Decorin binding protein essential peptides and methods of use

Details Decorin binding protein essential peptides and methods of use Decorin binding protein essential peptides and methods of use

2000-06-16

2003-02-11

Smith, Lynette R. F. (Department: 1645)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Amino acid sequence disclosed in whole or in part; or...

C424S184100, C424S234100, C424S130100, C530S324000, C435S007200

Reexamination Certificate

active

06517838

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of molecular biology and microbiology. More particularly, it concerns a method of use of residues necessary for ligand binding in a decorin binding protein, DbpA. More particularly, certain embodiments concern methods and compositions comprising DNA segments, and peptides derived from bacterial species.
2. Description of Related Art
Lyme disease (LD) is a chronic, multisystemic disease caused by the spirochete
Borrelia burgdorferi
(1). It is transmitted to humans and other mammals during the blood meal of Ixodes ticks (2, 3) and it is the most common vector-borne disease in the United States (1). This initial skin infection is often accompanied by a local rash (erythema migrans) which can be followed by a general flu-like illness. Untreated Lyme borreliosis can develop into a chronic, multisystemic disorder that may affect the joints (Lyme arthritis), skin, heart, and central nervous system (1).
Microbial adhesion to and colonization of host tissue is an early, critical event in an infection process. In the case of LD, host tissue adherence appears to be of importance during different stages of the disease process. Initially, during an infected tick's blood meal, a small number of spirochetes are deposited in the dermis of the host where the bacteria appear to colonize collagen fibers (32,33). As the infection disseminates to other tissues, bacteria may colonize additional extracellular matrix (ECM) structures and host cells may be involved. Adherence of
Borrelia burgdorferi
to collagen fibers involves a specific binding of the spirochete to decorin, a dermatan sulfate proteoglycan which is associated with and “decorates” collagen fibers (17, 18). A dermal route of entry into the host appears to be important for the development of disease. Spirochetes administered intravenously are rapidly and effectively cleared by Kupffer cells in the liver (34), whereas those inoculated intradermaly consistently establish infection (35). Perhaps the initial dermal colonization allows the organism to adapt to in vivo conditions before blood stream dissemination.
The genes coding for the two decorin-binding proteins (DbpA and DbpB) which are expressed at the surface of the spirochete as lipoproteins and act as adhesins of the MSCRAMM (Microbial Surface Component Recognizing Adhesive Matrix Molecule) family have been cloned and sequenced. Recombinant forms of DbpA and DbpB are capable of binding to decorin and DbpA effectively inhibits the adherence of
Borrelia burgdorferi
to a decorin substrate (17).
Active and passive immunization of mice using DbpA and DbpA antiserum, respectively, protected against challenge with
B. burgdofferi
(10,11). DbpA sequences vary significantly among different Borrelia strains. Nevertheless, antibodies to one recombinant form of DbpA can confer broad protection against various strains, suggesting that at least some immunoprotective epitopes are conserved.
During transmission from the tick's midgut to the vertebrate host, Borrelia spirochetes undergo antigenic modulations, which includes the up- and down-regulation of various surface-exposed proteins (e.g. OspA, B. and C) (4, 5). OspA has been the most widely studied Borrelia protein, and vaccine trials in both murine models of LD and in humans indicate that this protein can confer protection to infection against the Lyme spirochete (6-8).
However, some drawbacks in OspA-derived vaccine formulations exist. Since OspA expression occurs primarily in the tick vector, high anti-OspA titers need to be maintained to inhibit spirochete transmission (9-11) and anti-OspA antibodies lose efficacy against host-adapted Borrelia (12). Furthermore, protection against heterologous Borrelia strains is poor to nonexistent (13-16). Additionally, modulation of OspA expression by
B. burgdorferi
may limit the efficacy of anti-OspA antibodies to spirochetes residing in the midgut, and cross-reactivity of OspA with human lymphocyte function-associated antigen-1 (hLFA-1) may be a critical factor in treatment-resistant Lyme arthritis (5, 9, 10, 24).
The decorin binding proteins (DbpA and DbpB) of
Borrelia burgdorferi
are adhesins of the MSCRAMM family (17) which recognize the proteoglycan decorin and may play a role in the colonization of the skin and establishment of disease (17, 18). Unlike OspA, DbpA expression on
B. burgdorferi
is expressed in the vertebrate host (19). Recent work testing DbpA as a vaccine candidate against heterologous Borrelia strains demonstrated that DbpA-based formulations are capable of conferring protection against challenge with
B. burgdorferi
(19), and may be more effective than OspA formulations. (11).
Applicants' discovery of antigenic determinants on proteins capable of eliciting protective immunity are used to design novel peptides that may have the potential of mimicking the protective effects of native proteins. The risk of cross-reactivity against the host is reduced since peptide vaccines are chemically defined structures. Furthermore, peptides are stable and no infectious materials are used in their production (25).
SUMMARY OF THE INVENTION
Accordingly, the present invention provides several novel methods and related biological compositions for protecting against infection with
Borrelia burgdorferi.
Also provided are methods of identifying substances that alter or modulate the interaction of various decorin binding proteins with decorin, and the substances so identified.
The invention first provides a protein fragment or related peptide that spans the critical binding regions required for DbpA/decorin adhesion.
The invention also provides a method of inhibiting the binding of
B. burgdorferi
to decorin by contacting the decorin with a protein fragment or related peptide that spans the critical binding regions required for DbpA/decorin adhesion and thus preventing infection with
B. burgdorferi.
It is a further aspect of the present invention to provide isolated DbpA-derived peptide fragments or related peptides that are able to inhibit adhesion to the immobilized extracellular matrix of host cells or the surface of implanted biomaterials.
It is a further object of the present invention to provide a vaccine which can be used in generating an immonogenic reaction in a host and which thus can be used in treating or preventing infection by bacteria such as
B. burgdorferi.
It is still further an object of the present invention to generate antisera and antibodies to the decorin binding proteins from
B. burgdorferi bacteria
which can also be useful in methods of treatment which can inhibit binding of the
B. burgdorferi
bacteria to host cells or to implanted biomaterials and thus be employed in order to treat or prevent
B. burgdorferi
infections.
It is a further object of the present invention to provide improved materials and methods for detecting and differentiating decorin-binding proteins in
B. burgdorferi
in clinical and laboratory settings.
These and other objects are provided by virtue of the present invention which comprises a method of using an isolated decorin binding protein identified as the DbpA protein from
B. burgdorferi,
which has been determined to bind to decorin, along with the sequences governing the specific decorin-binding domains of this proteins. The isolated
B. burgdorferi
proteins of the present invention, or active portions or fragments thereof can thus be utilized in methods of treating or preventing
B. burgdorferi
infection through the inhibition of the ability of the bacteria to bind to decorin, or through the development of antibodies thereto which will prevent or inhibit the bacteria's ability to bind to host cells.
In another aspect of the present invention, there is also provided antisera and antibodies generated against the decorin binding proteins of the present invention which also can be utilized in methods of treatment which involve inhibition of the attachment of the DbpA proteins to decorin.
Accordingly, in

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