Compositions and methods for administering Borrelia DNA

FIELD OF THE INVENTION

This invention relates to compositions and methods for administering Borrelia genospecies DNA encoding antigen(s) in vivo or in vitro. More particularly, this invention relates to compositions and methods for administering Borrelia genospecies DNA encoding an antigen or antigens, e.g., OspA (outer surface protein A) and/or OspB (outer surface protein B), and/or OspC (outer surface protein C), or fragments thereof such as fragments thereof containing at least one epitope of interest, for expression thereof, in vivo, ex vivo or in vitro.

BACKGROUND OF THE INVENTION

Lyme disease is a multisystem illness, transmitted by ticks of the

Ixodes ricinus

complex. The spirochaete

Borrelia burgdorferi

sensu lato is the etiologic agent of Lyme disease, which is now the most common arthropod borne disease in the United States, and is endemic in Central Europe (Barbour and Fish 1993). More particularly, there are three genospecies of Borrelia associated with Lyme disease:

Borrelia burgdorferi, Borrelia afzelii

and

Borrelia garinii. Borrelia burgdorferi

is the etiologic agent of Lyme disease in North America, and some European Lyme disease is considered to be

Borrelia burgdorferi

sensu stricto.

Borrelia afzelii

and

Borrelia garinii

are the major cause of European Lyme disease and are considered

Borrelia burgdorferi

sensu lato.

Although Lyme disease is curable by antibiotic therapy in its early stages, if Lyme disease is allowed to progress, cardiac, neurological and joint abnormalities can arise. Investigations into the development of a human vaccine for Lyme disease are under way. The outer surface lipoprotein OspA of

Borrelia burgdorferi

is the current major candidate molecule for development of such a vaccine.

Recombinant OspA lipoprotein (rOspA) is known to elicit a protective immune response in mice against challenge by infectious

B. burgdorferi

(Fikrig et al., 1990; Erdile et al., 1993; U.S. Ser. No. 08/373,455). OspA is currently undergoing human field trials as a subcutaneously administered vaccine in the United States (Keller et al., 1994). Above-cited U.S. Pat. No. 5,688,512 relates to substantially pure OspA, vaccines including substantially pure OspA, and methods for inducing a protective immunological response against

B. burgdorferi

employing such vaccines, inter alia.

Above-cited applications Ser. No. 08/373,455 and PCT/US92/08697 relate to rOspA vaccines, especially lipidated rospA, and methods for expressing DNA encoding OspA. Above-cited applications Ser. No. 08/320,416 (now U.S. Pat. No. 5,582,990) and WO 90/04411 relate to DNA encoding OspA, the amino acid sequence of OspA, synthetic OspA, compositions containing OspA or synthetic OspA, and methods of using such compositions. Above cited application Ser. No. 08/137,175, filed Oct. 26, 1993 (now U.S. Pat. No. 5,777,095) relates to DNA coding for various OspAs and OspBs, OspAs and OspBs encoded by such DNA (including amino acid sequences therefor), and immunologically interesting fragments of OspAs and OspBs and DNA coding therefor.

In approximately half of the European isolates of

B. burgdorferi

, outer surface protein C (OspC) is the major surface antigen found on these spirochetes. Immunization of gerbils and mice with purified recombinant OspC produces protective immunity to

B. burgdorferi

strains expressing the homologous OspC protein (Preac-Mursic et al., INFECTION (1992) 20:342-349; Probert et al., INFECTION AND IMMUNITY (1994) 62:1920-1926). Published international patent application WO 91/09870 (Mikrogen Molekularbiologische Entwicklungs-GmbH) describes the DNA sequence of the ospC gene of

B. burgdorferi

strain Pko and the OspC protein encoded thereby of 22 kDa molecular weight (termed “pC” therein). This sequence reveals that OspC is a lipoprotein that employs a signal sequence similar to that used for OspA. As to DNA encoding OspC or recombinant OspC, reference is also made to WO96/05313 (Max-Planck Institute); Leuba-Garcia et al., Zentralbl Bakteriol. 287(4):475-84, 1998; Rauer et al., J. Clin. Microbiol. 36(4):857-61, 1998; Masuzawa et al., Clin. Diagn. Lab. Immunol. 4(1):60-63, 1997; Fukunaga et al. J. Clin. Microbiol. 33(9):2415-2420, 1995; Jauris-Heipke et al., J. Clin. Microbiol. 33(7):1860-66, 1995; Theisen et al., J. Bacteriol. 177(11):3036-3044, 1995; Stevenson et al. FEMS Microbiol. Lett. 124(3):367-72, 1994; and Padula et al., Infect. Immun. 61(12):5097-5105, 1993.

The other above-cited applications relate to DNA encoding other Borrelia antigens or other Osps, or to DNA encoding useful fragments of OspA or of other Osps, amino acid sequences thereof, compositions containing such fragments or other Osps, and methods for using such compositions; and, such DNA that can be used in the methods of Ser. No. 08/373,455 or PCT/US92/08697 to produce OspA, other Borrelia antigens or outer surface proteins (Osps), or fragments thereof, can be used in this invention. In regard to DNA useful in this invention, reference again made to V. Preac-Mursic et al., supra, W. S. Probert et al., supra, WO 91/09870, supra, as well as to all of the documents cited herein and also to Molecular Microbiology (1989), 3(4), 479-486, and PCT publications WO 93/04175, and WO 96/06165.

Alternative vaccination strategies are desirable as such provide alternative routes to administration or alternative routes to responses.

In particular, it is believed that heretofore the art has not taught or suggested administration to a eukaryotic cell in vitro or ex vivo, or to a mammalian host—domesticated or wild or human—susceptible to Lyme disease, of Borrelia genospecies DNA e.g., DNA encoding OspA and/or OspB, and/or OspC or expression thereof in vivo, especially as herein disclosed.

OBJECTS AND SUMMARY OF THE INVENTION

It is an object of the invention to provide methods and compositions for administering to a host, such as a mammalian host susceptible to Lyme Disease, Borrelia genospecies isolated and/or purified DNA encoding an antigen or antigens or a fragment or fragments thereof such as fragment or fragments containing at least one epitope of interest, e.g., isolated and/or purified DNA encoding an antigen or antigens or a fragment or fragments thereof such as fragment or fragments containing at least one epitope of interest from

Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii

or combinations thereof, such as isolated and/or purified DNA encoding OspA, and/or OspB and/or OspC, or isolated and/or purified DNA encoding at least one epitope of OspA and/or OspB and/or OspC; for instance, DNA encoding

Borrelia burgdorferi

OspA and/or OspB and/or OspC. The compositions can include a carrier or diluent. The DNA is administered in a form to be expressed by the host, and preferably in an amount sufficient to induce a response such as a protective immune response; and, the DNA can be administered without any necessity of adding any immunogenicity-enhancing adjuvant.

Accordingly, the present invention provides Borrelia genospecies antigen or epitope DNA plasmids for expression by eukaryotic cells, compositions containing the plasmids, and methods for using the compositions and for using the products from the compositions.

The plasmid of the invention can comprise from upstream to downstream (5′ to 3′): DNA encoding a promoter for driving expression in eukaryotic cells, DNA encoding a leader peptide for enabling secretion of a prokaryotic protein sequence from a mammalian cell, Borrelia genospecies antigen or epitope DNA, and DNA encoding a terminator.

The DNA encoding a promoter for driving expression in eukaryotic cells can be a eukaryotic, e.g., mammalian, viral promoter, such as a herpes virus promoter. A human cytomegalovirus promoter is presently preferred. The human cytomegalovirus promoter can be an immediate early human cytomegalovirus promoter such as HCMV-IE. The plasmid can contain the HCMV-IE gene 5′ untranslated region (UTR) which includes Intron A. This sequence can be 3′ to the HCMV-IE promoter and 5′ to the portion of the chimeric 5′ UTR sequence and leader peptide (the UTR and leader peptide coding sequence can be derived from the DNA encoding the human tissue plasminogen activator, as discussed below).

The DNA encoding a leader peptide is for facilitating secretion of a prokaryotic protein sequence from a mammalian cell. This DNA can be any DNA encoding a suitable or similar leader peptide for the purpose of secretion from a mammalian cell, e.g., DNA encoding a eukaryotic leader peptide. For instance, the DNA encoding a leader peptide can be from DNA encoding a peptide hormone, i.e., a peptide hormone leader peptide, such as from a mammal, e.g., a human peptide hormone leader peptide. Specific examples of DNA encoding leader peptides suitable use in the invention include the DNA encoding the leader peptide of insulin (human, bovine, porcine, etc.), renin, Factor VIII, and tissue plasminogen activator.

DNA encoding human tissue plasminogen activator (TPA) leader is presently preferred. The DNA encoding TPA is derived from the TPA gene and encodes a portion of the 5′ UTR and leader peptide from the gene. TPA DNA having a portion of the 5′ UTR and leader peptide can even increase eukaryotic cell expression. Without wishing to necessarily be bound by any one particular theory, increased expression can be due to the 5′ UTR.

The Borrelia genospecies antigen or epitope DNA is preferably without the natural leader sequence. The Borrelia genospecies antigen or epitope DNA can preferably encode at least one antigen selected from OspA, OspB, OspC, OspD, other Osps, and other antigens (see documents cited herein). Similarly, the Borrelia genospecies antigen or epitope DNA can preferably encode at least one epitope of interest from an antigen selected from OspA, OspB, OspC, OspD, other osps, and other antigens. DNA without the natural leader sequence encoding OspA and/or OspB and/or OspC and/or an epitope of interest from OspA and/or OspB and/or OspC is presently preferred. The DNA can be from

Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii

or from any combination thereof; with

Borrelia burdorferi

presently preferred.

The terminator can be any suitable terminator sequence for use in eukaryotic cells; for instance, a terminator sequence from a mammalian peptide hormone. The Bovine Growth Hormone (BGH) terminator is presently preferred.

The plasmid of the invention can contain genes for a fusion protein of the activator (e.g., TPA) and the Borrelia antigen (e.g., OspA, OspB, OspC) or at least an epitope thereof.

The invention comprehends compositions comprising the plasmid and a carrier. The carrier can be any suitable carrier which does not disrupt the DNA or the function of the plasmid. Saline is a presently preferred carrier.

The invention further comprehends methods of using the compositions, e.g., administering the composition to a host susceptible to Lyme disease to elicit a response or express an antigen. The response can be protective. The response can be simply an antibody response, and the method can further include recovering antibodies and/or antibody-producing cells (with the cells further useful for producing hybridoma cells and monoclonal antibodies, e.g., by fusing the antibody-producing cells to produce hybridoma cells, and then obtaining monoclonal antibodies from expression by the hybridoma cells). The host can be a mammal, such as a human.

Still further, the invention provides methods for expressing an antigen or epitope or preparing an antigen or epitope in vitro comprising transfecting eukaryotic cells with an inventive plasmid. The method can further comprise recovering the antigen or epitope from the cells.

Methods for preparing the plasmid by ligating the respective pieces of DNA (DNA molecules) from which it is comprised, and methods for using the antibodies and antigens or epitopes, as well as the antigens or epitopes and antibodies themselves, are contemplated by the present invention.

Other objects and embodiments are disclosed or are obvious from the following Detailed Description.

BRIEF DESCRIPTION OF DRAWINGS

In the following Detailed Description, reference is made to the accompanying Figures, incorporated herein by reference, wherein:

FIG. 1

shows a diagram of VR2210 with a few characteristic restriction sites;

FIGS. 2A

,

2

B,

2

C, and

2

D show the nucleotide sequence of VR2210 (SEQ ID NO:1) with the location of primer, used to amplify and insert the OspA coding sequence, underlined and overlined;

FIG. 3

shows a diagram of VR2211 with a few characteristic restriction sites;

FIGS. 4A

,

4

B,

4

C and

4

D show the nucleotide sequence of VR2211 (SEQ ID NO:2) with the location of primer, used to amplify and insert the OspB coding sequence, underlined and overlined;

FIGS. 5A

,

5

B, and

5

C show the nucleotide sequence of OspA (TRH43;

B. burgdorferi

strain 31) (SEQ ID NO:3);

FIGS. 6A

,

6

B, and

6

C show the nucleotide sequence of OspB (TRH46;

B. burgdorferi strain

B31) (SEQ ID NO:4);

FIGS. 7A

,

7

B,

7

C,

7

D,

7

E,

7

F,

7

G, and

7

H show a nucleotide sequence of ospA and ospB (SEQ ID NO:5) and a predicted amino acid sequence therefor (SEQ ID NO:18);

FIG. 8

shows a diagram of VR1012;

FIG. 9

shows the multiple cloning site sequence of VR1012 from nucleotides 1841 to 2001 (SEQ ID NO:6);

FIGS. 10A and 10B

show the nucleotide sequence of VR1012 (SEQ ID NO:7);

FIGS. 11A and 11B

show the nucleotide sequence of nkCMVintBL (SEQ ID NO:8);

FIG. 12

shows the nucleotide sequence of the nkCMVintBL cloning site with the TPA signal peptide sequence (SEQ ID NO:9);

FIG. 13

shows PCR primers for amplifying ospA (SEQ ID NO:10, 11);

FIG. 14

shows PCR primers for amplifying ospB (SEQ ID NO:12, 13);

FIG. 15

shows a partial nucleotide sequence of VR2210 (SEQ ID NO:16);

FIG. 16

shows a partial nucleotide sequence of VR2211 (SEQ ID NO:17);

FIGS. 17

,

18

A and

18

B show immunoblots;

FIG. 19

shows an immunoblot;

FIG. 20

shows a diagram of VR2212 with a few characteristic restriction sites;

FIG. 21

shows the nucleotide sequence of VR2212 (SEQ ID NO:19) (5626 bps DNA circular; B31 ospC with human TPA leader sequence; the region of bps 1-1621 is named CMVprom/enh/int″ which is CMVpromoter/enhancer/intron; the gene of bps 1631-1804 is named TPA signal which is 5′ UT and TPA signal; the gene of bps 1805-2390 is named B31 without its endogenous leader peptide ospC which is coding for the outer surface protein C from B.

burgdorferi

B31; the region of bps 2397-2944 is named BGH term. and is the BGH terminator; the gene of bps 5077-4262 is named Kan and is coding for Kanamycin resistance (as a selectable marker));

FIG. 22

shows a diagram of VR2214 with a few characteristic restriction sites;

FIG. 23

shows the nucleotide sequence of VR2214 (SEQ ID NO:20) (5632 bps DNA circular; Pko ospC with human TPA leader sequence; the region of bps 1-1621 is named CMvprom/enh/int″ which is CMVpromoter/enhancer/intron; the gene of bps 1631-1804 is named TPA signal which is 5′ UT and TPA signal and 5′ UT; the gene of bps 180-2396 is named PkoospC which is coding for the outer surface protein C from

B. burgdorferi

Pko with out its leader peptide; the region of bps 2403-2950 is named BGH term. and is the BGH terminator; the gene of bps 5083-4268 is named Kan and is coding for Kanamycin resistance [as a selectable marker]);

FIG. 24

shows a diagram of VR1020 with a few characteristic restriction sites;

FIG. 25

shows a diagram of VR1027 with a few characteristic restriction sites;

FIG. 26

shows the nucleotide sequence of VR1027 (SEQ ID NO:23) (5046 bps circular DNA); and

FIG. 27

shows a graph of ELISA GMT vs. days for Group 1 (diamonds: C3H/He mice that received B31 ospC DNA in plasmid form, i.e., VR2212), Group 2 (squares: ICR mice that received B31 ospC DNA in plasmid form, i.e., VR2212), Group 3 (triangles: C3H/He mice that received Pko ospC DNA in plasmid form, i.e., VR2214), and Group 4 (X's: ICR mice that received Pko ospC DNA in plasmid form, i.e., VR2214).

DETAILED DESCRIPTION

Direct injection of plasmid DNA has become a simple and effective method of vaccination against a variety of infectious diseases (see, e.g., Science, 259:1745-49, 1993; U.S. Pat. Nos. 5,703,055, 5,693,622, 5,589,466 and 5,580,859; Robinson et al., seminars in IMMUNOLOGY, 9:271-83, 1997; Luke et al., J. Infect. Dis., 175(1):91-97, 1997; Norman et al., Vaccine, 15(8):801-803, 1997; (Bourne al., The Journal of Infectious Disease, 173:800-7, 1996;) as well as allowed U.S. application Ser. No. 08/663,998; each of which, and the documents cited therein, are hereby incorporated herein by reference). It is potentially more potent and longer lasting than recombinant protein vaccination because it elicits both a humoral as well as a cellular immune response.

The present invention provides a DNA-based vaccine or immunological composition against Lyme disease (e.g.,

Borrelia burgdorferi, afzelii

, or

garinii

) and can elicit an immunological response, which can confer protection, even up to 100%, in mice against challenge with an infectious strain of

Borrelia burgdorferi

. Exemplary plasmids of the invention contain the human cytomegalovirus immediate early promoter driving expression of the B31 outer surface protein A (OspA), or outer surface protein B (OspB), or outer surface protein C (OspC). To facilitate expression in eukaryotic cells, the natural leader sequence of the gene encoding these Osps, e.g., OspA, OspB, OspC has been replaced with the human tissue plasminogen activator leader sequence.

Expression and secretion was demonstrated in transiently transfected UM449 and BHK cells by Western blot (See, e.g.,

FIGS. 17

,

18

A,

18

B).

Protection was demonstrated in C3H/HeN by injecting, intramuscularly, naked plasmid DNA and subsequently challenging with Sh2 spirochetes. Sera taken following vaccination contained high titers of antibody to OspA which inhibited spirochete growth in vitro. Immunized animals showed no sign of Lyme disease at 14 days after challenge. Moreover, all tissues examined were completely free of spirochetes.

Thus, a DNA vaccine or immunological composition, expressing a Borrelia antigen or epitope of interest, for instance a

Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii

antigen or epitope of interest or combinations thereof, e.g., OspA, OspB, OspC protein or epitopes therefrom or any combination thereof, can protect mice against infection by a Borrelia genospecies, the etiologic agent of Lyme disease. The composition is thus useful for eliciting a protective response in a host susceptible to Lyme Disease, as well as for eliciting antigens, epitopes, and antibodies, which also are useful in and of themselves.

Therefore, as discussed above, the invention in a general sense preferably provides methods for immunizing, or vaccinating, or eliciting an immunological response in a host, such as a host susceptible to Lyme disease, e.g., a mammalian host, against Borrelia and accordingly Lyme Disease, by administering DNA encoding a Borrelia antigen or epitope, for instance DNA encoding a

Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii

antigen or epitope of interest or combinations thereof, e.g., OspA and/or OspB, and/or OspC, preferably OspA, in a suitable carrier or diluent, such as saline; and, the invention provides plasmids and compositions for performing the method, as well as methods for making the plasmids, and uses for the expression products of the plasmids, as well as for antibodies elicited thereby.

From present dog and human trials based on efficacy studies with mice (Erdile et al., 1993; U.S. Ser. No. 08/373,455), it is clear that mice are now a suitable animal model with respect to Borrelia and Lyme disease for extrapolation to domestic animals, humans, and other animals susceptible to Lyme disease or Borrelia infection (e.g., wild animals such as deer).

In view of the broad nature of the invention, i.e., that the invention is applicable to Borrelia genospecies other than burgdorferi (i.e., the invention is also applicable to genospecies

afzelii

and

garinii

, and broadly to any Borrelia genospecies antigen or antigens or epitope or epitopes or immunologically active fragments of antigen or antigens), discussion herein directed to OspA is intended toward the broad nature of the invention, i.e., “OspA” is exemplary and can be read in this specification to include any Borrelia genospecies antigen or an immunological fragment thereof, including without limitation, OspA, OspB, OspC and epitopes or immunologically active fragments thereof.

In the present invention, the DNA encoding OspA, or broadly, the Borrelia genospecies antigen or epitope or immunologically active fragment thereof, can be administered in dosages and by techniques well known to those skilled in the medical or veterinary arts taking into consideration such factors as the age, sex, weight, species and condition of the particular patient, and the route of administration. DNA encoding OspA, or broadly the Borrelia genospecies antigen or epitope or immunologically active fragment thereof, can be administered alone, or can be co-administered or sequentially administered with other Borrelia antigens or epitopes, or with DNA encoding other Borrelia genospecies antigens or epitopes; and, the DNA encoding OspA or broadly the Borrelia genospecies antigen or epitope or immunologically active fragment thereof, can be sequentially administered, e.g., each Spring as the “Lyme Disease season” is about to begin.

As broadly discussed above, the invention comprehends plasmids comprising DNA including Borrelia genospecies antigen or epitope DNA for expression by eukaryotic cells. The DNA, from upstream to downstream (5′ to 3′), can comprise: DNA encoding a promoter for driving expression in eukaryotic cells, DNA encoding a leader peptide which enables secretion of a prokaryotic protein sequence from a mammalian cell, DNA encoding a Borrelia genospecies antigen (or antigens or epitope or epitopes), and DNA encoding a terminator.

For instance, the promoter can be a eukaryotic viral promoter such as a herpes virus promoter, e.g., human cytomegalovirus promoter DNA.

The DNA encoding a leader peptide which enables secretion of a prokaryotic protein sequence from a mammalian cell is any DNA encoding any suitable leader for this purpose such as DNA encoding a eukaryotic, preferably mammalian, leader sequence; for instance, DNA encoding a leader peptide of a peptide hormone, or, for example, of insulin, renin, Factor VIII, TPA, and the like, with DNA encoding human tissue plasminogen activator (TPA) leader peptide presently preferred.

The human cytomegalovirus promoter can be an immediate early human cytomegalovirus promoter such as HCMV-IE. As to HCMV promoter, reference is made to U.S. Pat. Nos. 5,168,062 and 5,385,839. The plasmid of the invention can contain the HCMV-IE gene 5′ untranslated region (UTR) which includes Intron A. This sequence can be 3′ to the HCMV-IE promoter and 5′ to the activator portion of the 5′ UTR sequence and leader peptide.

The TPA sequence can be derived from the TPA gene and can encode a portion of the 5′ UTR and leader peptide from that gene. The 5′ UTR of TPA may increase eukaryotic cell expression.

The Borrelia genospecies DNA can be from

B. burgdorferi, afzelii, garinii

or combinations thereof, e.g.,

B. burgdorferi

; and, can encode an antigen such as OspA, OspB, OspC, OspD, other outer surface proteins or an epitope or epitopes therefrom or a combination of antigens, e.g., OspA and/or OspB and/or OspC; preferably without the natural leader sequence.

The transcriptional terminator sequence can be any suitable terminator, such as a eukaryotic terminator, for instance, DNA encoding a terminator for a mammalian peptide, with the BGH terminator presently preferred.

The plasmid can be in admixture with any suitable carrier, diluent or excipient such as sterile water, physiological saline, and the like. Of course, the carrier, diluent or excipient should not disrupt or damage the plasmid DNA.

The plasmid can be administered in any suitable manner. The plasmid can be in a composition suitable for the manner of administration. The compositions can include: liquid preparations for orifice, e.g., oral, nasal, anal, vaginal, peroral, intragastric administration and the like, such as solutions, suspensions, syrups, elixirs; and liquid preparations for parenteral, subcutaneous, intradermal, intramuscular, intravenous administration, and the like, such as sterile solutions, suspensions or emulsions, e.g., for administration by injection. Intramuscular administration and compositions therefor are presently preferred.

The plasmids of the invention can be used for in vitro expression of antigens by eukaryotic cells. Recovery of such antigens can be by any suitable techniques; for instance, techniques analogous to the recovery techniques employed in the documents cited herein (such as the applications cited under Related Applications and the documents cited therein).

The thus expressed antigens or epitopes can be used in immunological, antigenic or vaccine compositions, with or without an immunogenicity-enhancing adjuvant (“expressed antigen compositions”). Such compositions can be administered in dosages and by techniques well known to those skilled in the medical or veterinary arts taking into consideration such factors as age, sex, weight, species, condition of the particular patient, and the route of administration. These compositions can be administered alone or with other compositions, and can be sequentially administered, e.g., each Spring as the “Lyme Disease season” is about to begin.

The route of administration for the expressed antigen compositions can be oral, nasal, anal, vaginal, peroral, intragastric, parenteral, subcutaneous, intradermal, intramuscular, intravenous, and the like.

The expressed antigen compositions can be solutions, suspensions, emulsions, syrups, elixirs, capsules (including “gelcaps”—gelatin capsule containing a liquid antigen or fragment thereof preparation), tablets, hard-candy-like preparations, and the like. The expressed antigen compositions may contain a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, adjuvants, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE”, 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.

Suitable dosages for plasmid compositions and for expressed antigen compositions can also be based upon the examples below, and upon the documents herein cited. For example, suitable dosages can be 0.5-500 μg antigen, preferably 0.5 to 50 μg antigen, for instance, 1-10 μg antigen in expressed antigen compositions. In plasmid compositions, the dosage should be a sufficient amount of plasmid to elicit a response analogous to the expressed antigen compositions; or expression analogous to dosages in expressed antigen compositions. For instance, suitable quantities of plasmid DNA in plasmid compositions can be 0.1 μg to 2 mg, preferably 1-10 μg. Dosages can also be varied by one skilled in the art, depending on typical factors, such as the age, sex, weight, general health, and species of the patient, without undue experimentation.

Thus, in a broad sense, the invention further provides a method comprising administering a composition containing plasmid DNA including DNA encoding a Borrelia genospecies antigen or antigens or epitope or epitopes: for expression of the antigen or antigens or epitope or epitopes in vivo for eliciting an immunological, antigenic or vaccine (protective) response by a eukaryotic cell; or, for ex vivo or in vitro expression (That is, the cell can be a cell of a host susceptible to Lyme Disease, i.e., the administering can be to a host susceptible to Lyme Disease such as a mammal, e.g., a human; or, the cell can be an ex vivo or in vitro cell). The invention further provides a composition containing a Borrelia genospecies antigen or antigens or epitope or epitopes from expression of the plasmid DNA by a eukaryotic cell, in vitro or ex vivo, and methods for administering such compositions to a host mammal susceptible to Lyme disease to elicit a response.

Since the methods can stimulate an immune or immunological response, the inventive methods can be used for merely stimulating an immune response (as opposed to also being a protective response) because the resultant antibodies (without protection) are nonetheless useful. From eliciting antibodies, by techniques well-known in the art, monoclonal antibodies can be prepared and, those monoclonal antibodies, can be employed in well known antibody binding assays, diagnostic kits or tests to determine the presence or absence of a Borrelia genospecies or to determine whether an immune response to the bacteria has simply been stimulated. Those monoclonal antibodies can also be employed in recovery or testing procedures, for instance, in immunoadsorption chromatography to recover or isolate a Borrelia genospecies antigen such as OspA, OspB, or OspC or epitope therefrom.

Monoclonal antibodies are immunoglobulins produced by hybridoma cells. A monoclonal antibody reacts with a single antigenic determinant and provides greater specificity than a conventional, serum-derived antibody. Furthermore, screening a large number of monoclonal antibodies makes it possible to select an individual antibody with desired specificity, avidity and isotype. Hybridoma cell lines provide a constant, inexpensive source of chemically identical antibodies and preparations of such antibodies can be easily standardized. Methods for producing monoclonal antibodies are well known to those of ordinary skill in the art, e.g., Koprowski, H. et al., U.S. Pat. No. 4,196,265, issued Apr. 1, 1989, incorporated herein by reference.

Uses of monoclonal antibodies are known. One such use is in diagnostic methods, e.g., David, G. and Greene, H. U.S. Pat. No. 4,376,110, issued Mar. 8, 1983; incorporated herein by reference. Monoclonal antibodies have also been used to recover materials by immunoadsorption chromatography, e.g., Milstein, C. 1980, Scientific American 243:66, 70, incorporated herein by reference.

Returning now to the preparation of the inventive plasmids, the DNA therein is preferably ligated together to form a plasmid. For instance, the promoter, leader sequence, antigen and terminator DNA is preferably isolated, purified and ligated together in a 5′ to 3′ upstream to downstream orientation. A three-way ligation, or a two-way ligation, as exemplified below, is presently preferred.

Accordingly, the inventive methods and products therefrom have several hereinstated utilities. Other utilities also exist for embodiments of the invention.

As to an epitope of interest from a Borrelia genospecies antigen, reference is made to the following discussion, and generally to Kendrew,

The Encyclopedia Of Molecular Biology

, Blackwell Science Ltd., 1995 and Sambrook, Fritsch and Maniatis,

Molecular Cloning, A Laboratory Manual,

2nd Ed., Cold Spring Harbor Laboratory Press, 1982 (“Maniatis et al., 1982”), U.S. Pat. Nos. 5,582,990, 5,688,512 and 5,777,095, and WO 91/09870, inter alia.

For instance, U.S. Pat. Nos. 5,582,990 and 5,688,512 provide the nucleotide and amino acid sequences of ospA and OspA, the hydropathic index of OspA (location of hydrophobic and hydrophilic amino acids in OspA), the hydrophilicity profile of OspA and the most hydrophilic region of the protein, a curve of the charge of OspA as a function of pH, the amino acid composition of OspA and the secondary structure of OspA, inter alia. U.S. Pat. No. 5,777,095 relates to DNA coding for various OspAs and OspBs, OspAs and OspBs encoded by such DNA (including amino acid sequences therefor), and immunologically interesting fragments of OspAs and OspBs and DNA coding therefor, inter alia. And, WO 91/09870 describes the DNA sequence of the ospC gene of

B. burgdorferi

strain Pko and the OspC protein encoded thereby of 22 kDa molecular weight (termed “pC” therein), inter alia.

More generally, one skilled in the art can determine an epitope or immunodominant region of a peptide or polypeptide, e.g., a Borrelia genospecies antigen such as OspA, OspB or OspC, and ergo the coding DNA therefor, from the knowledge of the amino acid and corresponding DNA sequences of the peptide or polypeptide, as well as from the nature of particular amino acids (e.g., size, charge, etc.) and the codon dictionary, without undue experimentation.

A general method for determining which portions of a protein, e.g., a Borrelia genospecies antigen such as OspA, OspB or OspC, to use in an immunological composition focuses on the size and sequence of the antigen of interest. “In general, large proteins, because they have more potential determinants are better antigens than small ones. The more foreign an antigen, that is the less similar to self configurations which induce tolerance, the more effective it is in provoking an immune response.” Ivan Roitt,

Essential Immunology

(Blackwell Scientific Publications, Oxford, 1988).

As to size: the skilled artisan can maximize the size of the protein, e.g., an epitope from a Borrelia genospecies antigen such as OspA, OspB or OspC, encoded by the DNA sequence to be inserted into the vector (keeping in mind the packaging limitations of the vector).

At a minimum, the DNA sequence can code for a peptide at least 8 or 9 amino acids long, i.e., at a minimum an epitope from a Borrelia genospecies antigen such as OspA, OspB or OspC, can be at least 8 or 9 amino acids long. This is the minimum length that a peptide needs to be in order to stimulate a CD8+T cell response (which recognizes virus infected cells or cancerous cells). A minimum peptide length of 13 to 25 amino acids is useful to stimulate a CD4+T cell response (which recognizes special antigen presenting cells which have engulfed the pathogen). See Kendrew, supra. However, as these are minimum lengths, these peptides are likely to generate an immunological response, i.e., an antibody or T cell response; but, for a protective response (as from a vaccine composition), an epitope from a Borrelia genospecies antigen such as OspA, OspB or OspC, is preferably a peptide having a greater length.

With respect to the sequence, the DNA sequence preferably encodes at least regions of the peptide, e.g., a Borrelia genospecies antigen such as OspA, OspB or OspC, that generate an antibody response or a T cell response. One method to determine T and B cell epitopes involves epitope mapping. The protein of interest is fragmented into overlapping peptides with proteolytic enzymes. The individual peptides are then tested for their ability to bind to an antibody elicited by the native protein or to induce T cell or B cell activation. This approach has been particularly useful in mapping T-cell epitopes since the T cell recognizes short linear peptides complexed with MHC molecules. Note that B-cell epitopes are often not linear amino acid sequence but rather result from the tertiary structure of the folded three-dimensional protein. Janis Kuby,

Immunology

, pp. 79-80 (W. H. Freeman, July 1992).

Another method for determining an epitope of interest is to choose the regions of the protein that are hydrophilic. Hydrophilic residues are often on the surface of the protein and are therefore often the regions of the protein which are accessible to the antibody. Janis Kuby,

Immunology,

p. 81 (W. H. Freeman, July 1992).

Yet another method for determining an epitope of interest is to perform an X-ray crystallographic analysis of the antigen (full length)-antibody complex. Janis Kuby,

Immunology

, p.80 (W. H. Freeman, July 1992).

Still another method for choosing an epitope of interest which can generate a T cell response is to identify from the protein sequence potential HLA anchor binding motifs (peptide sequences which are known to be likely to bind to the MHC molecule).

The peptide which is a putative epitope of interest, to generate a T cell response, should be presented in a MHC complex. The peptide preferably contains appropriate anchor motifs for binding to the MHC molecules, and should bind with high enough affinity to generate an immune response. Factors which can be considered are: the HLA type of the patient (vertebrate, animal or human) expected to be immunized, the sequence of the protein, the presence of appropriate anchor motifs and the occurrence of the peptide sequence in other vital cells.

An immune response is generated, in general, as follows: T cells recognize proteins only when the protein has been cleaved into smaller peptides and is presented in a complex called the “major histocompatability complex MHC” located on another cell's surface. There are two classes of MHC complexes—class I and class II, and each class is made up of many different alleles. Different patients have different types of MHC complex alleles; they are said to have a ‘different HLA type.’

Class I MHC complexes are found on virtually every cell and present peptides from proteins produced inside the cell. Thus, Class I MHC complexes are useful for killing cells when infected. T cells which have a protein called CD8 on their surface, bind specifically to the MHC class I/peptide complexes via the T cell receptor. This leads to cytolytic effector activities.

Class II MHC complexes are found only on antigen-presenting cells and are used to present peptides from circulating pathogens which have been endocytosed by the antigen-presenting cells. T cells which have a protein called CD4 bind to the MHC class II/peptide complexes via the T cell receptor. This leads to the synthesis of specific cytokines which stimulate an immune response.

An epitope from a Borrelia genospecies antigen such as OspA, OspB or OspC, is preferably a peptide having a length greater than 8 or 9 or 13 to 25 amino acids because cells may cut the expressed peptides. The peptide preferably contains an appropriate anchor motif which will enable it to bind to the various class I or class II molecules with high enough specificity to generate an immune response (See Bocchia, M. et al,

Specific Binding of Leukemia Oncogene Fusion Protein Peptides to HLA Class I Molecules

, Blood 85 (10):2680-2684, May 15, 1995; Englehard, VH,

Structure of peptides associated with class I and class II MHC molecules

, Ann. Rev. Immunol. 12:181 (1994)). This can be done, without undue experimentation, by comparing the sequence of the protein of interest with published structures of peptides associated with the MHC molecules. Protein epitopes recognized by T cell receptors are peptides generated by enzymatic degradation of the protein molecule and are presented on the cell surface in association with class I or class II MHC molecules.

Further, the skilled artisan can ascertain an epitope of interest by comparing the protein sequence with sequences listed in the protein data base.

Even further, another method is simply to generate or express portions of a protein of interest, generate monoclonal antibodies to those portions of the protein of interest, and then ascertain whether those antibodies inhibit growth in vitro. The skilled artisan can use the other guidelines set forth in this disclosure and in the art for generating or expressing portions of a protein of interest, e.g., a Borrelia genospecies antigen such as OspA, OspB or OspC, for analysis as to whether antibodies thereto inhibit growth in vitro. For example, the skilled artisan can generate portions of a protein of interest, e.g., a Borrelia genospecies antigen such as OspA, OspB or OspC, by: selecting 8 to 9 or 13 to 25 amino acid length portions of the protein, selecting hydrophylic regions, selecting portions shown to bind from X-ray data of the antigen (full length)-antibody complex, selecting regions which differ in sequence from other proteins, selecting potential HLA anchor binding motifs, or any combination of these methods or other methods known in the art.

Epitopes recognized by antibodies are expressed on the surface of a protein. To determine the regions of a protein most likely to stimulate an antibody response, e.g., regions of a Borrelia genospecies antigen such as OspA, OspB or OspC most likely to stimulate an antibody response, one skilled in the art can preferably perform an epitope map, using the general methods described above, or other mapping methods known in the art.

As can be seen from the foregoing, without undue experimentation, from this disclosure and the knowledge in the art, the skilled artisan can ascertain the amino acid and corresponding DNA sequence of an epitope of interest of a Borrelia genospecies antigen such as OspA, OspB or OspC, for obtaining a T cell, B cell and/or antibody response. Thus, without undue experimentation, one skilled in the art can make and use plasmids containing and expressing in vivo an immunologically active fragment or fragments or an epitope or epitopes of a Borrelia genospecies antigen, from this disclosure and the knowledge in the art.

A better understanding of the present invention and of its many advantages will be had from the following examples given by way of illustration.

EXAMPLES

Example 1 Plasmid Construction

DNA plasmids VR2210 (

FIGS. 1 and 2

) (SEQ ID NO:1) and VR2211 (

FIGS. 3 and 4

) (SEQ ID NO:2) containing, respectively, genes coding for OspA (

FIG. 5

) (SEQ ID NO:3) and OspB (

FIG. 6

) (SEQ ID NO:4) from

Borrelia burgdorferi

(see

FIG. 7

) (SEQ ID NO:5) were constructed by isolating, individually, three DNA molecules: (1) a first DNA molecule having a DNA sequence from restriction endonuclease (PstI and XbaI) digested VR1012 plasmid (

FIGS. 8

,

9

,

10

) (SEQ ID NOS:6, 7); (2) a second DNA molecule having a DNA sequence from PCR amplified TPA 5′ UTR and leader peptide coding sequence (from nkCMVintBL; see Manthorpe et al. (1993) Human Gene Therapy 4, 419-431; see also Chapman et al. (1991) Nucleic Acids Research 19, 3979-86), PstI and KpnI digested (subsequent to PCR amplification) (

FIGS. 11

,

12

) (SEQ ID NOS:8, 9); and (3) a third DNA molecule having a DNA sequence from PCR amplified Borrelia antigen coding sequence, KpnI and XbaI digested, e.g., PCR amplified DNA encoding OspA (

FIG. 5

; SEQ ID NO:3) (PCR primers:

FIG. 13

; SEQ ID NOS:10, 11) or OspB (

FIG. 6

; SEQ ID NO:5) (PCR primers:

FIG. 14

; SEQ ID NOS:12, 13) (see also

FIG. 7

; SEQ ID NO:5; and Howe et al., 1986, Infection and Immunity 54:207-212 (“Howe et al. 1986”); Bergstrom et al., 1989, Molecular Microbiology, 3(4), 479-486). The natural leader sequences of the DNA encoding the Borrelia antigen is not present so as to facilitate expression and secretion in mammalian cells. The constructs (VR2210, VR2211) express secreted proteins in vitro and elicit a specific antibody response in vivo.

In particular, the construct VR2210 contains DNA encoding OspA and was made by ligating three aforementioned DNA molecules (fragments) together (wherein the third DNA molecule or sequence in the foregoing paragraph is DNA encoding OspA, from a plasmid as in Howe et al. 1986, e.g., pTRH43); and, the construct VR2211 contains DNA encoding OspB and was made by ligating the three aforementioned DNA fragments together (wherein the third DNA molecule or sequence in the foregoing paragraph is DNA encoding OspB from a plasmid as in Howe et al., 1986, e.g., pTRH46).

More specifically, the DNA for encoding TPA 5′ UTR and leader peptide, ospA and ospB were PCR amplified. The TPA signal was PCR amplified from plasmid nKCMVintBL using the following primers:

The ospA gene was PCR amplified from pTRH43 using the primers shown in

FIG. 13

(SEQ ID NOS:10, 11) (“Forward” is ospA 5′ primer and “Reverse” is ospA 3′ primer). The ospb gene was PCR amplified from pTRH46 using the primers shown in

FIG. 14

(SEQ ID NOS:12, 13) (“Forward” is ospB 5′ primer and “Reverse” is ospB 3′ primer).

PCR program:

1st—Anneal primer and template

1. 94° C., 2 minutes

2. Ramp slowly, 10 minutes, down to 45° C.

3. 45° C., 5 minutes

2nd—Cycle program

1. 72° C., 3′

2. 93° C., 1′30″

3. Go to 1 32 cycles

4. 54° C., 2′30″

5. 72° C. 10′

6. 4° C. 1-24 hours

PCR REACTIONS

10x

Taq

Buf

H

2

O

BUFFER

dNTPS

5′ PRIMER

3′ PRIMER

DNA

fer

82 μL

10 μL

3 μL (25 mM)

TPA-5′

TPA-3′

ø

1 μL

10x Taq

2 μL (25 mM)

2 μL (25 pico

Buffer

pico-

mole/mM)

mole/μL)

80 μL

10 μL

3 μL (25 mM)

2 μL (25 pico

2 μL (25 pico

2 μL (5

1 μL

10x Taq

mole/μL)

mole/mM)

nano-

Buffer

grams/μL)

10 μL

3 μL (25 mM)

2 μL (25 pico

2 μL (25 pico

2 μL (5

1 μL

10x Taq

mole/μL)

mole/mM)

nano-

Buffer

grams/μL)

10 μL

3 μL (25 mM)

2 μL (25 pico

2 μL (25 pico

2 μL (5

1 μL

10x Taq

mole/μL)

mole/mM)

nano-

Buffer

grams/μL)

82 μL

10 μL

3 μL (25 mM)

ospA-5′

ospA-3′ 2 μL

ø

1 μL

10x Taq

2 μL (25 pico

(25 pico

Buffer

mole/μL)

mole/μL)

80 μL

10 μL

3 μL (25 mM)

ospA-5′

ospA-3′ 2 μL

2 μL (5

1 μL

10x Taq

2 μL (25 pico

(25 pico

nano-

Buffer

mole/μL)

mole/μL)

grams/μL)

72 μL

10 μL

3 μL (25 mM)

ospA-5′

ospA-3′ 2 μL

10 μL (1

1 μL

10x Taq

2 μL (25 pico

(25 pico

nano-

Buffer

mole/μL)

mole/μL)

grams/μL)

82 μL

10 μL

3 μL (25 mM)

ospB-5′

ospB-3′ 2 μL

1 μL

10x Taq

2 μL (25 pico

(25 pico

Buffer

mole/μL)

mole/μL)

80 μL

10 μL

3 μL (25 mM)

ospB-5′

ospB-3′ 2 μL

2 μL (5

1 μL

10x Taq

2 μL (25 pico

(25 pico

nano-

Buffer

mole/μL)

mole/μL)

grams/μL)

72 μL

10 μL

3 μL (25 mM)

ospB-5′

ospB-3′ 2 μL

10 μL (1

1 μL

10x Taq

2 μL (25 pico

(25 pico

nano-

Buffer

mole/μL)

mole/μL)

grams/μL)

The ospA PCR fragments were digested with KpnI/XbaI as follows:

A.

Mixed:

72 μL

ospA DNA

10 μL

New England Biolabs (NEB)

Buffer #1

10 μL

10x Bovine serum albumin (BSA)

8 μL

KDnI (10 units/μL)

100 μL

total.

The mixture was allowed to sit for 2 hrs at 37° C. and then subjected to phenol/chloroform extraction and spin column (G-50 Sephadex) for purification.

B.

Mixed:

100 μL

DNA (5 μg) (from A.)

11.5 μL

NEB Buffer #2 (New England Biobbs

(“NEB”) Buffer #2)

1 μL

BSA 100x

4 μL

XbaI (20 units/μL)

115 μL

total.

The mixture was incubated for 2 hrs at 37° C.

QIAquick (Qiagen) columns were used to purify the fragments (Final volume=50 μL in 10 mM Tris, pH 8.5).

The TPA fragments were digested with PstI/KpnI as follows:

A.

Mixed:

20 μL

TPA DNA (in Tris pH 8.5)

10 μL

NEB Buffer #1

1 μL

100x BSA

64 μL

TE (10 mM Tris, 1 mM EDTA, pH 8.0)

5 μL

KpnI (10 units/μL)

100 μL

total.

The mixture was incubated for 2 hrs at 37° C. and then purified by a QIAquick DNA spin column; and eluted with 50 μL H

2

O (and then subject to B).

B.

Mixed:

50 μL TPA DNA (from A.)

6 μL NEB Buffer #3

2 μL PstI (10 units/μL)

2 μL TE

60 μL total.

The mixture was incubated for 2 hrs at 37° C. Thereafter 40 μL TE was added; and then purified by phenol/chloroform extraction followed by G-50 spin column.

The ospb PCR reaction products that were uncut were consolidated and ethanol precipitated to concentrate them. Then, the ospB products were loaded on a 1% TBE—agarose gel and gel purified using QIAEXII gel extraction (Qiagen) as follows:

1. Excise bands and weigh.

2. Add 3 μL QX1/mg of gel slice.

3. Vortex QIAEX 1′ to completely resuspend.

4. Add 10 uL QIAEX for every 5 μg DNA or less Vortex.

5. Incubate at 50° C. for 10′ mix every 2 minutes.

6. Centrifuge for 30″.

7. Wash 2X in QX2; 500 μL wash. Resuspend by vortexing. Spin 30″ full speed.

8. Wash 2X in QX3; 500 μL wash.

9. Remove all traces of supernatant after last wash.

10. Allow to air dry 15 minutes.

11. Elute with 20 μL TE. Resuspend. Incubate 5′ at room temperature and spin down QIAEX. Remove supernatant.

The ospB fragments were digested with KpnI/BamHI as follows:

A.

Mixed:

5 μL TE

33 μL ospB (1.3 μg)

5 μL NEB Buffer #1

5 μL 10x BSA

2 μL KpnI (10 units/μL)

50 μL total.

The mixture was incubated for 2 hrs at 37° C. Thereafter the mixture was purified with a Qiaquick column and eluted in 30 μl H

2

O.

B.

Mixed:

30 μL DNA (1.3 mg; from A.)

4 μL BamHI Buffer (New England Biolabs)

1 μL BamHI (20 μg/μL) (20 units/μL)

5 μL TE

40 μL total.

The mixture was incubated for 2 hrs at 37° C. Thereafter the mixture was purified with a QIAquick column, and eluted in 3 μL H

2

O.

A.

Mixed:

5 μL DNA (5 μg) VR1012

3 μL 10X BSA

3 μL NEB Buffer #2

18 μL TE

1 μL XbaI (20 units/λ

30 μL total.

The mixture was incubated for 2 hrs at 37° C. 1 μL was then removed for gel analysis which confirmed digestion.

B. Added to product of A:

1.5 μL

1M Tris

3.5 μL

NaCl (500 mM)

1 μL

PstI (10 μ/λ)

35 μL

Total

The mixture in B. incubated sit for 2 hours at 37° C. Thereafter, 1 μL of EcoRV was added and the resultant mixture incubated at 37° C. for an additional hour (this cuts the small restriction fragment in half and can increase efficiency of removal by the spin column). The mixture was then subjected to a G50 spin column to remove the small insert.

To then construct VR2210, a mixture containing 1 μL of the PstI/XbaI digested VR1012 DNA (25 ng), 1 μL of the KpnI/XbaI digested ospA DNA (200 ng), 6 μL of the PstI/KpnI digested TPA DNA (50 ng), 2 μL Rapid DNA Ligation Buffer #2, 10 μL Rapid DNA Ligation Buffer #1, and 1 μL Rapid DNA ligase (Boehringer Mannheim) was prepared. To construct VR2211, a mixture containing 1 μL of the PstI/XbaI digested VR1012 DNA (25 ng), 5 μL of the KpnI/XbaI digested ospB DNA (150 ng), 3 μL of the PstI/KpnI digested TPA DNA, 2 μL Rapid DNA Ligation Buffer #2, 10 μL Rapid DNA Ligation Buffer #1, and 1 μL Rapid DNA ligase (Boehringer Mannheim) was prepared. Rapid, 3-way ligations occurred in each of the mixtures, with VR2210 and VR2211 resulting, respectively. Control mixtures without (i) the ospA and TPA DNA and (ii) the ospb and TPA were also prepared. The control mixtures were set up to test for the number of background clones due to uncut vector. A very low number (less than one-tenth) of clones were generated as a result of these control ligations clearly indicating that the three-way ligations worked efficiently.

FIG. 15

provides a partial sequence of VR2210 extending from a position 5′ to PstI site to an arbitrary point 3′ to the KpnI site (KpnI, 231) (through the PstI site, the TPA leader, the KpnI site, and into the ospA DNA) (SEQ ID NO: 16).

FIG. 16

provides a partial sequence of VR2211 extending from a position 5′ to PstI site to an arbitrary point 3′ to the KpnI site (KpnI, 266) (through the PstI site, the TPA leader, the KpnI site and into the ospB DNA) (SEQ ID NO: 17).

ANALYTICAL RESTRICTION DIGEST:

Enzyme(s)

Number and size of fragments

VR2210

1. KpnI

2 fragments: 1042/4804

2. HindIII

2 fragments: 2803/3043

3. PstI

2 fragments: 783/5063

VR2211

1. KpnI

2 fragments: 1091/4803

2. HindIII

2 fragments: 2460/3439

3. PstI

2 fragments: 834/5065

FIG. 7

provides the nucleotide sequence (SEQ ID NO: 5) of the ospA and ospB genes and the predicted amino acid sequences (SEQ ID NO: 18) of OspA and OspB. Numbers above each line refer to amino acid positions, whereas numbers below the sequence refer to nucleotide positions. The promoter regions P1 and P2 are indicated by horizontal lines. The respective -35 and -10 regions are also shown. The ribosomal binding sites (RBS) are shown by a horizontal line and bold lettering. A possible stem and loop structure at the end of the ospB sequence is indicated by horizontal broken arrows. Start codons of respective OspA and OspB proteins are indicated and stop codons are marked by asterisks.

FIG. 7

also shows the location of the KpnI, XbaI and BamHI sites, with arrows indicating direction (first KpnI to XbaI for OspA DNA, second KpnI to BamHI for OspB DNA are also indicated).

Example 2

Transfections

5μg of VR2210 was transfected into both baby hamster kidney (BHK) and UM449 human melanoma cells (from Mark Cameron, Univ. Mich.) according to the protocol of Felgner et al. (1994), J. Biol. Chem. 269, 2550-2561. The resultant supernatant and cell extract were analyzed for expression of OspA by Western blot, using two anti-OspA antibodies, 3TS at 1:100 dilution and H5332 at 1:10 dilution in 5% milk/BBS (as to the antibodies, reference is made to Barbour, A. G. et al., J. Infect. Dis. 1985, 152, 478-84; Barbour et al., Infect Immun. 1983, 41, 795-804).

As the Western blot (

FIG. 17

) shows, UM449 cells give a higher level of expression of OspA than BHK cells. Also, there is more OspA in the culture supernatant than in the cell lysate. Therefore, the prokaryotic protein is efficiently transported out of the mammalian cells by a eukaryotic signal peptide sequence (TPA). A summary of the results shown in

FIG. 17

is provided by the following tabulation.

Gel

sup/

DNA

DNA/

Amt.

lane

Sample

Cells

extract

Conc.

lipid

loaded

1

VR2211

UM449

extract

5 μg

1/1

40 μl

2

VR2210

UM449

extract

5 μg

1/1

40 μl

3

VR2211

BHK

extract

5 μg

1/1

40 μl

4

VR2210

BHK

extract

5 μg

1/1

40 μl

5

MW marker

15 μl

6

pos control

B31

1 μl

lysate

7

VR2211

UM449

sup

5 μg

1/1

20 μl

8

VR2210

UM449

sup

5 μg

1/1

20 μl

9

VR2211

BHK

sup

5 μg

1/1

20 μl

10

VR2210

BHK

sup

5 μg

1/1

20 μl

*VR2211 is a plasmid containing the OspB gene and is used as a negative control on the Western.

This Example also demonstrates that the inventive Borrelia antigen DNA plasmid compositions are additionally useful for in vitro expression of antigen(s) or epitope(s) (which by themselves are useful, e.g., for preparing antigenic, immunological or vaccine compositions, or for diagnostic, detection or assay purposes).

Example 3

Transfections

5 μg of VR2211 was transfected into UM449 human melanoma cells according to the protocol of Felgner et al. (1994), J. Biol Chem. 269, 2550-2561. Supernatants and cell extracts were analyzed for expression of OspB by Western blot using a monoclonal anti-OspB called H68 and by a monoclonal anti-OspB H6831 (as to the antibodies, reference is made to Barbour et al., Infect. Immun. 1984, 45, 94-100) (

FIGS. 18A and 18B

) with the ti-OspB H68 and H6831 at 1:10 in Blotto. These antibodies detect OspB in both supernatants and cell extracts. The monoclonal anti-OspB designated H6831 binds to spirochete OspB strain B31) and to unmodified OspB (in vitro translated) but not to modified OspB (i.e., glycosylated and/or phosphorylated by mammalian cellular machinery). Possibly the epitope for H6831 is blocked by eukaryotic post-translational modifications.

A summary of the results shown in

FIG. 18A

is provided following tabulation.

sup/

DNA

DNA/

Amt.

Lane

Sample

Cells

extract

Conc.

lipid

loaded

1

B31

spirochete

lysate

1 μ1

2

MW

15 μl

markers

3

VR2211

UM449

extract

5 μg

1/1

40 μl

clone #16

4

no DNA

UM449

extract

all

40 μl

lipid

5

VR2210

UM449

extract

5 μg

1/1

40 μl

6

VR2211

UM449

extract

5 μg

1/1

40 μl

clone #1

7

VR2211

UM449

extract

5 μg

1/1

40 μl

clone #2

8

VR2211

UM449

extract

5 μg

1/1

40 μl

clone #3

9

VR2211

UM449

extract

5 μg

1/1

40 μl

clone #4

10

VR2211

UM449

extract

5 μg

1/1

40 μl

clone #5

A summary of the results shown in

FIG. 18B

is provided following tabulation.

DNA

Amt

Lane

Sample

Cells

Supernatant

Conc.

DNA/lipid

loaded

1

B31

spirochete

lysate

1 μl

2

MW

15 μl

markers

3

VR2211

UM449

sup.

5 μg

1/1

20 μl

#16

4

no

UM449

sup.

all lipid

20 μl

DNA

5

VR2210

UM449

sup

5 μg

1/1

20 μl

6

VR2211

UM449

sup.

S μg

1/1

20 μl

#1

7

VR2211

UM449

sup.

5 μg

1/1

20 μl

#2

8

VR2211

UM449

sup.

5 μg

1/1

20 μl

#3

9

VR2211

UM449

sup.

5 μg

1/1

20 μl

#4

10

VR2211

UM449

sup.

5 μg

1/1

20 μl

#5

This Example further demonstrates that the inventive Borrelia antigen DNA plasmid compositions are additionally useful for in vitro expression of antigen(s) (which by themselves are useful, e.g., for preparing antigenic, immunological or vaccine compositions or for diagnostic, detection, or assay purposes).

Example 4

Animal Study With VR2210

Two groups of five mice were injected with VR2210 (i.e., ten mice; mice A1-A5, B1-B5), and two groups of five mice (i.e., ten mice) were injected with plasmid VR1020 negative control DNA. VR1020 does not contain a coding sequence for a Borrelia antigen. The plasmid and control DNA were diluted in standard saline. Three bilateral injections of DNA were given at two week intervals at a dosage of 50 μg/leg into the rectus femoris muscle. Sera were collected 12 days after each injection and analyzed by 1) Antibody ELISA and 2) Growth Inhibition of Spirochetes. Titers after the first and third injections (Titer #1, Titer #3) are set forth below.

Two weeks after the last injection, mice were challenged with 10

4

Sh2 spirochetes (same OspA serogroup as B31) injected intradermally in the tail. Sh2 is a virulent isolate of the same serogroup as B31. Mice were sacrificed 11 days following the challenge. Bladder, heart, plasma, and cross-cuttings of the tibiotarsal joints were cultured for 15 days at 34° C. in growth medium. Cultures were examined for the presence of spirochetes by phase-contrast microscopy and scored as negative if no spirochetes were seen in 50 high-power fields.

The antibody ELISA titers for the ten mice administered VR2210 are shown in the following Table. The titers were low after one injection and the group showed considerable variability in their immune response. After the third injection, however, the humoral immune response was uniformly high in 8 out of 10 mice with titers of greater than 1:40,000.

ANTIBODY ELISA TITERS

High passage B31 was the antigen used in the ELISA's.

Mouse

Immunogen

Titer #1

Titer #3

A1

ospA

640

10240

A2

ospA

640

10240

A3

ospA

40

>40,960

A4

ospA

1280

>40,960

A5

ospA

1280

>40,960

B1

ospA

<20

>40,960

B2

ospA

<20

>40,960

B3

ospA

2560

>40,960

B4

ospA

5120-10,240

>40,960

B5

ospA

1280

>40,960

No antibody binding to B31 (or immune response to B31) was observed for the mice administered the negative control DNA.

The following Table shows the Spirochete Growth Inhibition titers. The numbers indicate the maximal dilution of serum which inhibits spirochete growth in vitro. After the first injection of VR2210, growth inhibition was seen at serum dilutions of 32 to 128. After the third injection, however, inhibition of growth was detected at higher dilutions (up to 512), which is consistent with the antibody titer data.

Growth Inhibitory Titers

Method: Strain B31 spirochetes were mixed with an 8-fold dilution of serum and two-fold serially diluted in a 96-well plate. Guinea pig complement was added to each well to lyse the spirochetes which have bound antibody. The plates were covered with plastic seals and incubated for 72 hours at 34° C. Growth in each well was determined by observation of the phenol red indicator in the medium from red to yellow as well as by phase- contrast microscopy. The numbers were the maximal dilution of serum which inhibits spirochete growth in the well.

Mouse

Immunogen

Titer #1

Titer #3

A1

ospA

not

256-512

determined

A2

ospA

not

128-256

determined

A3

ospA

not

256

determined

A4

ospA

not

64

determined

A5

ospA

not

64

determined

B1

ospA

32

not determined

B2

ospA

64-128

not determined

B3

ospA

64

not determined

B4

ospA

64-128

not determined

B5

ospA

64

not determined

No inhibition was observed with the sera of the mice administered the negative control DNA. The organ culture data shown in the following Table indicates that all ten VR2210 vaccinated mice were completely free of spirochetes in all tissues examined whereas all ten negative control mice had spirochetes in their bladder and joints. Therefore, vaccination with three doses of VR2210 gives 100% protection against spirochete challenge in vivo.

PRESENCE OF SPIROCHETES IN CULTURED ORGANS

Combined culture data for all groups. # Positive cultures/Total

Immunogen

Plasma

Heart

Bladder

Joint

ospA

0/10

0/10

0/10

0/10

control

3/10

7/10

10/10

10/10

This is a demonstration that the Borrelia DNA plasmid is effective against bacterial pathogenic targets. Immunization with VR2210 encoding OspA protects mice completely against intradermal challenge with 10

4

virulent spirochetes. This in vivo protection correlates with the serum IgG response measured both by antibody ELISA and by an in vitro spirochete growth inhibition assay.

The ability of the inventive composition to elicit a protective response capable of inhibiting spirochete growth demonstrates that the critical immunogenic epitopes on the bacterial protein are conserved when genes encoding these proteins are expressed in mammalian cells. This is particularly interesting and surprising because native OspA contains a lipid moiety on its N-terminus which has been shown to be a crucial determinant for the immunogenicity of the recombinant protein vaccine made from

E. coli

. In contrast, in the DNA and expression product of the invention, the natural OspA leader sequence which contains the lipidation signal has been replaced by the human TPA leader sequence in VR2210. Since this lipidation event is part of a post-translational processing step found uniquely in prokaryotic cells, it is unlikely that the lipid moiety is present on the VR2210 protein product. And thus, the results obtained by the present invention are indeed surprising and unexpected.

This Example also demonstrates that plasmids encoding OspB, such as VR2211, or another Borrelia antigen, e.g., OspC, or encoding multiple Borrelia antigens, e.g., OspA and OspB and/or OspC, is within the scope of the invention, and are useful. For instance, this Example demonstrates that the inventive

Borrelia burgdorferi

antigen DNA composition of the invention is useful for eliciting antibodies which have in vivo and in vitro uses (e.g., protective response; diagnostic, detection or assay purposes).

Example 5

Nucleic Acid Immunization

Immunization of mice

6-10 week old female C3H/HeN mice (Harlan Laboratories, IN) were immunized with plasmid VR2210 or VR1020 (control) diluted in sterile standard saline using a collared 28 G ½″(12.7mm) needle. 50 μg of plasmid was administered intra-muscularly into the rectus femoris muscle of each leg in a volume of 0.05 ml composition. Mice were boosted with identical composition on day 14 and day 28.

Challenge with Infectious

Borrelia burgdorferi

13 days following the second boost, mice were challenged with

Borrelia burgdorferi

Sh-2-82 (Erdile et al., 1993). 10

4

B. burgdorferi

Sh-2-82 in 10% v/v BSKII in PBS (pH 7.4) were injected intra-dermally at the base of the tail. This inoculum is 100 times the ID

50

for this strain of

B. burdorferi

(Erdile et al., 1993). Mice were sacrificed 10 days following challenge. Bladder, heart and cross-cuttings of tibiotarsal joints were aseptically removed and were placed in 6 ml BSKII containing antibiotics. Cultures were incubated at 34° C. After 15 days, organ cultures were examined by phase contrast microscopy for the presence of spirochaetes. Cultures were considered negative if no spirochaetes were seen in 20 high power fields.

ELISA

Wet, whole cell ELISAs were carried out as previously described (Sadziene et al., 1991) using high passage

B. burgdorferi

B31, strain B311 (Sadziene et al., 1995) as the antigen. Serial dilutions of mouse sera were made in 1% w/v dried non-fat milk in PBS (pH 7.4). Secondary antibody was goat anti-mouse IgG+IgM+IgA (H+L) conjugated to alkaline phosphatase (Zymed Laboratories, Calif.) used at a dilution of 1:1000 in PBS/1% milk. Plates were developed as previously described (Sadziene et al., 1991). Absorbance was read at 490 nm on a Dynatech 580 plate reader. Samples were considered positive if the absorbance value was greater than the mean +3 standard deviations of the mean of that for non-immune and control sera (Burkot et al., 1994).

In vitro Growth Inhibition Assays

Growth inhibitory titers (GI titers) of the sera from the immunized mice were determined as previously described by Sadziene et al., 1993). Two (2) hemolytic units of unheated guinea pig complement (Calbiochem, Calif.) was added to each of the wells of the microtiter plate to give a final concentration of 10HU.ml

−1

of medium after the addition of antibody. Wells were monitored visually for changes in the color of the phenol red indicator in the medium and by phase contrast microscopy of set mounts of well contents. The GI titer was defined as the lowest dilution of antiserum that resulted in pink instead of yellow wells and represented at least 20-fold fewer cells than in wells with no serum added.

PAGE and Immunoblotting

PAGE and immunoblot were carried out as described previously (Sadziene et al., 1995). Twenty four micrograms of recombinant lipidated OspA (see Erdile et al., 1993, U.S. Ser. No. 08/373,455) or

B. burgdorferi

B31 were run on preparative polyacrylamide gels and were then transferred onto nitrocellulose membranes. Immunoblots were dried and stored at 4° C. until needed.

Immunization of Mice with VR2210 Resulted in an OspA-specific Antibody Response

Immunoblot of sera from the mice showed that those that received the plasmid bearing the

B. burgdorferi

B31 ospA gene, VR2210, developed an OspA-specific antibody response (FIG.

19

). ELISA of the sera also showed that these mice had mounted a significant antibody response against B31. The geometric mean reciprocal ELISA titers are given in the following Table. The sera from the mice that received the plasmid with no Borrelia gene inserted, VR 1020, did not demonstrate an antibody response to

B. burgdorferi

. The sera from the VR2210-immunized mice were found to inhibit growth of

B. burgdorferi

in vitro, to the same level as sera known to be protective. The geometric mean reciprocal GI titers of the sera are also given in the following Table.

The Table shows the reciprocal geometric mean ELISA titers of the sera from the VR2210-immunized mice when IP-90 (B. garinii) and ACA-I (B. afzelii) were used as antigen (sera are from the bleed taken 2 days before challenge).

Challenge with Infectious

B. burgdorferi

The results of the challenge of the DNA-immunized mice with

B. burgdorferi

Sh-2-82 are shown in the following Table. All mice which received VR2210 were protected against challenge, whereas Borrelias were recovered from all the mice that received the VR1020 plasmid, indicating no protection in these animals.

Table

Reciprocal geometric mean ELISA titers and GI titers and results of infectious challenge of mice immunized with DNA constructs VR2210 and VR1020.

ELISA TITER

GI

CULTURE

Immunogen

B31

IP-90

ACA-I

TITER

POSITIVE*

VR2210

54,696

2941

2389

388

0/10

(ospA)

VR1020

≦20

≦20

≦20

≦8

10/10

(no insert)

*Culture positive at one or more sites.

The results in this Table show that the invention is applicable to genospecies

burgdorferi, garinii

and

afzelii

; and need not be limited to

burgdorferi

or B31.

FIG. 19

is an immunoblot of

Borrelia burgdorferi

rospA probed with sera from mice immunized with either plasmid VR2210 (ospA) or VR1020. Sera were diluted 1:100. The positive control (+) was H5332 (anti-OspA) monoclonal hybridoma supernatant, diluted 1:10.

The immunization was repeated with the plasmid construct VR2211, containing the ospB of

Borrelia burgdorferi

B31. An additional boost of this plasmid was given two weeks after the second boost. The 5 mice that received this construct, along with three mice that received VR1020, were challenged, and are sacrificed later. OspB-specific antibodies in sera from the VR2211 (ospB) immunized mice were detected on Western blots.

Also, the VR2210 immunization was repeated, along with rOspA lipoprotein controls (immunized subcutaneously or intra-muscularly with 1 μg) (5 mice per group) and the mice are bled every 2 weeks to assess the duration of the immune response to OspA by ELISA and growth inhibition assay. The results parallel those provided above.

This Example demonstrates that Borrelia antigen or epitope DNA compositions of the invention are useful for eliciting an in vivo response, which can be protective against infection (and ergo against Lyme Disease); and, that the compositions of the invention are useful for merely eliciting antibodies, which by themselves are useful (e.g., for diagnostic, detection or assay purposes).

Example 6

OspC Plasmids and Nucleic Acid Immunization Therewith

OspC Cloning and Plasmid Constructions

DNA plasmids VR2212 (

FIGS. 20 and 21

) (SEQ ID NO:19) and VR2214 (

FIGS. 22 and 23

) (SEQ ID NO:20) containing genes coding for OspC from

Borrelia burgdorferi

strains B31 and Pko, respectively. These plasmids were constructed by isolating the DNA encoding OspC (i.e., the coding region for OspC, or the DNA coding for only the mature protein, without the leader sequence), and ligating that DNA with DNA for plasmid VR1027 by methods analogous to those in the foregoing examples (plasmid VR1027 is analogous to plasmid VR1020, discussed supra, insofar as VR1027 is VR1020 with KpnI site in the BGH term mutated to a PpuMI site; plasmid VR1020 is plasmid VR2210 without the

B. burgdorferi

DNA which is present in VR2210).

More specifically, plasmids pB319a4 and pPko9a were provided by Pasteur Merieux Connaught, Swiftwater Pa. These plasmids contained isolated nucleic acid molecules encoding OspC-lipidated proteins from

B. burgdorferi

B31 and Pko, respectively, with the ACA OspA signal. DNA sequences for OspC from

B. burgdorferi

strains are also known from the literature (see, e.g., V. Preac-Mursic et al., Infection 20:342-349, 1992; W. S. Probert et al., Infection and Immunity 62:1920-1926, 1994; published international patent application WO 91/09870 (Mikrogen Molekularbiologische Entwicklungs-GmbH); Leuba-Garcia et al., Zentralbl Bakteriol. 287(4):475-84, 1998; Rauer et al., J. Clin. Microbiol. 36(4):857-61, 1998; Masuzawa et al., Clin. Diagn. Lab. Immunol. 4(1):60-63, 1997; Fukunaga et al. J. Clin. Microbiol. 33(9):2415-2420, 1995; Jauris-Heipke et al., J. Clin. Microbiol. 33(7):1860-66, 1995; Theisen et al., J. Bacteriol. 177(11):3036-3044, 1995; Stevenson et al. FEMS Microbiol. Lett. 124(3):367-72, 1994; and Padula et al., Infect. Immun. 61(12):5097-5105, 1993).

The DNA encoding OspC was amplified by PCR reactions using the following primers:

The PCR reactions were performed in a manner analogous to that specified in the foregoing examples. The PCR reactions contained: 10 ng of plasmid DNA (pB319a4), 10 μL of Taq Buffer, 3 μL dNTPs, 50 picomoles of each primer, and 1 μL Taq; and 10 ng of plasmid DNA (pPko9a), 10 μL of Taq Buffer, 3 μL dNTPs, 50 picomoles of each primer, and 1 μL Taq.

The amplification products were separated by gel electrophoresis such that bands were obtained for a B31 ospC KpnI/BamHI fragment (“B31 ospC K/B”), and a Pko ospC KpnI/BamHI fragment (“Pko ospC K/B”). These bands were excised from the gel and purified using a QIAEX procedure, analogous to that described above in foregoing examples. Each band was eluted with TE to a total volume of 35 μL.

Restriction BamHI digestions were as follows:

a.

Mixed:

35.00 μL of B31 ospC K/B

4.50 μL of NEB #2

4.50 μL of NaCl (1M)

0.50 μL of 100X BSA

1.00 μL of BamHI (10 units/μL)

Total:

45.50 μL

b.

Mixed:

35.00 μL of Pko ospc K/B

4.50 μL of NEB #2

4.50 μL of NaCl (1M)

0.50 μL of 100X BSA

1.00 μL of BamHI (10 units/μL)

Total:

45.50 μL

a.

Mixed:

50.00 μL of B31 ospC product

2.40 μL TE

5.00 μL of New England Biolabs (NEB) #1

0.60 μL of 100X Bovine Serum Albumin (BSA)

2.00 μL of KpnI (10 units/μL)

Total:

60.00 μL

b.

Mixed:

50.00 μL of Pko ospC product

2.40 μL TE

5.00 μL of NEB #1

0.60 μL of 100X BSA

2.00 μL of KpnI (10 units/μL)

Total:

60.00 μL

Each of mixtures a. and b. was allowed to incubate for 2 hours at 37° C. to digest. The digests were extracted with phenol/chloroform and were then put through a spin column (G-50 Sephadex) to obtain B31 and Pko ospC (coding region, i.e., coding for only the mature OspC protein without the leader sequence).

As discussed above, VR1027 is analogous to VR1020. A diagram of VR1020 is provided in

FIG. 24. A

diagram of VR1027 is provided in FIG.

25

and the nucleotide sequence for VR1027 (SEQ ID NO:23) is provided in FIG.

26

.

a.

Mixed:

28.00 μL of TE

2.00 μL of VR1027 (5 μg)

5.00 μL of NEB #2

5.00 μL of NaCl (1M)

5.00 μL of 10X BSA

5.00 μL of BamHI (10 units/μL)

Total:

50.00 μL

Mixture a. was allowed to incubate for 2 hours at 37° C. to digest. The digest was extracted with phenol/chloroform and was then put through a spin column (G-50 Sephadex) to obtain a BamHI cut VR1027 product. This product was then subjected to KpnI digestion as follows:

b.

Mixed:

50.00 μL of DNA (BamHI cut VR1027 product)

6.00 μL of NEB #1

0.60 μL of 100X BSA

4.00 μL of KpnI (10 units/μL)

Total:

60.60 μL

The volume of the digest was brought to approximately 100 μL with TE by the addition of 40 μL TE. 1 μL calf intestine alkaline phosphatase (CIAP) was then added, and the resultant mixture was incubated for 10 min at 37° C. Thereafter the enzyme was heat-killed by bringing the mixture to 65° C. for 10 min. The digest was then extracted with phenol/chloroform and was then put through a spin column (G-50 Sephadex) to obtain a BamHI and KpnI cut VR1027 product.

The BamHI and KpnI cut VR1027 product was then used in ligations with the B31 and Pko ospc (coding region, i.e., coding for only the mature OspC protein without the leader sequence).

More in particular, to then construct VR2212, a mixture containing 1 μL of the BamHI and KpnI cut VR1027 product, 1 μL of the BamHI/KpnI cut B31 ospC DNA, 2 μL Rapid DNA Ligation Buffer (RDLB) #2, 10 μL Rapid DNA Ligation Buffer #1, 6 μL of TE, and 1 μL ligase (Boehringer Mannheim) was prepared. To construct VR2214, a mixture containing 1 μL of the BamHI and KDnI cut VR1027 product, 1 μL of the BamHI/KpnI cut Pko ospC DNA, 2 μL RDLB #2, 10 μL RPLB #1, 6 μL of TE, and 1 μL Rapid DNA ligase (Boehringer Mannheim) was prepared. Rapid ligations occurred in each of the mixtures, with VR2212 and VR2214 resulting, respectively. Restriction Endonuclease analysis confirmed that B31 ospC had been inserted into VR1027 to form VR2212 and that Pko ospC had been inserted into VR1027 to form VR2214.

Confirmation of OspC Expression by VR2212 and VR2214

Mice were immunized with lipidated OspC from both Pko and B31

B. Burgdorferi

strains (1 μg/antigen) on days 0, 21 and 42. At day 63 the mice were bled to obtain an antisera pool.

UM449 cells were transfected with plasmid VR2212 in duplicate wells of a 6-well dish. UM449 cells were also transfected with plasmid VR2214 in duplicate wells of a 6-well dish. The mouse antisera pool as described above was diluted to a 1/300 dilution. The transfected UM449 cells were subjected to Western blot analysis using the diluted mouse antisera. The Western blot analyses confirmed that VR2212 and VR2214 each express OspC.

In Vivo Study using VR2212 and VR2214

Formulation A: VR2212 @ 1000 μg/ml:

1.6 ml VR2212@6240 μg/ml+8.4 ml PBS.

Formulation B: VR2214 @ 1000 μg/ml:

2.05 ml VR2214@4870 μg/ml+7.95 ml PBS.

Formulation C: VR1027 @ 1000 μg/ml:

1.02 ml VR1027@9760 μg/ml+8.98 ml PBS (control).

Group 1: 8 C3H/He mice were each intramuscularly injected with 0.1 ml of Formulation A.

Group 2: 8 ICR mice were each intramuscularly injected with 0.1 ml of Formulation A.

Group 3: 8 C3H/He mice were each intramuscularly injected with 0.1 ml of Formulation B.

Group 4: 8 ICR mice were each intramuscularly injected with 0.1 ml of Formulation B.

Group 5: 8 C3H/He mice were each intramuscularly injected with 0.1 ml of Formulation C.

Group 6: 8 ICR mice were each intramuscularly injected with 0.1 ml of Formulation C.

Group 7: All three groups of C3H/He mice were bled on day 0 and their sera pooled.

Group 8: All three groups of ICR mice were bled on day 0 and their sera pooled.

The mice of Groups 1 to 6 were bled on days 21, 42, 63, 91 and 119 from the tail vein. Enough blood was removed from each mouse to generate 50 μL of serum, and each serum sample was kept separate.

The sera were tested for the presence of OspC using ELISA. The antigens used to assess the anti-OspC response were obtained as follows: Non-lipidated B31 OspC was used to assess the B31 OspC response and non-lipidated Pko OspC was used to assess the Pko OspC response. These non-lipidated versions of B31 and Pko OspC were purified using the method described in published international patent application WO 91/09870 (Mikrogen Molekularbiologische Entwicklungs-GmbH), with one modification; namely, the material eluted from the DEAE column was dialyzed into phosphate buffer instead of diluted into the buffer. The results of the ELISA are shown in FIG.

27

and are described below.

More in particular, the sera collected from Groups 1 to 8 were tested by ELISA for the presence of OspC. The sera from Groups 5 to 8 showed no OspC. Groups 1 to 4 tested positively for expression of OspC in vivo.

More specifically,

FIG. 27

shows a graph of ELISA GMT vs. days for Group 1 (diamonds: C3H/He mice that received B31 ospC DNA in plasmid form, i.e., VR2212), Group 2 (squares: ICR mice that received B31 ospC DNA in plasmid form, i.e., VR2212), Group 3 (triangles: C3H/He mice that received Pko ospC DNA in plasmid form, i.e., VR2214), and Group 4 (X's: ICR mice that received Pko ospC DNA in plasmid form, i.e., VR2214).

As can be seen from

FIG. 27

, VR2212 and VR2214 each resulted in expression in vivo of OspC and the resultant generation of antibodies to OspC.

For instance, VR2214 in ICR mice (Group 4) resulted in ELISA GMT of about 66.67 on all days tested; VR2212 in ICR mice (Group 2) resulted in ELISA GMT of about 66.67 on day 21, about 150 on day 42, about 700 on day 63, about 1466.67 on day 91, about 1200 on day 119, and about 1000 beyond day 140; VR2214 in C3H/He mice resulted in ELISA GMT of about 66.67 on days 21 and 42, about 100 on day 63, about 300 on day 82, about 190 on day 119 and about 66.67 beyond day 140; and, VR2212 in C3H/He mice resulted in ELISA GMT of about 66.67 on day 21, about 290 on day 42, about 250 on day 63, about 700 on day 82, about 400 on day 119 and about 250 beyond day 140.

Thus, VR2212 and 2214 are capable of having OspC expressed in vivo; and that the expressed OspC is capable of eliciting OspC specific antibodies. It is therefore expected that VR2212 and VR2214 will provide protection against Lyme disease in the same fashion that immunizations with OspC protein provide protection against Lyme disease.

Accordingly, this Example demonstrates that Borrelia antigen or epitope DNA compositions of the invention, especially such compositions wherein the DNA codes for OspC or an epitope of OspC, are useful for eliciting an in vivo response, which response may be protective against infection (and ergo against Lyme Disease); and, that the compositions of the invention are useful for merely eliciting antibodies, which by themselves are useful (e.g., for diagnostic, detection or assay purposes).

Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the appended claims is not to be limited by particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope thereof.

REFERENCES

1. Barbour et al., Infect Immun., 41, 795-804 (1983).

2. Barbour, A. G. and Fish, D., The biological and social phenomenon of Lyme Disease,

Science

260, 1610-1616 (1993).

3. Barbour, A. G. et al., J. Infect. Dis., 152, 478-84 (1985).

4. Barbour et al., Infect. Immun. 45, 94-100 (1984).

5. Bergstrom, S., Bundoc V. G., and Barbour, A. G., Molecular analysis of linear plasmid-encoded major surface proteins OspA and OspB, of the Lyme disease spirochaete

Borrelia burgdorferi

, Mol. Microbiol. 3:479-486 (1989).

6. Bocchia, M. et al.,

Specific Binding of Leukemia Oncogene Fusion Protein Peptides to HLA Class I Molecules

, Blood 85(10):2680-2684, May 15 (1995).

7. Bourne al., The Journal of Infectious Disease, 173:800-7, (1996).

8. Burkot, T. R., Piesman, J., and Wirtz, R. A. Quantitation of the

Borrelia burgdorferi

outer surface protein A in Ixodes scapularis: fluctuations during the tick life cycle, doubling times, and loss while feeding.

J. Infect. Dis.,

170, 883-889 (1994).

9. Chapman et al., Nucleic Acids Research 19, 3979-86 (1991).

10. Englehard, VH,

Structure of peptides associated with class I and class II MHC molecules

, Ann. Rev. Immunol. 12:181 (1994).

11. Erdile, L. f., Brandt, M., Warakomski, D. J., Westrack, G. J., Sadziene, A., Barbour, A. G. and Mays, J. P. Role of attached lipid in immunogenicity of

Borrelia burgdorferi

OspA.

Infect. Immun.

61, 81-90 (1993).

12. Felgner et al., J. Biol. Chem. 269, 2550-2561 (1994).

13. Fikrig, E., Barthold, S. W., Kantor, F. S. and Flavell, R. A. Protection of mice against the Lyme disease agent by immunizing with recombinant OspA.

Science,

250, 553-556 (1990). 14. Fukunaga et al. J. Clin. Microbiol. 33(9):2415-2420 (1995).

15. Howe et al., Infection and Immunity 54:207-212 (1986).

16. Jauris-Heipke et al., J. Clin. Microbiol. 33(7):1860-66, (1995).

17. Keller, D., Kister, F. T., Marks, D. H., Hosback, P., Erdile, L. F. and Mays, J. P. Safety and immunogenicity of a recombinant outer surface protein A Lyme vaccine,

J. Am. Med. Assoc.

271, 1764 (1994).

18. Kendrew, The Encyclopedia Of Molecular Biology, Blackwell Science Ltd. (1995).

19. Kuby, Immunology, pp. 79-80, W. H. Freeman, July (1992).

20. Leuba-Garcia et al., Zentralbl Bakteriol. 287(4):475-84 (1998).

21. Luke et al., J. Infect. Dis., 175(1):91-97 (1997).

22. Manthorpe et al., Human Gene Therapy 4, 419-431 (1993).

23. Masuzawa et al., Clin. Diagn. Lab. Immunol. 4(1):60-63, (1997).

24. Milstein, C., Scientific American 243:66, 70 (1980).

25. Norman et al., Vaccine, 15(8):801-803 (1997).

26. Padula et al., Infect. Immun. 61(12):5097-5105 (1993).

27. Preac-Mursic et al., INFECTION 20:342-349 (1992).

28. Probert et al., INFECTION AND IMMUNITY 62:1920-1926 (1994).

29. Rauer et al., J. Clin. Microbiol. 36(4):857-61 (1998).

30. Robinson et al., seminars in IMMUNOLOGY, 9:271-83 (1997).

31. Roitt, Essential Immunology, Blackwell Scientific Publications, Oxford (1988).

32. Sadziene, A., Thompson, P. A., and Barbour, A. G. In vitro inhibition of

Borrelia burgdorferi

growth by antibodies, J. Infect. Dis. 167, 165-172 (1993).

33. Sadziene, A., Thomas, D. D., and Barbour, A. G.

Borrelia burgdorferi

mutant lacing Osp: biological and immunological characterization,

Infect. Immun.

63, 1573-1580 (1995).

34. Sadziene, A., Thomas, D. D., Bundoc, V. G., Holt, S. H., and Barbour, A. G. A flagella-less mutant of

Borrelia burgdorferi.

J. Clin. Invest. 88, 82-92 (1991).

35. Sambrook, Fritsch and Maniatis, Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press (1982).

36. Stevenson et al. FEMS Microbiol. Lett. 124(3):367-72 (1994).

37. Theisen et al., J. Bacteriol. 177(11):3036-3044 (1995).

18

1

5845

DNA

Borrelia burgdorferi

1
gtcaccgtcg tcgaccagag ctgagatcct acaggagtcc agggctggag agaaaacctc 60
tgcgaggaaa gggaaggagc aagccgtgaa tttaagggac gctgtgaagc aatcatggat 120
gcaatgaaga gagggctctg ctgtgtgctg ctgctgtgtg gagcagtctt cgtttcgccc 180
agcggtacct gtaagcaaaa tgttagcagc cttgacgaga aaaacagcgt ttcagtagat 240
ttgcctggtg aaatgaaagt tcttgtaagc aaagaaaaaa acaaagacgg caagtacgat 300
ctaattgcaa cagtagacaa gcttgagctt aaaggaactt ctgataaaaa caatggatct 360
ggagtacttg aaggcgtaaa agctgacaaa agtaaagtaa aattaacaat ttctgacgat 420
ctaggtcaaa ccacacttga agttttcaaa gaagatggca aaacactagt atcaaaaaaa 480
gtaacttcca aagacaagtc atcaacagaa gaaaaattca atgaaaaagg tgaagtatct 540
gaaaaaataa taacaagagc agacggaacc agacttgaat acacagaaat taaaagcgat 600
ggatctggaa aagctaaaga ggttttaaaa ggctatgttc ttgaaggaac tctaactgct 660
gaaaaaacaa cattggtggt taaagaagga actgttactt taagcaaaaa tatttcaaaa 720
tctggggaag tttcagttga acttaatgac actgacagta gtgctgctac taaaaaaact 780
gcagcttgga attcaggcac ttcaacttta acaattactg taaacagtaa aaaaactaaa 840
gaccttgtgt ttacaaaaga aaacacaatt acagtacaac aatacgactc aaatggcacc 900
aaattagagg ggtcagcagt tgaaattaca aaacttgatg aaattaaaaa cgctcttaag 960
taaggagaat tttctagacc aggcgcctgg atccagatct gctgtgcctt ctagttgcca 1020
gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac 1080
tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat 1140
tctggggggt ggggtggggc agcacagcaa gggggaggat tgggaagaca atagcaggca 1200
tgctggggat gcggtgggct ctatgggtac ccaggtgctg aagaattgac ccggttcctc 1260
ctgggccaga aagaagcagg cacatcccct tctctgtgac acaccctgtc cacgcccctg 1320
gttcttagtt ccagccccac tcataggaca ctcatagctc aggagggctc cgccttcaat 1380
cccacccgct aaagtacttg gagcggtctc tccctccctc atcagcccac caaaccaaac 1440
ctagcctcca agagtgggaa gaaattaaag caagataggc tattaagtgc agagggagag 1500
aaaatgcctc caacatgtga ggaagtaatg agagaaatca tagaatttct tccgcttcct 1560
cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 1620
aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 1680
aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 1740
tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 1800
caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 1860
cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 1920
ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 1980
gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 2040
agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 2100
gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 2160
acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 2220
gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 2280
gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 2340
cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 2400
caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 2460
gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 2520
cagcgatctg tctatttcgt tcatccatag ttgcctgact ccgggggggg ggggcgctga 2580
ggtctgcctc gtgaagaagg tgttgctgac tcataccagg cctgaatcgc cccatcatcc 2640
agccagaaag tgagggagcc acggttgatg agagctttgt tgtaggtgga ccagttggtg 2700
atttgaactt ttgctttgcc acggaacggt ctgcgttgtc gggaagatgc gtgatctgat 2760
ccttcaactc agcaaaagtt cgatttattc aacaaagccg ccgtcccgtc aagtcagcgt 2820
aatgctctgc cagtgttaca accaattaac caattctgat tagaaaaact catcgagcat 2880
caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg 2940
tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta 3000
tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa 3060
aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa 3120
aagcttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa 3180
atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac 3240
gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac 3300
tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc 3360
tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg 3420
cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt 3480
aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt 3540
cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata 3600
cccatataaa tcagcatcca tgttggaatt taatcgcggc ctcgagcaag acgtttcccg 3660
ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt 3720
tcatgatgat atatttttat cttgtgcaat gtaacatcag agattttgag acacaacgtg 3780
gctttccccc cccccccatt attgaagcat ttatcagggt tattgtctca tgagcggata 3840
catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 3900
agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg 3960
tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat 4020
gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg 4080
tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga 4140
gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag 4200
aaaataccgc atcagattgg ctattggcca ttgcatacgt tgtatccata tcataatatg 4260
tacatttata ttggctcatg tccaacatta ccgccatgtt gacattgatt attgactagt 4320
tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt 4380
acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg 4440
tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg 4500
gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt 4560
acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg 4620
accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg 4680
gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 4740
ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 4800
tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 4860
tgggaggtct atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat 4920
ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg cggccgggaa 4980
cggtgcattg gaacgcggat tccccgtgcc aagagtgacg taagtaccgc ctatagagtc 5040
tataggccca cccccttggc ttcttatgca tgctatactg tttttggctt ggggtctata 5100
cacccccgct tcctcatgtt ataggtgatg gtatagctta gcctataggt gtgggttatt 5160
gaccattatt gaccactccc ctattggtga cgatactttc cattactaat ccataacatg 5220
gctctttgcc acaactctct ttattggcta tatgccaata cactgtcctt cagagactga 5280
cacggactct gtatttttac aggatggggt ctcatttatt atttacaaat tcacatatac 5340
aacaccaccg tccccagtgc ccgcagtttt tattaaacat aacgtgggat ctccacgcga 5400
atctcgggta cgtgttccgg acatgggctc ttctccggta gcggcggagc ttctacatcc 5460
gagccctgct cccatgcctc cagcgactca tggtcgctcg gcagctcctt gctcctaaca 5520
gtggaggcca gacttaggca cagcacgatg cccaccacca ccagtgtgcc gcacaaggcc 5580
gtggcggtag ggtatgtgtc tgaaaatgag ctcggggagc gggcttgcac cgctgacgca 5640
tttggaagac ttaaggcagc ggcagaagaa gatgcaggca gctgagttgt tgtgttctga 5700
taagagtcag aggtaactcc cgttgcggtg ctgttaacgg tggagggcag tgtagtctga 5760
gcagtactcg ttgctgccgc gcgcgccacc agacataata gctgacagac taacagactg 5820
ttcctttcca tgggtctttt ctgca 5845

2

5899

DNA

Borrelia burgdorferi

2
ctgtgcacaa aaaggtgctg agtcaattgg ttctcaaaaa gaaaatgatc taaaccttga 60
agactctagt aaaaaatcac atcaaaacgc taaacaagac cttcctgcgg tgacagaaga 120
ctcagtgtct ttgtttaatg gtaataaaat ttttgtaagc aaagaaaaaa atagctccgg 180
caaatatgat ttaagagcaa caattgatca ggttgaactt aaaggaactt ccgataaaaa 240
caatggttct ggaacccttg aaggttcaaa gcctgacaag agtaaagtaa aattaacagt 300
ttctgctgat ttaaacacag taaccttaga agcatttgat gccagcaacc aaaaaatttc 360
aagtaaagtt actaaaaaac aggggtcaat aacagaggaa actctcaaag ctaataaatt 420
agactcaaag aaattaacaa gatcaaacgg aactacactt gaatactcac aaataacaga 480
tgctgacaat gctacaaaag cagtagaaac tctaaaaaat agcattaagc ttgaaggaag 540
tcttgtagtc ggaaaaacaa cagtggaaat taaagaaggt actgttactc taaaaagaga 600
aattgaaaaa gatggaaaag taaaagtctt tttgaatgac actgcaggtt ctaacaaaaa 660
aacaggtaaa tgggaagaca gtactagcac tttaacaatt agtgctgaca gcaaaaaaac 720
taaagatttg gtgttcttaa cagatggtac aattacagta caacaataca acacagctgg 780
aaccagccta gaaggatcag caagtgaaat taaaaatctt tcagagctta aaaacgcttt 840
taaaataata tataggatcc agatctgctg tgccttctag ttgccagcca tctgttgttt 900
gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat 960
aaaatgagga aattgcatcg cattgtctga gtaggtgtca tctattctgg ggggtggggt 1020
ggggcagcac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt 1080
gggctctatg ggtacccagg tgctgaagaa ttgacccggt tcctcctggg ccagaaagaa 1140
gcaggcacat ccccttctct gtgacacacc ctgtccacgc ccctggttct tagttccagc 1200
cccactcata ggacactcat agctcaggag ggctccgcct tcaatcccac ccgctaaagt 1260
acttggagcg gtctctccct ccctcatcag cccaccaaac caaacctagc ctccaagagt 1320
gggaagaaat taaagcaaga taggctatta agtgcagagg gagagaaaat gcctccaaca 1380
tgtgaggaag taatgagaga aatcatagaa tttcttccgc ttcctcgctc actgactcgc 1440
tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 1500
tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 1560
ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 1620
agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 1680
accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 1740
ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct 1800
gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 1860
ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 1920
gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 1980
taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag 2040
tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 2100
gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 2160
cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 2220
agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 2280
cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 2340
cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 2400
ttcgttcatc catagttgcc tgactccggg gggggggggc gctgaggtct gcctcgtgaa 2460
gaaggtgttg ctgactcata ccaggcctga atcgccccat catccagcca gaaagtgagg 2520
gagccacggt tgatgagagc tttgttgtag gtggaccagt tggtgatttt gaacttttgc 2580
tttgccacgg aacggtctgc gttgtcggga agatgcgtga tctgatcctt caactcagca 2640
aaagttcgat ttattcaaca aagccgccgt cccgtcaagt cagcgtaatg ctctgccagt 2700
gttacaacca attaaccaat tctgattaga aaaactcatc gagcatcaaa tgaaactgca 2760
atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag 2820
gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc 2880
cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa 2940
gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagc ttatgcattt 3000
ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa 3060
ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa 3120
aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa 3180
caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga 3240
tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa 3300
gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa 3360
cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat 3420
agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag 3480
catccatgtt ggaatttaat cgcggcctcg agcaagacgt ttcccgttga atatggctca 3540
taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gatgatatat 3600
ttttatcttg tgcaatgtaa catcagagat tttgagacac aacgtggctt tccccccccc 3660
cccattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 3720
tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 3780
tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct 3840
ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga 3900
cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag 3960
cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga 4020
gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca 4080
gattggctat tggccattgc atacgttgta tccatatcat aatatgtaca tttatattgg 4140
ctcatgtcca acattaccgc catgttgaca ttgattattg actagttatt aatagtaatc 4200
aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt 4260
aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta 4320
tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg 4380
gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga 4440
cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt 4500
tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg 4560
gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc 4620
cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg 4680
taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat 4740
aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga 4800
cctccataga agacaccggg accgatccag cctccgcggc cgggaacggt gcattggaac 4860
gcggattccc cgtgccaaga gtgacgtaag taccgcctat agagtctata ggcccacccc 4920
cttggcttct tatgcatgct atactgtttt tggcttgggg tctatacacc cccgcttcct 4980
catgttatag gtgatggtat agcttagcct ataggtgtgg gttattgacc attattgacc 5040
actcccctat tggtgacgat actttccatt actaatccat aacatggctc tttgccacaa 5100
ctctctttat tggctatatg ccaatacact gtccttcaga gactgacacg gactctgtat 5160
ttttacagga tggggtctca tttattattt acaaattcac atatacaaca ccaccgtccc 5220
cagtgcccgc agtttttatt aaacataacg tgggatctcc acgcgaatct cgggtacgtg 5280
ttccggacat gggctcttct ccggtagcgg cggagcttct acatccgagc cctgctccca 5340
tgcctccagc gactcatggt cgctcggcag ctccttgctc ctaacagtgg aggccagact 5400
taggcacagc acgatgccca ccaccaccag tgtgccgcac aaggccgtgg cggtagggta 5460
tgtgtctgaa aatgagctcg gggagcgggc ttgcaccgct gacgcatttg gaagacttaa 5520
ggcagcggca gaagaagatg caggcagctg agttgttgtg ttctgataag agtcagaggt 5580
aactcccgtt gcggtgctgt taacggtgga gggcagtgta gtctgagcag tactcgttgc 5640
tgccgcgcgc gccaccagac ataatagctg acagactaac agactgttcc tttccatggg 5700
tcttttctgc agtcaccgtc gtcgaccaga gctgagatcc tacaggagtc cagggctgga 5760
gagaaaacct ctgcgaggaa agggaaggag caagccgtga atttaaggga cgctgtgaag 5820
caatcatgga tgcaatgaag agagggctct gctgtgtgct gctgctgtgt ggagcagtct 5880
tcgtttcgcc cagcggtac 5899

3

4016

DNA

Borrelia burgdorferi

misc_feature

(6)..(29)

N stands for A or G or C or T

3
gatccnnnnn nnnnnnnnnn nnnnnnnnna agcttaatta gaaccaaact taattaaaac 60
caaacttaat tgaagttatt atcattttat tttttttcaa ttttctattt gttattgtta 120
atcttataat ataattatac ttgtattaag ttatattaat ataaaaggag aatatattat 180
gaaaaaatat ttattgggaa taggtctaat attagcctta atagcatgta agcaaaatgt 240
tagcagcctt gacgagaaaa acagcgtttc agtagatttg cctggtgaaa tgaaagttct 300
tgtaagcaaa gaaaaaaaca aagacggcaa gtacgatcta attgcaacag tagacaagct 360
tgagcttaaa ggaacttctg ataaaaacaa tggatctgga gtacttgaag gcgtaaaagc 420
tgacaaaagt aaagtaaaat taacaatttc tgacgatcta ggtcaaacca cacttgaagt 480
tttcaaagaa gatggcaaaa cactagtatc aaaaaaagta acttccaaag acaagtcatc 540
aacagaagaa aaattcaatg aaaaaggtga agtatctgaa aaaataataa caagagcaga 600
cggaaccaga cttgaataca cagaaattaa aagcgatgga tctggaaaag ctaaagaggt 660
tttaaaaggc tatgttcttg aaggaactct aactgctgaa aaaacaacat tggtggttaa 720
agaaggaact gttactttaa gcaaaaatat ttcaaaatct ggggaagttt cagttgaact 780
taatgacact gacagtagtg ctgctactaa aaaaactgca gcttggaatt caggcacttc 840
aactttaaca attactgtaa acagtaaaaa aactaaagac cttgtgttta caaaagaaaa 900
cacaattaca gtacaacaat acgactcaaa tggcaccaaa ttagaggggt cagcagttga 960
aattacaaaa cttgatgaaa ttaaaaacgc tttaaaataa ggagaattta tgagattatt 1020
aataggattt gctttagcgt tagctttaat aggatgtgca caaaaaggtg ctgagtcnnn 1080
nnnnngttgg gaattcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 1140
tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 1200
gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 1260
tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 1320
ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 1380
cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 1440
gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 1500
tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 1560
agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 1620
tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 1680
cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 1740
ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 1800
ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 1860
ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 1920
ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 1980
cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 2040
gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 2100
atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 2160
ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 2220
gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 2280
tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 2340
ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 2400
taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 2460
gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 2520
gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 2580
ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 2640
aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 2700
gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 2760
cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 2820
actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 2880
caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 2940
gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 3000
ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 3060
caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 3120
tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 3180
gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 3240
cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa 3300
ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct 3360
gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac 3420
aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg 3480
catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg 3540
taaggagaaa ataccgcatc aggcgccatt cgccattcag gctgcgcaac tgttgggaag 3600
ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa 3660
ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca 3720
gtgccaagct tggctgcagg tcgacgctct cccttatgcg actcctgcat taggaagcag 3780
cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag 3840
atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg 3900
ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg 3960
ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagag 4016

4

3799

DNA

Borrelia burgdorferi

misc_feature

(1118)..(1125)

N stands for A or G or C or T

4
aattcaggca cttcaacttt aacaattact gtaaacagta aaaaaactaa agaccttgtg 60
tttacaaaag aaaacacaat tacagtacaa caatacgact caaatggcac caaattagag 120
gggtcagcag ttgaaattac aaaacttgat gaaattaaaa acgctttaaa ataaggagaa 180
tttatgagat tattaatagg atttgcttta gcgttagctt taataggatg tgcacaaaaa 240
ggtgctgagt caattggttc tcaaaaagaa aatgatctaa accttgaaga ctctagtaaa 300
aaatcacatc aaaacgctaa acaagacctt cctgcggtga cagaagactc agtgtctttg 360
tttaatggta ataaaatttt tgtaagcaaa gaaaaaaata gctccggcaa atatgattta 420
agagcaacaa ttgatcaggt tgaacttaaa ggaacttccg ataaaaacaa tggttctgga 480
acccttgaag gttcaaagcc tgacaagagt aaagtaaaat taacagtttc tgctgattta 540
aacacagtaa ccttagaagc atttgatgcc agcaaccaaa aaatttcaag taaagttact 600
aaaaaacagg ggtcaataac agaggaaact ctcaaagcta ataaattaga ctcaaagaaa 660
ttaacaagat caaacggaac tacacttgaa tactcacaaa taacagatgc tgacaatgct 720
acaaaagcag tagaaactct aaaaaatagc attaagcttg aaggaagtct tgtagtcgga 780
aaaacaacag tggaaattaa agaaggtact gttactctaa aaagagaaat tgaaaaagat 840
ggaaaagtaa aagtcttttt gaatgacact gcaggttcta acaaaaaaac aggtaaatgg 900
gaagacagta ctagcacttt aacaattagt gctgacagca aaaaaactaa agatttggtg 960
ttcttaacag atggtacaat tacagtacaa caatacaaca cagctggaac cagcctagaa 1020
ggatcagcaa gtgaaattaa aaatctttca gagcttaaaa acgctttaaa ataatatata 1080
agtaaacccc ctacaaggca tcagctagtg taggaagnnn nnnnnggccn nnnnnnngtt 1140
ggggatccgt cgacctgcag ccaagcttgg cgtaatcatg gtcatagctg tttcctgtgt 1200
gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag 1260
cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt 1320
tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 1380
gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 1440
ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 1500
caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 1560
aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 1620
atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 1680
cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 1740
ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 1800
gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 1860
accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 1920
cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 1980
cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct 2040
gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 2100
aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 2160
aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 2220
actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 2280
taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 2340
gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 2400
tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 2460
ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 2520
accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 2580
agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 2640
acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 2700
ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa 2760
gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca 2820
ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt 2880
tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt 2940
tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg 3000
ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga 3060
tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc 3120
agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg 3180
acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag 3240
ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg 3300
gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg 3360
acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat 3420
gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg 3480
gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc 3540
tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa 3600
ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc attcaggctg 3660
cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa 3720
gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt 3780
tgtaaaacga cggccagtg 3799

5

1917

DNA

Borrelia burgdorferi

5
ctagcttaat tagaaccaaa cttaattaaa accaaactta attgaagtta ttatcatttt 60
attttttttc aattttctat ttgttattta ttaatcttat aatataatta tacttgtatt 120
aagttatatt aatataaaag gagaatatat tatgaaaaaa tatttattgg gaataggtct 180
aatattagcc ttaatagcat gtaagcaaaa tgttagcagc cttgacgaga aaaacagcgt 240
ttcagtagat ttgcctggtg aaatgaaagt tcttgtaagc aaagaaaaaa acaaagacgg 300
caagtacgat ctaattgcaa cagtagacaa gcttgagctt aaaggaactt ctgataaaaa 360
caatggatct ggagtacttg aaggcgtaaa agctgacaaa agtaaagtaa aattaacaat 420
ttctgacgat ctaggtcaaa ccacacttga agttttcaaa gaagatggca aaacactagt 480
atcaaaaaaa gtaacttcca aagacaagtc atcaacagaa gaaaaattca atgaaaaagg 540
tgaagtatct gaaaaaataa taacaagagc agacggaacc agacttgaat acacagaaat 600
taaaagcgat ggatctggaa aagctaaaga ggttttaaaa ggctatgttc ttgaaggaac 660
tctaactgct gaaaaaacaa cattggtggt taaagaagga actgttactt taagcaaaaa 720
tatttcaaaa tctggggaag tttcagttga acttaatgac actgacagta gtgctgctac 780
taaaaaaact gcagcttgga attcaggcac ttcaacttta acaattactg taaacagtaa 840
aaaaactaaa gaccttgtgt ttacaaaaga aaacacaatt acagtacaac aatacgactc 900
aaatggcacc aaattagagg ggtcagcagt tgaaattaca aaacttgatg aaattaaaaa 960
cgctttaaaa taaggagaat ttatgagatt attaatagga tttgctttag cgttagcttt 1020
aataggatgt gcacaaaaag gtgctgagtc aattggttct caaaaagaaa atgatctaaa 1080
ccttgaagac tctagtaaaa aatcacatca aaacgctaaa caagaccttc ctgcggtgac 1140
agaagactca gtgtctttgt ttaatggtaa taaaattttt gtaagcaaag aaaaaaatag 1200
ctccggcaaa tatgatttaa gagcaacaat tgatcaggtt gaacttaaag gaacttccga 1260
taaaaacaat ggttctggaa cccttgaagg ttcaaagcct gacaagagta aagtaaaatt 1320
aacagtttct gctgatttaa acacagtaac cttagaagca tttgatgcca gcaaccaaaa 1380
aatttcaagt aaagttacta aaaaacaggg gtcaataaca gaggaaactc tcaaagctaa 1440
taaattagac tcaaagaaat taacaagatc aaacggaact acacttgaat actcacaaat 1500
aacagatgct gacaatgcta caaaagcagt agaaactcta aaaaatagca ttaagcttga 1560
aggaagtctt gtaggcggaa aaacaacagt ggaaattaaa gaaggtactg ttactctaaa 1620
aagagaaatt gaaaaagatg gaaaagtaaa agtctttttg aatgacactg caggttctaa 1680
caaaaaaaca ggtaaatagg aagacagtac tagcacttta acaattagtg ctgacagcac 1740
aaaaactaaa gatttggtgt tcttaacaga tggtacaatt acagtacaac aatacaacac 1800
agctggaacc agcctagaag gatcagcaag tgaaattaaa aatctttcag agcttaaaaa 1860
cgctttaaaa taatatataa gtaaaccccc ctacaaggca tcagctagtg taggaag 1917

6

320

DNA

Borrelia burgdorferi

6
gttcctttcc atgggtcttt tctgcagtca ccgtcgtcga caaggaaagg tacccagaaa 60
agacgtcagt ggcagcagct cacgtgtgat cagatatcgc ggccgctcta gaccaggcgc 120
gtgcacacta gtctatagcg ccgtcgagat ctggtccgcg ctggatccag atctgctgtg 180
ccttctagtt gccagccatc gacctaggtc tagacgacac ggaagatcaa cggtcggtag 240
tgttgtttgc ccctcccccg tgccttcctt gaccctggaa acaacaaacg gggagggggc 300
acggaaggaa ctgggacctt 320

7

4915

DNA

Borrelia burgdorferi

7
ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc 60
acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt 120
gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg 180
caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcagattg 240
gctattggcc attgcatacg ttgtatccat atcataatat gtacatttat attggctcat 300
gtccaacatt accgccatgt tgacattgat tattgactag ttattaatag taatcaatta 360
cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 420
gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 480
ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 540
ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca 600
atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gacctttcct 660
acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag 720
tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt 780
gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac 840
aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc 900
agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc atccacgctg ttttgacctc 960
catagaagac accgggaccg atccagcctc cgcggccggg aacggtgcat tggaacgcgg 1020
attccccgtg ccaagagtga cgtaagtacc gcctatagag tctataggcc cacccccttg 1080
gcttatgcat gctatactgt ttttggcttg gggtctatac acccccgctt cctcatgtta 1140
taggtgatgg tatagcttag cctataggtg tgggttattg accattattg accactcccc 1200
tattggtgac gatactttcc attactaatc cataacatgg ctctttgcca caactctctt 1260
tattggctat atgccaatac actgtccttc agagactgac acggactctg tatttttaca 1320
ggatggggtc tcatttatta tttacaaatt cacatataca acaccaccgt ccccagtgcc 1380
cgcagttttt attaaacata acgtgggatc tccacgcgaa tctcgggtac gtgttccgga 1440
catgggctct tctccggtag cggcggagct tctacatccg agccctgctc ccatgcctcc 1500
agcgactcat ggtcgctcgg cagctccttg ctcctaacag tggaggccag acttaggcac 1560
agcacgatgc ccaccaccac cagtgtgccg cacaaggccg tggcggtagg gtatgtgtct 1620
gaaaatgagc tcggggagcg ggcttgcacc gctgacgcat ttggaagact taaggcagcg 1680
gcagaagaag atgcaggcag ctgagttgtt gtgttctgat aagagtcaga ggtaactccc 1740
gttgcggtgc tgttaacggt ggagggcagt gtagtctgag cagtactcgt tgctgccgcg 1800
cgcgccacca gacataatag ctgacagact aacagactgt tcctttccat gggtcttttc 1860
tgcagtcacc gtcgtcgaca cgtgtgatca gatatcgcgg ccgctctaga ccaggcgcct 1920
ggatccagat ctgctgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg 1980
ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt 2040
gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcagcacagc 2100
aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatgggt 2160
acccaggtgc tgaagaattg acccggttcc tcctgggcca gaaagaagca ggcacatccc 2220
cttctctgtg acacaccctg tccacgcccc tggttcttag ttccagcccc actcatagga 2280
cactcatagc tcaggagggc tccgccttca atcccacccg ctaaagtact tggagcggtc 2340
tctccctccc tcatcagccc accaaaccaa acctagcctc caagagtggg aagaaattaa 2400
agcaagatag gctattaagt gcagagggag agaaaatgcc tccaacatgt gaggaagtaa 2460
tgagagaaat catagaattt cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 2520
tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc 2580
aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa 2640
aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 2700
tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 2760
ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 2820
cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag 2880
ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 2940
ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 3000
gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 3060
agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg 3120
cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 3180
aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 3240
aggatctcaa gaagatcctt tgatgttttc taccggggtc tgacgctcag tggaacgaaa 3300
actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 3360
taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 3420
gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 3480
tagttgcctg actccggggg gggggggcgc tgaggtctgc ctcgtgaaga aggtgttgct 3540
gactcatacc aggcctgaat cgccccatca tccagccaga aagtgaggga gccacggttg 3600
atgagagctt tgttgtaggt ggaccagttg gtgattttga acttttgctt tgccacggaa 3660
cggtctgcgt tgtcgggaag atgcgtgatc tgatccttca actcagcaaa agttcgattt 3720
attcaacaaa gccgccgtcc cgtcaagtca gcgtaatgct ctgccagtgt tacaaccaat 3780
taaccaattc tgattagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat 3840
caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac 3900
cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa 3960
catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac 4020
catgagtgac gactgaatcc ggtgagaatg gcaaaagctt atgcatttct ttccagactt 4080
gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat 4140
tcattcgtgc attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta 4200
caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca 4260
cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg 4320
agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat 4380
tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg 4440
ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca 4500
cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg 4560
gaatttaatc gcggcctcga gcaagacgtt tcccgttgaa tatggctcat aacacccctt 4620
gtattactgt ttatgtaagc agacagtttt attgttcatg atgatatatt tttatcttgt 4680
gcaatgtaac atcagagatt ttgagacaca acgtggcttt cccccccccc ccattattga 4740
agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 4800
aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc 4860
attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtc 4915

8

5215

DNA

Borrelia burgdorferi

8
aagctttttt tgcaaaagcc taggcctcca aaaaagcctc ctcactactt ctggaatagc 60
tcagaggccg aggcggcctc ggcctctgca taaataaaaa aaattagtca gccatggggc 120
ggagaatggg cggaactggg cgaagttagg ggcgggatgg gcggagtgaa ttattggcta 180
ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 240
aatatgaccg ccatgttgac attgattatt gactagttat taatagtaat caattacggg 300
gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taattggccc 360
gcctgctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata 420
gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc 480
cacttggcag tacatcaagt gtatcatatg ccaagtccgg ccccctattg acgtcaatga 540
cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttggtacttg 600
gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 660
caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 720
caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc 780
cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc 840
tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag 900
aagacaccgg gaccgatcca gcctccgcgg ccgggaacgg tgcattggaa cgcggattcc 960
ccgtgccaag agtgacgtaa gtaccgccta tagactctat aggcacaccc ctttggctct 1020
tatgcatgct atactgtttt tggcttgggg cctatacacc cccgctcctt atgctatagg 1080
tgatggtata gcttagccta taggtgtggg ttattgacca ttattgacca ctcccctatt 1140
ggtgacgata ctttccatta cttaatccat aacatggctc tttgccacaa ctatctctat 1200
tggctatatg ccaatactct gtccttcaga gactgacacg gactctgtat ttttacagga 1260
tggggtccca tttattattt acaaattcac atatacaaca acgccgtccc ccgtgcccgc 1320
agtttttatt aaacatagcg tgggatctcc acgcgaatct cgggtacgtg ttccggacat 1380
gggctcttct ccggtagcgg cggagcttcc acatccgagc cctggtccca tgcctccagc 1440
ggctcatggt cgctcggcag ctccttgctc ctaacagtgg aggccagact taggcacagc 1500
acaatgccca ccaccaccag tgtgccgcac aaggccgtgg cggtagggta tgtgtctgaa 1560
aatgagctcg gagattgggc tcgcaccgtg acgcagatgg aagacttaag gcagcggcag 1620
aagaagatgc aggcagctga gttgttgtat tctgataaga gtcagaggta actcccgttg 1680
cggttctgtt aacggtggag ggcagtgtag tctgagcagt actcgttgct gccgcgcgcg 1740
ccaccagaca taatagctga cagactaaca gactgttcct ttccatgggt cttttctgca 1800
gtcaccgtcg tcgaccagag ctgagatcct acaggagtcc agggctggag agaaaacctc 1860
tgcgaggaaa gggaaggagc aagccgtgaa tttaagggac gctgtgaagc aatcatggat 1920
gcaatgaaga gagggctctg ctgtgtgctg ctgctgtgtg gagcagtctt cgtttcgccc 1980
agcgctagag gatccagatc tctcgacatg ggcaaatatt atacgcaagg cgacaaggtg 2040
ctgatgccgc tggcgattca ggttcatcat gccgtctgtg atggcttcca tgtcggcaga 2100
atgcttaatg aattacaaca gtactgcgat gagtggcagg gcggggcgta atttttttaa 2160
ggcagttatt ggtgccctta aacgcctggt gctacgcctg aataagtgat aataagcgga 2220
tgaatggcag aaattcgccg gatctttgtg aaggaacctt acttctgtgg tgtgacataa 2280
ttggacaaac tacctacaga gatttaaagc tctaaggtaa atataaaatt tttaagtgta 2340
taatgtgtta aactactgat tctaattgtt tgtgtatttt agattccaac ctatggaact 2400
gatgaatggg agcagtggtg gaatgccttt aatgaggaaa acctgttttg ctcagaagaa 2460
atgccatcta gtgatgatga ggctactgct gactctcaac atttctactc ctccaaaaaa 2520
gaagagaaag gtagaagacc ccaaggactt tccttcagaa ttgctaagtt ttttgagtca 2580
tgctgtgttt agtaatagaa ctcttgcttg ctttgctatt tacaccacaa aggaaaaagc 2640
tgcactgcta tacaagaaaa ttatggaaaa atattctgta acctttataa gtaggcataa 2700
cagttataat cataacatac tgttttttct tactccacac aggcatagag tgtctgctat 2760
taataactat gctcaaaaat tgtgtacttt agctttttaa tttgtaaagg ggttaataag 2820
gaatatttga tgtatagtgc cttgactaga gatcataatc agccatacca catttgtaga 2880
ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa 2940
tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag 3000
catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa 3060
actcatcaat gtatcttatc atgtctggat cgatccccgg gtaccgagct cgaattcgta 3120
atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat 3180
acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt 3240
aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta 3300
atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc 3360
gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 3420
ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 3480
aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 3540
ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 3600
aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 3660
gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 3720
tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 3780
tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 3840
gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 3900
cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 3960
cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 4020
agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 4080
caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 4140
ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgaacaataa 4200
aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa 4260
cgtcttgctc gaggccgcga ttaaattcca acatggatgc tgattatatg ggtataaatg 4320
ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg gaagcccgat 4380
gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag 4440
atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa gcatttatcc 4500
gtactcctga tgatgcatgg ttactcacca ctgcgatccc cgggaaaaca gcattccagg 4560
tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc 4620
gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc 4680
tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg 4740
agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataagctt ttccattctc 4800
accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg 4860
gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct 4920
tgccatccta tggaactgcc tcggtgagtt ttctccttca tacagaaacg gctttttcaa 4980
aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcattgatg ctcgatgagt 5040
ttttctaaga attcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt 5100
gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag 5160
ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgcc 5215

9

211

DNA

Borrelia burgdorferi

9
ctgcagtcac cgtcgtcgac cagagctgag atcctacagg agtccagggc tggagagaaa 60
acctctgcga ggaaagggaa ggagcaagcc gtgaatttaa gggacgctgt gaagcaatca 120
tggatgcaat gaagagaggg ctctgctgtg tgctgctgct gtgtggagca gtcttcgttt 180
cgcccagcgc tagaggatcc agatctctcg a 211

10

30

DNA

Borrelia burgdorferi

10
gccttaggta cctgtaagca aaatgttagc 30

11

42

DNA

Borrelia burgdorferi

11
taataatcta gaaaattctc cttacttaag agcgttttta at 42

12

30

DNA

Borrelia burgdorferi

12
gctttaggta cctgtgcaca aaaaggtgct 30

13

30

DNA

Borrelia burgdorferi

13
agggggggat cctatatatt attttaaagc 30

14

21

DNA

Borrelia burgdorferi

14
tcttttctgc agtcaccgtc g 21

15

36

DNA

Borrelia burgdorferi

15
gagagatctg gatccggtac cgctgggcga aacgaa 36

16

839

DNA

Borrelia burgdorferi

16
tagctgacag actaacagac tgttcctttc catgggtctt ttctgcagtc accgtcgtcg 60
atcgactgtc tgattgtctg acaaggaaag gtacccagaa aagacgtcag tggcagcagc 120
accagagctg agatcctaca ggagtccagg gctggagaga aaacctctgc gaggaaaggg 180
tggtctcgac tctaggatgt cctcaggtcc cgacctctct tttggagacg ctcctttccc 240
aaggagcaag ccgtgaattt aagggacgct gtgaagcaat catggatgca atgaagagag 300
ttcctcgttc ggcacttaaa ttccctgcga cacttcgtta gtacctacgt tacttctctc 360
ggctctgctg tgtgctgctg ctgtgtggag cagtcttcgt ttcgcccagc ggtacctgta 420
ccgagacgac acacgacgac gacacacctc gtcagaagca aagcgggtcg ccatggacat 480
agcaaaatgt tagcagcctt gacgagaaaa acagcgtttc agtagatttg cctggtgaaa 540
tcgttttaca atcgtcggaa ctgctctttt tgtcgcaaag tcatctaaac ggaccacttt 600
tgaaagttct tgtaagcaaa gaaaaaaaca aagacggcaa gtacgatcta attgcaacag 660
actttcaaga acattcgttt ctttttttgt ttctgccgtt catgctagtt aacgttgtct 720
agacaagctt gagcttaaag gaacttctga taaaaacaat ggatctggag tacttgaaga 780
tctgttcgaa ctcgaatttc cttgaagact atttttgtta cctagacctc atgaacttc 839

17

840

DNA

Borrelia burgdorferi

17
gtactcgttg ctgccgcgcg cgccaccaga cataatagct gacagactaa cagactgttc 60
catgagcaac gacggcgcgc gcggtggtct gtattatcga ctgtctgatt gtctgacaag 120
ctttccatgg gtcttttctg cagtcaccgt cgtcgaccag agctgagatc ctacaggagt 180
gaaaggtacc cagaaaagac gtcagtggca gcagctggtc tcgactctag gatgtcctca 240
ccagggctgg agagaaaacc tctgcgagga aagggaagga gcaagccgtg aatttaaggg 300
ggtcccgacc tctcttttgg agacgctcct ttcccttcct cgttcggcac ttaaattccc 360
acgctgtgaa gcaatcatgg atgcaatgaa gagagggctc tgctgtgtgc tgctgctgtg 420
tgcgacactt cgttagtacc tacgttactt ctctcccgag acgacacacg acgacgacac 480
tggagcagtc ttcgtttcgc ccagcggtac ctgtgcacaa aaaggtgctg agtcaattgg 540
acctcgtcag aagcaaagcg ggtcgccatg gacacgtgtt tttccacgac tcagttaacc 600
ttctcaaaaa gaaaatgatc taaaccttga agactctagt aaaaaatcac atcaaaacgc 660
aagagttttt cttttactag atttggaact tctgagatca ttttttagtg tagttttgcg 720
taaacaagac cttcctgcgg tgacagaaga ctcagtgtct ttgtttaatg gtaataaaat 780
atttgttctg gaaggacgcc actgtcttct gagtcacaga aacaaattac cattatttta 840

18

569

PRT

Borrelia burgdorferi

18
Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala
1 5 10 15
Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val
20 25 30
Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys
35 40 45
Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys
50 55 60
Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys
65 70 75 80
Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln
85 90 95
Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys
100 105 110
Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu
115 120 125
Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg
130 135 140
Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu
145 150 155 160
Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr
165 170 175
Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser
180 185 190
Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala
195 200 205
Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr
210 215 220
Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu
225 230 235 240
Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu
245 250 255
Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu
260 265 270
Lys Met Arg Leu Leu Ile Gly Phe Ala Leu Ala Leu Ala Leu Ile Gly
275 280 285
Cys Ala Gln Lys Gly Ala Glu Ser Ile Gly Ser Gln Lys Glu Asn Asp
290 295 300
Leu Asn Leu Glu Asp Ser Ser Lys Lys Ser His Gln Asn Ala Lys Gln
305 310 315 320
Asp Leu Pro Ala Val Thr Glu Asp Ser Val Ser Leu Phe Asn Gly Asn
325 330 335
Lys Ile Phe Val Ser Lys Glu Lys Asn Ser Ser Gly Lys Tyr Asp Leu
340 345 350
Arg Ala Thr Ile Asp Gln Val Glu Leu Lys Gly Thr Ser Asp Lys Asn
355 360 365
Asn Gly Ser Gly Thr Leu Glu Gly Ser Lys Pro Asp Lys Ser Lys Val
370 375 380
Lys Leu Thr Val Ser Ala Asp Leu Asn Thr Val Thr Leu Glu Ala Phe
385 390 395 400
Asp Ala Ser Asn Gln Lys Ile Ser Ser Lys Val Thr Lys Lys Gln Gly
405 410 415
Ser Ile Thr Glu Glu Thr Leu Lys Ala Asn Lys Leu Asp Ser Lys Lys
420 425 430
Leu Thr Arg Ser Asn Gly Thr Thr Leu Glu Tyr Ser Gln Ile Thr Asp
435 440 445
Ala Asp Asn Ala Thr Lys Ala Val Glu Thr Leu Lys Asn Ser Ile Lys
450 455 460
Leu Glu Gly Ser Leu Val Val Gly Lys Thr Thr Val Glu Ile Lys Glu
465 470 475 480
Gly Thr Val Thr Leu Lys Arg Glu Ile Glu Lys Asp Gly Lys Val Lys
485 490 495
Val Phe Leu Asn Asp Thr Ala Gly Ser Asn Lys Lys Thr Gly Lys Trp
500 505 510
Glu Asp Ser Thr Ser Thr Leu Thr Ile Ser Ala Asp Ser Lys Lys Thr
515 520 525
Lys Asp Leu Val Phe Leu Thr Asp Gly Thr Ile Thr Val Gln Gln Tyr
530 535 540
Asn Thr Ala Gly Thr Ser Leu Glu Gly Ser Ala Ser Glu Ile Lys Asn
545 550 555 560
Leu Ser Glu Leu Lys Asn Ala Leu Lys
565

Number	Name	Date	Kind
5122458	Post et al.	Jun 1992	A
5558993	Champion et al.	Sep 1996	A
5571718	Dunn et al.	Nov 1996	A
5620896	Herrmann et al.	Apr 1997	A
5688512	Bergstrom et al.	Nov 1997	A
5736524	Content et al.	Apr 1998	A
5846946	Huebner et al.	Dec 1998	A
5942236	Lobet et al.	Aug 1999	A
5981505	Weiner et al.	Nov 1999	A
6090586	Bergstrom et al.	Jul 2000	A

Number	Date	Country
0 346 316	Dec 1989	EP
WO 9416737	Aug 1994	WO
WO 9425596	Nov 1994	WO
WO 9514781	Jun 1995	WO

	Number	Date	Country
Parent	08/663998	Jun 1996	US
Child	09/173053		US

Compositions and methods for administering Borrelia DNA

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

US Classifications

Field of Search

US

International Classifications

Abstract

Description

Claims

RELATED APPLICATIONS

STATEMENT OF GOVERNMENT SUPPORT

US Referenced Citations (10)

Foreign Referenced Citations (4)

Non-Patent Literature Citations (28)

Continuation in Parts (1)

Entry
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