Conserved metalloprotease epitopes

Details Conserved metalloprotease epitopes Conserved metalloprotease epitopes

1996-12-20

2001-08-07

Hutzell, Paula K. (Department: 1645)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Bacterium or component thereof or substance produced by said...

C424S184100, C424S190100, C424S234100, C424S130100, C424S094670, C424S261100, C424S246100, C424S185100, C424S197110, C530S300000, C530S324000, C530S325000, C530S326000, C530S327000, C530S328000, C530S350000, C530S387100

Reexamination Certificate

active

06270777

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the field of immunization against bacterial diseases. In particular, it relates to immunization against diseases caused by bacteria which secrete zinc metalloproteases and to therapeutic vaccines for treatment of such diseases.
BACKGROUND OF THE INVENTION
In the description which follows, references are made to certain literature citations which are listed at the end of the specification.
Many bacteria produce and secrete zinc metalloproteases. For example,
Pseudomonas aeruginosa
produces at least two zinc metalloproteases, elastase and alkaline protease. Elastase degrades several important biological substances including elastin, immunoglobulins, collagen, transferrin and complement components (1). Alkaline protease has been shown to degrade C1q and C3 proteins of serum complement (2) and gamma interferon (3)
P. aeruginosa
also secretes Las A protease, which has both elastolytic and staphylolytic activity and has some properties of a metalloprotease (4-6).
Burkholderia (Pseudomonas) cepacia
produces both a 36 kDa zinc metalloprotease (PSCP) which is immunologically related to elastase and a related 40 kDa protease (7).
Bacillus thermoproteolyticus
secretes thermolysin, a heat-stable neutral zinc metalloprotease. There is 28% sequence homology between
P. aeruginosa
elastase and thermolysin (8), with greater homology in certain regions, particularly around the active site. However, comparison of the three-dimensional structures of thermolysin and
P. aeruginosa
elastase reveals a striking similarity (9).
Vibrio cholerae
secretes a 33 kDa zinc metalloprotease, HA/protease, (10) which can cleave biologically important substrates such as mucin, fibronectin and bactoferritin (11). Hase and Finkelstein (12,13) have demonstrated that the
V. cholerae
HA/protease is related to
P. aeruginosa
elastase.
The bacterial metalloproteases have been shown to contribute to the virulence of many pathogenic organisms (14) which cause serious health problems.
For example, pulmonary dysfunction as a result of chronic airway infection is responsible for the vast majority of deaths in cystic fibrosis (CF) patients (15). Despite advances in microbial therapy, the treatment and prevention of infections due to
P. aeruginosa
and
B. cepacia
remains a clinical challenge (15). Although major efforts are underway to develop gene therapy as a treatment for CF patients, the practical applications of gene therapy and potential for success may not be determined for some time. Other avenues of infection control continue to be an important area for investigation.
P. aeruginosa
has been reported to be present in 60% of respiratory tract cultures from CF patients and once colonization has occurred,
P. aeruginosa
is difficult or impossible to eradicate (16). A large array of virulence factors have been identified and shown to contribute to the pathogenesis of
P. aeruginosa
infections. These include elastase, alkaline protease, exotoxin A, exoenzyme S, pyochelin, pyoverdin, phospholipase C, pili, outer membrane proteins, lipopolysaccharide (LPS) and alginate.
B. cepacia
is a nosocomial pathogen which has been isolated with increasing frequency from respiratory infections in CF patients over the past 20 years (15). Acquisition of
B. cepacia
can pose particular problems because of its resistance to many anti-pseudomonal antibiotics. For example, in one study of 55 CF patients with
B. cepacia
pulmonary infection, 39 (70%) had acquired multiresistant strains (17). Once acquired,
B. cepacia
is nearly impossible to eradicate.
The majority of strains (90%) of
B. cepacia
are protease positive (18). Proteases appear to be the major extracellular virulence factors produced by
B. cepacia
. McKevvit et al. (19) isolated a zinc metalloprotease, designated PSCP, which was produced by 90% of
B. cepacia
CF isolates. This protease was shown to degrade casein, gelatin, collagen, but not elastin and to cause bronchopneumonia when instilled intratracheally into rats (19).
Besides having a direct role in tissue destruction and injury, bacterial proteases such as elastase can function to modulate the host immune system and assist the bacterium in evading host defences (20-22).
Numerous studies since the late 1970's have analyzed the presence of serum antibodies to several
P. aeruginosa
antigens in CF patients (23-27). These studies all conclude that CF patients make antibodies to a variety of
P. aeruginosa
antigens and that elevated titres generally correlate with severity of disease.
Although CF patients produce high levels of antibodies to
P. aeruginosa
, several studies have indicated that these antibodies are not effective in clearance of the organism from the lungs.
These studies indicate that there remains a need for therapeutic strategies to improve the production of effective antibodies to combat infections with organisms such as
P. aeruginosa
and
B. cepacia.
SUMMARY OF THE INVENTION
The inventors have identified two regions of the
P. aeruginosa
elastase amino acid sequence, and within these regions, two epitopes, which are recognized by antibodies which neutralise the proteolytic activity of
P. aeruginosa
elastase and other thermolysin-like proteases.
Many pathogenic organisms secrete zinc metalloproteases which are thermolysin-like and their virulence is related to secretion of these enzymes.
Peptides corresponding to the two identified regions of amino acid sequence, and peptides comprising fragments of these sequences, provide immunogenic compositions which can be administered to a host to stimulate production of neutralising antibodies and protect the host against diseases caused by pathogens which secrete thermolysin-like metalloproteases.
Antibodies raised against the peptides of the invention also recognise serralysin-like proteases and immunization with these peptides may also be used to provide protection against pathogens secreting serralysin-like proteases.
In accordance with one embodiment, the invention provides a peptide comprising the amino acid sequence
VSHGFTEQNSGLIYRGQSGGMNEAF (Sequence ID NO:1)
or a fragment or analogue thereof.
In accordance with a further embodiment, the invention provides a peptide comprising the amino acid sequence
HGFTEQNSG (Sequence ID NO:3).
In accordance with a further embodiment, the invention provides a peptide comprising the amino sequence
SGALRYMDQPSRDGRSIDM (Sequence ID NO:11)
or a fragment or analogue thereof.
In accordance with a further embodiment, the invention provides a peptide comprising the amino acid sequence
RYMDQPSRD (Sequence ID NO:14).
In accordance with a further embodiment, the invention provides an immunogenic composition comprising at least one active component selected from the group consisting of:
(a) a peptide comprising the amino acid sequence HGFTEQNSG;
(b) a peptide comprising the amino acid sequence RYMDQPSRD;
(c) a peptide comprising the amino acid sequence VSHGFTEQNSGLIYRGQSGGMNEAF;
(d) a peptide comprising the amino acid sequence SGALRYMDQPSRDGRSIDM;
(e) a fragment or analogue of a peptide of (a), (b), (c) or (d);
(f) a purified and isolated nucleic acid molecule encoding a peptide of (a), (b), (c) or (d); and
(g) a nucleotide sequence which hybridises under stringent conditions to any of the nucleic acid molecules of (f)
and a pharmaceutically acceptable carrier, the at least one active component producing an immune response when administered to a host.
In accordance with a further embodiment, the invention provides an antibody or antiserum specific for a peptide selected from the group consisting of
(a) VSHGFTEQNSGLIYRGQSGGMNEAF;
(b) SGALRYMDQPSRDGRSIDM;
(c) HGFTEQNSG;
(d) RYMDQPSRD; and
(e) a fragment or analogue of a peptide of (a), (b), (c) or (d).
In accordance with a further embodiment, the invention provides a method for protecting a susceptible host against a disease caused by a bacterial pathogen which secretes a zinc metalloprotease.
In accordance with a further embodiment, the invention provides a purified isolated nucleic acid molecule

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