Link:  Pharm/Biotech Resources

Title:  Lyme disease vaccines

United States Patent:  6,902,893

Issued:  June 7, 2005

Inventors:  Choi; Gil H. (11429 Potomac Oaks Dr., Rockville, MD 20850); Erwin; Alice L. (7759 26th Ave., NW., Seattle, WA 98119); Hanson; Mark S. (7013 Woodscape Dr., Clarksville, MD 20109); Lathigra; Raju (19051 Steeple Pl., Germantown, MD 20874)

Appl. No.:  830230

Filed:  June 18, 1998

PCT Filed:  June 18, 1998

PCT NO:  PCT/US98/12718

371 Date:  September 27, 2001

102(e) Date:  September 27, 2001

PCT PUB.NO.:  WO98/59071

PCT PUB. Date:  December 30, 1998

The present invention relates to novel vaccines for the prevention or attenuation of Lyme disease. The invention further relates to isolated nucleic acid molecules encoding antigenic polypeptides of Borrelia burgdorferi. Antigenic polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the sane. The invention additionally relates to diagnostic methods for detecting, Borrelia gene expression.

FIELD OF THE INVENTION

The present invention relates to novel vaccines for the prevention or attenuation of Lyme disease. The invention further relates to isolated nucleic acid molecules encoding antigenic polypeptides of Borrelia burgdorferi. Antigenic polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. The invention additionally relates to diagnostic methods for detecting Borrelia gene expression.

BACKGROUND OF THE INVENTION

Lyme disease (Steere, A. C., Proc. Natl. Acad Sci. USA 91:2378-2383 (1991)), or Lyme borreliosis, is presently the most common human disease in the United States transmitted by an arthropod vector (Center for Disease Control, Morbid Mortal. Week Rep. 46(23):531-535 (1997)). Further, infection of house-hold pets, such as dogs, is a considerable problem.

While initial symptoms often include a rash at the infection point, Lyme disease is a multisystemic disorder that may include arthritic, cardiac, and neurological manifestations. While antibiotics are currently used to treat active cases of Lyme disease, B. burgdorferi persists even after prolonged antibiotic treatment. Further, B. burgdorferi can persist for years in a mammalian host in the presence of an active immune response (Straubinger. R. et al., J. Clin. Microbiol. 35:111-116 (1997); Steere, A., N. Engl. J. Med. 321:586-596 (1989)).

Lyme disease is caused by the related tick-borne-spirochetes classified as Borrelia burgdorferi sensu lato (including B. burgdorferi sensu stricto, B. afzelii, B. garinii). Although substantial progress has been made in the biochemical, ultrastructural, and genetic characterization of the organism; the spirochetal factors responsible for infectivity, immune evasion and disease pathogenesis remain largely obscure.

A number of antigenic B. burgdorferi cell surface proteins have been identified. These include the outer membrane surface proteins (Osp) OspA, OspB, OspC and OspD. OspA and OspB are encoded by tightly linked tandem genes which am transcribed as a single transcriptional unit (Brusca, J. et at, J. Bacteriol. 173:8004-8008 (1991)). The most-studied B. burgdorferi membrane protein is OspA, a lipoprotein antigen expressed by borreliae in testing ticks and the most abundant protein expressed in vitro by most borrelial isolates (Barbour, A. G., et al., Infection & Immunity 41:795-804 (1983); Howe, T. R. et al., Science 227:645 (1985)).

A number of different types of Lyme disease vaccines have been shown to induce immunological responses. Whole-cell B. burgdorferi vaccines, for example, have been shown to induce both immunological responses and protective immunity in several animal models (Reviewed in Wormser, G., Clin. Infect. Dis. 21:1267-1274 (1995)). Further, passive immunity has been demonstrated in both humans and other animals using B. burgdorferi specific antisera.

While whole-cell Lyme disease vaccines confer protective immunity in animal models, use of such vaccines presents the risk that responsive antibodies will produce an autoimmune response (Reviewed in Wormser, G., supra). This problem is at least partly the result of the production of B. burgdorferi specific antibodies which cross-react with hepatocytes and both muscle and nerve cells. B. burgdorferi heat shock proteins and the 41-kd flagellin subunit are believed to contain antigens which elicit production of these cross-reactive antibodies.

Single protein subunit vaccines for Lyme disease have also been-tested. The cell surface proteins of B. burgdorferi are potential candidates for use in such vaccines and several have been shown to elicit protective immune responses in mammals (Probert, W. et al., Vaccine 15:15-19 (1997); Fikrig, E. er al., Infect. Immun. 63: 1658-1662 (1995); Langerman S. et al., Nature 372:552-556 (1994); Fikrig, E. et al., J. Immunol. 148:2256-2260 (1992)). Experimental OspA vaccines, for example, have demonstrated efficacy in several animal models (Fikrig, E., et al., Proc. Natl. Acad. Sci. USA 89:5418-5421 (1992); Johnson, B. J., et al., Vaccine 13:1036-1094 (1996); Fikrig, E., et al., Infect. Immun. 60:657-661 (1992); Chang, Y. F., et al., Infection & Immunity 63:3543-3549 (1995)), and OspA vaccines for human use are under clinical evaluation (Keller, D., et al., J. Am. Med. Assoc. 271:1764-1768 (1994); Van Hoecke, C., et al., Vaccine 14:1620-1626 (1996)). Passive immunity is also conferred by antisera containing antibodies specific for the full-length OspA protein. Further, vaccination with plasmid DNA encoding OspA has been demonstrated to elicit protective immune responses in mice (Luke, C. et al., J. Infect. Dis. 175:91-97 (1997); Zhong, W. et al., Eur. J. Immunol. 26:2749-2757 (1996)).

Recent immunofluorescence assay observations indicate that during tick engorgement the expression of OspA by borreliae diminishes (deSilva, A. M., et al., J. Exp. Med. 183:271-275 (1996)) while expression of other proteins, exemplified by OspC, increases (Schwan, T. G., et al., Proc. Natl. Acad. Sci. USA 92:2909-2913 (1985)). By the time of transmission to hosts, spirochetes in the tick salivary glands express little or no OspA. This down-modulation of OspA appears to explain the difficulties in demonstrating immune responses to this antigen early in infection following tick bites (Kalish, R. A., et al., Infect. Immun. 63:2228-2235 (1995); Gem, L., et al., J. Infect. Dis. 167:971-975 (1993); Schiable, U. E., et al., Immunol. Lett. 36:219-226 (1993)) or following challenge with limiting doses of cultured borreliae (Schiable, U. E., et al., Immunol. Lett. 36:219-226 (1993); Barthold, S. W. and Bockenstedt, L. K., Infect. Immun. 61:4696-4702 (1993)).

Furthermore, OspA-specific antibodies are ineffective if administered after a borrelial challenge delivered by syringe (Schiable, U. E., et al., Proc. Natl. Acad. Sci. USA 87:3768-3772 (1990)) or tick bite (deSilva, A. M., et al., J. Exp. Med. 183:271-275 (1996)). To be efficacious, OspA vaccines must elicit protective levels of antibody which are maintained throughout periods of tick exposure in order to block borrelia transmission from the arthropod vector.

Vaccines in current use against other pathogens include in vivo-expressed antigens which could boost anamnestic responses upon infection, potentiate the action of immune effector cells and complement, and inhibit key virulence mechanisms. OspC is both expressed during infection (Montgomery, R. R., et al., J. Exp. Med. 183:261-269 (1996)) and a target for protective immunity (Gilmore, R. D., et al., Infect Immun. 64:2234-2239 (1996); Probert, W. S. and LeFebvre, R. B., Infect. Immun. 62: 1920-1926 (1994); Preac-Mursic, V., et. al., Infection 20:342-349 (1992)), but mice immunized with this protein were only protected against challenge with the homologous borrelial isolate (Probert, W. S., et al., J. Infect Dis. 175:400-405 (1997)). Identification of in vivo-expressed, and broadly protective, antigens of B. burgdorferi has remained elusive.

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid molecules comprising polynucleotides encoding the B. burgdorferi peptides having the amino acid sequences shown in Table 1 (referred to full patent). Thus, one aspect of the invention provides isolated nucleic acid molecules comprising polynucleotides having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding any of the amino acid sequences of the full-length polypeptides shown in Table 1 (referred to full patent); (b) a nucleotide sequence encoding any of the amino acid sequences of the full-length polypeptides shown in Table 1 (referred to full patent) but minus the N-terminal methionine residue, if present; (c) a nucleotide sequence encoding any of the amino acid sequences of the truncated polypeptides shown in Table 1 (referred to full patent); and (d) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), or (c) above.

Further embodiments of the invention include isolated nucleic acid molecules that comprise a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide sequences in (a), (b), (c), or (d) above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c), or (d) above. This polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues. Additional nucleic acid embodiments of the invention relate to isolated nucleic acid molecules comprising polynucleotides which encode the amino acid sequences of epitope-bearing portions of a B. burgdorferi polypeptide having an amino acid sequence in (a), (b), or (c) above.

The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using these vectors for the production of B. burgdorferi polypeptides or peptides by recombinant techniques.

The invention further provides isolated B. burgdorferi polypeptides having an amino acid sequence selected from the group consisting of: (a) an amino acid sequence of any of the full-length polypeptides shown in Table 1 (referred to full patent); (b) an amino acid sequence of any of the full-length polypeptides shown in Table 1 (referred to full patent) but minus the N-terminal methionine residue, if present; (c) an amino acid sequence of any of the truncated polypeptides shown in Table 1 (referred to full patent); and (d) an amino acid sequence of an epitope-bearing portion of any one of the polypeptides of (a), (b), or (c).

The polypeptides of the present invention also include polypeptides having an amino acid sequence with at least 70% similarity, and more preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similarity to those described in (a), (b), (c), or (d) above, as well as polypeptides having an amino acid sequence at least 70% identical, more preferably at least 75% identical, and still more preferably 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to those above; as well as isolated nucleic acid molecules encoding such polypeptides.

The present invention further provides a vaccine, preferably a multi-component vaccine comprising one or more of the B. burgdorferi polypeptides shown in Table 1 (referred to full patent), or fragments thereof, together with a pharmaceutically acceptable diluent, carrier, or excipient, wherein the B. burgdorferi polypeptide(s) are present in an amount effective to elicit an immune response to members of the Borrelia genus in an animal. The B. burgdorferi polypeptides of the present invention may further be combined with one or more immunogens of one or more other borrelial or non-borrelial organisms to produce a multi-component vaccine intended to elicit an immunological response against members of the Borrelia genus and, optionally, one or more non borrelial organisms.

The vaccines of the present invention can be administered in a DNA form, e.g., "naked" DNA, wherein the DNA encodes one or more borrelial polypeptides and, optionally, one or more polypeptides of a non-borrelial organism. The DNA encoding one or more polypeptides may be constructed such that these polypeptides are expressed fusion proteins.

The vaccines of the present invention may also be administered as a component of a genetically engineered organism. Thus, a genetically engineered organism which expresses one or more B. burgdorferi polypeptides may be administered to an animal. For example, such a genetically engineered organism may contain one or more B. burgdorferi polypeptides of the present invention intracellularly, on its cell surface, or in its periplasmic space. Further, such a genetically engineered organism may secrete one or more B. burgdorferi polypeptides.

The vaccines of the present invention may be co-administered to an animal with an immune system modulator (e.g., CD86 and GM-CSF).

The invention also provides a method of inducing an immunological response in an animal to one or more members of the Borrelia genus, e.g., B. burgdorferi sensu stricto, B. afzelii, and B. garinii, comprising administering to the animal a vaccine as described above.

The invention further provides a method of inducing a protective immune response in an animal, sufficient to prevent or attenuate an infection by members of the Borrelia genus, comprising administering to the animal a composition comprising one or more of the polypeptides shown in Table 1 (referred to full patent), or fragments thereof. Further, these polypeptides, or fragments thereof, may be conjugated to another immunogen and/or administered in admixture with an adjuvant.

The invention further relates to antibodies elicited in an animal by the administration of one or more B. burgdorferi polypeptides of the present invention.

The invention also provides diagnostic methods for detecting the expression of genes of members of the Borrelia genus in an animal. One such method involves assaying for the expression of a gene encoding Borrelia peptides in a sample from an animal. This expression may be assayed either directly (e.g., by assaying polypeptide levels using antibodies elicited in response to amino acid sequences shown in Table 1 (referred to full patent)) or indirectly (e.g., by assaying for antibodies having specificity for amino acid sequences shown in Table 1 (referred to full patent)). An example of such a method involves the use of the polymerase chain reaction (PCR) to amplify and detect Borrelia nucleic acid sequences.

The present invention also relates to nucleic acid probes having all or part of a nucleotide sequence shown in Table 1 (referred to full patent) which are capable of hybridizing under stringent conditions to Borrelia nucleic acids. The invention further relates to a method of detecting one or more Borrelia nucleic acids in a biological sample obtained from an animal, said one or more nucleic acids encoding Borrelia polypeptides, comprising:

Claim 1 of 9 Claims

1. An isolated polynucleotide which encodes at least 50 contiguous amino acid residues of SEQ ID NO:627.

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