1. Field of the Invention
This invention relates generally to an antigenic preparation and specifically to a Borrelia burgdorferi protein (EppA) which is used to induce a protective immune response in animals. This protein can be used immunologically as a vaccine for Lyme disease caused by this organism. Alternatively, diagnosis of Lyme disease can be performed by detecting the presence of the protein, antibody to the protein, or polynucleotide which encodes the protein.
2. Description of Related Art
Lyme disease is an infection with world-wide distribution caused by the spirochete Borrelia burgdorferi, and is the most commonly reported arthropod-borne disease in the United States. About 10,000 reported cases of Lyme disease occur every year in the United States, caused by deer-tick bites that transmit the Borrelia burgdorferi organism. If not identified early, by flu-like symptoms and the bull's-eye rash that usually appears at the site of infection, untreated Lyme disease can cause heart problems, arthritis, and neurological symptoms. A large group of patients suffer from lasting neurological symptoms, including vision loss, that can recur for years as a result of Lyme disease.
The genus Borrelia is unique among the pathogenic spirochetes in that it contains both linear and circular plasmids which account for approximately 150-kbp of the total genetic material (A. G. Barbour, J. Clin. Microbiol., 26:475-478, 1988; Hinnebusch, et al., J. Bacteriol., 174:5221-5227, 1992; Hinnebusch, et al., J. Bacteriol, 173:7233-7239, 1991; Hinnebusch, et al., Mol. Microbiol., 4:811-820, 1990; Howe, et al., Science, 82:151-154, 1985; Hyde, et al., J. Clin. Microbiol., 20:151-154, 1984; Schwan, et al., Infec. Immun., 56:1831-1836, 1988; Simpson, et al, J. Clin. Microbiol., 28:1329-1337, 1990; Simpson, et al., Microbiol. Path., 8:109-118, 1990). Only genes for the outer surface lipoproteins (Osps A, B, C, D, E, and F) have been mapped to these plasmids. The ospAB and ospEF operons have been mapped to linear plasmids of 49- and 45-kbp, respectively (Bergstron, et al., Mol. Microbiol., 3:479-486, 1986; Lam, et al., Infect. Immun., 62:290-298, 1994), while the gene encoding ospD resides on a 38-kbp linear plasmid (Norris, et al., Infec. Immun., 60: 4662-4672, 1992). The ospC locus has recently been localized to a 26-kbp circular plasmid and represents the first B. burgdorferi gene to be mapped to a circular plasmid (Fuchs, et al., Mol. Microbiol., 6:503-509, 1992; U. K. Laemmli, Nature, 227:680-685, 1970). Since many pathogenic bacteria harbor plasmids which encode genes whose expression is required for virulence, it is likely that genes encoding potential virulence determinants are also present on plasmids of B. burgdorferi.
The study of B. burgdorferi plasmids, with respect to virulence, has been limited by the lack of spirochete genetic exchange systems. Recently, one approach to studying these plasmids was developed based on the concept underlying TnphoA transposition (Boquet, et al., J. Bacteriol., 169:1663-1669, 1987; Hoffman, et al, Proc. Natl. Acad. Sci. USA, 62:5107-5111, 1985; Manoil, et al., Science, 233:1403-1408, 1986; Manoil, et al., J. Bacteriol., 172:515-518, 1990). The system utilizes a phoA expression vector termed pMG, that contains an alkaline phosphatase (AP) gene lacking its signal sequence, together with the E. coli mutant strain KS330 (Strauch, et al., Proc. Natl. Acad. Sci. USA, 85:1575-1580, 1988), which possesses a leaky outer membrane, to identify genes encoding signal peptide export-dependent proteins which may function as virulence determinants. The utility of this system has been confirmed for both Treponema pallidum (Blanco, et al., Mol. Microbiol., 5:2405-2415, 1991) and Leptospira alstoni in which signal peptide containing proteins from both organisms were shown to be exported in E. coli.
The pMG/KS330 system was utilized in identification of a B. burgdorferi B31 recombinant, termed Bb244, that was generated from a library of the 9.0-kbp circular plasmid (Giladi, et al., J. Bacteriol., 175:4129-4136, 1993). It had previously been reported by Schwan, et al. (Schwan, et al., supra) and later by Simpson, et al. (Simpson, et al., supra), that in B. burgdorferi strain SH-2-82, the loss of two similar sized circular plasmids (8.4- and 8.8-kbp) following 20 in vitro passages was correlated with the loss of virulence. Bb244 was shown to have an open reading frame, a typical signal peptide containing a type I leader peptidase cleavage site, translational, and transcriptional sequences upstream of the ATG start codon (Giladi, et al., supra).
There is a need to identify outer membrane proteins of Borrelia burgdorferi that may be associated with virulence of this spirochete. Such a protein would allow specific diagnosis of Borrefia infection and also be an excellent vaccine candidate for the prevention of such Borrelia associated diseases as Lyme disease.