WO1997042325A1

WO1997042325A1 - B. burgdorferi polypeptides expressed in vivo

Info

Publication number: WO1997042325A1
Application number: PCT/US1996/006610
Authority: WO
Inventors: Erol Fikrig; Kyoungho Suk; Stephen W. Barthold; Richard A. Flavell
Original assignee: Yale University
Priority date: 1996-05-08
Filing date: 1996-05-08
Publication date: 1997-11-13
Also published as: CA2253834A1; EP0915977A1; JP2000510339A

Abstract

Methods and compositions for the prevention, treatment and diagnosis of Lyme disease. Novel B. burgdorferi polypeptides, serotypic variants thereof, fragments thereof and derivatives thereof. Fusion proteins and multimeric proteins comprising same. Multicomponent vaccines comprising novel B. burgdorferi polypeptidesin addition to other immunogenic B. burgdorferi polypeptides. DNA sequences, recombinant DNA molecules and transformed host cells useful in the compositions and methods. Antibodies directed against the novel B. burgdorferi polypeptides, and diagnostic kits comprising the polypeptides or antibodies. A method for identifying bacterial genes that are selectively expressed in vivo.

Description

B. BURGDORFERI POLYPEPTIDES EXPRESSED IN VIVO

This invention was made with government support under Grant numbers AI30548, AI26815, AI49387 and AR40452 awarded by National

Institutes of Health The government has certain rights in the invention.

TECHNICAL FIELD OF THE INVENTION

This invention relates to compositions and methods useful for the prevention, diagnosis and treatment of Lyme disease. More particularly, this invention relates to novel B. burgdorferi polypeptides which are able to elicit in a treated animal, the formation of an immune response. This invention also relates to novel B. burgdorferi polypeptides that are expressed during infection of a host but are not expressed by B. burgdorferi in in vitro culture.

This invention also relates to multicomponent vaccines comprising one or more of the novel B. burgdorferi polypeptides. Also within the scope of this invention are DNA sequences encoding the novel B. burgdorferi polypeptides, antibodies directed against the novel polypeptides and diagnostic kits comprising the antibodies or the polypeptides Finally, this invention relates to novel methods for identifying bacterial genes that are selectively expressed in vivo. BACKGROUND OF THE INVENTION

Lyme borreliosis is the most common vector-borne infection in the United States [S.W. Barthold, et al., "An Animal Model For Lyme Arthritis", Ann. N.Y Acad. Sci., 539, pp. 264-73 (1988)]. It has been reported in every continent except Antarctica The clinical hallmark of Lyme disease is an early expanding skin lesion known as erythema migrans, which may be followed weeks to months later by neurologic, cardiac, and joint abnormalities.

The causative agent of Lyme disease is a spirochete known as Borreha burgdorferi, transmitted primarily by Ixodes ticks of the Ixodes acinus complex. B. burgdorferi has also been shown to be carried in other species of ticks and in mosquitoes and deer flies. But, it appears that only ticks of the I. ricinus complex are able to transmit the disease to humans.

Lyme disease generally occurs in three stages Stage one involves localized skin lesions (erythema migrans) from which the spirochete is cultured more readily than at any other time during infection [B. W. Berger et al. , "Isolation And Characterization Of The Lyme Disease Spirochete From The Skin Of Patients With Erythema Chronicum Migrans", J. Am. Acad. Dermatol., 3, pp.444-49 (1985)]. Flu-like or meningitis-like symptoms are common at this time Stage two occurs within days or weeks, and involves spread of the spirochete through the patient's blood or lymph to many different sites in the body including the brain and joints. Varied symptoms of this disseminated infection occur in the skin, nervous system, and musculoskeletal system, although they are typically intermittent.

Stage three, or late infection, is defined as persistent infection, and can be severely disabling. Chronic arthritis, and syndromes of the central and peripheral nervous system appear during this stage, as a result of the ongoing infection and perhaps a resulting auto-immune disease [R. Martin et al. , "Borreha burgdorferi -Specific And Autoreactive T-Cell Lines From Cerebrospinal Fluid In Lyme Radiculomyelitis", Ann NeuroL., 24, pp. 509-16 (1988)]. B. burgdorferi is much more difficult to culture from humans than from ticks. Therefore, at present, Lyme disease is diagnosed primarily by serology The enzyme-linked immunosorbent assay (ELISA) is a frequently used method of detection. Typically, sonicated whole cultured spirochetes are used as the antigen in such assays to detect anti-B. burgdorferi antibodies formed in the serum of infected individuals [J E. Craft et al., "The Antibody Response In Lyme Disease. Evaluation Of Diagnostic Tests", J. Infect. Dis. , 149, pp 789-95 (1984)].

However, false negative and, more commonly, false positive results are associated with currently available tests.

At present, all stages of Lyme disease are treated with antibiotics.

Treatment of early disease is usually effective. However, the cardiac, arthritic, and nervous system disorders associated with the later stages often do not respond to therapy [A.C. Steere, "Lyme Disease", New Eng J. Med.. 321, pp.586-96 (1989)]. Early intervention, thus, is crucial for effective therapy. Accordingly, there exists an urgent need to identify immunogenic B. burgdorferi proteins that are expressed early in infection.

Like Treponema palhdum, which causes syphilis, and leptospirae, which cause an infectious jaundice, Borrelia belong to the eubacterial phylum of spirochetes [A. G. Barbour and S F Hayes, "Biology Of Borrelia Species",

Microbiol. Rev., 50, pp. 381 -400 ( 1986)] Borrelia burgdorferi have a

protoplasmic cylinder that is surrounded by a cell membrane, then by flagella, and then by an outer membrane.

The B. burgdorferi outer surface proteins identified to date are believed to be lipoproteins, as demonstrated by labelling with [³H]palmitate [M.E. Brandt et al., "Immunogenic Integral membrane Proteins of Borrelia burgdorferi

Are Lipoproteins", Infect, Immun,, 58, pp. 983-91 (1990)]. The two major outer surface proteins are the 31 kDa outer-surface protein A (OspA) and the 34 kDa outer surface protein B (OspB). Both proteins have been shown to vary from different isolates or from different passages of the same isolate as determined by their molecular weights and reactivity with monoclonal antibodies. OspC is a 22 kDa membrane lipoprotein previously identified as pC [R. Fuchs et al , "Molecular Analysis and Expression of a Borrelia burgdorferi Gene Encoding a 22 kDa Protein (pC) in Escherichia coli", Mol. Microbiol.. 6, pp.503-09 (1992)] OspD is said to be preferentially expressed by low-passage, virulent strains of B. burgdorferi B31 [S.J Norris et al., "Low-Passage- Associated Proteins of Borrelia burgdorferi B31 Characterization and Molecular Cloning of OspD, A Surfaced-Exposed, Plasmid- Encoded Lipoprotein", Infect Immun.. 60, pp. 4662-4672 (1992)] OspE, a 19 kD protein, is expressed early in infection while OspF, a 26 kD protein, is expressed at a later stage [T T Lam et al , "Outer Surface Proteins E and F Of Borrelia burgdorferi, the Agent of Lyme Disease," Infect. Immun., 62, pp.290-298 (1994)].

Non-Osp B. burgdorferi proteins identified to date include the 41 kDa flagellin protein, which is known to contain regions of homology with other bacterial flagellins [G.S. Gassman et al , "Analysis of the Borrelia burgdorferi

GeHofla Gene and Antigenic Characterization of Its Gene Product", J. Bacteriol., 173, pp.1452-59 (1991)] and a 93 kDa protein said to be localized to the periplasmic space [D.J. Volkman et al , "Characterization of an Immunoreactive 93 kDa Core Protein of Borrelia burgdorferi With a Human IgG Monoclonal

Antibody", J. Immun.. 146, pp 3177-82 ( 1991 )].

B. burgdorferi is known to alter the antigens on its outer surface during different stages of its life cycle. For example, OspC is not expressed by spirochetes within unfed ticks. However, it is synthesized following engorgement and the introduction of a blood meal into the lumen of the tick's midgut. In contrast, OspA is a prominent surface antigen on spirochetes within the midguts of resting ticks. As spirochetes migrate from the midgut to the salivary gland during the tick feeding, OspA expression decreases The downregulation of OspA within ticks allows spirochetes to survive in the presence of an OspA antibody response, suggesting that selective antigen expression may be a mechanism by which B.

burgdorferi evade immune destruction.

It is known that the expression of other bacterial pathogen gene products is induced by environmental signals [J J Mekalanos, "Environmental Signal Controlling Expression of Virulence Determinants In Bacteria," J. Bacteriol., 174, pp.1 -7 (1992)]. A similar induction of gene expression may occur in the infected host where specific external signals are present. Thus, to understand the mechanism of pathogenesis, it is important to identify genes that are expressed in the host but not in in vitro culture and then to study the function of the gene product.

A genetic system using Salmonella typhimurium has been developed to identify bacterial genes induced in vivo [MJ. Mahan et al, "Selection of Bacterial Virulence Genes That Are Specifically Induced in Host Tissues," Science. 259, pp 686-688 (1993)]. However, this system may not be applied to pathogenic organisms for which a gene transfer system and a well-defined auxotroph are not available. Such systems are unavailable in B. burgdorferi Because effective treatment and prevention of Lyme disease requires an understanding of the mechanisms that allow B. burgdorferi to evade host defenses, cause disease and survive within the host, there is an urgent need for a method to identify B.

burgdorferi genes that are selectively expressed in vivo.

The humoral response to B. burgdorferi antigens that are expressed only within the vertebrate host may aid in the serologic diagnosis of Lyme disease. Such proteins are not present on spirochetes cultured in Barbour-Stoenner-Kelly (BSK) II medium Selective in vivo expression of some B. burgdorferi proteins may be one reason that current diagnostic tests for Lyme disease, based on whole- cell lysates of cultured B. burgdorferi, are unreliable Such tests cannot detect antibodies directed toward the in vivo expressed antigens Accordingly, there also exists a need to identify B. burgdorferi proteins that provide more reliable diagnostic tests for Lyme disease.

Recently, immunization of mice with recombinant OspA has been shown to be effective to confer long-lasting protection against subsequent infection with B. burgdorferi [E. Fikrig et al., "Long-Term Protection of Mice from Lyme Disease by Vaccination with OspA", Infec. Immun . 60, pp 773-77 (1992)].

However, protection by the OspA immunogens used to date appears to be somewhat strain specific, probably due to the heterogeneity of the OspA gene among different B. burgdorferi isolates For example, immunization with OspA from B. burgdorferi strain N40 confers protection against subsequent infection with strains N40, B31 and CD 16, but not against strain 25015 [E. Fikrig et al , "Borrelia burgdorferi Strain 25015 : Characterization of Outer Surface Protein A and Vaccination Against Infection", J. Immun.. 148, pp.2256-60 (1992)].

Immunization with OspB has also been shown to confer protection against Lyme disease but not to the same extent as that conferred by OspA [E

Fikrig et al , "Roles of OspA OspB, and Flagellin in Protective Immunity to Lyme Borreliosis in Laboratory Mice", Infec. Immun., 60, pp.657-61 (1992)] Moreover, some B. burgdorferi are apparently able to escape destruction in OspB-immunized mice via a mutation in the OspB gene which results in expression of a truncated OspB protein [E. Fikrig et al., "Evasion of Protective Immunity by Borrelia burgdorferi by Truncation of Outer Surface Protein B", Proc. Natl. Acad. Sci., 90, pp.4092-96 (1993)] OspC has also been shown to have protective effects in a gerbil model of B. burgdorferi infection However, the protection afforded by immunization with this protein appears to be only partial [V. Preac-Mursic et al , "Active Immunization with pC Protein of Borrelia burgdorferi Protects Gerbils against B. burgdorferi Infection", Infection, 20, pp.342-48 (1992)].

Immunization with OspF has also been shown to confer partial protection against infection [T. K. Nguyen et al , "Partial Destruction oϊ Borrelia burgdorferi Within Ticks That Engorged On OspE- Or OspF-Immunized Mice," Infect.. Immun.. 62, pp. 2079-2084 (1994)]. Both anti-OspE and anti-OspF antibodies have been shown to reduce the number of spirochetes in ticks [T. K. Nguyen et al , supra].

As prevention of tick infestation is imperfect, and Lyme disease may be missed or misdiagnosed when it does appear, there exists a continuing urgent need for the determination of additional antigens of B. burgdorferi and related proteins which are able to elicit a protective immune response and which may be useful in a broad-spectrum vaccine In addition, identification of additional B. burgdorferi antigens may enable the development of more reliable diagnostic reagents which are useful in various stages of Lyme borreliosis.

DISCLOSURE OF THE INVENTION

The present invention provides novel B. burgdorferi polypeptides which are substantially free of a B. burgdorferi spirochete or fragments thereof and, thus, are useful in compositions and methods for the diagnosis, treatment and prevention of B. burgdorferi infection and Lyme disease In one embodiment, this invention provides P21 polypeptides and compositions and methods comprising those polypeptides.

In another embodiment, this invention provides K2 polypeptides and compositions and methods comprising those polypeptides.

In another embodiment, this invention provides P35 polypeptides and compositions and methods comprising those polypeptides.

In another embodiment, this invention provides P37 polypeptides and compositions and methods comprising those polypeptides.

In another embodiment, this invention provides M30 polypeptides and compositions and methods comprising those polypeptides. In another embodiment, this invention provides V3 polypeptides and compositions and methods comprising those polypeptides.

In another embodiment, this invention provides J1 polypeptides compositions and methods comprising those polypeptides.

In another embodiment, this invention provides J2 polypeptides compositions and methods comprising those polypeptides.

The preferred polypeptides of each of the aforementioned embodiments are selectively expressed in vivo.

Also preferred are compositions and methods of each of the aforementioned embodiments are characterized by novel B. burgdorferi polypeptides which elicit in treated animals the formation of an immune response.

In another embodiment, this invention provides a multicomponent vaccine comprising one or more novel B. burgdorferi polypeptides of this invention in addition to one or more other immunogenic B. burgdorferi polypeptides. Such a vaccine is effective to confer broad protection against B. burgdorferi infection

In yet another embodiment, this invention provides antibodies directed against the novel B. burgdorferi polypeptides of this invention, and compositions and methods comprising those antibodies.

In another embodiment, this invention provides diagnostic means and methods characterized by one or more of the novel B. burgdorferi

polypeptides, or antibodies directed against those polypeptides. These means and methods are useful for the detection of Lyme disease and B. burgdorferi infect.ion. They are also useful in following the course of treatment against such infection. In patients previously inoculated with the vaccines of this invention, the detection means and methods disclosed herein are also useful for determining if booster inoculations are appropriate. In yet another embodiment, this invention provides methods for the identification and isolation of additional B. burgdorferi polypeptides, as well as compositions and methods comprising such polypeptides.

In yet another embodiment, this invention provides methods for identifying bacterial genes encoding an antigenic protein which is expressed during infection of a host but is not expressed during in vitro culture of the bacteria.

Finally, this invention provides DNA sequences that code for the novel B. burgdorferi polypeptides of this invention, recombinant DNA molecules that are characterized by those DNA sequences, unicellular hosts transformed with those DNA sequences and molecules, and methods of using those sequences, molecules and hosts to produce the novel B. burgdorferi polypeptides and multicomponent vaccines of this invention. DNA sequences of this invention are also advantageously used in methods and means for the diagnosis of Lyme disease and B. burgdorferi infection. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the DNA and amino acid sequences of the P21 polypeptide of B. burgdorferi strain N40.

Figure 2 depicts the DNA and amino acid sequences of the P35 polypeptide of B. burgdorferi strain N40.

Figure 3 depicts the DNA and amino acid sequences of the P37 polypeptide of B. burgdorferi strain N40.

Figure 4 depicts the DNA and amino acid sequences of the M30 polypeptide of B. burgdorferi strain N40.

Figure 5 depicts the DNA and amino acid sequences of the V3 polypeptide of B. burgdorferi strain N40.

Figure 6 depicts the hydrophilicity profiles of P35 and P37. Figure 7 depicts a comparison of the amino acid sequences of P21 and B. burgdorferi strain N40 OspE.

Figure 8 depicts a comparison of the control regions of transcription and translation among the DNA sequences encoding P21 and K2 and the DNA sequences of other known B. burgdorferi outer surface proteins.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to novel B. burgdorferi polypeptides, the DNA sequences which encode them, antibodies directed against those polypeptides, compositions comprising the polypeptides or antibodies, and methods for the detection, treatment and prevention of Lyme disease.

More specifically, in one embodiment, this invention relates to P21 polypeptides and compositions and methods comprising those polypeptides .

In another embodiment, this invention relates to K2 polypeptides and compositions and methods comprising those polypeptides.

In another embodiment, this invention relates to P35 polypeptides and compositions and methods comprising those polypeptides.

In another embodiment, this invention relates to P37 polypeptides and compositions and methods comprising those polypeptides.

In another embodiment, this invention relates to M30 polypeptides and compositions and methods comprising those polypeptides.

In another embodiment, this invention relates to V3 polypeptides and compositions and methods comprising those polypeptides.

In another embodiment, this invention relates to J1 and compositions and methods comprising those polypeptides.

In another embodiment, this invention relates to J2 and compositions and methods comprising those polypeptides. The preferred polypeptides, compositions and methods of each of the aforementioned embodiments are characterized by novel B. burgdorferi polypeptides that are immunogenic B. burgdorferi polypeptides.

In another embodiment, this invention relates to a multicomponent vaccine against Lyme disease comprising one or more of the novel B. burgdorferi polypeptides of this invention in addition to other immunogenic B. burgdorferi polypeptides Such vaccine is useful to protect against infection by a broad spectrum of B. burgdorferi organisms.

All of the novel B. burgdorferi polypeptides provided by this invention, and the DNA sequences encoding them, may be produced substantially free of B. burgdorferi spirochete or fragments thereof, and thus may be used in a variety of applications without the risk of unintentional infection or contamination with undesired B. burgdorferi components. Accordingly, the novel B. burgdorferi polypeptides of this invention are particularly advantageous in compositions and methods for the diagnosis and prevention of B. burgdorferi infection.

In another embodiment, this invention relates to compositions and methods comprising antibodies directed against the novel B. burgdorferi polypeptides of this invention Such antibodies may be used in a variety of applications, including to detect the presence of B. burgdorferi, to screen for expression of novel B. burgdorferi polypeptides, to purify novel B. burgdorferi polypeptides, to block or bind to the novel B. burgdorferi polypeptides, to direct molecules to the surface of B. burgdorferi, to prevent or lessen the severity, for some period of time, of B. burgdorferi infection, and to decrease the level of B. burgdorferi spirochetes in ticks.

In still another embodiment, this invention relates to diagnostic means and methods characterized by the novel B. burgdorferi polypeptides disclosed herein or antibodies directed against those polypeptides. In yet another embodiment, this invention relates to methods for identifying bacterial genes that are selectively expressed in vivo.

In order to further define this invention, the following terms and definitions are herein provided.

As used herein, an "immunogenic B. burgdorferi polypeptide" is any

B. burgdorferi polypeptide that, when administered to an animal, is capable of eliciting an immune response.

Immunogenic B. burgdorferi polypeptides are intended to include not only the novel B. burgdorferi polypeptides of this invention but also the OspA and OspB polypeptides disclosed in PCT patent application WO 92/00055; the OspC protein as described in R. Fuchs et al. , supra; the OspE and OspF

polypeptides disclosed in PCT patent application WO 95/04145, other

B. burgdorferi proteins, and fragments, serotypic variants and derivatives of any of the above In particular, immunogenic B. burgdorferi polypeptides are intended to include additional B. burgdorferi polypeptides which are identified according to the methods disclosed herein.

As used herein, a polypeptide which is "substantially free of a B. burgdorferi spirochete or fragments thereof is a polypeptide that, when introduced into modified Barbour-Stoener-Kelly (BSK-II) medium and cultured at 37°C for 7 days, fails to produce any B. burgdorferi spirochetes detectable by dark field microscopy or a polypeptide that is detectable as a single band on an immunoblot probed with polyclonal anti-B. burgdorferi anti-serum

As used herein, a B. burgdorferi polypeptide that is "selectively expressed in vivo" is a polypeptide encoded by a DNA sequence that corresponds to a B. burgdorferi gene that is expressed during infection of a host but is not expressed during in vitro culture of said B. burgdorferi A DNA sequence that "corresponds to a B. burgdorferi gene" is a DNA sequence that encodes a polypeptide that is the same as, a fragment of or a derivative of a naturally occurring B. burgdorferi polypeptide.

As used herein, a "P21 polypeptide" denotes a polypeptide which is selected from the group consisting of:

(a) a P21 polypeptide consisting of amino acids 1-182 of SEQ ID NO : 2;

(b) fragments comprising at least 15 amino acids taken as a block from the P21 polypeptide of (a); and

(c) a polypeptide that is selectively expressed in vivo and that :

(1) is a derivative of a P21 polypeptide of (a), said derivative being at least 80% identical in amino acid sequence to the corresponding polypeptide of (a);

(2) polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with B. burgdorferi, which antibodies are immunologically reactive with a P21 polypeptide of (a);

(3) polypeptides that are capable of eliciting antibodies that are immunologically reactive with B. burgdorferi and the P21 polypeptide of (a) and

(4) polypeptides that are immunologically reactive with antibodies elicited by immunization with the P21 polypeptide of (a).

As used herein, a "K2 polypeptide" denotes a polypeptide which is selected from the group consisting of:

(a) a polypeptide comprising the ammo acid sequence set forth in SEQ ID NO :3;

(b) derivatives of the polypeptide of (a), said derivative comprising a polypeptide having a block of amino acids at least 80% identical in sequence to SEQ ED NO : 3; and

(c) a polypeptide that is selectively expressed in vivo and that :

(1) is a derivative of a polyeptide of (a); said derivative being at least 80% identical in ammo acid sequence to the corresponding polypeptide of (a); (2) polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with B. burgdorferi, which antibodies are immunologically reactive with a polypeptide of (a);

(3) polypeptides that are capable of eliciting antibodies that are immunologically reactive with B. burgdorferi and the polypeptide of (a); and

(4) polypeptides that are immunologically reactive with antibodies elicited by immunization with the polypeptide of (a).

As used herein, a "P35 polypeptide" denotes a polypeptide which is selected from the group consisting of.

(a) a P35 protein comprising the amino acid sequence set forth in SEQ ID

NO: 5 and serotypic variants thereof,

(b) fragments comprising at least 8 amino acids taken as a block from the P35 polypeptide of (a);

(c) derivatives of the P35 polypeptide of (a) or (b), said derivatives being at least 80% identical in amino acid sequence to the corresponding polypeptide of (a) or (b);

(d) polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with B. burgdorferi. which antibodies are immunologically reactive with a P35 polypeptide of (a) or (b) or (c);

(e) polypeptides that are capable of eliciting antibodies that are

immunologically reactive with B. burgdorferi and the P35 polypeptide of (a) or (b) or (c); and

(f) polypeptides that are immunologically reactive with antibodies elicited by immunization with the P35 polypeptide of (a) or (b) or (c).

As used herein, a "P37 polypeptide" denotes a polypeptide which is selected from the group consisting of:

(a) a P37 protein having the amino acid sequence of SEQ ID NO : 7 and serotypic variants thereof; (b) fragments comprising at least 8 amino acids taken as a block from the P37 polypeptide of (a;

(c) derivatives of the P37 polypeptide of (a) or (b), said derivatives being at least 80% identical in amino acid sequence to the corresponding polypeptide of (a) or (b);

(d) polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with B. burgdorferi, which antibodies are immunologically reactive with a P37 polypeptide of (a) or (b) or (c);

(e) polypeptides that are capable of eliciting antibodies that are

immunologically reactive with B. burgdorferi and the P37 polypeptide of (a) or (b) or (c); and

As used herein, a "M30 polypeptide" denotes a polypeptide which is selected from the group consisting of:

(a) a M30 polypeptide having the amino acid sequence of SEQ ID NO : 9 and serotypic variants thereof;

(b) fragments comprising at least 8 amino acids taken as a block from the M30 polypeptide of (a);

(c) derivatives of the M30 polypeptide of (a) or (b), said derivatives being at least 80% identical in amino acid sequence to the corresponding polypeptide of (a) or (b);

(d) polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with B. burgdorferi, which antibodies are immunologically reactive with a M30 polypeptide of (a) or (b) or (c);

(e) polypeptides that are capable of eliciting antibodies that are

immunologically reactive with B. burgdorferi and the M30 polypeptide of (a) or (b) or (c); and (f) polypeptides that are immunologically reactive with antibodies elicited by immunization with the M30 polypeptide of (a) or (b) or (c).

As used herein, a "V3 polypeptide"denotes a polypeptide which is selected from the group consisting of :

(a) a V3 protein having an amino acid sequence encoded by SEQ ID NO : 10 and serotypic variants thereof;

(b) fragments comprising at least 8 amino acids taken as a block from the polypeptide of (a);

(c) derivatives of the polypeptide of (a) or (b), said derivatives being at least 80% identical in amino acid sequence to the corresponding polypeptide of (a) or

(b);

(d) polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with B. burgdorferi, which antibodies are immunologically reactive with a polypeptide of (a) or (b) or (c);

(e) polypeptides that are capable of eliciting antibodies that are

immunologically reactive with B. burgdorferi and the polypeptide of (a) or (b) or (c); and

(f) polypeptides that are immunologically reactive with antibodies elicited by immunization with the polypeptide of (a) or (b) or (c).

As used herein, a "V3 polypeptide" is intended to include a B.

burgdorferi polypeptide encoded in whole or in part by the B. burgdorferi DNA sequence contained in ATCC deposit No.__, which cross-hybridizes to the DNA sequence of SEQ ID NO: 10.

As used herein, a "J1 polypeptide" denotes a polypeptide which is selected from the group consisting of;

(a) a polypeptide encoded in whole or in part by the B. burgdorferi DNA sequence contained within ATCC deposit No. (2) and serotypic variants thereof; (b) fragments comprising at least 8 amino acids taken as a block from the polypeptide of (a);

(c) derivatives of the polypeptide of (a) or (b), said derivatives being at least 80% identical in amino acid sequence to the corresponding polypeptide of (a) or (b);

(e) polypeptides that are capable of eliciting antibodies that are

immunologically reactive with B. burgdorferi and the polypeptide of (a) or (b) or

(c); and

As used herein, a "J1 polypeptide" is intended to include a B.

burgdorferi polypeptide encoded in whole or in part by the B. burgdorferi DNA sequence contained within ATCC deposit No. (2A), which cross-hybridizes to the B. burgdorferi DNA sequence contained within ATCC deposit No. __

As used herein, a "J2 polypeptide" denotes a polypeptide which is selected from the group consisting of:

(a) a polypeptide encoded in whole or in part by the B. burgdorferi DNA sequence contained within ATCC deposit No. (3) and serotypic variants thereof,

(c) derivatives of the polypeptide of (a) or (b), said derivatives being at least 80%o identical in amino acid sequence to the corresponding polypeptide of (a) or

(b); (d) polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with B. burgdorferi, which antibodies are immunologically reactive with a polypeptide of (a) or (b) or (c);

(e) polypeptides that are capable of eliciting antibodies that are

As used herein, a "J2 polypeptide" is intended to include a B.

burgdorferi polypeptide encoded in whole or in part by the B. burgdorferi DNA sequence contained within ATCC deposit Nos (3A and 3B). which cross-hybridize to the B. burgdorferi DNA sequence contained within ATCC deposit No. (3)

As used herein, a "novel B. burgdorferi polypeptide" is a P21 polypeptide, a K2 polypeptide, a P35 polypeptide, a P37 polypeptide, an M30 polypeptide, a V3 polypeptide, a J1 polypeptide or a J2 polypeptide

As used herein, a "serotypic variant" of a novel B. burgdorferi polypeptide according to this invention is any naturally occurring polypeptide which may be encoded in whole or in part, by a DNA sequence which hybridizes, at 20- 27°C below Tm, to the DNA sequence encoding the novel B. burgdorferi polypeptide. One of skill in the art will understand that serotypic variants of a novel B. burgdorferi polypeptide according to this invention include polypeptides encoded by DNA sequences of which any portion may be amplified by using the polymerase chain reaction and oligonucleotide primers derived from any portion of the DNA sequence encoding the novel B. burgdorferi polypeptide.

As used herein, a "derivative" of a novel B. burgdorferi polypeptide according to his invention is a novel B. burgdorferi polypeptide in which one or more physical, chemical, or biological properties has been altered. Such modifications include, but are not limited to amino acid substitutions, modifications, additions or deletions; alterations in the pattern of lipidation, glycosylation or phosphorylation; reactions of free amino, carboxyl, or hydroxyl side groups of the amino acid residues present in the polypeptide with other organic and non-organic molecules; and other modifications, any of which may result in changes in primary, secondary or tertiary structure.

As used herein, a "protective antibody" is an antibody that confers protection, for some period of time, against any one of the physiological disorders associated with B. burgdorferi infection.

As used herein, a "protective B. burgdorferi polypeptide" is a polypeptide that comprises a protective epitope.

As used herein, a "protective epitope" is (1) an epitope which is recognized by a protective antibody, and/or (2) an epitope which, when used to immunize an animal, elicits an immune response sufficient to prevent or lessen the severity for some period of time, of B. burgdorferi infection.

Preventing or lessening the severity of infection may be evidenced by a change in the physiological manifestations of erythema migrans, arthritis, carditis, neurological disorders, and other Lyme disease related disorders It may be evidenced by a decrease in the level of spirochetes in the treated animal And, it may also be evidenced by a decrease in the level of spirochetes in infected ticks feeding on treated animals. A protective epitope may comprise a T cell epitope, a B cell epitope, or combinations thereof.

As used herein, a "T cell epitope" is an epitope which, when presented to T cells by antigen presenting cells, results in a T cell response such as clonal expansion or expression of lymphokines or other immunostimulatory molecules. A T cell epitope may also be an epitope recognized by cytotoxic T cells that may affect intracellular B. burgdorferi infection. A strong T cell epitope is a T cell epitope which elicits a strong T cell response. As used herein, a "B cell epitope" is the simplest spatial

conformation of an antigen which reacts with a specific antibody.

As used herein, a "therapeutically effective amount" of a polypeptide or of an antibody is the amount that, when administered to an animal, elicits an immune response that is effective to prevent or lessen the severity, for some period of time, of B. burgdorferi infection.

As used herein, an "antibody directed against a novel B. burgdorferi polypeptide" (also referred to as "an antibody of this invention") is an antibody directed against a P21 polypeptide, a K2 polypeptide, a P35 polypeptide, a P37 polypeptide, an M30 polypeptide, a V3 polypeptide, a J1 polypeptide or a J2 polypeptide. It should be understood that an antibody directed against a novel B. burgdorferi polypeptide may also be a protective antibody.

An antibody directed against a novel B. burgdorferi polypeptide may be an intact immunoglobulin molecule or a portion of an immunoglobulin molecule that contains an intact antigen binding site, including those portions known in the art as F(v), Fab, Fab' and F(ab')2 It may also be a genetically engineered or synthetically produced molecule.

The novel B. burgdorferi polypeptides disclosed herein are immunologically reactive with antisera generated by infection of a mammalian host with B. burgdorferi. Accordingly, they are useful in methods and compositions to diagnose and protect against Lyme disease, and in therapeutic compositions to stimulate immunological clearance of B. burgdorferi during ongoing infection. In addition, because at least some, if not all of the novel B. burgdorferi polypeptides disclosed herein are immunogenic surface proteins of B. burgdorferi, they are particularly useful in a multicomponent vaccine against Lyme disease, because such a vaccine may be formulated to more closely resemble the immunogens presented by replication-competent B. burgdorferi, and because such a vaccine is more likely to confer broad-spectrum protection than a vaccine comprising only a single B. burgdorferi polypeptide. Multicomponent vaccines according to this invention may also contain polypeptides which characterize other vaccines useful for immunization against diseases other than Lyme disease such as, for example, diphtheria, polio, hepatitis, and measles. Such multicomponent vaccines are typically incorporated into a single composition.

The preferred compositions and methods of this invention comprise novel B. burgdorferi polypeptides having enhanced immunogenicity. Such polypeptides may result when the native forms of the polypeptides or fragments thereof are modified or subjected to treatments to enhance their immunogenic character in the intended recicpient.

Numerous techniques are available and well known to those of skill in the art which may be used, without undue experimentation, to substantially increase the immunogenicity of the novel B. burgdorferi polypeptides herein disclosed. For example, the polypeptides may be modified by coupling to dinitrophenol groups or arsanilic acid, or by denaturation with heat and/or SDS. Particularly if the polypeptides are small polypeptides synthesized chemically, it may be desirable to couple them to an immunogenic carrier. The coupling of course, must not interfere with the ability of either the polypeptide or the carrier to function appropriately. For a review of some general considerations in coupling strategies, see Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, ed. E.

Harlow and D. Lane (1988). Useful immunogenic carriers are well known in the art. Examples of such carriers are keyhole limpet hemocyanin (KLH); albumins such as bovine serum albumin (BSA) and ovalbumin, PPD (purified protein derivative of tuberculin); red blood cells; tetanus toxoid, cholera toxoid; agarose beads; activated carbon, or bentonite.

Modification of the amino acid sequence of the novel B. burgdorferi polypeptides disclosed herein in order to alter the lipidation state is also a method which may be used to increase their immunogenicity and biochemical properties. For example, the polypeptides or fragments thereof may be expressed with or without the signal sequences that direct addition of lipid moieties.

As will be apparent from the disclosure to follow, the polypeptides may also be prepared with the objective of increasing stability or rendering the molecules more amenable to purification and preparation. One such technique is to express the polypeptides as fusion proteins comprising other B. burgdorferi or non- B. burgdorferi sequences.

In accordance with this invention, derivatives of the novel B.

burgdorferi polypeptides may be prepared by a variety of methods, including by in vitro manipulation of the DNA encoding the native polypeptides and subsequent expression of the modified DNA, by chemical synthesis of derivatized DNA sequences, or by chemical or biological manipulation of expressed amino acid sequences.

For example, derivatives may be produced by substitution of one or more amino acids with a different natural amino acid, an amino acid derivative or non-native amino acid, conservative substitution being preferred, e.g.,

3-methylhistidine may be substituted for histidine, 4-hydroxyproline may be substituted for proline, 5-hydroxylysine may be substituted for lysine, and the like.

Causing amino acid substitutions which are less conservative may also result in desired derivatives, e.g., by causing changes in charge, conformation and other biological properties. Such substitutions would include for example, substitution of a hydrophilic residue for a hydrophobic residue, substitution of a cysteine or proline for another residue, substitution of a residue having a small side chain for a residue having a bulky side chain or substitution of a residue having a net positive charge for a residue having a net negative charge. When the result of a given substitution cannot be predicted with certainty, the derivatives may be readily assayed according to the methods disclosed herein to determine the presence or absence of the desired characteristics. In a preferred embodiment of this invention, the novel B.

burgdorferi polypeptides disclosed herein are prepared as part of a larger fusion protein For example, a novel B. burgdorferi polypeptide of this invention may be fused at its N-terminus or C-terminus to a different immunogenic B. burgdorferi polypeptide, to a non-5, burgdorferi polypeptide or to combinations thereof, to produce fusion proteins comprising the novel B. burgdorferi polypeptide.

In a preferred embodiment of this invention, fusion proteins comprising novel B. burgdorferi polypeptides are constructed comprising B cell and/or T cell epitopes from multiple serotypic variants of B. burgdorferi, each variant differing from another with respect to the locations or sequences of the epitopes within the polypeptide In a more preferred embodiment, fusion proteins are constructed which comprise one or more of the novel B. burgdorferi polypeptides fused to other immunogenic B. burgdorferi polypeptides. Such fusion proteins are particularly effective in the prevention, treatment and diagnosis of Lyme disease as caused by a wide spectrum of B. burgdorferi isolates.

In another preferred embodiment of this invention, the novel B. burgdorferi polypeptides are fused to moieties, such as immunoglobulin domains, which may increase the stability and prolong the in vivo plasma half-life of the polypeptide. Such fusions may be prepared without undue experimentation according to methods well known to those of skill in the art, for example, in accordance with the teachings of United States patent 4,946,778, or United States patent 5, 116,964. The exact site of the fusion is not critical as long as the polypeptide retains the desired biological activity. Such determinations may be made according to the teachings herein or by other methods known to those of skill in the art.

It is preferred that the fusion proteins comprising the novel B.

burgdorferi polypeptides be produced at the DNA level, e g , by constructing a nucleic acid molecule encoding the fusion, transforming host cells with the molecule, inducing the cells to express the fusion protein, and recovering the fusion protein from the cell culture. Alternatively, the fusion proteins may be produced after gene expression according to known methods.

The novel B. burgdorferi polypept s may also be part of larger multimeric molecules which may be produced recombinantly or may be synthesized chemically. Such multimers may also include the polypeptides fused or coupled to moieties other than amino acids, including lipids and carbohydrates.

Preferably, the multimeric proteins will consist of multiple T or B cell epitopes or combinations thereof repeated within the same molecule, either randomly, or with spacers (amino acid or otherwise) between them.

In the most preferred embodiment of this invention, the novel B burgdorferi polypeptides of this invention which are also immunogenic B.

burgdorferi polypeptides are incorporated into a multicomponent vaccine which also comprises other immunogenic B. burgdorferi polypeptides. Such a multicomponent vaccine, by virtue of its ability to elicit antibodies to a variety of immunogenic B. burgdorferi polypeptides, will be effective to protect against Lyme disease as caused by a broad spectrum of different B burgdorferi isolates, even those that may not express one or more of the Osp proteins.

The multicomponent vaccine may contain the novel B. burgdorferi polypeptides as part of a multimeric molecule in which the various components are covalently associated. Alternatively, it may contain multiple individual components For example, a multicomponent vaccine may be prepared comprising two or more of the novel B. burgdorferi polypeptides, or comprising one novel B. burgdorferi polypeptide and one previously identified B. burgdorferi polypeptide, wherein each polypeptide is expressed and purified from independent cell cultures and the polypeptides are combined prior to or during formulation.

Alternatively, a multicomponent vaccine may be prepared from heterodimers or tetramers wherein the polypeptides have been fused to immunoglobulin chains or portions thereof. Such a vaccine could comprise, for example, a P35 polypeptide fused to an immunoglobulin heavy chain and an OspA polypeptide fused to an immunoglobulin light chain, and could be produced by transforming a host cell with DNA encoding the heavy chain fusion and DNA encoding the light chain fusion. One of skill in the art will understand that the host cell selected should be capable of assembling the two chains appropriately.

Alternatively, the heavy and light chain fusions could be produced from separate cell lines and allowed to associate after purification.

The desirability of including a particular component and the relative proportions of each component may be determined by using the assay systems disclosed herein, or by using other systems known to those in the art Most preferably, the multicomponent vaccine will comprise numerous T cell and B cell epitopes of immunogenic B. burgdorferi polypeptides, including the novel B. burgdorferi polypeptides of this invention.

This invention also contemplates that the novel B. burgdorferi polypeptides of this invention, either alone or with other immunogenic B.

burgdorferi polypeptides, may be administered to an animal via a liposome delivery system in order to enhance their stability and/or immunogenicity. Delivery of the novel B. burgdorferi polypeptides via liposomes may be particularly advantageous because the liposome may be internalized by phagocytic cells in the treated animal Such cells, upon ingesting the liposome, would digest the liposomal membrane and subsequently present the polypeptides to the immune system in conjunction with other molecules required to elicit a strong immune response.

The liposome system may be any variety of unilamellar vesicles, multilamellar vesicles, or stable plurilamellar vesicles, and may be prepared and administered according to methods well known to those of skill in the art, for example in accordance with the teachings of United States patents 5,169,637, 4,762,915, 5,000,958 or 5,185,154. In addition, it may be desirable to express the novel B. burgdorferi polypeptides of this invention, as well as other selected B. burgdorferi polypeptides, as lipoproteins, in order to enhance their binding to liposomes.

Any of the novel B. burgdorferi polypeptides of this invention may be used in the form of a pharmaceutically acceptable salt.. Suitable acids and bases which are capable of forming salts with the polypeptides of the present invention are well known to those of skill in the art, and include inorganic and organic acids and bases.

According to this invention, we describe a method which comprises the steps of treating an animal with a therapeutically effective amount of a novel B. burgdorferi polypeptide, or a fusion protein or a multimeric protein comprising a novel B. burgdorferi polypeptide, in a manner sufficient to prevent or lessen the severity, for some period of time, of B. burgdorferi infection. The polypeptides that are preferred for use in such methods are those that contain protective epitopes. Such protective epitopes may be B cell epitopes, T cell epitopes, or combinations thereof.

According to another embodiment of this invention, we describe a method which comprises the steps of treating an animal with a multicomponent vaccine comprising a therapeutically effective amount of a novel B. burgdorferi polypeptide, or a fusion protein or multimeric protein comprising such polypeptide in a manner sufficient to prevent or lessen the severity, for some period of time, of B. burgdorferi infection. Again, the polypeptides, fusion proteins and multimeric proteins that are preferred for use in such methods are those that contain protective epitopes, which may be B cell epitopes, T cell epitopes, or combinations thereof.

The most preferred polypeptides, fusion proteins and multimeric proteins for use in these compositions and methods are those containing both strong T cell and B cell epitopes. Without being bound by theory, we believe that this is the best way to stimulate high titer antibodies that are effective to neutralize B. burgdorferi infection. Such preferred polypeptides will be internalized by B cells expressing surface immunoglobulin that recognizes the B cell epitope(s). The B cells will then process the antigen and present it to T cells. The T cells will recognize the T cell epitope(s) and respond by proliferating and producing lymphokines which in turn cause B cells to differentiate into antibody producing plasma cells. Thus, in this system, a closed autocatalytic circuit exists which will result in the amplification of both B and T cell responses, leading ultimately to production of a strong immune response which includes high titer antibodies against the novel B. burgdorferi polypeptide.

One of skill in the art will also understand that it may be advantageous to administer the novel B. burgdorferi polypeptides of this invention in a form that will favor the production of T-helper cells type 2 (T_H2), which help B cells to generate antibody responses Aside from administering epitopes which are strong B cell epitopes, the induction of T_H2 cells may also be favored by the mode of administration of the polypeptide for example by administering in certain doses or with particular adjuvants and immunomodulators, for example with interleukin-4.

To prepare the preferred polypeptides of this invention, in one embodiment, overlapping fragments of the novel B. burgdorferi polypeptides of this invention are constructed. The polypeptides that contain B cell epitopes may be identified in a variety of ways for example by their ability to (1 ) remove protective antibodies from polyclonal antiserum directed against the polypeptide or (2) elicit an immune response which is effective to prevent or lessen the severity of B.

burgdorferi infection.

Alternatively, the polypeptides may be used to produce monoclonal antibodies which are screened for their ability to confer protection against B.

burgdorferi infection when used to immunize naive animals. Once a given monoclonal antibody is found to confer protection, the particular epitope that is recognized by that antibody may then be identified. As recognition of T cell epitopes is MHC restricted, the polypeptides that contain T cell epitopes may be identified in vitro by testing them for their ability to stimulate proliferation and/or cytokine production by T cell clones generated from humans of various HLA types, from the lymph nodes, spleens, or peripheral blood lymphocytes of C3H/He mice, or from domestic animals Compositions comprising multiple T cell epitopes recognized by individuals with different Class II antigens are useful for prevention and treatment of Lyme disease in a broad spectrum of patients.

In a preferred embodiment of the present invention, a novel B.

burgdorferi polypeptide containing a B cell epitope is fused to one or more other immunogenic B. burgdorferi polypeptides containing strong T cell epitopes. The fusion protein that carries both strong T cell and B cell epitopes is able to participate in elicitation of a high titer antibody response effective to neutralize infection with B. burgdorferi.

Strong T cell epitopes may also be provided by non-B. burgdorferi molecules For example, strong T cell epitopes have been observed in hepatitis B virus core antigen (HBcAg). Furthermore, it has been shown that linkage of one of these segments to segments of the surface antigen of Hepatitis B virus, which are poorly recognized by T cells, results in a major amplification of the anti-HBV surface antigen response, [D.R. Milich et al , "Antibody Production To The

Nucleocapsid And Envelope Of The Hepatitis B Virus Primed By A Single

Synthetic T Cell Site", Nature. 329, pp.547-49 (1987)].

Therefore, in yet another preferred embodiment, B cell epitopes of the novel B. burgdorferi polypeptides are fused to segments of HBcAG or to other antigens which contain strong T cell epitopes, to produce a fusion protein that can elicit a high titer antibody response against B. burgdorferi In addition, it may be particularly advantageous to link a novel B. burgdorferi polypeptide of this invention to a strong immunogen that is also widely recognized, for example tetanus toxoid.

It will be readily appreciated by one of ordinary skill in the art that the novel B. burgdorferi polypeptides of this invention, as well as fusion proteins and multimeric proteins containing them, may be prepared by recombinant means, chemical means, or combinations thereof.

For example, the polypeptides may be generated by recombinant means using the DNA sequences of B. burgdorferi strain N40 as set forth in the sequence listings contained herein DNA encoding serotypic variants of the polypeptides may likewise be cloned, e. g , using PCR and oligonucleotide primers derived from the sequences herein disclosed.

In this regard, it may be particularly desirable to isolate the genes encoding novel B. burgdorferi polypeptides from strain 25015 and other strains of B. burgdorferi that are known to differ antigenically from strain N40, in order to obtain a broad spectrum of different epitopes which would be useful in the methods and compositions of this invention For example, the OspA gene of B. burgdorferi strain 25015 is known to differ from the OspA gene of B. burgdorferi strain N40 to the extent that anti-OspA antibodies, which protect against subsequent infection with strain N40, appear ineffective to protect against infection with strain 25015.

Oligonucleotide primers and other nucleic acid probes derived from the genes encoding the novel B. burgdorferi polypeptides may also be used to isolate and clone other related surface proteins from B. burgdorferi and related spirochetes which may contain regions of DNA sequence homologous to the DNA sequences of this invention In addition, the DNA sequences of this invention may also be used in PCR reactions to detect the presence of B. burgdorferi in a suspected infected sample.

If the novel B. burgdorferi polypeptides of this invention are produced recombinantly, they may be expressed in unicellular hosts . As is well known to one of skill in the art, in order to obtain high expression levels of foreign DNA sequences in a host, the sequences are generally operatively linked to transcriptional and translational expression control sequences that are functional in the chosen host. Preferably, the expression control sequences, and the gene of interest, will be contained in an expression vector that further comprises a selection marker.

The DNA sequences encoding the polypeptides of this invention may or may not encode a signal sequence. If the expression host is eukaryotic, it generally is preferred that a signal sequence be encoded so that the mature protein is secreted from the eukaryotic host.

An amino terminal methionine may or may not be present on the expressed polypeptides of this invention. If the terminal methionine is not cleaved by the expression host, it may, if desired, be chemically removed by standard techniques.

A wide variety of expression host/vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors for eukaryotic hosts, include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, adeno- associated virus, cytomegalovirus and retroviruses including lentiviruses . Useful expression vectors for bacterial hosts include bacterial plasmids, such as those from E. coli, including pBluescript, pGEX-2T, pUC vectors, col E1, pCR1, ρBR322, pMB9 and their derivatives, pET-15, wider host range plasmids, such as RP4, phage DNAs, e.g., the numerous derivatives of phage lambda, e.g λGT10 and λGT1 1, and other phages Useful expression vectors for yeast cells include the 2μ plasmid and derivatives thereof Useful vectors for insect cells include pVL 941.

In addition, any of a wide variety of expression control sequences— sequences that control the expression of a DNA sequence when operatively linked to it— may be used in these vectors to express the DNA sequences of this invention. Such useful expression control sequences include the expression control sequences associated with structural genes of the foregoing expression vectors. Examples of useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the T3 and T7 promoters, the major operator and promoter regions of phage lambda, the control regions of fd coat protein, the promoter for 3- phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast α-mating system and other constitutive and inducible promoter sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

In a preferred embodiment, DNA sequences encoding the novel B. burgdorferi polypeptides of this invention are cloned in the expression vector lambda ZAP II (Stratagene, La Jolla, CA), in which expression from the lac promoter may be induced by IPTG.

In another preferred embodiment, DNA encoding the novel B.

burgdorferi polypeptides of this invention is inserted in frame into an expression vector that allows high level expression of the polypeptide as a glutathione S- transferase fusion protein. Such a fusion protein thus contains amino acids encoded by the vector sequences as well as amino acids of the novel B. burgdorferi polypeptide.

A wide variety of unicellular host cells are useful in expressing the DNA sequences of this invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus,

Streptomyces, fungi, yeast, insect cells such as Spodoptera frugiperda (SF9), animal cells such as CHO and mouse cells, African green monkey cells such as COS 1, COS 7, BSC 1, BSC 40, and BMT 10, and human cells, as well as plant cells. It should of course be understood that not all vectors and expression control sequences will function equally well to express the DNA sequences of this invention. Neither will all hosts function equally well with the same expression system. However, one of skill in the art may make a selection among these vectors, expression control sequences and hosts without undue experimentation and without departing from the scope of this invention. For example, in selecting a vector, the host must be considered because the vector must be replicated in it. The vector's copy number, the ability to control that copy number, the ability to control integration, if any, and the expression of any other proteins encoded by the vector, such as antibiotic or other selection markers, should also be considered.

In selecting an expression control sequence, a variety of factors should also be considered. These include, for example, the relative strength of the promoter sequence, its controllability, and its compatibility with the DNA sequence of this invention, particularly with regard to potential secondary structures.

Unicellular hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded for by the DNA sequences of this invention, their secretion characteristics, their ability to fold the polypeptide correctly, their fermentation or culture requirements, and the ease of purification from them of the products coded for by the DNA sequences of this invention.

Within these parameters, one of skill in the art may select various vector/expression control sequence/host combinations that will express the DNA sequences of this invention on fermentation or in other large scale cultures.

The molecules comprising the novel B. burgdorferi polypeptides encoded by the DNA sequences of this invention may be isolated from the fermentation or cell culture and purified using any of a variety of conventional methods including liquid chromatography such as normal or reversed phase, using HPLC, FPLC and the like, affinity chromatography (such as with inorganic ligands or monoclonal antibodies); size exclusion chromatography; immobilized metal chelate chromatography; gel electrophoresis; and the like. One of skill in the art may select the most appropriate isolation and purification techniques without departing from the scope of this invention.

In addition, the novel B. burgdorferi polypeptides may be generated by any of several chemical techniques For example, they may be prepared using the solid-phase synthetic technique originally described by R. B. Merrifield, "Solid Phase Peptide Synthesis. I. The Synthesis Of A Tetrapeptide", J. Am. Chem. Soc., 83, pp.2149-54 (1963), or they may be prepared by synthesis in solution A summary of peptide synthesis techniques may be found in E. Gross & H. J.

Meinhofer, 4 The Peptides : Analysis, Synthesis, Biology, Modern Techniques Of Peptide And Amino Acid Analysis, John Wiley & Sons, (1981) and M Bodanszky, Principles Of Peptide Synthesis, Springer- Verlag (1984).

Typically, these synthetic methods compπse the sequential addition of one or more amino acid residues to a growing peptide chain. Often peptide coupling agents are used to facilitate this reaction. For a recitation of peptide coupling agents suitable for the uses described herein see M. Bodansky, supra. Normally, either the amino or carboxyl group of the first amino acid residue is protected by a suitable, selectively removable protecting group. A different protecting group is utilized for amino acids containing a reactive side group, e. g., lysine. A variety of protecting groups known in the field of peptide synthesis and recognized by conventional abbreviations therein, may be found in T. Greene, Protective Groups In Organic Synthesis, Academic Press (1981).

According to another embodiment of this invention, antibodies directed against the novel B. burgdorferi polypeptides are generated. Such antibodies are immunoglobulin molecules or portions thereof that are

immunologically reactive with a novel B. burgdorferi polypeptide of the present invention. It should be understood that the antibodies of this invention include antibodies immunologically reactive with fusion proteins and multimeric proteins comprising a novel B. burgdorferi polypeptide.

Antibodies directed against a novel B. burgdorferi polypeptide may be generated by a variety of means including infection of a mammalian host with B. burgdorferi, or by immunization of a mammalian host with a novel B.

burgdorferi polypeptide of the present invention. Such antibodies may be polyclonal or monoclonal, it is preferred that they are monoclonal. Methods to produce polyclonal and monoclonal antibodies are well known to those of skill in the art.. For a review of such methods, set Antibodies, A Laboratory Manual, supra, and D.E. Yelton, et al., Ann. Rev, of Biochem., 50, pp.657-80 (1981).

Determination of immunoreactivity with a novel B. burgdorferi polypeptide of this invention may be made by any of several methods well known in the art, including by immunoblot assay and ELISA.

An antibody of this invention may also be a hybrid molecule formed from immunoglobulin sequences from different species (e. g., mouse and human) or from portions of immunoglobulin light and heavy chain sequences from the same species. It may be a molecule that has multiple binding specificities, such as a bifunctional antibody prepared by any one of a number of techniques known to those of skill in the art including the production of hybrid hybridomas; disulfide exchange, chemical cross-linking; addition of peptide linkers between two monoclonal antibodies; the introduction of two sets of immunoglobulin heavy and light chains into a particular cell line, and so forth.

The antibodies of this invention may also be human monoclonal antibodies produced by any of the several methods known in the art. For example, human monoclonal antibodies may produced by immortalized human cells, by SCID-hu mice or other non-human animals capable of producing "human" antibodies, by the expression of cloned human immunoglobulin genes, by phage- display, or by any other method known in the art. In addition, it may be advantageous to couple the antibodies of this invention to toxins such as diphtheria, pseudomonas exotoxin, ricin A chain, gelonin, etc., or antibiotics such as penicillins, tetracyclines and chloramphenicol.

In sum, one of skill in the art, provided with the teachings of this invention, has available a variety of methods which may be used to alter the biological properties of the antibodies of this invention including methods which would increase or decrease the stability or half-life, immunogenicity, toxicity, affinity or yield of a given antibody molecule, or to alter it in any other way that may render it more suitable for a particular application.

One of skill in the art will understand that antibodies directed against a novel B. burgdorferi polypeptide may have utility in therapeutic and prophylactic compositions and methods directed against Lyme disease and B. burgdorferi infection. For example, the level of B. burgdorferi in infected ticks may be decreased by allowing them to feed on the blood of animals immunized with the novel B. burgdorferi polypeptides of this invention.

The antibodies of this invention also have a variety of other uses For example, they are useful as reagents to screen for expression of the B.

burgdorferi polypeptides, either in libraries constructed from B. burgdorferi DNA or from other samples in which the proteins may be present. Moreover, by virtue of their specific binding affinities, the antibodies of this invention are also useful to purify or remove polypeptides from a given sample, to block or bind to specific epitopes on the polypeptides and to direct various molecules, such as toxins, to the surface of B. burgdorferi.

To screen the novel B. burgdorferi polypeptides and antibodies of this invention for their ability to confer protection against Lyme disease or their ability to lessen the severity of B. burgdorferi infection, C3H/He mice are preferred as an animal model. Of course, while any animal that is susceptible to infection with B. burgdorferi may be useful, C3H/He mice are not only susceptible to B. burgdorferi infection but are also afflicted with clinical symptoms of a disease that is remarkably similar to Lyme disease in humans. Thus, by administering a particular polypeptide or antibody to C3H/He mice, one of skill in the art may determine without undue experimentation whether that polypeptide or antibody would be useful in the methods and compositions claimed herein.

The administration of the novel B. burgdorferi polypeptide or antibody of this invention to the animal may be accomplished by any of the methods disclosed herein or by a variety of other standard procedures. For a detailed discussion of such techniques, see Antibodies, A Laboratory Manual, supra Preferably, if a polypeptide is used, it will be administered with a pharmaceutically acceptable adjuvant, such as complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulating complexes) Such adjuvants may protect the polypeptide from rapid dispersal by sequestering it in a local deposit, or they may contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system Preferably, if a polypeptide is being administered, the immunization schedule will involve two or more administrations of the polypeptide, spread out over several weeks.

Once the novel B. burgdorferi polypeptides or antibodies of this invention have been determined to be effective in the screening process, they may then be used in a therapeutically effective amount in pharmaceutical compositions and methods to treat or prevent Lyme disease which may occur naturally in various animals.

The pharmaceutical compositions of this invention may be in a variety of conventional depot forms These include, for example, solid, semi-solid and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, liposomes, capsules, suppositories, injectable and infusible solutions. The preferred form depends upon the intended mode of administration and prophylactic application.

Such dosage forms may include pharmaceutically acceptable carriers and adjuvants which are known to those of skill in the art. These carriers and adjuvants include, for example, RIBI, ISCOM, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, and polyethylene glycol Adjuvants for topical or gel base forms may be selected from the group consisting of sodium carboxymethylcellulose, polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wood wax alcohols.

The vaccines and compositions of this invention may also include other components or be subject to other treatments during preparation to enhance their immunogenic character or to improve their tolerance in patients.

Compositions comprising an antibody of this invention may be administered by a variety of dosage forms and regimens similar to those used for other passive immunotherapies and well known to those of skill in the art

Generally, the novel B. burgdorferi polypeptides may be formulated and

administered to the patient using methods and compositions similar to those employed for other pharmaceutically important polypeptides (e.g., the vaccine against hepatitis).

Any pharmaceutically acceptable dosage route, including parenteral, intravenous, intramuscular, intralesional or subcutaneous injection, may be used to administer the polypeptide or antibody composition. For example, the composition may be administered to the patient in any pharmaceutically acceptable dosage form including those which may be administered to a patient intravenously as bolus or by continued infusion over a period of hours, days, weeks or months,

intramuscularly -- including paravertebrally and periarticularly— subcutaneously, intracutaneously, intra-articularly, intrasynovially, intrathecally, intralesionally, periostally or by oral or topical routes. Preferably, the compositions of the invention are in the form of a unit dose and will usually be administered to the patient intramuscularly.

The novel B. burgdorferi polypeptides or antibodies of this invention may be administered to the patient at one time or over a series of treatments. The most effective mode of administration and dosage regimen will depend upon the level of immunogenicity, the particular composition and/or adjuvant used for treatment, the severity and course of the expected infection, previous therapy, the patient's health status and response to immunization, and the judgment of the treating physician. For example, in an immunocompetent patient, the more highly immunogenic the polypeptide, the lower the dosage and necessary number of immunizations. Similarly, the dosage and necessary treatment time will be lowered if the polypeptide is administered with an adjuvant Generally, the dosage will consist of 10 μg to 100 mg of the purified polypeptide, and preferably, the dosage will consist of 10-1000 μg. Generally, the dosage for an antibody will be 0.5 mg-3.0 g.

In a preferred embodiment of this invention, the novel B.

burgdorferi polypeptide is administered with an adjuvant, in order to increase its immunogenicity. Useful adjuvants include RIBI, and ISCOM, simple metal salts such as aluminum hydroxide, and oil based adjuvants such as complete and incomplete Freund's adjuvant When an oil based adjuvant is used, the polypeptide usually is administered in an emulsion with the adjuvant.

In yet another preferred embodiment, E.coli expressing proteins comprising a novel B. burgdorferi polypeptide are administered orally to non- human animals to decrease or lessen the severity of B. burgdorferi infection. For example, a palatable regimen of bacteria expressing a novel B. burgdorferi polypeptide, alone or in the form of a fusion protein or multimeric protein, may be administered with animal food to be consumed by wild mice or deer, or by domestic animals. Ingestion of such bacteria may induce an immune response comprising both humoral and cell-mediated components See J. C. Sadoff et al., "Oral

Salmonella Typhimurium Vaccine Expressing Circumsporozoite Protein Protects Against Malaria", Science, 240, pp.336-38 (1988) and K. S. Kim et al.,

"Immunization Of Chickens With Live Escherichia coli Expressing Eimeria acervulina Merozoite Recombinant Antigen Induces Partial Protection Against

Coccidiosis", Inf. Immun., 57, pp.2434-40 (1989). In fact, oral vaccination with bacteria expressing OspA has been shown to be effective. See, M. Dunne et al., "Oral Vaccination Against Lyme Disease Using Salmonella Expressing OspA," Inf. and Immun.. 63 : 1611 (1995), E. Fikrig et al., "Protection of Mice From Lyme Borreliosis By Oral Vaccination With Escherichia coli Expressing OspA," J. Infec. Pis., 164 : 1224 (1991). Moreover, the level of B. burgdorferi infection in ticks feeding on such animals will be lessened or eliminated, thus inhibiting transmission to the next animal.

According to yet another embodiment, the antibodies of this invention as well as the novel B. burgdorferi polypeptides of this invention, and the DNA sequences encoding them are useful as diagnostic agents for detecting infection with B. burgdorferi, because the polypeptides are capable of binding to antibody molecules produced in animals, including humans that are infected with B. burgdorferi, and the antibodies are capable of binding to B. burgdorferi or antigens thereof.

Such diagnostic agents may be included in a kit which may also comprise instructions for use and other appropriate reagents, preferably a means for detecting when the polypeptide or antibody is bound For example, the polypeptide or antibody may be labeled with a detection means that allows for the detection of the polypeptide when it is bound to an antibody, or for the detection of the antibody when it is bound to B. burgdorferi or an antigen thereof.

The detection means may be a fluorescent labeling agent such as fluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC), and the like, an enzyme, such as horseradish peroxidase (HRP), glucose oxidase or the like, a radioactive element such as ¹²⁵ I or ⁵¹ Cr that produces gamma ray emissions, or a radioactive element that emits positrons which produce gamma rays upon encounters with electrons present in the test solution, such as ¹¹C, ¹⁵O, or ¹³N . Binding may also be detected by other methods, for example via avidin-biotin complexes.

The linking of the detection means is well known in the art For instance, monoclonal antibody molecules produced by a hybridoma can be metabolically labeled by incorporation of radioisotope-containing amino acids in the culture medium, or polypeptides may be conjugated or coupled to a detection means through activated functional groups.

The diagnostic kits of the present invention may be used to detect the presence of a quantity of B. burgdorferi or anti-B. burgdorferi antibodies in a body fluid sample such as serum, plasma or urine. Thus, in preferred embodiments, a novel B. burgdorferi polypeptide or an antibody of the present invention is bound to a solid support typically by adsorption from an aqueous medium. Useful solid matrices are well known in the art, and include crosslinked dextran, agarose, polystyrene, polyvinylchloride, cross-linked polyacrylamide; nitrocellulose or nylon- based materials, tubes, plates or the wells of microtiter plates. The polypeptides or antibodies of the present invention may be used as diagnostic agents in solution form or as a substantially dry powder, e g., in lyophilized form.

Novel B. burgdorferi polypeptides and antibodies directed against those polypeptides provide much more specific diagnostic reagents than whole B. burgdorferi and thus may alleviate such pitfalls as false positive and false negative results.

In particular, one of skill in the art would understand that novel B. burgdorferi polypeptides of this invention that are selectively expressed in the infected host and not in cultured B. burgdorferi, and antibodies directed against the polypeptides, allow detection of antigens and antibodies in samples that are undetectable by diagnostic methods using lysates of cultured spirochetes as the antigen.

One skilled in the art will realize that it may also be advantageous in the preparation of detection reagents to utilize epitopes from other B. burgdorferi proteins, including the flagella-associated protein, and antibodies directed against such epitopes. Antibodies to P35 and P37 tend to occur early in the course of B. burgdorferi infection while antibodies against P21 and OspF tend to appear later Accordingly, it may be particularly advantageous to use P35 or P37 epitopes in combination with epitopes from other B. burgdorferi proteins that elicit antibodies that occur in the later stages of Lyme disease. Diagnostic reagents containing multiple epitopes which are reactive with antibodies appearing at different times are useful to detect the presence of anti-5. burgdorferi antibodies throughout the course of infection and to diagnose Lyme disease at all stages.

The polypeptides and antibodies of the present invention, and compositions and methods comprising them, may also be useful for detection, prevention, and treatment of other infections caused by spirochetes which may contain surface proteins sharing amino acid sequence or conformational similarities with the novel B. burgdorferi polypeptides of the present invention. These other spirochetes include Borrelia Hermsii and Borrelia Recunentis, Leptospira, and Treponema.

According to another embodiment of this invention, we describe a method for identifying bacterial genes encoding an antigenic proteins that are expressed during infection of a host but that are not expressed during in vitro culture of the bacteria, the method comprising the steps of:

(a) constructing an expression library for the bacteria;

(b) screening the library with antisera from an animal infected with the bacteria;

(c) screening the library with antisera from an animal immunized with non-viable bacteria or components thereof: and

(d) identifying clones that react with the first antisera but not with the second antisera.

It will be readily apparent to one of skill in the art that an expression library for use in the methods of this invention may be constructed using any techniques known in the art.

To generate antisera for use in the methods of this invention, any animal capable of generating an immune response is useful. Antisera may be generated by any of the wide variety of techniques that are well known to those of skill in the art.

As used herein, bacteria include any pathogenic or non-pathogenic bacteria that are capable of proliferating in a host. In a preferred embodiment, the bacteria are pathogenic bacteria

As used herein, a host is any living organism that may be infected by bacteria, including plant and animal hosts. In a preferred embodiment, the host is a mammal.

As used herein, non-viable bacteria are bacteria that are incapable of synthesizing proteins. In a preferred embodiment, the bacteria are heat-killed bacteria. However, according to the methods of this invention, the bacteria may be rendered non-viable by any method known in the art. As used herein, components of non-viable bacteria include lysates, homogenates, or subcellular fractions thereof such as cell membrane containing fractions.

To screen the expression library for clones that react with the antisera, any of the techniques known to those of skill in the art are useful. In a preferred embodiment, binding of the antisera is detected with a secondary antibody coupled to a detection means. One of skill in the art will readily appreciate that any of the wide variety of detection means known in the art is useful Examples of useful detection means are set forth supra.

In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only, and are not to be construed as limiting the scope of the invention in any manner.

Example I - Construction and screening of a

B. burgdorferi expression library A. Construction of An Expression Library

We began with a B. burgdorferi genomic DNA expression library constructed in Lambda ZAP II by Stratagene (La Jolla, CA) [T.T. Lam et al., Inf. Immun.. 62, pp.290-298 (1994)]. Briefly, we grew B. burgdorferi strain N40 in modified Barbour-Stoenner-Kelly (BSK) II medium at 32°C for 7 days, harvested by centrifugation at 16,000 rpm for 30 minutes, and lysed with SDS [A.G. Barbour, "Isolation and Cultivation of Lyme Disease Spirochetes", Yale J. Biol. Med., 57, pp.521-25 (1984)]. We then isolated the genomic DNA from the spirochetes and purified it by phenol/chloroform extraction.

To construct the library, 200 μg of DNA was randomly sheared, blunt-ended with S1 nuclease, and the EcoR1 sites were methylated with EcoR1 methylase. EcoR1 linkers were then ligated to the ends of the DNA molecules, the DNA was digested with EcoR1 and the fragments were purified over a sucrose gradient. Fragments of 1 to 9 kb were isolated and ligated to EcoR1 digested Lambda ZAP II arms.

We prepared E. coli SURE bacteria (Stratagene) for phage infection as follows. We picked a single colony into LB media supplemented with 0.2% maltose and 10 mM magnesium sulfate and cultured overnight at 30°C with vigorous shaking. We then centrifuged the cells at 2000 rpm for 10 minutes and resuspended in lOmM magnesium sulfate. The cells were further diluted to

O.D.₆₀₀ = 0.5 for bacteriophage infection.

B. Preparation of Anti-B. burgdorferi Antisera

We prepared anti- B. burgdorferi N40 antisera for differentially screening the expression library as follows.

1. Immune Antisera

We prepared "immune" mouse anti- B. burgdorferi N40 antiserum as follows We injected 3 to 5 groups of five three-week old female C3h/HeNC_r or J (C3H) mice subcutaneously with an inoculum of 10⁷ heat-killed (1 hour at 60° C) B. burgdorferi N40 in complete Freund's adjuvant (CFA). We were unable to infect mice with the heat inactivated preparation or to culture spirochetes from the preparation placed in BSKII medium, thus confirming that all of the heat-inactivated spirochetes were killed We boosted the mice with the same dosage of B.

burgdorferi in incomplete Freund's adjuvant (IF A) at two weeks and four weeks Two weeks after the last boost, we sacrificed and bled the mice and separated the anti-B. burgdorferi antiserum by centrifuging the blood at 2000 rpm for 15 minutes.

To remove antibodies in the serum that would recognize E. coli and phage proteins, we absorbed the antiserum with an E. co///phage lysate (Stratagene) as follows. We diluted the lysate 1 :10 in Tris-buffered saline with 0.05% Tween-20 (TBST). We then incubated 0.45 μM pore size nitrocellulose filters (Millipore, Bedford, MA) in the lysate for 30 minutes at room temperature, removed and air dried the filters on Whatman filter paper (Whatman International Ltd , Maidstone, England), and washed 3 times (5 minutes each) with TBS. We blocked the filters by immersing in 1% Bovine Serum Albumin (BSA) in TBS for 1 hour at room temperature and rinsing 3 times with TBST. We then diluted the mouse antiserum 1 : 5 in TBST, incubated it with the filters with shaking for 10 minutes at 37°C, and removed and discarded the filters.

2. Infected Antisera

We injected three C3H/HeJ mice by intradermal inoculation with 10⁴ B. burgdorferi N40 spirochetes. We documented infection by culturing spirochetes from the spleen, bladder and skin (ear punches) of the challenged mice and by histopathologic examination of the joints and heart for evidence of inflammation. We collected serum from the infected mice at various times after infection.

Both immune and infected antisera contained a high titer of anti-B. burgdorferi antibodies directed against whole cell lysates. We detected antibodies in the sera of immunized and infected mice at a dilution of 1 15,000 and 1 10,000 by immunoblot and 1 : 6400 and 1 3200 by ELISA, respectively Moreover, both antisera recognized many B. burgdorferi antigens by immunoblot, with different intensities.

After absorption, we diluted the antiserum to a final dilution of 1 : 100 and used it to screen the nitrocellulose filters containing the expressed proteins from the lambda ZAP library according to manufacturer's instructions

C. Differential Screening of A Genomic

B. burgdorferi N40 Expression Library

To screen the library, we used the picoBIue Immunoscreening Kit (Stratagene). We plated 4 x 10⁴ plaque forming units of recombinant phage on a lawn of bacteria, induced protein expression with 10mM IPTG and transferred the proteins to duplicate plaque lifts on nitrocellulose filters according to methods well known in the art.

We incubated one set of plaque lifts with pooled sera from mice immunized with heat-killed spirochetes (immune sera) and the other set with sera from mice infected for nine months (infected sera). After washing, we incubated the filters with a 1 : 5000 dilution of alkaline phosphatase-conjugated goat anti- mouse IgG antibody (Organon Teknika Corp., West Chester, PA), and used nitro blue tetrazolium (NBT) (Stratagene) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Stratagene) for color development. We selected clones that reacted with infected sera but not with immune sera for further study.

Example II - Cloning of the p21 Ik2 Operon

Differential screening of a B. burgdorferi N40 genomic expression library, as described in Example I, revealed one hundred seventy-two clones that reacted with sera from the infected mice and one hundred sixty-nine clones that reacted with sera from immunized mice. We subjected the three phage clones that reacted differentially with the two sera to another round of screening with identical results.

We excised the pBluescript plasmid from one of those clones, clone 1, by infection of XL 1 -Blue E. coli cells and rescued with R408 helper phage according to the manufacturer's instructions. Using the recovered plasmid, we used

T3 and T7 universal primers to obtain an intial sequence of the plasmid. From that initial sequence of 100-300 bp, we made new primers which used to extend the sequence 100-300 bp at a time until we obtained the entire sequence.

Alternatively, we generated a nested set of deletions in the DNA insert of clone 1 with the Erase- A-Base System (Promega, Madison, WI) (using Smal to generate the 5' blunt end and BstXI to generate a 3' overhang). We then sequenced the subclones using the Sequenase Kit (United States Biochemical Corp., Cleveland, OH) and reconstructed the entire sequence using Mac Vector

(International Biotechnology, Inc., New Haven, CT).

We determined the nucleotide sequence of the plasmid insert using the Circumvent Thermal cycle Dideoxy DNA sequencing kit (New England Biolabs). Conditions for denaturation, annealing and extension were: 94° C for 30 sec. , 55° C for 20 sec, and 72° C for 20 sec, respectively.

Analysis of the DNA sequence of the insert revealed that we had isolated a clone containing a complete open reading frame and a partial open reading frame having the sequence set forth in SEQ ID NO: 1. We conducted a search of GenBank (December 1994) with the Genetics Computer Group Program (University of Wisconsin Biotechnology Center, Madison, WI). Our search revealed that we had isolated a novel, bicistronic B. burgdorferi operon We designated the complete open reading frame p21 and the partial open reading frame k2. We designated the antigens encoded by the two genes in the operon P21 and K2, respectively.

Example III - Sequence analysis of the p21/k2 operon

As shown in SEQ ID NO: 1, the p21 gene, at the 5' end of the operon, contains a 546 nucleotide open reading frame capable of encoding a 182- amino acid protein (SEQ ID NO: 2). The deduced amino acid sequence of P21 contains a typical prokaryotic signal sequence for posttranslational processing by cleavage and lipidation, suggesting that the gene product is a lipoprotein of approximately 20.7 kDa. P21 has 71% amino acid sequence identity to B.

burgdorferi OspE (Figure 7).

The ATG start codon for the k.2 gene is located 27 nucleotides downstream of the TAG stop codon of the p21 gene. The k2 gene in clone 1 contains a partial open reading frame of 32 nucleotides, capable of encoding the first 10 amino terminal amino acids (SEQ ID NO: 3) However, based on the last two nucleotides of the K2 sequence of SEQ ID NO: 3, the eleventh amino acid must be valine Accordingly, as used herein, a K2 polypeptide is a polypeptide that comprises the 1 1-amino acid sequence of SEQ ID NO: 3 The amino terminal amino acids of K2 are 64% homologous with the amino terminal sequence of OspF. Therefore, we would expect that the full-length protein encoded by the k2 gene would have similar homology to full-length OspF protein.

A consensus ribosome binding site with the sequence -GGAG- (Shine-Dalgarno sequence) is located 10 bp upstream of the p21 ATG start codon Further upstream of this translational initiation sequence are the promoter segments known as the "-10" region and the "-35" region, which are similar to those found in E. coli and other B. burgdorferi genes. (See Figure 8 for a comparison of these regions between various B. burgdorferi genes) . An additional ribosome binding site with the sequence -GGAG- is located 1 1 bp upstream of the ATG start codon of the k2 gene. The location of these sequence elements suggests that both the p21 and k2 genes are controlled by a single promoter The homology of P21 and K2 to OspE and OspF and their location in a bicistronic operon suggests that a recombinational event has most likely occurred between these genes in recent evolutionary time.

Like OspA, OspB, OspD, Osp E and OspF, the protein encoded by thep21 gene appears to be a surface lipoprotein. As shown in SEQ ID NO: 2, the protein begins with a basic N-terminal peptide of five amino acids, followed by an amino-terminal hydrophobic domain of about 20 amino acids that corresponds to the leader peptide found in typical prokaryotic lipoprotein precursors [M E Brandt et al., supra and C. H. Wu and M. Tokunaga, "Biogenesis of Lipoproteins in Bacteria". Current Topics in Microbiology and Immunology. 125. pp.127-157 (1986)]. The carboxyl terminus of the hydrophobic domain contains a cleavage site presumably recognized by a B. burgdorferi signal peptidase In P21 , as in OspF, the potential cleavage site is located between Ser₁₇ and Cys_18.

The consensus sequence of typical bacterial lipoprotein precursors recognized and cleaved by signal peptidase II is a Leu and a Cys separated usually by two small neutral amino acids [C.H. W u et al., supra]. Indeed, the OspA and OspB genes of B. burgdorferi B31 contain signal sequences of -L-I-A-C- and -L-I- G-C-, respectively [S. Bergstrom et al., "Molecular Analysis of Linear Plasmid- Encoded Major Surface Proteins, OspA and OspB, of the Lyme Disease

Spirochaete Borrelia burgdorferi", Mol Microbiol ., 3, 479-86 ( 1989)].

In contrast, the signal sequences of the B. burgdorferi N40p21 gene (-L-I-S-S-C-), like the OspE (-L-I-G-A-C-), OspF (-L-I-V-S-C-), OspC-PKo (-L-F-I-S-C-) and OspD-B31 (-L-S-I-S-C-) genes, contains three amino acids between the leucine and cysteine instead of two (See R.S. Fuchs et al and S.J. Norris et al., supra ) However, despite this variation in the signal sequence, OspA,

OspB and OspD have been shown to be lipoproteins by the established,

[³H]-palmitate labelling procedure (See M.E. Brandt et al and S.J. Norris et al , supra.) The leader signal sequence of P21 suggests that this surface protein may be processed as a lipoprotein as well The addition of a lipid moiety at the cysteine residue could serve to anchor the protein to the outer surface of the spirochetes (see H.C. Wu and M Tokunaga, supra).

Finally, P21 contains a long hydrophilic domain separated by short stretches of hydrophobic segments.

A comparison of the DNA sequences indicates that p21 and ospE are closely related but distinct genes within the B burgdorferi genome, with identical -35 and -10 promoter sequences and ribosome binding sites The 5' upstream regions oϊp21 and ospE are identical upstream from the -10 sequence to the boundary of the 5' flanking DNA which has been sequenced (189 nt 5' of the ATG)(Figure 7). Only eight nucleotide differences between p21 and ospE are evident in the area between the -10 region and the ATG start codon Upstream of the ATG, the following differences are noted in ospE, when compared with p21 :- 54, G, -45, C; -32, T; -30, G; -24, A; -15, C; -6, T; -3, C (where -1 is the A in the ATG codon) All of the differences are located in the region likely to contain the 5' untranslated region of p21 mRNA.

In view of this homology between P21 and OspE, one of skill in the art would understand that in formulating therapeutic and diagnostic compositions, it may be desirable to select epitopes of P21 that do not cross-react with OspE. Example IV - Analysis of p21 Expression In Cultured

B. burgdorferi By Northern Blotting

To determine whether p21 is transcribed during in vitro culture of spirochetes, we assessed its expression by Northern blot analysis We isolated total RNA from cultured B. burgdorferi by acid guanidium thiocyanate/phenol/ chloroform extraction [cite] We electrophoresed 20 μg of isolated RNA in a 1% formaldehyde-agarose gel and blotted onto Hybond-N® membrane (Amersham). We generated biotinylated p21 and ospA (control) probes with a Phototope® random-primer biotin-labeling kit (New England Biolabs) The p21 and ospA probes contained the entire p21 and ospA sequences, respectively We used amplified PCR products oϊp21 or ospA as templates for the random octamer- primed labeling reaction.

We conducted hybridization and signal detection with a Photope® chemiluminescent kit (New England Biolabs) Briefly, we prehybridized the blotted membrane in SSC for 1 hour at 68° C and hybridized with biotinylated probes for p21 or ospA (control) at 68° C overnight We washed the membrane at a final stringency of 0 IX standard saline citrate (SSC)/0 1% SDS at 68° C. We detected biotin-labeled probe by a series of incubations with streptavidin, biotinylated alkaline phosphatase, and lumigen-PPD.

We detected ospA RNA but no p21 RNA from cultured B.

burgdorferi. Example V - Southern Dot Blot Analysis and

PCR of Cultured B. burgdorferi Genome

Because in vitro culture of B. burgdorferi is often associated with the loss of genes or plasmids [cite], we used dot blot analysis and PCR to examine the genome of the cultured B. burgdorferi from which RNA was obtained for Northern blot analysis described in Example IV for the presence of the p21 gene.

A. Southern Dot Blot Analysis

For dot blot analysis, we spotted 2 μg of denatured λ phage (control) or cultured B. burgdorferi DNA onto Hybond-N® membrane. We first stained the dried membrane with ethidium bromide to confirm that an equal amount of DNA was present. We then hybridized with the p21 and ospA probes described in Example IV for Northern blot analysis. Both probes hybridized strongly to B. burgdorferi genomic DNA but not to bacteriophage DNA, confirming the presence of the p21 gene in the cultured B. burgdorferi.

B. PCR Analysis

We subjected 10 ng of genomic DNA from cultured B. burgdorferi to PCR using primers derived from the p21 gene . We used the 5' and 3' primers shown in SEQ ID NO: 1 1 and SEQ ID NO: 12, respectively These primers are specific for p21 and do not amplify ospE. We used the following conditions for PCR of cultured B. burgdorferi DNA : 30 cycles with denaturing, annealing and extension temperatures of 94° C for 1 min., 65°C for 1 min , 72° C for 2 min_., respectively. Using these primers, we obtained a 513 bp PCR product of the p21 coding region, further confirming that the p21 gene is present in the genome of the cultured B. burgdorferi used for Northern blot studies.

Example VI - Examination of p21 Expression By

B. burgdorferi In Ticks

To determine whether P21 is expressed by B. burgdorferi in Ixodes ticks, we examined lysates of flat and engorged ticks containing the spirochetes by indirect immunofluorescence. Using the same methods, one of skill in the art could readily determine without undue experimentation whether other novel B.

burgdorferi polypeptides of this invention are expressed in ticks.

Briefly, we allowed B. burgdorferi N40-infected ticks to feed to repletion on C3H/He mice. We lightly homogenized each unfed and engorged tick in 100 μl PBS and spotted a 10 μl aliquot onto a sylilated glass slide We air-dried the slides and fixed them with 4% paraformaldehyde and saponin. We incubated the specimens in a 1 10 dilution of antisera from mice immunized with the P21 - specific peptide prepared as in Example VII and as shown in SEQ. ID NO: _, for 1 hour We washed the slides and incubated them in anti-mouse IgG coupled to FITC (1 : 500 dilution) for 1 hour and viewed the slides under a Zeiss Axioskop® fluorescent microscope. We used anti-OspA monoclonal antibody CIII.78, which recognizes B. burgdorferi within unfed ticks but does not readily detect spirochetes within engorged ticks as a positive control [De Silva et al., (1996)]. We used anti- flagellin monoclonal antibody H9724, which recognizes B. burgdorferi in both flat and engorged ticks as a second positive control [Cite]. We used anti BSA sera as a negative control.

Consistently with previous studies, spirochetes were readily detected by flagellin-specific monoclonal antibody in both flat and engorged ticks while OspA-specific monoclonal antibody detected spirochetes in flat but not in engorged ticks. However, no P21 -specific immunofluorescence was detected in either flat or engorged ticks.

To confirm that the P21 -specific antisera could react with P21, we used the antisera to probe recombinant P21, prepared as in Example 12, or recombinant OspE. As expected, P21 antisera readily recognized recombinant P21 but not OspE. These results indicate that P21 is also not expressed in infected ticks.

Example VII - Confirmation oϊp21 Expression in Infected Mice By

Dot Immunoblot Analysis and RNA-PCR A. Dot Immunoblot Analysis

We next confirmed that p21 is expressed in mice infected with B. burgdorferi by demonstrating the presence of antibodies against P21 in sera from two infected mice.

We compared the amino acid sequences of P21 and OspE and chose a region of P21 comprising amino acids 31-40 which is unique to P21. We had Quality Control Biochemicals (Hopkinton, MA) synthesize the peptide coupled to bovine serum albumin (BSA). (A cysteine was added to the amino terminus of the peptide for the BSA coupling reaction). The amino acid sequence of the peptide is set forth in SEQ ID NO: 13.

We spotted 3 μg of BSA or the synthetic P21 -derived peptide coupled to BSA onto nitrocellulose membranes. We incubated the dried membranes with either serum from mice immunized with heat-killed B. burgdorferi or serum from infected mice. We detected bound antibody by incubating with a second antibody conjugated to horseradish peroxidase (ECL Western blot detection system, Amersham). Finally, we stained the membranes with amido black to demonstrate that an equal quantity of protein was present in all of the test samples. Sera from infected mice but not from mice immunized with heat- killed B. burgdorferi reacted with the P21 peptide. Thus, P21 is selectively expressed in vivo.

B. RNA PCR

We further demonstrated expression ofp21 in infected mice using

RNA PCR to detect p21 RNA. We used acid guanidium thiocyanate/phenol/ chloroform extraction (Micro RNA Isolation Kit, Stratagene) to isolate total RNA from spleens of the mice infected with B. burgdorferi via tick transmission and RNA from in vitro cultured B. burgdorferi. We allowed five B. burgdorferi N40- infected ticks to feed to repletion on the mice. To remove any residual DNA, we treated 10 μg of pooled RNA with RNase-free DNase (Promega) for 3 hours at 37° C with HPRI and the Rnase inhibitor. We conducted the RNA PCR with and without reverse transcriptase to exclude the possibility that residual DNA might also be amplified We synthesized cDNA by reverse transcription with Moloney murine leukemia virus reverse transcriptase (Stratagene) and 3' primers for either p21 (murine tissue and cultured B. burgdorferi), γ-actin (murine tissue control), or ospA (cultured B. burgdorferi control). We subsequently inactivated the reverse transcriptase by heating for 5 min. at 95° C. We then added 5' primer for p21, γ- actin or ospA and carried out PCR for 45 cycles of 94° C for 1 min., 55° C for 1 min and 72° C for 2 min.

We obtained a 513 bp product from RNA PCR ofp21 only in the presence of reverse transcriptase. To confirm the identity of the amplified product asp21, we denatured and electrophoresed RNA PCR products, transferred them to Hybond-N® membrane and hybridized with p21 probes as described in Example IV for Northern blot analysis. The absence of product without reverse transcriptase confirms that DNA was not amplified. We obtained no amplification with p21- specific primers from RNA prepared from uninfected mice or from RNA PCR of B. burgdorferi cultured in vitro. Example VIII - Sequence Analysis of the p35 and p37 Genes

We differentially screened the lambda Zap II B. burgdorferi expression library exactly as described in Example I but using sera from mice immunized with heat-killed B. burgdorferi and mice infected for 90 days with live B. burgdorferi. We identified 14 phage clones that reacted with antibodies in the sera from infected mice but not with antibodies in sera from mice immunized with heat-killed spirochetes.

We selected two of the clones that reacted strongly to the infected antisera, excised the plasmids and sequenced the inserts as described in Example I. One insert contained an open reading frame of 927 nucleotides encoding a 309 amino acid protein (SEQ ID NO 5) We conducted a search of GenBank (July 1995) with the Genetics Computer Group Program (University of Wisconsin Biotechnology Center, Madison, WI). Our search revealed that we had isolated a novel, B. burgdorferi gene which we designated p35. We designated the antigen encoded by the gene P35.

The other insert contained an open reading frame of 996 nucleotides encoding a 332 amino acid protein (SEQ ID NO: 7) A search of GenBank (July 1995)revealed that we had isolated a second novel, B. burgdorferi gene which we designated p37. We designated the antigen encoded by the gene P37.

As is evident from SEQ ID NO: 7, the deduced amino acid sequence of P37 reveals a leader peptide similar to those found in typical prokaryotic lipoprotein precursors. At the carboxy terminus of the hydrophobic core is a potential signal peptidase II cleavage site between Ser₁₉ and Cys_20. P35, however, has a potential cleavage site with five amino acids intervening between the Leu and the Cys, making a lipoprotein less likely It will be necessary to look for further evidence of to confirm that P35 is a lipoprotein. Finally, P37 contains a long hydrophilic domain separated by short hydrophobic segments. The hydrophilicity profiles of P35 and P37, shown in Figure 6 suggest that both are hydrophilic proteins. We identified -35 and -10 regions as well as ribosome binding sites upstream of the respective open reading frames. Example IX - Mapping of the p21. p35 and p37 Genes

We mapped the p21, p35 and p37 genes by pulsed-field electrophoresis (PFGE) with total B. burgdorferi N40 DNA using a modification of the technique described in M.S. Ferdows and A..G. Barbour, "Megabase-Sized Linear DNA in the Bacterium Borrelia burgdorferi, the Lyme Disease Agent", Proc. Natl. Acad. Sci., 86, pp.5969-5973 (1989). Briefly, we treated DNA plugs containing approximately 10 B. burgdorferi N40 with sarkosyl, lysed overnight with proteinase K and then separated the chromosomal and plasmid DNA by loading onto a 0.8% agarose gel. We electrophoresed the DNA in Tris-borate- EDTA (TBE) buffer (0.025 M Tris, 0.5 mM EDTA 0.025 M boric acid) using the Chef-DRII® system (Bio-Rad Laboratories, Richmond, Calif.) at 14°C for 18 hours at 198V, with ramped pulse times from 1 to 30 sec. For two-dimensional electrophoresis of the B. burgdorferi DNA, we changed the direction 90 degrees and electrophoresed again at a constant voltage of 80v for 6 hours.

We transferred the pulsed-field B. burgdorferi DNA to nitrocellulose membrane and probed with PCR-amplified radiolabelled p21, p35, p37 probes. We used p30, ospA and ospD probes as controls in the Southern blot. We generated p35 and p37 probes labeled with [a-³²P]dCTP, using the Prime-It® random primer kit according to the manufacturer's protocol (Stratagene).

As expected, the ospA and ospD probes hybridized to plasmids migrating at 49 kb and 38 kb, respectively [A.G. Barbour and C.F. Garon, "Linear Plasmids of the Bacterium Borrelia burgdorferi Have Covalently Closed Ends", Science, 237, pp. 409-41 1 (1987) and S.J. Norris et al., supra]. The p30 probe identified the chromosome. The full-length p21 probe bound at three locations but a p 21-specific probe (SEQ ID NO: 14) recognized a circular plasmid The P35 probe bound to a plasmid which appeared to migrate with the same mobility as a linear plasmid of around 42 kb The P37 probe bound to a plasmid which appeared to migrate with the same mobility as a linear plasmid of around 16 kb. Example X - Analysis of Cultured

B. burgdorferi For p35 or p37 Expression

To determine whether p35 or p37 are transcribed in vitro, we performed the same analyses as set forth in examples IV and V The 5' and 3' primers used for PCR analysis are shown in SEQ ID NOS : 15 and 16 (for p35) and in SEQ ID NOS : 17 and 18 (for p37).

The results of these analyses confirmed that p35 and p37 are transcribed in vitro

Example XI - Confirmation oϊp35 And p37 Expression In Infected

Mice by Immunoblot Analysis and RNA-PCR

We used the same dot blot and RNA PCR methods employed in

Example 6 and the primers used in Example 9. We confirmed that p35 and p37 are expressed in infected mice Therefore, p35 and p37 are selectively expressed in vivo.

Example XII - Expression of P21. P35 and P37 Polypeptides

To express the novel B. burgdorferi genes of this invention, we utilized the pMX vector, which is capable of directing expression of cloned inserts as glutathione S-transferase fusion proteins [see J. Sears et al., "Molecular Mapping of Osp A-Mediated Immunity to Lyme Borreliosis", J. Immunol., 147, pp.1995- 2000 ( 1991 )]. The PMX vector also contains a thrombin cleavage site immediately following the GT protein, thus, allowing the recovery of recombinant proteins without the GT fusion partner.

We first used PCR to amplify the P37 gene lacking the sequences encoding the hydrophobic leader peptides. We chose to delete that sequence to ensure that the polypeptide would be expressed as soluble fusion protein rather than as a lipoprotein, which would be anchored to the cell membrane or might aggregate elsewhere in the cell during or after biosythesis.

To facilitate subcloning, we amplified the genes using primers with additional restriction enzyme digestion sites. We amplified the p21 gene using a 5' primer with an additional BamHl site and a 3'primer with a Hind III site (SEQ ID NO : 21 and 22). We amplified the p35 gene using a 5' primer with an additional Xhol restriction enzyme digestion site and a 3' primer with a supplementary Hind III site [SEQ ID NO : 23 and 24].

We amplified the p37 gene using a 5' primer with an additional BamHl restriction enzyme digestion site and a 3' primer with a supplementary Xhol site [SEQ ID NO: 25 and 26]. We used 50 ng of plasmid DNA excised from initial phage colonies using the R408 helper phage as a template for the genes.

We performed the PCR for 30 cycles with initial template denaturation at 94°C for 1 minute, annealing at 40°C for 2 minutes and extension at 72° C for 3 minutes.

We digested the amplified gene products with BamHl (p21), Xhol and Bam HI (p35) or Hind III and Xhol (p37) and cloned onto the corresponding sites in the PMX plasmid. We then used the ligation mixture to transform

Escherichia coli DH5α according to methods well known to those of skill in the art. We isolated colonies containing the recombinant plasmid on Luria broth supplemented with ampicillin and cultured the cells.

We induced expression of the genes as glutathione S-transferase fusion proteins by growing the transformed bacteria to logarithmic phase and adding 1 mM isopropyl-1-thi-beta-galactoside (IPTG) for 3 hours. One of skill in the art could readily express the other B. burgdorferi polypeptides of this invention without undue experimentation following the above-described techniques.

Example XIII - Purification of Recombinant Fusion Proteins After inducing protein expression as described in Example XI, we placed the E. coli in phosphate buffered saline (PBS) with 1% Triton and subjected them to sonication. We purified the glutathione S-transferase- B. burgdorferi polypeptide fusion proteins (GT-P21, GT-P35, GT-P37 and GT-M30) from cell lysates as follows.

We separated the cell supernatant and pellet by centrifugation at

1000x for 8 mins and passed the supernatant containing the recombinant fusion proteins over a glutathione-Sepharose 4B column (Pharmacia) according to the manufacturer's instructions. We eluted the fusion protein from the column using a solution containing excess glutathione and quantified using the Bradford assay.

In addition, to purify the B. burgdorferi proteins without the glutathione S-transferase, we loaded the glutathione S-transferase fusion proteins over the glutathione-Sepharose 4B column, added 25 units of thrombin to cleave the recombinant B. burgdorferi protein from the GT and incubated overnight at room temperature. We then eluted the proteins with 50 mM Tris-CaCl₂-NaCl, treated the eluent with anti-thrombin beads for 1.5 to 2 hours and centrifuged at 13,000 rpm.

One of skill in the art would understand that other novel B.

burgdorferi polyeptides of this invention may be readily purified without undue experimentation using these procedures. Example XIV - Preparation Of Antibodies Directed Against The

B. burgdorferi Polypeptides Of This Invention

We generated antibodies directed against the novel B. burgdorferi polypeptides of this invention as follows. We immunized C3H/He mice (Frederick Cancer Research Center, Frederick, MD) subcutaneously with 10 micrograms of either GT-P21, P21 -specific peptide of SEQ ID NO: 13 bound to BSA GT-P35 or GT-P37 in complete Freund's adjuvant (CFA) and boosted with the same amount of antigen in incomplete Freund's adjuvant (IFA) at 14 and 28 days according to published protocols. We immunized control mice in the same manner with either recombinant glutathione S-transferase or BSA.

Fourteen days after the last boost, we collected sera from the immunized animals and used it to hybridize to Western blots of SDS-PAGE gels of recombinant GT-P21, BSA-linked P21 -specific peptide, P35 or P37 polypeptides

Recombinant P35 and P37 elicited antibodies in mice that were detectable by immunoblotting at a dilution of up to 1 :5000. We also detected binding by ELISA.

Example XV - Isolation of the Full-Length K2 Polypeptide

The full-length K2 polypeptide and DNA encoding it may be isolated by a variety of methods available to one of skill in the art. For example, antiserum raised against the peptide set forth in SEQ ID NO: 3 may be used to screen a

B. burgdorferi expression library for clones capable of expressing the protein. Alternatively, an expression library could be constructed in which smaller fragments of B. burgdorferi DNA are cloned in frame into an expression vector from which they would be expressed as glutathione S-transferase fusion proteins, such as pGEX-2T, pMX, or pGEMEX. Such a library would have a high likelihood of expressing the sequence as a fusion protein, even if it is normally linked to a promoter that is not transcriptionally active in E. coli. Alternatively, the DNA encoding the peptide set forth in SEQ ID NO: 3 may be used as the basis of an oligonucleotide probe to screen a small cDNA library.

Example XVI - Characterization of the Immune Response

To Novel B. burgdorferi Polypeptides

A. Murine Humoral Response

To characterize the immune response to the B. burgdorferi polypeptides of this invention, we infected C3H/He mice by intradermal inoculation with 10⁴ B. burgdorferi N40 or by tick-transmission using B. burgdorferi N40 infected I. scapularis ticks (Harvard School of Tropical Public Health). In the tick transmission studies, we exposed mice to 5 ticks infected with B. burgdorferi N40. We allowed the ticks to feed to repletion and collected them over a water bath for examination.

We collected sera from infected mice at day 7, 14, 30, 90 and day 180 after infection WE stored the samples overnight in test tubes for clot formation and isolated the sera by centrifugation for 30 min at 900X g. We then used the sera in ELISA with purified GT-P21 , BSA-linked P21 -specific peptide, GT-P35 or GT-P37 polypeptides as follows.

We coated duplicate sets of 96- well microtiter plates with the various recombinant polypeptides (200 micrograms { 1 μg/ml, 200 ml/well} and incubated overnight at 4° C. To prevent non-specific binding, we blocked with 100 μl/ml of 10% fetal calf serum in PBS for 1 hour We washed the plates three times with 0.05% PBS Tween (PBST). We added triplicate samples of sera (200 microliters/ well, diluted 1 :100) to the coated plates and incubated for 1 hour at room temperature/ 8 hours at 4°C. We then washed the plates 3 times with PBST and added goat anti-mouse IgM or goat anti-mouse IgG, each diluted 1 : 2000 and linked to alkaline phosphatase, to each well. We incubated the plates at room temperature for 1 hour and washed 3 times with PBST. Finally, we added 200 microliters of freshly prepared p-nitrophenol phosphate (1 mg/ml in glycine buffer {pH 10.5}) to each well and monitored the color change at 405 nanometers. We stopped the reaction with 3M NaOH.

We detected high titers of antibodies to both P35 and P37 as early as

14 days after infection. The response peaked 30 days after infection, diminished by 60-90 days after infection and almost disappeared by 180 days P21 -specific antibodies appeared in sera of mice on day 28 and persisted throughout the course of infection.

One of skill in the art can readily determine without undue experimentation the murine humoral response to other novel b. burgdorferi polypeptides of this invention using the procedures taught herein.

B. Human Humoral Response

We also characterized the human immune response to the P21, P35 and P37 proteins. For the P21 study, we obtained a panel of 82 patients' sera from the Yale Lyme Disease Clinic and a panel of 40 patients' sera from the Centers for Disease Control (CDC). Patients were classified as having early or late stage Lyme disease based on the clinical presentation, as documented by a physician, and serum antibodies to B. burgdorferi, according to CDC-defined disease criteria. Over 60% of the patients that donated samples to the CDC were culture positive for B.

burgdorferi. Patients from the Yale clinic were not routinely assessed for infection by culture.

We used the sera in ELISA with recombinant GT-P21, BSA-linked P21 -specific peptide. We found that 20 of the 82 sera (24%) from the Yale clinic had IgG antibodies to recombinant P21 and 8 of those 20 also had anti-P21 IgM antibodies. Out of the 20 sera with anti-P21 antibodies, 4 had IgM and 16 had IgG antibodies that bound to P21 -specific peptide. We found that 13 of the 40 sera (33%) from the CDC had IgG and/or IgM antibodies to P21 Of those 13 sera, 1 1 had IgG and 4 had IgM antibodies that bound to P21 -specific peptide. In general, we detected IgM responses in patients with Lyme disease of 3 months or less duration. We detected IgG antibodies in patients with a disease course of greater than 3 months and in 56% of the patients with Lyme arthritis.

For the P35 and P37 studies, we used the 40 sera from the Centers for Disease Control and sera from an additional 25 patients with well-documented Lyme disease who were seen at the Yale clinic and at the Connecticut Agricultural Research Station.

We used the sera in ELISA with recombinant GT-P35 and GT-P37 as described above, using goat anti-human IgG and IgM as the secondary antibodies.

We found that all of the sera from the CDC had IgG responses to P35 and P37. Because of the high reactivity to recombinant P35 and P37, we tested sera from an additional 25 patients with well-documented Lyme disease who were seen in our clinic and Lyme disease laboratory at Yale University Medical School and the Connecticut Agricultural Research Station Of these, 22 sera had antibody response to P35 and 20 sera had antibody response to P37.

Exa mple XVII - Ability of Novel B. burgdorferi

Polypeptides To Protect Against

B. burgdorferi Infection

To determine whether the novel B. burgdorferi polypeptides of this invention were able to elicit an immune response that would be effective to protect against B. burgdorferi infection, we actively immunized C3H/He mice

subcutaneously with 10 micrograms of recombinant GT-P35 or recombinant GT- P37 polypeptides in CFA and boosted at 14 and 28 days with the same amount of antigen in IFA according to published protocols. We immunized control mice in the same manner with recombinant GT. We then attempted to infect the immunized mice with B. burgdorferi N40. We grew a low passage isolate of B. burgdorferi N40 with demonstrated infectivity and pathogenicity in C3H/He mice, to log phase at 33° C in BSK II medium and counted with a hemocytometer under dark-field microscopy.

We challenged the actively immunized mice approximately 14 days after the last b oost with iinnttrradermal inoculations of 10⁴ spirochetes and sacrificed fourteen days after infection.

At sacrifice, we aseptically collected the blood, spleen, bladder and ear punches, cultured the tissues in BSK II medium for two weeks and examined by darkfield microscopy for spirochetes. At the same time we sectioned, formalin fixed and paraffin embedded, and then examined joints and hearts for inflammation We examined the heart and tibiotarsi blindly. We characterized arthritis by edema and synovial infiltration with neutrophils and lymphocytes. We characterized carditis by the presence of aortitis, myocarditis or pericarditis.

Preliminary results generated using these methods suggest that P35 or P37 may confer protection.

Example XVIII - Protection against tick-mediated

transmission

We also determined whether the novel B. burgdorferi polypeptides of this invention were able to elicit an immune response that would be effective to protect against tick-mediated transmission of the spirochete. We obtained spirochete-free Ixodes dammini ticks from the Harvard School of Public Health, which maintains a laboratory colony derived from an Ipswich, MA population. We infected the ticks (at the larval stage) by allowing them to feed to repletion on outbred CD-1 mice that had been previously infected (three weeks prior to serving as hosts) by intradermal inoculation of 10³ B. burgdorferi N40 spirochetes. Upon repletion, we collected engorged larvae, pooled them in groups of 100-200, and permitted them to molt to the nymphal stage at 21 °C and 95% relative humidity. We determined the prevalence of infection in each pool by immunofluorescence of a representative sample (10 ticks) three weeks after molting We used only those pools having an infection prevalence of greater than 70% for challenge experiments

We actively immunized mice with GT-P35, GT-P37, or both, GT- P21 or GT (control) as described in Example XVII. Two weeks after the last boost, we placed 5/15 infected nymphal ticks on each mouse, allowed them to feed to repletion and then allowed them to detach naturally over water. Two weeks later we sacrificed the mice and cultured the tissues for spirochetes and examine the organs, as described above.

Immunization with GT-P21 did not protect mice from infection or disease. Each mouse in the control and treatment group developed specific antibody titer of at least 1 : 5000 which have been found to be sufficient to protect mice from infection and disease in cases of protective antibodies like OspA (Fikrig et al., 1992). Mice were challenged with spirochetes at the peak antibody titer period which is a week after the final boost. It is possible that P21 is not expressed in high quantity in the early stages of infection We have shown the appearance of P21 -specific antibody 28 days post infection when it may be expressed in very low quantity It is also possible that immunization with P21 did not produce sufficient protective antibodies in mice or that P21 was not expressed in sufficient quantity on the surface of the spirochete to make them vulnerable to antibody-mediated killing

Example XIX - Decrease in spirochete load in ticks

feeding on immunized animals

Previous studies have shown that immunization of mice with recombinant OspA can eliminate the spirochetes from ticks feeding on the immunized animals [E. Fikrig et al., "Elimination of Borrelia burgdorferi from vector ticks feeding on OspA-immunized mice", Proc. Natl, Acad. Sci., 89, pp. 5418-5421 (1992)]. Thus, to determine if spirochetes also are killed when infected ticks fed on animals immunized with the novel B. burgdorferi polypeptides of this invention we conduct the following experiment..

We place five Ixodes dammini ticks, infected as described in Example XVIII, on each of 12 control mice immunized with GT or 12 mice immunized with GT-P21. After feeding to repletion, the ticks are allowed to naturally detached over water. Only a portion of the ticks are recovered from each group, the remainder apparently having been ingested by the mice. Ten days post- repletion, we homogenized individual ticks in 100 μl of PBS in a 1.5 ml microfuge tube and spotted 10 μl aliquots on each of three slides. We allowed the slides to air-dry, fixed in cold acetone for 10 minutes, and assayed by direct or indirect immunofluorescence .

For the direct immunofluorescence assay, we incubated the slides with FITC-conjugated rabbit anti- B. burgdorferi N40 antiserum at a dilution of 1 : 100, mounted under a coverslip and examined on a Zeiss Axioscop® Fluorescent Microscope. We quantified the spirochetes by counting the number of fluorescing cells in approximately 20 fields per slide. B. burgdorferi infection rates were similar within ticks that fed on immunized and control mice indicating that immunization with GT-P21 does not protect against infection.

One of skill in the art would understand that the effect of immunization with other novel B. burgdorferi polypeptides of this invention can be readily determined without undue experimentation using the methods taught herein.

Example XX - Passive Immunization of Mice With

Anti-P35 or Anti-P37 Antiserum

To determine if antiserum from animals immunized with

recombinant B. burgdorferi polypeptides would confer protection, we passively immunized mice with 0.2 ml of GT-P35, GT-P37 or anti-P35/P37 antisera. We then challenged the passively immunized mice with 10⁴ B. burgdorferi N40 at one day after the immunization.

Preliminary results indicate that the frequency of B. burgdorferi infection and the disease course in passively immunized mice appeared to be the same as in the control mice.

In a separate study, we inoculated three groups of 5 scid mice with 10³ B. burgdorferi N40 and then injected 0.5 ml of antiserum (diluted 1 : 10) from either GT-P21 immunized, GT immunized or 90 day infected mice on days 1, 4, 8 and 12 post-inoculation. We sacrificed the mice on day 15 and cultured blood, bladder, spleen and skin from the inoculation site in BSK II medium. We also examined the tibiotarsi and heart of each mouse for inflammation. The rate of infection and disease in mice passively immunized with P21 antiserum was similar to the rates in control mice. Mice passive immunized with 90 day antiserum from B. burgdorferi infected mice were substantially protected from infection.

Again, one of skill in the art would understand that to detect a protective effect, one could various of the experimental conditions. For example, one could obtain antiserum by immunization with a recombinant polypeptide without GT, collect antiserum at a different time point when the titer is higher, passively immunize with more antiserum, decrease the spirochete dose, or other means known in the art Example XXI - Additional Clones of In Vivo

Expressed B. burgdorferi Polypeptides

We have performed preliminary analyses of two additional clones produced by the screening set forth in Example 1. We designated those clones VI and V3. We deposited plasmids pVl and pV3, contained in VI and V3

respectively, on May 7, 1996 at the American Type Culture Collection, 12301

Parklawn Drive, Rockville, MD. Clone V3 has been sequenced (SEQ ID NO: 10). One of skill in the art could conduct similar experiments as set forth above to confirm that the polypeptides encoded by these clones are selectively expressed in vivo.

We have also performed preliminary analyses of the remaining clones identified in the screening set forth in Example VII. Based on the ability of each clone to cross-hybridize to the others, we separated those clones into five groups. At least three genes were identified in addition to those encoding P35 and P37 The DNA and amino acid sequences of one of those genes, designated M30, is set forth in SEQ ID NOS. 8 and 9 We designated the other genes J1 and J2. Plasmids from clones corresponding to J1 have been deposited as pi 5 and p5 under ATCC accession numbers___. Plasmids from clones corresponding to p2, p7 and p9 have been deposited under ATCC accession numbers_____.

Example XXII - Determination of Protective Epitopes

We construct recombinant genes which will express fragments of the novel B. burgdorferi polypeptides in order to determine which fragments contain protective epitopes. First, we produce overlapping 200-300 bp fragments which encompass the entire nucleotide sequence of each of the genes, either by restriction enzyme digestion, or by amplification of specific sequences of using PCR and oligonucleotide primers containing restriction endonuclease recognition sequences, as described supra. We then clone these fragments into an appropriate expression vector, preferably a vector from which the fragments will be expressed as fusion proteins, in order to facilitate purification and increase stability. For example, the gene fragments could be cloned into pGEMEX (Promega, Madison, WS) and expressed as T7 gene 10 fusion proteins. Such proteins would be insoluble and thus easily purified by recovery of the insoluble pellet fraction followed by solubilization in denaturants such as urea. Alternatively, the fragments could be expressed as glutathione S-transferase fusion proteins as described above. We then transform appropriate host cells and induce expression of the fragments. One way to identify fragments that contain protective B-cell epitopes is to use the individual purified fragments to immunize C3H/HeJ mice, as described above After challenge of the mice with B. burgdorferi, we determine the presence of infection by blood and spleen cultures and by histopathologic examination of the joints and heart.

Another technique to identify protective epitopes is to use the various fragments to immunize mice, allow ticks infected with B. burgdorferi to feed on the mice, and then determine, as set forth in Example VIII, whether the immune response elicited by the fragments is sufficient to cause a decrease in the level of B. burgdorferi in the ticks. Any epitopes which elicit such a response, even if they are not sufficient by themselves to confer protection against subsequent infection with B. burgdorferi, may be useful in a multicomponent vaccine.

Once we have localized various epitopes to particular regions of the fusion proteins, we conduct further analyses using short synthetic peptides of 5-35 amino acids. The use of synthetic peptides allows us to further define each epitope, while eliminating any variables contributed by the non-B. burgdorferi portion of the fusion protein.

Example XXIII - Preparation of a multicomponent vaccine

We determine which of the protective epitopes is able to elicit antibodies that will protect against subsequent infection with strains of B.

burgdorferi other than the strain from which the Osp gene was cloned. We then design a vaccine around those epitopes If none of the protective epitopes is able to confer protection against infection with other strains of B. burgdorferi, it may be particularly advantageous to isolate the corresponding novel B. burgdorferi polypeptides from those strains. A multicomponent vaccine may then be constructed that comprises multiple epitopes from several different B. burgdorferi isolates. Such a vaccine will, thus, elicit antibodies that will confer protection against a variety of different strains.

Example XXIV - Identification of T cell epitopes

Stimulation in animals of a humoral immune response containing high titer neutralizing antibodies will be facilitated by antigens containing both T cell and B cell epitopes. To identify those polypeptides containing T cell epitopes, we infect C3H/HeJ mice with B. burgdorferi strain N40 in complete Freund's adjuvant, as described supra. Ten days after priming, we harvest the lymph nodes and generate in vitro T cell lines. These T cell lines are then cloned using limiting dilution and soft agar techniques. We use these T cell clones to determine which polypeptides contain T cell epitopes. The T cell clones are stimulated with the various polypeptides and syngeneic antigen presenting cells. Exposure of the T cell clones to the polypeptides that contain T cell epitopes in the presence of antigen presenting cells causes the T cells to proliferate, which we measure by ³H- Thymidine incorporation. We also measure lymphokine production by the stimulated T cell clones by standard methods.

To determine T cell epitopes of the polypeptides recognized by human T cells, we isolate T cell clones from B. burgdorferi-infected patients of multiple HLA types. T cell epitopes are identified by stimulating the clones with the various polypeptides and measuring ³H-Thymidine incorporation. The various T cell epitopes are then correlated with Class II HLA antigens such as DR, DP, and

DQ. The correlation is performed by utilization of B lymphoblastoid cell lines expressing various HLA genes. When a given T cell clone is mixed with the appropriate B lymphoblastoid cell line and a novel B. burgdorferi polypeptide, the B cell will be able to present the polypeptide to the T cell Proliferation is then measured by ³H-Thymidine incorporation. Alternatively, T cell epitopes may be identified by adoptive transfer of T cells from mice immunized with various of the novel B. burgdorferi polypeptides of this invention to naive mice, according to methods well known to those of skill in the art. [See, for example M.S. DeSouza et al , "Long-Term Study of Cell-Mediated Responses to Borrelia burgdorferi in the Laboratory Mouse", Infect. Immun., 61, pp. 1814-22 (1993)].

We then synthesize a multicomponent vaccine based on different T cell epitopes. Such a vaccine is useful to elicit T cell responses in a broad spectrum of patients with different HLA types.

We also identify stimulating T cell epitopes in other immunogenic B. burgdorferi polypeptides or in non-B. burgdorferi polypeptides and design multicomponent vaccines based on these epitopes in conjunction with B cell and T cell epitopes from the novel B. burgdorferi polypeptides of this invention.

Example XXV - Construction of fusion proteins

comprising T and B cell epitopes

After identifying T cell epitopes of the novel B. burgdorferi polypeptides, we construct recombinant proteins comprising these epitopes as well as the B cell epitopes recognized by neutralizing antibodies. These fusion proteins, by virtue of containing both T cell and B cell epitopes, permit antigen presentation to T cells by B cells expressing surface immunoglobulin. These T cells in turn stimulate B cells that express surface immunoglobin, leading to the production of high titer neutralizing antibodies.

We also construct fusion proteins from the novel B. burgdorferi polypeptides by linking regions of the polypeptides determined to contain B cell epitopes to strong T cell epitopes of other antigens. We synthesize an

oligonucleotide homologous to amino acids 120 to 140 of the Hepatitis B virus core antigen. This region of the core antigen has been shown to contain a strong T cell epitope [D. R. Millich, et al., supra]. The oligonucleotide is then ligated to the 5' and 3' ends of segments of DNA encoding the B cell epitopes recognized by neutralizing antibodies, as in Example XI. The recombinant DNA molecules are then used to express a fusion protein comprising a B cell epitope from the novel B. burgdorferi polypeptide and a T cell epitope from the core antigen, thus enhancing the immunogenicity of the polypeptide.

We also construct fusion proteins comprising epitopes of the novel B. burgdorferi polypeptides as well as epitopes of the tetanus toxoid protein.

We also construct a plasmid containing the B cell epitopes of various of the novel B. burgdorferi polypeptides incoφorated into the flagellin protein of Salmonella. Bacterial flagellin are potent stimulators of cellular and humoral responses, and can be used as vectors for protective antigens [S.M.C. Newton, C. Jacob, B. Stocker, "Immune Response To Cholera Toxin Epitope Inserted In Salmonella Flagellin", Science. 244, pp 70-72 (1989)] We cleave the cloned H 1- d flagellin gene of Salmonella muenchens at a unique Eco RV site in the hypervariable region. We then insert blunt ended DNAs encoding protective B cell epitopes of the polypeptides using T4 DNA ligase. The recombinant plasmids are then used to transform non-flagellate strains of Salmonella for use as a vaccine. Mice are immunized with live and formalin killed bacteria and assayed for antibody production. In addition spleen cells are tested for proliferative cellular responses to the peptide of interest. Finally the mice immunized with this agent are challenged with B. burgdorferi as described supra.

We also construct fusion proteins comprising B cell epitopes from one of the novel B. burgdorferi polypeptides and T cell epitopes from a different novel B. burgdorferi polypeptide or other immunogenic B. burgdorferi

polypeptides. Additionally, we construct fusion proteins comprising T cell epitopes from novel B. burgdorferi polypeptides and B cell epitopes from a novel B.

burgdorferi polypeptide and/or other immunogenic B. burgdorferi polypeptides. Construction of these fusion proteins is accomplished by recombinant DNA techniques well known to those of skill in the art. Fusion proteins and antibodies directed against them, are used in methods and composition to detect, treat, and prevent Lyme disease as caused by infection with B. burgdorferi.

While we have described a number of embodiments of this invention, it is apparent that our basic constructions may be altered to provide other embodiments which utilize the processes and products of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims, rather than by the specific embodiments which have been presented by way of example.

Claims

We claim.

1. An isolated DNA molecule comprising a DNA sequence which encodes a B. burgdorferi polypeptide, wherein said polypeptide is selected from the group consisting of:

(a) a P35 polypeptide encoded by SEQ ID NO: 4;

(b) a P37 polypeptide encoded by SEQ ID NO: 6;

(c) an M30 polypeptide encoded by SEQ ID NO: 8;

(d) a V3 polypeptide encoded by SEQ ID NO: 10;

(e) a J1 polypeptide encoded in whole or in part by the B. burgdorferi DNA sequence contained within ATCC deposit #_;

(f) a J2 polypeptide encoded in whole or in part by the B. burgdorferi DNA sequence contained within ATCC deposit #_;

(g) serotypic variants of any one of the polypeptides of (a)-

(f);

(h) fragments comprising at least 8 amino acids taken as a block from any one of the polypeptides of (a)-(g);

(i) derivatives of any one of the polypeptides of (a)-(h), said derivatives being at least 80% identical in amino acid sequence to the corresponding polypeptide of (a)-(h); (j) polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with B. burgdorferi, which antibodies are immunologically reactive with any one of the polypeptides of (a)-(i),

(k) polypeptides that are capable of eliciting antibodies that are immunologically reactive with B. burgdorferi and any one of the polypeptides of (a)-(i); and

(I) polypeptides that are immunologically reactive with antibodies elicited by immunization with any one of the polypeptides of (a)-(i).

2 An isolated DNA molecule comprising a DNA sequence which encodes a B. burgdorferi polypeptide, wherein said polypeptide is selected from the group consisting of.

(a) a P21 polypeptide consisting of amino acids 1-182 of SEQ ID NO : 2

(c) a polypeptide that is selectively expressed in vivo and that

(1) is a derivative of a P21 polyeptide of (a), said derivative being at least 80% identical in amino acid sequence to the corresponding polypeptide of (a); (2) polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with B. burgdorferi, which antibodies are immunologically reactive with a P21 polypeptide of (a);

(3) polypeptides that are capable of eliciting antibodies that are immunologically reactive with B. burgdorferi and the P21 polypeptide of (a); and

3 An isolated DNA molecule comprising a DNA sequence which encodes a B. burgdorferi polypeptide, wherein said polypeptide is selected from the group consisting of.

(a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3;

(b) derivatives of the polypeptide of (a), said derivative comprising a polypeptide having a block of amino acids at least 80% identical in sequence to SEQ ID NO. 3; and

(c) a polypeptide that is selectively expressed in vivo and that

(1) is a derivative of a polyeptide of (a), said derivative being at least 80% identical in amino acid sequence to the corresponding polypeptide of (a); (2) polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with B. burgdorferi, which antibodies are immunologically reactive with a polypeptide of (a);

(3) polypeptides that are capable of eliciting antibodies that are immunologically reactive with B. burgdorferi and the polypeptide of (a), and

4. The DNA molecule according to any one of claims 1 to 3, wherein the polypeptide comprises a protective epitope.

5. An isolated DNA molecule comprising a DNA sequence encoding a fusion protein comprising a B. burgdorferi polypeptide according to any one of claims 1 to 4.

6. An isolated DNA molecule comprising a DNA sequence encoding a multimeric protein, which multimeric protein comprises a B. burgdorferi polypeptide according to any one of claims 1 to 4.

7. A DNA molecule according to any one of claims 1 -6, further comprising an expression control sequence operatively linked to the DNA sequence.

8 A host cell transformed with a DNA molecule according to any one of claims 1 to 7.

9. A polypeptide encoded by a DNA molecule according to any one of claims 1 to 6.

10. A method for producing a polypeptide according to claim 9, comprising the step of culturing a host cell transformed with a DNA molecule according to claim 7.

1 1. A fusion protein comprising a B. burgdorferi polypeptide according to claim 9.

12. The fusion protein according to claim 11, wherein said fusion protein comprises two or more B. burgdorferi polypeptides according to claim 9, each derived from a different strain of B. burgdorferi.

13. The fusion protein according to claim 11, wherein said fusion protein further comprises an immunogenic B. burgdorferi polypeptide different than the polypeptide according to claim 9.

14. A multimeric protein comprising a polypeptide according to claim 9.

15. An antibody that binds to a polypeptide according to claim 9.

16. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a component selected from the group consisting of: a polypeptide according to claim 9; a fusion protein according to any one of claims 11 to 13; and a multimeric protein according to claim 14.

17. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of an antibody according to claim 15.

18 The pharmaceutical composition according to claim 16, further comprising at least one additional immunogenic B. burgdorferi polypeptide.

19. The pharmaceutical composition according to claim 16, further comprising at least one additional non-5, burgdorferi polypeptide.

20. A method for treating or preventing B. burgdorferi infection or Lyme disease comprising the step of administering to a patient a therapeutically effective amount of a pharmaceutical composition according to any one of claims 16 to 19.

21. A diagnostic kit comprising a component selected from the group consisting of a polypeptide according to claim 9, a fusion protein according to any one of claims 11-13, and a multimeric protein according to claim 14; and also comprising a means for detecting binding of said component to an antibody.

22. A method for detecting B. burgdorferi infection comprising the step of contacting a body fluid of a suspected infected mammalian host with a polypeptide according to claim 9, a fusion protein according to any one of claims 1 1 -13, and a multimeric protein according to claim 14.

23. A diagnostic kit comprising an antibody according to claim 15.

24. A method for detecting B. burgdorferi infection comprising the step of contacting a body fluid of a mammalian host with an antibody according to claim 15.

25. A method for identifying a bacterial gene encoding an antigenic protein which is expressed during infection of a host but is not expressed during in vitro culture of the bacteria, comprising the steps of.

(a) constructing an expression library from the bacterial DNA,

(b) screening the expression library with a first antiserum from an animal infected with the bacteria;

(c) screening the expression library with a second antiserum from an animal immunized with non-viable bacteria or components thereof, and

(d) identifying clones that react with the first antiserum but not with the second antiserum.

26. The method according to claim 25, wherein the non-viable bacteria is obtained from in vitro culture of the bacteria.

27. The method according to claim 25, wherein the non-viable bacteria is obtained from an infected host vector.

28. The method according to any one of claims 25 to 27, wherein the bacteria is a spirochete.

29. The method according to claim 28, wherein the bacteria is B. burgdorferi.

30. The method according to claim 29, wherein the host is a tick.