Surface antigens and proteins useful in compositions for diagnosis and prevention of lyme disease

A technology of composition and fusion protein, applied in the direction of peptide/protein composition, antibacterial immunoglobulin, drug combination, etc.

Inactive Publication Date: 2000-08-02
TULANE EDUCATIONAL FUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, repeated administration of the OspA vaccine may be required to maintain effective antibody titers

Method used

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  • Surface antigens and proteins useful in compositions for diagnosis and prevention of lyme disease
  • Surface antigens and proteins useful in compositions for diagnosis and prevention of lyme disease
  • Surface antigens and proteins useful in compositions for diagnosis and prevention of lyme disease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0144] Example 1 Borrelia strains and antibodies

[0145] A. Bacterial strains

[0146] The B. burgdorferi (narrow sense) JD1 strain was obtained from the Center for Disease Control and Prevention [J. Piesman et al., J. Clin. Microbiol., 25:557-558 (1987) and T.G. Schwan et al. , J. Clin. Microbiol., 27:1734-1738 (1989)] and low passage (<7) frozen stocks were preserved. B31 is also a strain of B. burgdorferi (narrow sense) purchased from CDC and deposited as described above. IP90 is a strain of B. garinii, which was also provided by CDC.

[0147] B. burgdorferi organisms were grown at 34°C in BSK-H medium (Sigma Chemical Co., St. Louis, Mo).

[0148] B. Monkey Antibody

[0149] Serum antibodies were incubated with live spirochetes from macaques that had been infected with B. burgdorferi strain JD1 through pupal bites of Ixodes scapularis. Unbound antibody was removed by washing from the spirochetes, and bound antibody was removed with a low pH buffer. Monkey antibodies ...

Embodiment 2

[0153] Example 2 Preliminary Identification of P39.5 of IP90

[0154] Antibodies from part B of Example 1 were reacted with Western blots of whole extracts of JD1, B31 and IP90 spirochetes. Western blotting was performed by electrophoresis of antigen preparations on 15% acrylamide mini gels (10 x 10 x 0.1 cm) and 5% acrylamide stacking gels. Dispense 20 µl per lane containing 7 x 10 8 Bacterial lysate or 25 micrograms of protein (measured at 280nm OD) (there were 16 single lanes across the preparative lane; therefore 400 micrograms of protein were loaded on each preparative gel). Electrophoresis was performed using a buffer in U. Laemmli, Nature, 227:680-685 (1970) at a constant pressure of 23 mA using a mini gel apparatus (Integrated Separation Systems, Hyde Park, MA). For immunoblotting, 22 volts were used in a Mighty Small transfer unit (Hoeffer Scientific Instruments, San Francisco, CA) as described by H. Towbin et al., Proc. Natl. Acad. Sci. USA 76:4350-4354 (1979). Pr...

Embodiment 3

[0157] Example 3 Antibody-dependent complement-mediated killing of JD1, B31 and IP90 spirochetes

[0158] Tick-inoculated monkey sera were used in the ADCK assay as described below. Thaw frozen B. burgdorferi samples rapidly at 37°C and incubate until they reach mid-log phase (approximately 3 days, 1-2 x 10 7 Spirochetes / ml), centrifuged at 8000×g for 2 minutes, resuspended in BSK-H medium and fixed. ADCK assays were performed in duplicate in 96-well tissue culture plates (Costar). Dissolve a total of 5-6 x 10 in 25 µl of BSK-H medium 5 Spirochetes were added to each well containing 50 microliters of heat-inactivated (56°C, 30 minutes) serum samples (diluted 1:10 in the same medium). Before adding 25 μl of complement (normal monkey serum), in 3% CO 2 , 5% O 2 and balance N 2 Incubate the plate at 34°C for 20 minutes in the gas mixture. After 18-24 hours of incubation under the same conditions, the total number of dead (immobile) and live (motile) bacteria was quantified...

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Abstract

A novel isolated Borrielia burgdorferi sensu lato surface antigen is characterized by a relative molecular mass of 39.5 kDa. This antigen is expressed in vitro by spirochetes of a B. burgdorferi sensu lato strain. This antigen induces antibodies which kill spirochetes of a B. burgdorferi sensu lato strain by ADCK in vitro. Novel Borrelia cassette string protein or fragments thereof are also useful, as is the P39.5 protein in diagnosing Lyme disease and in compositions for treatment or prophylaxis thereof.

Description

[0001] This invention was supported in part by National Institutes of Health Grant Nos. ROI AI35027 and RR / AE00164-32. The US Government has certain rights in this invention. field of invention [0002] The present invention relates generally to the field of medicaments and diagnostic compositions for the diagnosis, treatment and prevention of Lyme borreliosis. More specifically, the present invention provides isolated Borrelia native surface antigens and antibodies thereof for use in the diagnosis, treatment or prevention of Lyme disease. Background of the invention [0003] Borrelia burgdorferi (broad sense) is a generic term that includes several Borrelia species associated with Lyme borreliosis (Lyme disease) and thought to be the main cause: B. burgdorferi (narrow sense), B. garinii, and B. afezlii. The disease is transmitted by the bite of various ticks of the genus Ixodes that carry spirochetes. In the United States, the primary host of infection is the white-legge...

Claims

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Application Information

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IPC IPC(8): G01N33/53A61K38/00A61K39/00A61K39/02A61K39/395A61P31/04C07K14/20C07K16/12C07K16/42C12N15/09C12N15/31C12P21/02G01N33/569
CPCA61K38/00C07K14/20A61K39/00A61P19/02A61P31/04Y02A50/30
Inventor M·T·菲利浦
Owner TULANE EDUCATIONAL FUND
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