Antibodies to cloned Leptospira outer membrane protein and...

Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Monoclonal antibody or fragment thereof

Reexamination Certificate

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C530S388400

Reexamination Certificate

active

06309641

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to an antigenic preparation and specifically to a Leptospira outer membrane protein (OmpL2) which is used to induce a protective immune response in animals. Such a protein can be used immunologically as a vaccine for leptospirosis caused by this organism. Alternatively, diagnosis of leptospirosis 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
Leptospirosis is a widespread zoonotic disease caused by pathogenic strains of Leptospira which are capable of infecting most mammalian species. At present, there are six pathogenic species and three nonpathogenic species within the genus Leptospira. Infection occurs either through direct contact with an infected animal or indirect contact with contaminated soil or water. In livestock, the disease causes economic losses due to abortion, stillbirth, infertility, decreased milk production, and death.
Efforts to control leptospirosis have been hampered because virulent leptospires have the capacity for both long-term survival in the environment as well as persistent infection and shedding by wildlife and livestock. Currently available leptospiral vaccines produce short-term immunity and do not provide cross-protection against many of the 170 serovars of pathogenic Leptospira (Thiermann, et al.,
J. Am. Vet. Med. Assoc
. 184:722, 1984). These vaccines consist of inactivated whole organisms or outer envelope preparations which produce seroreactivity as determined by microscopic agglutination of intact organisms. The nature of the protective immunogens in these vaccine preparations has not been conclusively elucidated, although several lines of evidence suggest that lipopolysaccharide-like substance (LLS) may confer a degree of protection.
The pathhogeanesis of leptospircsls is veny similar to that of other spirochetal diseases, including syphillis (caused by
Treponema pallidum
) and Lyme borreliosis (caused by
Borrelia burgdorferi
). Both syphilis and Lyme borreliosis are characterized by widespread dissemination early in the course of disease, including invasion of the central nervous system. Leptospira share this ability with other pathogenic spirochetes such that meningitis is a common manifeslation of leptospirosis. Another feature of spirochetal infections is the ability to persist chronically in the host, as manifested in cases of tertiary syphilis and chronic Lyme arthritis.
In attempting to identify leptospiral outer membrane proteins (OMPs), previous research was unsuccessful due to such problems as: 1) the techniques used to identify surface-exposed proteins probably involved damage to the fragile leptospiral outer membrane resulting in exposure of subsurface structures; 2) putative surface-exposed proteins that were identified included a 35-36 kD doublet corresponding to Leptospira endoflagella (Kelson, et al.,
J. Med. Microbiol
. 26:47, 1988), which are subsurface structures in spirochetes; and 3) use of SDS which nonselectively solubilizes proteins irrespective of their native cellular location.
Nunes-Edwards, et al. (
Infect. Immun
. 48:492, 1985) introduced the use of radioimmunoprecipitation and cell fractionation schemes based on the use of SDS in an effort to identify leptospiral OMPs. The leptospires used in their radioimmunoprecipitation procedure were subjected to high speed centrifugation (20,000×g) prior to the addition of antibody. Such high centrifugal forces cause mechanical disruption of the leptospiral outer membrane. Niikura, et al. (
Zbl. Bakt. Hyg. A
. 266:453, 1987) immunoprecipitated SDS-solubilized extracts of virulent and avirulent strains of
L. interrogans
serovar copenhageni that had been labeled by lactoperoxidase-catalyzed surface radioiodination. Since both of these studies precipitated a 35-36 kD doublet consistent with leptospiral endoflagella, there was a concern as to whsther the other prot-ins identified might also have a subsurface rather than a surface location.
Jost, et al. (
J. Med. Microbiol
. 27:143) characterized a monoclonal antibody with specificity for a 35 kD proteinase K sensitive antigen which was present in a leptospiral outer envelope preparation. However, to demonstrate binding of the monoclonal antibody by immunoelectron microscopy, the leptospiral outer membrane had to be disrupted. Doherty, et al. (
J. Med. Microbiol
. 28:143) cloned two leptospiral proteins represented in an SDS-generated outer membrane preparation of
L. interrogans
, but did not provide corroborating evidence that these proteins are either constituents of the outer membrane or are surface-exposed.
Unsuccessful research on the identification of Leptospira and
T. pallidum
OMPs has shown the importance of taking into account spirochetal outer membrane fragility and the lack of outer membrane selectivity of ionic detergents such as sodium dodecyl sulfate (SDS) (Cunningham, et al.,
J. Bacteriol
. 170:5789, 1988; Penn, et al.,
J. Gen. Microbiol
. 131:2349, 1985; Stamm, et al.,
Infect. Immun
. 55:2255, 1987). Outer membrane proteins are of great importance because they play a key role in bacterial pathogenesis. The identification of outer membrane proteins involved in Leptospira pathogenesis is significant to understanding not only leptospiral outer membrane proteins and their involvement in pathogenesis, but also to understanding other spirochetal outer membrane proteins and their role in pathogenesis.
SUMMARY OF THE INVENTION
The present invention is based on the identification of OmpL2 as a leptospiral outer membrane protein which is associated with pathogenic strains of Leptospira. Due to spirochetal outer membrane fragility and the fact that outer membrane proteins are present in small amounts, there have been no definitive reports of membrane spanning spirochetal outer membrane proteins until the present invention. The invention describes a 63 kD outer membrane protein from Leptospira and the gene encoding the protein. The deduced amino acid sequence has a typical leader peptidase I cleavage site, implying export beyond the inner membrane. The 63 kD protein has been designated OmpL2 for outer membrane protein of Leptospira. This immunogenic polypeptide is useful for inducing an immune response to pathogenic Leptospira as well as providing a dianostic target for leptospirosis.


REFERENCES:
patent: 5679353 (1997-10-01), Hall et al.
P.L. Nunes-Edwards et al., Infection and Immunity 48(2):492-497, May 1985.*
K.W. Ruby et al.,Biologicals20:259-266, 1992.*
Dunn et al., “Outer Surface Protein A (OspA) from the Lyme Disease Spirochete, Borrelia burgdorferi: High Level Expression and Purification of a Soluble Recombinant Form of OspA,”Protein Expression and Purification,vol. 1, No.2, (2 pgs.).
Kida et al., “Immunological and Morphological Analysis of Sodium Dodecyl Sulfate Extract of Leptospira,”Zbl. Bakt. HY6 I. ABT. Orig. A236, pp. 328-335 (1976).
Nicholson, Vivian and Prescott, John, “Outer Membrane proteins of three pathogenic Leptospira species,”Veterinary Microbiology,36, pp. 123-138 (1993).
Zuerner et al., “Characterization of outer membrane and secreted proteins of Leptospira interrogans serovar pomona,”Microbial Pathogenesis,10:pp. 311-322, (1991).
Brown et al., “Protein and Antigen Profiles of Prevalent Serovars of Leptospira interrogans,”Infection and Immunity,vol. 59, No. 5, pp. 1772-1777 (May, 1991).
Haake et al., “Molecular Cloning and Sequence Analaysis of the Gene Encoding OmpL1, a Transmembrane Outer Membrane Protein of Pathogenic Leptospira spp.,”Journal of Bacteriology,vol. 175, No. 13, pp. 4225-4234, (Jul., 1993).
Haake et al., “Molecular Cloning and Sequence Analaysis of the Gene Encoding OmpL1, a Transmembrane Outer Membrane Protein of Pathogenic Leptospira spp.,”Journal of Bacteriology,vol. 175, No. 13, pp. 4225-4234, (Jul., 1993).
Haake et al., “Molecular Cloning of 1-11 the Gene Encoding OmpL2, a putative TonB-dependent Outer Membrane Protein of Leptospira Alstoni”,Abstracts of the G

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