CA2253834A1 - B. burgdorferi polypeptides expressed in vivo - Google Patents
B. burgdorferi polypeptides expressed in vivo Download PDFInfo
- Publication number
- CA2253834A1 CA2253834A1 CA002253834A CA2253834A CA2253834A1 CA 2253834 A1 CA2253834 A1 CA 2253834A1 CA 002253834 A CA002253834 A CA 002253834A CA 2253834 A CA2253834 A CA 2253834A CA 2253834 A1 CA2253834 A1 CA 2253834A1
- Authority
- CA
- Canada
- Prior art keywords
- burgdorferi
- polypeptide
- polypeptides
- lys
- antibodies
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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- C07K16/1207—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
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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
CA 022~3834 1998-11-06 WO97/42325 PCTrUS96/06610 B. BUl~GDORFFRI POLYPEPTIDES E~'RESSED IN VIVO
This invention was made with government support under Grant numbers AI30548, AI26815, AI49387 and AR40452 awarded by National S ~nctitutes of Health. The govellllllenl has certain rights in the invention.
TFCHNICAL FTFT n OF THE INVENTION
This invention relates to compositions and methods useful for the prevention, diagnosis and tre~tm~nt of Lyme disease. More particularly, this invention relates to novel B. burgdorferi polypeptides which are able to elicit in a l C treated animal, the forrnation of an immlme response. This invention also relates to novel B. burgdorferi polypeptides that are expressed during infection of a host but are not l..A~JI essed 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 di-ecled 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.
CA 022~3834 1998-11-06 WO97/42325 PCT~US96/~6610 ~ACKGROUND OF THE I~VENTION
Lyme borreliosis is the most common vector-borne infection in the United States [S.W. Barthold, et al., "An Animal Model For Lyme Arthritis", nn.
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 exp~n-lin~ skin lesion known as erythema migrans, which may be followed weeks to months later by neurologic, cardiac, and joint abnorrnalities.
The causative agent of Lyme disease is a spirochete known as Borrelia burgdorferi, transmitted primarily by Ixodes ticks of the Ixodes ricinus 10 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 menin~itis-like symptoms are common at this time. Stage twooccurs within days or weeks, and involves spread of the spirochete through the patient's blood or Iymph to many dirrel enl sites in the body including the brain and joints. Varied symptoms of this ~ s~min~ted infection occur in the skin, nervoussystem, 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 2s system appear during this stage, as a result ofthe ongoing infection and perhaps a resulting auto-immune disease [R. Martin et al., "Borrelia b~rgdorferi-Specific And Autoreactive T-Cell Lines From Cerebrospinal Fluid In Lyme Radiculomyelitis", Ann Neurol., 24, pp. 509-16 (1988)].
CA 02253834 1998-ll-06 W097/4232S PC~/U$3~ 10 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 immllnosorbent 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 I,yme Disease:
Evaluation Of Diagnostic Tests", J. Infect. I)is., 149, pp. 789-95 (1984)].
However, false negative and, more colllmollly, 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 F~ 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 pallidum, which causes syphilis, and leptospirae, which cause an infectious jaundice, Borrelia belong to the eubacterial phylum ofspirochetes [A.G. Barbour and S.F. Hayes, "Biology Of Borrelia Species", Microbiol. Rev., 50, pp. 381-400 (1986)]. Borrelia b1lrgdorferi 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 [ H]p~lmit~te [M.E.
Brandt et al., "Immunogenic Integral membrane Proteins of Borrelia b1lrgdorferi Are Lipoproteins", Infect. lmmlln, 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 CA 022~3834 1998-11-06 W 097/42325 PCT~US96/06610 di~renl isolates or from di~el enl passages of the same isolate as determined bytheir 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 pr~el el~lially expressed by low-passage, virulent strains of B. burgdorferi B3 1 [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
10 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. G~sm~n et al., "Analysis of the Borrelia burgdorferi GeHo.~qa 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. ~mm--n, 146, pp. 3177-82 (1991)].
B. burgdorferi is known to alter the antigens on its outer surface during di~rel en~ stages of its life cycle. For example, OspC is not expressed by spirochetes within unfed ticks. However, it is synthesized following engorgementand 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 ~A,ulession decreases. The downregulation of OspA within ticks allows spirochetes to survive in the presence of an OspA antibody response, ... .
CA 022~3834 1998-ll-06 W097/42325 PCT~US96/06610 suggestine that selective antigen ~ ression may be a mech~ni~m by which B.
burgdorferi evade imml Ine destruction.
It is known that the eAI)res~ion of other bacterial pathogen gene products is in~nced by envilo~u~lelllal signals ~J.J. Mekalanos, ~'Environm~nt~lSignal Controlling Expression of Virulence Dete, llflllallls In Bacteria," J. Bacteriol, 174, pp. 1-7 (1992)]. A similar induction of gene e"l,lession may occur in the infected host where specific external signals are present. Thus, to understand the mech~nism 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 [M. J. Mahan et al, "Selection of Bacterial Virulence Genes That Are Specifically lnduced in Host Tissues," Science, 259, pp 686-688 (1993)]. However, this system may not be applied to pathogenic orgalllslns 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 me~h~icm.s 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 medi~lm Selective in vivo expression of some B. burgdorferi proteins may be one reason that current diagnostic tests for Lyme disease, based on whole-cell Iysates of cultured B. burgdorferi, are unreliable. Such tests cannot detect antibodies directed toward the in vivo expressed antigens. Accordingly, there also CA 022~3834 1998-ll-06 W 097/42325 PCT~US96/06610 exists a need to identify B. burgdorferi proteins that provide more reliable diagnostic tests for Lyme disease.
Recently, imm-mi7~tion of mice with recombinant OspA has been shown to be effective to confer long-lasting protection against subsequent infection with B. bz ,~dorJre, i [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 imm--nogens used to date appears to be somewhat strain specific, probably due to the heterogeneity of the OspA gene among dirrele,ll B. burgdorferi isolates. For example, immlmi7:~tion with OspA
1 C from B. burgdorferi strain N40 confers protection against subsequent infection with strains N40, B31 and CD16, but not against strain 25015 ~E. Fikrig et al., "Borrelia burgdorferi Strain 2501 ~: Characterization of Outer Surface Protein A and Vaccination Against Infection", J. Immun., 148, pp. 2256-60 (1992)].
Tmmlmi7~tion 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 Tmmllnity to Lyme Borreliosis in Laboratory Mice", Infec. Immun., 60, pp. 657-61 (1992)]. Moreover, some B. burgdorferi are appare,l~ly able to escape destruction in OspB-imm-mi7edmice via a mutation in the OspB gene which results in expression of a truncated OspB protein [E. Fikrig et al., "Evasion of Protective Immllnity 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 immlmi7.~tion with this protein appears to be only partial ~V. Preac-Mursic et al., 25 "Active Tmmlmi7:~tion with pC Protein of Borrelia burgdorferi Protects Gerbils against B. burgdorferi Infection", Infection, 20, pp. 342-48 (1992)].
Tmmlmi7~tion with OspF has also been shown to confer partial protection against infection [T. K. Nguyen et al., "Partial Destruction of Borrelia W097/42325 PCTrUS96/06610 burgdorferi Within Ticks That Engorged On OspE- Or OspF-lmml-ni~ed 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., ~].
As prevention of tick infestation is imperfect, and Lyme disease may be missed or mi~ gnosed when it does appear, there exists a contin-ling urgent need for the delellninalion of additional antigens of B. burgdorferi and relatedproteins which are able to elicit a protective imm~me response and which may be useful in a broad-spectrum vaccine. In addition, identification of additional B.lC burgdorferi antipen~ may enable the development of more reliable diagnostic reagents which are useful in various stages of Lyme borreliosis.
DISCLOSURE OF T~lF INVENTION
The present invention provides novel B. burgdorferi polypeptides which are substantially free of a B. burgdorferi spirochete or fr~gmçnts thereof and, thus, are useful in compositions and methods for the diagnosis, tre~tment and prevention of B. burgdorfeff infection and Lyme disease. In one embodiment, thisinvention provides P2 1 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.
CA 022~3834 1998-ll-06 W097/42325 PCT~US96/06610 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 plefelled polypeptides of each of the aforementioned embodiments are selectively expressed in vivo.
Also p, t;~lled are compositions and methods of each of the o 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 im mllnr~genic 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 andmethods are useful for the detection of Lyme disease and B. burgdorferi infection.
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 po}ypeptides.
In yet another embodiment, this invention provides methods for 5 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 10 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.
BRTFF DESCRIPTION OF THE DRAWINGS
Figure I depicts the DNA and amino acid sequences of the P2 1 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 P3 7 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 2 5 polypeptide of B. burgdorferi strain N40.
Figure 6 depicts the hydrophilicity profiles of P35 and P37.
W 097142325 PCTrUS96/06610 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 ~ slalion among the DNA sequences encoding P2 1 and K2 and the DNA
5 sequences of other known B. burgdorferi outer surface proteins.
DETAILED DFSCRIPTION OF T~F. 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 co-llposilions and methods comprising those polypeptides.
In another embodiment, this invention relates to K2 polypeptides and 15 compositions and methods culllplis;l1g 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 embodiment1 this invention relates to M30 polypeptides and compositions and methods COlll,ul;S;llg 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 2~ and methods colllplisil1g those polypeptides.
In another embodiment, this invention relates to J2 and compositions and methods comprising those polypeptides.
., . . . . _ .
CA 022~3834 1998-11-06 W097/42325 PCT~US96/06610 The pl e~e,l ed 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 CO~ JI ish~g 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 organi~lns.
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 l~nintentional infection or cont~min~tion with undesired B. burgdorferi components. Accordingly, the novel B. burgdorferi polypeptides of this invention are particularly advantageous in compositions andmethods 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, in~.h]ding to detect the presence of B. burgdorferi, to screen forexpression 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.
CA 022~3834 1998-ll-06 W 097/42325 PCT~US96/06610 In yet another embodim~nt, 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 "im mllnQgenic B. burgdorferi polypeptide" is any B. burgdorferi polypeptide that, when ~dministered to an animal, is capable of eliciting an irnmune response.
Immunogenic B. burgdorferi polypeptides are int~n-led 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 fr~gm~nts, serotypic variants and derivatives of any of the above. ~n particular, immllnl genic B. burgdorferi polypeptides are intl~n~led to 5 include additional B. burgdorferi polypeptides which are identified according to the rnethods disclosed herein.
As used herein, a polypeptide which is "substantially free of a B.
burgdorferi spirochete or fragm~nt~ thereof'' is a polypeptide that, when introduced into modified Barbour-Stoener-Kelly (BSK-II) medium and cultured at 37~C for 7 2 o days, fails to produce any B. burgdorferi spirochetes detect~hle by dark field microscopy or a polypeptide that is detectable as a single band on an immunoblotprobed with polyclonal anti-B. burgdorferi anti-serum.
As used herein, a B. burgdorferi polypeptide that is "selectively expressed in ~ivo" is a polypeptide encoded by a DNA sequence that corresponds 2 5 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 CA 022~3834 1998-11-06 WO 97/42325 PCT/U~G/OG~10 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 con~;sLing of:
(a) a P21 polypeptide consisting of amino acids 1-182 of SEQ ID NO: 2;
(b) fr~gmçnt~ 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 P2 1 polypeptide of (a), said derivative being 10 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 m~mm~ n host with B. burgdorferi, which antibodies are immunologically reactive with a P2 1 polypeptide of (a)~
This invention was made with government support under Grant numbers AI30548, AI26815, AI49387 and AR40452 awarded by National S ~nctitutes of Health. The govellllllenl has certain rights in the invention.
TFCHNICAL FTFT n OF THE INVENTION
This invention relates to compositions and methods useful for the prevention, diagnosis and tre~tm~nt of Lyme disease. More particularly, this invention relates to novel B. burgdorferi polypeptides which are able to elicit in a l C treated animal, the forrnation of an immlme response. This invention also relates to novel B. burgdorferi polypeptides that are expressed during infection of a host but are not l..A~JI essed 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 di-ecled 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.
CA 022~3834 1998-11-06 WO97/42325 PCT~US96/~6610 ~ACKGROUND OF THE I~VENTION
Lyme borreliosis is the most common vector-borne infection in the United States [S.W. Barthold, et al., "An Animal Model For Lyme Arthritis", nn.
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 exp~n-lin~ skin lesion known as erythema migrans, which may be followed weeks to months later by neurologic, cardiac, and joint abnorrnalities.
The causative agent of Lyme disease is a spirochete known as Borrelia burgdorferi, transmitted primarily by Ixodes ticks of the Ixodes ricinus 10 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 menin~itis-like symptoms are common at this time. Stage twooccurs within days or weeks, and involves spread of the spirochete through the patient's blood or Iymph to many dirrel enl sites in the body including the brain and joints. Varied symptoms of this ~ s~min~ted infection occur in the skin, nervoussystem, 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 2s system appear during this stage, as a result ofthe ongoing infection and perhaps a resulting auto-immune disease [R. Martin et al., "Borrelia b~rgdorferi-Specific And Autoreactive T-Cell Lines From Cerebrospinal Fluid In Lyme Radiculomyelitis", Ann Neurol., 24, pp. 509-16 (1988)].
CA 02253834 1998-ll-06 W097/4232S PC~/U$3~ 10 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 immllnosorbent 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 I,yme Disease:
Evaluation Of Diagnostic Tests", J. Infect. I)is., 149, pp. 789-95 (1984)].
However, false negative and, more colllmollly, 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 F~ 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 pallidum, which causes syphilis, and leptospirae, which cause an infectious jaundice, Borrelia belong to the eubacterial phylum ofspirochetes [A.G. Barbour and S.F. Hayes, "Biology Of Borrelia Species", Microbiol. Rev., 50, pp. 381-400 (1986)]. Borrelia b1lrgdorferi 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 [ H]p~lmit~te [M.E.
Brandt et al., "Immunogenic Integral membrane Proteins of Borrelia b1lrgdorferi Are Lipoproteins", Infect. lmmlln, 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 CA 022~3834 1998-11-06 W 097/42325 PCT~US96/06610 di~renl isolates or from di~el enl passages of the same isolate as determined bytheir 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 pr~el el~lially expressed by low-passage, virulent strains of B. burgdorferi B3 1 [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
10 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. G~sm~n et al., "Analysis of the Borrelia burgdorferi GeHo.~qa 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. ~mm--n, 146, pp. 3177-82 (1991)].
B. burgdorferi is known to alter the antigens on its outer surface during di~rel en~ stages of its life cycle. For example, OspC is not expressed by spirochetes within unfed ticks. However, it is synthesized following engorgementand 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 ~A,ulession decreases. The downregulation of OspA within ticks allows spirochetes to survive in the presence of an OspA antibody response, ... .
CA 022~3834 1998-ll-06 W097/42325 PCT~US96/06610 suggestine that selective antigen ~ ression may be a mech~ni~m by which B.
burgdorferi evade imml Ine destruction.
It is known that the eAI)res~ion of other bacterial pathogen gene products is in~nced by envilo~u~lelllal signals ~J.J. Mekalanos, ~'Environm~nt~lSignal Controlling Expression of Virulence Dete, llflllallls In Bacteria," J. Bacteriol, 174, pp. 1-7 (1992)]. A similar induction of gene e"l,lession may occur in the infected host where specific external signals are present. Thus, to understand the mech~nism 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 [M. J. Mahan et al, "Selection of Bacterial Virulence Genes That Are Specifically lnduced in Host Tissues," Science, 259, pp 686-688 (1993)]. However, this system may not be applied to pathogenic orgalllslns 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 me~h~icm.s 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 medi~lm Selective in vivo expression of some B. burgdorferi proteins may be one reason that current diagnostic tests for Lyme disease, based on whole-cell Iysates of cultured B. burgdorferi, are unreliable. Such tests cannot detect antibodies directed toward the in vivo expressed antigens. Accordingly, there also CA 022~3834 1998-ll-06 W 097/42325 PCT~US96/06610 exists a need to identify B. burgdorferi proteins that provide more reliable diagnostic tests for Lyme disease.
Recently, imm-mi7~tion of mice with recombinant OspA has been shown to be effective to confer long-lasting protection against subsequent infection with B. bz ,~dorJre, i [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 imm--nogens used to date appears to be somewhat strain specific, probably due to the heterogeneity of the OspA gene among dirrele,ll B. burgdorferi isolates. For example, immlmi7:~tion with OspA
1 C from B. burgdorferi strain N40 confers protection against subsequent infection with strains N40, B31 and CD16, but not against strain 25015 ~E. Fikrig et al., "Borrelia burgdorferi Strain 2501 ~: Characterization of Outer Surface Protein A and Vaccination Against Infection", J. Immun., 148, pp. 2256-60 (1992)].
Tmmlmi7~tion 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 Tmmllnity to Lyme Borreliosis in Laboratory Mice", Infec. Immun., 60, pp. 657-61 (1992)]. Moreover, some B. burgdorferi are appare,l~ly able to escape destruction in OspB-imm-mi7edmice via a mutation in the OspB gene which results in expression of a truncated OspB protein [E. Fikrig et al., "Evasion of Protective Immllnity 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 immlmi7.~tion with this protein appears to be only partial ~V. Preac-Mursic et al., 25 "Active Tmmlmi7:~tion with pC Protein of Borrelia burgdorferi Protects Gerbils against B. burgdorferi Infection", Infection, 20, pp. 342-48 (1992)].
Tmmlmi7~tion with OspF has also been shown to confer partial protection against infection [T. K. Nguyen et al., "Partial Destruction of Borrelia W097/42325 PCTrUS96/06610 burgdorferi Within Ticks That Engorged On OspE- Or OspF-lmml-ni~ed 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., ~].
As prevention of tick infestation is imperfect, and Lyme disease may be missed or mi~ gnosed when it does appear, there exists a contin-ling urgent need for the delellninalion of additional antigens of B. burgdorferi and relatedproteins which are able to elicit a protective imm~me response and which may be useful in a broad-spectrum vaccine. In addition, identification of additional B.lC burgdorferi antipen~ may enable the development of more reliable diagnostic reagents which are useful in various stages of Lyme borreliosis.
DISCLOSURE OF T~lF INVENTION
The present invention provides novel B. burgdorferi polypeptides which are substantially free of a B. burgdorferi spirochete or fr~gmçnts thereof and, thus, are useful in compositions and methods for the diagnosis, tre~tment and prevention of B. burgdorfeff infection and Lyme disease. In one embodiment, thisinvention provides P2 1 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.
CA 022~3834 1998-ll-06 W097/42325 PCT~US96/06610 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 plefelled polypeptides of each of the aforementioned embodiments are selectively expressed in vivo.
Also p, t;~lled are compositions and methods of each of the o 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 im mllnr~genic 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 andmethods are useful for the detection of Lyme disease and B. burgdorferi infection.
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 po}ypeptides.
In yet another embodiment, this invention provides methods for 5 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 10 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.
BRTFF DESCRIPTION OF THE DRAWINGS
Figure I depicts the DNA and amino acid sequences of the P2 1 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 P3 7 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 2 5 polypeptide of B. burgdorferi strain N40.
Figure 6 depicts the hydrophilicity profiles of P35 and P37.
W 097142325 PCTrUS96/06610 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 ~ slalion among the DNA sequences encoding P2 1 and K2 and the DNA
5 sequences of other known B. burgdorferi outer surface proteins.
DETAILED DFSCRIPTION OF T~F. 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 co-llposilions and methods comprising those polypeptides.
In another embodiment, this invention relates to K2 polypeptides and 15 compositions and methods culllplis;l1g 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 embodiment1 this invention relates to M30 polypeptides and compositions and methods COlll,ul;S;llg 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 2~ and methods colllplisil1g those polypeptides.
In another embodiment, this invention relates to J2 and compositions and methods comprising those polypeptides.
., . . . . _ .
CA 022~3834 1998-11-06 W097/42325 PCT~US96/06610 The pl e~e,l ed 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 CO~ JI ish~g 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 organi~lns.
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 l~nintentional infection or cont~min~tion with undesired B. burgdorferi components. Accordingly, the novel B. burgdorferi polypeptides of this invention are particularly advantageous in compositions andmethods 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, in~.h]ding to detect the presence of B. burgdorferi, to screen forexpression 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.
CA 022~3834 1998-ll-06 W 097/42325 PCT~US96/06610 In yet another embodim~nt, 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 "im mllnQgenic B. burgdorferi polypeptide" is any B. burgdorferi polypeptide that, when ~dministered to an animal, is capable of eliciting an irnmune response.
Immunogenic B. burgdorferi polypeptides are int~n-led 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 fr~gm~nts, serotypic variants and derivatives of any of the above. ~n particular, immllnl genic B. burgdorferi polypeptides are intl~n~led to 5 include additional B. burgdorferi polypeptides which are identified according to the rnethods disclosed herein.
As used herein, a polypeptide which is "substantially free of a B.
burgdorferi spirochete or fragm~nt~ thereof'' is a polypeptide that, when introduced into modified Barbour-Stoener-Kelly (BSK-II) medium and cultured at 37~C for 7 2 o days, fails to produce any B. burgdorferi spirochetes detect~hle by dark field microscopy or a polypeptide that is detectable as a single band on an immunoblotprobed with polyclonal anti-B. burgdorferi anti-serum.
As used herein, a B. burgdorferi polypeptide that is "selectively expressed in ~ivo" is a polypeptide encoded by a DNA sequence that corresponds 2 5 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 CA 022~3834 1998-11-06 WO 97/42325 PCT/U~G/OG~10 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 con~;sLing of:
(a) a P21 polypeptide consisting of amino acids 1-182 of SEQ ID NO: 2;
(b) fr~gmçnt~ 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 P2 1 polypeptide of (a), said derivative being 10 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 m~mm~ n host with B. burgdorferi, which antibodies are immunologically reactive with a P2 1 polypeptide of (a)~
(3) polypeptides that are capable of eliciting antibodies that are irnmunologically reactive with B. burgdorferi and the P2 1 polypeptide of (a) and (4) polypeptides that are immunologically reactive with antibodies elicited by i~nmllni~:~tion with the P21 polypeptide of(a).
As used herein, a "K2 polypeptide" denotes a polypeptide which is 20 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 25 SEQ I:D 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), CA 022~3834 1998-11-06 W097/42325 PCTrUS96/06610 (2) polypeptides that are immllnl~logically reactive with antibodies generated by infection of a m~mm~ n host with B. burgdorferi, which antibodies are immlmologically reactive with a polypeptide of (a);
(3) polypeptides that are capable of çli~iting antibodies that are imm--nologically reactive with B. burgdorferi and the polypeptide of (a); and (4) polypeptides that are imml~nologically reactive with antibodies elicited by immllni7~tion with the polypeptide of (a).
As used herein, a "P35 polypeptide" denotes a polypeptide which is s~lected from the group consicting of:
(a) a P35 protein comprising the amino acid sequence set forth in SEQ ID
NO: 5 and serotypic variants thereof;
(b) fr~gm~nts comprising at least 8 amino acids taken as a block from the P35 polypeptide of (a);
(c) derivatives of the P3 5 polypeptide of (a) or (b), said derivatives being atleast 80% id~ntic~l 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 m~mm~ n host with B. burgdorferi which antibodies are immlmQlogically reactive with a P35 polypeptide of (a) or (b) or (c);
2 o (e) polypeptides that are capable of eliciting antibodies that are immlm- logically reactive with ~. burgdorferi and the P3 5 polypeptide of (a) or (b) or (c); and (f) polypeptides that are immunologically reactive with antibodies elicited by immllni7:~tion 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;
.
CA 022~3834 1998-ll-06 5 PCT~US96/06610 (b) fr~ement~ 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 seq~]ence to the corresponding polypeptide of (a) or (b);
(d) polypeptides that are immunologically reactive with antibodies generated by infection of a m~mm~ n 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 immllnologically reactive with B. burgdorferi and the P37 polypeptide of (a) or (b) or (c); and (f) polypeptides that are immunologically reactive with antibodies elicited by immuni7~tion with the P35 polypeptide of (a) or (b) or (c).
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 M3 0 polypeptide of (a) or (b), said derivatives being atleast 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 m~mm~ n 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 CA 022~3834 1998-11-06 (f~ polypeptides that are immunQlogically reactive with antibodies elicited by imml-ni7~tion 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 m~mm~ n host with B. burgdorferi, which antibodies are imrnunologically reactive with a polypeptide of (a) or (b) or (c);
(e) polypeptides that are capable of eliciting antibodies that are imrnunologically reactive with B. burgdorferi and the polypeptide of (a) or (b) or (c); and (f) polypeptides that are immunologically reactive with antibodies elicited by imml~ni7~ion with the polypeptide of (a) or (b) or (c) 2c 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 2 5 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 ~ and serotypic variants thereof;
CA 022~3834 1998-11-06 W 097/42325 PCTrUS96/06610 (b) fr~gment~ comprising at lezst 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% idçntic~l in amino acid sequence to the corresponding polypeptide of (a) or (b);
(d) polypeptides that are imml-nologically reactive with antibodies generated by infection of a m~mm~ n host with B. burgdorferi, which antibodies are irnmllnologically 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 immllnologically reactive with antibodies elicited by immllni7~tion with the polypeptide of (a) or (b) or (c).
As used herein, a "Jl 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 'IJ2 polypeptide" denotes a polypeptide which is selected from the group consisting of:
2 o (a) a polypeptide encoded in whole or in part by the B. burgdorferi DNA
sequence contained within ATCC deposit No. ~ and serotypic variants thereof;
(b) fr~m~nt~ 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 2 5 80% identical in amino acid sequence to the corresponding polypeptide of (a) or (b), CA 022~3834 l998-ll-06 W097/4232S PCTrUS96/06610 (d) polypeptides that are immunologically reactive with antibodies generated by infection of a m~mm~ n host with B. bu~d~"~eri, which antibodies are imml]nologically 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 imml~ni7~tion with the polypeptide of (a) or (b) or (c).
As used herein, a "J2 polypeptide" is intçn-led 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 seq~l~nce contained within ATCC deposit No.
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 ormore physical, chemical, or biological pl opel Lies has been altered. Such modifications include, but are not limited to: amino acid substitutions, CA 022=,3834 1998-ll-06 WO97/4232~ PCTrUS96/06610 _ 19 _ modifications, additions or deletions; alterations in the pattern of lipidation,glycosylation or phosphorylation; reactions of free amino, carboxyl, or hydroxylside 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 10 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 immllni7e an animal, elicits an immune response sufficient to prevent or lessen the severity for some period of time, of B. burgdorferi infection.
Preventing or lessçning 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 pres~nting cells, results in a T cell response such as clonal expansion or expression of iymphokines or other immlmnstimulatory molecules. A T cell epitope may also be an epitope recognized by cytotoxic T cells that may affect intr~cf~ r B. burgdorferi infection. A strong T cell epitope is a T cell epitope which elicits a strong T cell response.
CA 022~3834 1998-ll-06 W097/42325 PCTrUS96/06610 As used herein, a "B cell epitope" is the simplest spatial co,~ll,laLion 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 arnount that, when admini~tered 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 10 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 m~mm~ n host with B. burgdorferi. Accordingly, they are useful in methods and compositions todiagnose and protect against Lyme disease, and in therapeutic compositions to stim~ te 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. hurgdorferi, they are particularly useful in a multicomponent vaccine against Lyme disease, because such a vaccine may be formlll~ted 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.
CA 022~3834 1998-11-06 W097/42325 PCT~US96/06610 burgdorferi polypeptide. Multicomponent vaccines according to this invention mayalso contain polypeptides which characterize other vaccines useful for immlmi7~tion against ~lise~c other than Lyme disease such as, for ~,Aalllplc, diphtheria, polio, hep~titi~, and me~les. Such multicomponent vaccines are typically incorporated into a single composition.
The plt;felled compositions and methods ofthis invention comprise novel B. burgdorferi polypeptides having enh~n-,ed immunogenicity. Such polypeptides may result when the native forms of the polypeptides or fra~ments thereof are modified or subjected to lr~ ,l.c to enhance their imml~nQgenic character in the int~.n-~ed recipient.
Numerous techniques are available and well known to those of skill in the art which may be used, without undue experimçnt~tion, to substantially increase the imm~lnogenicity of the novel B. burgdorferi polypeptides herein disclosed. For example, the polypeptides may be modifled by coupling to dinitrophenol groups or arsanilic acid, or by denaturation with heat and/or SDS.Particularly if the polypeptides are small polypeptides synthe~i7ed 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 M~n~l~l Cold Spring Harbor Laboratory, ed. E.
Harlow and D. Lane (1988). Useful immllnogenic 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 25 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 imm~lnogenicity and biochemical properties.
CA 02253834 1998-ll-06 WO 97t42325 PCT~US96/06610 For example, the polypeptides or fraEnl~nt~ thereof may be expressed with or without the signal sequences that direct addition of lipid moieties.
As will be appalenl from the disclosure to follow, the polypeptides may also be prepared with the objective of increasing stability or rendering themolecules more amenable to purification and prepa, ~lion. 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 prepal ed by a variety of methods, inrhl-ling by in lo vitro manipulation of the DNA encoding the native polypeptides and subse~uent 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 di~enl natural amino acid, an amino acid derivative or non-native amino acid, conservative substitution being plef~lled, e.g, 3-methylhistidine may be substituted for histidine, 4-hydroxyproline may be substituted for proline, S-hydroxylysine may be substituted for Iysine, 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 agiven 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.
WO g7142325 PCTrUS96/06610 In a p.~re,~ed embodiment of this invention, the novel B.
burgdorferi polypeptides disclosed herein are prepared as part of a larger fusion protein. For e,~a.llple, a novel B. burgdorferi polypeptide of this invention may be fused at its N-terminus or C-terminus to a di~el ~nL immunogenic B. burgdorferi polypeptide, to a non-B. burgdorferi polypeptide or to combinations thereof, to produce fusion proteins COI~l~)l ising 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 di~ering from another with respect to the locations or sequences of the epitopes within the polypeptide. In a more pl~re.. ~d embodiment, fusion proteins are constructed which comprise one or more of the novel B. burgdorferi polypeptides fused to other immllnogenic 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. burgdor~eri 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 thepolypeptide. 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 te~ching~ 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 te~ching~ herein or by other methods known to those of skill in the art.
It is preferred that the fusion proteins comprising the novel ~.
burgdorferi polypeptides be produced at the DNA level, e.g., by constructing a nucleic acid molecule encoding the fusion, transforming host cells with the CA 022~3834 1998-ll-06 W097/42325 PCTrUS96/06610 molecule, ind~lcing 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 t;..l,ression accolding to known methods The novel B. burgdorferi polypeptides may also be part of larger 5 mllltim~ric molecules which may be produced recombinantly or may be synthesized chemically. Such mllltimers may also include the polypeptides fused or coupled to moieties other than amino acids, incllldin~ 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 10 randomly, or with spacers (amino acid or otherwise) between them.
In the most p- ~re. I ed embodiment of this invention, the novel B.
burgdorferi polypeptides of this invention which are also immunQgenic B.
burgdorferi polypeptides are incorporated into a multicomponent vaccine which also comprises other immllnogenic B. burgdorferi polypeptides. Such a 5 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 di~erenl 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 2 o 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. burgdorferipolypeptide and one previously identified B. burgdorferi polypeptide, wherein each 2 5 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 WO 97/42325 PCTrUS96/06610 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 imm-ln~globulin light chain, and could be produced by transforming a host cell with DNA encoding the heavy chain filsion 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 appropliately.
Alternatively, the heavy and light chain filsions could be produced from separate cell lines and allowed to associate after purification.
The desirability of inchlAinE 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 immlmogenic B. burgdorferi polypeptides, including the novel B.
b2~rgdorferi 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 ~dmini~tered to an animal via a liposome delivery system in order to enh~nce their stability and/or immllnQgenicity Delivery of the novel B. burgdorferi polypeptides via liposomes may be particularly advantageousbecause the liposome may be intemalized by phagocytic cells in the treated animal.
Such cells, upon ingesting the liposome, would digest the liposomal n~elllbl~le 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 llnil~mellat vesicles, multilamellar vesicles, or stable plurilamell~r vesicles, and may be prepared and aAmini.~tered according to methods well known to those of skill in the art, for example in accordance with the teaching~ 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 CA 022~3834 1998-ll-06 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 5 be used in the form of a pharm~ceutic~lly 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 polypeptidesthat are pl ere" ~d 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 20 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 mllltim~ric proteins that are pl~f~l,ed 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 prerelled polypeptides, fusion proteins and multimeric proteins for use in these compositions and methods are those cont~inin~ both strong T cell and B cell epitopes. Without being bound by theory, we believe that this is the best way to stim~ te high titer antibodies that are effective to neutralize . . .
CA 022~3834 1998-11-06 W097/42325 PCT~US96/06610 B. burgdorferi infection. Such prer~l I ed 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 ~ cells will recognize the T cell epitope(s) and respond by proliferating and producing Iymphokines which in turn cause B cells to di~l ~lltiate into antibody producingplasma 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 incllldes high titer antibodies against the novel B. burgdorferi polypeptide.
One of skill in the art will also understand that it may be advantageous to ~(lminister the novel B. burgdorferi polypeptides of this invention in a form that will favor the production of T-helper cells type 2(TH2), which help B
cells to generate antibody responses. Aside from ~(lmini.ctering epitopes which are strong B cell epitopes, the induction of TH2 cells may also be favored by the mode of ~dmini.~tration ofthe polypeptide for example by admini~tering in certain doses or with particular adjuvants and imm~n~modulators, for example with interleukin-4.
To prepare the pr~r~lled polypeptides ofthis invention, in one embodiment, overlapping fr~gmPnts 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 ( I ) 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 immuni7e 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.
CA 02253834 1998-ll-06 W O97/42325 PCTrUS96/06610 As recognition of T cell epitopes is MHC restricted, the polypeptides that contain T cell epitopes may be identified in vi~ro by testing them for their ability to stim.ll~te proliferation and/or cytokine production by T cell clones generated from humans of various HLA types, from the Iymph nodes, spleens, or peripheral blood Iymphocytes of C3H/He mice, or from domestic animals. Compositions comprising multiple T cell epitopes recognized by individuals with di~elen~ Class II
~ntigçn.c are useful for prevention and tre~tment of Lyme disease in a broad spectrum of patients.
In a pl ~re~l ~d embodiment of the present invention, a novel B.
lo burgdorferi polypeptide cont~ining a B cell epitope is fused to one or more other im m~nogenic 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. burgdorfeff.
Strong T cell epitopes may also be provided by non-B. burgdorferi molecules. For example, strong T cell epitopes have been observed in hepatitis Bvirus core antigen (HBcAg). Furthermore, it has been shown that linkage of one of these egm~nts to segments of the surface antigen of Hepatitis B virus, which arepoorly 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 segment.~ 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 CA 022~3834 1998-11-06 W 097/4232S PCT~US96/06610 invention to a strong immlln~ gen that is also widely recognized, for example tetanus toxoid.
It will be readily appl e~;ated by one of ordinary skill in the art that the novel B. burgdorferi polypeptides of this invention, as well as fusion proteins 5 and mllltim~ric proteins col~ e them, may be prepared by recombinant means, ~h~mic~l meàns, 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 primersderived from the sequences herein disclosed.
In this regard, it may be particularly desirable to isolate the genes encoding novel B. burgdorferi polypeptides from strain 2501 S 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. burgdorferistrain 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 infectionwith strain N40, appear ineffective to protect against infection with strain 25015.
2 o 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 relatedspirochetes 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. burgd~orferi polypeptides of this invention are produced recombinantly, they may be expressed in unicellular hosts. As is well W097/42325 PCTrUS96106610 known to one of skill in the art, in order to obtain high t;A~ures~ion 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. Pler~l~bly, the eA,u~es~ion control sequences, and the gene of 5 interest, will be contained in an e ,~pression vector that further comprises a selection marker.
The DNA sequences encoding the polypeptides of this invention may or may not encode a signal sequPnce. If the expression host is eukaryotic, it generally is pl erel l ed 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 teç~ln~ es A wide variety of expression host/vector combinations may be employed in ~A~,.essillg 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, inc~ 1in~ pBluescript, pGEX-2T, pUC vectors, col E1, pCRl, pBR322, 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. AGT10 and ~GT11, and other phages. Useful expression vectors for yeast cells include the 2plasmid 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 ,, CA 022~3834 1998-11-06 W097/42325 PCT~US96/06610 invention. Such useful eAI)I ession control sequences include the ~I.ression control sequen~es associated with structural genes of the foregoing eApression vectors.
Examples of useful expression control sequences include, for example, the early and late promoters of SV40 or adenovin~s, the 1~ system, the ~ system, the ~ or ~ 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 phosph~t~.ce, e.g., Pho5, the promoters of the yeast a-mating system and other constitutive and inducible promoter sequences known to control the eAI.)I es~ion of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
ln a preferred embodiment, DNA sequences encoding the novel B.
burgdorferi polypeptides of this invention are cloned in the expression vector lambda ZAP II (Str~t~g~ne, 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 e~pl ession vector that allows high level ~,~p,ession 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 c"~ressi,lg the DNA sequences of this invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, 2 5 Streptomyces, fungi, yeast, insect cells such as Spodoptera frugiperda (SF~), 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.
CA 022~3834 1998-ll-06 W097t4232S PCT~US96/06610 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 e~ es~ion system. However, one of skill in the art may make a selection among these vectors, e,~l)ression control sequences and hosts ~vithout undue expelh,l~lltalion 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 t~AI~l es~ion 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
seql1ences 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 conventionalmethods including: liquid chromatography such as normal or reversed phase, usingHPLC, FPLC and the like; affinity chromatography (such as with inorganic ligandsor monoclonal antibodies); size exclusion chromatography; immobilized metal CA 022~3834 1998-11-06 W 097/42325 PCTrUS96/06610 chelate chromatography; gel electrophoresis; and the like. One of skill in the art may select the most ap?rop-iate 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 chernical techni~ues. For example, they may be prepared using the solid-phase synthetic techni~ue 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 prepa-ed by synthesis in solution. A
summary of peptide synthesis techniques may be found in E. Gross & H. J.
1C Meinhofer, 4 The Peptides: Analysis, Synthesis, Biology; Modern Techniques OfPeptide And Arnino Acid Analysis, John Wiley & Sons, (1981) and M. Bodanszky, Principles Of Peptide Synthesis, Springer-Verlag (1984).
Typically, these synthetic methods comprise the sequential addition of one or more amino acid residues to a growing peptide chain. Often peptide 5 coupling agents are used to facilit~te 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 .ii~lel,l protecting group is utilized for amino acids cont~ining 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 immllnoglobulin molecules or portions thereofthat are im~nunologically reactive with a novel B. burgdorferi polypeptide of the presentinvention. It should be understood that the antibodies of this invention include CA 022~3834 1998-ll-06 W097/42325 PC~rUS96/06610 antibodies immunologically reactive with fusion proteins and mllltimPric proteins co~ ,is;ilg a novel B. burgdorferi polypeptide.
Antibodies directed against a novel B. burgdorferi polypeptide may be generated by a variety of means in~.lu~line infection of a m~mm~ n host with B. burgdorferi, or by immuni7:~tion of a m~mm~ n host with a novel B.
burgdorferi polypeptide of the present invention. Such antibodies may be polyclonal or monoclonal, it is p~ ~rel I ~d that they are monoclonal. Methods to produce polyclonal and monoclonal antibodies are well known to those of skill inthe art. For a review of such methods, see An~ibodies, A Laborafo~ Manual, lo supra, and D.E. Yelton, et al., Ann. Rev. of Biochem.~ 50, pp. 657-80 (19~1).
Determination of immllnnreactivity with a novel B. burgdorferi polypeptide of this invention may be made by any of several methods well known in the art, includingby immllnoblot assay and ELISA.
An antibody of this invention may also be a hybrid molecule formed from imm-mclglobulin sequences from di~erel.L species (e.g., mouse and human) orfrom portions of immllnoglobulin light and heavy chain sequences from the same species. It may be a molecule that has multiple binding specificities, such as abifunctional 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.
W097/42325 PCT/U'53.'~GC10 ln 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 te~çhin~.c of this 5 invention, has available a variety of methods which may be used to alter the biological p~upel~ies ofthe antibodies ofthis invention inrlllding methods whichwould 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 infectiûn. For example, the level of B. burgdorferi in infected ticks may be decreased by allowing them to feed on the blood of animals immllni7ed with the 5 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 2 0 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 2 5 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 prerel ~ ~d 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.
CA 022~3834 1998-ll-06 WO 97/42325 PCT~US96/06610 burgdorferi infection but are also afflicted with clinical symptoms of a disease that is remarkably similar to Lyme disease in humans. Thus, by a~mini~tering a particular polypeptide or antibody to C3H/He mice, one of skill in the art may determine without undue e~cli~ne~ tion whether that polypeptide or antibody 5 would be useful in the methods and compositions claimed herein.
The a~imini~tration 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 ~timinictered with a pharmaceutically acceptable adjuvant, such as complete or incomplete Freund's adjuvant, RIBI
(muramyl dipeptides) or ISCOM (immunostim~ ting complexes). Such adjuvants may protect the polypeptide from rapid dispersal by sequestering it in a local deposit, or they may contain substances that stim~ te 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 immnni7~tion schedule will involve two or more ~mini.~trations 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 pharrnaceutical 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 tablels, pills, powders, liquid solutions or suspensions, liposomes, capsules, suppositories, injectable and infusible solutions.
CA 02253834 1998-ll-06 The pleÇ~Iled form depends upon the intçn-1ed mode of atlmini~tration and prophylactic application.
Such dosage forms may include pharm~cel~tically acceptable carriers and adjuvants which are known to those of skill in the art. These carriers and adjuvants include, for exarnple, RIBI, ISCOM, ion exchangers, all-min~ lminl]m 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 prota,llllle sulfate, disodium hydrogen phosphate, sodium chloride, zinc salts, 10 colloidal silica, m~ene~ium tri~ilicate, 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 glycol1 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 enhancetheir immunogenic character or to improve their tolerance in patients.
Compositions comprising an antibody of this invention may be a~minictçred by a variety of dosage forms and regimen.c similar to those used for 2 o other passive immunotherapies and well known to those of skill in the art.
Generally, the novel B. burgdorferi polypeptides may be formul~ted and a~mini.ct~red 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, intr~mll~c~ r, intralesional or subcutaneous injection, may be used to a(lmini.~ter the polypeptide or antibody composition. For example, the composition may be arlminictered to the patient in any pharmaceutically acceptable dosage form CA 022=,3834 1998-ll-06 in~.lutline those which may be ~(lminictered to a patient intravenously as bolus or by continued infusion over a period of hours, days, weeks or months, intramllcc~ rly -- incl~lrling paravertebrally and periarticularly -- subcutaneously, intr~c.ut~neously, 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 a~lminictered to the patient intr~m-lscul~rly.
The novel B. burgdorferi polypeptides or antibodies of this invention may be a~mini.ctçred to the patient at one time or over a series of Lle~l~,.e~,lc. The 1 c most effective mode of a~l".lnic~ ion and dosage regimen will depend upon the level of immunogenicity, the particular composition and/or adjuvant used for tre~tm~nt, the severity and course of the expected infection, previous therapy, the patient's health status and response to imm~ni7.~tion~ 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 immlmi7~tions Similarly, the dosage and necçcs~ry treatment time will be loweredif the polypeptide is ~flmini.ct~?red with an adjuvant. Generally, the dosage will consist of 10 ,ug to 100 mg of the purified polypeptide~ and preferably, the dosage will consist of 10-1000 ~lg. Generally, the dosage for an antibody will be 0.5 mg-3.0g.
In a p,~rel led embodiment of this invention, the novel B.
burgdorferi polypeptide is ~r~minictered with an adjuvant, in order to increase its imm~ln~,genicity. Useful adjuvants include RIBI, and ISCOM, simple metal salts such as al--min~lm hydroxide. and oil based adjuvants such as complete and incomplete Freund's adjuvant. When an oil based adjuvant is used, the polypeptide usually is a-lminictered in an emulsion with the adjuvant.
In yet another preferred embodiment, E.coli expressing proteins comprising a novel B. burgdorferi polypeptide are at~mini~tered orally to non-CA 022~3834 1998-11-06 W 097/42325 PCTrUS96/06610 human animals to decrease or lessen the severity of B. burgdorferi infection. For example, a palatable regimen of bacteria c, I~l essh~g a novel B. burgdorferi polypeptide, alone or in the form of a fusion protein or multim~ric protein, may be ~tlminictered with animal food to be consumed by wild mice or deer, or by domestic animals. Ingestion of such bacteria may induce an immllne response comprising both humoral and cell-me~i~ted components. See J.C. Sadoffet al., "Oral Salmonella ~yphimurium Vaccine Expressing Circumsporozoite Protein Protects Against Malaria", Science. 240,pp.336-38 (1988) and K.S. Kim et al., "Tmmllni7~ion Of Chickens With Live Esc*erichia coli E~pres~ing Eimeria acervulina Merozoite Recombinant Antigen Induces Partial Protection Against Coccidiosis", Inf. Immun., 57, pp. 2434-40 (1989). In fact, oral vaccination with bacteria ~ ,ressing OspA has been shown to be effective. See, M. Dunne et al., "Oral Vaccination Against Lyme Disease Using Salmonella Expressing OspA," I~
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.
Dis., 164:1224(1991). Moreover, the level of B. burgdorferi infection in ticks feeding on such animals will be lessened or eliminated, thus inhibiting tr~n~mis~ion 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 ~nim~l~ inçlu~ing humans that are infected with B. burgdorferi, and the antibodies are capable of binding to B. burgdorferi or 25 ~nti~n~ 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 CA 022~3834 1998-ll-06 W097/42325 PCTrUS96/06610 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 I or Cr that produces gam na ray emissions, or a radioactive element that emits positrons which produce gamma rays upon encounters with electrons present in the test solution, such as C, O, or 13N.
10 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-cont~ining amino acids in the culture m~-1inml 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 solutionform or as a substantially dry powder~ e.g., in Iyophilized form.
Novel B. burgdorferi polypeptides and antibodies directed against those polypeptides provide much more specific diagnostic reagents than whole CA 022~3834 1998-ll-06 W 097/4232S PCT~US96/06610 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 theinfected 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 Iysates 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, inclur~ing 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 P2 1 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 cont~ining multiple epitopes which are reactive with antibodies appearing at di~Terenl times are useful to detect the presence of anti-B. 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 other2 5 spirochetes include Borrelia Hermsii and Borrelia Recurientis, I,ep~ospira, and Treponema.
According to another embodiment of this invention, we describe a method for identifying bacterial genes encoding an antigenic proteins that are CA 022~3834 1998-ll-06 W097t42325 PCT~US96/06610 expressed during infection of a host but that are not expressed during in vitro culture of the bacteria, the method co~ ,-ish~g the steps of:
(a) constructing an e~ ess.on library for the bacteria;
(b) SCIeel~~ng the library with antisera from an animal infected with the bacteria;
(c) screening the library with antisera from an animal immlmi7ed with non-viable bacteria or components thereof; and (d) identifying clones that react with the first antisera but not with the second antisera.
1C It will be readily appare-ll 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, inchl~ling plant and animal hosts. In a ple~el-ed embodiment, the host is a m~mm~l 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.
CA 022~3834 1998-11-06 W097/42325 PCTrUS96/06610 As used herein, components of non-viable bacteria include Iysates, homogen~tes, or subcellular fractions thereof such as cell membrane cont~ining 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 libr~y 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 20 by centrifugation at 16,000 rpm for 30 minlltes~ and Iysed 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 llg of DNA was randomly sheared, blunt-ended with S 1 nuclease, and the EcoRI sites were methylated with EcoRl methylase. EcoRl linkers were then ligated to the ends of the DNA molecules, theDNA was digested with EcoRl and the fr~gm~ntc were purified over a sucrose CA 022~3834 1998-11-06 W097/42325 PCTAUS96tO6610 gradient. Fragments of 1 to 9 kb were isolated and ligated to EcoR1 ~igested Lambda ZAP II arms.
We prepared E. coli SU~ bacteria (Stratagene) for phage infection as follows. We picked a single colony into LB media suppl~mçnted with 0.2%
5 maltose and 10 mM magnesium sulfate and cultured overnight at 30~C with vigorous ~h~king 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 1 n We prepared anti-B. burgdorferi N40 antisera for differentially screening the expression library as follows.
1 . Tmml Ine Anti~ra We prepared "immllne" mouse anti-B. burgdorferi N40 antiserum as follows. We injected 3 to 5 groups of five three-week old female C3h/HeNCr or J
5 (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.
20 b2lrgdorferi in incomplete Freund's adjuvant (rFA) 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. coli/phage Iysate (Stratagene) 2 5 as follows. We diluted the Iysate 1 :10 in Tris-buffered saline with 0. 05% Tween-20 (TBST). We then incubated 0.45 ~M pore size nitrocellulose filters (Millipore, Bedford, MA) in the Iysate for 30 minutes at room temperature, removed and air . _ CA 02253834 1998-ll-06 W O97/42325 PCT~US96/06610 dried the filters on Whatman filter paper (Whatman International Ltd., Maidstone, F.n~1~nd), and washed 3 times (5 minutes each) with TBS. We blocked the filters by irnmersing in 1% Bovine Serum Albumin (BSA) in TBS for I hour at room te.n,~elal~lre and rinsing 3 times with TBST. We then diluted the mouse antiserum 5 1:5 in TBST, incubated it with the filters with shaking for 10 minutes at 37CC, and removed and discarded the filters.
2. lnfected Antisera We injected three C3H/HeJ mice by intradermal inoculation with 104 B. burgdorferi N40 spirochetes. We docum~nted infection by culturing spirochetesfrom the spleen, bladder and skin (ear punches) of the challenged mice and by histopathologic examination of the joints and heart for evidence of infl~mm~tionWe collected serum from the infected mice at various times a~er infection Both immlln~ and infected antisera contained a high titer of anti-B.
burgdorferi antibodies directed against whole cell Iysates. We detected antibodies in the sera of immllni7ed 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 difre, e.
intensities.
After absorption, we diluted the antiserum to a final dilution of 1:100 and used it to screen the nitrocellulose filters cont~ininE the expressed proteins from the lambda ZAP library according to m~nuf~ctllrer's instructions.
C. Differential Screening of A Genomic B. burgdorferi N40 Expression Library To screen the library, we used the picoBlue Immunoscreening Kit (Stratagene). We plated 4 x 104 plaque forming units of recombinant phage on a lawn of bacteria, induced protein expression with 1 OmM IPTG and transferred the CA 022~3834 1998-ll-06 W O97142325 PCTrUS96/06610 proteins to duplicate plaque lifts on nitrocellulose filters according to methods well known in the art.
We incllb~ed one set of plaque lifts with pooled sera from mice imml-ni7ed with heat-killed spirochetes (immllne sera) and the other set with sera from rnice infected for nine months (infected sera). A~er 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 10 infected sera but not with immune sera for further study.
Example II - Cloning ofthep21/k.? Operon Di~l e:nLial screening of a B. burgdorferi N40 genomic ~Ayression library, as described in Example I, revealed one hundred seventy-two clones thatreacted with sera from the infected mice and one hundred sixty-nine clones that reacted with sera from immllni7ed mice. We subjected the three phage clones thatreacted di~l en~ially 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 m~nllf~ctllrer'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
2~ insert of clone I with the Erase-A-Base System (Promega, Madison, WI) (using SmaI 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, WO 97/42325 PCT~US96/06610 C~leveland, OH) and recolls~ cted the entire sequence using MacVector (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 Fnel~n~l Biolabs). Conditions for denaturation, ~nne~ling 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 cont~ining a complete open reading frame and a partial open reading frame having the sequ~nce set forth in SEQ ID NO: 1. We conducted a 10 search of GenBank (December 1994) with the Genetics Computer Group Program (University of Wisconsin Biotechnology Center, Madison, W~). Our search revealed that we had isolated a novel, bicistronic B. burgdorferi operon. We de~ign~ted the complete open reading framep21 and the partial open reading framek2. We d~sign~ted the ~ntig~ni 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, thep21 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 ded~lced amino acid seqllence of P2]
2 o contains a typical prokaryotic signal sequence for posttranslational processing by cleavage and lipidation, s~ggesting that the gene product is a lipoprotein of apl)lox""ately 20.7 kDa. P21 has 71% amino acid sequence identity to B.
burgdorferi OspE (Figure 7).
The ATG start codon for the k2 gene is located 27 nucleotides downstream of the TAG stop codon of the p21 gene. The k2 gene in clone I
contains a partial open reading frame of 32 nucleotides, capable of encoding thefirst 10 amino terminal amino acids (SEQ ID NO: 3). However, based on the last CA 02253834 1998-ll-06 W 097/42325 PCTrUS96/06610 two nucleotides of the K2 sequ~nr,e 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 11-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 up~Llea"~ ofthis translational initiation sequence are the promoter segments 10 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 I I bp upstream of the ATG start codon of the k2 gene. The location of these sequence elements suggests that both thep21 and k2 genes are controlled by a single promoter. The homology of P2 1 and K2 toOspE 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 arnino 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 Microbiolo~y and Immunology, 125, pp. 127-157 (1986)].
CA 022~3834 1998-11-06 WO 97/42325 PCT~US96/06610 The carboxyl terminus of the hydrophobic domain contains a cleavage site presumably recognized by a B. burgdorferi signal peptidase. In P2 1, as in OspF, the potential cleavage site is located between Serl7 and Cysl8.
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. Wu et al., supra~ Indeed, the OspA and OspB genes of B. burgdorferi B3 1 contain signal sequences of -L-I-A-C- and -L-I-G-C-, respectively [S. Bel g~ll o,n et al., "Molecular Analysis of Linear Plasmid-Encoded Major Surface Proteins, OspA and OspB, of the Lyme Disease SpirochaeteBorreliaburgdorferi", 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-B3 1 (-L-S-I-S-C-) genes, contains three amino acids between the leucine and cysteine instead oftwo. (See R.S. Fuchs et al. and S.~.
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 2 o residue could serve to anchor the protein to the outer surface of the spirochetes (see H.C. Wu and M. Tokunaga, supra).
Finally, P2 1 contains a long hydrophilic domain separated by short stretches of hydrophobic segments.
A comparison of the DNA sequences indicates that p21 and ospE
2 5 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 of p21 and ospE are identical upstream from the -10 sequence tothe boundary ofthe 5' fl~nkin~ DNA which has been sequenced (189 nt 5' ofthe CA 022~3834 1998-ll-06 - SO -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 di~el~nces 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' untran~l~ted region ofp21 mRNA.
In view of this homology between P2 1 and OspE, one of skill in the art would understand that in form~ ting therapeutic and diagnostic compositions, it may be desirable to select epitopes of P21 that do not cross-react with OspE.
1C Example IV - Analysis ofp21 Expression In Cultured B. burgdorJeri By Northern Blotting To determine whetherp21 is transcribed during in vitro culture of spirochetes, we assessed its ~ es~ion by Northern blot analysis. We isolated total RNA from cultured B. burgdorferi by acid guanidium thiocyanate/phenol/
chloroform extraction [cite]. We electrophoresed 20 llg of isolated RNA in a 1%
formaldehyde-agarose gel and blotted onto Hybond-N~ membrane (Amersham).
We generated biotinylated p21 and ospA (contro1) probes with a Phototope~
random-primer biotin-labeling kit (New England Biolabs). Thep21 and ospA
probes contained the entirep21 and ospA sequences, respectively. We used amplified PCR products ofp21 or ospA as templates for the random octamer-primed labeling reaction.
We condllctçd hybridization and signal detection with a Photope~
ch~mill-minescçnt kit (New Fn~l~nd Biolabs). Briefly, we prehybridized the blotted membrane in SSC for 1 hour at 68~ C and hybridized with biotinylated probes for p21 or osp,4 (control) at 68~ C overnight. We washed the membrane at a final stringency of 0.1X standard saline citrate (SSC)/0.1% SDS at 68~ C. We detected CA 02253834 1998-ll-06 WO 97/42325 PCTrUS96/06610 biotin-labeled probe by a series of inc~bations with streptavidin, biotinylated alkaline phosphatase, and lumigen-PPD.
We detected ospA RNA but nop21 RNA from cultured B.
burgdorferi.
Example V - Southern Dot Blot Analysis and PCR of Cultured ~. burgdorferi Genome Because in vitro culture of B. burgdorferi is often associated with the loss of genes or pl~smi(lc [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 ofthep21 gene A. Southern Dot Blot Analysis For dot blot analysis, we spotted 2 ~g of denatured 1 phage (control) or cultured B. burgdorferi DNA onto Hybond-N~) membrane. We first stained the dried membrane with ethi~illm bromide to confirrn 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, conrll llling the presenceof the p21 gene in the cultured B. burgdorferi .
B. PCR ~n~lysis 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: 11 and SEQ ID NO: 12, respectively. These primers are specific forp21 and do not amplify osp~ We used the following conditions for PCR of cultured B. burgdorferi DNA: 30 cycles with denaturing, ~nne~l;n~e and 2 5 extension temperatures of 94~ C for I min., 65 ~ C for 1 min., 72 ~ C for 2 min., respectively.
CA 022~3834 1998-ll-06 WO 97/42325 PCTrUS96/06610 Using these primers, we obtained a 513 bp PCR product ofthep21 coding region, further co~ lling that the p21 gene is present in the genome of the cultured B. burgdorferi used for Northern blot studies.
Example VI - E~min~tion of p21 Expression By B. burgdorferi In Ticks To determine whether P2 1 is expressed by B. burgdorferi in Ixodes ticks, we exarnined Iysates of flat and engorged ticks cont~ining the spirochetes by indirect immllnofluorescence. Using the same methods, one of skill in the art could readily deterrnine without undue experimçnt~tion whether other novel B.
lo burgdorferi polypeptides of this invention are expressed in ticks.
Briefly, we allowed B. burgdorferi N40-infected ticks to feed to repletion on C3HlHe mice. We lightly homogenized each unfed and engorged tick in 100 1ll PBS and spotted a 10 ~1 aliquot onto a sylilated glass slide. We air-dried the slides and fixed them with 4% pal~fo-l-laldehyde and saponin. We incubated the specimens in a 1:10 dilution of antisera from mice immllni7ed with the P21-specific peptide prepared as in Example VII and as shown in SEQ. ID NO:_, for I
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-fl~g~llin monoclonal antibody H9724, which recognizes B. burgdorferi in both flat and engorged ticlcs as a second positive control [Cite]. We used anti BSA sera as a negative control.
Con~ictçntly 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 CA 02253834 l998-ll-06 W 097/42325 PCT/U55~ 10 ticks. However, no P21-specific immllnofluorescence was detected in either flat or engorged ticks.
To confirm that the P2 1 -specific antisera could react with P2 1, we used the antisera to probe lecol.lbinalll P21, prepared as in Example 12, or reco,nl,inallt OspE. As expected, P2 1 antisera readily recognized recombinant P2 1 but not OspE. These results indicate that P21 is also not expressed in infected ticks.
Example VlI - Co,.r~-~l-aLion of p21 Expression in Infected Mice By Dot Immunoblot Analysis and R~A-PCR
1 c A. Dot Tmmunoblot Analysis We next confirmed that p21 is expressed in mice infected with B. burgdorferi by demonstrating the presence of antibodies against P2 1 in sera from two infected mice.
We compared the amino acid sequences of P2 1 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 isset forth in SEQ ID NO: 13.
We spotted 3 ,ug of BSA or the synthetic P21-derived peptide coupled to BSA onto nitrocellulose n~elllbl~nes We incubated the dried membranes with either serum from mice immllni7ed with heat-killed B. burgdorferior serum ~rom infected mice. We detected bound antibody by incubating with a second antibody conjugated to horseradish peroxidase (ECL Western blot detectionsystem, 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.
CA 022~3834 1998-ll-06 Sera from infected mice but not from mice imm~lni7ed with heat-killed B. burgdorferi reacted with the P2 1 peptide. Thus, P2 1 is selectively expressed in vivo.
B. RNA PCR
We further demonstrated expression of p21 in infected mice using RNA PCR to detectp21 RNA. We used acid guanidium thiocyanate/phenoV
chloroform extraction (Micro RNA Isolation Kit, Stratagene) to isolate total RNAfrom spleens of the mice infected with B. burgdorferi via tick tr~n~mi~ion 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 llg of pooled RNA with RNase-free DNase (Promega) for 3 hours at 3 7 ~
C with HPRI and the Rnase inhibitor. We conducted the RNA PCR with and without reverse transcriptase to exclude the possibility that residual DNA mightalso be amplified. We synthçsi7e(1 cDNA by reverse transcription with Moloney murine leukemia virus reverse ll~ns~ ase (Stratagene) and 3' primers for either p21 (murine tissue and cultured B. burgdor~eri), ~-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 S' primer forp21, y-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 of p21 only in the presence of reverse ~l ~ns~ ase. To confirm the identity of the amplified product as p21, we denatured and electrophoresed RNA PCR products, transferred them to Hybond-N~ membrane and hybridized withp21 probes as described in Example IV
for Northern blot analysis. The absence of product without reverse transcriptaseconfirms that DNA was not amplified. We obtained no amplification withp21-specific primers from RNA prepared from uninfected mice or from RNA PCR of B.
burgdorferi cultured in vitro.
CA 022~3834 1998-ll-06 W097t42325 PCT~US96/06610 Example VIII - Sequence An~lysis of the p35 ~ntl p37 Genes - We dirrel enlially screened the lambda Zap II B. burgdorferi expression library exactly as described in Example I but using sera from mice immllni7ed 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 immuni~ed withheat-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.
10 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 design~ted p35. We desi~n~ted the antigen 5 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 de~i~n~tedp37. We design~ted 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 Ser,g and Cys2" 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 CA 022~3834 1998-11-06 W097/42325 PCTrUS96/06610 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 upsllean~ of the respective open reading frames.
Example IX - Mapping of the p21. p35 and p37 Genes We mapped the p21, p35 and p3 7 genes by pulsed-field electrophoresis (PFGE) with total B. burgdorferi N40 DNA using a modification ofthe 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 cont~ining ap,()loxilllately 10 B. burgdorferi N40 with sarkosyl, Iysed 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 radiolabelledp21, p35, p37 probes. We usedp30, ospA and ospD probes as controls in the Southern blot. We generated p35 andp37 probes labeled with [a-32P]dCTP, using the Prime-lt~ random primer kit according to the m~mlf~ctllrer'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, "LinearPlasmids of the Bacterium Borrelia burgdorferi Have Covalently Closed Ends", Science, 237, pp. 409-411 (19~7) and S.J. Norris et al., supra]. Thep30 probe identified the chromosome. The full-lengthp21 probe bound at three locations but CA 022~3834 1998-11-06 WO 97/42325 PCT~US96/06610 ap21-specific probe (SEQ ID NO: 14) recognized a circular plasmid. The P35 probe bound to a plasrnid 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.
5 Example X - Analysis of Cultured B. burgdorferi Forp35 orp37 Expression To determine whetherp35 orp37 are transcribed in vitro, we perforrned 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 (forp35) and 1C in SEQ ID NOS. 17 and 18 (for p37).
The results ofthese analyses confirmed that p35 andp37 are transcribed in vitro.
Example XI - Confirrnation of p35 And p37 Expression In Infected Mice by ~mmunoblot An~lysis 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 p3 7 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 OspA-Mediated lmmunity to Lyme Borreliosisl', J. lmmunol, 147, pp. 1995-2000 (1991)]. The PMX vector also contains a thrombin cleavage site immedi~tely CA 02253834 1998-ll-06 following the GT protein, thus, allowing the recovery of r ecolllbina.ll 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 s ensure that the polypeptide would be expressed as soluble fusion protein rather than as a lipoprotein, which would be anchored to the cell Ille.nb.~ne or might aggregate elsewhere in the cell during or after biosythesis.
To f~çilit~te subcloning, we amplified the genes using primers with additional restriction enzyme digestion sites. We amplified thep21 gene using a 5' primer with an additional BarnHI 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 XhoI restriction enzyme digestion site and a 3' primer with a supplementary HindIII site [SEQ ID NO: 23 and 24].
We amplified thep37 gene using a 5' primer with an additional BamHI restriction enzyme digestion site and a 3' primer with a supplementary XhoI
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 I minute, ~nn~ling at 40~C for 2 minutes and extension at 72~C for 3 minlltes.
We digested the amplified gene products with BamHI ~721), XhoI
and Bam HI (p35) or Hind III and XhoI ~D37) and cloned onto the corresponding sites in the PMX plasmid. We then used the ligation mixture to transform Escherichia coli DH5a according to methods well known to those of skill in the art. We isolated colonies cont~ining 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 logaliLhlllic phase and W097t42325 PCT~US96/06610 adding 1 mM isopropyl-l-thi-beta-galactoside (IPTG) for 3 hours. One of skill inthe art could readily express the other B. burgdorferi polypeptides of this invention without undue experim~nt~tion following the above-described te~hniques.
Example XIII - Pnr;fication of Recoll,binarll Fusion Protein~
After in~ çing protein expression as described in Example XI, we placed the ~. coli in phosphate buffered saline (PBS) with 1% Triton and subjected them to sonication. We purified the glutathione S-transferase-B burgdorferl polypeptide fusion proteins (GT-P21, GT-P35, GT-P37 and GT-M30) from cell Iysates as follows.
We separated the cell supernatant and pellet by centrifugation at 1000x for 8 mins and passed the supernatant cont~ining the recombinant fusion proteins over a glutathione-Sepharose 4B column (Pharmacia) according to the m~nl~f~ctnrer's instructions. We eluted the fusion protein from the column using a solution cont~ining excess glutathione and quantified using the Bradford assay.
In addition, to purify the B. burgdorferi proteins without the glutathione S-~-~ns~e-ase, we loaded the glutathione S-transferase fusion proteins over the glutathione-Sepharose 4B column, added 25 units ofthlo-.-l)i.l to cleave the recombinant B. burgdorferi protein from the GT and incubated overnight at room temperature. We then eluted the proteins with 50 rnM Tris-CaC12-NaCI, 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 experim~nt~tion using these procedures.
CA 022~3834 l998-ll-06 W~ 97/4232~ PCT~US96/06610 Example XlV - Preparation Of Antibodies Directed Against The B. hurgdorferi Polypeptides Of This Invention We generated antibodies directed against the novel B. burgdorferi polypeptides of this invention as follows. We imm--ni7e~l C3H/He mice (FrederickCancer 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 immllni7ed control mice in the same manner with either 10 recombinant glutathione S-transferase or BSA.
Fourteen days after the last boost, we collected sera from the immlmi~ed 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 ~:Apl essing 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 se~uence as a filsion protein, even if it is normally linked to apromoter that is not transcriptionally active in E coli.
CA 022~3834 1998-11-06 W 097/42325 PCTnUS96/06610 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 ofthe ~mmllne Response To Novel B. burgdorferi Polypeptides A. Murine H~m~-ral Response To characterize the immllne 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-tr~ncmic.~ion using B. burgdorferi N40 infected 1. scapularis ticks (Harvard School of Tropical Public Health). ln the tick tr~n~mi~sion 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 a~[er 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 thenused 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 ,ug/ml, 200 ml/well3 and incub~ted overnight at 4~ C. To prevent non-specific binding, we blocked with 100 ,ul/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 l hour at room tel~,pt~ re/ 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 te~ )e~ re for 1 hour and washed 3 times with PBST. Finally, we added 200 microliters of freshly prepaled 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, ~imini.~hed by 60-90 days after infection and almost disappeared by 180 days. P2 l-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 experiment~tion the murine humoral response to other novel b. burgdorferi polypeptides of this invention using the procedures taught herein.
B. Human Humoral Respon~
We also characteri~ed the human immllne 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%
2 o 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 P2 1 -specific peptide. We found that 20 of the 82 sera (24%) from the Yale clinic had IgG antibodies to recolllbh~alll P21 and 8 ofthose 20 also had anti-P21 IgM
antibodies. Out of the 20 sera with anti-P2 1 antibodies, 4 had IgM and 16 had IgG
antibodies that bound to P2 1 -specific peptide. We found that 13 of the 40 sera(33%) from the CDC had IgG and/or IgM antibodies to P2 1. Of those 13 sera, 11 CA 022~3834 1998-11-06 WO 97/42325 PCT~US96/06610 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-documçntedLyme 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. ~3ecause ofthe high reactivity to recombinant P35 and P37, we tested sera from an additional 25 patients with well-documented Lyme disease whowere 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.
Example 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 immuni7ed C3H/He mice subcutaneously with 10 micro~rams of recombinant GT-P35 or recombinant GT-P37 polypeptides in CFA and boosted at 14 and 28 days with the same amount ofantigen in IFA according to published protocols. We imm~ni7ed control mice in the same manner with recombinant GT. We then attempted to infect the immllni7ed mice with B. burgdorferi N40.
CA 022~3834 1998-ll-06 W097/42325 PCT~US96/06610 We grew a low passage isolate of B. burgdorferi N40 with demonstrated infectivity and pathogenicity in C3HlHe mice, to log phase at 33~ Cin BSK II me~ m and counted with a hemocytometer under dark-field microscopy.
We challenged the actively immllni7ed mice approximately 14 days after the last boost with intradermal inoculations of 10 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 me~ m for two weeks and ex~mined by darkfield microscopy for spirochetes. At the same time we sectioned, formalin 10 fixed and paraffln embedded, and then examined joints and hearts for infl~mm~tion We examined the heart and tibiotarsi blindly. We characterized arthritis by edema and synovial infiltration with neutrophils and Iymphocytes. We characterized carditis by the presence of aortitis, myocarditis or pericarditis.
Preliminary results generated using these methods suggest that P3 5 or P37 may confer protection.
Example XVIII - Protection against tick-medi~ted tr~ncrnission 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 tr~n~mi~ion of the spirochete. We obtained spirochete-free Ixodes dammini ticks from the Harvard School of Public Health, which m~int~in.~ 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 C~- 1 mice that had been previously infected (three weeks prior to serving as hosts) by intradermal inoculation of 10 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 21CC and 95% relative humidity.
CA 022~3834 1998-11-06 W097/42325 PCT/Ug~6,'0GC10 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 imml-ni7ed mice with GT-P35, GT-P37, or both, GT-P21 or GT (control) as described in Example XVI~. Two weeks after the last boost, we placed 5/15 infected nymphal ticks on each mouse, allowed them to feedto repletion and then allowed them to detach naturally over water. Two weeks later we sacrificed the mice and cultured the tissues for spirochetes and exarnine theorgans, as described above.
Tmmnni7~tion 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 immllni7~tion with P21 did not produce sufficient protective antibodies in rnice or that P21 was not expressed in sufficient quantity on the surface of the spirochete to make them vulnerable to antibody-mediated killing.
Example X~X - Decrease in spirochete load in ticks feeding on imm~1ni?ed animals Previous studies have shown that immllni7~tion of mice with recombinant OspA can eliminate the spirochetes from ticks feeding on the imm~lni7ed animals [E. Fikrig et al., "Flimin~tion of Borrelia burgdorferi from vector ticks feeding on OspA-imm~ni7ed mice", Proc. Natl. Acad. Sci., 89,pp.
5418-5421(1992)]. Thus, to determine if spirochetes also are killed when infected CA 02253834 1998-ll-06 WO 97/4232~ PCT~US96/06610 ticks fed on animals imm-mi7~d 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 imm-mi7.ed with GT or 12 mice immllni7:ed with GT-P21. After feeding to repletion, the ticks are allowed to naturally det~hed over water. Only a portion of the ticks are recovered from each group, the remainder appa,~ ly having been ingested by the mice. Ten days post-repletion, we homogenized individual ticks in 100 Ill of PBS in a 1.5 ml microfuge tube and spotted 10 ~,11 aliquots on each of three slides. We allowed the slides to air-dry, fixed in cold acetone for 10 mimltes, and assayed by direct or indirect imml Inofluorescence.
For the direct immnnofluorescence 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 ~ppro~ ately 20 fields per slide. B. burgdorferi infection rates were similar within ticks that fed on immllni7ed and control mice indicating that immlmi7~tion with GT-P21 does not protect against infection.
One of skill in the art would understand that the effect of immnni7~tion with other novel B. burgdorferi polypeptides of this invention can be readily deterrnined without undue experimçnt~tion using the methods taught herein.
Example XX - Passive Tmm~lni7~tion of Mice With Anti-P35 or Anti-P37 Antisen~m To determine if antiserum from animals immllni7ed with recombinant B. burgdorferi polypeptides would confer protection, we passively immnni7.od mice with 0.2 ml of GT-P35, GT-P37 or anti-P35/P37 antisera. We .... . .
CA 022~3834 1998-11-06 W 097/42325 PCT~US96/06610 then challenged the passively imml~ni7ed mice with 10 B. burgdorferi N40 at one day after the irnml~ni7~tiQn.
Plelilninaly results indicate that the frequency of B. burgdorferi infection and the disease course in passively immuni7ed mice appeared to be the same as in the control mice.
In a separate study, we inoculated three groups of S scid mice with 10 B. burgdorferi N40 and then injected 0.S ml of antiserum (diluted 1:10) from either GT-P21 immllni7e(1, GT imml-ni7ed or 90 day infected mice on days 1, 4, 8and 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 i,lflal",llation. The rate of infection and disease in mice passively immllni7ed with P2 1 antiserum was similar to the rates in control mice. Mice passive imml1ni7ed 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 imm--ni7~fion with a reco,llbinanl polypeptide without GT, collect antiserum at a di~elelll time point when the titer is higher, passively immllni7e with more antiserum, decrease the spirochete dose, or other means known in the art.
2 o Example XXI - Additional Clones of In Vivo Expressed ~. burgdorferi Polypeptides We have performed preliminary analyses of two additional clones produced by the screening set forth in Example 1. We designated those clones V1 and V3. We deposited plasmids pVI and pV3, contained in Vl 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 CA 022~3834 1998-11-06 WO97/42325 PCT~US96/06610 confirrn that the polypeptides encoded by these clones are selectively expressed in vlvo.
We have also pel ~rl-lcd preliminary analyses of the r~m~ining 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, design~ted M30, is set forthin SEQ ID NOS: 8 and 9. We design~ted the other genes Jl and J2. Plasmids from clones corresponding to J1 have been deposited as plS and p5 under ATCC
accession numbers Plasmids from clones corresponding to p2, p7 and p9 have been deposited under ATCC accession numbers Example XXII - Deterrnination of Protective Epitopes We construct recombinant genes which will express fragments of the novel B. burgdorferi polypeptides in order to determine which fragments contain 5 protective epitopes. First, we produce overlapping 200-300 bp fr~gm~nts which encompass the entire nucleotide sequçnce of each of the genes, either by restriction enzyme digestion, or by amplification of specific sequences of using PCR and oligonucleotide primers cont~ining restriction endonuclease recognition sequences, as described st~pra. We then clone these fr~gm~nts into an appropriate expression vector, preferably a vector from which the fr~gment~ will be expressed as fusionproteins, in order to façilit~te purification and increase stability. For example, the gene fr~gment~ 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 2 5 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.
CA 022~3834 1998-11-06 WO 97142325 PCT~US96/06610 One way to identify fragments that contain protective B-cell epitopes is to use the individual purified fr~gment~ to immuni7e C3H/HeJ mice, as described above. Af~Ler ~h~llP~lge of the mice with B. burgdorferi, we deterrnine the presence of infection by blood and spleen cultures and by histopathologic ex~min~tion of the 5 joints and heart.
Another technique to identify protective epitopes is to use the various fragm~nts to immuni7~ 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 subsequentinfection 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 5 amino acids. The use of synthetic peptides allows us to further define each epitope, while elimin~ting 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 CA 022~3834 1998-11-06 W097/4232~ PCTAUS96/06610 isolates. Such a vaccine will, thus, elicit antibodies that will confer protection against a variety of di~lelll strains.
Example XXIV - Identification of T cell epitopes Stimulation in animals of a humoral immune response co~
5 high titer neutralizing antibodies will be facilitated by antigens cont~ining both T cell and B cell epitopes. To identify those polypeptides cont~ining 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 Iymph nodes and generate in vitro T cell lines. These T cell lines are then cloned using limiting o dilution and soft agar techniques. We use these T cell clones to determine which polypeptides contain T cell epitopes. The T cell clones are stim~ ted with the various polypeptides and syngeneic antigen p~eselllh~g cells. Exposure ofthe T cell clones to the polypeptides that contain T cell epitopes in the presence of antigen pres~nting cells causes the T cells to proliferate, which we measure by H-Thymidine incorporation. We also measure Iymphokine production by thestimulated 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 ofmultiple HLA types. T cell epitopes are identified by stim~ ting the clones with the various polypeptides and measuring H-Thymidine incorporation. The various T
cell epitopes are then correlated with Class II HLA antigens such as DR, DP, andDQ. The correlation is performed by utilization of B Iymphoblastoid cell lines expressing various HLA genes. When a given T cell clone is mixed with the appropliate B Iymphoblastoid 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 H-Thymidine incorporation.
. ~ .
CA 02253834 1998-ll-06 W097/4232S PCTrUS96/06610 Alternatively, T cell epitopes may be identified by adoptive transfer of T cells from mice immllni7ed with various of the novel B. burgdorferi polypeptides of this invention to naive mice, according to methods well known tothose of skill in the art. [See, for example, M.S. DeSouza et al., "Long-Term Study of Cell-~edi~ted Responses to Borrelia bz~r~,do,J~ri in the Laboratory Mouse", Infect. Immun., 61, pp. 1814-22 (1993)].
We then synth-osi7e a multicomponent vaccine based on di~lent T
cell epitopes. Such a vaccine is useful to elicit T cell responses in a broad spectrum of patients with di~erent HLA types.
We also identify stimlll~ting 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 Tcell epitopes from the novel B. burgdorferi polypeptides of this invention.
Example XXV - Construction of fusion proteins comprising T ~ntl E3 cell epitopes A~er 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 filsion proteins, by virtue of cont~ining both T cell and B cell epitopes, permit antigen presenlalion 2 0 to T cells by B cells exp- essing surface immunt~globulin. These T cells in turn stimlll~te B cells that express surface imm~lnoglobin, 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 cellepitopes to strong T cell epitopes of other antigens. We synthe.ci7e an oligonucleotide homologous to amino acids 120 to 140 ofthe 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' CA 022=,3834 1998-11-06 WO97/42325 PCTrUS96/06610 and 3' ends of segm~nts 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 co~ ~sing a B cell epitope from the novel B.
burgdorferi polypeptide and a T cell epitope from the core antigen, thus enh~n~ing the immllnogenicity ofthe 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 co~ g the B cell epitopes of various of the novel B. burgdorferi polypeptides incorporated into the fl~eell;n protein of Salmonella. Bacterial 11~gçllin 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 cellepitopes 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 imm~lni7,ed with live and formalin killed bacteria and assayed for antibody production. In addition spleen cells are tested for proliferative cellular responses to 2 o the peptide of interest. Finally the mice immuni~ed 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 di~e~el.L
novel B. burgdorferi polypeptide or other immunogenic B. burgdorferi 25 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
CA 022s3834 1998-ll-06 WO 97/4232S PCT~US96/06610 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, andprevent Lyme disease as caused by infection with B. burgdorferi.
While we have described a number of embodiments of this invention, s it is appare-lL 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.
CA 022~3834 1998-11-06 W097/42325 PCT~US96/06610 SEQUENCE LISTING
(1~ GENERAL INFORMATION:
(i) APPLICANT: Flavell, Richard A.
Fikrig, Erol Barthold, Stephen W.
Suk, Kyoungho (ii) TITLE OF INVENTION: B. BURGDORFERI POLYPEPTIDES EXPRESSED IN
VIVO
(iii) NUMBER OF SEQUENCES: 28 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Fish & Neave (B) STREET: 1251 Avenue of the Americas (C) CITY: New York (D) STATE: New York (E) COUNTRY: USA
(F) ZIP: 10020 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Verslon #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Haley Jr., James F.
(B) REGISTRATION NUMBER: 27,794 (C) REFERENCE/DOCKET NUMBER: YU-103 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 212-596-9000 (B) TELEFAX: 212-596-9090 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 752 base pairs (B) TYPE: nuclelc acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA (genomlC) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
tA) NAME/KEY: CDS
(B) LOCATION: 145..690 (ix) FEATURE:
(A) NAME/KEY: CDS
SU~S 111 UTE SHEET (RULE 26) CA 022~3834 1998-11-06 W O 97/42325 PCT/U~3G,~ 10 (B) LOCATION: 721..750 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Met Asn Lys Lys Met Phe Ile Val Cys Ala Val Phe Ala Leu Ile Ser Ser Cys Lys Ile His Thr Leu Ser Met Tyr Asp Glu Gln Ser Asn Asn Glu Leu Lys Val Lys Gln Ser Asn Gly Glu Val Lys Val Lys Lys Ile Glu Phe Ser Glu Phe Thr Val Lys Ile Lys Tyr Lys Lys Asp Asn Ser Ser Asn Trp Glu Asp Leu Gly Thr Leu Val Val Arg Lys Glu Val Asp Gly Ile Asp Thr Gly Leu Asn Val Gly Lys Gly Tyr Ser Ala Thr Phe Phe Ser Leu Glu Glu Ser Glu Val Asn go 95 100 105 Asn Phe Ile Lys Ala Met Thr Lys Gly Gly Thr Phe Lys Thr Ser Leu Tyr Tyr Gly Tyr Lys Glu Glu Gln Ser Gly Glu Asn Gly Ile Gln Asn Lys Lys Ile Ile Thr Lys Ile Glu Lys Ile Asp Asp Phe Glu Tyr Ile ACA TTT TTA GGA &AT AAA ATT AAG GAT TCA GGA GAT AAA GTT GTT GAA 651 Thr Phe Leu Gly Asp Lys Ile Lys Asp Ser Gly Asp Lys Val Val Glu Tyr Ala Ile Leu Leu Glu Asp Leu Lys Lys Asn Leu Lys Met Asn Gln Lys Ala Phe Ile Ile Cys Ala SUBSTITUTE SHEET (RULE 26) CA 022~3834 1998-11-06 W097/42325 PCT~US96/06610 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 182 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (li) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Asn Lys Lys Met Phe Ile Val Cys Ala Val Phe Ala Leu Ile Ser ~er Cys Lys Ile His Thr Leu Ser Met Tyr Asp Glu Gln Ser Asn Asn Glu Leu Lys Val Lys Gln Ser Asn Gly Glu Val Lys Val Lys Lys Ile Glu Phe Ser Glu Phe Thr Val Lys Ile Lys Tyr Lys Lys Asp Asn Ser Ser Asn Trp Glu Asp Leu Gly Thr Leu Val Val Arg Lys Glu Val Asp ~ly Ile Asp Thr Gly Leu Asn Val Gly Lys Gly Tyr Ser Ala Thr Phe ~he Ser Leu Glu Glu Ser Glu Val Asn Asn Phe Ile Lys Ala Met Thr Lys Gly Gly Thr Phe Lys Thr Ser Leu Tyr Tyr Gly Tyr Lys Glu Glu Gln Ser Gly Glu Asn Gly Ile Gln Asn Lys Lys Ile Ile Thr Lys Ile Glu Lys Ile Asp Asp Phe Glu Tyr Ile Thr Phe Leu Gly Asp Lys Ile Lys Asp Ser Gly Asp Lys Val Val Glu Tyr Ala Ile Leu Leu Glu Asp ~eu Lys Lys Asn Leu Lys ~2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (iil MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Met Asn Gln Lys Ala Phe Ile Ile Cys Ala SU~S 111 UTE SHEET (RULE 26) CA 022~3834 1998-11-06 WO 97/42325 PCT~US96/06610 (2) INFORMATION FOR SEQ ID NO:4:
li) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1353 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOl~rlCAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 181..1107 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Thr Met Ile Thr Pro Ser Ser Lys Leu Thr Leu Thr Lys Gly Asn Lys Ser Trp Ser Ser Thr Ala Val Ala Ala Ala Leu Glu Leu Val Asp Pro Pro Gly Cys Arg Asn Ser Lys Ser Asn Phe Leu Gln Lys Asn Val Ile Leu Glu Glu Glu Ser Leu Lys Thr Glu Leu Leu Lys Glu Gln Ser SUbS 111 UTE SHEET (RULE 26) -CA 022~3834 l998-ll-06 WO g7/42325 PCTrUS96/06610 Glu Thr Arg Lys Glu Lys Ile Gln Lys Gln Gln Asp Glu Tyr Lys Gly 75 80 85 go Met Thr Gln Gly Ser Leu Asn Ser Leu Ser Gly Glu Ser Gly Glu Leu 95 lO0 105 Glu Glu Pro Ile Glu Ser Asn Glu Ile Asp Leu Thr Ile Asp Ser Asp Leu Arg Pro Lys Ser Phe Leu Gln Gly Ile Ala Gly Ser Asn Ser Ile Ser Tyr Thr Asp Glu Ile Glu Glu Glu Asp Tyr Asp Arg Tyr Tyr Leu Asp Glu Asp Asp Glu Asp Asp Glu Glu Asp Glu Glu Glu Ile Arg Leu Ser Asn Arg Tyr Gln Ser Tyr Leu Glu Gly Val Lys Tyr Asn Val Asp Ser Ala Ile Gln Thr Ile Thr Lys Ile Tyr Asn Thr Tyr Thr Leu Phe Ser Thr Lys Leu Thr Gln Met Tyr Ser Thr Arg Leu Asp Asn Phe Ala Lys Ala Lys Ala Lys Glu Glu Ala Ala Lys Phe Thr Lys Glu Asp Leu Glu Lys Asn Phe Lys Thr Leu Leu Asn Tyr Ile Gln Val Ser Val Lys Thr Ala Ala Asn Phe Val Tyr Ile Asn Asp Thr His Ala Lys Arg Lys Leu Glu Asn Ile Glu Thr Glu Ile Lys Thr Leu Ile Ala Lys Ile Lys Glu Lys Pro Asp Leu Tyr Gln Ala Tyr Lys Ala Ile Val Thr Pro Ile SUt~S 111 IJTE SHEET (RULE 26) CA 022~3834 1998-ll-06 W O97/42325 PCT~US96/06610 - 79 -TTA TTA ATG AGG GAT TCT CTT A~A GAA GTG CAA AGT GCC ATT GAC AAG 1092 Leu Leu Met Arg Asp Ser Leu Lys Glu Val Gln Ser Ala Ile Asp Lys Asn Gly Ile Trp Tyr (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 309 amino acids (B) TYPE: amlno acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Met Thr Met Ile Thr Pro Ser Ser Lys Leu Thr Leu Thr Lys Gly Asn ~ys Ser Trp Ser Ser Thr Ala Val Ala Ala Ala Leu Glu Leu Val Asp Pro Pro Gly Cys Arg Asn Ser Lys Ser Asn Phe Leu Gln Lys Asn Val Ile Leu Glu Glu Glu Ser Leu Lys Thr Glu Leu Leu Lys Glu Gln Ser Glu Thr Arg Lys Glu Lys Ile Gln Lys Gln Gln Asp Glu Tyr Lys Gly ~et Thr Gln Gly Ser Leu Asn Ser Leu Ser Gly Glu Ser Gly Glu Leu ~lu Glu Pro Ile Glu Ser Asn Glu Ile Asp Leu Thr Ile Asp Ser Asp Leu Arg Pro Lys Ser Phe Leu Gln Gly Ile Ala Gly Ser Asn Ser Ile Ser Tyr Thr Asp Glu Ile Glu Glu Glu Asp Tyr Asp Arg Tyr Tyr Leu Asp Glu Asp Asp Glu Asp Asp Glu Glu Asp Glu Glu Glu Ile Arg Leu SU~ ~111 UTE SHEET (RULE 26) CA 022~3834 1998-11-06 W 097/42325 PCT~US96/06610 Ser Asn Arg Tyr Gln Ser Tyr Leu Glu Gly Val Lys Tyr Asn Val Asp ~er Ala Ile Gln Thr Ile Thr Lys Ile Tyr Asn Thr Tyr Thr Leu Phe Ser Thr Lys Leu Thr Gln Met Tyr Ser Thr Arg Leu Asp Asn Phe Ala Lys Ala Lys Ala Lys Glu Glu Ala Ala Lys Phe Thr Lys Glu Asp Leu Glu Lys Asn Phe Lys Thr Leu Leu Asn Tyr Ile Gln Val Ser Val Lys ~hr Ala Ala Asn Phe Val Tyr Ile Asn Asp Thr His Ala Lys Arg Lys ~eu Glu Asn Ile Glu Thr Glu Ile Lys Thr Leu Ile Ala Lys Ile Lys Glu Lys Pro Asp Leu Tyr Gln Ala Tyr Lys Ala Ile Val Thr Pro Ile Leu Leu Met Arg Asp Ser Leu Lys Glu Val Gln Ser Ala Ile Asp Lys Asn Gly Ile Trp Tyr (2) INFORMATION FOR SEQ ID NO:6:
(i~ SEQUENCE CHARACTERISTICS:
~A) LENGTH: 1490 base palrs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(ivJ ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 121..1116 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
ATG AAT TTA ATA ATT A~A GTG ATG TTG ATA TCC AGT TTA TTT TCT AGC 168 Met Asn Leu Ile Ile Lys Val Met Leu Ile Ser Ser Leu Phe Ser Ser Phe Ile Ser Cys Lys Leu Tyr Glu Lys Leu Thr Asn Lys Ser Gln Gln SUBSTITUTE SHEET (RULE 26) W097/42325 PCT~US96/06610 Ala Leu Ala Lys Ala Phe Val Tyr Asp Lys Asp Ile Ala Asp Asn Lys Ser Thr Asn Ser Thr Ser Lys Leu Asp Asn Ser Ser Leu Asp Ser Ile Lys Asp Asn Asn Arg Ser Gly Arg Thr Ser Arg Ala Leu Asp Asp Ala Glu Glu Ile Gly Val Lys Glu Ser Asn Gln Asn Arg Asn Asp Gln Gln Gln Asn Asn Glu Ser Lys Val Lys Glu Ser Glu Lys Asn Asn Ser Ser Gly Ile Gln Ala Asp Asp Ser Val Leu Gly Thr Ala His Ser Asp Ala Ser Glu Val Glu Asn Lys Lys His Asp Thr Ser Arg Gln Pro Gln Leu Leu Asn Lys Asp Ser Ser Glu Ala Arg Glu Ala Ser Lys Ile Ile Gln Lys Ala Ser Thr Ser Leu Glu Glu Ala Glu Lys Val Asn Val Ala Leu 470 475 480 q85 Lys Glu Thr Arg Ser Lys Leu Asp Lys Ile Lys Arg Leu Ala Asp Ser Ala Lys Ser Tyr Leu Asn Asn Ala Arg Lys Asn Ser Arg Thr Asn Gly Ser Ile Leu Glu Ile Leu Pro Asn Leu Asp Lys Ala Ile Glu Lys Ala Ile Ser Ser Tyr Ala Ser Leu Asn Val Cys Tyr Thr Asp Ala Ile Ala Ala Leu Ala Lys Ala Lys Asn Asp Phe Glu His Ala Lys Arg Lys Ala Asn Asp Ala Leu Glu Glu Ala Leu Lys Asp Ile Thr His Phe Arg Gly Tyr Asn Tyr Leu Tyr His Tyr Arg Ile Asn Asn Ala Asn Asp Ala Met SUt~S 111 UTE SHEET (RULE 26) CA 022~3834 1998-11-06 W 097/42325 PCT~US96/06610 Glu Ser Ala Lys Ser Leu Leu Glu Val Ala Lys Asn Lys Gln Lys Glu Leu Asn Glu Asn Ile Thr Lys Thr Asn Lys Asp Phe Gln Glu Leu Asn Asp Ile Tyr Lys Lys Leu Gln Asp Met Asp Ser Arg ~lc~ lCT AAACAACACT TTATTTTCTC TTAACTTTAT AGTTTGACTT AAAAAGTCAT 1246 TATTTTTAAA TTATTACAT& AATTGCCTTG AATATCTTTA TTTTTATATT ATAATTATTA 1306 T~ .rlGA TTTGA~AGTT GTTCTGGTTT TCTATCTAAA AAATCTATAG AACAGTTTGC 1486 (2) INFORMATION EOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 332 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID No:7:
Met Asn Leu Ile Ile Lys Val Met Leu Ile Ser Ser Leu Phe Ser Ser Phe Ile Ser Cys Lys Leu Tyr Glu Lys Leu Thr Asn Lys Ser Gln Gln Ala Leu Ala Lys Ala Phe Val Tyr Asp Lys Asp Ile Ala Asp Asn Lys Ser Thr Asn Ser Thr Ser Lys Leu Asp Asn Ser Ser Leu Asp Ser Ile SU~5 111 lJTE SHEET (RULE 26) CA 022~3834 1998-11-06 W097/42325 PCT~US96/06610 Lys Asp Asn Asn Arg Ser Gly Arg Thr Ser Arg Ala Leu Asp Asp Ala ~lu Glu Ile Gly Val Lys Glu Ser Asn Gln Asn Arg Asn Asp Gln Gln ~ln Asn Asn Glu Ser Lys Val Lys Glu Ser Glu Lys Asn Asn Ser Ser Gly Ile Gln Ala Asp Asp Ser Val Leu Gly Thr Ala Hls Ser Asp Ala Ser Glu Val Glu Asn Lys Lys His Asp Thr Ser Arg Gln Pro Gln Leu Leu Asn Lys Asp Ser Ser Glu Ala Arg Glu Ala Ser Lys Ile Ile Gln ~ys Ala Ser Thr Ser Leu Glu Glu Ala Glu Lys Val Asn Val Ala Leu ~ys Glu Thr Arg Ser Lys Leu Asp Lys Ile Lys Arg Leu Ala Asp Ser Ala Lys Ser Tyr Leu Asn Asn Ala Arg Lys Asn Ser Arg Thr Asn Gly Ser Ile Leu Glu Ile Leu Pro Asn Leu Asp Lys Ala Ile Glu Lys Ala Ile Ser Ser Tyr Ala Ser Leu Asn Val Cys Tyr Thr Asp Ala Ile Ala ~la Leu Ala Lys Ala Lys Asn Asp Phe Glu His Ala Lys Arg Lys Ala ~sn Asp Ala Leu Glu Glu Ala Leu Lys Asp Ile Thr His Phe Arg Gly Tyr Asn Tyr Leu Tyr His Tyr Arg Ile Asn Asn Ala Asn Asp Ala Met Glu Ser Ala Lys Ser Leu Leu Glu Val Ala Lys Asn Lys Gln Lys Glu Leu Asn Glu Asn Ile Thr Lys Thr Asn Lys Asp Phe Gln Glu Leu Asn ~sp Ile Tyr Lys Lys Leu Gln Asp Met Asp Ser Arg (2) INFORMATION FOR SEQ ID NO:8:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 825 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) ~iii) HYPOTHETICAL: NO
SIJ~S ~ )TE SHEET (RULE 26) CA 022~3834 l998-ll-06 W 097/42325 PCT~US96/06610 (iv) ANTI-SENSE: NO
(ix) FEATURE:
(A~ NAME~KEY: CDS
(B) LOCATION: 1..825 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Met Cys Ala Phe Leu Leu Leu Asn Leu Val Asn Cys Lys Phe Asp Ser Leu Asn Leu Ser Thr Lys Ser Val Asp Asp Lys Asn Asn Ser Ile Ala Lys Leu Leu Gln His Leu Ser Lys Ser Glu Asp Gln Ala Asn Lys Thr TCT ACC TCA GAA GAC CAA AAG GAA TTA GAA ATT ACG GAA AAC A~A GAA 192 Ser Thr Ser Glu Asp Gln Lys Glu Leu Glu Ile Thr Glu Asn Lys Glu Gln Glu His Glu Lys Leu Ser Gln Val Ala Gln His Ala Pro Asn Ser Lys Ile Glu Lys Val Lys Ser Asp Gly Lys Pro Val Pro Gly Asp Lys Ile Leu Ser Ser Asn Lys Asp Ile Tyr Asn Ser Tyr Ile Pro Glu Val 430 q35 440 Lys Glu Glu Ile Val Tyr Glu Ile Leu Glu Glu Val Ile Ile Pro Glu Thr Lys Ile Pro Glu Ile Thr Glu Glu Val Ile Met Pro Ile Pro Gln Thr Ile Asp Phe Tyr Ile Glu Pro Arg Pro Ile Ser Ser Phe Leu Thr &ln Gly Thr Ser Pro Ser Ile Thr Ser Thr Ile Lys Ser Tyr Lys Glu SU~;~ 111 UTE SHEET (RULE 26) CA 022~3834 l998-ll-06 W 097/4232~ PCT~US96/06610 Leu Ala Lys Glu Lys Ile Asn Asn Gly Leu Asn Ile Val Gln Lys Ile Thr Gln Asn Ile Asp Asn Ile Thr Glu Asn Leu Asn Ser Lys Glu Thr Pro Lys Glu Ile Ser Gly Lys Glu Val Glu Glu Lys Ile Thr His Pro Ile Phe Asp His Ile Thr Gly Ser Gly Asn Asn Pro Gly Gln Asp Ser Ile Ser Asn Thr Trp Gly Glu Gly Leu Glu Ile Gly Gly Asp Ser Asn Phe Phe Thr Asn Leu Glu Glu Val Arg Ser Ser Ile Arg Thr Lys Ile Lys Val Ser (2~ INFORMATION FOR SEQ ID NO:9:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 275 amlno acids (B~ TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Met Cys Ala Phe Leu Leu Leu Asn Leu Val Asn Cys Lys Phe Asp Ser ~eu Asn Leu Ser Thr Lys Ser Val Asp Asp Lys Asn Asn Ser Ile Ala Lys Leu Leu Gln His Leu Ser Lys Ser Glu Asp Gln Ala Asn Lys Thr Ser Thr Ser Glu Asp Gln Lys Glu Leu Glu Ile Thr Glu Asn Lys Glu Gln Glu His Glu Lys Leu Ser Gln Val Ala Gln His Ala Pro Asn Ser ~ys Ile Glu Lys Val Lys Ser Asp Gly Lys Pro Val Pro Gly Asp Lys SU~S 1 1 1 UTE SHEET (RULE 26) CA 022~3834 l998-ll-06 W097/42325 PCT~US96/06610 Ile Leu Ser Ser Asn Lys Asp Ile Tyr Asn Ser Tyr Ile Pro Glu Val Lys Glu Glu Ile Val Tyr Glu Ile Leu Glu Glu Val Ile Ile Pro Glu Thr Lys Ile Pro Glu Ile Thr Glu Glu Val Ile Met Pro Ile Pro Gln Thr Ile Asp Phe Tyr Ile Glu Pro Arg Pro Ile Ser Ser Phe Leu Thr ~ln Gly Thr Ser Pro Ser Ile Thr Ser Thr Ile Lys Ser Tyr Lys Glu ~eu Ala Lys Glu Lys Ile Asn Asn Gly Leu Asn Ile Val Gln Lys Ile Thr Gln Asn Ile Asp Asn Ile Thr Glu Asn Leu Asn Ser Lys Glu Thr Pro Lys Glu Ile Ser Gly Lys Glu Val Glu Glu Lys Ile Thr His Pro Ile Phe Asp His Ile Thr Gly Ser Gly Asn Asn Pro Gly Gln Asp Ser ~le Ser Asn Thr Trp Gly Glu Gly Leu Glu Ile Gly Gly Asp Ser Asn ~he Phe Thr Asn Leu Glu Glu Val Arg Ser Ser Ile Arg Thr Lys Ile Lys Val Ser ~2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 1221 base palrs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPB: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
SU~S 111 ~JTE SHEET (RULE 26) CA 022~3834 l998-ll-06 W097/42325 PCT~US96/06610 (2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single SUBSTITUTE SHEET (RULE 26) CA 022~3834 1998-11-06 WO 97/42325 PCT~US96106610 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
TGTACAAGCT TCTATTTTAA AllllllLlA AGATC 35 (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ~iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Cys Asn Asn Glu Leu Lys Val Lys Gln Ser Asn (2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
~ (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:14:
SIJ~S 111 ~ITE SHEET (RULE 26) CA 022~3834 1998-11-06 W 097/4232S PCT~US96/06610 (2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
TCTGATTTAA GGCCA~AG 18 (2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucle1c acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
SU~ JTE SHEET (RULE 26) CA 022~3834 l998-ll-06 W097/4232~PCTrUS96/06610 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs IB) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
~iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (il) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
SlJ~S 111 UTE SHEET (RULE 26) CA 022~3834 1998-11-06 W097/42325 PCTrUS96/06610 (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
AGTCAAGCTT CTATTTTAAA ll L 1 1 1 1 l~AA GATC 34 (2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acld (C) STRANDEDNESS: single (D) TOPOLOGY: linear SU~S 111 IJTE SHEET (RULE 26) CA 022~3834 1998-ll-06 W097142325 PCT~US96/06610 (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base palrs (B) TYPE: nuclelc acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
liv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
SU~;> 111 UTE SHEET (RULE 26) CA 022~3834 1998-ll-06 W097/42325 PCT~US96/06610 (2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs (B) TYPE: nuclelc acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 24 base palrs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICA~: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
SlJt~ 111 UTE SHEET (RULE 26) INDICATION'S RELATING TO A DEPOSITED MICROOR(~ANISM
(PCT Ru le 1 3bis) A. i 11-~ indicillions ~nade below rel~le to Ihe microoreanism relerred to in the descriplion on paee , llne B. IDENTIFICATION OF DEPOSIT plasmid ~'urther deposits arc idenlilied on iln iiddilion~71 sheet ~3 ~ -r ~ l -k~
Nameofdeposilar~ institution American Type Culture Collection Address ol depoiitilr! inslilulion ~inclu~lin~,~ poslal code a~l~i coZ~nrr 12301 Parklawn Drive Rockville, Maryland 20852 Unlted ~tates of Amerlca l);uc ol dcpo~il i Accc~sl0n Nulllhcr 7 May 1996 (07.05.96) C ADDITION'AL INDICATIONS lleal~ h/i7n~ o~ ap/~llcahlc/ I his inlonll;llioll I~ ~onthlucd on Ull ~dditiom71 shcct O
In respect of the designatlon of the EPO, samples of the de-posited microorganisms will be made available until the publicatio~
of the mention of the grant of the European patent or until the da'-e on whlch the application is refused or withdrawn or is deemed to be withdrawn, as provided in Rule 28(3) of the Implementing Regu-lations under the EPC only by the issue of a sample to an expert nominated b~ the requester (Rule 28(4) ~PC~.
D. DESIG~ATED STATES FOR WHICH INi)lCATlONS ARE ~IADE /~ .ol~ c ~ 11 oe~ 'lla/e~/.S~lle~J
EP
E. SEPAR~TE FIJRNISHING OF INDICATIONS H~ hlal7~ 11 nol applicabl-l'hc in~ilcinions lis(ed belo\~ ~ ill he suhmitlcd to Ihe Internc7lioll~1 3urcilu l~lcr l~ clt\ Ibe yL'/7~aH7a~ 'C o/rll,~ ind7ca~io~L~ e.~ lo :\'7~ h~ po511"J
Accession number of deposit.
r r~cci~ e Ollicc usc onl! I-or Inl.rniuiol~ urcilu use onh nee~ s recei~ed ~ith Ihe internilliom~ pplici~lh~n ~ -I his sheel ~s rccel~ed h\ the Intermllion~ ureill7 oll Authori~e~l olficer Authori;~cd ollicer .-orm I'CT RO 13~ Hl71! 199_) CA 02253834 1998-ll-06 INDICATIONS RELATING TO A DEPOSITED MICROOR~;ANISM
(PCTRule l3~,is) A. f hc indieations madc belo-~ rclate to the l"i,~uv~ "i~l" relerrcd to in thc dcseription on pa~c ~ q , linc 2 ~_ B. IDENTIFICATION OF DEPOSIT plasmid p Vl l:unhcr dcposils are identified on an additional shcc Narne of depositarv instilution American Type Culture Collection Addrcss ot'deposltar~ instltution /~neludin~e poslal cod~ and counrr 12301 Parklawn Drlve Rockville, Maryland 20852 Unlted States of America l)at. u~ dcposil ~ .\c~esslo~l l\iumh.r 7 May 1996 (07.05.96) C. .~DDlTlC~l~'.tL INDIC.~TIONS lleal L7 hlan~ 1/ nr71 ~Ippllcahl. ~ I hB informanon is contilluc-l on an a-fdilion~l ~heel O
In respect of the deslgnation of the EPO, samples of the de-posited mlcroorganlsms wlll be made available until the publlcation of the mention of the grant of the European patent or until the da~e on whlch the application is refused or withdrawn or lS deemed to be withdrawn, as provided in Rule 28(3) oi the Implementlng Regu-lations under the EPC only by the issue of a sample to an expert nominated by the requester (Rule 28(4) ~PC) D. DESIC,N.~TEI) ST~TES FOR S~'HI( II I~DIC.-~TIONS AKF .~ilADE /~J ~r/~ lo~l~ alc /lot lo~ all (Dslen(71.~ 51an EP
E. SEPARATE FURNISHING OF ll~il)lC:ATlONS /1-7ale hlan~ or ~pllLahl.~, I hl: indiciltlons list~d hclo~ dl be sllhmltted lo Ille Intematiorl;ll 13ureau latcr /sp-~C/h ~ eL~nLraDranlr~ otrlle ~ carlLrnsLl e ~ l~cL~
rlt7 ot L)r?pOS/I /
Accesslon number of deposlt.
- l or rcccl~ In!! ()f lic-~ us~ ~U~ H Ir Inlern;ltiunal Burcall use onh~is shcel ~a~ rcecl~cd ~Htll the In~crnali()nal ;Ippllcatlon O I his sh-el ~ r.cel~ed h! Ihe Inlernn~ioll;ll Uurea(l .u:
i~Ulh~r~'dt5~c''r ~,~ .\ulllori~ed olliccr l'orm PCT.ROil3~ Hul~ 199'~
CA 02253834 1998-ll-06 W097/42325 93~3 PCT~US96tO6610 INDICATIONS RELATING TO A DEPOSITED MICROORC~ANISM
(PCT Rule I ~bis) A I hc indicnlions made hclo~v rel tte to the ~ oo~ ~:d"ism relerred to in the descrlption on pa~c p. 1(O~ 20 ~ line B IDENTIFICATION OF DEPOSIT plasmid p V3 'r:urthcr deposits are idenliiied on 3n ndditional shect [~3 Nrlmc of depositalv instilution American Type Culture Collection Address of deposilar! institution ~includmg postal code and cortnrn 12301 Parklawn Drive Rockville, Maryland 20852 ~nited ~tates of America l);nc o~ depo~ A~c~s~on Numbcr 7 May 1996 (07.05.96) C' ADDlTlONAL li~DlCATlO~'S ~ bla~11; I/ nl71 ap/711caole/ I hi~ inlorm31ion is con~hluc~ on Ull ~ddition;ll shect O
In respect of the designatlon of the EPO, samples of the de-posited microorganisms will be made available until the publication of the mention of the grant of the European patent or until the da e on whlch the application is refused or withdrawn or is deemed to be withdrawn, as provided in Rule 28(3) of the Implementing Regu-lations under the EPC only by the issue of a sample to an expert nominated by the requester (Rule 28(4) ~PC).
1) DESIC.~ATED STAT~S FOR ~ HICII INDICATIO~S ARE ~IADE /~ rl.~ (Jr. ~ "11 llcsl~r~nar,~/.SIam.~, EP
E SEPAR~TE Fl,RNISHING OF I~DICATIONS rlea\ e bla~ J nor a/~pllcable~
1 hc illdlc:ltions listcd bclo~ ill hc submittcd to the Intcrnallon31 l~ure;~u httcr ~5/lccih //1L7~eL'~ r~ Mtlr~C oJrlre al~licarlo~ L 6 ' IL'C~L~SSI~nl 7l l~posit';
Accession number of deposit.
i:or rccel~ mo ( )i1icc ~l~c ~nl! hur Intcrn3tiol1al l~ure3u usc ~nl!
,hcct ~s rccel~d ~-ith ~hc imem3~ionl1 tppllc;~noll ~ O I nis ,ilc,~ 5 rcccl~cd b! ~he Intcrn3tion tl ~ure;~u on A~7ed~1iccr ~ '\ulhorizcd of'liccr l:orrnl'CTRC)'13~JUl! 199-1 W097/42325 93~4 PCTrUS96/06610 INDICATIO~'S RELATING TO A DEPOSITED MICROOR(~ANISM
(PCT Rule I ~bis) A 1 hc indicrttions madc bclo~ rel tte to thc ~ ,o, ~ l- retcrrcd lo in thc dcscription on pa~c p \~ ~ ~ ~7 ;p ~ ~ ) ~ ,1 B. IDENTIFICATION OF DEPOSIT plasmid p 15 I ur~hcr dcposils arc identified on ;tn tddition~tl sheet Nameofdepositar~ inslitulion American Type Culture Collection Address of dcposit;lr~ instllution ~inclu~ng postal code and countn 12301 Parklawn Drive Rockville, Maryldnd 20852 ~nlted States of America l):nc ot'dcposi~ C~'L'SSlOn IS,umhcr 7 May 1996 (07.05.96) ADDlTlOi' ~L INDICATIONS ~/L~I~e hl~a~ lic~lhll l l hl~ Inlorm t~ion j~ COnI;I1UC(J On illl Idd;~;I1I1LtI Sh~'CI O
In respect of the deslgnation of the EPO, samples of the de-posited mlcroorganiSmS will be made avallable until the publlcatio:n of the mention of the grant of the European patent or until the da-e on which the appllcation is refused or wlthdrawn or is deemed to be withdrawn, as provided in Rule 28(3) of the Implementing Regu-lations under the EPC only by the issue of a sample to an expert nominated by the requester (Rule 28(4) ~PC~.
D DESIGNATED STATES FOR W~IIC~ TIONS ARE I~IADF llt ~ IJ~7/~ L' ~l0l /0~ all lle~ al~ SIale~
EP
E SEPARATE F- RNISHINC. OF INDlC' ~TIONS tlca~e hla~ ol appllcabll~J
l hc indicittions lis~ b~ l hc ~uhmit~Ld lo ~h~ lntcrnatlon~ ur~;lu ~ cr ~ !h l/lL~x~lerolnah~r~ îc l~l~llca~lo~u ~ c~ ~s .~ t~ h~ epo~
Accession number of deposit.
r r.-cl\ m~ Dt~icc usc~ ol~ r Inlcrna~mll;~l Uurc tu us~ 01)l!
e~S shcct \\ ;~ rcccl\ c 1 ~ ith Ihc inlcrn;tll(ln~l ~pphc~ l O I hl~ shc~ rccci~ cLI h! [hc lntcrn;tlion;~ urcLtll ~n Au3~ ~ ~uthorl~cd olticc~
Form PCT ROH3~ I!ul~ 199 ) W097/42325 93~5 PCTrUS96/06610 INDICATII~.~S RELATING TO A DEPOSITED MICROORG
(PCTRule 13bi.~) A. Th~ indicmiuns made belo~v rela~e ~o the microorcanism reterred to in th~ d~scription on p,t~ p, ~7 ~ \~L l~o ~; ; pQ4~ q B. IDENTIFICATION OF DEPOSIT plasmid P 5 I-urthcr d~posils dre idenlit;ed on ~n ~ddilionul shcet Name ol'deposilar~Hnslitution American Type Culture Collection Addrcss ol'deposil;~r~- instilulion /inCIud~n,L~' poslul code ~tl~l COUn~r~') 12301 Parklawn Drlve Rockville, Maryland 20852 ~nlted ~tates of Amerlca l):u-~ 01 del-osi~ C~II)II Numhcr -7 May 1996 (07.05.96) C'. ADDITIONAL l.'NDICATIONS l~'O\'L' hlunA ~ Ot oppllo(lt7ic H ~ mlorrn3tl0n IS COlllinUt~d on 3n tddllion~l .sll~cl C~
In respect of the deslgnatlon of the EPO, samples of the de-posited microor~anisms will be made available until the publication of the mention of the grant of the European patent or until the da'-e on which the application is refused or wlthdrawn or is deemed to be withdrawn, as provided in Rul~ 28(3) of the ImplemenL ng Regu-lations under the EPC only by the issue of a sample to an expert nominated b~ the requester (Rule 28(4) ~PC).
D. DESICIN.~TED ST,~TES FOR ~ 'HICH IN'DIC.-~TIO~S .~Rt: ,~IADE /1/ ~b~ nO/I~ 101~ 1 Hicslr!l7~lr~cl .51 EP
E. SEP.~RATE Fl, RNISHINC OF l?NDICATIONS ~ o~ u hlor~ lo~ o/)pllcabl~J
l n~ aicltlons li~l~d beln~ ill be suhmltt~ o lh~ lnlcrn~llol~3l l3(lr~3u l;llcr ~ h f~ r(li~7~ n~ L7t~o~L~ c~ o~
lli L'~' 0/ D~posi("l Accesslon number of deposit.
P nr recel~ Inu OlliC u~u nrll! I or Inlcrn;l~ n;ll L3urc;(u use onb E~shc~ r~:C~ ilh th~ lnl~rn~7lion;~ r'l'~ 3~ 0 I'hi~ r~ l h! Ih~ rll;llloll;ll 13ur-:
~UI~ ~ ulhon~(:d olticrr l orm PC'r R()'13~ (Hll! 199'7) CA 02253834 1998-ll-06 W097/42325 93/6 PCT/U',~/QC610 I~IDICATIO!~S RELATING TO A DEPOSlTEl) MICROORGANISM
(PCT Rule I il1is) A. The indications made belo~v relate to the microort~nnism reterred lo in the description on pa~c p ~
B. IDENTIFICATION OF DEPOSIT plasmid p 2 I:unher deposits ;lre idcntitied on an additionai sh~e~ ~3 Namc of depositr~,Hnstitution American Type Culture Collection Address of deposit~r! institution ~mcludlng poslal corJe anrl cormrr~J
12301 Parklawn Drive Rockville, Maryland 20852 United States of Amerlca ol'~t~l-Oi~ c~ ioll ~unlh.r 7 May 1996 (07.05.96) ( . ~DDITIONAL INDICATIONS /i.~a\. blar7L ~/ no~ pllcabl~ / I hi~ inli)rnuulon i~ conlillu_d on ;n~ nddilional shccl O
In respect of the designation of the EPO, samples of the de-posited microorganisms will be made available until the publicatio~
of the mention of the grant of the European patent or until the da-e on which the application is refused or withdrawn or is deemed to be withdrawn, -s provided ln Rule 28(3) of the Implementing Regu-lations under the EPC only by the issue of a sample to an expert nominated bv the requester (Rule 28(4~ FPC).
1). DESlG~i~TED STATES FOR ~ HICH INDlE.i~ TIONS ARE ~ DE ~ r~rlloll~ ot i~ all (/esir~nar~ r EP
E. SEPARATE FURNISHING OF INDICATIONS r/ea-L~ blan'- i¦nor opplrcablc indicDIiollslistcdbclo~ h~suhmltlc~tlolh~lnlcrnrlliollal~llrc;Jul~ r~sp~ h~ wn(r~/o/l~lel~ carlo~se~r~ c.
.~ I/r~lh~r o/ /~eposir~r Accession number of deposit.
I:or n:c~ hlL! ()tlice USi onl~ l-or In~c-n~lloll;ll L3ure~u use ~nl!; E3--s shecl ~;IS recei~e(l ~HIh Illc Intcrn;nlon~l ~ppllc;llion ~ l hl~ ShLC~ reccl~ed h! Ihe lnlern tlioll~l ~(lrc~u l~n ~~i7ed~lcer ~ uthori7ed olticer ~orm PCT/~Oil3~ (Jul! 199~
W097142325 93~7 PCTrUS96106610 INDICATIONS RELATING TO A DEPOSITED MICROORC~ANISM
(PCT ~ule 1 3bis) A. I'he indieations mude bclo~ relate to the mieroorf2amsm referred to in the dcserip~ion on pa~ ,,~P p, tc~ f~
B. IDENTIFICATION OF DEPOSIT plasmid p 7 I:unhcr deposits are identified on an ~dditionrl sheel ~3 Name of depositarv insu~ution American Type Culture Collection Addr~ss of' deposit~r~ insti~ulion ~Includin~ postal code an~l caunrr! J
12301 Parklawn Drive Roc~ville, Maryland 20852 United States of America .' 0~ d~pnslt ~ C~1011 Numbcr 7 May 1996 (07.05.96) ('. ADDITIONAL INDICATION'S ~le~\'t' hlunl; ~ ol apr~llcahll l l hi~ mlorm.llion 1~ con~ ucd oll url ~ddiliull~l shccl O
In respect of the designation of the EPO, samples of the de-posited microorganisms will be made avallable until the publicatlon of the mention of the grant of the European patent or until the da-e on which the appllcation is refused or wlthdrawn or is deemed to be withdrawn, as provided in Rul~ 28(3) of the Implementlng Regu-lations under the EPC only by the issue of a sample to an expert nominated by the requester (Rule 28(4) F.PC ) .
D. DEslG-~ ~TEl) ~ ~TE~s FoR ~ lcfl lNDlc ~Tlo~ RE ~lADE ~ o~ la EP
E. SEPARATE FD'RrNlSillNG OF INDICATIONS lle(l~ blal~ ; nol ~Ippll(abllu -lh~indic:llionslist~dhc~ lb~ubmit~dlolhcln~rnalio~ lr~lul~ltcrl~r~lc!hlll~ r(llnalllreo~t~lel~ ica~ 'C(?SSI~
.\lu~loer ol Deposl~"
Accession number of deposit.
I:or recel~nn~ ()fli;e u~ oul\ I or In~crn31l0n~1 f~ure~u use olll ~/is sh~t ~ s r~cei\ ed ~ hl~ e inlern;llion;ll ~pplie~lioll O I'hi~ ~hee( ~.n~ rccei~d h~ th~ Inlern.llioll,ll Uure;lu o Al~) ed ~fAecr () ~ ulhorii~ed o~'ficer I:orm l'CT RO 134 1 .lul~ 199' 1 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule I ~hr.~ ) A. ~r he indie3lions made below relate to Ihe mierooreanism reterred lo in the deserip~ion on pagc ~ t lS, ~ 1~
B. IDENTIFICATIONOFDEPOSIT plasmid p 9 ~urtherdepositsareidenljf~edon~nndditionalsheet O
Narne of depositary inStitNtiOn American Type Culture Collectlon Addre~s ordeposit3r~ institution (includingpos~al code and countrL~J
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In respect of the deslgnation of the EPO, samp~es of the de-posited mlcroorganisms will be made available until the publicatio~
of the mention of the grant of the European patent or until the da~e on which the application is refused or withdrawn or is deemed to be withdrawn, as provided in Rule 28(3) of the Implementiny Regu-lations under the EPC only by the lssue of a sample to an expert nominated by the requester (Rule 28~4) ~P~
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As used herein, a "K2 polypeptide" denotes a polypeptide which is 20 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 25 SEQ I:D 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), CA 022~3834 1998-11-06 W097/42325 PCTrUS96/06610 (2) polypeptides that are immllnl~logically reactive with antibodies generated by infection of a m~mm~ n host with B. burgdorferi, which antibodies are immlmologically reactive with a polypeptide of (a);
(3) polypeptides that are capable of çli~iting antibodies that are imm--nologically reactive with B. burgdorferi and the polypeptide of (a); and (4) polypeptides that are imml~nologically reactive with antibodies elicited by immllni7~tion with the polypeptide of (a).
As used herein, a "P35 polypeptide" denotes a polypeptide which is s~lected from the group consicting of:
(a) a P35 protein comprising the amino acid sequence set forth in SEQ ID
NO: 5 and serotypic variants thereof;
(b) fr~gm~nts comprising at least 8 amino acids taken as a block from the P35 polypeptide of (a);
(c) derivatives of the P3 5 polypeptide of (a) or (b), said derivatives being atleast 80% id~ntic~l 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 m~mm~ n host with B. burgdorferi which antibodies are immlmQlogically reactive with a P35 polypeptide of (a) or (b) or (c);
2 o (e) polypeptides that are capable of eliciting antibodies that are immlm- logically reactive with ~. burgdorferi and the P3 5 polypeptide of (a) or (b) or (c); and (f) polypeptides that are immunologically reactive with antibodies elicited by immllni7:~tion 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;
.
CA 022~3834 1998-ll-06 5 PCT~US96/06610 (b) fr~ement~ 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 seq~]ence to the corresponding polypeptide of (a) or (b);
(d) polypeptides that are immunologically reactive with antibodies generated by infection of a m~mm~ n 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 immllnologically reactive with B. burgdorferi and the P37 polypeptide of (a) or (b) or (c); and (f) polypeptides that are immunologically reactive with antibodies elicited by immuni7~tion with the P35 polypeptide of (a) or (b) or (c).
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 M3 0 polypeptide of (a) or (b), said derivatives being atleast 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 m~mm~ n 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 CA 022~3834 1998-11-06 (f~ polypeptides that are immunQlogically reactive with antibodies elicited by imml-ni7~tion 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 m~mm~ n host with B. burgdorferi, which antibodies are imrnunologically reactive with a polypeptide of (a) or (b) or (c);
(e) polypeptides that are capable of eliciting antibodies that are imrnunologically reactive with B. burgdorferi and the polypeptide of (a) or (b) or (c); and (f) polypeptides that are immunologically reactive with antibodies elicited by imml~ni7~ion with the polypeptide of (a) or (b) or (c) 2c 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 2 5 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 ~ and serotypic variants thereof;
CA 022~3834 1998-11-06 W 097/42325 PCTrUS96/06610 (b) fr~gment~ comprising at lezst 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% idçntic~l in amino acid sequence to the corresponding polypeptide of (a) or (b);
(d) polypeptides that are imml-nologically reactive with antibodies generated by infection of a m~mm~ n host with B. burgdorferi, which antibodies are irnmllnologically 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 immllnologically reactive with antibodies elicited by immllni7~tion with the polypeptide of (a) or (b) or (c).
As used herein, a "Jl 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 'IJ2 polypeptide" denotes a polypeptide which is selected from the group consisting of:
2 o (a) a polypeptide encoded in whole or in part by the B. burgdorferi DNA
sequence contained within ATCC deposit No. ~ and serotypic variants thereof;
(b) fr~m~nt~ 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 2 5 80% identical in amino acid sequence to the corresponding polypeptide of (a) or (b), CA 022~3834 l998-ll-06 W097/4232S PCTrUS96/06610 (d) polypeptides that are immunologically reactive with antibodies generated by infection of a m~mm~ n host with B. bu~d~"~eri, which antibodies are imml]nologically 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 imml~ni7~tion with the polypeptide of (a) or (b) or (c).
As used herein, a "J2 polypeptide" is intçn-led 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 seq~l~nce contained within ATCC deposit No.
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 ormore physical, chemical, or biological pl opel Lies has been altered. Such modifications include, but are not limited to: amino acid substitutions, CA 022=,3834 1998-ll-06 WO97/4232~ PCTrUS96/06610 _ 19 _ modifications, additions or deletions; alterations in the pattern of lipidation,glycosylation or phosphorylation; reactions of free amino, carboxyl, or hydroxylside 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 10 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 immllni7e an animal, elicits an immune response sufficient to prevent or lessen the severity for some period of time, of B. burgdorferi infection.
Preventing or lessçning 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 pres~nting cells, results in a T cell response such as clonal expansion or expression of iymphokines or other immlmnstimulatory molecules. A T cell epitope may also be an epitope recognized by cytotoxic T cells that may affect intr~cf~ r B. burgdorferi infection. A strong T cell epitope is a T cell epitope which elicits a strong T cell response.
CA 022~3834 1998-ll-06 W097/42325 PCTrUS96/06610 As used herein, a "B cell epitope" is the simplest spatial co,~ll,laLion 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 arnount that, when admini~tered 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 10 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 m~mm~ n host with B. burgdorferi. Accordingly, they are useful in methods and compositions todiagnose and protect against Lyme disease, and in therapeutic compositions to stim~ te 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. hurgdorferi, they are particularly useful in a multicomponent vaccine against Lyme disease, because such a vaccine may be formlll~ted 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.
CA 022~3834 1998-11-06 W097/42325 PCT~US96/06610 burgdorferi polypeptide. Multicomponent vaccines according to this invention mayalso contain polypeptides which characterize other vaccines useful for immlmi7~tion against ~lise~c other than Lyme disease such as, for ~,Aalllplc, diphtheria, polio, hep~titi~, and me~les. Such multicomponent vaccines are typically incorporated into a single composition.
The plt;felled compositions and methods ofthis invention comprise novel B. burgdorferi polypeptides having enh~n-,ed immunogenicity. Such polypeptides may result when the native forms of the polypeptides or fra~ments thereof are modified or subjected to lr~ ,l.c to enhance their imml~nQgenic character in the int~.n-~ed recipient.
Numerous techniques are available and well known to those of skill in the art which may be used, without undue experimçnt~tion, to substantially increase the imm~lnogenicity of the novel B. burgdorferi polypeptides herein disclosed. For example, the polypeptides may be modifled by coupling to dinitrophenol groups or arsanilic acid, or by denaturation with heat and/or SDS.Particularly if the polypeptides are small polypeptides synthe~i7ed 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 M~n~l~l Cold Spring Harbor Laboratory, ed. E.
Harlow and D. Lane (1988). Useful immllnogenic 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 25 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 imm~lnogenicity and biochemical properties.
CA 02253834 1998-ll-06 WO 97t42325 PCT~US96/06610 For example, the polypeptides or fraEnl~nt~ thereof may be expressed with or without the signal sequences that direct addition of lipid moieties.
As will be appalenl from the disclosure to follow, the polypeptides may also be prepared with the objective of increasing stability or rendering themolecules more amenable to purification and prepa, ~lion. 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 prepal ed by a variety of methods, inrhl-ling by in lo vitro manipulation of the DNA encoding the native polypeptides and subse~uent 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 di~enl natural amino acid, an amino acid derivative or non-native amino acid, conservative substitution being plef~lled, e.g, 3-methylhistidine may be substituted for histidine, 4-hydroxyproline may be substituted for proline, S-hydroxylysine may be substituted for Iysine, 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 agiven 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.
WO g7142325 PCTrUS96/06610 In a p.~re,~ed embodiment of this invention, the novel B.
burgdorferi polypeptides disclosed herein are prepared as part of a larger fusion protein. For e,~a.llple, a novel B. burgdorferi polypeptide of this invention may be fused at its N-terminus or C-terminus to a di~el ~nL immunogenic B. burgdorferi polypeptide, to a non-B. burgdorferi polypeptide or to combinations thereof, to produce fusion proteins COI~l~)l ising 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 di~ering from another with respect to the locations or sequences of the epitopes within the polypeptide. In a more pl~re.. ~d embodiment, fusion proteins are constructed which comprise one or more of the novel B. burgdorferi polypeptides fused to other immllnogenic 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. burgdor~eri 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 thepolypeptide. 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 te~ching~ 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 te~ching~ herein or by other methods known to those of skill in the art.
It is preferred that the fusion proteins comprising the novel ~.
burgdorferi polypeptides be produced at the DNA level, e.g., by constructing a nucleic acid molecule encoding the fusion, transforming host cells with the CA 022~3834 1998-ll-06 W097/42325 PCTrUS96/06610 molecule, ind~lcing 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 t;..l,ression accolding to known methods The novel B. burgdorferi polypeptides may also be part of larger 5 mllltim~ric molecules which may be produced recombinantly or may be synthesized chemically. Such mllltimers may also include the polypeptides fused or coupled to moieties other than amino acids, incllldin~ 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 10 randomly, or with spacers (amino acid or otherwise) between them.
In the most p- ~re. I ed embodiment of this invention, the novel B.
burgdorferi polypeptides of this invention which are also immunQgenic B.
burgdorferi polypeptides are incorporated into a multicomponent vaccine which also comprises other immllnogenic B. burgdorferi polypeptides. Such a 5 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 di~erenl 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 2 o 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. burgdorferipolypeptide and one previously identified B. burgdorferi polypeptide, wherein each 2 5 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 WO 97/42325 PCTrUS96/06610 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 imm-ln~globulin light chain, and could be produced by transforming a host cell with DNA encoding the heavy chain filsion 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 appropliately.
Alternatively, the heavy and light chain filsions could be produced from separate cell lines and allowed to associate after purification.
The desirability of inchlAinE 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 immlmogenic B. burgdorferi polypeptides, including the novel B.
b2~rgdorferi 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 ~dmini~tered to an animal via a liposome delivery system in order to enh~nce their stability and/or immllnQgenicity Delivery of the novel B. burgdorferi polypeptides via liposomes may be particularly advantageousbecause the liposome may be intemalized by phagocytic cells in the treated animal.
Such cells, upon ingesting the liposome, would digest the liposomal n~elllbl~le 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 llnil~mellat vesicles, multilamellar vesicles, or stable plurilamell~r vesicles, and may be prepared and aAmini.~tered according to methods well known to those of skill in the art, for example in accordance with the teaching~ 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 CA 022~3834 1998-ll-06 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 5 be used in the form of a pharm~ceutic~lly 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 polypeptidesthat are pl ere" ~d 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 20 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 mllltim~ric proteins that are pl~f~l,ed 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 prerelled polypeptides, fusion proteins and multimeric proteins for use in these compositions and methods are those cont~inin~ both strong T cell and B cell epitopes. Without being bound by theory, we believe that this is the best way to stim~ te high titer antibodies that are effective to neutralize . . .
CA 022~3834 1998-11-06 W097/42325 PCT~US96/06610 B. burgdorferi infection. Such prer~l I ed 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 ~ cells will recognize the T cell epitope(s) and respond by proliferating and producing Iymphokines which in turn cause B cells to di~l ~lltiate into antibody producingplasma 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 incllldes high titer antibodies against the novel B. burgdorferi polypeptide.
One of skill in the art will also understand that it may be advantageous to ~(lminister the novel B. burgdorferi polypeptides of this invention in a form that will favor the production of T-helper cells type 2(TH2), which help B
cells to generate antibody responses. Aside from ~(lmini.ctering epitopes which are strong B cell epitopes, the induction of TH2 cells may also be favored by the mode of ~dmini.~tration ofthe polypeptide for example by admini~tering in certain doses or with particular adjuvants and imm~n~modulators, for example with interleukin-4.
To prepare the pr~r~lled polypeptides ofthis invention, in one embodiment, overlapping fr~gmPnts 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 ( I ) 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 immuni7e 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.
CA 02253834 1998-ll-06 W O97/42325 PCTrUS96/06610 As recognition of T cell epitopes is MHC restricted, the polypeptides that contain T cell epitopes may be identified in vi~ro by testing them for their ability to stim.ll~te proliferation and/or cytokine production by T cell clones generated from humans of various HLA types, from the Iymph nodes, spleens, or peripheral blood Iymphocytes of C3H/He mice, or from domestic animals. Compositions comprising multiple T cell epitopes recognized by individuals with di~elen~ Class II
~ntigçn.c are useful for prevention and tre~tment of Lyme disease in a broad spectrum of patients.
In a pl ~re~l ~d embodiment of the present invention, a novel B.
lo burgdorferi polypeptide cont~ining a B cell epitope is fused to one or more other im m~nogenic 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. burgdorfeff.
Strong T cell epitopes may also be provided by non-B. burgdorferi molecules. For example, strong T cell epitopes have been observed in hepatitis Bvirus core antigen (HBcAg). Furthermore, it has been shown that linkage of one of these egm~nts to segments of the surface antigen of Hepatitis B virus, which arepoorly 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 segment.~ 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 CA 022~3834 1998-11-06 W 097/4232S PCT~US96/06610 invention to a strong immlln~ gen that is also widely recognized, for example tetanus toxoid.
It will be readily appl e~;ated by one of ordinary skill in the art that the novel B. burgdorferi polypeptides of this invention, as well as fusion proteins 5 and mllltim~ric proteins col~ e them, may be prepared by recombinant means, ~h~mic~l meàns, 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 primersderived from the sequences herein disclosed.
In this regard, it may be particularly desirable to isolate the genes encoding novel B. burgdorferi polypeptides from strain 2501 S 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. burgdorferistrain 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 infectionwith strain N40, appear ineffective to protect against infection with strain 25015.
2 o 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 relatedspirochetes 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. burgd~orferi polypeptides of this invention are produced recombinantly, they may be expressed in unicellular hosts. As is well W097/42325 PCTrUS96106610 known to one of skill in the art, in order to obtain high t;A~ures~ion 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. Pler~l~bly, the eA,u~es~ion control sequences, and the gene of 5 interest, will be contained in an e ,~pression vector that further comprises a selection marker.
The DNA sequences encoding the polypeptides of this invention may or may not encode a signal sequPnce. If the expression host is eukaryotic, it generally is pl erel l ed 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 teç~ln~ es A wide variety of expression host/vector combinations may be employed in ~A~,.essillg 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, inc~ 1in~ pBluescript, pGEX-2T, pUC vectors, col E1, pCRl, pBR322, 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. AGT10 and ~GT11, and other phages. Useful expression vectors for yeast cells include the 2plasmid 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 ,, CA 022~3834 1998-11-06 W097/42325 PCT~US96/06610 invention. Such useful eAI)I ession control sequences include the ~I.ression control sequen~es associated with structural genes of the foregoing eApression vectors.
Examples of useful expression control sequences include, for example, the early and late promoters of SV40 or adenovin~s, the 1~ system, the ~ system, the ~ or ~ 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 phosph~t~.ce, e.g., Pho5, the promoters of the yeast a-mating system and other constitutive and inducible promoter sequences known to control the eAI.)I es~ion of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
ln a preferred embodiment, DNA sequences encoding the novel B.
burgdorferi polypeptides of this invention are cloned in the expression vector lambda ZAP II (Str~t~g~ne, 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 e~pl ession vector that allows high level ~,~p,ession 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 c"~ressi,lg the DNA sequences of this invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, 2 5 Streptomyces, fungi, yeast, insect cells such as Spodoptera frugiperda (SF~), 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.
CA 022~3834 1998-ll-06 W097t4232S PCT~US96/06610 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 e~ es~ion system. However, one of skill in the art may make a selection among these vectors, e,~l)ression control sequences and hosts ~vithout undue expelh,l~lltalion 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 t~AI~l es~ion 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
seql1ences 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 conventionalmethods including: liquid chromatography such as normal or reversed phase, usingHPLC, FPLC and the like; affinity chromatography (such as with inorganic ligandsor monoclonal antibodies); size exclusion chromatography; immobilized metal CA 022~3834 1998-11-06 W 097/42325 PCTrUS96/06610 chelate chromatography; gel electrophoresis; and the like. One of skill in the art may select the most ap?rop-iate 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 chernical techni~ues. For example, they may be prepared using the solid-phase synthetic techni~ue 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 prepa-ed by synthesis in solution. A
summary of peptide synthesis techniques may be found in E. Gross & H. J.
1C Meinhofer, 4 The Peptides: Analysis, Synthesis, Biology; Modern Techniques OfPeptide And Arnino Acid Analysis, John Wiley & Sons, (1981) and M. Bodanszky, Principles Of Peptide Synthesis, Springer-Verlag (1984).
Typically, these synthetic methods comprise the sequential addition of one or more amino acid residues to a growing peptide chain. Often peptide 5 coupling agents are used to facilit~te 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 .ii~lel,l protecting group is utilized for amino acids cont~ining 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 immllnoglobulin molecules or portions thereofthat are im~nunologically reactive with a novel B. burgdorferi polypeptide of the presentinvention. It should be understood that the antibodies of this invention include CA 022~3834 1998-ll-06 W097/42325 PC~rUS96/06610 antibodies immunologically reactive with fusion proteins and mllltimPric proteins co~ ,is;ilg a novel B. burgdorferi polypeptide.
Antibodies directed against a novel B. burgdorferi polypeptide may be generated by a variety of means in~.lu~line infection of a m~mm~ n host with B. burgdorferi, or by immuni7:~tion of a m~mm~ n host with a novel B.
burgdorferi polypeptide of the present invention. Such antibodies may be polyclonal or monoclonal, it is p~ ~rel I ~d that they are monoclonal. Methods to produce polyclonal and monoclonal antibodies are well known to those of skill inthe art. For a review of such methods, see An~ibodies, A Laborafo~ Manual, lo supra, and D.E. Yelton, et al., Ann. Rev. of Biochem.~ 50, pp. 657-80 (19~1).
Determination of immllnnreactivity with a novel B. burgdorferi polypeptide of this invention may be made by any of several methods well known in the art, includingby immllnoblot assay and ELISA.
An antibody of this invention may also be a hybrid molecule formed from imm-mclglobulin sequences from di~erel.L species (e.g., mouse and human) orfrom portions of immllnoglobulin light and heavy chain sequences from the same species. It may be a molecule that has multiple binding specificities, such as abifunctional 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.
W097/42325 PCT/U'53.'~GC10 ln 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 te~çhin~.c of this 5 invention, has available a variety of methods which may be used to alter the biological p~upel~ies ofthe antibodies ofthis invention inrlllding methods whichwould 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 infectiûn. For example, the level of B. burgdorferi in infected ticks may be decreased by allowing them to feed on the blood of animals immllni7ed with the 5 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 2 0 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 2 5 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 prerel ~ ~d 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.
CA 022~3834 1998-ll-06 WO 97/42325 PCT~US96/06610 burgdorferi infection but are also afflicted with clinical symptoms of a disease that is remarkably similar to Lyme disease in humans. Thus, by a~mini~tering a particular polypeptide or antibody to C3H/He mice, one of skill in the art may determine without undue e~cli~ne~ tion whether that polypeptide or antibody 5 would be useful in the methods and compositions claimed herein.
The a~imini~tration 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 ~timinictered with a pharmaceutically acceptable adjuvant, such as complete or incomplete Freund's adjuvant, RIBI
(muramyl dipeptides) or ISCOM (immunostim~ ting complexes). Such adjuvants may protect the polypeptide from rapid dispersal by sequestering it in a local deposit, or they may contain substances that stim~ te 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 immnni7~tion schedule will involve two or more ~mini.~trations 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 pharrnaceutical 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 tablels, pills, powders, liquid solutions or suspensions, liposomes, capsules, suppositories, injectable and infusible solutions.
CA 02253834 1998-ll-06 The pleÇ~Iled form depends upon the intçn-1ed mode of atlmini~tration and prophylactic application.
Such dosage forms may include pharm~cel~tically acceptable carriers and adjuvants which are known to those of skill in the art. These carriers and adjuvants include, for exarnple, RIBI, ISCOM, ion exchangers, all-min~ lminl]m 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 prota,llllle sulfate, disodium hydrogen phosphate, sodium chloride, zinc salts, 10 colloidal silica, m~ene~ium tri~ilicate, 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 glycol1 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 enhancetheir immunogenic character or to improve their tolerance in patients.
Compositions comprising an antibody of this invention may be a~minictçred by a variety of dosage forms and regimen.c similar to those used for 2 o other passive immunotherapies and well known to those of skill in the art.
Generally, the novel B. burgdorferi polypeptides may be formul~ted and a~mini.ct~red 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, intr~mll~c~ r, intralesional or subcutaneous injection, may be used to a(lmini.~ter the polypeptide or antibody composition. For example, the composition may be arlminictered to the patient in any pharmaceutically acceptable dosage form CA 022=,3834 1998-ll-06 in~.lutline those which may be ~(lminictered to a patient intravenously as bolus or by continued infusion over a period of hours, days, weeks or months, intramllcc~ rly -- incl~lrling paravertebrally and periarticularly -- subcutaneously, intr~c.ut~neously, 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 a~lminictered to the patient intr~m-lscul~rly.
The novel B. burgdorferi polypeptides or antibodies of this invention may be a~mini.ctçred to the patient at one time or over a series of Lle~l~,.e~,lc. The 1 c most effective mode of a~l".lnic~ ion and dosage regimen will depend upon the level of immunogenicity, the particular composition and/or adjuvant used for tre~tm~nt, the severity and course of the expected infection, previous therapy, the patient's health status and response to imm~ni7.~tion~ 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 immlmi7~tions Similarly, the dosage and necçcs~ry treatment time will be loweredif the polypeptide is ~flmini.ct~?red with an adjuvant. Generally, the dosage will consist of 10 ,ug to 100 mg of the purified polypeptide~ and preferably, the dosage will consist of 10-1000 ~lg. Generally, the dosage for an antibody will be 0.5 mg-3.0g.
In a p,~rel led embodiment of this invention, the novel B.
burgdorferi polypeptide is ~r~minictered with an adjuvant, in order to increase its imm~ln~,genicity. Useful adjuvants include RIBI, and ISCOM, simple metal salts such as al--min~lm hydroxide. and oil based adjuvants such as complete and incomplete Freund's adjuvant. When an oil based adjuvant is used, the polypeptide usually is a-lminictered in an emulsion with the adjuvant.
In yet another preferred embodiment, E.coli expressing proteins comprising a novel B. burgdorferi polypeptide are at~mini~tered orally to non-CA 022~3834 1998-11-06 W 097/42325 PCTrUS96/06610 human animals to decrease or lessen the severity of B. burgdorferi infection. For example, a palatable regimen of bacteria c, I~l essh~g a novel B. burgdorferi polypeptide, alone or in the form of a fusion protein or multim~ric protein, may be ~tlminictered with animal food to be consumed by wild mice or deer, or by domestic animals. Ingestion of such bacteria may induce an immllne response comprising both humoral and cell-me~i~ted components. See J.C. Sadoffet al., "Oral Salmonella ~yphimurium Vaccine Expressing Circumsporozoite Protein Protects Against Malaria", Science. 240,pp.336-38 (1988) and K.S. Kim et al., "Tmmllni7~ion Of Chickens With Live Esc*erichia coli E~pres~ing Eimeria acervulina Merozoite Recombinant Antigen Induces Partial Protection Against Coccidiosis", Inf. Immun., 57, pp. 2434-40 (1989). In fact, oral vaccination with bacteria ~ ,ressing OspA has been shown to be effective. See, M. Dunne et al., "Oral Vaccination Against Lyme Disease Using Salmonella Expressing OspA," I~
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.
Dis., 164:1224(1991). Moreover, the level of B. burgdorferi infection in ticks feeding on such animals will be lessened or eliminated, thus inhibiting tr~n~mis~ion 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 ~nim~l~ inçlu~ing humans that are infected with B. burgdorferi, and the antibodies are capable of binding to B. burgdorferi or 25 ~nti~n~ 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 CA 022~3834 1998-ll-06 W097/42325 PCTrUS96/06610 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 I or Cr that produces gam na ray emissions, or a radioactive element that emits positrons which produce gamma rays upon encounters with electrons present in the test solution, such as C, O, or 13N.
10 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-cont~ining amino acids in the culture m~-1inml 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 solutionform or as a substantially dry powder~ e.g., in Iyophilized form.
Novel B. burgdorferi polypeptides and antibodies directed against those polypeptides provide much more specific diagnostic reagents than whole CA 022~3834 1998-ll-06 W 097/4232S PCT~US96/06610 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 theinfected 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 Iysates 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, inclur~ing 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 P2 1 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 cont~ining multiple epitopes which are reactive with antibodies appearing at di~Terenl times are useful to detect the presence of anti-B. 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 other2 5 spirochetes include Borrelia Hermsii and Borrelia Recurientis, I,ep~ospira, and Treponema.
According to another embodiment of this invention, we describe a method for identifying bacterial genes encoding an antigenic proteins that are CA 022~3834 1998-ll-06 W097t42325 PCT~US96/06610 expressed during infection of a host but that are not expressed during in vitro culture of the bacteria, the method co~ ,-ish~g the steps of:
(a) constructing an e~ ess.on library for the bacteria;
(b) SCIeel~~ng the library with antisera from an animal infected with the bacteria;
(c) screening the library with antisera from an animal immlmi7ed with non-viable bacteria or components thereof; and (d) identifying clones that react with the first antisera but not with the second antisera.
1C It will be readily appare-ll 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, inchl~ling plant and animal hosts. In a ple~el-ed embodiment, the host is a m~mm~l 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.
CA 022~3834 1998-11-06 W097/42325 PCTrUS96/06610 As used herein, components of non-viable bacteria include Iysates, homogen~tes, or subcellular fractions thereof such as cell membrane cont~ining 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 libr~y 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 20 by centrifugation at 16,000 rpm for 30 minlltes~ and Iysed 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 llg of DNA was randomly sheared, blunt-ended with S 1 nuclease, and the EcoRI sites were methylated with EcoRl methylase. EcoRl linkers were then ligated to the ends of the DNA molecules, theDNA was digested with EcoRl and the fr~gm~ntc were purified over a sucrose CA 022~3834 1998-11-06 W097/42325 PCTAUS96tO6610 gradient. Fragments of 1 to 9 kb were isolated and ligated to EcoR1 ~igested Lambda ZAP II arms.
We prepared E. coli SU~ bacteria (Stratagene) for phage infection as follows. We picked a single colony into LB media suppl~mçnted with 0.2%
5 maltose and 10 mM magnesium sulfate and cultured overnight at 30~C with vigorous ~h~king 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 1 n We prepared anti-B. burgdorferi N40 antisera for differentially screening the expression library as follows.
1 . Tmml Ine Anti~ra We prepared "immllne" mouse anti-B. burgdorferi N40 antiserum as follows. We injected 3 to 5 groups of five three-week old female C3h/HeNCr or J
5 (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.
20 b2lrgdorferi in incomplete Freund's adjuvant (rFA) 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. coli/phage Iysate (Stratagene) 2 5 as follows. We diluted the Iysate 1 :10 in Tris-buffered saline with 0. 05% Tween-20 (TBST). We then incubated 0.45 ~M pore size nitrocellulose filters (Millipore, Bedford, MA) in the Iysate for 30 minutes at room temperature, removed and air . _ CA 02253834 1998-ll-06 W O97/42325 PCT~US96/06610 dried the filters on Whatman filter paper (Whatman International Ltd., Maidstone, F.n~1~nd), and washed 3 times (5 minutes each) with TBS. We blocked the filters by irnmersing in 1% Bovine Serum Albumin (BSA) in TBS for I hour at room te.n,~elal~lre and rinsing 3 times with TBST. We then diluted the mouse antiserum 5 1:5 in TBST, incubated it with the filters with shaking for 10 minutes at 37CC, and removed and discarded the filters.
2. lnfected Antisera We injected three C3H/HeJ mice by intradermal inoculation with 104 B. burgdorferi N40 spirochetes. We docum~nted infection by culturing spirochetesfrom the spleen, bladder and skin (ear punches) of the challenged mice and by histopathologic examination of the joints and heart for evidence of infl~mm~tionWe collected serum from the infected mice at various times a~er infection Both immlln~ and infected antisera contained a high titer of anti-B.
burgdorferi antibodies directed against whole cell Iysates. We detected antibodies in the sera of immllni7ed 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 difre, e.
intensities.
After absorption, we diluted the antiserum to a final dilution of 1:100 and used it to screen the nitrocellulose filters cont~ininE the expressed proteins from the lambda ZAP library according to m~nuf~ctllrer's instructions.
C. Differential Screening of A Genomic B. burgdorferi N40 Expression Library To screen the library, we used the picoBlue Immunoscreening Kit (Stratagene). We plated 4 x 104 plaque forming units of recombinant phage on a lawn of bacteria, induced protein expression with 1 OmM IPTG and transferred the CA 022~3834 1998-ll-06 W O97142325 PCTrUS96/06610 proteins to duplicate plaque lifts on nitrocellulose filters according to methods well known in the art.
We incllb~ed one set of plaque lifts with pooled sera from mice imml-ni7ed with heat-killed spirochetes (immllne sera) and the other set with sera from rnice infected for nine months (infected sera). A~er 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 10 infected sera but not with immune sera for further study.
Example II - Cloning ofthep21/k.? Operon Di~l e:nLial screening of a B. burgdorferi N40 genomic ~Ayression library, as described in Example I, revealed one hundred seventy-two clones thatreacted with sera from the infected mice and one hundred sixty-nine clones that reacted with sera from immllni7ed mice. We subjected the three phage clones thatreacted di~l en~ially 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 m~nllf~ctllrer'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
2~ insert of clone I with the Erase-A-Base System (Promega, Madison, WI) (using SmaI 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, WO 97/42325 PCT~US96/06610 C~leveland, OH) and recolls~ cted the entire sequence using MacVector (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 Fnel~n~l Biolabs). Conditions for denaturation, ~nne~ling 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 cont~ining a complete open reading frame and a partial open reading frame having the sequ~nce set forth in SEQ ID NO: 1. We conducted a 10 search of GenBank (December 1994) with the Genetics Computer Group Program (University of Wisconsin Biotechnology Center, Madison, W~). Our search revealed that we had isolated a novel, bicistronic B. burgdorferi operon. We de~ign~ted the complete open reading framep21 and the partial open reading framek2. We d~sign~ted the ~ntig~ni 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, thep21 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 ded~lced amino acid seqllence of P2]
2 o contains a typical prokaryotic signal sequence for posttranslational processing by cleavage and lipidation, s~ggesting that the gene product is a lipoprotein of apl)lox""ately 20.7 kDa. P21 has 71% amino acid sequence identity to B.
burgdorferi OspE (Figure 7).
The ATG start codon for the k2 gene is located 27 nucleotides downstream of the TAG stop codon of the p21 gene. The k2 gene in clone I
contains a partial open reading frame of 32 nucleotides, capable of encoding thefirst 10 amino terminal amino acids (SEQ ID NO: 3). However, based on the last CA 02253834 1998-ll-06 W 097/42325 PCTrUS96/06610 two nucleotides of the K2 sequ~nr,e 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 11-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 up~Llea"~ ofthis translational initiation sequence are the promoter segments 10 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 I I bp upstream of the ATG start codon of the k2 gene. The location of these sequence elements suggests that both thep21 and k2 genes are controlled by a single promoter. The homology of P2 1 and K2 toOspE 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 arnino 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 Microbiolo~y and Immunology, 125, pp. 127-157 (1986)].
CA 022~3834 1998-11-06 WO 97/42325 PCT~US96/06610 The carboxyl terminus of the hydrophobic domain contains a cleavage site presumably recognized by a B. burgdorferi signal peptidase. In P2 1, as in OspF, the potential cleavage site is located between Serl7 and Cysl8.
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. Wu et al., supra~ Indeed, the OspA and OspB genes of B. burgdorferi B3 1 contain signal sequences of -L-I-A-C- and -L-I-G-C-, respectively [S. Bel g~ll o,n et al., "Molecular Analysis of Linear Plasmid-Encoded Major Surface Proteins, OspA and OspB, of the Lyme Disease SpirochaeteBorreliaburgdorferi", 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-B3 1 (-L-S-I-S-C-) genes, contains three amino acids between the leucine and cysteine instead oftwo. (See R.S. Fuchs et al. and S.~.
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 2 o residue could serve to anchor the protein to the outer surface of the spirochetes (see H.C. Wu and M. Tokunaga, supra).
Finally, P2 1 contains a long hydrophilic domain separated by short stretches of hydrophobic segments.
A comparison of the DNA sequences indicates that p21 and ospE
2 5 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 of p21 and ospE are identical upstream from the -10 sequence tothe boundary ofthe 5' fl~nkin~ DNA which has been sequenced (189 nt 5' ofthe CA 022~3834 1998-ll-06 - SO -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 di~el~nces 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' untran~l~ted region ofp21 mRNA.
In view of this homology between P2 1 and OspE, one of skill in the art would understand that in form~ ting therapeutic and diagnostic compositions, it may be desirable to select epitopes of P21 that do not cross-react with OspE.
1C Example IV - Analysis ofp21 Expression In Cultured B. burgdorJeri By Northern Blotting To determine whetherp21 is transcribed during in vitro culture of spirochetes, we assessed its ~ es~ion by Northern blot analysis. We isolated total RNA from cultured B. burgdorferi by acid guanidium thiocyanate/phenol/
chloroform extraction [cite]. We electrophoresed 20 llg of isolated RNA in a 1%
formaldehyde-agarose gel and blotted onto Hybond-N~ membrane (Amersham).
We generated biotinylated p21 and ospA (contro1) probes with a Phototope~
random-primer biotin-labeling kit (New England Biolabs). Thep21 and ospA
probes contained the entirep21 and ospA sequences, respectively. We used amplified PCR products ofp21 or ospA as templates for the random octamer-primed labeling reaction.
We condllctçd hybridization and signal detection with a Photope~
ch~mill-minescçnt kit (New Fn~l~nd Biolabs). Briefly, we prehybridized the blotted membrane in SSC for 1 hour at 68~ C and hybridized with biotinylated probes for p21 or osp,4 (control) at 68~ C overnight. We washed the membrane at a final stringency of 0.1X standard saline citrate (SSC)/0.1% SDS at 68~ C. We detected CA 02253834 1998-ll-06 WO 97/42325 PCTrUS96/06610 biotin-labeled probe by a series of inc~bations with streptavidin, biotinylated alkaline phosphatase, and lumigen-PPD.
We detected ospA RNA but nop21 RNA from cultured B.
burgdorferi.
Example V - Southern Dot Blot Analysis and PCR of Cultured ~. burgdorferi Genome Because in vitro culture of B. burgdorferi is often associated with the loss of genes or pl~smi(lc [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 ofthep21 gene A. Southern Dot Blot Analysis For dot blot analysis, we spotted 2 ~g of denatured 1 phage (control) or cultured B. burgdorferi DNA onto Hybond-N~) membrane. We first stained the dried membrane with ethi~illm bromide to confirrn 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, conrll llling the presenceof the p21 gene in the cultured B. burgdorferi .
B. PCR ~n~lysis 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: 11 and SEQ ID NO: 12, respectively. These primers are specific forp21 and do not amplify osp~ We used the following conditions for PCR of cultured B. burgdorferi DNA: 30 cycles with denaturing, ~nne~l;n~e and 2 5 extension temperatures of 94~ C for I min., 65 ~ C for 1 min., 72 ~ C for 2 min., respectively.
CA 022~3834 1998-ll-06 WO 97/42325 PCTrUS96/06610 Using these primers, we obtained a 513 bp PCR product ofthep21 coding region, further co~ lling that the p21 gene is present in the genome of the cultured B. burgdorferi used for Northern blot studies.
Example VI - E~min~tion of p21 Expression By B. burgdorferi In Ticks To determine whether P2 1 is expressed by B. burgdorferi in Ixodes ticks, we exarnined Iysates of flat and engorged ticks cont~ining the spirochetes by indirect immllnofluorescence. Using the same methods, one of skill in the art could readily deterrnine without undue experimçnt~tion whether other novel B.
lo burgdorferi polypeptides of this invention are expressed in ticks.
Briefly, we allowed B. burgdorferi N40-infected ticks to feed to repletion on C3HlHe mice. We lightly homogenized each unfed and engorged tick in 100 1ll PBS and spotted a 10 ~1 aliquot onto a sylilated glass slide. We air-dried the slides and fixed them with 4% pal~fo-l-laldehyde and saponin. We incubated the specimens in a 1:10 dilution of antisera from mice immllni7ed with the P21-specific peptide prepared as in Example VII and as shown in SEQ. ID NO:_, for I
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-fl~g~llin monoclonal antibody H9724, which recognizes B. burgdorferi in both flat and engorged ticlcs as a second positive control [Cite]. We used anti BSA sera as a negative control.
Con~ictçntly 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 CA 02253834 l998-ll-06 W 097/42325 PCT/U55~ 10 ticks. However, no P21-specific immllnofluorescence was detected in either flat or engorged ticks.
To confirm that the P2 1 -specific antisera could react with P2 1, we used the antisera to probe lecol.lbinalll P21, prepared as in Example 12, or reco,nl,inallt OspE. As expected, P2 1 antisera readily recognized recombinant P2 1 but not OspE. These results indicate that P21 is also not expressed in infected ticks.
Example VlI - Co,.r~-~l-aLion of p21 Expression in Infected Mice By Dot Immunoblot Analysis and R~A-PCR
1 c A. Dot Tmmunoblot Analysis We next confirmed that p21 is expressed in mice infected with B. burgdorferi by demonstrating the presence of antibodies against P2 1 in sera from two infected mice.
We compared the amino acid sequences of P2 1 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 isset forth in SEQ ID NO: 13.
We spotted 3 ,ug of BSA or the synthetic P21-derived peptide coupled to BSA onto nitrocellulose n~elllbl~nes We incubated the dried membranes with either serum from mice immllni7ed with heat-killed B. burgdorferior serum ~rom infected mice. We detected bound antibody by incubating with a second antibody conjugated to horseradish peroxidase (ECL Western blot detectionsystem, 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.
CA 022~3834 1998-ll-06 Sera from infected mice but not from mice imm~lni7ed with heat-killed B. burgdorferi reacted with the P2 1 peptide. Thus, P2 1 is selectively expressed in vivo.
B. RNA PCR
We further demonstrated expression of p21 in infected mice using RNA PCR to detectp21 RNA. We used acid guanidium thiocyanate/phenoV
chloroform extraction (Micro RNA Isolation Kit, Stratagene) to isolate total RNAfrom spleens of the mice infected with B. burgdorferi via tick tr~n~mi~ion 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 llg of pooled RNA with RNase-free DNase (Promega) for 3 hours at 3 7 ~
C with HPRI and the Rnase inhibitor. We conducted the RNA PCR with and without reverse transcriptase to exclude the possibility that residual DNA mightalso be amplified. We synthçsi7e(1 cDNA by reverse transcription with Moloney murine leukemia virus reverse ll~ns~ ase (Stratagene) and 3' primers for either p21 (murine tissue and cultured B. burgdor~eri), ~-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 S' primer forp21, y-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 of p21 only in the presence of reverse ~l ~ns~ ase. To confirm the identity of the amplified product as p21, we denatured and electrophoresed RNA PCR products, transferred them to Hybond-N~ membrane and hybridized withp21 probes as described in Example IV
for Northern blot analysis. The absence of product without reverse transcriptaseconfirms that DNA was not amplified. We obtained no amplification withp21-specific primers from RNA prepared from uninfected mice or from RNA PCR of B.
burgdorferi cultured in vitro.
CA 022~3834 1998-ll-06 W097t42325 PCT~US96/06610 Example VIII - Sequence An~lysis of the p35 ~ntl p37 Genes - We dirrel enlially screened the lambda Zap II B. burgdorferi expression library exactly as described in Example I but using sera from mice immllni7ed 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 immuni~ed withheat-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.
10 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 design~ted p35. We desi~n~ted the antigen 5 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 de~i~n~tedp37. We design~ted 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 Ser,g and Cys2" 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 CA 022~3834 1998-11-06 W097/42325 PCTrUS96/06610 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 upsllean~ of the respective open reading frames.
Example IX - Mapping of the p21. p35 and p37 Genes We mapped the p21, p35 and p3 7 genes by pulsed-field electrophoresis (PFGE) with total B. burgdorferi N40 DNA using a modification ofthe 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 cont~ining ap,()loxilllately 10 B. burgdorferi N40 with sarkosyl, Iysed 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 radiolabelledp21, p35, p37 probes. We usedp30, ospA and ospD probes as controls in the Southern blot. We generated p35 andp37 probes labeled with [a-32P]dCTP, using the Prime-lt~ random primer kit according to the m~mlf~ctllrer'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, "LinearPlasmids of the Bacterium Borrelia burgdorferi Have Covalently Closed Ends", Science, 237, pp. 409-411 (19~7) and S.J. Norris et al., supra]. Thep30 probe identified the chromosome. The full-lengthp21 probe bound at three locations but CA 022~3834 1998-11-06 WO 97/42325 PCT~US96/06610 ap21-specific probe (SEQ ID NO: 14) recognized a circular plasmid. The P35 probe bound to a plasrnid 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.
5 Example X - Analysis of Cultured B. burgdorferi Forp35 orp37 Expression To determine whetherp35 orp37 are transcribed in vitro, we perforrned 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 (forp35) and 1C in SEQ ID NOS. 17 and 18 (for p37).
The results ofthese analyses confirmed that p35 andp37 are transcribed in vitro.
Example XI - Confirrnation of p35 And p37 Expression In Infected Mice by ~mmunoblot An~lysis 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 p3 7 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 OspA-Mediated lmmunity to Lyme Borreliosisl', J. lmmunol, 147, pp. 1995-2000 (1991)]. The PMX vector also contains a thrombin cleavage site immedi~tely CA 02253834 1998-ll-06 following the GT protein, thus, allowing the recovery of r ecolllbina.ll 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 s ensure that the polypeptide would be expressed as soluble fusion protein rather than as a lipoprotein, which would be anchored to the cell Ille.nb.~ne or might aggregate elsewhere in the cell during or after biosythesis.
To f~çilit~te subcloning, we amplified the genes using primers with additional restriction enzyme digestion sites. We amplified thep21 gene using a 5' primer with an additional BarnHI 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 XhoI restriction enzyme digestion site and a 3' primer with a supplementary HindIII site [SEQ ID NO: 23 and 24].
We amplified thep37 gene using a 5' primer with an additional BamHI restriction enzyme digestion site and a 3' primer with a supplementary XhoI
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 I minute, ~nn~ling at 40~C for 2 minutes and extension at 72~C for 3 minlltes.
We digested the amplified gene products with BamHI ~721), XhoI
and Bam HI (p35) or Hind III and XhoI ~D37) and cloned onto the corresponding sites in the PMX plasmid. We then used the ligation mixture to transform Escherichia coli DH5a according to methods well known to those of skill in the art. We isolated colonies cont~ining 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 logaliLhlllic phase and W097t42325 PCT~US96/06610 adding 1 mM isopropyl-l-thi-beta-galactoside (IPTG) for 3 hours. One of skill inthe art could readily express the other B. burgdorferi polypeptides of this invention without undue experim~nt~tion following the above-described te~hniques.
Example XIII - Pnr;fication of Recoll,binarll Fusion Protein~
After in~ çing protein expression as described in Example XI, we placed the ~. coli in phosphate buffered saline (PBS) with 1% Triton and subjected them to sonication. We purified the glutathione S-transferase-B burgdorferl polypeptide fusion proteins (GT-P21, GT-P35, GT-P37 and GT-M30) from cell Iysates as follows.
We separated the cell supernatant and pellet by centrifugation at 1000x for 8 mins and passed the supernatant cont~ining the recombinant fusion proteins over a glutathione-Sepharose 4B column (Pharmacia) according to the m~nl~f~ctnrer's instructions. We eluted the fusion protein from the column using a solution cont~ining excess glutathione and quantified using the Bradford assay.
In addition, to purify the B. burgdorferi proteins without the glutathione S-~-~ns~e-ase, we loaded the glutathione S-transferase fusion proteins over the glutathione-Sepharose 4B column, added 25 units ofthlo-.-l)i.l to cleave the recombinant B. burgdorferi protein from the GT and incubated overnight at room temperature. We then eluted the proteins with 50 rnM Tris-CaC12-NaCI, 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 experim~nt~tion using these procedures.
CA 022~3834 l998-ll-06 W~ 97/4232~ PCT~US96/06610 Example XlV - Preparation Of Antibodies Directed Against The B. hurgdorferi Polypeptides Of This Invention We generated antibodies directed against the novel B. burgdorferi polypeptides of this invention as follows. We imm--ni7e~l C3H/He mice (FrederickCancer 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 immllni7ed control mice in the same manner with either 10 recombinant glutathione S-transferase or BSA.
Fourteen days after the last boost, we collected sera from the immlmi~ed 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 ~:Apl essing 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 se~uence as a filsion protein, even if it is normally linked to apromoter that is not transcriptionally active in E coli.
CA 022~3834 1998-11-06 W 097/42325 PCTnUS96/06610 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 ofthe ~mmllne Response To Novel B. burgdorferi Polypeptides A. Murine H~m~-ral Response To characterize the immllne 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-tr~ncmic.~ion using B. burgdorferi N40 infected 1. scapularis ticks (Harvard School of Tropical Public Health). ln the tick tr~n~mi~sion 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 a~[er 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 thenused 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 ,ug/ml, 200 ml/well3 and incub~ted overnight at 4~ C. To prevent non-specific binding, we blocked with 100 ,ul/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 l hour at room tel~,pt~ re/ 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 te~ )e~ re for 1 hour and washed 3 times with PBST. Finally, we added 200 microliters of freshly prepaled 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, ~imini.~hed by 60-90 days after infection and almost disappeared by 180 days. P2 l-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 experiment~tion the murine humoral response to other novel b. burgdorferi polypeptides of this invention using the procedures taught herein.
B. Human Humoral Respon~
We also characteri~ed the human immllne 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%
2 o 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 P2 1 -specific peptide. We found that 20 of the 82 sera (24%) from the Yale clinic had IgG antibodies to recolllbh~alll P21 and 8 ofthose 20 also had anti-P21 IgM
antibodies. Out of the 20 sera with anti-P2 1 antibodies, 4 had IgM and 16 had IgG
antibodies that bound to P2 1 -specific peptide. We found that 13 of the 40 sera(33%) from the CDC had IgG and/or IgM antibodies to P2 1. Of those 13 sera, 11 CA 022~3834 1998-11-06 WO 97/42325 PCT~US96/06610 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-documçntedLyme 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. ~3ecause ofthe high reactivity to recombinant P35 and P37, we tested sera from an additional 25 patients with well-documented Lyme disease whowere 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.
Example 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 immuni7ed C3H/He mice subcutaneously with 10 micro~rams of recombinant GT-P35 or recombinant GT-P37 polypeptides in CFA and boosted at 14 and 28 days with the same amount ofantigen in IFA according to published protocols. We imm~ni7ed control mice in the same manner with recombinant GT. We then attempted to infect the immllni7ed mice with B. burgdorferi N40.
CA 022~3834 1998-ll-06 W097/42325 PCT~US96/06610 We grew a low passage isolate of B. burgdorferi N40 with demonstrated infectivity and pathogenicity in C3HlHe mice, to log phase at 33~ Cin BSK II me~ m and counted with a hemocytometer under dark-field microscopy.
We challenged the actively immllni7ed mice approximately 14 days after the last boost with intradermal inoculations of 10 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 me~ m for two weeks and ex~mined by darkfield microscopy for spirochetes. At the same time we sectioned, formalin 10 fixed and paraffln embedded, and then examined joints and hearts for infl~mm~tion We examined the heart and tibiotarsi blindly. We characterized arthritis by edema and synovial infiltration with neutrophils and Iymphocytes. We characterized carditis by the presence of aortitis, myocarditis or pericarditis.
Preliminary results generated using these methods suggest that P3 5 or P37 may confer protection.
Example XVIII - Protection against tick-medi~ted tr~ncrnission 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 tr~n~mi~ion of the spirochete. We obtained spirochete-free Ixodes dammini ticks from the Harvard School of Public Health, which m~int~in.~ 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 C~- 1 mice that had been previously infected (three weeks prior to serving as hosts) by intradermal inoculation of 10 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 21CC and 95% relative humidity.
CA 022~3834 1998-11-06 W097/42325 PCT/Ug~6,'0GC10 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 imml-ni7ed mice with GT-P35, GT-P37, or both, GT-P21 or GT (control) as described in Example XVI~. Two weeks after the last boost, we placed 5/15 infected nymphal ticks on each mouse, allowed them to feedto repletion and then allowed them to detach naturally over water. Two weeks later we sacrificed the mice and cultured the tissues for spirochetes and exarnine theorgans, as described above.
Tmmnni7~tion 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 immllni7~tion with P21 did not produce sufficient protective antibodies in rnice or that P21 was not expressed in sufficient quantity on the surface of the spirochete to make them vulnerable to antibody-mediated killing.
Example X~X - Decrease in spirochete load in ticks feeding on imm~1ni?ed animals Previous studies have shown that immllni7~tion of mice with recombinant OspA can eliminate the spirochetes from ticks feeding on the imm~lni7ed animals [E. Fikrig et al., "Flimin~tion of Borrelia burgdorferi from vector ticks feeding on OspA-imm~ni7ed mice", Proc. Natl. Acad. Sci., 89,pp.
5418-5421(1992)]. Thus, to determine if spirochetes also are killed when infected CA 02253834 1998-ll-06 WO 97/4232~ PCT~US96/06610 ticks fed on animals imm-mi7~d 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 imm-mi7.ed with GT or 12 mice immllni7:ed with GT-P21. After feeding to repletion, the ticks are allowed to naturally det~hed over water. Only a portion of the ticks are recovered from each group, the remainder appa,~ ly having been ingested by the mice. Ten days post-repletion, we homogenized individual ticks in 100 Ill of PBS in a 1.5 ml microfuge tube and spotted 10 ~,11 aliquots on each of three slides. We allowed the slides to air-dry, fixed in cold acetone for 10 mimltes, and assayed by direct or indirect imml Inofluorescence.
For the direct immnnofluorescence 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 ~ppro~ ately 20 fields per slide. B. burgdorferi infection rates were similar within ticks that fed on immllni7ed and control mice indicating that immlmi7~tion with GT-P21 does not protect against infection.
One of skill in the art would understand that the effect of immnni7~tion with other novel B. burgdorferi polypeptides of this invention can be readily deterrnined without undue experimçnt~tion using the methods taught herein.
Example XX - Passive Tmm~lni7~tion of Mice With Anti-P35 or Anti-P37 Antisen~m To determine if antiserum from animals immllni7ed with recombinant B. burgdorferi polypeptides would confer protection, we passively immnni7.od mice with 0.2 ml of GT-P35, GT-P37 or anti-P35/P37 antisera. We .... . .
CA 022~3834 1998-11-06 W 097/42325 PCT~US96/06610 then challenged the passively imml~ni7ed mice with 10 B. burgdorferi N40 at one day after the irnml~ni7~tiQn.
Plelilninaly results indicate that the frequency of B. burgdorferi infection and the disease course in passively immuni7ed mice appeared to be the same as in the control mice.
In a separate study, we inoculated three groups of S scid mice with 10 B. burgdorferi N40 and then injected 0.S ml of antiserum (diluted 1:10) from either GT-P21 immllni7e(1, GT imml-ni7ed or 90 day infected mice on days 1, 4, 8and 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 i,lflal",llation. The rate of infection and disease in mice passively immllni7ed with P2 1 antiserum was similar to the rates in control mice. Mice passive imml1ni7ed 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 imm--ni7~fion with a reco,llbinanl polypeptide without GT, collect antiserum at a di~elelll time point when the titer is higher, passively immllni7e with more antiserum, decrease the spirochete dose, or other means known in the art.
2 o Example XXI - Additional Clones of In Vivo Expressed ~. burgdorferi Polypeptides We have performed preliminary analyses of two additional clones produced by the screening set forth in Example 1. We designated those clones V1 and V3. We deposited plasmids pVI and pV3, contained in Vl 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 CA 022~3834 1998-11-06 WO97/42325 PCT~US96/06610 confirrn that the polypeptides encoded by these clones are selectively expressed in vlvo.
We have also pel ~rl-lcd preliminary analyses of the r~m~ining 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, design~ted M30, is set forthin SEQ ID NOS: 8 and 9. We design~ted the other genes Jl and J2. Plasmids from clones corresponding to J1 have been deposited as plS and p5 under ATCC
accession numbers Plasmids from clones corresponding to p2, p7 and p9 have been deposited under ATCC accession numbers Example XXII - Deterrnination of Protective Epitopes We construct recombinant genes which will express fragments of the novel B. burgdorferi polypeptides in order to determine which fragments contain 5 protective epitopes. First, we produce overlapping 200-300 bp fr~gm~nts which encompass the entire nucleotide sequçnce of each of the genes, either by restriction enzyme digestion, or by amplification of specific sequences of using PCR and oligonucleotide primers cont~ining restriction endonuclease recognition sequences, as described st~pra. We then clone these fr~gm~nts into an appropriate expression vector, preferably a vector from which the fr~gment~ will be expressed as fusionproteins, in order to façilit~te purification and increase stability. For example, the gene fr~gment~ 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 2 5 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.
CA 022~3834 1998-11-06 WO 97142325 PCT~US96/06610 One way to identify fragments that contain protective B-cell epitopes is to use the individual purified fr~gment~ to immuni7e C3H/HeJ mice, as described above. Af~Ler ~h~llP~lge of the mice with B. burgdorferi, we deterrnine the presence of infection by blood and spleen cultures and by histopathologic ex~min~tion of the 5 joints and heart.
Another technique to identify protective epitopes is to use the various fragm~nts to immuni7~ 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 subsequentinfection 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 5 amino acids. The use of synthetic peptides allows us to further define each epitope, while elimin~ting 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 CA 022~3834 1998-11-06 W097/4232~ PCTAUS96/06610 isolates. Such a vaccine will, thus, elicit antibodies that will confer protection against a variety of di~lelll strains.
Example XXIV - Identification of T cell epitopes Stimulation in animals of a humoral immune response co~
5 high titer neutralizing antibodies will be facilitated by antigens cont~ining both T cell and B cell epitopes. To identify those polypeptides cont~ining 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 Iymph nodes and generate in vitro T cell lines. These T cell lines are then cloned using limiting o dilution and soft agar techniques. We use these T cell clones to determine which polypeptides contain T cell epitopes. The T cell clones are stim~ ted with the various polypeptides and syngeneic antigen p~eselllh~g cells. Exposure ofthe T cell clones to the polypeptides that contain T cell epitopes in the presence of antigen pres~nting cells causes the T cells to proliferate, which we measure by H-Thymidine incorporation. We also measure Iymphokine production by thestimulated 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 ofmultiple HLA types. T cell epitopes are identified by stim~ ting the clones with the various polypeptides and measuring H-Thymidine incorporation. The various T
cell epitopes are then correlated with Class II HLA antigens such as DR, DP, andDQ. The correlation is performed by utilization of B Iymphoblastoid cell lines expressing various HLA genes. When a given T cell clone is mixed with the appropliate B Iymphoblastoid 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 H-Thymidine incorporation.
. ~ .
CA 02253834 1998-ll-06 W097/4232S PCTrUS96/06610 Alternatively, T cell epitopes may be identified by adoptive transfer of T cells from mice immllni7ed with various of the novel B. burgdorferi polypeptides of this invention to naive mice, according to methods well known tothose of skill in the art. [See, for example, M.S. DeSouza et al., "Long-Term Study of Cell-~edi~ted Responses to Borrelia bz~r~,do,J~ri in the Laboratory Mouse", Infect. Immun., 61, pp. 1814-22 (1993)].
We then synth-osi7e a multicomponent vaccine based on di~lent T
cell epitopes. Such a vaccine is useful to elicit T cell responses in a broad spectrum of patients with di~erent HLA types.
We also identify stimlll~ting 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 Tcell epitopes from the novel B. burgdorferi polypeptides of this invention.
Example XXV - Construction of fusion proteins comprising T ~ntl E3 cell epitopes A~er 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 filsion proteins, by virtue of cont~ining both T cell and B cell epitopes, permit antigen presenlalion 2 0 to T cells by B cells exp- essing surface immunt~globulin. These T cells in turn stimlll~te B cells that express surface imm~lnoglobin, 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 cellepitopes to strong T cell epitopes of other antigens. We synthe.ci7e an oligonucleotide homologous to amino acids 120 to 140 ofthe 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' CA 022=,3834 1998-11-06 WO97/42325 PCTrUS96/06610 and 3' ends of segm~nts 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 co~ ~sing a B cell epitope from the novel B.
burgdorferi polypeptide and a T cell epitope from the core antigen, thus enh~n~ing the immllnogenicity ofthe 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 co~ g the B cell epitopes of various of the novel B. burgdorferi polypeptides incorporated into the fl~eell;n protein of Salmonella. Bacterial 11~gçllin 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 cellepitopes 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 imm~lni7,ed with live and formalin killed bacteria and assayed for antibody production. In addition spleen cells are tested for proliferative cellular responses to 2 o the peptide of interest. Finally the mice immuni~ed 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 di~e~el.L
novel B. burgdorferi polypeptide or other immunogenic B. burgdorferi 25 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
CA 022s3834 1998-ll-06 WO 97/4232S PCT~US96/06610 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, andprevent Lyme disease as caused by infection with B. burgdorferi.
While we have described a number of embodiments of this invention, s it is appare-lL 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.
CA 022~3834 1998-11-06 W097/42325 PCT~US96/06610 SEQUENCE LISTING
(1~ GENERAL INFORMATION:
(i) APPLICANT: Flavell, Richard A.
Fikrig, Erol Barthold, Stephen W.
Suk, Kyoungho (ii) TITLE OF INVENTION: B. BURGDORFERI POLYPEPTIDES EXPRESSED IN
VIVO
(iii) NUMBER OF SEQUENCES: 28 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Fish & Neave (B) STREET: 1251 Avenue of the Americas (C) CITY: New York (D) STATE: New York (E) COUNTRY: USA
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(A) NAME: Haley Jr., James F.
(B) REGISTRATION NUMBER: 27,794 (C) REFERENCE/DOCKET NUMBER: YU-103 (ix) TELECOMMUNICATION INFORMATION:
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(A) LENGTH: 752 base pairs (B) TYPE: nuclelc acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA (genomlC) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
tA) NAME/KEY: CDS
(B) LOCATION: 145..690 (ix) FEATURE:
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Met Asn Lys Lys Met Phe Ile Val Cys Ala Val Phe Ala Leu Ile Ser Ser Cys Lys Ile His Thr Leu Ser Met Tyr Asp Glu Gln Ser Asn Asn Glu Leu Lys Val Lys Gln Ser Asn Gly Glu Val Lys Val Lys Lys Ile Glu Phe Ser Glu Phe Thr Val Lys Ile Lys Tyr Lys Lys Asp Asn Ser Ser Asn Trp Glu Asp Leu Gly Thr Leu Val Val Arg Lys Glu Val Asp Gly Ile Asp Thr Gly Leu Asn Val Gly Lys Gly Tyr Ser Ala Thr Phe Phe Ser Leu Glu Glu Ser Glu Val Asn go 95 100 105 Asn Phe Ile Lys Ala Met Thr Lys Gly Gly Thr Phe Lys Thr Ser Leu Tyr Tyr Gly Tyr Lys Glu Glu Gln Ser Gly Glu Asn Gly Ile Gln Asn Lys Lys Ile Ile Thr Lys Ile Glu Lys Ile Asp Asp Phe Glu Tyr Ile ACA TTT TTA GGA &AT AAA ATT AAG GAT TCA GGA GAT AAA GTT GTT GAA 651 Thr Phe Leu Gly Asp Lys Ile Lys Asp Ser Gly Asp Lys Val Val Glu Tyr Ala Ile Leu Leu Glu Asp Leu Lys Lys Asn Leu Lys Met Asn Gln Lys Ala Phe Ile Ile Cys Ala SUBSTITUTE SHEET (RULE 26) CA 022~3834 1998-11-06 W097/42325 PCT~US96/06610 (2) INFORMATION FOR SEQ ID NO:2:
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Met Asn Lys Lys Met Phe Ile Val Cys Ala Val Phe Ala Leu Ile Ser ~er Cys Lys Ile His Thr Leu Ser Met Tyr Asp Glu Gln Ser Asn Asn Glu Leu Lys Val Lys Gln Ser Asn Gly Glu Val Lys Val Lys Lys Ile Glu Phe Ser Glu Phe Thr Val Lys Ile Lys Tyr Lys Lys Asp Asn Ser Ser Asn Trp Glu Asp Leu Gly Thr Leu Val Val Arg Lys Glu Val Asp ~ly Ile Asp Thr Gly Leu Asn Val Gly Lys Gly Tyr Ser Ala Thr Phe ~he Ser Leu Glu Glu Ser Glu Val Asn Asn Phe Ile Lys Ala Met Thr Lys Gly Gly Thr Phe Lys Thr Ser Leu Tyr Tyr Gly Tyr Lys Glu Glu Gln Ser Gly Glu Asn Gly Ile Gln Asn Lys Lys Ile Ile Thr Lys Ile Glu Lys Ile Asp Asp Phe Glu Tyr Ile Thr Phe Leu Gly Asp Lys Ile Lys Asp Ser Gly Asp Lys Val Val Glu Tyr Ala Ile Leu Leu Glu Asp ~eu Lys Lys Asn Leu Lys ~2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
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Met Asn Gln Lys Ala Phe Ile Ile Cys Ala SU~S 111 UTE SHEET (RULE 26) CA 022~3834 1998-11-06 WO 97/42325 PCT~US96/06610 (2) INFORMATION FOR SEQ ID NO:4:
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(B) LOCATION: 181..1107 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Thr Met Ile Thr Pro Ser Ser Lys Leu Thr Leu Thr Lys Gly Asn Lys Ser Trp Ser Ser Thr Ala Val Ala Ala Ala Leu Glu Leu Val Asp Pro Pro Gly Cys Arg Asn Ser Lys Ser Asn Phe Leu Gln Lys Asn Val Ile Leu Glu Glu Glu Ser Leu Lys Thr Glu Leu Leu Lys Glu Gln Ser SUbS 111 UTE SHEET (RULE 26) -CA 022~3834 l998-ll-06 WO g7/42325 PCTrUS96/06610 Glu Thr Arg Lys Glu Lys Ile Gln Lys Gln Gln Asp Glu Tyr Lys Gly 75 80 85 go Met Thr Gln Gly Ser Leu Asn Ser Leu Ser Gly Glu Ser Gly Glu Leu 95 lO0 105 Glu Glu Pro Ile Glu Ser Asn Glu Ile Asp Leu Thr Ile Asp Ser Asp Leu Arg Pro Lys Ser Phe Leu Gln Gly Ile Ala Gly Ser Asn Ser Ile Ser Tyr Thr Asp Glu Ile Glu Glu Glu Asp Tyr Asp Arg Tyr Tyr Leu Asp Glu Asp Asp Glu Asp Asp Glu Glu Asp Glu Glu Glu Ile Arg Leu Ser Asn Arg Tyr Gln Ser Tyr Leu Glu Gly Val Lys Tyr Asn Val Asp Ser Ala Ile Gln Thr Ile Thr Lys Ile Tyr Asn Thr Tyr Thr Leu Phe Ser Thr Lys Leu Thr Gln Met Tyr Ser Thr Arg Leu Asp Asn Phe Ala Lys Ala Lys Ala Lys Glu Glu Ala Ala Lys Phe Thr Lys Glu Asp Leu Glu Lys Asn Phe Lys Thr Leu Leu Asn Tyr Ile Gln Val Ser Val Lys Thr Ala Ala Asn Phe Val Tyr Ile Asn Asp Thr His Ala Lys Arg Lys Leu Glu Asn Ile Glu Thr Glu Ile Lys Thr Leu Ile Ala Lys Ile Lys Glu Lys Pro Asp Leu Tyr Gln Ala Tyr Lys Ala Ile Val Thr Pro Ile SUt~S 111 IJTE SHEET (RULE 26) CA 022~3834 1998-ll-06 W O97/42325 PCT~US96/06610 - 79 -TTA TTA ATG AGG GAT TCT CTT A~A GAA GTG CAA AGT GCC ATT GAC AAG 1092 Leu Leu Met Arg Asp Ser Leu Lys Glu Val Gln Ser Ala Ile Asp Lys Asn Gly Ile Trp Tyr (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 309 amino acids (B) TYPE: amlno acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Met Thr Met Ile Thr Pro Ser Ser Lys Leu Thr Leu Thr Lys Gly Asn ~ys Ser Trp Ser Ser Thr Ala Val Ala Ala Ala Leu Glu Leu Val Asp Pro Pro Gly Cys Arg Asn Ser Lys Ser Asn Phe Leu Gln Lys Asn Val Ile Leu Glu Glu Glu Ser Leu Lys Thr Glu Leu Leu Lys Glu Gln Ser Glu Thr Arg Lys Glu Lys Ile Gln Lys Gln Gln Asp Glu Tyr Lys Gly ~et Thr Gln Gly Ser Leu Asn Ser Leu Ser Gly Glu Ser Gly Glu Leu ~lu Glu Pro Ile Glu Ser Asn Glu Ile Asp Leu Thr Ile Asp Ser Asp Leu Arg Pro Lys Ser Phe Leu Gln Gly Ile Ala Gly Ser Asn Ser Ile Ser Tyr Thr Asp Glu Ile Glu Glu Glu Asp Tyr Asp Arg Tyr Tyr Leu Asp Glu Asp Asp Glu Asp Asp Glu Glu Asp Glu Glu Glu Ile Arg Leu SU~ ~111 UTE SHEET (RULE 26) CA 022~3834 1998-11-06 W 097/42325 PCT~US96/06610 Ser Asn Arg Tyr Gln Ser Tyr Leu Glu Gly Val Lys Tyr Asn Val Asp ~er Ala Ile Gln Thr Ile Thr Lys Ile Tyr Asn Thr Tyr Thr Leu Phe Ser Thr Lys Leu Thr Gln Met Tyr Ser Thr Arg Leu Asp Asn Phe Ala Lys Ala Lys Ala Lys Glu Glu Ala Ala Lys Phe Thr Lys Glu Asp Leu Glu Lys Asn Phe Lys Thr Leu Leu Asn Tyr Ile Gln Val Ser Val Lys ~hr Ala Ala Asn Phe Val Tyr Ile Asn Asp Thr His Ala Lys Arg Lys ~eu Glu Asn Ile Glu Thr Glu Ile Lys Thr Leu Ile Ala Lys Ile Lys Glu Lys Pro Asp Leu Tyr Gln Ala Tyr Lys Ala Ile Val Thr Pro Ile Leu Leu Met Arg Asp Ser Leu Lys Glu Val Gln Ser Ala Ile Asp Lys Asn Gly Ile Trp Tyr (2) INFORMATION FOR SEQ ID NO:6:
(i~ SEQUENCE CHARACTERISTICS:
~A) LENGTH: 1490 base palrs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(ivJ ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 121..1116 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
ATG AAT TTA ATA ATT A~A GTG ATG TTG ATA TCC AGT TTA TTT TCT AGC 168 Met Asn Leu Ile Ile Lys Val Met Leu Ile Ser Ser Leu Phe Ser Ser Phe Ile Ser Cys Lys Leu Tyr Glu Lys Leu Thr Asn Lys Ser Gln Gln SUBSTITUTE SHEET (RULE 26) W097/42325 PCT~US96/06610 Ala Leu Ala Lys Ala Phe Val Tyr Asp Lys Asp Ile Ala Asp Asn Lys Ser Thr Asn Ser Thr Ser Lys Leu Asp Asn Ser Ser Leu Asp Ser Ile Lys Asp Asn Asn Arg Ser Gly Arg Thr Ser Arg Ala Leu Asp Asp Ala Glu Glu Ile Gly Val Lys Glu Ser Asn Gln Asn Arg Asn Asp Gln Gln Gln Asn Asn Glu Ser Lys Val Lys Glu Ser Glu Lys Asn Asn Ser Ser Gly Ile Gln Ala Asp Asp Ser Val Leu Gly Thr Ala His Ser Asp Ala Ser Glu Val Glu Asn Lys Lys His Asp Thr Ser Arg Gln Pro Gln Leu Leu Asn Lys Asp Ser Ser Glu Ala Arg Glu Ala Ser Lys Ile Ile Gln Lys Ala Ser Thr Ser Leu Glu Glu Ala Glu Lys Val Asn Val Ala Leu 470 475 480 q85 Lys Glu Thr Arg Ser Lys Leu Asp Lys Ile Lys Arg Leu Ala Asp Ser Ala Lys Ser Tyr Leu Asn Asn Ala Arg Lys Asn Ser Arg Thr Asn Gly Ser Ile Leu Glu Ile Leu Pro Asn Leu Asp Lys Ala Ile Glu Lys Ala Ile Ser Ser Tyr Ala Ser Leu Asn Val Cys Tyr Thr Asp Ala Ile Ala Ala Leu Ala Lys Ala Lys Asn Asp Phe Glu His Ala Lys Arg Lys Ala Asn Asp Ala Leu Glu Glu Ala Leu Lys Asp Ile Thr His Phe Arg Gly Tyr Asn Tyr Leu Tyr His Tyr Arg Ile Asn Asn Ala Asn Asp Ala Met SUt~S 111 UTE SHEET (RULE 26) CA 022~3834 1998-11-06 W 097/42325 PCT~US96/06610 Glu Ser Ala Lys Ser Leu Leu Glu Val Ala Lys Asn Lys Gln Lys Glu Leu Asn Glu Asn Ile Thr Lys Thr Asn Lys Asp Phe Gln Glu Leu Asn Asp Ile Tyr Lys Lys Leu Gln Asp Met Asp Ser Arg ~lc~ lCT AAACAACACT TTATTTTCTC TTAACTTTAT AGTTTGACTT AAAAAGTCAT 1246 TATTTTTAAA TTATTACAT& AATTGCCTTG AATATCTTTA TTTTTATATT ATAATTATTA 1306 T~ .rlGA TTTGA~AGTT GTTCTGGTTT TCTATCTAAA AAATCTATAG AACAGTTTGC 1486 (2) INFORMATION EOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 332 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID No:7:
Met Asn Leu Ile Ile Lys Val Met Leu Ile Ser Ser Leu Phe Ser Ser Phe Ile Ser Cys Lys Leu Tyr Glu Lys Leu Thr Asn Lys Ser Gln Gln Ala Leu Ala Lys Ala Phe Val Tyr Asp Lys Asp Ile Ala Asp Asn Lys Ser Thr Asn Ser Thr Ser Lys Leu Asp Asn Ser Ser Leu Asp Ser Ile SU~5 111 lJTE SHEET (RULE 26) CA 022~3834 1998-11-06 W097/42325 PCT~US96/06610 Lys Asp Asn Asn Arg Ser Gly Arg Thr Ser Arg Ala Leu Asp Asp Ala ~lu Glu Ile Gly Val Lys Glu Ser Asn Gln Asn Arg Asn Asp Gln Gln ~ln Asn Asn Glu Ser Lys Val Lys Glu Ser Glu Lys Asn Asn Ser Ser Gly Ile Gln Ala Asp Asp Ser Val Leu Gly Thr Ala Hls Ser Asp Ala Ser Glu Val Glu Asn Lys Lys His Asp Thr Ser Arg Gln Pro Gln Leu Leu Asn Lys Asp Ser Ser Glu Ala Arg Glu Ala Ser Lys Ile Ile Gln ~ys Ala Ser Thr Ser Leu Glu Glu Ala Glu Lys Val Asn Val Ala Leu ~ys Glu Thr Arg Ser Lys Leu Asp Lys Ile Lys Arg Leu Ala Asp Ser Ala Lys Ser Tyr Leu Asn Asn Ala Arg Lys Asn Ser Arg Thr Asn Gly Ser Ile Leu Glu Ile Leu Pro Asn Leu Asp Lys Ala Ile Glu Lys Ala Ile Ser Ser Tyr Ala Ser Leu Asn Val Cys Tyr Thr Asp Ala Ile Ala ~la Leu Ala Lys Ala Lys Asn Asp Phe Glu His Ala Lys Arg Lys Ala ~sn Asp Ala Leu Glu Glu Ala Leu Lys Asp Ile Thr His Phe Arg Gly Tyr Asn Tyr Leu Tyr His Tyr Arg Ile Asn Asn Ala Asn Asp Ala Met Glu Ser Ala Lys Ser Leu Leu Glu Val Ala Lys Asn Lys Gln Lys Glu Leu Asn Glu Asn Ile Thr Lys Thr Asn Lys Asp Phe Gln Glu Leu Asn ~sp Ile Tyr Lys Lys Leu Gln Asp Met Asp Ser Arg (2) INFORMATION FOR SEQ ID NO:8:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 825 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) ~iii) HYPOTHETICAL: NO
SIJ~S ~ )TE SHEET (RULE 26) CA 022~3834 l998-ll-06 W 097/42325 PCT~US96/06610 (iv) ANTI-SENSE: NO
(ix) FEATURE:
(A~ NAME~KEY: CDS
(B) LOCATION: 1..825 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Met Cys Ala Phe Leu Leu Leu Asn Leu Val Asn Cys Lys Phe Asp Ser Leu Asn Leu Ser Thr Lys Ser Val Asp Asp Lys Asn Asn Ser Ile Ala Lys Leu Leu Gln His Leu Ser Lys Ser Glu Asp Gln Ala Asn Lys Thr TCT ACC TCA GAA GAC CAA AAG GAA TTA GAA ATT ACG GAA AAC A~A GAA 192 Ser Thr Ser Glu Asp Gln Lys Glu Leu Glu Ile Thr Glu Asn Lys Glu Gln Glu His Glu Lys Leu Ser Gln Val Ala Gln His Ala Pro Asn Ser Lys Ile Glu Lys Val Lys Ser Asp Gly Lys Pro Val Pro Gly Asp Lys Ile Leu Ser Ser Asn Lys Asp Ile Tyr Asn Ser Tyr Ile Pro Glu Val 430 q35 440 Lys Glu Glu Ile Val Tyr Glu Ile Leu Glu Glu Val Ile Ile Pro Glu Thr Lys Ile Pro Glu Ile Thr Glu Glu Val Ile Met Pro Ile Pro Gln Thr Ile Asp Phe Tyr Ile Glu Pro Arg Pro Ile Ser Ser Phe Leu Thr &ln Gly Thr Ser Pro Ser Ile Thr Ser Thr Ile Lys Ser Tyr Lys Glu SU~;~ 111 UTE SHEET (RULE 26) CA 022~3834 l998-ll-06 W 097/4232~ PCT~US96/06610 Leu Ala Lys Glu Lys Ile Asn Asn Gly Leu Asn Ile Val Gln Lys Ile Thr Gln Asn Ile Asp Asn Ile Thr Glu Asn Leu Asn Ser Lys Glu Thr Pro Lys Glu Ile Ser Gly Lys Glu Val Glu Glu Lys Ile Thr His Pro Ile Phe Asp His Ile Thr Gly Ser Gly Asn Asn Pro Gly Gln Asp Ser Ile Ser Asn Thr Trp Gly Glu Gly Leu Glu Ile Gly Gly Asp Ser Asn Phe Phe Thr Asn Leu Glu Glu Val Arg Ser Ser Ile Arg Thr Lys Ile Lys Val Ser (2~ INFORMATION FOR SEQ ID NO:9:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 275 amlno acids (B~ TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Met Cys Ala Phe Leu Leu Leu Asn Leu Val Asn Cys Lys Phe Asp Ser ~eu Asn Leu Ser Thr Lys Ser Val Asp Asp Lys Asn Asn Ser Ile Ala Lys Leu Leu Gln His Leu Ser Lys Ser Glu Asp Gln Ala Asn Lys Thr Ser Thr Ser Glu Asp Gln Lys Glu Leu Glu Ile Thr Glu Asn Lys Glu Gln Glu His Glu Lys Leu Ser Gln Val Ala Gln His Ala Pro Asn Ser ~ys Ile Glu Lys Val Lys Ser Asp Gly Lys Pro Val Pro Gly Asp Lys SU~S 1 1 1 UTE SHEET (RULE 26) CA 022~3834 l998-ll-06 W097/42325 PCT~US96/06610 Ile Leu Ser Ser Asn Lys Asp Ile Tyr Asn Ser Tyr Ile Pro Glu Val Lys Glu Glu Ile Val Tyr Glu Ile Leu Glu Glu Val Ile Ile Pro Glu Thr Lys Ile Pro Glu Ile Thr Glu Glu Val Ile Met Pro Ile Pro Gln Thr Ile Asp Phe Tyr Ile Glu Pro Arg Pro Ile Ser Ser Phe Leu Thr ~ln Gly Thr Ser Pro Ser Ile Thr Ser Thr Ile Lys Ser Tyr Lys Glu ~eu Ala Lys Glu Lys Ile Asn Asn Gly Leu Asn Ile Val Gln Lys Ile Thr Gln Asn Ile Asp Asn Ile Thr Glu Asn Leu Asn Ser Lys Glu Thr Pro Lys Glu Ile Ser Gly Lys Glu Val Glu Glu Lys Ile Thr His Pro Ile Phe Asp His Ile Thr Gly Ser Gly Asn Asn Pro Gly Gln Asp Ser ~le Ser Asn Thr Trp Gly Glu Gly Leu Glu Ile Gly Gly Asp Ser Asn ~he Phe Thr Asn Leu Glu Glu Val Arg Ser Ser Ile Arg Thr Lys Ile Lys Val Ser ~2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 1221 base palrs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPB: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
SU~S 111 ~JTE SHEET (RULE 26) CA 022~3834 l998-ll-06 W097/42325 PCT~US96/06610 (2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single SUBSTITUTE SHEET (RULE 26) CA 022~3834 1998-11-06 WO 97/42325 PCT~US96106610 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
TGTACAAGCT TCTATTTTAA AllllllLlA AGATC 35 (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ~iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Cys Asn Asn Glu Leu Lys Val Lys Gln Ser Asn (2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
~ (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:14:
SIJ~S 111 ~ITE SHEET (RULE 26) CA 022~3834 1998-11-06 W 097/4232S PCT~US96/06610 (2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
TCTGATTTAA GGCCA~AG 18 (2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucle1c acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
SU~ JTE SHEET (RULE 26) CA 022~3834 l998-ll-06 W097/4232~PCTrUS96/06610 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs IB) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
~iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (il) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
SlJ~S 111 UTE SHEET (RULE 26) CA 022~3834 1998-11-06 W097/42325 PCTrUS96/06610 (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
AGTCAAGCTT CTATTTTAAA ll L 1 1 1 1 l~AA GATC 34 (2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acld (C) STRANDEDNESS: single (D) TOPOLOGY: linear SU~S 111 IJTE SHEET (RULE 26) CA 022~3834 1998-ll-06 W097142325 PCT~US96/06610 (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base palrs (B) TYPE: nuclelc acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
liv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
SU~;> 111 UTE SHEET (RULE 26) CA 022~3834 1998-ll-06 W097/42325 PCT~US96/06610 (2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs (B) TYPE: nuclelc acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 24 base palrs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICA~: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
SlJt~ 111 UTE SHEET (RULE 26) INDICATION'S RELATING TO A DEPOSITED MICROOR(~ANISM
(PCT Ru le 1 3bis) A. i 11-~ indicillions ~nade below rel~le to Ihe microoreanism relerred to in the descriplion on paee , llne B. IDENTIFICATION OF DEPOSIT plasmid ~'urther deposits arc idenlilied on iln iiddilion~71 sheet ~3 ~ -r ~ l -k~
Nameofdeposilar~ institution American Type Culture Collection Address ol depoiitilr! inslilulion ~inclu~lin~,~ poslal code a~l~i coZ~nrr 12301 Parklawn Drive Rockville, Maryland 20852 Unlted ~tates of Amerlca l);uc ol dcpo~il i Accc~sl0n Nulllhcr 7 May 1996 (07.05.96) C ADDITION'AL INDICATIONS lleal~ h/i7n~ o~ ap/~llcahlc/ I his inlonll;llioll I~ ~onthlucd on Ull ~dditiom71 shcct O
In respect of the designatlon of the EPO, samples of the de-posited microorganisms will be made available until the publicatio~
of the mention of the grant of the European patent or until the da'-e on whlch the application is refused or withdrawn or is deemed to be withdrawn, as provided in Rule 28(3) of the Implementing Regu-lations under the EPC only by the issue of a sample to an expert nominated b~ the requester (Rule 28(4) ~PC~.
D. DESIG~ATED STATES FOR WHICH INi)lCATlONS ARE ~IADE /~ .ol~ c ~ 11 oe~ 'lla/e~/.S~lle~J
EP
E. SEPAR~TE FIJRNISHING OF INDICATIONS H~ hlal7~ 11 nol applicabl-l'hc in~ilcinions lis(ed belo\~ ~ ill he suhmitlcd to Ihe Internc7lioll~1 3urcilu l~lcr l~ clt\ Ibe yL'/7~aH7a~ 'C o/rll,~ ind7ca~io~L~ e.~ lo :\'7~ h~ po511"J
Accession number of deposit.
r r~cci~ e Ollicc usc onl! I-or Inl.rniuiol~ urcilu use onh nee~ s recei~ed ~ith Ihe internilliom~ pplici~lh~n ~ -I his sheel ~s rccel~ed h\ the Intermllion~ ureill7 oll Authori~e~l olficer Authori;~cd ollicer .-orm I'CT RO 13~ Hl71! 199_) CA 02253834 1998-ll-06 INDICATIONS RELATING TO A DEPOSITED MICROOR~;ANISM
(PCTRule l3~,is) A. f hc indieations madc belo-~ rclate to the l"i,~uv~ "i~l" relerrcd to in thc dcseription on pa~c ~ q , linc 2 ~_ B. IDENTIFICATION OF DEPOSIT plasmid p Vl l:unhcr dcposils are identified on an additional shcc Narne of depositarv instilution American Type Culture Collection Addrcss ot'deposltar~ instltution /~neludin~e poslal cod~ and counrr 12301 Parklawn Drlve Rockville, Maryland 20852 Unlted States of America l)at. u~ dcposil ~ .\c~esslo~l l\iumh.r 7 May 1996 (07.05.96) C. .~DDlTlC~l~'.tL INDIC.~TIONS lleal L7 hlan~ 1/ nr71 ~Ippllcahl. ~ I hB informanon is contilluc-l on an a-fdilion~l ~heel O
In respect of the deslgnation of the EPO, samples of the de-posited mlcroorganlsms wlll be made available until the publlcation of the mention of the grant of the European patent or until the da~e on whlch the application is refused or withdrawn or lS deemed to be withdrawn, as provided in Rule 28(3) oi the Implementlng Regu-lations under the EPC only by the issue of a sample to an expert nominated by the requester (Rule 28(4) ~PC) D. DESIC,N.~TEI) ST~TES FOR S~'HI( II I~DIC.-~TIONS AKF .~ilADE /~J ~r/~ lo~l~ alc /lot lo~ all (Dslen(71.~ 51an EP
E. SEPARATE FURNISHING OF ll~il)lC:ATlONS /1-7ale hlan~ or ~pllLahl.~, I hl: indiciltlons list~d hclo~ dl be sllhmltted lo Ille Intematiorl;ll 13ureau latcr /sp-~C/h ~ eL~nLraDranlr~ otrlle ~ carlLrnsLl e ~ l~cL~
rlt7 ot L)r?pOS/I /
Accesslon number of deposlt.
- l or rcccl~ In!! ()f lic-~ us~ ~U~ H Ir Inlern;ltiunal Burcall use onh~is shcel ~a~ rcecl~cd ~Htll the In~crnali()nal ;Ippllcatlon O I his sh-el ~ r.cel~ed h! Ihe Inlernn~ioll;ll Uurea(l .u:
i~Ulh~r~'dt5~c''r ~,~ .\ulllori~ed olliccr l'orm PCT.ROil3~ Hul~ 199'~
CA 02253834 1998-ll-06 W097/42325 93~3 PCT~US96tO6610 INDICATIONS RELATING TO A DEPOSITED MICROORC~ANISM
(PCT Rule I ~bis) A I hc indicnlions made hclo~v rel tte to the ~ oo~ ~:d"ism relerred to in the descrlption on pa~c p. 1(O~ 20 ~ line B IDENTIFICATION OF DEPOSIT plasmid p V3 'r:urthcr deposits are idenliiied on 3n ndditional shect [~3 Nrlmc of depositalv instilution American Type Culture Collection Address of deposilar! institution ~includmg postal code and cortnrn 12301 Parklawn Drive Rockville, Maryland 20852 ~nited ~tates of America l);nc o~ depo~ A~c~s~on Numbcr 7 May 1996 (07.05.96) C' ADDlTlONAL li~DlCATlO~'S ~ bla~11; I/ nl71 ap/711caole/ I hi~ inlorm31ion is con~hluc~ on Ull ~ddition;ll shect O
In respect of the designatlon of the EPO, samples of the de-posited microorganisms will be made available until the publication of the mention of the grant of the European patent or until the da e on whlch the application is refused or withdrawn or is deemed to be withdrawn, as provided in Rule 28(3) of the Implementing Regu-lations under the EPC only by the issue of a sample to an expert nominated by the requester (Rule 28(4) ~PC).
1) DESIC.~ATED STAT~S FOR ~ HICII INDICATIO~S ARE ~IADE /~ rl.~ (Jr. ~ "11 llcsl~r~nar,~/.SIam.~, EP
E SEPAR~TE Fl,RNISHING OF I~DICATIONS rlea\ e bla~ J nor a/~pllcable~
1 hc illdlc:ltions listcd bclo~ ill hc submittcd to the Intcrnallon31 l~ure;~u httcr ~5/lccih //1L7~eL'~ r~ Mtlr~C oJrlre al~licarlo~ L 6 ' IL'C~L~SSI~nl 7l l~posit';
Accession number of deposit.
i:or rccel~ mo ( )i1icc ~l~c ~nl! hur Intcrn3tiol1al l~ure3u usc ~nl!
,hcct ~s rccel~d ~-ith ~hc imem3~ionl1 tppllc;~noll ~ O I nis ,ilc,~ 5 rcccl~cd b! ~he Intcrn3tion tl ~ure;~u on A~7ed~1iccr ~ '\ulhorizcd of'liccr l:orrnl'CTRC)'13~JUl! 199-1 W097/42325 93~4 PCTrUS96/06610 INDICATIO~'S RELATING TO A DEPOSITED MICROOR(~ANISM
(PCT Rule I ~bis) A 1 hc indicrttions madc bclo~ rel tte to thc ~ ,o, ~ l- retcrrcd lo in thc dcscription on pa~c p \~ ~ ~ ~7 ;p ~ ~ ) ~ ,1 B. IDENTIFICATION OF DEPOSIT plasmid p 15 I ur~hcr dcposils arc identified on ;tn tddition~tl sheet Nameofdepositar~ inslitulion American Type Culture Collection Address of dcposit;lr~ instllution ~inclu~ng postal code and countn 12301 Parklawn Drive Rockville, Maryldnd 20852 ~nlted States of America l):nc ot'dcposi~ C~'L'SSlOn IS,umhcr 7 May 1996 (07.05.96) ADDlTlOi' ~L INDICATIONS ~/L~I~e hl~a~ lic~lhll l l hl~ Inlorm t~ion j~ COnI;I1UC(J On illl Idd;~;I1I1LtI Sh~'CI O
In respect of the deslgnation of the EPO, samples of the de-posited mlcroorganiSmS will be made avallable until the publlcatio:n of the mention of the grant of the European patent or until the da-e on which the appllcation is refused or wlthdrawn or is deemed to be withdrawn, as provided in Rule 28(3) of the Implementing Regu-lations under the EPC only by the issue of a sample to an expert nominated by the requester (Rule 28(4) ~PC~.
D DESIGNATED STATES FOR W~IIC~ TIONS ARE I~IADF llt ~ IJ~7/~ L' ~l0l /0~ all lle~ al~ SIale~
EP
E SEPARATE F- RNISHINC. OF INDlC' ~TIONS tlca~e hla~ ol appllcabll~J
l hc indicittions lis~ b~ l hc ~uhmit~Ld lo ~h~ lntcrnatlon~ ur~;lu ~ cr ~ !h l/lL~x~lerolnah~r~ îc l~l~llca~lo~u ~ c~ ~s .~ t~ h~ epo~
Accession number of deposit.
r r.-cl\ m~ Dt~icc usc~ ol~ r Inlcrna~mll;~l Uurc tu us~ 01)l!
e~S shcct \\ ;~ rcccl\ c 1 ~ ith Ihc inlcrn;tll(ln~l ~pphc~ l O I hl~ shc~ rccci~ cLI h! [hc lntcrn;tlion;~ urcLtll ~n Au3~ ~ ~uthorl~cd olticc~
Form PCT ROH3~ I!ul~ 199 ) W097/42325 93~5 PCTrUS96/06610 INDICATII~.~S RELATING TO A DEPOSITED MICROORG
(PCTRule 13bi.~) A. Th~ indicmiuns made belo~v rela~e ~o the microorcanism reterred to in th~ d~scription on p,t~ p, ~7 ~ \~L l~o ~; ; pQ4~ q B. IDENTIFICATION OF DEPOSIT plasmid P 5 I-urthcr d~posils dre idenlit;ed on ~n ~ddilionul shcet Name ol'deposilar~Hnslitution American Type Culture Collection Addrcss ol'deposil;~r~- instilulion /inCIud~n,L~' poslul code ~tl~l COUn~r~') 12301 Parklawn Drlve Rockville, Maryland 20852 ~nlted ~tates of Amerlca l):u-~ 01 del-osi~ C~II)II Numhcr -7 May 1996 (07.05.96) C'. ADDITIONAL l.'NDICATIONS l~'O\'L' hlunA ~ Ot oppllo(lt7ic H ~ mlorrn3tl0n IS COlllinUt~d on 3n tddllion~l .sll~cl C~
In respect of the deslgnatlon of the EPO, samples of the de-posited microor~anisms will be made available until the publication of the mention of the grant of the European patent or until the da'-e on which the application is refused or wlthdrawn or is deemed to be withdrawn, as provided in Rul~ 28(3) of the ImplemenL ng Regu-lations under the EPC only by the issue of a sample to an expert nominated b~ the requester (Rule 28(4) ~PC).
D. DESICIN.~TED ST,~TES FOR ~ 'HICH IN'DIC.-~TIO~S .~Rt: ,~IADE /1/ ~b~ nO/I~ 101~ 1 Hicslr!l7~lr~cl .51 EP
E. SEP.~RATE Fl, RNISHINC OF l?NDICATIONS ~ o~ u hlor~ lo~ o/)pllcabl~J
l n~ aicltlons li~l~d beln~ ill be suhmltt~ o lh~ lnlcrn~llol~3l l3(lr~3u l;llcr ~ h f~ r(li~7~ n~ L7t~o~L~ c~ o~
lli L'~' 0/ D~posi("l Accesslon number of deposit.
P nr recel~ Inu OlliC u~u nrll! I or Inlcrn;l~ n;ll L3urc;(u use onb E~shc~ r~:C~ ilh th~ lnl~rn~7lion;~ r'l'~ 3~ 0 I'hi~ r~ l h! Ih~ rll;llloll;ll 13ur-:
~UI~ ~ ulhon~(:d olticrr l orm PC'r R()'13~ (Hll! 199'7) CA 02253834 1998-ll-06 W097/42325 93/6 PCT/U',~/QC610 I~IDICATIO!~S RELATING TO A DEPOSlTEl) MICROORGANISM
(PCT Rule I il1is) A. The indications made belo~v relate to the microort~nnism reterred lo in the description on pa~c p ~
B. IDENTIFICATION OF DEPOSIT plasmid p 2 I:unher deposits ;lre idcntitied on an additionai sh~e~ ~3 Namc of depositr~,Hnstitution American Type Culture Collection Address of deposit~r! institution ~mcludlng poslal corJe anrl cormrr~J
12301 Parklawn Drive Rockville, Maryland 20852 United States of Amerlca ol'~t~l-Oi~ c~ ioll ~unlh.r 7 May 1996 (07.05.96) ( . ~DDITIONAL INDICATIONS /i.~a\. blar7L ~/ no~ pllcabl~ / I hi~ inli)rnuulon i~ conlillu_d on ;n~ nddilional shccl O
In respect of the designation of the EPO, samples of the de-posited microorganisms will be made available until the publicatio~
of the mention of the grant of the European patent or until the da-e on which the application is refused or withdrawn or is deemed to be withdrawn, -s provided ln Rule 28(3) of the Implementing Regu-lations under the EPC only by the issue of a sample to an expert nominated bv the requester (Rule 28(4~ FPC).
1). DESlG~i~TED STATES FOR ~ HICH INDlE.i~ TIONS ARE ~ DE ~ r~rlloll~ ot i~ all (/esir~nar~ r EP
E. SEPARATE FURNISHING OF INDICATIONS r/ea-L~ blan'- i¦nor opplrcablc indicDIiollslistcdbclo~ h~suhmltlc~tlolh~lnlcrnrlliollal~llrc;Jul~ r~sp~ h~ wn(r~/o/l~lel~ carlo~se~r~ c.
.~ I/r~lh~r o/ /~eposir~r Accession number of deposit.
I:or n:c~ hlL! ()tlice USi onl~ l-or In~c-n~lloll;ll L3ure~u use ~nl!; E3--s shecl ~;IS recei~e(l ~HIh Illc Intcrn;nlon~l ~ppllc;llion ~ l hl~ ShLC~ reccl~ed h! Ihe lnlern tlioll~l ~(lrc~u l~n ~~i7ed~lcer ~ uthori7ed olticer ~orm PCT/~Oil3~ (Jul! 199~
W097142325 93~7 PCTrUS96106610 INDICATIONS RELATING TO A DEPOSITED MICROORC~ANISM
(PCT ~ule 1 3bis) A. I'he indieations mude bclo~ relate to the mieroorf2amsm referred to in the dcserip~ion on pa~ ,,~P p, tc~ f~
B. IDENTIFICATION OF DEPOSIT plasmid p 7 I:unhcr deposits are identified on an ~dditionrl sheel ~3 Name of depositarv insu~ution American Type Culture Collection Addr~ss of' deposit~r~ insti~ulion ~Includin~ postal code an~l caunrr! J
12301 Parklawn Drive Roc~ville, Maryland 20852 United States of America .' 0~ d~pnslt ~ C~1011 Numbcr 7 May 1996 (07.05.96) ('. ADDITIONAL INDICATION'S ~le~\'t' hlunl; ~ ol apr~llcahll l l hi~ mlorm.llion 1~ con~ ucd oll url ~ddiliull~l shccl O
In respect of the designation of the EPO, samples of the de-posited microorganisms will be made avallable until the publicatlon of the mention of the grant of the European patent or until the da-e on which the appllcation is refused or wlthdrawn or is deemed to be withdrawn, as provided in Rul~ 28(3) of the Implementlng Regu-lations under the EPC only by the issue of a sample to an expert nominated by the requester (Rule 28(4) F.PC ) .
D. DEslG-~ ~TEl) ~ ~TE~s FoR ~ lcfl lNDlc ~Tlo~ RE ~lADE ~ o~ la EP
E. SEPARATE FD'RrNlSillNG OF INDICATIONS lle(l~ blal~ ; nol ~Ippll(abllu -lh~indic:llionslist~dhc~ lb~ubmit~dlolhcln~rnalio~ lr~lul~ltcrl~r~lc!hlll~ r(llnalllreo~t~lel~ ica~ 'C(?SSI~
.\lu~loer ol Deposl~"
Accession number of deposit.
I:or recel~nn~ ()fli;e u~ oul\ I or In~crn31l0n~1 f~ure~u use olll ~/is sh~t ~ s r~cei\ ed ~ hl~ e inlern;llion;ll ~pplie~lioll O I'hi~ ~hee( ~.n~ rccei~d h~ th~ Inlern.llioll,ll Uure;lu o Al~) ed ~fAecr () ~ ulhorii~ed o~'ficer I:orm l'CT RO 134 1 .lul~ 199' 1 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule I ~hr.~ ) A. ~r he indie3lions made below relate to Ihe mierooreanism reterred lo in the deserip~ion on pagc ~ t lS, ~ 1~
B. IDENTIFICATIONOFDEPOSIT plasmid p 9 ~urtherdepositsareidenljf~edon~nndditionalsheet O
Narne of depositary inStitNtiOn American Type Culture Collectlon Addre~s ordeposit3r~ institution (includingpos~al code and countrL~J
12301 Parklawn Drive Rockville, Maryland 20852 Un1ted States of America D;lte ol depo~u Ac-e~lorl Numher 7 May 199~ (07.05.96) C ADDITIONAl. INDICATIONS /lea~e 1Slank 1/ ~101 appllcaolel H ~ Inlorm tllon ~i conlinueLd on ;In 3dditl0lIat sheel G
In respect of the deslgnation of the EPO, samp~es of the de-posited mlcroorganisms will be made available until the publicatio~
of the mention of the grant of the European patent or until the da~e on which the application is refused or withdrawn or is deemed to be withdrawn, as provided in Rule 28(3) of the Implementiny Regu-lations under the EPC only by the lssue of a sample to an expert nominated by the requester (Rule 28~4) ~P~
D DESIC~ATED ST~TES FOR WHICH INDICATIONS ARE l IADE ~ earloll~ rrr~ nor n ~ ieslr~l1arcLI~5tar~ ~J
EP
E SEPARATE FliRNlSHlNG OF INDICATIONS ~lea-e blank 1/ ~lor appllcableJ
'I'hcindiculionshstedbclo~ illbesuhmlllcdtothclnlem3tlon~1~ure;1ul:11erl~r~L~ re~e~r~lDlan~rL7o~ emdlcatlonse~ "Icce.~slo \ll~nbL~ "ll~eposlr"l Accession number of deposit.
t or r.. ~n~ mr~ Ottice USE orll\ H lr InlL rn71iol1ul 13ure~u u~e onl~
~hee~ recel~ed~iththemtL~m3tl0n~ ppllc;ulon O Ihl~slleel\~ recei~u:dh\ IheIntern;llion;~ ure~ll on :
Au~ ~ Aulhoriz~d otliccr ~orm PCTiR() 13~ (Jul~ 1997)
Claims (35)
1. An isolated DNA molecule comprising a DNA sequence which encodes a B.burgdorfen 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) an 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) a naturally occurring polypeptide encoded by a DNA
sequence which hybridizes to one of the DNA sequences of (a)-(f) under hybridization conditions which are 20-27°C below the Tm of the DNA sequence of (a)-(f);
(h) fragments comprising at least 8 amino acids taken as a block from any one of the polypeptides of (a)-(f);
(i) derivatives of any one of the polypeptides of (a)-(f), said derivatives being at least 80% identical in amino acid sequence to the corresponding polypeptide of (a)-(f); and (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)-(f) and (h).
(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) an 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) a naturally occurring polypeptide encoded by a DNA
sequence which hybridizes to one of the DNA sequences of (a)-(f) under hybridization conditions which are 20-27°C below the Tm of the DNA sequence of (a)-(f);
(h) fragments comprising at least 8 amino acids taken as a block from any one of the polypeptides of (a)-(f);
(i) derivatives of any one of the polypeptides of (a)-(f), said derivatives being at least 80% identical in amino acid sequence to the corresponding polypeptide of (a)-(f); and (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)-(f) and (h).
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 (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).
(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).
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 expresses in vivo and that;
(1) is a derivative of a 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 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).
(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 expresses in vivo and that;
(1) is a derivative of a 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 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).
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.
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.
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.
molecule according to claim 7.
11. 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 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-B. 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 11-13; 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.
(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 the 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 spirochere.
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.
31. The antibody according to claim 15 that is monoclonal antibody.
32. The antibody according to claim 15 that is isolated from other antibodies.
33. An isolated antibody which binds P35 having the amino acid sequence of SEQ ID NO: 5.
34. An isolated antibody which binds P37 having the amino acid sequence of SEQ ID NO: 7.
35. The use of a pharmaceutical composition of any one of claims 16 to 19 for manufacture of a medicament for treating or preventing B. burgdorferi infection or Lyme disease.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/US1996/006610 WO1997042325A1 (en) | 1996-05-08 | 1996-05-08 | B. burgdorferi polypeptides expressed in vivo |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2253834A1 true CA2253834A1 (en) | 1997-11-13 |
Family
ID=22255070
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002253834A Abandoned CA2253834A1 (en) | 1996-05-08 | 1996-05-08 | B. burgdorferi polypeptides expressed in vivo |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP0915977A1 (en) |
JP (1) | JP2000510339A (en) |
CA (1) | CA2253834A1 (en) |
WO (1) | WO1997042325A1 (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2283494A1 (en) * | 1998-01-08 | 1999-07-15 | Biomerieux, Inc. | Recombinant p37/flaa as a diagnostic reagent |
DE19960113A1 (en) * | 1999-12-08 | 2001-06-13 | Wita Proteomics Ag | Means and procedures for the diagnosis of Lyme disease and Lyme disease vaccine |
FI112544B (en) * | 2001-11-26 | 2003-12-15 | Bortech Oy | Procedure for the diagnosis of early and late Lyme Borreliosis |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DK590288D0 (en) * | 1988-10-24 | 1988-10-24 | Symbicom Ab | CHEMICAL COMPOUNDS |
-
1996
- 1996-05-08 JP JP09539871A patent/JP2000510339A/en active Pending
- 1996-05-08 CA CA002253834A patent/CA2253834A1/en not_active Abandoned
- 1996-05-08 WO PCT/US1996/006610 patent/WO1997042325A1/en not_active Application Discontinuation
- 1996-05-08 EP EP96915650A patent/EP0915977A1/en not_active Withdrawn
Also Published As
Publication number | Publication date |
---|---|
JP2000510339A (en) | 2000-08-15 |
EP0915977A1 (en) | 1999-05-19 |
WO1997042325A1 (en) | 1997-11-13 |
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Legal Events
Date | Code | Title | Description |
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EEER | Examination request | ||
FZDE | Discontinued |