B. burgdorferi polypeptides

Details B. burgdorferi polypeptides B. burgdorferi polypeptides

1998-09-14

2004-04-06

Helms, Larry R. (Department: 1642)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S007100, C435S007200, C530S350000, C530S387100, C530S389100

Reexamination Certificate

active

06716591

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
This invention relates to compositions and methods useful for the prevention, diagnosis and treatment of Lyme disease. More particularly, this invention relates to novel
B. burgdorferi
polypeptides which are able to elicit in a treated animal, the formation of an immune response which is effective to prevent or lessen the severity, for some period of time, of
B. burgdorferi
infection. This invention also relates to multicomponent vaccines comprising one or more of the novel
B. burgdorferi
polypeptides. Also within the scope of this invention are antibodies directed against the novel
B. burgdorferi
polypeptides and diagnostic kits comprising the antibodies or the polypeptides.
BACKGROUND OF THE INVENTION
Lyme borreliosis is the most common vector-borne infection in the United States [S. W. Barthold, et al., “An Animal Model For Lyme Arthritis”,
Ann. N.Y. Acad. Sci
., 539, pp. 264-73 (1988)]. It has been reported in every continent except Antarctica. The clinical hallmark of Lyme Disease is an early expanding skin lesion known as erythema migrans, which may be followed weeks to months later by neurologic, cardiac, and joint abnormalities.
The causative agent of Lyme disease is a spirochete known as Borrelia burgdorferi, transmitted primarily by Ixodes ticks of the Ixodes ricinus complex.
B. burgdorferi
has also been shown to be carried in other species of ticks and in mosquitoes and deer flies, but it appears that only ticks of the I. ricinus complex are able to transmit the disease to humans.
Lyme disease generally occurs in three stages. Stage one involves localized skin lesions (erythema migrans) from which the spirochete is cultured more readily than at any other time during infection [B. W. Berger et al., “Isolation And Characterization Of The Lyme Disease Spirochete From The Skin Of Patients With Erythema Chronicum Migrans”,
J. Am. Acad. Dermatol
., 3, pp. 444-49 (1985)]. Flu-like or meningitis-like symptoms are common at this time. Stage two occurs within days or weeks, and involves spread of the spirochete through the patient's blood or lymph to many different sites in the body including the brain and joints. Varied symptoms of this disseminated infection occur in the skin, nervous system, and musculoskeletal system, although they are typically intermittent. Stage three, or late infection, is defined as persistent infection, and can be severely disabling. Chronic arthritis, and syndromes of the central and peripheral nervous system appear during this stage, as a result of the ongoing infection and perhaps a resulting auto-immune disease [R. Martin et al., “
Borrelia burgdorferi
-Specific And Autoreactive T-Cell Lines From Cerebrospinal Fluid In Lyme Radiculomyelitis”,
Ann Neurol
., 24, pp. 509-16 (1988)].
B. burgdorferi
is much easier to culture from the tick than from humans, therefore at present, Lyme disease is diagnosed primarily by serology. The enzyme-linked immunosorbent assay (ELISA) is one method of detection, using sonicated whole spirochetes as the antigen [J. E. Craft et al., “The Antibody Response In Lyme Disease: Evaluation Of Diagnostic Tests”,
J. Infect. Dis
., 149, pp. 789-95 (1984)]. However, false negative and, more commonly, false positive results are associated with currently available tests.
At present, all stages of Lyme disease are treated with antibiotics. Treatment of early disease is usually effective, however the cardiac, arthritic, and nervous system disorders associated with the later stages often do not respond to therapy [A. C. Steere, “Lyme Disease”,
New Eng. J. Med
., 321, pp. 586-96 (1939)].
Like
Treponema pallidum
, which causes syphilis, and leptospirae, which cause an infectious jaundice, Borrelia belong to the eubacterial phylum of spirochetes [A. G. Barbour and S. F. Hayes, “Biology Of Borrelia Species”,
Microbiol. Rev
., 50, pp. 381-400 (1986)].
Borrelia burgdorferi
have a protoplasmic cylinder that is surrounded by a cell membrane, then by flagella, and then by an outer membrane.
The
B. burgdorferi
outer surface proteins identified to date are believed to be lipoproteins, as demonstrated by labelling with [
3
H]palmitate [M. E. Brandt et al., “Immunogenic Integral membrane Proteins of
Borrelia burgdorferi
Are Lipoproteins”,
Infect. Immun
., 58, pp. 983-91 (1990)]. The two major outer surface proteins are the 31 kd outer-surface protein A (OspA) and the 34 kd outer surface protein B (OspB). Both proteins have been shown to vary from different isolates or from different passages of the same isolate as determined by their molecular weights and reactivity with monoclonal antibodies OspC is a 22 kDa membrane lipoprotein previously identified as pC. [R. Fuchs et al., “Molecular Analysis and Expression of a
Borrelia burgdorferi
Gene Encoding a 22 kDa Protein (pC) in
Escherichia coli”, Mol. Microbiol
., 6, pp. 503-09 (1992)]. OspD is said to be preferentially expressed by low-passage, virulent strains of
B. burgdorferi
B31 [S. J. Norris et al., “Low-Passage-Associated Proteins of
Borrelia burgdorferi
B31: Characterization and Molecular Cloning of OspD, A Surfaced-Exposed, Plasmid-Encoded Lipoprotein”,
Infect. Immun
., 60, pp. 4662-4672 (1992)].
Non-Osp
B. burgdorferi
proteins identified to date include the 41 kD flagellin protein, which is known to contain regions of homology with other bacterial flagellins [G. S. Gassman et al., “Analysis of the
Borrelia burgdorferi
GeHo fla Gene and Antigenic Characterization of Its Gene Product.”,
J. Bacteriol
., 173, pp. 1452-59 (1991)] and a 93kDa protein said to be localized to the periplasmic space [D. J. Volkman et al., “Characterization of an Immunoreactive 93 kDa Core Protein of
Borrelia burgdorferi
With a Human IgG Monoclonal Antibody”,
J. Immun
., 146, pp. 3177-82 (1991)].
Recently, immunization of mice with recombinant OspA has been shown to be effective to confer long-lasting protection against subsequent infection with
B. burgdorferi
[E. Fikrig et al., “Long-Term Protection of Mice from Lyme Disease by Vaccination:with OspA”,
Infec. Immun
., 60, pp. 773-77 (1992)]. However, protection by the OspA immunogens used to date appears to be somewhat strain specific, probably due to the heterogeneity of the OspA gene among different
B. burgdorferi
isolates. For example, immunization with OspA from
B. burgdorferi
strain N40 confers protection against subsequent infection with strains N40, B31 and CD16, but not against strain 25015 [E. Fikrig et al., “
Borrelia burgdorferi
Strain 25015: Characterization of Outer Surface Protein A and Vaccination Against Infection”,
J. Immun
., 148, pp. 2256-60 (1992)].
Immunization with OspB has also been shown to confer protection against Lyme disease but not to the same extent as that conferred by OspA [E. Fikrig et al., “Roles of OspA, OspB, and Flagellin in Protective Immunity to Lyme Borreliosis in Laboratory Mice”,
Infec. Immun
., 60, pp. 657-61 (1992)]. Moreover, some
B. burgdorferi
are apparently able to escape destruction in OspB-immunized mice via a mutation in the OspB gene which results in expression of a truncated OspB protein [E. Fikrig et al., “Evasion of Protective Immunity by
Borrelia burgdorferi
by Truncation of Outer Surface Protein B”,
Proc. Natl. Acad. Sci
., 90, pp. 4092-96 (1993)]. OspC has also been shown to have protective effects in a gerbil model of
B. burgdorferi
infection. However, the protection afforded by immunization with this protein appears to be only partial [V. Preac-Mursic et al., “Active Immunization with pC Protein of
Borrelia burgdorferi
Protects Gerbils against
B. burgdorferi
Infection”,
Infection
, 20, pp. 342-48 (1992)].
As prevention of tick infestation is imperfect, and Lyme disease may be missed or misdiagnosed when it does appear, there exists a continuing urgent need for the determination of additional antigens of
B. burgdorferi
and related proteins

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