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Title:  VMP-like sequences of pathogenic borrelia

United States Patent:  6,437,116

Issued:  August 20, 2002

Inventors:  Norris; Steven J. (Houston, TX); Zhang; Jing-Ren (Houston, TX); Hardham; John M. (Houston, TX); Howell; Jerrilyn K. (Houston, TX); Barbour; Alan G. (Irvin, CA); Weinstock; George M. (Houston, TX)

Assignee:  Board of Regents, The University of Texas System (Austin, TX)

Appl. No.:  125619

Filed:  January 27, 1999

PCT Filed:  February 20, 1997

PCT NO:  PCT/US97/02952

371 Date:  January 27, 1999

102(e) Date:  January 27, 1999

PCT PUB.NO.:  WO97/31123

PCT PUB. Date:  August 28, 1997

Abstract

The present invention relates to DNA sequences encoding Vmp-like polypeptides of pathogenic Borrelia, the use of the DNA sequences in recombinant vectors to express polypeptides, the encoded amino acid sequences, application of the DNA and amino acid sequences to the production of polypeptides as antigens for immunoprophylaxis, immunotherapy, and immunodiagnosis. Also disclosed are the use of the nucleic acid sequences as probes or primers for the deletion of organisms causing Lyme disease, relapsing fever, or related disorders, and kits designed to facilitate methods of using the described polypeptides, DNA segments and antibodies.

SUMMARY OF THE INVENTION

Partial and complete DNA sequences have been determined for several recombinant clones containing DNA encoding VMP-like sequences. The identification and characterization of these sequences now allows: (1) identification of the expressed gene(s) in B. burgdorferi; (2) expression of these gene(s) by a recombinant vector in a host organism such as E. coli; (3) immunization of laboratory animals with the resulting polypeptide, and determination of protective activity against B. burgdorferi infection; (4) use of antibodies against the expressed protein to identify the reactive polypeptide(s) in B. burgdorferi cells; (5) use of the expressed protein(s) to detect antibody responses in infected humans and animals; (6) determination of the presence, sequence differences, and expression of the VMP-like DNA sequences in other Lyme disease Borreliae.

The invention is contemplated to be useful in the immunoprophylaxis, diagnosis, or of Lyme disease, relapsing fever, or related diseases in humans or animals. It is expected that recombinant or native proteins expressed by the VMP-like genes (or portions thereof) will be useful for (a) immunoprophylaxis against Lyme disease, relapsing fever, or related disorders in humans and animals; (b) immunotherapy of existing Lyme disease, relapsing fever, or related illnesses, by way of immunization of injection of antibodies directed against VMP-like proteins; and (c) immunodiagnosis of Lyme disease, relapsing fever, or related diseases, including their use in kits in which the VMP-like proteins are the sole antigen or one of multiple antigens. The DNA may be employed in: (a) production of recombinant DNA plasmids or other vectors capable of expressing recombinant polypeptides; and (b) design and implementation of nucleic acid probes or oligonucleotides for detection and/or amplification of VMP-like sequences. The latter is expected to have application in the diagnosis of infection with Borrelial organisms.

Similar sequences in B. burgdorferi and other Lyme disease Borreliae have not been reported previously, as determined by BLAST searches of current nucleotide and amino acid databases including Genbank, the EMBL DNA database, and the Swiss Protein database. Although there is some similarity between the B. burgdorferi deduced amino acid sequences with previously published B. hermsii VMP deduced amino acid sequences, the degree of identity and similarity is only .about.30% and .about.50%, respectively. Outer surface protein C (OspC) of Lyme disease organisms has been reported to have sequence similarities to VMPs, but the highest similarity is to a different subgroup of VMPs than the sequences reported here (Carter et al., 1994). The VMP-like sequences such as those contained in pJRZ53-31 have a low degree of homology with OspC from some Lyme disease organisms (e.g. B. burgdorferi 2591), as indicated by a BLASTP homology score of 60 and a probability of 0.0013. Thus, the B. burgdorferi VMP-like DNA sequences are unique, although they have an apparent evolutionary relationship with other Borrelia genes.

Another aspect of the invention is the method for identification of possible virulence factors. This approach entails subtractive hybridization of target DNA from high infectivity organisms with driver DNA from low-infectivity strains or clones. This procedure greatly enriches for sequences which differ between the high- and low-infectivity strains and thus may encode proteins important in virulence. Of particular utility is the use of closely related isogenic clones that differ in their infectivity; in this case, the DNA differences should be restricted more stringently to those related to infectivity.

Open reading frames in a B. burgdorferi plasmid that encode hypothetical proteins resembling the VMP proteins of relapsing fever organisms have now been identified. The inventors have found that the presence of the plasmid containing these VMP-like sequences in B. burgdorferi clones correlates strongly with infectivity. Thus it is likely that the proteins encoded by the VMP-like sequences are important in immunoprotection and pathogenesis. Proteins encoded by the VMP-like sequences of B. burgdorferi may provide protection when used either alone or in combination with other antigens. They may also be useful for immunodiagnosis.

The invention is considered to include DNA segments corresponding to 20, 30, and 40 base pairs of the VMP-like sequences; DNA segments inclusive of the entire open leading frames of the VMP-like sequences; amino acid sequences corresponding to both conserved and variable regions of the VMP-like sequences; recombinant vectors encoding an antigenic protein corresponding to the above amino acid sequences; recombinant cells where extrachromosomal DNA expresses a polypeptide encoded by the DNA encoding Borrelia VMP-like sequences; a recombinant B. burgdorferi or E. coli cell containing the DNA encoding VMP-like sequences; methods of preparing transformed bacterial host cells using the DNA encoding the VMP-like polypeptides; methods using the plasmid or vector to transform the bacterial host cell to express B. burgdorferi polypeptides encoded by the DNA sequences; methods for immunization of humans or animals with the native B. burgdorferi polypeptide or polypeptides expressed by recombinant cells that include DNA encoding the VMP-like polypeptides; and methods for identifying potential virulence factors using subtractive hybridization between target DNA from high-infectivity cells and driver DNA from low-infectivity cells.

Also included in the invention are primer sets capable of priming amplification of the VMP-like DNA sequences; kits for the detection of B. burgdorferi nucleic acids in a sample, the kits containing a nucleic acid probe specific for the VMP-like sequences, together with a means for detecting a specific hybridization with the probe; kits for detection of antibodies against the VMP-like sequences of B. burgdorferi and kits containing a native or recombinant VMP-like polypeptide, together with means for detecting a specific binding of antibodies to the antigen.

Claim 1 of 48 Claims

What is claimed is:

1. An isolated and purified nucleic acid segment that encodes a polypeptide comprising an amino acid residue sequence of SEQ ID NO:2, or its complement.
 


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