CA2112466A1 - Methods and compositions for diagnosing lyme disease - Google Patents

Abstract

A chromosomal gene of Borrelia burgdorferi which encodes a conserved antigen of approximately 79 kD has been isolated and sequenced. The chromosomal gene, the gene product, and antibodies to the gene product may be used in diagnostic methods for the detection of Borrelia burgdorferi infection. The antigen and fragments thereof are suitable for use in vaccine compositions and methods.

Description

~9~ 2i~5~ PCI/US92/05~39 - '.

,a M~5T~OD8 AND CO~P08ITIO~B ~FOR DIAt;~O~ING 1.~l5 DI~EA8 BAC~GRO~ID QF~THE I~YXN~ION
l of th~ I~tion ~:
Lyme disease was f irst described in the late 1970 ' s as a unique grouping of arthritic symptoms in patients from Lyme, Corm~cticut. Subsequent investigati~n demonstrated that the disease is caused by inf ectiot with Borrelia burgdorferi, a spirc)chete, following exposure to deer ( ixsdid) ticks . It is now known that lyme disease in humans is a multi-systemic disorder charact~ized by dematologic, rheumatologic, cardi~c, and neurologic manife~tations.
~.- While several isolates of B. bur~orferi from ~orth America and Europe have been characterized at both the genetic and antigenic: level, the une~uivocal diagnosis of lyme disease remains problematic. Isolation and cultivation of borrelia from infected patients is di~fficult and not practical for routine diagnosis.

2 0 : I mmunological and genetic: detection methods have not become generally availabl~, at least in part because the known major antigens~ of B. burgdorferi hav~ previously bee.n l:hought to be coded on linear plasmid and have demonstrated signif lcant antigenic variation with regard to the level of expression and molecular weight. Th~re have als bePn reports of serological cross-reactivity ~.
with related human pathogel s, such a~ B. h~rmsii ~the causative agent of tick-born relapsing f~ver) and ~
~rep~nema pall~dium (the causative agent of syphilis). ~:
It would therefore be desirable to provide improved methQ:~s for diagnosing lyme disease and in particular for unequivocally determining t ~ presence of :~
B . burgdorferi in pa'c ient samples . To provide suc:h methods, it would be desirable to identify genetic and ~.
antigeni-:: information which is widely conserve,d :amorlg urgdo~feri strains and which can b~ used in a variety of detection protocols. More specificallyr it would be w~ g3/~ 2 ~ ht ~ ~ 2 PCT/US92/0 desirable to identify a conserved chromosomal gene which encodes a major antigenic protein, where the gene can ser~e as the basis for genetic screening assays and the antigen can serve as the basis for immunologic screening assays.
2. De~criptio~ of th~ ~ackqrou~ Art Immunochemical analyses of B. burgdorferf have revealed the presence of several immuncdominant ant~gens that are reco~nized during the described stages of human infection. ~Craft, et al. (1986). Clin. Invest. 78:934-938; Grodzicki, et al. (1988) J. Infect. Dis. 157:790-797; Nadal, et al. (1989) Pediatr. Res. 26O377-382) One of the immunodominant antigens was reported to have a ~-~ mQlecu~ar weight of approximately 83 kD. No descrip~ion or characterization of the gene encoding this antigen had baen reported priox to th~ work which is the subject of : the pr~s~nt appIication~ The da~a included in the Experim~ntal ection h~r~inafter was r~ported in LeFebvre et al. (1990) J. Clin. ~icrobiol. 28:1673-1675 and in Perng et al. ~1991) Infect. Immun. 5g:2070 2074.
8~AaY OF ~ I ~ ION
~ The methods and compositions of the present : ~inventio~ ar~:based on the discovery that an approximately 79 kilodalton (kD) imm~nodomînant antigen of Borrelia burgdorferi is encoded by a chromosomal gene which is widely conserved amsng strains throughout Europe and No~th America. Based on this discovery, the present invention pro~id~s screening assays for Borre I ia burgdor rî inf ection, where the assays detect the .30 presence of the chromosomal gene, the gene product ~79 kD
antigen), or ~ntibodies to the 79 kD antigen, in a patient sa~ple.
The present invention al50 provides isolated polynucleogides which encode the approxi~ately 79 kD
antigen or fragments thereof, as well as oligonucleotides capable of hybridizing under string~nt conditions to ~Ae chromosomal gene. The oligonucleotides may be labeled ~ WO93/~ ~ 8 .- 2 ~ :1 2 ~1 ~ S PCT/US92/05~39 for use in hybridization assays or may be present as primer pairs for use in polymerase chain reaction assays.
The present invention further provides di~gnostic kits which contain a reagent capable of binding to the gene, gene product (antigen), or antibodies to the antigen under conditions which permit detection of binding. The kits will further contain instructions which set forth the corresponding detection protocol, such as hybridization assays, polymerase chain reaction assay, or immunoassay.
The present învention still further Gomprises isolated and optionally purified polypeptides whi~h comprise a sequence analogous to at l~ast six contiguou~
~- a~ino acids of the approximately 79 kD antigen~ Such polypep~ides may be produced by expres~ion of a heterologou~ gene in cul~ured cells and will usually be reactive with a~tibodies produced by a host in response to infection by Borrelia burgdorferi . Pr~ferably, the : polypeptid~s will be capable of eliciting a protective immunity when admini~tered to a susceptible host.
: Vaccine compo~itio~s according to the present invention will comprise a polypeptide which is i ~ unologicall~ cross reactive with t~e conserved 79 kD
immunodominant antigen. The polypept~.de wi}l be pr~s~nt in a:physiologically acceptable carrier in an a~ount effective to elicit protec~ive immunity when admini~tered to a su~ceptible host. The polypeptide may comprise subst~ntially the ~ntire 79 kD antig~n, or may be a portion thereof. Frequently, the polypeptide will be produced by expression in a heterologous gene in cultured cells.
The present invention further comprises monospecific antibodies for the conserved 79kD antigen of ~orrelia bursdor~eri . The monospecific antibodies may be monoclonal or polyclonal.

. .

WO 93/00448 ~ 4 PCI/US92/o~- ~9 BRIEF DERCRI~?TION OF T~E D~ R~WING8 Fig. 1 is an immunoblot demonstra~ing the rea ::tivity of the approximately 79kD antigen to rabbit antiserum raised against whole-cell Borrella burgdorferi. -~
Fig. 2 i~ an immunoblot demonstrating the reactivity of the approximately 79kD antigen to human seru~ from seropositive Lyme borreliosis patients.
Fig. 3A is a polyacrylamide gel of whole-cell Borre 7 ia burgdorferi . Fig . 3B is a Southern blot of the gel in Fig. 3P,. ~ ~
Fig.. ~4 is an autoradiograph of whole c:ell DPJA
.
from B . burgdor eri digested ~ith Cl aI and probed with a ~: labeled fragment; ~of the 79.8kD gene.
,~ Fig. 5:~ ~is an immunoblot of whole cell lysates of representative strains of B . bu~gdorf eri reac:ted with antisera raised against the 79 . 8kD antigen.
DE~RIP~ION OF q~ PRE~FERRED E:I~BODI~
The present invention pro~rides novel ~i:
compositions ~ a`nd:~methods for diagnosing, treating, and 20: ~ vaccinating against infection by Borrelia burgdorferi, the~ ~ pirochete which causes I.yme disease in humans . The invention is~ based, in~ part, on the discovery of a widely onserved approximately ~9 }cilodalton (kD) immunodominant :antigen; which~is encoded ~y a chr~mosomal~ gene. As :~25 : discussed above,~ the existence of the chromosomal gene en~oding an immunodomin nt antigen is~ surprising since previous genes encoding ~ajor antigens of Borre~ia ~ .
burgd~rferi were :~believed to be located on linear plasmids, and wouId theref ore not be expected to be as widely conser~ed as c:hromosomal genes.
The present invention provides a complete sequence of the chromosomal gene encoding the 79kD
antigen as well as the corresponding amino acid s~Euence of the antigen itself. Both the polynucleotide and the

3 S sequence and he amino acid sequence are set f orth in - 5EQ ID NO: 1 and SEQ ID NO: 2, respectively. TAe 79kD
antigen gene is contained within a 2 . 5 kilobase pair .--W093/~ 2 1 ~ 2 ~ PCT~VS~2/05539 ~ ~ 5 (kbp) FoKI fragment of the chromosomal DNA of Borrelia burgdorferi reference strain B31, which is ~vailable from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, under the designation ATCC 35210.
It will be appreciated that the DNA s~yuence set for~h in SEQ ID N0:1 represents ~he particular allele which is present in the reference strain B31 and that other alleles will exist in nature. The co~positions of the present invention may be derived frsm such other al}eles and from the nucleic acid and amino acid sequences of such alleles according to th~ general methods as described hereinafter.
~ In accordance with the present invention, nucleic acid ~equences encoding the conserved 79kD
antigen have been isolated and characterizedO An : isolated nucleic acid is one which has been removed from i~s natural chromosomal location. Isolated nucleic a~i~s may ~e produced by excision from the chromosomal D~ of 20 : cultured Borrelia burgdorfer~ cells, ~.g., using restriction enzymes. Alternatively, the isolated nucleic acids may be produced by the synthesis t~chniques set forth h~reinbelow.
~: Isolated nucleic acid sequ~nces, including polynucleotides and oligonucleotide~, encoding the : antigen and por~ions thereof may be expressed in cultured . ~ : cel}s to provide isolatable quan~ities of polypeptides ~: ~ displaying biological (e.g. immunological) properties of the naturally occurring 79kD antigen. Oth~r useful nucleic acids are substantially homologous to the sequences encoding the antigen~ can be either natural or syr~hetic. Such homologou~. nucleic acids may find use as pro~es or primers:for locating or characterizing natural or synthetic nucleic acids encoding the 79kD antigen.
3S Substantial homology of a nucleic acid sequence means i~her that (a~ there is greater than about 65%, typically greater than about 75%, more typically greater W093/~M~8 2 1 1 ~ PCT~US92/0'--~

than about 85%, preferably greater than about 95%, and more preferably greater tha~ about 98~ homology with a comparPd segment of at least about lo contiguous nucleotides or ~b) the homologous nucleic acid sequence will hybridize to the compared sequence or its complementary strand under stringent conditions of the temperature and salt concentration. These stringent conditions will generally be a temperature greater than about 22C, usually greater than about 30C and more usually greater than about 45 , and a salt concentration generally less than about lM, usually less than about 500mM, and preferably less than about 200mM. The combination of temperature and salt concentration is more ~-important in defining stringency than either the temperature or tha salt concentration alone. Other conditions which ~ffect stringency include GC content of the compared sequence, extent of complementarity of the ~sequences, and length of the sequences involved in the ~ hybridization, as::well as the composition of buffer ~ solution(s) used in the hybridization mixture. These and other factors affecting stringency are well described in ~:~ the scientifia and~patent literature.
Nucleic acids according to the present invention can~ be synthesized based on the DNA sequence Z5 ~ set forth in~SEQ ID NO.1 using well known:synthesis :~ techniques~ For example, short, single-stranded DNA
fragments (oligonucleotides) may be prepared by the phosphoramadite method described by Beaucage and Carruthers ~1981) Tett. Lett. 22:1859-1862. A double-stranded fra~ment may then be obtained by either synthesizing the complementary strand and annealing the strands together under appropriate conditions or by adding the complementary strand using DNA polymerase and an appropriate primer sequence.
Poly~erase chain reaction techniques may also be used for the pro~uction of probes and/or the amplification of polynucleotides for synthetio purposes.

-.WO 93/0~ 2 ~ i 2 ~1 6 S PCT/~$92/~53g Suitable primer pairs for use in polymerase chain reaction techniques may be obtained based on the DNA
sequence of SEQ ID N0:1. See, Innis et al., Ed., PCR
Protocols, Academic Press, New York, 1990, the dis ~osure of which is incorporated herein by reference.
. Natural or synthetic DN~ fragment encoding the 79kD antig~n or portions thereof may be incorporated in DNA constructs, usually self-replicating ~onstruct, capable of introductiQn to and expression in in vitro cell culture. Usually, the DNA construct~ will be capab~e of repIication in a u~icellular host, such as bacteria or yeast, but may also be intended for introdu~tion to and the integration wit~in the genome of ~f- cultured mammalian or other eukaryotic cell lines. DNA
constructs prepared for intro~uction into bactaria usually include replication ~ystem recognized by the host, the de~ired coding sequenc~ for the 79kD anti~en or a portion ther~of, transcriptional and translational initiation regulatory sequences joined to the 5' end of the antigen coding sequence, and transcriptional and translational termination regulatory sequences drawing to the 3'-end of the antigen coding sequence. The transcriptional regulatory sequ~nc~s will typically include a heterologous promoter which is recogniz~d by the:host. Conveniently, available expression veCtors which include the replication system and transcriptional and translational regulatory sequences together wi~ an ins~rtion site for the antigen coating regicn may be empl~yed.
3 0 The present invention further provides polypeptides which correspond to the 79kD antigen or a fragment there~f. The polypeptides will usually b~
either haptenic or antigenic, typically including a~
least six amino ac:ids, usually ine:luding at l~ast nine amino acids, and more usually at least twel~re amino ac:ids found contiguously wit~in a natural form of the ~9kD
antigen. The contiguous amino acids may be located W093/~8 PCT/US92/~ 9 within any re~ions of the antigen. L~nger peptides up to and including subs~antially the full length of the natural protein, and larger, may also find use.
Usually, the polypeptides will define or correspond to at least one determinant site (also known as epitopic site) which is oharacteristic of a naturally-occurring 79kD antigen of Borrelia burgdorferi. By characteristic, it is meant that the determinant site will allow immunolo~ic detection of the exposed polypeptide segment in a biological sample with reasonable assurance, in most cases allowing the B~rrelia burgdorf~ri to~be immunoloqically distinguished from other related or~anis~s, particularly~pathogenic organisms.
The polype~tides will be usDful as a reagents for use in diagnostic~methods and kits, for preparing poly~lon~l and~:monoclonal antibodie~, and for use as the immunogenic co~ponent in vaccine compositiQns. Each of these uses will~ be described in more detail hereinafter.
~ In a preferred aspect, the polypep~ides o~ the present invention will be identical to or equivalen~ ~o a~lea t a portion of the amino acid sequence set forth ;~in~SEQ ID N0:20: By equivalent, it is meant that a :~substantial identity ~of amino acids exist over a series of at least 10 contiguous residues, or that each posi~ion ~of~the corresponding sequences is either identical to or ::~has conservative substitution in at:least about 60~ of the residues~, preferably at least about 70% of the residues, and more preferably at least about 80% of the residues. The polypeptide sequences, howevert may be modified by occasional deletions, add:îtions, or ~rep~acements in accordance with known methods for comparison. See, for example, Sequence Analysis Software P~ckage ~ uniYersity of Wisconcin Biotechnology Center, ~adison, Wisc~nsin. Conservative substitutions or replacements within the groups (a) gly~ al.; ~b) val, 2112~S'~
" ~W093/~ PCT~USg2/05539 g ile, leu; (c~ asp, glu, (d~ asn, gln; (e) ser, thr;
(f) lys, arg; and ~g~ phe, tyr.
Synthetic polypeptides which are - immunologically cro~s-reactive with a natural 79kD
antigen may be produced by ~ither of at least two general - approaches. First, polypeptides having fewer than about 100 amino acids, more usually having few~r than about 50 amino acids, can be synthesized by the Merrifeld solid-pha~e cynthesis method where amino acids sequentially added to a growing chain. See, Nerrifield ~1963~ Journal of the ~merican Chemical Society J. Am. Chem. Soc.
85:Z149-214~. Conveniently, the aminQ acid sequences may be taken directly ~ro~ the listing in Fig. 4. E~uipment b-~' for automAtically ~ynthesizing polypep~ides is commercially available from suppliers such as Applied Biosystems, Inc., Foster City, California; DuPon~
Co~pany, Bio~e~hnology Systems, Wil~ington, Delaware; and MilliGen/Biosearch, a division of ~illipore, Burlington, :Mas~achus~tts, An alternativa for synthesizing polypeptides of ths~pres~nt invention involves ~he expres~ion in cultured : cells of rec~mbinant DNA molecules encoding ~he 79kD
antigen or a desired portion th~reof. The gene itself may ba natural or synthetiG, as described above. The na~ural gene is obtainable fr~m c~N~ or gen~mic libraries using standard techniques. In particular, probes . synthesized ba~ed on the nucl~otide sequ~nc~ of SEQ ID
N0:1 may be us~d to id~ntify and isolate genes representing other allelPs of the 79kD antigen gene using well known methods.
To be useful in t~e detection and therapeutic methods of the present invention, the polypeptides are usually obtained in substantially pure form. That is, the polyp~ptides will be a~ }ea~t about 50% pure by weight and be substantially free of interfering proteins and contaminants. Preferably, the polypeptides of the pres~nt invention will be isolated or synthesized to a W~93/0~ PCT/US92/Or 9 t S - 10 purity of at least about 80% by weight, more preferably being at least about 95% by weight or greater. Using conventional protein purification techniques, homogeneous polypeptide c~mpositions of at least 99~ by weight can be obtained. F~r example, the polypeptides may be purified by use of the antibodies described hereinafter in affinity chromatography prosedures. Such affinity chromatography is performed by first linking antibodies : to a solid support and then contacting the solid support IO with the source of the polypeptides, e.g. lysates of the : B~rrelia burgdorferi which naturally produce the 79kD
antigen or lysates of cultured cells in which the 79kD
;anti~en or fragment thereof have been.recombinantly ~ produced.
:~ 15 Production of antibodies to the apprsximately ; 79kD antigen o~ Borrelia burgdorferi or antigenic portions thereof~may be accomplished using the purified ~, ~ : . . :
or~:synthetic;polypeptides of th~ present invention. Once :` a::sufficient~quan~ity of the polypeptide has been ~ : obtained:by any:of the methods described above, polyclonal antibodies specific for the determinant regions~may be:produced~ in in vivo or in :vitro ec~niques. In vivo techniques rely~on the exposure of ;a~tiqenic~polypeptides~or fragments:to any of a wide:: : 25~ variety of vertebra~es, suitable vertebrates are typically:non-h~man, such as mice, rats, rabbits, sheep, goàts, and the like. Polypeptides~having at least about 5~a~ino acids, usually at least about 30 amino acids, and preferably at least about 50 amino cids~may serve ~ directly as immunogens. If the~polypeptide is smaller than about lOkD,~particularly smaller than about 6kV, it may be necessary~to ~oin the polypeptide to a larger : molecule to elicit the desired immune response. The ~ immunogens are then injected into the animal host 35 ~according to a predet~rmined schedule, and the animals are bled periodically, with successive bleeds generally having improved titer and specificity. Such antisera PCI'/US9~/05~39 will then ~e purified in order to produce the desired monospecific antibodies of the present invention.
Con~eniently, purification can be accomplished by affinity chromatography using the polypeptides of the present invention coupled to a suitable solid phase.
- Such affinity chromatography for the purification of antibodies is well described in the patent and scientific literature. See, for example, Hudson and Hay, Practical Immunol ogy, Blackwell Scientific Publications, Oxford, 1980, ~he relevant portions of which are incorporated herein ~y reference. Suitable in vitro techniques for producing ~he monospecific antibodies of the present invention in~olYe the in vitro exposu~e of lymphocytes to ~ the antigenic polypeptidec corresponding the 79kD antigen of Borrel ia ~urgdorf eri . Such techniques produce monoclonal antibodie:s and are described in Huse et al.
(1989) Science 246:1275-12~1; and Ward et al. (1989) Nature 341:544-546, both of which are incorporated herein ~by r~ference~
~20 Monoclonal antibodies may also ~ prepared by : the:now classic technique of Kohler and Milstein, a~ well : as by improvements on their basic technique, Briefly, these methods rely on preparing i~mortalized cell lines ;capable of producing antibodies haYing the desired : 25 specificity, i.e., reactivity with the determinant : regions of the 79kD antigen. Such immortalized cell l:ine~ may be produced by a variety of techniques.
~ Conveniently, a small vertebraté, such as mouse, is : hyperimmunized with the desired antigen by the method just described in connection with the preparation of polyclonal antibodies. The vartebrate is then killed, usually several days after the final immunization, the spleen removed, and the spleen cells immortalized. The manne~ of immortalization is not critical~ Presentl~, 3S the most common technique is fusion with a myeloma cell fusion partner, as first described by Kohler and Milstein ~1976) Eur. J. Immunol. 6:511-519, Other techni~ues W~93/~ 2 ~ PCT/US92/0~-~9 ~ 12 include ~V~ transformation, transformation with bare DNA, e.g., oncogenes, retroviruses, and the like, ~nd any other method which provides for the stable maintenance of the cell line and production of monoclonal antibodies.
Common techniques are described in Lane and Harlow, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1988; and Goding, Monoclonal Antibodi~s:
Principles and Practi~e, 2nd Edition, Academic Pres~, New York, 1986, both of which are incorporated herein by reference.
W~en employing fusion with a fusion partner, the manner of fusion i5 usually not critical and various techniques may be employed. Conveniently, the spleen ~-~ cells and myeloma cells are combined i~ the presence of a nonionic datergent, usually polye~hylene glycol, and other additives such as Dulbecco's Modified Eaglels ~ M~dium, for a few ~inutes. At the end of the fusion, the - nonionic detergent is rapidly moved by washing the cells.
The fu~ed cells are promptly dispen~ed in small cul~ure wells (usually in a micro. ~er plate) at relatively low ~ density, ranging from about 1 to 5xlOs per well, in a selectiv~ medium chos~n to support growth of the hybrid c~lls while being lethal to th~ myeloma cells~ Usually, the ~yel~ma cell line has been mutated to be sensitiYe to a lethal age~t, typically being H~T sensitive. After a sufficient time,:usually from about 1 to 2 weeks, colonies of hybrids are observed and plates containing hybrid positive wells are identified. The plates and wells having only one colony per well are selected, and supernatants from these wells are tested for binding activity against the desired 79kD antigen or i~olated portion thereof. Once positive hybridomas are iden~ified, the cell line can be maintained as a viable culture and/or by lyophilization ~nd frozen storage7 Depending on the desired use for the ~ntibodies, fur~her screening of the hybridomas may be de~irable. Hybrid~mas providing high titers are 2112~S
3/ ~ PCT/US92/05539 desixable. Furthermore, cytotoxic antibodies, e.g., IgG2b, IgG3 and Ig~, may b~ selected for use in therapeutic treatment of Borrelia burgd~rferi infPction.
For use in immunodiagnostic assays as described in more detail hereinbelow, antibodies having very high specificity for corresponding determinants ar~ desirable~
Onc~ th~ d~sired hybridomas hav~ been selected, monoclonal antibodies may be isolated from the supernatants of the growing colonies. The yisld of antibodies obtained, however, is usually low. The yield may be enhanced by various technique~, such as injection of the hybridoma rell line into the peritoneal cavity of a ~er~ebrat~ h~st which will accept the cellsO
~ Monocl~nal antibodies may then be harvested from the ascites f~uid or the blood. Proteinaceous and other contaminants will usually be removed fr~m the monoclonal antibodies prior to use by conventional techniques, e.g., chr~matography, gel filtration r precipitation, extraction, or the lik~. ~
By properly selecting the polypeptides used as ~ the immunogen, antibodies having high specificity and : a~finity for the~desired 79 kD antigen epitope can be obtaine.~. The polypeptide selected should repr~sent one or more epitopic sites which are unique to the antigen 25: protein and which can distinguish Borrelia burgdorferi from closely related pathoqens and other potentially cross-reactive proteins . Such uni~ e epitopes are found on polypeptides expr2ssed by cells .ontaining sequences disclosed in SEQ ID NO:2.
The po~ypeptides and antibodies of the pre ent invention ~ay be used with or without modification.
Frequently, the polypeptides and antibodies will be labeled by joining, either covalent~y or non-covalently, a substance which provides ~or a detectable si~nal. A
wide variety of labels and conjugation techniques are known and ar~ reported extensively in both the scientific ~nd patent literature. Suitable labels include w093~ 2 ~ 1? ~ PCT/US92/0'-~9 radionurlides, en~ymes, substrates, cofactors, inhibi-tors, fluoresce~s, chemiluminescers, magnetic particles, biotin, and the like. Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752;
3,939,350; 3,996,345; 4,277,437; ~,275,149; and

4,366,241, the disclosures of which are incorporated herein by reference.
An~ibodies and polypeptid2s prepared as describPd ab~ve can be used in various immunological techniques for detecting the 79 KD antigen of Borrelia burgdorf eri in biological specimens, particularly body fluid samples, including blood, pla~m~, serum, urine, stool, and the like as well as cell sa~ples, such as ~ tissue biopsy sampl~s. The use of blood, plas~a and serum samples will prsferably be pr~ferred.
A first type of immunoaæ~ay will involve combining the pa~i~nt sa~ple with monosp~cific antibody, : ei~h~r polyclonal or monoclonal, which is capable of specifically binding to the 79kD antige~ in the sample.
The antibody will usually be labeled or immobilized, as describ~d above, and detection of binding between the antibody and antig~n will b indicative of the presence o~ Borrel ia burgd~rf eri in the sample. Numerous specific : protocols are available for performing su h antibody-based immunoassays and are well des~ribed in the scientifi~ and patent literature. Such protocols can : . generally be clas~ified as:heterog~neous (~aquiring separation of the an~igen from a sample)' or homogenous ~not requiring separation); competitiYe or noncompeti~ive; and the like.
A second type of immunoassays relies on the combination of the patient sample with the isolated 7gkD
antigen polypeptide or a fragment thereof. The polypep ide will be able to bind to a patient antibody which has been produced in response to infection with Borrelia burgdorferi. The speci~ic protocols for detecting patient antibody with the polypeptides will - wo 93/~48 2 1 1 2 4 ~ S PCT~US92/0~39 generally rely on use of immobilized polypeptide to capture and remove the antibody from the sample. The captured antibody can be detected in using labeled anti-human antibody in a sandwich technique or using a labeled polypeptide in a semi-competitive technique.
An exemplary polypeptide-based assay for detecting infection by Borre~i2 burgdorferi is an enzyme linked immunosorbent assay (ELISA) where the polyp~ptide `~ is immobilized on a solid phase, usually a test well in a microtiter plate, although polystyrene beads, glass beads, agarQse beads, and other solid phases may also ~ind use. After axposure to sample and binding of antibody (if~one is present) the solid phase is exposed ,~ to labeled antibody directcd against the patient anti~ody, e.g.~ labeled;anti-human antibody. The label is an enzyme which may~b~ detected by exposure to appropriate substrates.~ Such ELISA techniques are amply ; described in;the~patent and scientific literature.
` Of~course, numerous other assay protocols exist which may combine use of the polypeptide antigens and antibodies of the present invention. The above descriptions~of polypeptide-based and anti~ody-based assays are intended~to be exemplary~only.
Tn addition to immunoassays, the presence of 25 ~ Borrelia burgdorf~i in a patient sample may be detected using~nucleic acid hybridization probes prepared a~
described hereinabove. The samples~will ~e treated to release chromosomal DNA from any Borrel~ia burgdorferi which may be present in the samp}e~ Optionally, but not necessarily, the patient sample may be cultured to expand the amount of Borrelia burgdorferi present prio~ to treatment to release the chromoæomal DNA. In a first paxt~cular protocol, the B~rrelia burgdorferi will be lysed to free the chromosomal DNA, and the released DNA
~ixed on a solid phase support. The DNA is denatured so that single stranded fragments remain on the solid phase, and the solid phase is contacted with the labeled .

W093/~8 ~ l 2 ~ ~ ~ PCT/US92/~--9 oligonucleotide probes of the present invention under hybridizing conditions. The hybridizing conditions are sufficiently stringent to assure binding specificity.
Hybridization of the nucleic acid probes to the solid phase is thus diagnostic of the presence of the Borrelia burgdorferi. Such nurleic acid hybridization methods are described in U.S. Patent Nou 4,358,535, the disclosure of which i~ incorporated herein by referenca.
An alternative protocol permits the Borrelia burgdorf~ri to be lysed in a liquid phase without prior culturing to expand the amcunt of the microorganisms present. ~ysing is effected using a detergent to free ~ the nucleic acids under denaturing c~nditiGns. The lysed : ~-~ solution is then contacted with the solid phasa which selectively binds a single strand of nucleic acids which : may:then be detected using the oligonucleotide probes of the:present inven~ion. Such an alternati~e protocol described in PCT Application 86/00139~,~the~disclosùre of which:is incorporated~herein by reference. Methods for ~ performing such nucleic acid hybridization assays are described in general in Hanes and Higgins, Nucleic Acid ~ybridization:;~ A~Practical Approach,~ RL Press, 1985 ; the~-disclosure of which is incorporated herein by ref~erence. : ~
25~ The~use of hybridization assays ~ay:be combined with~polymerase:ch~in reaction techniques to amplify any amount of a:gene encoding the 79kD~antigen~which may be present in the patient sample. Polymerase chain reaction employ pairs of nucleotides, usually from about 10 to 50 3 0 ~ nucleotides in length, and which are spaced f rom about 5 O
; : to 500 nucl~otides apart on either of the two complementary strands of the 79kD gene. Specimen ~ ~ preparation will include cellular lysis, typically :; ~ followed by separation of the released nucleic acids on a solid phase, usually for example, glass beads or silicone : particles such as those employed in the commercial kit : GENECLEAN, available from BiolO1, La Jolla, California.

~ ~093/U~X 2 1 i 2 4 S ~ PCT/US92/~5539 After the sing~e stranded nucleic acids are bound to the solid pha~e, they can be amplified by thermal cycling in the pres~nce of the nucleotide primers and DNA
polymerase. Typically, ~he temperature will be cycled between abou~ 9~C and 60C, for ti~e periods as short as 1 or 2 minutes per cycle. Cycling can be con~inued for as long as the desire to achieve amplification of a portion of the gene bounded by the two primers. The amplified gene fragm~nt may then be detected using labeled cligonucleotide probes generally as described above.
V~ccines against Borrelia burgdorferi infections may be prepared using the compositions of the ~- present invention. These vaccines may be pas ive, consisting of Ig supplementation-which interferes with Borrelia burgdorferi growth or toxicity. Alternatively, v~ccines may be.active pro~ucing a humoral and/or cellular response providing protective immunity by active suppression of Borrelia burgdorferi infections.
~ P. vaccine prepared utilizing the 79 kD
polypeptide antigen or immunogenic equivalents will usualIy comprise: (a) fixed s:ells ei~her recombinantly : : altered to produce tbese antigen proteins or cells from ~ :the Bor~elia burgdorferi itself; (b~ a crude cell ~Ytra:ct; or (c) a partially or completely purifiecl antig:en preparation. Fusion proteins combining a segment , of ~he 79 kD antigen will be readily prepared. In some embodimar.t--, a;multiple vaccination may be achieved by fusing these antigens with other target antigens on a single protein, inducing p_otection against multiple infectant vectors. Alternatively, a "cocktail" of different immunogens may be simultaneously administered or inoculated. These immunogens can be prepared in vaccine dose form by well-known procedures. These 3 5 vac ines can be in the form of an inj ectable dose and may ~e administered intramus~ularly, intravenously, or subcutaneously. These vaccines can also be administered W093/0~8 2 ~ PCT/US92/0~`9 intranasally by aspiration, or orally by mixing the active components with food or water, providing a tablet form, and the like. Means for administering, or more typically inoculating, these vaccines should be apparent to those skilled in the art from the teachings herein;
accordingly, the scope of the invention is not limited to any particular delivery form.
For parenteral administration, such as subcutaneous injectionl these immunogens can be combined with a suitable p~ysiologically acceptable carrier, for "
e~ample, it can~be administered in water, saline, alcohol, f~ts, waxes, or buffered vehicles with or with~ut various adjuvants or immunomodulating agents.
Suitable immunological adjuvants or agents include, but are not limited to, aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfatet silica,~kaolin, carbon, water-in-oil e~ulsions, oil-in-water emulsions, muramyl dipeptide,~ bacterial endotoxin, lipid X,~Corynebacterium parvum~
~ Proplonobacterium~acnes),~Bordetolla~pertussis, polyribonucleo~ide~,;sodium alginate, lanolin, lysolecithin,~ vitamin A, saponin, liposomes, levamisole, DEAE-dextran,~bloaked copolymers or othêr~synthetic adjuvants.~ Such adjuvants~are available~;c~ommercially Z~5 ~ from~various~sourc~s, for example, Merck~Adjuvant 65 erck and Company, Inc., Rahway, N.J.~)~`or Freund's Incomplete Adjuvant and Complete Adjuvant ~(Difco ;Laboratories, Detroit, Michigan). Other suitable adjuvants are Amphigen (oil-in-water), Alhydrogel 30 ~ (aluminum hydroxide), or a mixture of Amphigen and Alhydrogel.
; The~proportion of immunogen and adjuvant can be ~aried over a broad range so long as both are~present in ~ ~ ~ effective a~ounts. On a per-dose basis, the amount of ;~ 35 ~ the immunogen can range broadly from about 1.0 pg to ~ :
about 100 ~g pe~ ~g of host, u~ually at least about 1~
pg, typically at least about 100 pg, and preferably at ! . WO 93/o~ 2 1 1 2 ~ ~ S P ~/US92/0~53~
}9 least about 1 ng per kg of host weight, and usually less than about 1 mg, typically less tha~ about 10 ~g, and more typically less than about 1 ~g, and preferably less - than about 100 ng per kg of host. A preferabl~ range is from about 10 pg to about 100 ng per dose. A suitable dose size will usually be between about 0.01 and 5 ml, preferably about 0,5 ml for a 20-59 kg organism.
Comparable dose forms can also be prepared for parQnteral administration to smallar or larger patients but the amount of immunog~n per do e will usually be smaller, for a smaller patients.
For the initial vaccination of immunologically naive patientæ, a regiment of between 1 and 4 dos~ can ~ be used with the injections spaced out over a 2 to 6-week period. Typically, a two-dose regimen is used. The seco~d dose of the vaccine then should be administered :~ s~me weeks after the first dose, for example, about 2 to : ~ 4 weeks later. Patients that have been previously exposed to Borrelia burgdorferi.
20~ Th vaccine may also be combined with other vacclnes for other diseases to produce multivalent ~:vaccines. It may also be combined with other : medicamen~s, for ~xample, antibiotics. A
: pharmaceutically effective amount~of the vaccin~ can be : 25 : employed with~:a pharmaceutica}ly a ceptable carrier such : as viral capsid prot~in complex or diluent understood to . ~ be useful for the vaccination of animals.
The~foIlowing examples are offered by way of : iIlustration, not by way of limitation~
EXP~RIMENTAL
~T~RIj~LB ~ND M~T~OD~
B~c~orial ~tr~is~. The isolates used in this study were chosen to represent a wide geographical distribution of : B. burgdorf~rl strains. The B. burgdor~ri i~o}ates Son 335, Son 188,~ Hum 3336, and Ala 410 were received from M.
Bis6ett (California Department of Public Health, Berkeley, California). Isolates RSL20, CA2, and CA3 were W0~3/~ PCr/US~2/0' ? ~ 20 received from R.S. Lane ~Department of Entomological Sciences, U.C. Berkeley, California). The B. burgdorferi isolates B31 (Bb), B3s (At), Wisconsin, Massachusetts, 297, B914, and X48, B. h~rmsii, B. coriaceae and B.
duttonii strain~ were supplied by R.C. Johnson (University of ~innesota, Minneapolis, Minnesota).
Strain PGU was received from V. Preac-Mursic (University of Munich, Munich). The B56 isolate was received from A.
. .
Schonberg (Institute fur Veterinarmedizin des : 10 Bundesgesundheitsamtes, West Germany), B . anserina strain was obtained from R. Walker (U.C. Davis, Davis, California) (see Table 1). The spirochetes were maintained in BSKII (Barbour-Stoenner-Kelly) medium (Barbour (1984) Yale J. Biol. Med. 57:521-525) at 33C.
The cells were harvested at mid-logphase of growth ~(10~/ml), washed in PBS, and prepared for DNA or protein ~analysis as:described below. :
A~LE l~ De-cription of~Organi~ms ol~t- ~ource ~ Lo~tion 20 ~ B~: burgdorf~
B31 ~TCC35210 Ixodes dammini New York ;:
, ~ ~
Wis ~:~human skin isolate Wisconsin : Ma human skin isolate Mass~chusetts ;;B35 reference strain ~ TCC35211 Ixodes ricinus Swit2erland K48 Ixodes ricinus Czechoslovakia V297 hum~n cerebral Connecticut spinal fluid ~
B56 Ixodes ricinus Germany B914 Ixodes ricinus Germany R/GU human skin isolate Germany Cal~for~olat~s : : cou~ty Son335Ix~des pacificus 50noma Sonl88: Ixodes pacificus Sonoma ;: H~m3336 Ixodes pacificus Humboldt Ala410Ixodes pacificus Alameda .

2 121~S
t ~-~W093J0~8 PCT/US92/0~539 C~ Ixodes neotomae Mendocino CA3 Ixodes pacificus Marin RSL20 Ixodes pacificus Marin B . cor~ ac~a~3 - ATCC Ornithodoros coriaceae California 43381 (B.c.) B,. he~sii ATCC Or~ithodoros hermsii Washington 35209 (B-h-) B . t~n~e~i~a poultry . California B. duttonii mouse Minnesota losi~g of th~ ~hro~o~o~al D~ ~o~ stra~ B31.
~: Chromosomal DNA was isolated as follows. A 20~ml portion : ~of logarithmic-gr~wthapha3e cells was wa~hed three times in~phosphate-buffered saline (pH 7.2) plus 5 mM MgCl~.
The pellets were ~uspended in 100 ~1 of 15% sucro~e in 50 mM Tris hydrochlQride, pH 800-50 m~ E~TA, to which an ~:equal Yolume of 5 M~NaCl wa~ added. To this solution wa~
added~1/10 volume (~20 ~1) of 1~ sodium deoxycholate. The : ~solut:ion was placed on ice ~or 20 minutes, followed by centrifugation in ~ microcentrifuge for 5 min. ~The ~uperna~ant fluid, containiri -he plas~id DNA, wa~ saved : for other studies.~ The pe .-_, containing the chromosomal DNA was suspended in 100 ~1 of 15% sucr~se in 50 m~ Tris hydrochloride, p~ 8.0-50 m~ EDTA. To this was added 50 ~1 of 100% sodium dodecyl sulfate and 25 ~1 ~of 20 mg of pro~einase K per ml~ The solution wa incubated for 3Q min at 37C, followed by phenol extr~ctisn and e~hanol precipitation. The chromosomal D~A was digested with ClaI and ligated into a ClaI and ligated in~o a ClaI-digested P~v-Vrf e ~ ression vector (Crowl e~ al. ~1985) Gene 38:31-38). E. coli R~I t~

2112~1~SS
W093/0~ ^ PCT/US92/0 cI857) was ~ransformed wit~ the recombinant plasmids.
Expression of cloned genes into this vectdr was controlled by heat shock (4~C ~or 2 h). Recombinant colonies were screened for expression of B. burgdorferi antigens by colony ~lot immunoassay ~Craft et al. (19B6) Clin. Invest. 7B:934-938). Positive clones were further characterized by immunoblot assay (Burne~t ~1981) Anal.
: Biochem. 112:195-203, Towbin et al. (1979) Proc. Natl~
Acad. Sci. USA 76:4350-4354~ by using sodium dodecyl sulfate-polyacryla~ide gel electrophoresis: (12% gels) ~(Laemmli (1~70)~Nature 227:680-685). Recombinant plasmids from clones iden~ified to be expressing B. burgdor~eri antigens:were purified~.~Birnbolm ~1983) ~ J. Clin. Microbiol. 25-2296-2301), radiolabeled (Feinberg: 15 - et al~ (1983) Anal. Biochem. 132:6-13, Maniatis ~t al.
(19B2) ~Molecular Clonin~: A Lab~ratory~ Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), and used to~probe S~uthern blots (Southern (1975) J. Mol.
Biol.:98:503-51B)~ of pulsed-field-gels ~Schwartz et al.
Z0~ (19B4) Cell 37:67-75:containing undigested, whole-cell B. bur~dorferi~ ;DNA. The electrophoresis was performed as :pr~viously described (LeFebvre et al.~(1989) J. Clin.
icrobiol. 27:38g-393.
8ubclo~i~g an~s-gu-~eing. The gene expressing the 79.8kD antigen~(L~Febvre;et al. (19903 J. Clin.
:Mic~o~iol. 28:1673~-1675) was determined~ to be contained < ~ within a 2.5kbp FoKI fragment of the original 6.5~bp ClaI
insert in the pLPI plasmid based on DNA:sequence ~: ~nalysis. This fragment was subclonzd~into a pUC~9 : 30 plasmid according to standard procedures (Maniatis et al.
; ~ ( 1982 ) supra . ) and was designated pLP4. The gene was sequencea according to standard procedures tSanger et al.
(1977) Proc. Natl. Acad. Sci. USA 74:5463-$467). Within this 2~5Xbp fragment were several ~indIII sites. A 500bp H~ndIII fragment o~ the gene was subcloned, radiolabeled t~einberg et al. (1983) Anal. Biochem. 132:6-13, Maniatis (1982) sup~a. ), and used as a probe to determine the ;~`V093/0~8 2 1 1 2 Rr ~ s PCT/US92tO5~39 - presence and configuration of the gene in other B . bur~dorf eri isolates from North America and Europe as well as other Borrel ia spp.
- ~ ino aci~ 3~que~c~ ana ~ 8i~ of the ~ntigen. The amino acid sequence of the antigen, hydrophobic and - hydrophilic domains, and codon usage were derived from the nucleic acid sequence using the Genetics Computer Group (GCG) software (Devereux (1989) Version 6.0, Genetics Computer ~roup,:University of Wisconsin, ~ Biotechno}ogy Center, Madison, Wisconsin).
~DetectiQ~ of th- ~bro~osoma~l ge~ e~pr~ing the 75.8~D
~tige~ i~ North a~ri~a~ ~ud ~uropean i~ol~tes. Whole cell DN~ from~the Borrelia spp. was isolated as previously~described ~:Anderson et al. tl989) J~ Clin.
~icrobiol. 27il3-20j. The purified ~NA was digested with ClaI (the restriction enzyme used initially to clone the 79.8kD gene (LeFebvre et al.:(199Q) supra.): according to ;the: manuf acturer's specif ications (BRL).~: The digested fragments were~fractionated in a 0.7% agarose gel at 60V
20~ ;for~15.5 hr in Tris-Borate buffer ~TBE, 0.089M Tris, 0.~0~89~boric ac:id, 0.002M EDTA,~ pH8.0). The gel was :stained with athidium bromide:and photographed under ultraviolet illumination.~ The DNA was transferred to a nylon~membrane~by the method of Soutbern (Southern (1975) 25~ :J~ Mol. Biol~.~47::61-66). ;The hybridization and subsequent washes~were carried out;under stringent conditions according to standard procedures (Maniatis (1982)~ supra.):.~
~ I~u~oblot a~ly~i~ for expre~io~ of th~ 7~.8~D ~tigen : 30 i~ North Amer~c~n an~ ~urop~ olat~. Expression of : the 79.8kD antigen in the B. burgdorferi isolates ~:~d :
Borrelia spp. was determined. Sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as previously described (Laemmli (1970) Nature 227::680-685, Maniatis et al. (1982) supra.).
Approximately 20 ~g of whole cell lysates of each isolate was added to a 12% gel. The gel was run at 60 mA

W093/0~ PCT/VS92/Or 9 ~ 3~ 24 constant current for 4.5-5 hr. Transfer of the electrophoresed proteins to nitroce}lulose membranes was performed with a Pharmacia/LRB semi-dry transfer system according to manufacturer's specifications (Pharmacia, Uppsala, Sweden). The membrane was blocked and probed by standard procedures (Maniatis et al. (1982) supra. ) .
Rabbit anti erum raised against B. burgdorferi (titered at 1:10,000 ~y enzyme-linked immunosorbent assay (ELISA~) : was used at a dilution of 1:5000 to probe the blot.
Biotinylated goat anti-rabbit antibody (Vector Laboratories,~Burlingame, California~ waæ added, followed ~; by alk:aline phosphatase:conjugated to streptavidin (K~irkegaard and~Perry:Laboratories, Gaithersburg, Maryland). The~enzyme substrate for ~he alkaline phosphatase was 5-bromo-4-chloro-3-indolylphosphate.
: ~ ~ RE8~LT~
Cloni~g of Chro~o~o~al;DN~ from ~trai~:B31.
, O~approximately 10 clones~which were identified as~expressing B. bur~dorferi antigens in E.
2~0~ coli ~ce:lls, 3 were found to express an 83-kDa protein later~determined to be :approxima~ely 79.8kD), as détermined with~rabbit~polyclonal antiserum~r~ised àgainst whol--cell B. burgdorferi (Fig. 1):. The protein had~the same~:molecular size and antigenic reactiYity as 25:~ an~83-kDa an~igen~in~:B. burgd~rferi cells.~ Several lower-molecular-weight antigens were also expressed by t~e~clone. These antigens are not discussed further herein:.~
: Fig.::l~is an immunoblot demonstrating the r activity of the putative 83 kDa antigen to rabbit : antiserum raised against whole-cell B. burgdorferi. The :~ : whole-cell lysat:es:were fractionated in 12~ acrylamide ~: gels prior to transfer to nitrocellulose membranes. Lane : 1, B. burqdorferi B31; lane 2, E. coli RRI (~ cI857) 3S (negative contFol); lane 3, induced:E. coli RRI ~ cI857) containing the~Pev-Vrf expression vector (negative control); lane;4, ~. coli RRI (~ cI857) containin~ a 2112'161~
~V093/N~* PCT/US92/0~539 recombinan~ Pev-Vrf ligated to a randomly cloned piece of B . burgdorferi chromosomal DNA; lanes 5 to 7, ~. coli RRI
~ cI857) induced with recombinants of Pev-Vrf containing B. bur~orferi chromo~omal DNA inserts expressing the 83-kDa antigen.
One of the recombinant plasmids expressing the putative 83-~Da an~igen, designated pLPl, was further characterized. Fig r 2 represents an immunoblot of lysed ~: whole-cell B. burgdor~eri an~ E. coli cells transformed with either pLPl~or pUC18 plasmids. The blot was exposed ~to human antisera from two patients presenting with symptoms of ~yme borreliosis and determined to have been exposed to~the:agent by using enzymè-linked immunosorbent assay ~Voller:~et;al:. ~1980) Manual o~ Clinical Immunology, 2nd~ Ed:., American Society for Microbiology, Washington, D.C.~) with sera diluted 1:200 (data not shown~ The 83-kDa antigen expressed in the E. co~i cells~containing the pLPl plas~id reactéd w~ith the sPra from~both patients. No~reaction at~this~molecular size~: 20 : ~ was~seen in the;pUC18 control cells~ These results demonstrat,e the fact that the 83-kDa a~tiqen expressed by the~cloned chromos~al gene in E.;coli~represents a major antigen~of B.~burgd~rferi which induces:an immune r~esponse in the~huma~ host. No~reaction:to the antigen ~ was~observed with~antisera from humans~who served as negative controls (data not shown).~
The~ unoblot of Fig. 2~demonstrates the reactivity of~the putative 83-kDa~antigen to human serum from seropositive Lyme borreliosis~patients. Lanes 1 to ~ 3 were reacted with~serum from patient A~ and lanes 4 and : : 5 were reacted with patient B antise~um. Lane 1, B.
burgdorferi B31 whole cells; lanes 2 and 4, F. coli ~RI
(A c1857) induced with the Pev-Vrf expression ve~tor : (negative control), lanes 3 and 5~, E. coli RRI t~ c~857J
35 ~ transformed with the recombinant plasmid pLPl expr~ssing the 83-kDa antiget2.

,~ ~

W093/~ PCT/US92/0~9 2 ~ 26 Fig. 3A is a`photograph of an ethidium bromide-stained pulsed-field gel illuminated by W irradiation.
Several bands, representing linear and circular plasmids as well as chromosomal DNA, are evident. Of importance in this figure is the largest band which entered the gel (-l.c.) and the DNA remaining in the loading well. These :~
two regio~s represent broken or linearized chromosomal DNA and intact circular chromosomal DNA, re-~pectively.
: As de~onstrated hereinafter, both of these bands ~10~ represent chrsmosomal DNA of B. burgdorferi. Fig. 3B is an autoradiograph demonstrating the results of a Southern blot hybridization o~ this gel. The probe used in lane 1 ~: was labeled whole~cell B31 DNA. RadiQlabeled recombinant plasmid pLPl;(Maniatis et al. ~1982) supra.) was used as the probe in lane 2. The pLPl probe hybridized specifically and exclusively to the two bands representlng B. burgdorferi chromosomal DNA, as described ;~
above.~ Thus, pLPl:~represents the f~rst description of a ;chro~oso~al gene e ~ reosing an antigen in B. burgdorferiO
20 : ~; Only B . coriaceae has been shown to encode ma~or antigens n~chromosomal~DNA (Perng et al. ~1990) Infect. Immun.
58:1744-1748)~ ;All:other reports:describing antigen expressio~-in Borrclia species have located the genes on linear~:plasmids~(Barbour (1988) J.~;Clin.~:Microbiol.
25 ~ 26~475-478; Barbour et al. (1987) Science 237:409-411;
N~we:~et al.:(~1986) Infect. Immun. 54:207-212; Howe et al.
(1984;)~ Science 2~27:645-646; Meier et al. (1985) Cell 41::4~03-409; and Plasterk et al. ~1985) Nature 318:257-262). ;~ ~
Fig. 3A is an ethidium bromide-stained pulsed-field gel of:whole-cell B. burgdo ff eri B31 DNA, cc., Circular chromosomal DNA which did not enter the gel;
~ l.c. broken or~linearized chromosomal DNA. Fig. 3B is a :~ Southern blot of the gel in Fig. 3A. Lane 1 was probed with radiolabeled whole-cell B31 DNA. Lane 2 was probed with the recombinant plasmid pLPl ~ontaining the 83-kDa antigen gene.

2112~ ~ S
0~3/~ PC~/US~2/05539 ~7 DNA and ~i~o acid .~eque~c~. The gene expressing the 79.8kD antigen wa~ determined to be contained within the -2.5kbp ClaI fragment in the original pLP1 clone. The gene was sequenced and a functional TATA box (Pribnow Box) and ribosome binding site (Shine Dalgarno se~uence) were identified (5EQ ID NO:13. SEQ ID NO:l lists the DNA
sequence of the gene, and SEQ ID NO:2 lists the amino acid sequence of the expressed antigen. The : hydrophobicity profile revea~e~ that the 79.8kD protein is a hydrophilic protein (78%). Two large hydrophobic domains were identified; one located at the carboxy-terminal portion (about 30 amino acid residues), and the other at the amino~terminal portion ~about 20 amino acid ~-residue~). The central domain of the protein consisted of hydrophilic amino acids (about 350 amino acid reæidues). On the basis of its amino acid composition, the 79.8kD protein is an a~idic protein with an isoelectric point of 4.92. The calculated molecular mass (79.8kD) was so~ewhat lower than the molecular mas~
; 20 ~estimated from~:SDS-PAGE (83kD). No cysteine residues, two~tryptophan residues,~and three histidine r~sidue~
were found: in~the deduced~amino acid~sequence. In a comparison with previously~published protein sequences from a search~of a protein data base, a low degree of ho~ology (<15%3 was~observed. Table 2 1ists the c~don frequency and amino acid usage for the antigen.

WO 93~Q044B 2. 1 1 2 4 ~ ~ 2 8 PCI /US92/~ 9 TABI,E: 2 . Codon f requency and amino acid usage in the B31 79 . ~kDa antigen gene.

A . A . L Codon Numberb A . A . Codon Number ~;ly GGG 3 Thr ACG 0 :~
1 t) Gly GGA 8 Thr ACA 11 Gly GGT 10 Thr ACT 13 Gly GGC 4 Thr ACC 2 Glu GAG 23 Tyr TAT 19 Glu G~A . 40 Tyr T~C 0 2 0 A~p GAT 60 Leu TTG 14 A3p GAt: 8 Lau TTA . 31 Val GTG 5 Phe TTT 2 3 2~i Val GTA 6 Phe TTC 2 Val GTT 30 Ser TCG 0 3 0 ~ Val 5TC 0 Ser TCA 9 Ala GCG 0 Ser TCT 23 : Ala GCA 11 Ser TCC 3 ~ ~
Ala GCT 13 ~ Arq CGG
Ala GCC: 3 Arg CGA
4 0 Ala~ GCC 3 Arg CGA
Arg AG~: 5 Arg CGT
Ars~ AGA 12 Arg CGC 0 Ser M:~ ~ 22 Gln CAG 12 Ser A~C 9 Cln CA~ 18 ~ Ly~ AAG 28 His CAT 3 Ly~ A~ 63 Hi8 CAC 0 ASN AAT 4 0 Leu CTG
Asn AAC 5 Leu CTA
Met ATA 9. Leu CTT 21 6C) Xle ATA 11 Leu CTC 0 Ils~ ATT 40 Pro CCG 2 I le ATC 2 Pro CCA 3 2 1 1'~
. .~WO 93J0~448 PCl/US92/05539 Table 2 - contimled :: Cys TGT O Pro CCT 11 Cy8 TaC O Pro ::CC O
End TAG O 'rrp TGG
End TAA 1 End TGA O
~ ...... .
~ A . A.: al; ino a~id .
b Number indicate~ the ~um u~age of a codon in the 79 . 8kD
antigen ge~ne.

D~t~ctio~ of t~e 79.8~D a~t~y~ ge~ i~ B . bu~gdorferi isol~e~ Borrelia 9pp. Whole cells DNA, digested ~-with Cl~I, was electrophoresed, blotted, and probed with the lab~led 500bp subclone of the 79.8kD protein gane.
Fi~. 4 displays an autoradiograph of the ClaI digested whole c~ll DNA hybridized t~ the probe. A single major band of approxi~ately 6.5 kb was seen in B31, the strain from which the gena was cloned. A single ~ajor band of the same size was~also obser~ed in the Wisc, 297, MA, Sonl~8, RSL20, AT, and CA2 B. burgdorfe~i isolates and i~
B . ~anserina .
The B914, B56, and K48 strains were similar to each other in that they revealed 2 major band which hybridized to the probe. The weaker of these 2 bands resol~ed at 2Okb and the stronger reso~ved at 3kb. The ~S P/Gu strain demo~strated 4 band which hybridized to the probe at 6.8, 4.1, 2.1, and l.O kbp. The B. coriac~ae, : B. ~rmsii, B. duttoni did not hybridize to the probe.
I~u~o~ay o B. burgdor~eri ~trai~ ~d Borrelia s~p~
for ~xpr~io~ o . th~ 79 .8kD ant~g~ i~ vitro. Fig. 5 is an:immunoblot of whole cell lysates of representative strains of the B. burgdorferi isolates and other Borrel ia spp. discussed in Table l. It is apparent from Fig. 5 that the 79.8kD antigen is a commonly expressed antigen amongst B. burgdorferi isolates. All of ~he strains assayed exhibited a reacting antigen at approximately W093/0~ 2 1 ~ S 30 PCT/US92/~ 9 7~.8kD. Previous rPports have also identified the presence of this antigen in other B. burgdorferi isolates (Craft et al. (1986) supra. j LeFebvre et al. (1990) supr~.; LeFe~vre et al. (1990) J. Clin. Microbiol.
28:700-707; Maniatis et al. (1982) supra .; Muhlemann et al. (1982) Lancet i:553-554; and Nadal et al. (1989) Pediatr. Res. 2~:377-38~ The B914 strain exhibited a slightly higher molecular weight antigen. Of the non-B. burgdor~eri spp., only B. anserina ~xpressed a reacting antigen at 79.8kD, In Coomassie blue or silver stained gels of ~. burgdorferi the 79.8kD protein could not be vi~ualized (data not shown) thus suggesting that it i~ expressed at low levels within the cells. This ~ ~inding, in turn, indicates that the protein is a poten~
immunogen based on its low level of éxpression within B. burgdorferi iso}ates and its ~trong reactivity to anti-B. burgdo~feri immune sera.
Although the foregoing invention has been dPscribed in d~tail fur purposes of clarity of - 20 under~tanding, it will be obvious that certain ^ modifications may be practiced within the scope of the appended claim~.

:

~ ; .

21i2~ ~,S
`~W~93/~k~ P~T/~92~39 .

SEQUENCE LISTING

~') GENERAL INFORMATION:
'~) APPLICANT: LeFebvre, Rance B.
Perng, Guey-Chen i_) TITLE OF INVENTION: Methods and Compositions for Diagnosing Lyme Disease ~iii) NUMBER OF SEQUENCES: ~
i~J~ CORR~SPONDENCE ADDRESS:
(A)-ADDRESSEE: James M. Heslin . (B) 5TREE~: 379 Lytton Avenue (C) CITY: Palo Alt~
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(B) FILIN~ DATE:
(C~ CLASSIFICATION:
(viii:) ATTO~NEY~AGENT INFORMATION:
~A) NAME: Heslin, JamaR M.
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(A) TELEPHONE: 415-326-2400 ~B) TELEFAX: 415-326-2422 .

WO 93/00448 . PCl~/US92/(~ 9 ~ .L t 2 ~ 3 2 2 ) I~FORMAT~ON FOR SEQ D NO: l:
(~) SEOUENCe CXARAC'rERISTTeS:
( A ~ LcNC;'r~: 2258 base pas rs ( El ) TYPE: nucleic acid ( C ) ST~ EDNESS: single ( D ) TOPOLOGY: linear ( ii ) MCSLECULE ~PE: DNA ( s~nom~ c ) ( LX ) FEATURE
,~ . ( A ) NAME/KE~F: CDS
~~ ( B ) LOC~TION: 51. . 2151 (.xi) SE~UENCE DESC~IP~ION: SQ ID NO:1:
ATT~AGTTGt; TAII~GGTAGAT T~TAATTAAA TTTATA5t;AG AATTTcrTTT ATG AAA 56 Met Lys ~: AAA ATG TTA CTA ATt:: TTT Al:;T TTT TTT CTT ATT TTC 'TG AAT GGA TTT 104 ~ . ~
Lys Met Leu Leu Il~ Phe Ser Ph~ Phe Lau Ile Phe Leu Asn Gly Phe

5 10 15 CCT GTT ~T GC:A A~ t;AA GTT G~T AGG G~A AAA TTA AAG G~C TTT GTT 152 Pro ~Val Ser Ala: ~ Glu Val Asp Arg Ç:lu Lys Leu Lys Asp Phe Val 2:5 3~) x,~r ATG G~ C:TT GAS; T~T GTA AAT TAT AAA GGC C~ 'rAT GAT TCr ACA 2.0Q
Asn M0t Asp Le~ Glu P~e~ Val A~z~ Tyr Lys Gly Pro Tyr Asp Se~ Thr 5~
~AT AC~ TAT GA~ C~A ATA GTG &GT ATT GGG t;AG T~T TTA GCA A~;A CCG 248 Asn Thr Tyr Glu Gln Ile Val Gly Ile Gly Glu Ph~ L~u Ala Arg Pro TTG ACC AAT TCC AAT AGt:: AAC TCA AGT TAT TAT GGT AAA TAT TTT A~T 296 r eu Thr Asn Ser Asn Ser As~ Ser Ser ryr Tyr Gly Ly~ Tyr Phe Ile 7~ 7~i 80 ~AT AG~ TTT ATT GAT G~T C~A G~r AAA AAA GCA AGC t;TT GAT G$T TTT 344 Asn Arg Phe Ile Asp ~LSp Gln Asp ~ys Lys Ala Ser Val Asp Val Pl~e ~5 90 95 2112~53 S
r ~ 93/~0448 PC~/US92/05539 TCT ATT GGT AGT AAG TCA GAG CTT GAC AGT A'rA TTG AAT T"'A AG~ AG~ 392 Se- ~' e Gly Se~ Lvs Ser Glu Leu Asp Se~ Ile Leu Asn Leu ~S Ar~
;00 lOS 1' 0 ATT CTT A~A GGv T~T TTA ATA AAG TCT 'rTC GAT TAT G~C At;G ~C~ AGT ~40 eu Thr Gly ryr L~U I1B Ly-~s Ser Phe Asp q~ Asp Ars~ Ser Ser 11; 120 125 130 G;~ GAA ~TA AT~ GCT AAG GTT ATT ACA ATA ~T AAT GCT GTT T~T AGA ~86 A;a Glu Leu ~le Ala Lys Val Il~a Thr Il~ Tyr Asn Ala Val T~rr Arg 135 140 14a GGA G~T TTG G~T ~rAT TAT AAA GGC; TTT TAT ATT GAG GGT GCT TT2. AAG ~36 Gly Asp L~u A-~p Tyr Tyr Lys Gly Ph~ Tyr Tle Glu Ala Ala Zeu Lys I50 lS5 160 T~ TTA AGT AAA G~A AAT Gt:A GGT CrT TCT AGG GTT TAT AGT CAG TGv 384 S~F L6~U Ser I,ys Glu Asn Ala ~ly ~u Ser Arg Val 'ryr Ser Gln Trp GC . GGA AAG ACA C~A ATA TTT ATT ccr CTT AAA AAG GAT ATT TTG TCT 632 Ala Gly Lys: Thr Gln Ile Phe Ile Pro Leu Lys Lys Asp Ile Lau Ser 180 lP'5 190 GGA A~T ATT GAG TC r GAC ATT GA~ ATT GAC AGT TTA G~T At:A GAT A~AG 680 Gly Asn Il~ Glu Ses Asp Ile Asp Tle Asp Ser Leu Val Thr As~ Lys 195 200 205 ~lO
GTG GTG GCA GCT CTT TTA AGT GAA AAT G~A GCA ~;GT GST AAC ~TT GCA 728 Val Val Ala Ala Leu Leu Ser Glu Asn Glu Ala Gly Val Asn Phe Ala 215 2~0 225 AGA GP~T ATT ACA GAT ATT CAA GGC GAA ACT CAT AAG GCA :;AT CA~ GAT 775 Arg Asp ~le Th: Asp Ile Gln Gly Glu Thr His Lys Ala Asp Gln Asp ~AA ATT GAT ~TT ~AA TTA GAC AAT ATT CAT GAA AGT GAT TCC AAT ATA 824 Lys Ile Asp Il~o Glu Leu Asp Asn Ile His Glu Ser ~sp Ser Asn Ile ACA G~A A~r ATT GAA AAT TTA ~GG GAT CAt; CTT GAA A~ Gt:T AC~ GAT 872 Thr Glu Thr lle Glu Asn ~eu Arg Asp Gln Leu Glu Lys Ala Thr Asp 260 265 27~

WO ~0448 PCl'/VS92/0 9 ~ -~ J~ S 3 4 G~A GAG C~T AA~ AAA GAG ATT G~ AGT C~G GTT GAT G~, . ~A AAG AAA ~ .^
G1U G;U HiS LVS LtS GiU I1e G1U Ser G1:~ Va1 ASP A1a LYS LJS L S

C~A ;~AG G~A G~G C~.~ GAT AAA AAG GCA ATA AAT crT GAT AAA GCT C~G 368 Gl~ Lys alu Glu Leu Asp ~ys Lys Ala Ile Asn Leu Asp Lys Ala G;n 2gS 3QO ^05 C~A AAA TTA ~T TCT G~ G~A GAT AAT TTA GAT GST CAA AGA AAT ~CT
G1n LYS LeU ASP Ser A1~ G1U ASP ASn ~eU ASP Va1 G1n Arg Asn Thr 310 315 320 `
GTT AGA G~ AAA ~TT C;~A GAG GAT ATT AAC G;U~ ATT AAC AP.G G~A AAG :^6d, ,~.
~ Val ~rg G;lu I,ys Il~ Gln Glu Asp Ile Asn Glu Il~ Asn Lys Glu L~s ~.

AA~ TTA CCA ~ CCT GGT G~ GlrA AGT TCS CCT ~A GTT GA'r A~G t:~ 1'i2 Asn ~eu Pro Lys Pro Gly Asp Val Ser Ser Pro r.ys Val Asp Lys Gln 340: 345 50 ~ AT~ AAA GAG AGC C:~G l:;AA GAT T'rG CAG GAG CAG C~T AAA GAA 7160 Leu :Gl~ ~la I-2'5 Glu Se~ ~u Glu Asp Leu Gln Glu Gln 1eu Lys Blu 55 ~ 360 365 370 ACT GGT GAT GAA AA'r C~ AA AGA Ç~ ATT GAA AAG CAA AST G~ ATC ' 2~8 : i ~Thr Gly Asp Glu Asn Gln Lys Ar~ Glu Ile Glu Lys Gln Ile Glu Ile : 37S 380 385 : - AAA AAA ABT G~T GAP AAG CTT TTA AAA AGT AA~ GAT GAT ~AA GCA AGT 125 hys Lys Ser Asp Glu Lys Leu I.eu I,ys Ser Ly~ Asp Asp Lys Ala Ser . 390 395 400 AAA GAT GGT ~ Gr'- TTG GAT c'r~ l;AT CGA GAA TTA AAT TCT AAA GCT .304 ~ys Asp Gly Lys Ala Leu Asp Leu Asp Arg Glu Leu Asn S~ Lys Al2 TCT AGC P~ GAA AAA AGT AAA GCC AAG GAA GAA GAA ATA ACC AAG GGT 13 Ser Ser Lys Glu Lys Ser I.ys Ala Lys Glu Glu Glu Ile ~hr Lys Glv 4;~C~ 425 430 21~2 1 6~
JO 93J~10448 PCI'/VS~2/05539 ~AG TCA CAG AAA AGC TTA G:;C G~T TTG ~AT .~AT GAT G~A AAT CTm ATG 1400 _ Js Ser Gln L ys Ser Leu G~y As~ Leu Asn Asn ~s~ Glu Asn Leu ~et ~3a ~40 445 ~50 ~ G CC~ GAi~ GAT CAA AAA TTA CC'" GAG GTm AAA A~A TT~ GAT AGC AAA 1448 Met Pro Glu Asp Gln Lys Leu Pro Glu Val Lys Lys Leu Asp Ser Lys ~55 460 46a G~ TTT AA~ GT &T~ TG-- G~G G,m G~G A~A TTA G~T AAG AT"' TTm 1~56 ;,ys Glu Phe Lys Pro V~l Ser ~;lu Val Glu Lys ~eu Aso Lys Il~ Phe AAG TCT AAT AAC AA~ GTT GG~ GA~ TTA TCA CCG TTA G~T AAA TC~ TG~ 1544 Lys S~r Asn Asn Asn Val Gly Glu Leu Ser P--o Leu Asp Lys Ser Ser : :~85 490 495 TAT A~A :GAC ~T~ G~T TCA AAA G~G G~G AC~ GTT ~T A~ GAT GTT AAT ' S92 Tyr r,ys ~sp Ile Asp Ser Ly~ Glu Glu Thr Val Asn l.ys Asp Val ~sn 500 505 510~ :
TT~ C~A AAG ACT AAG CC~ C~G GTT AAA G~C l::~A GTT ACT TCT 'TTG AAT 1640 :: L~U G12~ LY5 Thr LY5 P~O G~n Va1 LY~ ASP G1n Va1 Thr Ser LeU Asn : Si5 , 5~0 525 ~ 530 GA~;;GAT T~G ACr ACr ATG rcr ATA GAT TCC AGT AGT l CS GT~ TTm TT~ 1688 G1U ASP LeU Thr Thr Met S3r r1e ASP Ser Ser S~r ~rO Va1 Phe LeU

G~G ~rT ATT GAT C~ ATT ACA AAT ~TA GC;A ACT c~r C~A ~rT AT~ GAT 1736 G1U Va1 I1e ASP PrO I1e ~'nr ASn t.eU G1Y Thr Lau Gln Leu Ile Asp 55~ 5~ 560 TTA AA~ ACT GGT GTT AGC C~ AAA GAA AGC ACT CAG CAA GGC ATT C~G 1784 Leu Asn Thr Gly Va1 Ser L2U LYS G1U S~r T~r Gln Gln Gly Ile G1n CGG TAT GG~ ATT TAT GAA CVT G~A AA~ GAT ~TG GTT GTT ATT AAA ATG 1882 Arg q~S?r G1Y I1e TYr G1U Arg G1U LYS ASP LeU Va1 Va1 I18 LYS Met 580 585 5~

GAT T~ GGA AAA GCT A~G CTT CAG ATA Cr~ GAT AAA CTT G~A AAT 'rTA 1880 ASD Ser G1Y LYS A1a Lys LeU G1n I1a LeU ASD L~rs ~eu Glu ASn Leu 5~5 600 605 610 W~ 93/00~ PCI/US92/0~:-'9

6 J . 3 6 ~A GTG GTP. TCA GAG TCT A~r T TT GAG ATT ~T A~ ~T TCA TCT CTm q 328 .ys Yal Val Ser Glu Ser ~sn Pha G~u Ile Asn Lys Asn Ser Se- _eu ~15 620 525 T~'r GTT G~T TCT AA~ ASG ATT TTA G-'A GCT GTT AGG GAT AAA GAT AGT 1976 Syr Val Asp S~r Lys M~t 'le LBU Val Ala Val Arg Asp Lys Rso Ser 630 ~ 635 640 AGT AAS GAT TGv AGA TT~ GCC, AAA TTT TCT CCT AAA AAT TTA GAT G;~G 2024 b- Ser P sn Asp Ts~ Arg Leu Ala Lys Ph~ S-r Pro Lys Asrl Leu As~ Glu TTT ATT CTT TC~ G~G AAT AAA ATT ATG ccr T~'r ACr AGC TTT TCT GTG 2072 Phe Ile Le~ ser Glu Asn Lys I12 Met Pro Phe T~r Ser Phe Ser Val 660 66~ 670 ~: AGA AAA A~T TTT ATT T~ TTG C;~A GAT GAG 'rTT A~ AGT CTt~ GT~ ATT 2120 Arg Lys Asn Phe Ile Tyr :Leu Gln Asp Glu Phe Lys Ser Leu Val Ile T~ ~ GAT GTA AAT ACT T~A AAG AAA GTT AAG T AACTTTP.CAG TTAGATTTAA 2171 `~
- : . : :~
r.eu ~p Val Asn T~r l.s~u Ly~ r y8 Yal r~y8 695 ~ 700 TTTGTASAAA TCGTTAA~A ACC~A~0 C'r~AQA ~AGTTTT~G CTSTTTT~AT 2231 --~W~ 93/00448 2 PCI'/US92/OS539 - ~ 2 ) INFORMATION FOR SEQ ID NO: ~:
( i ) SEQUENt:E CKARACTERISTICS:
t A ) LENGT~: 700 amino acids B ) rYPE: amino acid ( D ) TOPOLOGY: linear ( ii ) MOLF :UI.E: rYPE: protein:
(xi) SEQUENCE DESCRI~TION: SFQ ID NO:2:
~et Lys Lys Met Leu Le~s Ile Phe S~r Phe Phe Leu Ile Phe Leu Asn lS
~-~ly Phe Pro Val Ser Ala Arg Glu Val Asp Arg Glu Lys Leu Lys Asp ~Phe Val Asn ~Met Asp Leu G1u Phe Yal Asn Tyr Lys Gly Pro T~r Asp Sèr ~hr~ ~Asn T~.r T~r Glu Gln Ile Val Gly Ile G}y ~Glu Phe Leu Ala : ~ 50 : ~ 55 60 Pro :Leu Thr Asn Se r Asn Ser Asn Ser Ser T~ Tyr Gly Lys Tyr 65~ 7~ 75 8~:1 Phe: Ile Asn Ar~ Phe Ile Asp Asp Gln Asp Lys Lys Ala S~ Yal Asp : ~ 85 90 ~ 9~
Val Phe~ Ser ~le GIy Ser Lys Ser Glu ~eu ~;p Se r Ile L~u Asn r,eu : ~ 100 105 : : : 110 Arg ~ ~g~ Ile Leu Th~ Gly Tyr Leu Ile L~s Ser Phl3: Asp Tyr Asp Arg 115 ~ 120 ~ 12S:
S~r Ser Ala Glu Leu Ile ~la Lys Y~l Ile Thr Ile ~yr Asn Ala Val 130 :: 135 ~ 0 Tyr Arg Gly Asp Leu Asp ~ Tyr Lys Gly Phe T~r Ile. Glu Ala A~a 145 150 155 ~ }60 Leu l,ys Ser Leu Ser Lys Glu Asn Ala Gly Leu Ser Arg Val Ty:r Ser 165 170 ~ ~75 Gln ~2 Ala Gly L~s Thr ~;ln Ile Phe Ile Pro Leu Lys Lys Asp I~le ~eu Ser Gly Asn Ile Glu Ser Asp Ile Asp Ile Asp Ser Leu V'al Thr Asp Lys Val Val Ala Ala Leu Leu Ser Glu Asn Glu Ala Gly Val Asn 21~ 215 220 W0 93/0~448 P~/U~,921~ g 2 1 1 ~ 38 Phe Ala Arg Asp Ile ~.r Asp Ile Gln Gly G}u Th_ His Lys ~la As~
225 230 235 2~0 Gln Asp Lys 'le P~.s;:~ _' e Glu Leu Asp Asn Ile His Glu Se_ As~ Ser 245 250 25:
Asn Ile Thr :;lu Thr Ile Glu Asn Leu Arg Asp Gln Leu Glu Lys Ala Thr Asp G u Glu His r,ys Lys Glu Ile Glu Ser Gln Va Asp Ala Lys Ly~; Lys- Gln Lys Glu G~.u Leu Asp Lys Lys Ala ~ e Asn L~u Asp Lys Ala Gln Gln Lys Leu Asp Ser Ala G~ u Asp Asn L~u Asp Val Gln Arsl ~sn Thr Val ~rg Glu Lys Il~ Gln Glu ~sp ~ Asn Glu Ile Asn Lys 3~5 330 335 Glu~ Lys Asn Leu Pæo I.y5 Pro Gly Asp Val S~r Ser Pro Lys Val ~p 3~Q 345 35~ :
I.~s Gln Leu Gln Ile Lys Glu Ser Leu ~lu Asp I.eu Gln ~lu l;ls~ l.eu Lys u Thr G~y Asp~ G1U AS G1A r.ys Arg Glu 1 Glu Lys Gln Ile : ~ :
G :Ile ~ Lyo Lys S--r A-p Glu Lys I.ou LGU LYS Se,r LYS ASP ASP YS

Aia Se~ ~ys ~sp Gly Lys Ala Leu Asp Leu Asp Arg Glu Leu ~sn Ser 405 ~ 41CI 415 : Lys Ala Se~ S~r Lys Glu Lys Ser Lys Ala Lys Glu Glu Glu I1Q Thr ~20 4;~ 430 Lys Gly ~ys Ses Gln ~ys Ser Leu &ly Asp L2U Asn Asn As p Gl u A~n ~35 440 44~
Leu Met Me~t Pro Glu Asp Gln l.ys Leu Pro Glu Val Lys Lys Le~ ~sp 450 455 4~0 Se2~ Lys Lys S~lu Phe Lys Pro Val Ser Glu Val Glu Lys Leu Asp Lys 46~ 470 475 430 Il~ Phe l.ys Ser Asn Asn Asn Val Gly ~lu L~u Ser P~o l.eu ~sp l.ys Ser Ser Tyr, I.ys Asp Ile Asp Ser Lys Glu Glu Thr Val Asn Lys Asp 51:~0 505 51~) .~ ~0 93/0~448 2 1 1 2 ~ 6 ~
3~
. ,s .~ .

. .

Val Asn Leu Gln l:ys T~r Lys Pro Gln Val Lys Asp Gln Val ~h- Ser Lau As;~ Glu Asp Leu Thr Thr Met Ser Ile Asp Ser Ser Ser Pro Val 530 535 54~) -Phe Leu Glu Val Ile Asp ~ro Ile Thr Asn LQU Gly T~r Leu Gln Leu 5~5 550 555 5~0 Ile Asp Leu A~n Tnr Gly Val Ser Leu r.ys Glu Ser Thr Gln Gln Gly 565 57~ 575 Ile~ Gln A~g Tyr Gly Ile ~ ;lu Arg Glu Lys Asp Leu Val Yal Il~

I,ys Met ~Yp Ser Gly Lys Ala Lys L~u Gln I1~ I.eu P sp Lys Leu Glu Asn; Leu hys Val eral Ses Glu Ser Asn Pho Glu IIe ~sn Lys Asn Ser S~ Leu Ty~ Val P,sp Ser Lys Met ~le L~u Val Ala Val ~rg Asp Lvs 625~ 630 635 64 A-~:p Ser Ser Asn Asp Trp Arg L~u Ala ys ~h~ Ser Pro Lys Asn L~u ~ ~.
Asp Glu Ph~ Ile L~u S~r GIu As~ L~s Ile Met Pro P~e Thr Ssr Phe 660 ~ 665 670 ser Val A~y Lys Asn Ph~ Tyr I eu Gln A~p Glu he ~ys S~r Leu Val ~le Lau A~p Val As~ Thr Leu Lys Lys Val Lys ~90: ~ 695 700 , .
. :

.~ .

Claims (12)

PCT/US92/0?53?

WHAT IS CLAIMED IS:

1. An assay for detecting the presence of

Borrelia burgdorferi in a patient sample, said assay comprising detecting in the sample the presence of a chromosomal gene of Borrelia burgdorferi which encodes a conserved antigen of approximately 79kD.

3. An assay for detecting the presence of Borrelia burgdorferi in a patient sample, said assay comprising contacting the patient sample with monospecific antibody which binds to an approximately 79kD antigen encoded by a chromosomal gene of Borrelia burgdorferi and detecting binding between antigen in the sample and said antibody.

4. An assay for detecting the presence of Borrelia burgdorferi in a patient sample, said assay comprising contacting the patient sample with an isolated polypeptide which is cross-reactive with an approximately 79kD antigen encoded by a chromosomal gene of Borrelia burgdorferi and detecting binding between antibodies in the sample and said polypeptide.

5. An isolated polynucleotide molecule which encodes an approximately 79kD conserved antigen of Borrelia burgdorferi or a fragment thereof.

6. An oligonucleotide comprising at least about ten nucleotides and being capable of binding under stringent conditions to a chromosomal gene of Borrelia burgdorferi which encodes a conserved antigen of approximately 79kD.

7. An oligonucleotide as in claim 6, further comprising a detectable label.

8. An oligonucleotide as in claim 6, having a sequence which is the same as or complementary to at least ten contiguous nucleotides of the sequence of Fig. 4.

9. A diagnostic kit comprising a labeled oligonucleotide in a container, wherein the oligonucleotide binds to a chromosomal gene of Borrelia burgdorferi which encodes a conserved antigen of approximately 79 kD under conditions sufficiently stringent to assure specific binding, and instructions setting forth a detection method including a step of contacting the labeled oligonucleotide with a patient sample.

10. An isolated polypeptide comprising a sequence homologous to at least six continuous amino acids in the listing of Fig. 4.

11. A vaccine composition for protection of a host against infection by Borrelia burgdorferi, said vaccine comprising a polypeptide which is immunologically cross-reactive with a conserved antigen of approximately 79kD encoded by a chromosomal gene of Borrelia burgdorferi, said polypeptide being present in a physiologically acceptable carrier in an amount effective to elicit a protective immunity when administered to the host.

12. An antibody which is monospecific for an approximately 79kD antigen encoded by a chromosomal gene of Borrelia burgdorferi.