpAB[see below]), which are found in both infectious and noninfec-tious B. burgdorferi, would seem to invalidate the selectivity ofour screen, the isolation of erpX in our antibody-based screencan be explained by differential expression of B. burgdorferigenes in vitro relative to arthropod or mammalian hosts. Thatis, if erpX is not expressed during in vitro cultivation in eitherinfectious or noninfectious B. burgdorferi but is derepressed ininfectious B. burgdorferi upon infection of the mammalian hostand is antigenic, antibodies would be generated against ErpX.Subsequent adsorption against noninfectious B. burgdorferi,which does not express erpX, would not remove antibodiesagainst ErpX, resulting in their presence in the final adsorbedIRS reagent. Use of this reagent against a B. burgdorferi ex-pression library could then result in the identification of erpXclones.

It is curious that additional erp genes other than erpM and

FIG. 3—Continued.

VOL. 67, 1999 B. BURGDORFERI PLASMID-ENCODED ANTIGENS 4413

on March 22, 2014 by guest

http://iai.asm.org/

Dow

nloaded from

erpX were not also identified in our screen, inasmuch as thesegenes, or their alleles in other strains of B. burgdorferi, havebeen identified as encoding in vivo-induced antigens by otherinvestigators (11, 46). Additionally, Erp proteins have signifi-cant similarity at the amino acid level, implying that antigenicErp proteins should have common epitopes. One possible ex-planation for the limited number of Erp proteins identified inour screen is the lack of a comprehensive screen. This seemsunlikely since we had significant redundancy for clones con-taining rev (i.e., the pEXS series clones). Another possibility isrelated to differential expression of erp genes in the rabbitrelative to murine or human hosts. Nevertheless, it is interest-ing that ErpM is the most closely related to ErpX of all of theErp proteins characterized, suggesting that these proteins haverelated epitopes and that perhaps the identification of clonesencoding these antigens may be due to the recognition ofcommon epitopes. Again, we are not certain that continuedscreening of immunoreactive clones would not yield insertsencoding ErpB2, ErpJ, and ErpD, since all three of these Erpproteins are also similar to both ErpM and ErpX (40 to 46%identity and 49 to 56% similarity relative to ErpX [data notshown]). All of these Erp proteins also contain repetitive se-quences, and therefore, in light of the identification of anotherhighly repetitive antigen in our screen (VraA), it is tempting tospeculate that the repeat units are defining the epitopes rec-ognized by the adsorbed IRS. For example, ErpX contains fiveconsecutive 5-amino-acid domains (DATGK) located at resi-dues 23 to 47 of the precursor protein that distinguishes it fromErpM (48) as well as from ErpB2, ErpJ, and ErpD. Alterna-tively, the stretch between residues 91 and 154 of ErpX differssignificantly from that in the other Erp proteins, suggestingthat this may be an immunoreactive domain (48). Furthercharacterization is required to determine which of these re-gions specify the ErpX-specific epitope(s) as well as theepitope(s) that defines the recognition of ErpM in our screen.

(iv) dbpAB (BBA24 and BBA25; lp54). Clones containingdbpAB were identified twice independently in our screen (en-coded by pEXL4 and pEXC8). It should be noted that clonepEXC8 encodes an in-frame fusion of the T7 gene 10 proteinto most of DbpB, resulting in the production of the approxi-mately 50-kDa species observed in Fig. 2 (Table 1 gives de-

tails). Inasmuch as the genes encoding decorin binding proteinA and B are observed in both infectious and noninfectiousisolates of B. burgdorferi (Fig. 3), the identification of dbpABclones in our screen would seem to contradict the infectivity-associated specificity of our screen. However, our data suggestthat DbpB is made at very low levels in noninfectious cellsgrown at 37°C yet is derepressed significantly in infectious B.burgdorferi cells grown at 37°C, i.e., conditions that simulatevertebrate infection (Fig. 4C). Therefore, when infectious B.burgdorferi enters the mammalian host, dbpAB is probably up-regulated and DbpA and DbpB are synthesized and processedby the rabbit immune system, resulting in a significant humoralresponse to these antigens. In contrast, during in vitro cultiva-tion at 32 or 37°C, noninfectious cells make very low levels ofDbpB (Fig. 4) yet synthesize an appreciable amount of DbpA.Thus, when noninfectious cells with little DbpB on their sur-face were used to adsorb out common antibodies, antibodiesgenerated against DbpB were not eliminated. Antibodies toDbpB presumably resulted in the isolation of the dbpAB-con-taining clones pEXL4 and pEXC8 (Fig. 2). Recently, Hagmanet al. demonstrated that dbpA and dbpB are contained on asingle polycistronic mRNA, suggesting that the low levels ofDbpB made may be due to translation-regulatory mechanisms(25). As indicated above for erpX, the identification of genesfound in both infectious and noninfectious B. burgdorferi doesnot invalidate our screen. We contend that the different classesof clones identified expose the bias inherent to our antigen-based screen and further highlight the complexity of geneexpression when in vitro cultivated B. burgdorferi is comparedwith host-adapted B. burgdorferi.

(v) Additional, uncharacterized infection-associated loci.The additional three loci identified in this report have beendesignated BBK19, BBK45, and BBJ34 by TIGR (18). TheTIGR genome sequence indicates that BBK19 and BBK45map to lp36 and that BBJ34 maps to lp38. Our assignment ofthe genetic loci encoding these antigens is based on the corre-lation between the molecular mass of these antigens observedin Fig. 2 and the potential open reading frames present onthese cloned inserts (Table 1) (18). The BBJ34 antigen shows24% identity and 40% similarity to a 50-kDa surface-exposedlipoprotein from Mycoplasma hominis that has adhesin activity

FIG. 4. Temperature regulation of dbpA and dbpB. Protein lysates derived from B. burgdorferi B31 noninfectious cells (lanes 1 through 3) and infectious cells(passage 4; lanes 4 through 6) grown at 23°C (lanes 1 and 4), 32°C (lanes 2 and 5), or 37°C (lanes 3 and 6) were separated by SDS-PAGE and stained with Coomassieblue (A) or immunoblotted and probed with antiserum specific for either DbpA (B) or DbpB (C). Numbers on the left refer to the molecular mass of protein markers(in kilodaltons).

4414 SKARE ET AL. INFECT. IMMUN.

on March 22, 2014 by guest

http://iai.asm.org/

Dow

nloaded from

and is a phase-variable antigen (7). Whether BBJ34 functionsas an adhesin in B. burgdorferi has yet to be determined. Nohomologues have been identified, and no function has beenattributed to the gene product of BBK19. By comparison,BBK45 is one of four genes that comprise a paralogous genefamily (18). Paralogous family 75 (18) contains BBK45,BBK46, BBK48, and BBK50. Of these loci, only BBK50 hasbeen previously characterized and corresponds to the p37 locuscharacterized by Fikrig et al. (13). Alignment of BBK45 andBBK50 indicates that these two P37 proteins have 38% identityand 52% similarity at the amino acid level (data not shown).Fikrig et al. demonstrated that BBK50 is preferentially ex-pressed within an infected mouse but not during in vitro cul-tivation of B. burgdorferi and confers passive protection againstB. burgdorferi challenge. In contrast, our RT-PCR results indi-cate that BBK45 is expressed during in vitro cultivation ofinfectious B. burgdorferi (data not shown). Since we do nothave a monospecific antibody reagent against BBK45 P37, wedo not know whether our P37 protein is synthesized in vitro.Alternatively, it is possible that p37 transcription is similar tothat reported for the erp genes, where transcription does notnecessarily guarantee that translation of the correspondingprotein will ensue (46). Whether the P37 homologue encodedby BBK45 is regulated in this manner remains to be elucidated.

Southern blot data obtained with BBJ34 and BBK19 asprobes indicates that these genes are definitive infection-asso-ciated loci (Fig. 3, panels J34 and K19). In contrast, Southernblots obtained with pEXC5 as a probe indicate that p37-likesequences are also present in noninfectious B. burgdorferi. Thisis most probably due to the additional genes present on theBBK45-containing pEXC5 plasmid, including BBK40, BBK41,BBK42, BBK43, and BBK44. Of these five additional loci,BBK40 belongs to the paralogous gene family 113, which con-tains numerous genetic loci, some of which are present onplasmids common to noninfectious B. burgdorferi strains (48).

Relation of infection-associated and non-infection-associ-ated antigens to pathogenesis and protective immunity. Re-cently, several investigators have demonstrated that the ospAlocus is downregulated in vertebrates (6, 9, 30) or under con-ditions where ticks have fed to repletion on a murine host (12,38). Experimental evidence supports the contention that anti-bodies directed against OspA kill B. burgdorferi within themidgut of an infected tick when ticks obtain a blood meal froma mammalian host (14). As such, OspA is now purported to bean arthropod-specific vaccinogen (12) and has been tested inhuman trials (39, 45). Recently, it was determined that OspAand a human leukocyte antigen share a cross-reactive epitope,suggesting that antibodies against OspA recognize an autoan-tigen which may contribute to some of the autoimmune patho-logic findings associated with chronic arthritic manifestations(22). Since OspA is downregulated in the vertebrate host andsince the kinetics of OspA turnover are not well understood,we, along with others (11, 13, 17, 26, 33–35), have suggestedthat additional vaccine candidates, either separately or in com-bination with OspA, should be evaluated to determine theirability to protect against the population of OspA-less B. burg-dorferi that would not be cleared by OspA antibodies. The typeof screen described in this study, involving antibodies directedagainst antigenic proteins in the rabbit, i.e., expressed within arabbit mammalian host that develops complete immunity tochallenge reinfection (16), provides an alternate approach toidentify potential second-generation vaccine candidates. Fur-thermore, the rabbit humoral response appears to be qualita-tively and quantitatively more robust than the immune re-sponse elicited by other animal models, most notably C3H/HeJand HeN mice, as well as chronically infected humans. This can

be assessed qualitatively by evaluating the immunoreactivity ina Western blot assay of the recombinant antigens identified inthis screen with antiserum from chronically infected humans orpooled mouse sera (Fig. 5) relative to the antigens recognizedby the adsorbed rabbit antiserum (Fig. 2). Differences in thehumoral response between these serum samples include (i) thetwo Erp antigens (ErpM and ErpX), which are weakly recog-nized by the human and pooled mouse sera, and (ii) the cp9Rev antigen, which is recognized by the human serum (Fig.5A) but is identified only in clones S3, S4, and S7 when probedwith the pooled mouse sera (Fig. 5B). Three of the antigensrecognized by the adsorbed rabbit serum but not detected byserum from a chronically infected human or by pooled mousesera are the p37 homologue (BBK45), the antigen encoded bythe BBJ34 locus, and what we have described as VraA(BBI16). Our preliminary data indicates that the VraA antigenalso protects against B. burgdorferi infection, suggesting thatthe clearance of B. burgdorferi from infected rabbits may bemediated in part by the immune response against this and

FIG. 5. Recognition of B. burgdorferi recombinant antigens by infection-de-rived human sera (A) and pooled mouse sera (B). The samples shown wereprocessed as described in Materials and Methods. Each lane is labeled with thepEX clone designation assigned to each immunoreactive recombinant (see Fig.2 and Table 1 for a summary). Vertical numbers indicate the molecular massesof markers in kilodaltons.

VOL. 67, 1999 B. BURGDORFERI PLASMID-ENCODED ANTIGENS 4415

on March 22, 2014 by guest

http://iai.asm.org/

Dow

nloaded from

perhaps other antigens identified in our screen (data notshown). Along these lines, Hanson et al. have recently shownthat decorin binding protein A, an antigen found in both in-fectious and noninfectious B. burgdorferi strains and also iden-tified in this study, protects against experimental Lyme borre-liosis in the mouse model of infection (26).

With the advent of the B. burgdorferi genome project and theexisting DNA array technology, it is now possible to assessdifferential gene expression in B. burgdorferi by simulating con-ditions within the arthropod vector or mammalian hosts. Weare in the process of addressing these issues experimentally,more specifically in regard to the expression of the infection-associated genes identified in this study.

ACKNOWLEDGMENTS

We thank Ellen Shang, Cheryl Champion, and Maria Labandeira-Rey for helpful discussions and Yi-Ping Wang, Xiao-Yang Wu, andJason Barranco for excellent technical assistance.

This work was supported by National Research Service Award AI-09117 from the National Institute of Allergy and Infectious Diseases(NIAID) (to J.T.S.), a Scientist Development Grant from the Ameri-can Heart Association (to J.T.S.), NIAID training grant AI-07323 (toJ.T.S. and D.M.F.), Public Health Service (PHS) grants AI-21352 andAI-29733 (both to M.A.L.), and PHS grant AI-37312 (to J.N.M.).

REFERENCES

1. Akins, D. R., K. W. Bourell, M. J. Caimano, M. V. Norgard, and J. D. Radolf.1998. A new animal model for studying Lyme disease spirochetes in amammalian host-adapted state. J. Clin. Investig. 101:2240–2250.

2. Akins, D. R., S. F. Porcella, T. G. Popova, D. Shevchenko, S. I. Baker, M. Li,M. V. Norgard, and J. D. Radolf. 1995. Evidence for in vivo but not in vitroexpression of a Borrelia burgdorferi outer surface protein F (OspF) homo-logue. Mol. Microbiol. 18:507–520.

3. Baker, E. A., and J. T. Skare. Unpublished data.4. Barbour, A. G. 1984. Isolation and cultivation of Lyme disease spirochetes.

Yale J. Biol. Med. 57:521–525.5. Barthold, S. W., and L. K. Bockenstedt. 1993. Passive immunizing activity of

sera from mice infected with Borrelia burgdorferi. Infect. Immun. 61:4696–4702.

6. Barthold, S. W., E. Fikrig, L. K. Bockenstedt, and D. H. Persing. 1995.Circumvention of outer surface protein A immunity by host-adapted Borreliaburgdorferi. Infect. Immun. 63:2255–2261.

7. Boesen, T., J. Emmersen, L. T. Jensen, S. A. Ladefoged, P. Thorsen, S.Birkelund, and G. Christiansen. 1998. The Mycoplasma hominis vaa genedisplays a mosaic gene structure. Mol. Microbiol. 29:97–110.

8. Carroll, J. A., and F. C. Gherardini. 1996. Membrane protein variationsassociated with in vitro passage of Borrelia burgdorferi. Infect. Immun. 64:392–398.

9. Cassatt, D. R., N. K. Patel, N. D. Ulbrandt, and M. S. Hanson. 1998. DbpA,but not OspA, is expressed by Borrelia burgdorferi during spirochetemia andis a target for protective antibodies. Infect. Immun. 66:5379–5387.

10. Champion, C. I., D. R. Blanco, J. T. Skare, D. A. Haake, M. Giladi, D. Foley,J. N. Miller, and M. A. Lovett. 1994. A 9.0-kilobase-pair circular plasmid ofBorrelia burgdorferi encodes an exported protein: evidence for expressiononly during infection. Infect. Immun. 62:2653–2661.

11. Das, S., S. W. Barthold, S. S. Giles, R. R. Montgomery, S. R. Telford III, andE. Fikrig. 1997. Temporal pattern of Borrelia burgdorferi p21 expression inticks and the mammalian host. J. Clin. Investig. 99:987–995.

12. de Silva, A. M., S. R. Telford III, L. R. Brunet, S. W. Barthold, and E. Fikrig.1996. Borrelia burgdorferi OspA is an arthropod-specific transmission-block-ing Lyme disease vaccine. J. Exp. Med. 183:271–275.

13. Fikrig, E., S. W. Barthold, W. Sun, W. Feng, S. R. Telford III, and R. A.Flavell. 1997. Borrelia burgdorferi P35 and P37 proteins, expressed in vivo,elicit protective immunity. Immunity 6:531–539.

14. Fikrig, E., S. R. Telford III, S. W. Barthold, F. S. Kantor, A. Spielman, andR. A. Flavell. 1992. Elimination of Borrelia burgdorferi from vector ticksfeeding on OspA-immunized mice. Proc. Natl. Acad. Sci. USA 89:5418–5421.

15. Foley, D. M., D. R. Blanco, M. A. Lovett, and J. N. Miller. Unpublished data.16. Foley, D. M., R. J. Gayek, J. T. Skare, E. A. Wagar, C. I. Champion, D. R.

Blanco, M. A. Lovett, and J. N. Miller. 1995. Rabbit model of Lyme borre-liosis: erythema migrans, infection-derived immunity, and identification ofBorrelia burgdorferi proteins associated with virulence and protective immu-nity. J. Clin. Investig. 96:965–975.

17. Foley, D. M., Y. P. Wang, X. Y. Wu, D. R. Blanco, M. A. Lovett, and J. N.Miller. 1997. Acquired resistance to Borrelia burgdorferi infection in the

rabbit. Comparison between outer surface protein A vaccine- and infection-derived immunity. J. Clin. Investig. 99:2030–2035.

18. Fraser, C. M., S. Casjens, W. M. Huang, G. G. Sutton, R. Clayton, R.Lathigra, O. White, K. A. Ketchum, R. Dodson, E. K. Hickey, M. Gwinn, B.Dougherty, J. F. Tomb, R. D. Fleischmann, D. Richardson, J. Peterson, A. R.Kerlavage, J. Quackenbush, S. Salzberg, M. Hanson, R. van Vugt, N.Palmer, M. D. Adams, J. Gocayne, J. C. Venter, et al. 1997. Genomicsequence of a Lyme disease spirochaete, Borrelia burgdorferi. Nature 390:580–586.

19. Garcia-Monco, J. C., and J. L. Benach. 1995. Lyme neuroborreliosis. Ann.Neurol. 37:691–702.

20. Giladi, M., C. I. Champion, D. A. Haake, D. R. Blanco, J. F. Miller, J. N.Miller, and M. A. Lovett. 1993. Use of the “blue halo” assay in the identi-fication of genes encoding exported proteins with cleavable signal peptides:cloning of a Borrelia burgdorferi plasmid gene with a signal peptide. J. Bac-teriol. 175:4129–4136.

21. Gilmore, R. D., Jr., and M. L. Mbow. 1998. A monoclonal antibody gener-ated by antigen inoculation via tick bite is reactive to the Borrelia burgdorferiRev protein, a member of the 2.9 gene family locus. Infect. Immun. 66:980–986.

22. Gross, D. M., T. Forsthuber, M. Tary-Lehmann, C. Etling, K. Ito, Z. A. Nagy,J. A. Field, A. C. Steere, and B. T. Huber. 1998. Identification of LFA-1 asa candidate autoantigen in treatment- resistant Lyme arthritis. Science 281:703–706.

23. Gruber, A., and B. Zingales. 1995. Alternative method to remove antibac-terial antibodies from antisera used for screening of expression libraries.BioTechniques 19:28–30.

24. Guo, B. P., S. J. Norris, L. C. Rosenberg, and M. Hook. 1995. Adherence ofBorrelia burgdorferi to the proteoglycan decorin. Infect. Immun. 63:3467–3472.

25. Hagman, K. E., P. Lahdenne, T. G. Popova, S. F. Porcella, D. R. Akins, J. D.Radolf, and M. V. Norgard. 1998. Decorin-binding protein of Borrelia burg-dorferi is encoded within a two-gene operon and is protective in the murinemodel of Lyme borreliosis. Infect. Immun. 66:2674–2683.

26. Hanson, M. S., D. R. Cassatt, B. P. Guo, N. K. Patel, M. P. McCarthy, D. W.Dorward, and M. Hook. 1998. Active and passive immunity against Borreliaburgdorferi decorin binding protein A (DbpA) protects against infection.Infect. Immun. 66:2143–2153.

27. Johnson, R. C., C. Kodner, and M. Russell. 1986. Passive immunization ofhamsters against experimental infection with the Lyme disease spirochete.Infect. Immun. 53:713–714.

28. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature 227:680–685.

29. Logigian, E. L., R. F. Kaplan, and A. C. Steere. 1990. Chronic neurologicmanifestations of Lyme disease. N. Engl. J. Med. 323:1438–1444.

30. Montgomery, R. R., S. E. Malawista, K. J. Feen, and L. K. Bockenstedt.1996. Direct demonstration of antigenic substitution of Borrelia burgdorferiex vivo: exploration of the paradox of the early immune response to outersurface proteins A and C in Lyme disease. J. Exp. Med. 183:261–269.

31. Norris, S. J., C. J. Carter, J. K. Howell, and A. G. Barbour. 1992. Low-passage-associated proteins of Borrelia burgdorferi B31: characterization andmolecular cloning of OspD, a surface-exposed, plasmid-encoded lipoprotein.Infect. Immun. 60:4662–4672.

32. Norris, S. J., J. K. Howell, S. A. Garza, M. S. Ferdows, and A. G. Barbour.1995. High- and low-infectivity phenotypes of clonal populations of in vitro-cultured Borrelia burgdorferi. Infect. Immun. 63:2206–2212.

33. Philipp, M. T. 1998. Studies on OspA: a source of new paradigms in Lymedisease research. Trends Microbiol. 6:44–47.

34. Philipp, M. T., and B. J. Johnson. 1994. Animal models of Lyme disease:pathogenesis and immunoprophylaxis. Trends Microbiol. 2:431–437.

35. Philipp, M. T., Y. Lobet, R. P. Bohm, Jr., E. D. Roberts, V. A. Dennis, Y. Gu,R. C. Lowrie, Jr., P. Desmons, P. H. Duray, J. D. England, P. Hauser, J.Piesman, and K. Xu. 1997. The outer surface protein A (OspA) vaccineagainst Lyme disease: efficacy in the rhesus monkey. Vaccine 15:1872–1887.

36. Porcella, S. F., T. G. Popova, D. R. Akins, M. Li, J. D. Radolf, and M. V.Norgard. 1996. Borrelia burgdorferi supercoiled plasmids encode multicopytandem open reading frames and a lipoprotein gene family. J. Bacteriol.178:3293–3307.

37. Schwan, T. G., W. Burgdorfer, and C. F. Garon. 1988. Changes in infectivityand plasmid profile of the Lyme disease spirochete, Borrelia burgdorferi, as aresult of in vitro cultivation. Infect. Immun. 56:1831–1836.

38. Schwan, T. G., J. Piesman, W. T. Golde, M. C. Dolan, and P. A. Rosa. 1995.Induction of an outer surface protein on Borrelia burgdorferi during tickfeeding. Proc. Natl. Acad. Sci. USA 92:2909–2913.

39. Sigal, L. H., J. M. Zahradnik, P. Lavin, S. J. Patella, G. Bryant, R. Haselby,E. Hilton, M. Kunkel, D. Adler-Klein, T. Doherty, J. Evans, P. J. Molloy,A. L. Seidner, J. R. Sabetta, H. J. Simon, M. S. Klempner, J. Mays, D.Marks, and S. E. Malawista. 1998. A vaccine consisting of recombinantBorrelia burgdorferi outer-surface protein A to prevent Lyme disease. Re-combinant Outer-Surface Protein A Lyme Disease Vaccine Study Consor-tium. N. Engl. J. Med. 339:216–222.

40. Skare, J. T., E. S. Shang, D. M. Foley, D. R. Blanco, C. I. Champion, T.

4416 SKARE ET AL. INFECT. IMMUN.

on March 22, 2014 by guest

http://iai.asm.org/

Dow

nloaded from

Mirzabekov, Y. Sokolov, B. L. Kagan, J. N. Miller, and M. A. Lovett. 1995.Virulent strain associated outer membrane proteins of Borrelia burgdorferi.J. Clin. Investig. 96:2380–2392.

41. Steere, A. C. 1989. Lyme disease. N. Engl. J. Med. 321:586–596.42. Steere, A. C. 1995. Musculoskeletal manifestations of Lyme disease. Am. J.

Med. 98:44S–51S.43. Steere, A. C., W. P. Batsford, M. Weinberg, J. Alexander, H. J. Berger, S.

Wolfson, and S. E. Malawista. 1980. Lyme carditis: cardiac abnormalities ofLyme disease. Ann. Intern. Med. 93:8–16.

44. Steere, A. C., S. E. Malawista, D. R. Snydman, R. E. Shope, W. A. Andiman,M. R. Ross, and F. M. Steele. 1977. Lyme arthritis: an epidemic of oligoar-ticular arthritis in children and adults in three connecticut communities.Arthritis Rheum. 20:7–17.

45. Steere, A. C., V. K. Sikand, F. Meurice, D. L. Parenti, E. Fikrig, R. T. Schoen,J. Nowakowski, C. H. Schmid, S. Laukamp, C. Buscarino, and D. S. Krause.1998. Vaccination against Lyme disease with recombinant Borrelia burgdor-feri outer-surface lipoprotein A with adjuvant. Lyme Disease Vaccine StudyGroup. N. Engl. J. Med. 339:209–215.

46. Stevenson, B., J. L. Bono, T. G. Schwan, and P. Rosa. 1998. Borrelia burg-

dorferi Erp proteins are immunogenic in mammals infected by tick bite, andtheir synthesis is inducible in cultured bacteria. Infect. Immun. 66:2648–2654.

47. Suk, K., S. Das, W. Sun, B. Jwang, S. W. Barthold, R. A. Flavell, and E.Fikrig. 1995. Borrelia burgdorferi genes selectively expressed in the infectedhost. Proc. Natl. Acad. Sci. USA 92:4269–4273.

48. The Institute for Genomic Research Web Site. 16 October 1998, copyrightdate. [Online.] http://www.tigr.org/tdb/CMR/gbb/htmls/SplashPage.html. [14July 1999, last date accessed.]

49. Wallich, R., C. Brenner, M. D. Kramer, and M. M. Simon. 1995. Molecularcloning and immunological characterization of a novel linear-plasmid-en-coded gene, pG, of Borrelia burgdorferi expressed only in vivo. Infect. Immun.63:3327–3335.

50. Xu, Y., C. Kodner, L. Coleman, and R. C. Johnson. 1996. Correlation ofplasmids with infectivity of Borrelia burgdorferi sensu stricto type strain B31.Infect. Immun. 64:3870–3876.

51. Zhang, J. R., J. M. Hardham, A. G. Barbour, and S. J. Norris. 1997.Antigenic variation in Lyme disease borreliae by promiscuous recombinationof VMP-like sequence cassettes. Cell 89:275–285.

Editor: P. E. Orndorff

VOL. 67, 1999 B. BURGDORFERI PLASMID-ENCODED ANTIGENS 4417

on March 22, 2014 by guest

http://iai.asm.org/

Dow

nloaded from